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MUSHROOMS AS
FUNCTIONAL FOODS



MUSHROOMS AS
FUNCTIONAL FOODS



Edited by

Peter C. K. Cheung

The Chinese University of Hong Kong




WILEY

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Library of Congress Cataloging-in-Publication Data:

Cheung, Peter C. K.

Mushrooms as fuctional foods / Peter C.K. Cheung,
p. cm.

Includes bibliographical references and index.
ISBN 978-0-470-05406-2 (cloth)

1. Mushrooms — Therapeutic use. 2. Functional foods. I
[DNLM: 1. Agaricales — chemistry. 2. Food Analysis. 3.
QW 180.5.B2. c526m 2008]
RM666.M87C45 2008
615'.3296— dc22



. Title.
Food Technology.



2007050403



Printed in the United States of America



10 98765432 1



This book is dedicated to my family members:
Carmen, Timothy, Rebekah, and Anthony Cheung



CONTENTS



Foreword xv

Preface xvii

Acknowledgments xix

Contributors xxi

1 Overview of Mushroom Cultivation and Utilization

as Functional Foods 1

Shu-Ting Chang

1.1. Introduction 1

1.2. What Are Mushrooms? 3

1.2.1. Definition of a Mushroom 3

1 .2.2. Ecological Classification of Mushrooms 4

1.2.3. Identification of Mushrooms 4

1.3. Concept of Mushroom Biology and Applied

Mushroom Biology 6

1.3.1. Mushroom Biology 6

1.3.2. Applied Mushroom Biology 7

1.3.3. Impact of Applied Mushroom Biology 9

1.3.3.1. Nongreen Revolution 9

1.3.3.2. Mushroom Bioremediation 11

1.4. Mushroom Cultivation 11

1 .4. 1 . Major Phases of Mushroom Cultivation 1 2

1.4.2. Cultivation of Several Selected Mushrooms 13

1 .4.2. 1 . Cultivation of it Agaricus 14

1.4.2.2. Cultivation of Lentinula edodes 14

1.4.2.3. Cultivation of Pleurotus sajor-caju 17

1.4.2.4. Cultivation of Volvariella 17

1.4.2.5. Cultivation of Agaricus brasiliensis 18



vii



Viii CONTENTS



1.4.2.6. Cultivation of Ganoderma lucidum 19

1.4.3. Utilization of Mushroom Germplasm 20

1.5. World Mushroom Production 21

1.6. Mushroom Biotechnology 23

1.6.1. Nutritional and Medicinal Value of Mushrooms 23

1.6.2. Nutriceuticals and Dietary Supplements 24

1 .7. Development of World Mushroom Industry Movements 25

1.7.1. International Movement for Edible Mushrooms 26

1.7.2. International Movement for Medicinal Mushrooms 27

1.7.3. International Movement for Wild Mushrooms 27

1.8. Concluding Remarks 28
References 29

2 Molecular Analysis and Genomic Studies of Shiitake

Mushroom Lentinula edodes 35

Hoi-Shan Kxvan and Winnie W. Y. Chum

2. 1 . Introduction 35

2.2. Isolation of Genes 36

2.2.1. Growth 36
2.2.1.1. Substrate-Utilizing Genes 36

2.2.2. Development 37

2.2.2.1. Mating-Type Genes 38

2.2.2.2. Genes Differentially Expressed in

Dikaryotic Mycelium 38

2.2.2.3. Genes for Initial Fruiting Bodies/Primordium
Formation 38

2.2.2.4. Genes for Mature Fruiting

Bodies Formation 44

2.2.3. Physiological Processes in Lentinula edodes 47

2.2.3.1. Signal Transduction 47

2.2.3.2. Energy Production 47

2.2.3.3. Structural Proteins in Development 48

2.3. Molecular Genetics 48

2.3.1. Generation of Markers 49

2.3.2. Typing/Fingerprinting 50

2.3.3. Genetic Mapping 50



CONTENTS ix



2.4. Functional Genomic Approaches for Gene

Expression Analysis 50

2.4.1. Differential Display: RAP-PCR 51

2.4.2. cDNA Representation Difference Analysis 52

2.4.3. SAGE and LongSAGE 52

2.4.3. 1 . SAGE Profiles: Mycelium to Primordium 53

2.4.3.2. SAGE Profiles: Fruiting Bodies 53

2.4.4. cDNA Microarray 53

2.4.5. Expressed Sequence Tag 54

2.4.6. Yeast Two-Hybrid System 54

2.4.7. Sequencing-by-Synthesis Approach

(454 Life Science) 54

2.5. Transcriptional Regulation 55

2.5.1. Transcriptional Factors 55

2.5.2. Promoter Analysis 55

2.6. Transformation 56

2.6.1. Transformation Methods 56

2.6.1.1. PEG-Mediated Transformation 56

2.6.1.2. Restriction Enzyme-Mediated Integration 57

2.6.1.3. Others 58

2.6.2. Lentinula edodes Genes Used in Transformation 58

2.7. Process Analysis 59

2.7.1. Postharvest Studies 59

2.7.2. Stress Responses 59

2.7.2. 1 . Studies of Temperature Stress in Mushrooms 59

2.7.2.2. Studies of Molecular Chaperones in Fungi 59

2.7.3. Lignocellulose Degradation 60

2.7.4. Meiosis 60

2.8. Conclusion 61
References 61

3 Nutritional Value and Health Benefits of Mushrooms 71

Peter C. K. Cheung

3.1. Introduction 71

3.2. Wild and Cultivated Edible Mushrooms 72

3.3. Production of Cultivated Mushrooms 72



X CONTENTS



3.4. Nutritional Composition 73
3.4.1. Conventional Edible Mushrooms 73

3.4.1.1. Moisture 73

3.4.1.2. Protein and Amino Acids 74

3.4.1.3. Fat 75

3.4.1.4. Ash and Minerals 75

3.4.1.5. Vitamins 76

3.4.1.6. Dietary Fiber 77

3.4.1.7. Carbohydrates 78

3.4.1.8. Energy 78

3.4.1.9. Other Components 78

3.5. Newly Cultivated/Nonconventional Mushrooms 79

3.6. Nutritional Evaluation 80

3.6.1. General Aspects 80

3.6.2. Biological Methods for Nutritional Evaluation 80

3.6.3. Mushroom Protein Quality 87

3.7. Health Benefits of Edible Mushrooms 89

3.7.1. General Aspects 89

3.7.2. Antioxidants in Mushrooms 89

3 .7.2. 1 . Bioactive Components and Their

Antioxidative Activities 89

3.7.2.2. Characterization of Mushroom Phenolic
Antioxidants 91

3.7.2.3. Biosynthesis of Phenolic Compounds

from Mushrooms or Fungi 93

3.7.3. Hypocholesterolemic Effect of Mushrooms 94

3.7.4. Hypoglycemic Effect of Mushrooms 97

3.8. Conclusion 99
References 99

4 Sclerotia: Emerging Functional Food Derived from Mushrooms 111

Ka-Hing Wong and Peter C. K. Cheung

4. 1 . Introduction 111

4.2. Concepts of Mushroom Sclerotia 112

4.3. Ontogeny of Sclerotia 112

4.3.1. Morphological Aspects 112

4.3.2. Physiological Aspects 114



CONTENTS Xi



4.3.2.1. Translocation 114

4.3.2.2. Exudation 115

4.4. Structure of Sclerotia 115

4.4.1. Rind 115

4.4.2. Cortex 116

4.4.3. Medulla 117

4.5. Cultivation of Mushroom Sclerotia 117

4.5.1. Sclerotia of Pleurotus tuber-regium (Fries) Singer 118

4.5.2. Sclerotia of Polyporus rhinocerus Cooke 1 19

4.5.3. Sclerotia of Wolfiporia cocos (Schw.) Ryv. Et Gilbn

[Poria cocos (Schw.) Wolf] 120

4.6. Biochemical, Nutritional, and Technological Characteristics of
Mushroom Sclerotia 121

4.6.1. Biochemical Components of Mushroom Sclerotia 121

4.6.1.1. Cell Walls 121

4.6.1.2. Extracellular Matrix 122

4.6.1.3. Cytoplasmic Reserves 122

4.6.2. Nutritional Evaluation of Mushroom Sclerotia 123

4.6.2.1. Proximate Composition 123

4.6.2.2. Sclerotial Dietary Fiber 124

4.6.3. Physicochemical and Functional Properties

of Mushroom Sclerotial DF 126

4.7. Biopharmacological Values of Mushroom Sclerotia of

P. tuber-regium, P. rhinocerus, and W. cocos 128

4.7.1. In Vitro Mineral Binding Capacity 128

4.7.2. In Vitro Fermentability 129

4.7.3. In Vivo Ca and Mg Absorption 131

4.7.4. Antitumor and Immunomodulatory Activities 132

4.8. Conclusion 134
References 134

5 Antitumor and Immunomodulatory Activities of

Mushroom Polysaccharides 147

Vincent E. C. Ooi

5.1. Introduction 1 47

5.2. Antitumor Polysaccharides from Mushrooms

(Higher Fungi) 149



Xii CONTENTS



5.3. Mechanisms of Antitumor Action of Mushroom Polysaccharides 153

5.3.1. Antiproliferation of Cancer Cells and Induction

of Apoptosis 153

5.3.2. Immunomodulation 161

5.3.2.1. Effects of Mushroom Polysaccharides

on Macrophages and Spleen Cells 163

5.3.2.2. Effects of Mushroom Polysaccharides

on NK Cells 167

5.3.2.3. Effects of Mushroom Polysaccharides

on DCs 168

5.3.2.4. Effects of Mushroom Polysaccharides

on Hematopoietic Stem Cells 170

5.3.3. Antimetastasis 171

5.3.4. Antiangiogenesis 172

5.4. Structure and Antitumor Activity Relationship of
Polysaccharides 173

5.4.1. Effect of Molecular Mass 174

5.4.2. Impact of Branching Configuration 174

5.4.3. Relationship of Antitumor Activity and Conformation 175

5.4.4. Improvement of Antitumor Activity by Chemical
Modifications 176

5.5. Conclusions 178
References 179

6 Regulatory Issues of Mushrooms as Functional Foods

and Dietary Supplements: Safety and Efficacy 199

Solomon P. Wasser and Eden Akavia

6.1. Introduction 199

6.2. Legal and Regulatory Issues of Introducing and
Controlling Dietary Supplements from Medicinal

Mushrooms in Different Countries 202

6.2. 1 . World Health Organization Guidelines 202

6.2.2. Codex Alimentarius 202

6.2.3. United States 203

6.2.4. European Union 208

6.2.5. Canada 210

6.2.6. Australia and New Zealand 212

6.2.7. Japan 213

6.2.8. Israel 215



CONTENTS Xiii



6.3. Safety and Diversity of Dietary Supplement Types from



Culinary-Medicinal Mushrooms 216

6.4. Submerged Culturing as Best Technique for Obtaining

Consistent and Safe Mushroom Products 220

6.5. Experiences of Seven Countries in Consolidating

Their Food Safety Systems 220

6.6. Summary 221
References 221



Index



227



FOREWORD



It has been over twenty years since the concept of "functional foods" was first
introduced as a factor in the analysis of foods after nutrients. Consumers are now
deeply interested in food bioactives that provide beneficial effects to humans in
terms of health promotion and disease risk reduction. They also demand more
detailed information about food factors in order to obtain appropriate functional
food products.

In Asian countries, like China and Japan, mushrooms have been collected and
cultivated for hundred of years. They have a long history of use for their health
promotion benefits. In recent years reports on the chemistry, and the nutritional
and functional properties of mushroom have been overwhelming. In the Journal
of Agricultural and Food Chemistry alone there have been more than 300 articles
related to mushrooms published since 1990. However, there is no in-depth com-
prehensive reference book of mushrooms as functional food available. The current
book of Professor C. K. Cheung, Mushrooms as Functional Foods, is a timely
and well welcomed book for scientists and students working in functional food
research.

Besides covering the agricultural production, nutritional values, and health ben-
efits of mushrooms, this book also introduces emerging molecular analysis and
functional genomics to the study of mushroom. Health benefits of mushrooms,
such as, antioxidative, hypocholesterolemic, and hypoglycemic effects are dis-
cussed in depth. Polysaccharides are the best known and potent mushroom-derived
substances with immunomodulating and antitumor activities and this topic has
been treated extensively in a separate chapter. Included also is a unique and useful
chapter on regulatory issues of mushrooms as functional foods in different coun-
tries.

Scientists and students who research mushrooms will certainly benefit from
reading this comprehensive monograph to gain in-depth knowledge for the devel-
opment of mushrooms into functional foods.

Chi-Tang Ho

Rutgers University



xv



PREFACE



Mushrooms have been known for their nutritional and culinary values as well as
viewed as tonics and used as medicines by humans for ages. In modern terms, they
can be considered as functional foods which can provide health benefits beyond the
traditional nutrients they contain. There are monographs that cover the medicinal
and healing properties of some individual traditional mushrooms and fungi such as
the Ganoderma, Shiitake mushroom, and Cordyceps for the general public. How-
ever, there are very few in-depth and up-to-date comprehensive reference books
in the scientific literature of both the basic and applied aspects of mushrooms as
functional foods.

This book is an integration of the recent research conducted on the biologi-
cal and chemical aspects of mushrooms when being utilized as a functional food.
Topics that are covered in this book range from the agricultural production of
mushrooms to the use of molecular biological techniques like functional genomics,
from nutritional values of newly cultivated mushroom species to the multifunc-
tional effects of the unconventional form of the mushroom (sclerotium), and from
the mechanistic actions of the physiological benefits and pharmacological prop-
erties of bioactive components in mushrooms to the regulations of their uses as
functional foods and dietary supplements in different parts of the world.

This comprehensive book should serve as a reference for scientists; chemists;
biologists; food manufacturers; students majoring in food science, nutrition, biol-
ogy, and biochemistry, to name a few; and all those who are interested in obtaining
a stronger background in the development of mushrooms and edible fungi into
functional foods.

Peter C. K. Cheung

The Chinese University of Hong Kong



xvii



ACKNOWLEDGMENTS



I want to thank the many colleagues and research collaborators who have
graciously given support and advice in the development of this book: Professor
Shu-ting Chang and Marilyn M. L. Yu, The Chinese University of Hong Kong;
Professor Lina Zhang and Dr. Mei Zhang, Wuhan University; and Nian-Lai
Huang, Sanming Mycological Institute. The clerical support provided by Yuk-fan
Ng and Kit-fong Tong is deeply appreciated.

P. C. K. C.



xix



CONTRIBUTORS



Eden Akavia, International Center of Biotechnology and Biodiversity of Fungi,
Institute of Evolution, University of Haifa, Haifa, Israel

Shu-Ting Chang, Department of Biology, The Chinese University of Hong Kong,
Hong Kong, China

Peter C. K. Cheung, Food and Nutritional Sciences Programme, Department of
Biology, The Chinese University of Hong Kong, Hong Kong, China

Winnie W. Y. Chum, Department of Biology, The Chinese University of Hong
Kong, Hong Kong, China

Hoi-Shan Kwan, Department of Biology, The Chinese University of Hong Kong,
Hong Kong, China

Vincent E. C. Ooi, Department of Biology and Institute of Chinese Medicine,
The Chinese University of Hong Kong, Hong Kong, China

Solomon P. Wasser, International Center of Biotechnology and Biodiversity of
Fungi, Institute of Evolution, University of Haifa, Haifa, Israel

Ka-Hing Wong, Department of Biology, The Chinese University of Hong Kong,
Hong Kong, China



xxi




(c) Pleurotus cornucopiae (Pco) (d) Pleurotus djamor (Pd)

Figure 3.2 Photos of dried samples of newly developed cultivated mushrooms.



(e) Pleurotus eryngii (Pe)



(f) Pleurotus eryngii var. ferulae (Pevf)






(I) Agrocybe aegehta (Aa) (m) Agaricus blazei (Ab)




(n) Agrocybe chaxinggu (Ac)
Figure 3.2 (Continued)




(q) Grifola frondosa (Gf)
Figure 3.2 (Continued)




(r) GK1 6 (s) Hericium erinaceus (He)




(t) Hypsizigus marmoreus (Hm)
Figure 3.2 (Continued)



(u) Hericium ramosum (Hr)



(v) Lentinula giganteus (Lg)




(w) LPK 15
Figure 3.2 (Continued)



(x) Pholiota adipose (Pa)



(y) Pholiota nameko (Pn)




(z) Strophaha rugoso-annulata (Sra)
Figure 3.2 (Continued)



CHAPTER 1



Overview of Mushroom Cultivation
and Utilization as Functional Foods

Shu-Ting Chang

Department of Biology, The Chinese University of Hong Kong, Hong Kong, China

CONTENTS

1 . 1 Introduction

1.2 What Are Mushrooms?

1.4 Mushroom Cultivation

1 . 5 World Mushroom Production

1.6 Mushroom Biotechnology
1 . 8 Concluding Remarks
References



1.1 INTRODUCTION

In 1957, R. Gordon Wasson, a world known amateur mycologist, proposed the
division of people into two classes for which he coined the following terms:

Mycophiles — Those who love and know their mushrooms intimately.
Mycophobes — Those who fear, dislike, and do not know their mushrooms.

I think all readers of this book belong to the former and not the latter.

Knowledge of numerous new mushroom species has accumulated through
time. The number of recognized mushroom species has been reported to be
14,000, which is about 10% of the total estimated mushroom species on the
earth (Hawksworth, 2001). China is estimated to have about 1500-2000 edible
mushroom species with 981 species identified. By 2002, 92 species have been
domesticated while 60 of these have been commercially cultivated (Mau et al.,



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



1



2 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

2004). However, mushrooms have nearly always been around, with a very long
and interesting history. Mushrooms have been found in fossilized wood that is
estimated to be 300 million years old, and almost certainly prehistoric man used
mushrooms collected in the wild as food. Recently, the importance of the role of
mushrooms in history was evidenced by the fact that the desert truffle, Terfezia
arnenari, was described in the Bible as "bread from heaven" and also "manna of
the Israelites" (Pegler, 2002).

It may be interesting to have a charming mushroom poem as a beginning for
this chapter: "Without leaves, without buds, without flowers, yet, they form fruit;
as a food, as a tonic, as a medicine, the entire creation is precious" (Chang and
Miles, 1989, p. 345). The first part describes the morphological and physiological
characteristics of mushrooms while the second states the nutritional and medicinal
properties of mushrooms.

Our attitudes to the phenomenon of nature are seldom based on simple
observation. There are, however, examples throughout history where certain
living things have inspired fear and loathing simply because they are regarded as
ugly species with peculiar behavior and supposedly evil. For example, in some
communities, bats, snakes, spiders, toads, and owls have all been associated with
the devil or regarded as harbingers of illness and even of death. This is one of the
reasons why some refer to the poisonous mushroom as a "toadstool." Actually,
the name has no scientific basis at all and should not be used in any situations,
although it is possible to find a toad sitting beside or even on top of a mushroom.
Mushrooms attract toads, not due to the mushroom itself, but because of the
various insects which are harbored in them. Insects certainly are interested in
mushrooms as a source of food (Chang, 2005).

It cannot be denied that some mushrooms, even though they represent less
than about 1% of the world's known mushrooms, are dangerous if eaten. Some
are deadly poisonous. But perhaps a more likely explanation for the widespread
abhorrence of wild mushrooms in communities is that they are by nature a rather
strange and mysterious group of organisms, quite unlike the green plants. In some
ancient communities, the seemingly miraculous manner of its growth without seed,
without leaf, and without bud, its fruiting body's sudden appearance after rain,
especially after lightning and thunderstorms, its equally rapid disappearance, and
its curious umbrellalike shape gave rise to a wealth of illusions and mythologies.

Fungi are found just about everywhere. Mushrooms, a special group of macro-
fungi, are rather more selective than other fungi in that the size of the fruiting body
requires the availability of more nutrients than are required for the production of
asexual spores by microfungi. Nevertheless, in damp places, such as tree-fern gul-
lies and areas of rain forest, plentiful moisture leads to mushroom formation and
mushrooms can be collected during most of the year. There may be a particular
flora of mushroom species associated with the seasons of autumn, summer, and
spring. Relatively few mushrooms are produced during the cold winter months,
although there are perennial fruiting bodies that persist during the winter. But in
drier regions mushrooms occur only after seasonal rains. Formation of mushroom



WHAT ARE MUSHROOMS? 3



fruiting bodies depends very much on the pattern of rain and, in some years, there
may be virtually a complete lack of fruiting.

There has been a recent upsurge of interest in mushrooms not only as a health
vegetable (food) which is rich in protein but also as a source of biologically active
compounds of medicinal value. Uses include complementary medicine/dietary
supplements for anticancer, antiviral, immunopotentiating, hypocholesterolemic,
and hepatoprotective agents. This new class of compounds, termed mushroom
nutriceuticals (Chang and Buswell, 1996), are extractable from either the
mushroom mycelium or fruiting body and represent an important component of
the expanding mushroom biotechnology industry. It has been shown that constant
intake of either mushrooms or mushroom nutriceuticals (dietary supplements) can
make people fitter and healthier. In addition, mushroom cultivation can also help
to convert agricultural and forest wastes into useful matter and reduce pollution
in the environment. Therefore, mushroom cultivation can make three important
contributions: production of health food, manufacture of nutriceuticals, and
reduction of environmental pollution.

1.2 WHAT ARE MUSHROOMS?
1.2.1 Definition of a Mushroom

Mushrooms along with other fungi are something special in the living world, being
neither plant nor animal. They have been placed in a kingdom of their own, called
Myceteae (Miles and Chang, 1997). But what are mushrooms? The word mush-
room may mean different things to different people and countries. It was reported
(Chang and Miles, 1992) that specialized studies and the economic value of mush-
rooms and their products had reached a point where a clear definition of the term
mushroom was warranted. In a more broad sense "mushroom is a macrofungus
with a distinctive fruiting body, which can be either epigeous or hypogeous and
large enough to be seen with naked eye and to be picked by hand" (Chang and
Miles, 1992). Thus, mushrooms need not be Basidiomycetes, or aerial, or fleshy,
or edible. Mushrooms can be Ascomycetes, grow underground, have a nonfleshy
texture, and need not be edible. This definition is not a perfect one but can be
accepted as a workable term to estimate the number of mushrooms on the earth
(Hawks worth, 2001). The most common type of mushrooms is umbrella shaped
with a pileus (cap) and a stipe (stem), that is, Lentinula edodes. Other species
additionally have a volva (cup), that is, Volvariella volvacea, or an annulus (ring),
that is, Agaricus campestris, or both, that is, Amanita muscaria. Furthermore, some
mushrooms are in the form of pliable cups; others are round like golf balls. Some
are in the shape of small clubs; some resemble coral; others are yellow or orange
jellylike globs; and some even very much resemble the human ear. In fact, there is
a countless variety of forms.

The structure that we call a mushroom is in reality only the fruiting body of the
fungus. The vegetative part of the fungus, called the mycelium, comprises a system
of branching threads and cordlike strands that branch out through soil, compost,



4 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

wood log, or other lignocellulosic material on which the fungus may be growing.
After a period of growth and under favorable conditions, the established (matured)
mycelium could produce the fruit structure which we call the mushroom. Accord-
ingly mushrooms can be grouped into four categories: (1) those which are fleshy
and edible fall into the edible mushroom category (e.g., Agaricus bisporus); (2)
mushrooms which are considered to have medicinal applications are referred to as
medicinal mushrooms (e.g., Ganoderma lucidum); (3) those which are proven to be
or suspected of being poisonous are named poisonous mushrooms (e.g., Amanita
phalloides); and (4) a miscellaneous category, which includes a large number of
mushrooms whose properties remain less well defined, may tentatively be grouped
together as "other mushrooms." Certainly, this approach of classifying mushrooms
is not absolute and not mutually exclusive. Many kinds of mushrooms are not only
edible but also possess tonic and medicinal qualities.

1.2.2 Ecological Classification of Mushrooms

Mushrooms can be ecologically classified into three categories: saprophytes, par-
asites, and mycorrhiza.

There are only a few parasitic mushrooms. Most of the cultivated gourmet
mushrooms are saprophytic fungi. Some are mycorrhizal mushrooms, for
example, Perigold black truffle (Tuber melanosporum) and matsutake mushroom
(Tricholoma matsutake). It is difficult to bring these pricey wild gourmet species
into cultivation because they are mycorrhiza. These mushroom species have a
symbiotic relationship with some vegetation, particularly trees, that is, there is a
relationship of mutual need. Therefore, the substratum (host) should be carefully
recorded, as this can be an important feature in identification and in classification,
for example, whether the mushroom is growing on dung, wood, bark, living
trees, litter, or soil. If the mushroom is growing on a living plant or on dead parts
recognizable as belonging to a nearby plant, flowers, fruits, or other parts of the
plant, these should be collected for identification of the host or substrate if its
name is not known.

Saprophytes obtain nutrients from dead organic materials; parasites derive food
substances from living plants and animals, causing harm to the hosts; and mycor-
rhiza live in a close physiological association with host plants and animals, thereby
forming a special partnership where each partner enjoys some vital benefits from
the other.

However, some mushrooms do not fall neatly within these man-made categories
and can share two of these categories (Figure 1.1). For example, some Ganoderma
spp., including G. lucidum, are common saprophytes but can be pathogenic too;
also T. matsutake, while initially appearing to be mycorrhizal on young roots, soon
becomes pathogenic and finally exhibits some saprophytic ability.

1.2.3 Identification of Mushrooms

Successful identification of wild mushrooms requires a basic knowledge of the
structure of fungi and of the way in which they live. To identify a given mushroom,



WHAT ARE MUSHROOMS? 5



Mycorrhizal



Saprobe



Cantharellus
cibarius




Tricholoma
matsutake; Tubur
\ melanosporum



Pathogen



Ganoderma species
including G. lucidum



Figure 1.1 Modified triangular model for ecological classification of mushrooms (Hall
et al., 2003b).

it is necessary to examine the fruiting bodies with the utmost care. A fresh fruiting
body is much easier to identify than a pickled (preserved in formalin) or a dried
one. A good reference material, usually a book with color, pictures of the different
mushrooms known, is a basic requirement. A key is usually provided to simplify
identification in most reference texts (Arora, 1986; Carlucccio, 2003; Chang and
Mao, 1995; Fuhrer, 2005; Shepherd and Totterdell, 1988; van der Westhuizen and
Eicker, 1994).

In using the reference, it is essential that one knows some specific characteristics
of the mushroom being identified. These characteristics are (1) size, color, and
consistency of the cap and the stalk; (2) mode of attachment of the gills to the
stalk; (3) spore color in mass; and (4) chemical tests or reactions.

Although the color of the gills is a good indication of the spore color, there are
instances when the experienced mycologist will have to resort to what is called
"spore print" examination to determine the real color of the spores. For specimens
with a distinct cap and stem, the cap is removed and placed fertile-side down,
preferably on a microscope slide, but in the absence of such, on white paper, black
paper, or cellophane. Then it is covered with a bowl or similar object to prevent
air currents. A thin spore print is often visible after as little as a half hour, but a
useful deposit usually requires longer time (up to 2 hours or more). The print is
necessary to determine overall spore color. It is also a source of mature spores for
microscopic examination and measurements.

The mode of gill attachment to the stem indicates the genus of the mushroom
and should be carefully noted. To determine the mode of attachment, the mush-
room is cut longitudinally through the cap, exposing the point of attachment of the
gills to the stem.

The environment in which the mushroom was picked should also be noted. It
is important to know whether the mushroom grows directly on the ground, on
decaying wood, on a living tree trunk, or on compost. One should not overlook the
species of those on which the mushrooms are found growing or the type of grasses
or moss present in the area where the mushrooms are collected.



6 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

There is no single reference work in which all mushrooms are illustrated or
described. In most cases, mushroom species in publications are grouped by region
or locality, for example, North American mushrooms, mushrooms of the Western
Hemisphere, and mushrooms of South Africa. While certain mushrooms are
easy to identify, many are not. In fact, there is a great number of look-alikes. To
avoid any unpleasant experiences, especially when identifying mushrooms for
the purpose of determining edibility, experts should always be consulted (Quimio
et al, 1990).

Collectors should always remember when using keys that the mushroom they
have in hand might not be included in the book they are consulting (or in any other
book, for that matter). Once they have obtained a name with a key, they must read
the detailed description provided for the mushroom and compare it with the one
they are trying to identify. If the description does not fit the specimen, then they
must go back to the key and try again, following a different route. If they exhaust
all of the possible routes and still cannot find a description that fits, they should
assume that the fungus in hand is not in the books being consulted. Using the
information gained, they may then consult other appropriate references that may
be available or they may seek the assistance of specialists working with the group
in question. They should never attempt to force a specimen into a category where
it does not fit.

Some mushrooms are very palatable due to their exotic taste, but some mush-
rooms are very poisonous. Unfortunately, there are no general guidelines for dis-
tinguishing between the poisonous and edible species. The only means by which
a nonspecialist can determine the edibility or toxicity of a given mushroom is
to carry out an accurate identification of the specimen. Such identification may
be obtained by consulting the relevant literature, preferably with illustrations, or
experts in the subject. Identification of a mushroom at the generic level is inad-
equate since, within a given genus (e.g., Lepiota) some species are edible while
other species are highly poisonous.

Several species of Amanita are extremely poisonous, but obvious symptoms do
not appear until 8-12 hours after ingestion. The poisonous compound, amatoxin, is
not destroyed by boiling or processing. Some less poisonous mushrooms produce
only nausea or gastric upset within 30-60 minutes of ingestion (Hall et al., 2003a;
Quimio et al., 1990). Mushrooms partially eaten by animals or insects are not nec-
essarily fit for human consumption. When the mushroom is in doubt, throw it out.
If you are not absolutely sure whether a given mushroom is edible or otherwise, do
not touch it. Leave the strange mushroom alone.

1 .3 CONCEPT OF MUSHROOM BIOLOGY AND
APPLIED MUSHROOM BIOLOGY

1.3.1 Mushroom Biology

The biological science that is concerned with fungi is called mycology. Mushroom
biology is the branch of mycology that deals with mushrooms in many disciplines.



CONCEPT OF MUSHROOM BIOLOGY AND APPLIED MUSHROOM BIOLOGY 7



When knowledge increases and areas of specialization develop within the disci-
pline, it is convenient to indicate that area of specialization with a self-explanatory
name. In biology, there are such specializations as neurobiology, bacteriology,
plant pathology, pomology, molecular biology, virology, fungal physiology,
embryology, endocrinology, phycology, and entomology. These names indicate
either a group of organisms (e.g., bacteria, algae, and insects) and/or an approach
to the study (e.g., disease, development, and physiology).

Although several terms for this important branch of mycology that deals with
mushroom have been used, and each of these has its merit, when we get down to
the matter of definitions, it seems that there is a place for a new term — mushroom
biology (Chang and Miles, 1992). Mushroom biology is a new discipline concerned
with any aspect of the scientific study of mushrooms, such as taxonomy; physiol-
ogy, and genetics.

1.3.2 Applied Mushroom Biology

Applied mushroom biology is concerned with all aspects of the application of
mushroom biology. It consists of three main components: mushroom science,
mushroom biotechnology (Chang, 1993), and mushroom bioremediation
(Figure 1.2). Mushroom science deals with mushroom cultivation and produc-
tion (mushrooms themselves) and encompasses the principles of mushroom
biology/microbiology, bioconversion/composting technology, and environmental
engineering (Figure 1.3); Mushroom biotechnology is concerned with mushroom
products (mushroom derivatives) and encompasses the principles of mushroom
biology/microbiology, fermentation technology, and bioprocess (Figure 1.4).
Mushroom biotechnology, both as a technology and as the basis for new
mushroom products, requires industrial development. It, like many bioscience




Figure 1.2 Applied mushroom biology consists of three components: mushroom science,
mushroom biotechnology, and mushroom bioremediation.



8



OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS




Figure 1.3 Mushroom science: concerned with mushroom cultivation and production.




Figure 1.4 Mushroom biotechnology: concerned with mushroom products (mushroom
nutriceuticals/dietary supplements).



industries, operates at the cutting edge of science and involves numerous
regulatory issues. The third component of applied mushroom biology has been
developed in recent years. This is mushroom bioremediation and is concerned
with the beneficial impacts of mushrooms on the environment (from mushroom
mycelia) and encompasses principles of mushroom biology/microbiology,
ecology, and bioconversion technology (Figure 1.5).

Therefore, the aims of the discipline of applied mushroom biology (Figure 1 .2)
are to tackle the three basic problems — shortage of food, diminishing quality of



CONCEPT OF MUSHROOM BIOLOGY AND APPLIED MUSHROOM BIOLOGY 9




Figure 1.5 Mushroom bioremediation: concerned with beneficial impacts of mushrooms
on environment.



human health, and pollution of the environment — which human beings still face,
and will continue to face, due to the continued increase of the world population.
The twentieth century began with a world population of 1 .6 billion and ended with
6 billion inhabitants. The world's population is likely to reach 9.2 billion in 2050
from the current 6.7 billion with most of the growth occurring in developing coun-
tries. The growing world population is increasing by about 80 million people per
year. At present, about 900 million people in the world are living in poverty. On the
other hand, it has been observed that over 70% of agricultural and forest products
have not been put to total productivity and have been discarded as waste. Applied
mushroom biology not only can convert these huge lignocellulosic biomass wastes
into human food but also can produce notable nutriceutical products that have
many health benefits. Another significant aspect of applied mushroom biology is
using the biota in creating a pollution-free and beneficial environment. The three
components of applied mushroom biology are closely associated with three aspects
of well-being — food, health, and pollution.

The discipline that is concerned with the principles and practice of mushroom
cultivation is known as mushroom science (Chang and Miles, 1982). The establish-
ment of principles requires facts arrived at through systematic investigation. The
systematic investigation must involve the practical aspects of mushroom cultiva-
tion as well as scientific studies. The consistent production of successful mush-
room crops necessitates both practical experience and scientific knowledge.

1.3.3 Impact of Applied Mushroom Biology

1.3.3.1 Nongreen Revolution The world population has reached over 6
billion now. It is expected to continue increasing in the twenty-first century.



10 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

The amount of food and the level of medical care available to each individual,
especially those in less developed countries, will decrease. Environmental
pollution and greenhouse gas effects will also become a more serious problem.
However, the world has an immense amount of lignocellulosic material resource
that, like solar energy, is sustainable. Lignocellulosic material is a kind of biomass
which is estimated to amount to 1.09 x 10 11 1 dry matter on land annually (Chang,
1989), which consists of mainly three components: cellulose, hemicellulose, and
lignin. Lignocellulose is a major component of wood and other plant materials.
The world's annual yield of cereal straws in 1999 is estimated to be 3570 x 10 6 t.
Since such a large amount of energy is in lignocellulosic biomass (3020 EJ solar
energy fixed in biomass per year), it can constitute principal objects for conversion
into useful products by man's activities. Note that E is the metric prefix for exa
(10 18 ) and joule is the unit of energy.

Although various strategies have been developed to utilize part of the vast
quantities of waste lignocellulose generated annually through the activities of
agricultural, forestry, and food processing industries, one of the most significant,
in terms of producing a higher value product from the waste, is the cultivation
of edible mushrooms by solid-state fermentation. More recently, attention has
focused on a second area of exploitation following the discovery that many
of these mushrooms produce a range of metabolites of intense interest to the
pharmaceutical/nutriceutical (e.g., antitumor, immunomodulation agents, and
hypocholesterolemic agents), and food (e.g., flavor compounds) industries.
Mushrooms, like all other fungi, lack chlorophyll and are nongreen organisms.
They cannot convert solar energy through the process of photosynthesis to
organic matter as green plants do, but they can produce extensive enzymes
that can degrade lignocellulosic materials for their own nutrients for growth
and fruiting. Different mushrooms have different lignocellulolytic enzyme
profiles (Buswell and Chang, 1994; Buswell et al., 1996b). These are reflected
in qualitative variations in the major enzymatic determinants (i.e., cellulases,
ligninases) required for substrate bioconversion. For example, L. edodes, which
is cultivated on highly lignified substrates such as wood or sawdust, produces
two extracellular enzymes that have been associated with lignin depolymerization
in other fungi (manganese peroxidase and laccase) (Buswell et al., 1995).
Conversely, V. volvacea, which prefers high-cellulose, low-lignin-containing
substrates, produces a family of cellulolytic degrading enzymes, including at least
five endoglucanases, five cellobiohydrolases, and two /J-glucosidases (Cai et al.,
1994, 1998, 1999). Pleurotus sajor-caju, the grey oyster mushroom, exhibits both
cellulase and ligninase secretions (Buswell et al., 1996a) and therefore is the
most adaptable of the three species. It can grow on a wide variety of agricultural
waste materials of differing composition in terms of the polysaccharide-lignin
ratio. This demonstrates the impressive capacities of mushrooms for biosynthesis,
which is different from photosynthesis by green plants. The species of mushroom
fungi not only can convert the agricultural and forestry lignocellulosic wastes
through solid fermentation technology into the high-quality protein consumed
directly in the form of the mushroom fruiting body but also can convert food



MUSHROOM CULTIVATION 1 1



processing biomass wastes (e.g., soybean wastes using submerged culture) into
fungal protein (Buswell and Chang 1994) or "mycomeat" (Miles and Chang
1988). Soybean waste materials (slurries) are generated in large quantities during
the processing of soybean milk and "tofu" (bean curd), which are popular foods
in many countries now and are, in some places, discarded without treatment,
thereby constituting an environmental pollutant. In addition, mushrooms and
their mycelia can provide nutriceutical and pharmaceutical products. As outlined
above, by blending the advances in basic biological knowledge with that of
practical technology, a mushroom-related industry based on utilization of the
lignocellulosic waste materials that are abundantly available in rural and urban
areas can have positive global impacts on long-term food nutrition, health,
environmental conservation and regeneration, and economic and social change.
Therefore, the significant impact of applied mushroom biology on human welfare
has been named a "nongreen revolution" (Chang, 1999).

1.3.3.2 Mushroom Bioremediation This component of applied mushroom
biology deals mainly with the aspects of benefits to the earth from the activities
of mushroom mycelium. Environmental contamination can be ameliorated by the
application of mushroom mycelial technologies. For example:

1 . The use of bioconversion processes to transform the polluting substances
into valuable foodstuffs, for example, the proper treatment and reutilization
of spent substrates/composts in order to eliminate the pollution problems
(Beyer, 2005; Noble, 2005). One of the most intriguing opportunities offered
by mushroom mycelia in the area of bioconversion is the exploitation of
their ability to degrade pollutants, many of which are highly carcinogenic,
released into the environment as a consequence of human activity.

2. The use of fungi/mushroom mycelia as tools for healing soil, what Stamets
(2005) called "mycorestoration," which is the use of fungi/mushrooms to
repair or restore the weakened or damaged biosystems of environment.
The processes of mycorestoration include the selective use of mushrooms
for mycofiltration to filter water, mycoforestry to enact ecoforestry policy,
mycoremediation to denature toxic wastes, and mycopesticides to control
insect pests. Mycorestoration recognizes the primary role fungi/mushrooms
play in determining the balance of biological populations.



1.4 MUSHROOM CULTIVATION

Mushroom cultivation is both a science and an art. The science is developed
through research; the art is perfected through curiosity and practical experience.
Mushroom growth dynamics involve some technological elements that are in
consonance with those exhibited by our common agricultural crop plants. For
example, there is a vegetative growth phase, when the mycelia grow profusely,
and a reproductive (fruiting) growth phase, when the umbrella-like body that



12



OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS



Triggers



Vegetative Phase



C0 2 »

Temperature ,
Light



Reproductive Phase
0 2



Figure 1.6 Two major phases of mushroom growth and development: vegetative and
reproductive. The triggers for the transition from the vegetative phase to the reproductive
phase are usually regulated by environmental factors.



we call mushroom develops. In agricultural plants (e.g., sunflowers), when the
plants switch from vegetative growth to reproductive growth, retarding tips for
further growth (elongation) is an obvious phenomenon of maturity. It is the
same principle in mushroom production. After the vegetative (mycelial) phase
has reached maturity, what the mushroom farmer needs next is the induction
of fruiting. This is the time the mycelial growth tips should be retarded by
regulating the environmental factors. These factors, generally called "triggers" or
"environmental shocks," such as switching on the light, providing fresh air, and
lowering temperature, can trigger fruiting (Figure 1.6).



1.4.1 Major Phases of Mushroom Cultivation

Mushroom farming is a complex business that requires precision. Indeed, it is not
as simple as what some people often loosely stipulate. It calls for adherence to pre-
cise procedures. The major practical steps/segments of mushroom cultivation are
(1) selection of an acceptable mushroom species, (2) secretion of a good-quality
fruiting culture, (3) development of robust spawn, (4) preparation of selective sub-
strate/compost, (5) care of mycelial (spawn) running, (6) management of fruiting
and mushroom development, and (7) careful harvesting of mushrooms (Chang and
Chiu, 1992; Chang 1998). If you ignore one critical step/segment, you are invit-
ing trouble, which could lead to a substantially reduced mushroom crop yield and
mushroom marketing value:

1. Before any decision to cultivate a particular mushroom is made, it is
important to determine if that species possesses organoleptic qualities



MUSHROOM CULTIVATION 13



acceptable to the indigenous population or to the international market,
if suitable substrates for cultivation are plentiful, and if environmental
requirements for growth and fruiting can be met without excessively costly
systems of mechanical control.

2. A "fruiting culture" is defined as a culture with the genetic capacity to form
fruiting bodies under suitable growth conditions. The stock culture selected
should be acceptable in terms of yield, flavor, texture, fruiting time, and so
on.

3. A medium through which the mycelium of a fruiting culture has grown and
which serves as the inoculum of "seed" for the substrate in mushroom cul-
tivation is called the "mushroom spawn." Failure to achieve a satisfactory
harvest may often be traced to unsatisfactory spawn used. Consideration
must also be given to the nature of the spawn substrate since this influ-
ences rapidity of growth in the spawn medium as well as the rate of mycelial
growth and filling of the beds following inoculation.

4. While a sterile substrate free from all competitive microorganisms is the
ideal medium for cultivating edible mushrooms, systems involving such
strict hygiene are generally too costly and impractical to operate on a large
scale. Substrates for cultivating edible mushrooms normally require varying
degrees of pretreatment in order to promote growth of the mushroom
mycelium to the practical exclusion of other microorganisms. The substrate
must be rich in essential nutrients in forms which are readily available to the
mushroom and be free of toxic substances that inhibit growth of the spawn.
Moisture content, pH, and good gaseous exchange between the substrate
and the surrounding environment are important physical factors to consider.

5. Following composting, the substrate is placed in beds where it is generally
pasteurized by steam to kill off potential competitive microorganisms. After
the compost has cooled, the spawn may be broadcast over the bed surface
and then pressed down firmly against the substrate to ensure good contact or
inserted 2-2.5 cm deep into the substrate. Spawn running is the phase during
which mycelium grows from the spawn and permeates into the substrate.
Good mycelial growth is essential for mushroom production.

6. Under suitable environmental conditions, which may differ from those
adopted for spawn running, primordial formation occurs and then is fol-
lowed by the production of fruiting bodies. The appearance of mushrooms
normally occurs in rhythmic cycles called "flushes."

7. Harvesting is carried out at different maturation stages depending upon the
species and consumer preferences and market value.



1.4.2 Cultivation of Several Selected Mushrooms

The cultivation of edible mushrooms can be divided into two major stages. The
first stage involves the preparation of the fruiting culture, stock culture, mother
spawn, and planting spawn, while the second stage entails the preparation of



14 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

the growth substrates for mushroom cultivation. Cultivation conditions for a few
selected mushroom species are briefly described in the following sections.

1.4.2.1 Cultivation of Agaricus Composting is prepared in accordance with
well-documented commercial procedures (van Griensven, 1988; Chang and Hayes,
1978; Kaul and Dhar, 2007). In phase I of the process (outdoor composting),
locally available raw materials are arranged into piles that are periodically turned
and watered. The initial breakdown of the raw ingredients by microorganisms takes
place in phase I. This phase is usually complete within 9-12 days, when the mate-
rials have become pliable, dark brown in color, and capable of holding water.
There is normally a strong smell of ammonia. Phase II (indoor fermentation) is
pasteurization, when undesirable organisms are removed from the compost. This
is carried out in a steaming room where the air temperature is held at 60�� C for at
least 4 hours. The temperature is then lowered to 50��C for 8-72 hours depending
upon the nature of the compost. Carbon dioxide is maintained at 1.5-2% and the
ammonia level drops below 10 PPM. Following phase II composting, the substrate
is cooled to 30��C for Agaricus bitorquis and to 25��C for A. bisporus for spawning.
Production of phase III or IV composts for growing Agaricus mushrooms has been
an advanced technological development in recent years in Western countries. The
production of phase III compost is phase II compost spawn run in a bulk tunnel
and ready for casing when delivered to the grower. If the phase III compost is then
cased and spawn developed into the casing layer before dispatching to the growing
unit or delivering to growers, it is named as phase IV compost. The successes of
bulk phase III and IV depend a lot on the quality of phase I and II processes. Phase
II on shelves produces an average of 4.1 crops per year. Since 1999, growers using
Phase III production enjoyed an average of 7.1 crops per year. In recent years,
phase IV can generate 10-12 crops per year (Dewhurst, 2002; Lemmers, 2003).

1.4.2.2 Cultivation of Lentinula edodes Lentinula edodes (xiang gu in
Chinese and shiitake in Japanese) was the second most important cultivated
edible mushroom, but since 2002 it has become the world number one cultivated
mushroom. It can be cultivated either on wood log or on synthetic substrate logs
(Quimio et al., 1990; Stamets, 2000; Chang and Miles, 2004).

1 . Biological Nature Lentinula edodes is a heterothallic mushroom. Its sex-
uality is controlled by two mating factors, A and B, with multiple alleles, and
therefore, its life history is a tetrapolar or bifactorial mating system (Chang and
Miles, 1984).

Its life cycle starts the germination of basidiospores. After selected mating
between two compatibility germinative mycelium, the dikaryon mycelium or fruit-
ing culture is established. From the fruiting culture, the stock culture, mother
spawn, and commercially planting spawn can be made. When the spawn is planted
on a suitable substrate, under good climatic conditions the fruiting bodies of the
mushroom are developed. Then when the mature stage is reached, the spores are
discharged and its life cycle is completed.



MUSHROOM CULTIVATION 15



Lentinula edodes is kind of wood rot fungus. In nature, it grows on dead tree
trunks or stumps. In general, the wood for the mushroom growth consists of crude
protein 0.38%, fat 4.5%, soluble sugar 0.56%, total nitrogen 0.148%, cellulose
52.7%, lignin 18.09%, and ash 0.56%. Generally speaking, the carbon-nitrogen
ratio in substrate should be in the range of 25 : 1 -40 : 1 in the vegetative growth
stage and from 40 : 1 to 73 : 1 in the reproductive stage. If the nitrogen source is
too rich in the reproductive phase, fruiting bodies of the mushroom are usually not
formed and developed.

The optimum temperature of spore germination is 22-26��C. The temperature
for mycelial growth ranges from 5 to 35��C, but the optimum temperature is
23-25��C. Generally speaking, L. edodes belongs to low-temperature mushrooms;
the initial and development temperature of fruiting body formation is in the range
of 10-20��C and the optimum temperature of fructification for most varieties of
the mushroom is about 15��C. Some varieties can fruit in higher temperatures (e.g.,
20-23��C). These high-temperature mushrooms usually grow faster and have a
bigger and thinner cap (pileus) and a thin and long stalk (stipe). Their fruiting
bodies are easily opened and become flat-grade mushrooms, which are considered
to be low quality. The optimum pH of the substrate used in making the mushroom
bag/log is about 5.0-5.5.

2. Culture Media and Preparation The mushroom can grow on a variety of cul-
ture media and on different agar formulations, both natural and synthetic, depend-
ing on the purpose of the cultivation. Synthetic media are often expensive and time
consuming in preparation; hence they are not commonly used for routine purposes.

The potato dextrose agar, or PDA, is the simplest and the most popular medium
for growing the mycelium of the mushroom. It is prepared as follows:

(a) Ingredients: Diced potato, 200 g; dextrose (or ordinary white cane sugar),
20 g; powdered agar (or agar bars), 20 g; and distilled water (or tap water),
1L.

(b) Procedure: Peeled potatos are washed, weighed, and cut into cubes. They
are boiled in a casserole with at least 1 L of water until they become soft
(around 15 minutes). The potatos are removed and water is added to the
broth to make exactly 1 L. The broth is returned to the casserole, and dex-
trose and the agar are added. The solution is heated and stirred occasionally
until the agar is melted. The hot solution is then poured into clean flat bot-
tles. For pure or stock cultures, the test tubes are filled with at least 10 mL of
liquid agar solution. The bottles or test tubes are plugged with cottonwool.
When petri dishes are available, these can be used to produce mycelial plugs
for inoculation of mother spawn.

Examples of the different formulas for spawn substrates are described below.
Mother grain spawn: (i) Wheat/rye grain + 1.5% gypsum or slaked lime, (ii) Cot-
ton seed hull 40%, sawdust 38%, wheat bran 20%, sugar 1%, and gypsum 1%. (iii)
Sugar cane bagasse 40%, sawdust 38%, wheat bran 20%, sugar 1%, and gypsum
1%. Planting spawn: A number of materials, mostly agricultural and forest wastes,
can be used to prepare mushroom planting spawn. Three of them are given here



16 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

as examples: sawdust 78%, rice/wheat bran 16%, sugar 1.5%, corn flour 1.7%,
ammonium sulfate 0.3%, calcium superphosphate 0.5%, and gypsum 2%; saw-
dust 64%, wheat bran 15%, spent coffee grounds 20%, and gypsum/lime 1%; and
sawdust 78%, sucrose 1%, wheat bran 20%, and calcium carbonate 1%.

The L. edodes mushroom is produced on both a cottage and a commercial scale.
Some issues associated with the different cultivation styles are summarized below:

1 . Cottage-Scale Cultivation There are many formulas for the composition of
the substrate. The ingredients can be variable from place to place and country to
country depending upon the raw materials available and local climatic conditions.
In general, after mixing the dry ingredients by hand or with a mechanical mixer,
water is added to the mixture so that the final moisture content of the substrate is
between 55 and 60%, depending on the capacity of the sawdust to absorb water.
The ingredients are then packed into autoclavable polypropylene or high-density
polyethylene bags. Although they are more expensive, polypropylene bags are the
most popular since polypropylene provides greater clarity than polyethylene. After
the bags have been filled (1.5-4 kg wet weight) with the substrate, the end of the
bag can be closed either by strings or plugged with a cottonwool stopper. Four
formulas in the preparation of the substrate for the cultivation of the mushroom
are given here as reference, (i) Sawdust 82%, wheat bran 16%, gypsum 1.4%,
potassium phosphate, dibasic 0.2%, and lime 0.4%. (ii) Sawdust 54%, spent cof-
fee grounds 30%, wheat bran 15%, and gypsum 1%. (iii) Sawdust 63%, corncob
powder 20%, wheat bran 15%, calcium superphosphate 1%, and gypsum 1%. (iv)
Sawdust 76%, wheat bran 18%, corn powder 2%, gypsum 2%, sugar 1.2%, calcium
superphosphate 0.5%, and urea 0.3%.

2. Commercial-Scale Cultivation In general, the operation can use oak or other
hardwood sawdust medium to grow the mushroom. The basic steps are (i) mix the
sawdust, supplements, and water; (ii) bag the mixture; (iii) autoclave the bags to
121��C and cool the bags; (iv) inoculate and seal the bags; (v) incubate for 90 days
to achieve full colonization of the sawdust mixture, in other words, to allow the
mycelium to be established for ready fructification; (vi) fruit the colonized and
established sawdust logs/bags/blocks 6 times using a 21-day cycle at 16-18��C;
and (vii) harvest, clip steps, grade, box, and cold store for fresh market or harvest,
dry, cut steps, grade, and dry again before boxing for dry market.

The major equipment used in production consists of a mixer/conveyor, auto-
clave, gas boiler, cooling tunnel, laminar flow cabinet, bag sealer, air compressor
for humidification, and shelves to incubate.

Incubation can be done in two rooms and in two shipping containers. The two
shipping containers can be installed near the fruiting rooms. The temperature dur-
ing incubation is held between 18 and 25��C.

Fruiting can be done in six rooms so that the blocks/logs can be moved as a
unit. With compartmentalization, blocks in each room can be subjected to a cycle
of humid cold, humid heat, and dry heat.



MUSHROOM CULTIVATION 1 7



1.4.2.3 Cultivation of Pleurotus sajor-caju Pleurotus sajor-caju (grey
oyster mushroom) is comparable to the high-temperature species in the group of
Pleurotus (oyster) mushrooms, with high temperatures required for fructification.
This mushroom has a promising prospect in tropical/subtropical areas. Its
cultivation is easy with relatively less complicated procedures (Chang and
Miles, 2004; Kaul and Dhar, 2007):

1. Biological Nature The temperature for growth of mycelium is 10-35��C.
The optimum growing temperature of the mycelium is 23-28��C. The optimum
developmental temperature of the fruiting body is 18-24��C. The optimum pH of
the substrate used in making the mushroom bag/bed is 6.8-8.0. The C/N ratio
in the substrate is in the range of 30 : 1-60 : 1. A large circulation of air and
reasonable light are required for the development of the fruiting bodies.

2. Spawn Substrate (i) Wheat grain + 1.5% gypsum or lime, (ii) Cotton
seed hull 88%, wheat bran 10%, sugar 1%, and gypsum 1%. (hi) Sawdust 78%,
wheat bran 20%, sugar 1%, and gypsum 1%. (vi) Sawdust 58%, spent coffee
grounds/spent tea leaves 20%, water hyacinth/cereal straw 20%, sugar 1%, and
gypsum 1%.

3. Cultivation Substrate (i) Cotton seed hull 95%, gypsum 2%, lime 1%, and
calcium superphosphate 2%. (ii) Rice straw 80%, cotton waste 18%, gypsum 1%,
and lime 1%. (iii) Water hyacinth 80%, cereal straw 17%, gypsum 2%, and lime
1%.

For demonstration purpose, this mushroom can be nurtured to grow into a tree-
like shape (Chang and Li, 1982). The cultivation method, which has been tested to
be successful, is as follows: Cotton waste or rice straw mixed with water hyacinth
is used as the substrate. Tear large pieces of cotton waste into small parts or cut
the straw and water hyacinth into small segments. Add 2% (w/w) lime and mix
with sufficient water to get moisture content of about 60-65%. Pile the materials
up, cover with plastic sheets, and leave to stand overnight. Load the substrate into
small baskets or on shelves for pasteurization or cook the substrate with boiled
water for 15 minutes. After cooling to approximately 25��C, mix around 2% (w/w)
spawn thoroughly with the substrate and pack into columns of 60-cm-long tubes
which have hard plastic [polyvinyl chloride (PVC)] tubing of 100 cm (4 cm in
diameter) as central support and with plastic sheets as outside wrapping.

Incubate these columns at around 24— 28��C, preferably in the dark. When the
mycelium of the mushroom has ramified the entire column of substrate after three
to four weeks, remove the plastic wrapping and switch on white light. Watering
occasionally is needed to keep the surface from drying. In around three to four
days white primordia start to appear over the whole surface. After another two to
three days, the Pleurotus mushrooms are ready for harvesting. During the cropping
period watering is very important if many flushes are required.

1.4.2.4 Cultivation of Volvariella The edible straw mushroom Volvariella
volvacea is a fungus of the tropics and subtropics and has been traditionally culti-
vated in rice straw for many yeas in China and South East Asian countries. In 1971,



18 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

cotton wastes were first introduced as heating material for growing the straw mush-
room (Yau and Chang, 1972), and in 1973, cotton wastes had completely replaced
the traditional paddy straw to grow the mushroom (Chang, 1974). This was a turn-
ing point in the history of straw mushroom cultivation because the cotton waste
compost through pasteurization brought the cultivation of the mushroom into an
industrial scale first in Hong Kong and then in Taiwan, Thailand, and elsewhere
in China. Several techniques are adopted for the cultivation of the mushroom,
which thrives in the temperature range of 28-36��C and a relative humidity of
75-85%. Detailed descriptions of the various methods are given by Chang and
Quimio (1982), Chang and Miles (2004), Kaul and Dhar (2007), and Quimio et al.
(1990). Choice of technologies usually depends on personal preference and the
availability of substrates and resources. While the more sophisticated indoor tech-
nology is recommended for the industrial-scale production of the mushroom, most
of the other technologies are low cost and appropriate for rural area development,
especially when production is established at the community level.

1.4.2.5 Cultivation of Agaricus brasiliensis In recent years, A. brasilien-
sis, formerly called Agaricus blazei Murill (Wasser et al., 2002), has rapidly
become a popular mushroom. It has been proved to be not only a good-tasting
and highly nutritious mushroom but also an effective medicinal mushroom,
particularly for antitumour active polysaccharides.

Agaricus brasiliensis was a wild mushroom in southeastern Brazil, where it
was consumed by the people as a part of their diet. The culture of the mushroom
was brought to Japan in 1965 and an attempt to cultivate this mushroom commer-
cially was made in 1978. In 1992, this mushroom was introduced to China for
commercial cultivation (Chang and Miles, 2004).

1. Biological Nature Agaricus brasiliensis belongs to middle-temperature
mushrooms. The growth temperature for mycelium ranges from 15 to 35��C
and the optimum growth temperature range is 23-27��C. The temperature for
fruiting can be from 16 to 30��C and the optimum developmental temperature of
fruiting bodies is 18-25��C. The ideal humidity for casing soil is 60-65%. The air
humidity in a mushroom house is preferably 60-75% for mycelium growth and
70-85% for fruiting body formation and development. The optimum pH of the
compost used in making the mushroom bed is 6.5-6.8. The optimum pH of the
casing soil is 7.0. A good circulation of air is required for the development of the
fruiting bodies. These conditions are similar to those needed for the cultivation
of A. bisporus. Under natural conditions, the mushroom can be cultivated for
two crops each year. Each crop can harvest three flushes. According to the local
climates, the farmer can decide the spawning time in the year in order to have
mushrooms for harvest within 50 days after spawning.

2. Preparation of Mushroom Bed (Stamets, 2000) Agaricus brasiliensis is a
kind of mushroom belonging to straw-dung fungi and prefers to grow on sub-
strate rich in cellulose. The waste/by-product agro-industrial materials [e.g., rice
straw, wheat straw, bagasse (squeezed residue of sugar cane), cotton seed hull,



MUSHROOM CULTIVATION 19



corn stalks, sorghum stover, and even wild grasses] can be used as the principal
component of the compost for cultivation of the mushroom. It should be noted
that these materials have to be air dried first and then mixed with cattle dung,
poultry manure, and some chemical fertilizers. The following formulas for making
compost are for reference only. They should be modified according to the local
available materials and climatic conditions, (i) Rice straw 70%, air-dry cattle dung
15%, cottonseed hull 12.5%, gypsum 1%, calcium superphosphate 1%, and urea
0.5%. (ii) Corn stalks 36%, cottonseed hull 36%, wheat straw 11.5%, dry chicken
manure 15%, calcium carbonate 1%, and ammonium sulfate or urea 0.5%. (iii)
Rice straw 90.6%, rice bran 2.4%, fowl droppings 3.6%, slaked lime 1.9%, super-
phosphate 1.2%, and ammonium sulfate/urea 0.3%. (iv) Bagasse 75%, cottonseed
hull 13%, fowl droppings 10%, superphosphate 0.5%, and slaked lime 1.5%.



1.4.2.6 Cultivation of Ganoderma lucidum Although the medicinal value
of G. lucidum has been treasured in China for more than 2000 years, the mushroom
was found infrequently in nature. This lack of availability was largely responsi-
ble for the mushroom being so highly cherished and expensive. During ancient
times in China, any person who picked the mushroom from the natural environ-
ment and presented it to a high-ranking official was usually well rewarded (Chang
and Miles, 2004).

Artificial cultivation of this valuably mushroom was successfully achieved in
the early 1970s and, since 1980 and particularly in China, production of G. lucidum
has developed rapidly. Currently, the methods most widely adopted for commercial
production are the wood log, short wood segment, tree stump, sawdust bag, and
bottle procedures (Hsu, 1994; Mizuno et al., 1996; Hung, 1996; Mayzumi et al.,
1997; Chang and Buswell, 1999; Stamets, 2000).

Log cultivation methods include the use of natural logs and tree stumps which
are inoculated with spawn directly under natural conditions. The third alternative
technique involves the use of sterilized short logs about 12 cm in diameter and
approximately 15 cm long which allow for good mycelial running. This method
provides for a short growing cycle, higher biological efficiency, good-quality fruit-
ing bodies, and, consequently, superior economical benefit. However, this pro-
duction procedure is more complex and the production costs much higher than
the natural log and tree stump methods. For this production procedure, the wood
logs should be prepared from broad-leaf trees, preferably from oak. Felling of the
trees is usually carried out during the dormant period, which is after defoliation in
autumn and prior to the emergence of new leaves the following spring. The opti-
mum moisture content of the log is about 45-55%. The flowchart for the short-log
cultivation method is as follows: selection and felling of the tree, sawing/cutting
the log into short segments, transfering segments to plastic bags, sterilization, inoc-
ulation, spawn running, burial of the log in soil, tending the fruiting bodies during
development from the pinhead stage to maturity, harvesting the fruiting bodies,
drying the fruiting bodies by electrical driers, and packaging. It should be noted
that the prepared logs/segments are usually buried in soil inside a greenhouse or



20 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

plastic shed. The soil should allow optimum conditions of drainage, air permeabil-
ity, and water retention, but excessive humidity should be avoided.

Examples of cultivation substrates using plastic bags or bottles as containers
include the following (note that these examples are for reference purposes only
and can be modified according to the strains selected and the materials available
in different localities): (i) sawdust 78%, wheat bran 20%, gypsum 1%, and soy-
bean powder 1%; (ii) bagasse 75%, wheat bran 22%, cane sugar 1%, gypsum 1%,
and soybean powder 1%; (iii) cotton seed hull 88%, wheat bran 10%, cane sugar
1%, and gypsum 1%; (iv) sawdust 70%, corn cob powder 14%, wheat bran 14%,
gypsum 1%, and cereal straw ash 1%; (v) corn cob powder 78%, wheat/rice bran
20%, gypsum 1%, and straw ash 1%. After sterilization, the plastic bags can be
laid horizontally on beds or the ground for fruiting.

1.4.3 Utilization of Mushroom Germplasm

The item of mushroom germplasm is a selective subject only and is not an exhaus-
tive approach to mushroom utilization. It is concerned with the broadening of
available mushroom resources in nature in order to conduct successful domesti-
cating and breeding programs, with the aim at developing the cultivation of wild
mushrooms and improving all desirable mushroom traits. It cannot be overempha-
sized that, to fully exploit the opportunities offered by mushrooms which have
been properly collected and characterized, it is necessary to ensure a continuous
exchange of information between scientists from different disciplines engaged in
different areas of mushroom research. Included among the many possible examples
that could be quoted are the analytical chemists who analyze the many existing and
potentially new growth substrates used for mushroom cultivation in order to certify
their suitability from an alimentary standpoint; biochemists who study the fungal
enzymes involved in the degradation of the individual components constituting the
different substrates; fungal physiologists who focus their attention on the mecha-
nisms underlying carpophore formation; geneticists who are able to comprehend
the life cycles of different mushrooms as well as to undertake breeding programs
and select strains with desirable characteristics; and growers who transfer to the
field scale the knowledge and techniques obtained in the laboratory.

One of the basic requirements for breeding better quality mushrooms in
higher yields is the wider availability of a large reserve of phenotypic variation
(traits) which can be used for selection purposes by both researchers and the
mushroom industry. Since all these phenotypic differences are ultimately under
genetic control, mushroom strains with different traits actually possess distinctive
gene combinations which can be generated artificially by conventional crossing
methods, by protoplast fusion technology, and by transformation with genes
cloned using recombinant deoxyribonucleic acid (rDNA) technology. Since the
mushrooms themselves are the only source of this genetic material, the genes
contained in existing mushroom strains and species represent the total genetic
resource, that is, the entire pool of mushroom germplasm. Extinction of a single
strain or species would mean the potential loss of many thousands of unique genes
that could be used for breeding desirable new strains.



WORLD MUSHROOM PRODUCTION 21



Mushroom germplasm can be preserved by in situ conservation and ex situ
preservation. The maintenance of mushrooms in natural preserves as part of a
strategy for protecting an ecosystem constitutes in situ conservation. Although this
approach is clearly important, it will not be considered here. Mushroom germplasm
can also be preserved ex situ as fungal spores or tissue in the form of a culture col-
lection or gene bank. The collection and classification of information pertaining to
the morphological, physiological, biochemical, and genetic characteristics of indi-
vidual mushroom strains can be stored in computer databases called germplasm
databases. Such databases would provide valuable and readily accessible informa-
tion for future breeding programs and academic research (Chang et al., 1995). This
emerging mushroom germplasm science will address aspects relating to the col-
lection, identification, characterization, utilization and preservation of mushroom
germplasm.

Mushrooms have the potential for multipurpose usages ranging from protein
enrichment of the human diet, the selective delignification of lignocellulosic mate-
rials as part of the recycling process and reinsertion into the food chain and dietary
supplements markets, and a contribution to environmental decontamination. The
realization of this potential depends upon the availability of mushrooms which
possess the characteristics necessary to achieve specified objectives. The pool of
available selective mushrooms from nature through the processes of collection,
identification, and utilization should be as large as possible in order to ensure that
the most appropriate choice of mushroom germplasm can be caught and utilized.



1.5 WORLD MUSHROOM PRODUCTION

The world market for the mushroom industry in 2001 was valued at over U.S.$40
billion (Chang, 2006a). The mushroom industry can be divided into three main
categories: edible mushrooms, medicinal mushroom products, and wild mush-
rooms (Chang, 2006b). International bodies/forums have developed for each of
these segments of the mushroom industry that have helped to bring them to the
forefront of international attention: (1) International Society of Mushroom Sci-
ence (ISMS) for edible mushrooms, (2) World Society for Mushroom Biology and
Mushroom Products (WSMBMP) for mushroom biology and medicinal mushroom
products, and (3) International Workshops on Edible Mycorrhizal Mushrooms for
some wild mushrooms. The three international bodies/forums have done much to
promote each of their respective fields, not the least of which is bringing scien-
tists together for useful discussions, encouraging research and the dissemination
of valuable information. The outlook for many of the known mushroom species is
bright. Production of mushrooms worldwide has been steadily increasing, mainly
due to contributions from developing countries, such as China, India, Poland, Hun-
gary, and Vietnam. There are also increasing experimentally based evidence to
support centuries of observations regarding the nutritional and medicinal benefits
of mushrooms. The value of mushrooms has recently been promoted to tremendous
levels with medicinal mushrooms trials conducted for human immunodeficiency



22 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

virus/acquired immunodeficiency syndrome (HIV/AIDS) patients in Africa, gen-
erating encouraging results (Chang and Mshigeni, 2000). However, harvests of
highly prized edible mycorrhizal mushrooms are continuously decreasing. This has
triggered research into devising methods for improved cultivation of wild mush-
room. It is hoped that there will be even more research into this area, so that larger
quantities of wild mushrooms can be massively harvested through semicultiva-
tion methods. Technological development in the mushroom industry in general
has seen increasing production capacities, innovations in cultivation technologies,
improvements to final mushroom goods, capitalizing the nutritional and medic-
inal properties of mushrooms, and utilizing the natural qualities of mushrooms
for environmental benefits. However, there is always the need to maintain current
trends and to continue to seek out new opportunities. The challenge is to recog-
nize opportunities such as increasing consumption capabilities with the increase in
world population and to take advantage of this by promoting the consumption of
more mushrooms.

Generally, cultivated mushrooms should play a greater role in the endeavor
to increase food protein. This is especially true in developing countries, since
growth substrates for mushrooms are basically agricultural and industrial discards
that are inedible for humans (Chang and Miles, 1984). Biological (bioconversion)
efficiency, that is, the yield of fresh weight mushrooms in proportion to the spawn-
ing compost in Agaricus or to the air-dried substrates in other noncomposting
mushrooms, can reach 60-100% for Agaricus and 15-100% depending on the
cultivation conditions for other species.

The statistics in Table 1.1 illustrate the dramatic increase in the production of
farmed mushrooms during the period 1960-2002 (Chang 1999, 2006b; Delcaire,
1978).



TABLE 1.1 World Mushroom Production, 1960-2002





World Production


Year


(x 1000 t)


1960


170


1965


301


1970


484


1975


922


1978


1,060


1981


1,257


1983


1,453


1986


2,182


1990


3,763


1994


4,909


1997


6,158


2002


12,250



MUSHROOM BIOTECHNOLOGY 23



Whereas in 1997, Asia contributed 74.4% of the total world mushroom tonnage,
Europe 16.3%, and North America 7.0%, both Africa and Latin America's shares
were less than 1%. This is largely due to lack of know-how, lack of understanding
that mushrooms can play vital roles toward enhancing human health when used
as dietary food supplements, lack of reliable sources of good-quality mushroom
spawn for supporting the efforts of local mushroom growers, lack of venture capital
to support mushroom farming entrepreneurs, and absence of systematic govern-
ment support toward promoting mushroom farming as a valuable nontraditional
new food and cash crop (comparable to coffee, tea, cotton, tobacco, etc.).



1.6 MUSHROOM BIOTECHNOLOGY

It has been pointed out that mushroom biotechnology is concerned with mushroom
products and encompasses the principles of fermentation technology, mushroom
biology/microbiology, and bioprocess. The products have a more generalized or
tonic effect, which in some cases may act prophylactically by increasing resistance
to disease in humans from the balancing of nutrients in the diet and the enhancing
of the immune systems.

1.6.1 Nutritional and Medicinal Value of Mushrooms

The greatest difficulty in feeding humans is to supply a sufficient quantity of the
body-building material protein. The other three nutritional categories are the source
of energy (carbohydrates and fats); accessory food factors (vitamins); and inor-
ganic compounds which are indispensable to good health. Of course, water, too, is
essential.

The moisture content of fresh mushrooms varies within the range 70-95%
depending upon the harvest time and environmental conditions, whereas it is about
10-13% in dried mushrooms. The protein content of cultivated species ranges
from 1.75 to 5.9% of their fresh weight. It has been estimated that an average value
of 3.5-4.0% would be more representative. This means that the protein content
of edible mushrooms in general is about twice that of onion (1.4%) and cabbage
(1.4%) and 4 and 12 times those of oranges (1.0%) and apples (0.3%), respec-
tively. In comparison, the protein content of common meats is as follows: pork,
9-16%; beef, 12-20%; chicken, 18-20%; fish, 18-20%; and milk, 2.9-3.3%. On
a dry-weight basis, mushrooms normally contain 19-35% protein, as compared to
7.3% in rice, 12.7% in wheat, 38.1% in soybean, and 9.4% in corn. Therefore, in
terms of the amount of crude protein, mushrooms rank below animal meats but
well above most other foods, including milk, which is an animal product. Further-
more, mushroom protein contains all the nine essential amino acids required by
humans.

In addition to their good protein, mushrooms are a relatively good source of the
following individual nutrients: fat, phosphorus, iron, and vitamins, including thi-
amine, riboflavin, ascorbic acid, ergosterol, and niacin. They are low in calories,



24 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

carbohydrates, and calcium. It has also been reported that a total lipid content vary-
ing between 0.6 and 3.1% of the dry weight is found in the commonly cultivated
mushrooms. At least 72% of the total fatty acids are found to be unsaturated in all
the four tested mushrooms (Huang et al., 1985). It should be noted that unsaturated
fatty acids are essential and significant for our diet and our health.

In recent years, there has been a trend toward discovering ways of treating
mushrooms so as to give them added value. For example, Wermer and Beelman
(2002) have reported on growing mushrooms enriched in selenium. By adding
sodium selenite to compost over a range of 30-300 PPM, they found that the mush-
rooms increasingly absorbed selenium according to the amount in the compost, so
that it is possible to grow mushrooms containing a desired concentration. Selenium
is an essential micronutrient that has generated much recent interest in nutritional
and medical research — and more recently within the food industry (Beelman and
Royse, 2006). Selenium has numerous physiological functions but is best known
as a necessary cofactor for the glutathione peroxidase enzyme system. This system
is responsible for removing free radicals from the body, thus reducing oxidative
damage.

The desirability of a food product does not necessarily bear any correlation
to its nutritional value. Instead, its appearance, taste, and aroma sometimes can
stimulate one's appetite (preference). In addition to nutritional value, mushrooms
have some unique color, taste, aroma, and texture characteristics which attract their
consumption by humans.

The second major attribute of mushrooms, their medicinal properties, has
also been drawn to our attention for study, for example, for hypotensive and
renal effects (Yip et al., 1987), immunomodulatory and antitumor activities of
polysaccharide -protein complex (PSPC) from mycelial cultures (Liu et al., 1995,
1996; Wang et al., 1995a, 1996b, c), immunomodulatory and antitumor activities
of lectins from edible mushrooms (Wang et al., 1995b, 1996a, 1997), isolation
and characterization of a type I ribosome inactivation protein from V. volvacea
(Yao et al., 1998), and medicinal effects of G. lucidum (Chang and Buswell,
1999; Chang and Miles, 2004). For more detailed coverage of this aspect and
comprehensive lists of mushrooms used in dietary supplements and in medicines,
the reader is referred to later chapters.

1.6.2 Nutriceuticals and Dietary Supplements

There has been a recent upsurge of interest in mushrooms not only as health
vegetables (food) but also as a source of biological active compounds of medicinal
value, including use as complementary medicine/dietary supplements for anti-
cancer, antiviral, immunopotentiating, hypocholesterolemic, and hepatoprotective
agents. These new compounds, termed mushroom nutriceuticals (Chang and
Buswell, 1996), are extractable from either the fungal mycelium or fruiting body
and represent an important component of the expanding mushroom biotechnology
industry.

Of the 14,000-15,000 species of so-called mushrooms in the world, around
400 have known medicinal properties. However, it has been estimated that there



DEVELOPMENT OF WORLD MUSHROOM INDUSTRY MOVEMENTS 25

are about 1800 species of mushrooms with the potential of medicinal properties.
Both these mushrooms and their rootlike structure (called mycelium) produce sev-
eral medicinal or nutriceutical (general immune-enhancing) compounds, central
of which are the polysaccharides (high-molecular-weight strings of sugars), triter-
penes, and immunomodulatory proteins. Although virtually all mushrooms and
many foods have polysaccharides in their cell walls, certain mushroom species
have been found to contain polysaccharides which are particularly effective in
retarding the progress of various cancers and other diseases and in alleviating the
side effects of chemotherapy and radiation treatment (through cell-level regen-
erative effects). There are now many studies in Asia, and particularly in China
and Japan, documenting life-span increases of cancer patients undergoing conven-
tional cancer treatment plus mushroom extract consumption or injection (Mizuno
et al., 1995; Liu, 1999). At the same time, due to the enhancement of the immune
systems, it can help people reduce the possibility of being infected by other dis-
eases.

Between 80 and 85% of all medicinal mushroom products are derived from
the fruiting bodies, which have been either commercially farmed or collected
from the wild, for example, Lentinan, a high-molecular-weight (1 — > 3)-/?-D-
glucan from L. edodes and various products from G. lucidum. Only about 15%
of all products are based on extracts from mycelia. Notable examples are PSK
(trade name Krestin) of a polysaccharide peptide and PSP (polysaccharide-
bound peptide) extracted from Coriolus versicolor. A smaller percent of
mushroom products are obtained from culture filtrates, for example, schizo-
phyllan, a high-molecular-weight (1 -> 3),(1 6)-/3-D-glucan prepared from
Schizophyllum commune Fr., and PSPC (a protein-bound polysaccharide complex)
from Tricholoma lobayense Hein. However, due to increased quality control and
year-round production, mycelial products are the wave of the future.

The market value of medicinal mushrooms and their derivative dietary supple-
ments worldwide was about U.S. $1.2 billion in 1991 and about U.S. $3.6 billion
in 1994 (Chang, 1996). In 1999, it was estimated to be U.S.$6.0 billion. The
market value of Ganoderma-based nutriceuticals alone in 1995 was estimated at
U.S. $1628.4 million (Chang and Buswell, 1999). The corresponding monetary val-
ues were generated by another famous mushroom, L. edodes. Ninety-nine percent
of all sales of medicinal mushrooms and their derivatives occurred in Asia and
Europe with less than 0.1% in North America. The 1999 U.S. market for dietary
supplements based mainly on mushrooms was estimated to be U.S. $35 million.
However, in recent years, the North American demand is increasing between 20
and 40% annually, depending upon species.



1 .7 DEVELOPMENT OF WORLD MUSHROOM
INDUSTRY MOVEMENTS

Although mushrooms have been collected from the wild and cultivated artificially
for human food and for medicine uses for hundreds and thousands of years, it is



26



OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS



Cultivated edible
mushrooms



m




Medicinal
mushrooms



Figure 1.7 Mushroom industry can be considered to be composed of cultivated edible
mushrooms, medicinal mushrooms, and wild mushrooms Single arrows indicate that num-
ber of both edible and medicinal mushroom species increases from time to time through
identification and domestication of unknown and wild mushrooms. Double arrow indicates
that most edible species also possess medicinal properties while many medicinal mush-
rooms can be artificially cultivated.



only recently that the three main segments of the mushroom industry could be
identified. These three segments have received international recognition as impor-
tant interrelated components (Figure 1.7), with each deserving its own special
patronage and paths of development: (1) cultivated edible mushrooms (mushroom
themselves used directly or indirectly as food), (2) medicinal mushrooms (mush-
room derivatives used as nutriceutical therapy/dietary supplements), and (3) wild
mushrooms, including edible mycorrhizal, symbiotic, and poisonous mushrooms
(collected, up to now, only from the wild). The development of three important
international bodies/forums has helped to bring each of these three components of
the mushroom industry to the forefront of international attention, showcasing their
positive contributions to human welfare (Chang, 2006b).



1.7.1 International Movement for Edible Mushrooms

The movement mainly concerned with mushroom production (mushrooms them-
selves) was initiated during the First International Conference on Mushroom Sci-
ence held in Peterborough, the United Kingdom, May 3-11, 1950. Chairman F. C.
Atkins with P. J. Bels, E. B. Lambert, and R. L. Edwards were on the organizing
committee. The committee members later formed the International Commission
on Mushroom Science, which eventually developed into the ISMS.

The Seventeenth International Congress of ISMS will be held May 21-24,
2008, in Cape Town, South Africa. Traditionally, the focus of the ISMS has been
on the A. bisporus mushroom industry. In recent years, the interests of the ISMS
have become more diversified, but A. bisporus is still its main concern.



DEVELOPMENT OF WORLD MUSHROOM INDUSTRY MOVEMENTS



27



1.7.2 International Movement for Medicinal Mushrooms

The movement mainly concerned with mushroom products (mushroom deriva-
tives) was instituted during the First International Conference on Mushroom Biol-
ogy and Mushroom Products held in Hong Kong, August 23-26, 1993. Chairman
S. T. Chang with J. A. Buswell, V. E. C. Ooi, K. W. K. Liu, and S. W. Chiu were
on the organizing committee.

The WSMBMP was launched in January 1994 in response to strong interest
expressed at the conference in Hong Kong the previous year. The object of the
WSMBMP is to promote the enhancement and application of knowledge related
to the basic and applied aspects of mushroom biology and mushroom products
(mushroom derivatives possessing medicinal properties from edible, medicinal,
and wild mushrooms) through publications, meetings, and other means deemed
appropriate. The WSMBMP holds the International Conference for Mushroom
Biology and Mushroom Products (ICMBMP) every three years. The sixth one is
to be held in Bonn, Germany, in 2008.

The international movement for the medicinal segment of the mushroom indus-
try was given a further boost with the launch of the International Journal of Medic-
inal Mushrooms (IJMM) in 1999 by Solomon P. Wasser as editor-in-chief with
Takashi Mizuno, Shu-Ting Chang, and Alexander L. Weis as editors. This then led
to the inaugural International Medicinal Mushroom Conference (IMMC) held in
Kiev, Ukraine, September 12-14, 2001. It has been agreed that there is an IMMC
after an interval of two years. The second IMMC was held in Pattaya, Thailand,
July 17-19, 2003, and the third in Port Townsend, Washington, the United States,
October 12- 17, 2005. IMMC 4 will be in Slovenia in 2007 and IMMC 5 in China
in 2009.



1.7.3 International Movement for Wild Mushrooms

The movement, mainly concerned with edible mycorrhizal mushrooms, was
born as a pre-Congress activity during the second International Conference
on Mycorrhizas in Uppsala, Sweden, in 1999. Two years later, the second
International Workshop on Edible Mycorrhizal Mushrooms (IW-EMM) was held
in Christchurch, New Zealand, July 3-6, 2001. The third IW-EMM was hosted by
the University of Victoria, Canada, August 16-22, 2003, and the fourth was held
in Murcia, Spain, November 29-December 2, 2005. The fifth IW-EMM was held
in Yunnan, China, in 2007. It should be noted that edible mycorrhizal mushrooms
belong to a special group of wild mushrooms which include other symbiotic
mushrooms, for example, termite, hallucinogenic, and poisonous mushrooms.

These three international bodies/forums have done much to promote each of
their respective fields, not the least of which is bringing together scientists in inter-
national forums for useful discussions, encouraging research and the dissemination
of valuable information. These three segments of the mushroom-based industry are
not for competition but for complementation.



28 OVERVIEW OF MUSHROOM CULTIVATION AND UTILIZATION AS FUNCTIONAL FOODS

1.8 CONCLUDING REMARKS

As the population of the world is expected to continue increasing in the twenty-first
century, so will the amount of food and the level of medical care required by each
individual, especially those living in less developed countries. The level of environ-
mental pollution will also become a serious problem. However, the world has an
immense amount of lignocellulosic biomass resource which, like solar energy, is
sustainable. Currently, the bulk of the lignocellulosic biomass is, to a large extent,
considered insignificant or of no commercial value and certainly of no food value,
at least in its original form. It should be noted that large amounts of research funds
have been set aside to search for increased productivity of the core product, like
the oil in the coconut, the cellulose in the tree, the fiber in the sisal, the coffee in
the coffee berry, or the grain in the cereal crop. However, little research funding
has been reserved for the search for the reuse of many by-products (wastes) from
the core products, which are usually considered waste materials. When they are
carelessly disposed to the surrounding environment by dumping or burning, these
so-called wastes are bound to lead to environmental pollution and consequently to
health hazards. It should be emphasized that these lignocellulosic wastes are actu-
ally a kind of new natural resource or new raw material. If they could be properly
managed and utilized, then eventually economic growth would be promoted. In
other words, the by-products in processing the core products can be used/treated
as raw materials for the production of second- or third-core products. For example,
cereal straw, coffee pulp, spent coffee ground, and sisal waste can be used to grow
mushrooms. After harvesting mushrooms, some spent substrates can be used as
feeding materials for animals or used for growing earthworms, and afterward, the
residues can be used as soil conditioners or crop fertilizers. In the whole exercise,
there is no waste produced. This is the concept of zero emissions or total produc-
tivity of raw materials (Pauli, 1996). Therefore, the significant impact of applied
mushroom biology on human welfare in the twenty-first century could be consid-
ered globally as "nongreen revolution."

Since mushrooms, like all other fungi, lack chlorophyll and are nongreen
organisms, they cannot convert solar energy through the process of photo-
synthesis to organic matter, as the green plants do. But they can produce a
wide range of enzymes which can degrade lignocellulosic materials for their
growth and for fruiting. This serves to demonstrate the magnificent capacities
of the mushrooms for biosynthesis, which is different from photosynthe-
sis affected by green plants. Mushrooms not only can become nutritious
protein-rich food (through mushroom science) but also can provide nutriceu-
tical and pharmaceutical products (through mushroom biotechnology). In
addition, through mushroom bioremediation, the recycling of the by-products
(wastes) in the course of each stage of mushroom production using mushroom
mycelia can create a pollution-free environment. Therefore, mushrooms,
with their great variety of species, can constitute a cost-effective means of
supplementing the food nutrition of humankind through the production of
edible mushrooms; alleviate the suffering caused by certain kinds of illnesses



REFERENCES 29



through medicinal mushrooms and their derivatives as nutriceuticals/dietary
supplements (Chang and Buswell, 1996, 2003; Chang and Mshigeni, 2001)
and mycomedicinals (Stamets and Yao, 1998); and reduce environmental
pollution as well as heal the soils (Stamets, 2005) through mushroom mycelial
activities.

The implementation of applied mushroom biology through a well-designed
package of multidisciplinary technologies has already had an impact on human
welfare at national and regional levels in the twentieth century. It is believed that by
blending advances in basic biology with practical technology, mushroom-related
industries can have a global and positive impact on long-term food nutrition, health
benefits, environmental conservation and regeneration, and economic and social
changes.

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CHAPTER 2



Molecular Analysis and Genomic
Studies of Shiitake Mushroom
Lentinula edodes

Hoi-Shan Kwan and Winnie W. Y. Chum

Department of Biology, The Chinese University of Hong Kong, Hong Kong, China

CONTENTS

2. 1 Introduction

2.2 Isolation of Genes

2.3 Molecular Genetics

2.4 Functional Genomic Approaches for Gene Expression Analysis

2.5 Transcriptional Regulation

2.6 Transformation

2.7 Process Analysis

2.8 Conclusion
References



2.1 INTRODUCTION

Lentinula edodes (Berk.) Pegler (L. edodes) is named xianggu in Chinese
and shiitake in Japanese. It is classified in the genus Lentinula, the family
Tricholomataceae, the order Agaricales, and the subphylum Homobasidiomycetes
of the phylum Basidiomycota. Lentinula edodes is largely cultivated in China,
Japan, and other Asian countries and is one of the most popular edible mushrooms
in the world because of its taste and nutritional value. It also contains components,
such as lentinan, that are well-known for their medicinal utility. Due to its
high economic value, many researchers have carried out studies on the strain
improvement of L. edodes. To achieve this improvement, we have to ascertain



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



35



36 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

its biological characteristics and understand the molecular mechanisms involved
in the growth and development of L. edodes at the molecular level. Many
molecular methods have been used to type strains of L. edodes, to isolate
developmentally regulated genes or genes that are expressed in different phys-
iological processes in L. edodes, and to carry out more advanced transcriptome
analysis. For genetic approaches, the methods have included gene cloning,
differential displays with ribonucleic acid (RNA) fingerprinting by arbitrarily
primed polymerase chain reaction (RAP-PCR), expressed sequence tags (ESTs),
complementary deoxyribonucleic acid (cDNA) representational difference
analysis (cDNA-RDA), serial analysis of gene expression (SAGE), cDNA
microarray, and the sequencing-by-synthesis approach (454 Life Science). Using
these methods, many genes have been reported to participate in the fruiting body
development of L. edodes. The genes that are differentially expressed at initiation
of the fruiting body can be categorized into (1) the initiation — stress response
and specific signal transduction; (2) the reconstruction of proteome — protein
degradation, modification, and biosynthesis; and (3) the switching of biochemical
pathways and structural components. In this chapter, we discuss the findings of
these molecular studies, which aim to investigate the growth and developmental
processes of L. edodes.

2.2 ISOLATION OF GENES
2.2.1 Growth

2.2.1.1 Substrate-Utilizing Genes Lentinula edodes generates many
extracellular enzymes to degrade extracellular substrates for energy production.
It utilizes the cellulose and hemicellulose in wood as its major carbon sources.
The enzymes that degrade cellulose and its partial degradation products,
exo-(\ — > 4)-/3-D-glucansase (exo-cellobiohydrolase) and endo-{\ — > 4)-/3-D-
glucansase (<?n<io-cellobiohydrolase), have been isolated, as have the enzymes
that degrade starch, hemicelluloses, and other water-soluble polysaccha-
rides, (gluco)amylase, hemicellulase, a-L-arabinosidase, /J-D-xylosidase, /J-D-
galactosidase, /J-D-mannosidase, and polygalacturonase-pectinase. To degrade
the carbohydrate polymers, L. edodes has to remove lignin with a lignolytic
system. Acid protease is used to degrade proteins. Some water-insoluble
fungal cell wall polysaccharides and their partial degradation products, such
as laminarinase or endo-{\ 6)-/6-D-glucanae (or both), /J-D-glucosidase,
)6-iV-acetyl-D-glucosamindase, and chitinase, have been detected by assays. All of
these enzymes have been extracted and identified, but only a few genes have been
isolated. The molecular studies of the lignocellulytic enzymes that are produced
by L. edodes are discussed in Section 2.7.4.

Cellulase activity increases during colonization and peaks at the veil break of
fruiting body development, which suggests that cellulase is also important for
fruiting body development (Ohga and Royse, 2001). Starch is available for fungi
growing on plants or plant residues that use enzymes, including glucoamylase



ISOLATION OF GENES 37




Mature fruit bodies
during spore formation

Figure 2.1 Life cycle of Lentinula edodes (L-54) grown in sawdust.



(Zhao et al., 2000). Glucoamylase-encoding genes have been cloned from several
fungi, for example, Neurospora crassa (Stone et al., 1993). The glucoamylase gene
(glal) from L. edodes has also been cloned to show that its expression is induced
by starch and increases during fruiting body formation (Zhao et al., 2000).

2.2.2 Development

Lentinula edodes follows a typical basidiomycete life cycle studied for many
years (Figure 2.1) (Carlile and Watkinson, 1997a; Kiies, 2000; Kiies and Liu,
2000; Miles, 1993; Moore, 1998; Wessels, 1993): Under specific environmental
conditions, it produces haploid basidiospores for reproduction. The basidiospores
germinate to form monokaryotic mycelium (monokaryon). Two compatible
monokaryons mate to form dikaryotic mycelium. Clamp connection occurs to
maintain the dikaryotic condition in each hyphal cell after mitosis. When the
mycelium has stored enough nutrients and the environmental conditions allow, it
can proceed to the fruiting cycle. There are four consecutive stages in a fruiting
cycle: induction, pinning, fruiting, and resting. During fruiting, hyphae aggregate
to form primordia and then differentiate into specialized mushroom tissues.
After growing into fruiting bodies, nuclear fusion occurs at the basidium of



38 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

the mushroom gills to form karyogamy. Immediately after karyogamy, meiosis
proceeds to generate four genetically unique haploid basidiospores. These
basidiospores are then dispersed by air currents, and the life cycle begins again
(Figure 2.1). The period from dikaryotic mycelium to primordium is critical to
fruiting. Lowering the temperature can induce pinning in secondary mycelium.
Once primordia start to emerge, mature fruiting bodies can develop.

2.2.2.1 Mating-Type Genes Only two haploid monokaryotic mycelia with
different but compatible mating types can fuse to form dikaryon and then pri-
mordium (Table 2.1). The mating types are controlled by two main loci: mating
types A and B. In related mushrooms, such as Schizophyllum commune, mating
type A locus is responsible for genes that encode different homeodomains of HD1
and HD2 proteins for transcriptional regulation, whereas mating type B locus is
responsible for the genes that regulate pheromone and the pheromone receptor for
the signal transduction of mating and fruiting. The mating type genes found in
L. edodes include a STE3-like pheromone receptor gene (Li et al., 2007). STE3
was reported to be the a-factor receptor gene for mating of Saccharomyces cere-
visiae (Hagen et al. 1986). Details of the mating systems of L. edodes are still
unclear and require more molecular studies.

2.2.2.2 Genes Differentially Expressed in Dikaryotic Mycelium In the

mycelial stages, the genes responsible for energy production and structural com-
ponents are expressed at higher levels (Table 2.1, Figure 2.2). The small G-protein
RAS regulates these growth processes (Hori et al., 1991; Tanaka et al., 2005). Two
transport genes, LeDep and LeStr, may be associated with the transportation of
the signal and sugar, respectively, for signal transduction and energy production
(Leung et al., 2000). Apparently, simple but intensive growth to occupy more nutri-
ents and places is most important in the mycelial stages.

2.2.2.3 Genes for Initial Fruiting Bodies I Primordium Formation

Induced by certain environmental factors, such as cold shock, the dikaryotic
mycelium of L. edodes aggregates and differentiates into primordium (Table 2.1,
Figure 2.3). The genes for signal transduction and transcriptional regulation,
including Gy, cAMP, Le.MAPK, Le.DRMIP, Le.nikl, PriA, PriB, LeJun, LeNotl,
and Le.cdc5, are highly expressed in primordium. The active signal transduction
and transcriptional regulation process are important to initiate and regulate a
variety of physiological activities for primordium formation. It has been suggested
that the high level of intracellular cyclic adenosine monophosphate (cAMP) is
closely related to the onset of fruiting body development and/or primordium
formation (Takagi et al., 1988). The PriA gene encodes a DNA-binding transcrip-
tion factor and has higher expression in primordia/immature fruiting bodies than
in preprimordial mycelia and mature fruiting bodies (Kajiwara et al., 1992). It
may function during fruiting body initiation (Kajiwara et al., 1992). The 565a.a.
PRIB protein is a zinc (II) cys6 zinc cluster DNA-binding motif (Endo et al.,
1994; Miyazaki et al., 1997) and may control expression of the gene(s) correlated



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ISOLATION OF GENES 43



Le.ras
LeDep, LeStr

M *

Structural development Energy production/Lignin degradation
Le.hyd2 LeGpd-M, Exg1, Exg2

Figure 2.2 Genes that may be related to biological processes in dikaryotic mycelium
growth.



Gy

i

cAMP



Le.MAPK, Le.DRMIP, Le.nikl
PriA, PriB, LeJun, LeNott Le.cdc5



t



LeStr

M JC M * X

Cell cycle Protein Stress Fruiting bodies Energy production

control trafficking response morphogenesis LeGpd-M, TCA
LeClb Ubiquitin Le.-FAD1 Hyd1, Le.cypl & cycle, alcohol

2, Le.-FAD2 fermentation, Gla1

Figure 2.3 Genes that may be related to different biological pathways in primordium
development.



to the onset of fruiting body development and/or primordia formation (Endo
et al., 1994; Miyazaki et al., 1997, 2004b). The Le.CDC5 protein, encoded by
the cDNA homologue to Schizosaccharomyces pombe cdc5(+), contains two
putative phosphorylation sites of the cAMP-dependent protein kinase (A kinase)
in its C-terminus (Miyazaki et al., 2004a). Transcripts of Le.cdc5 are highly
expressed in primordia and immature fruiting bodies, which implies that they may
play a role in the beginning and in the early stages of fruiting body development
(Miyazaki et al., 2004a). The Le.MAPK and Le.DRMIP genes are important in
the signal transduction pathway, which is discussed in Section 2.2.3.1 (Szeto
et al., 2007). The gene for vegetative growth is depressed, whereas the gene for
active primordium growth is expressed. LeNotl and LeClb are highly expressed in
primordium to suppress the expression of the genes involved in vegetative growth
and to increase cell numbers for the rapid growth of primordium, respectively
(Leung et al., 2000). Ubiquitins for the reconstruction of proteome-protein



44 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

degradation, modification, and biosynthesis are highly expressed in primordium
(Leung et al., 2000). Both genes and proteins are regulated for initial fruiting body
formation.

Increasing energy demand is necessary for the initial formation of the fruiting
bodies. Different metabolic mechanisms, such as the tricarboxylic acid (TCA)
cycle, alcohol fermentation, and the utilization of lipid and starch, are active.
The gene that encodes the beta subunit of the mitochondrial processing peptidase
(/6-MPP) expresses higher during the fruiting body formation than during that
of the vegetative mycelium (Zhang et al., 1998). Thus, higher mitochondrial
activities may be required to meet the energy demands of the rapid growth of
the fruiting bodies (Zhang et al., 1998). Details are discussed in Section 2.2.3.2.
The expression of the Le.-FDAl gene is induced by a lower temperature and
fruiting body formation (Sakai and Kajiwara, 2003). Alteration of the fatty acid
composition by Le.-FADl may play a role in the stress response for fruiting (Sakai
and Kajiwara, 2003). The morphogenesis of fruiting bodies is a more complex
mechanism than is mycelial structural development. Different hydrophobins
are involved in dikaryotic mycelium and primordium (Hydl and Le.Hyd.2) (Ng
et al., 2000). The Le.cypl, Le.cyp2, and Le.-FAD2 genes for stipe elongation are
successively increased in the expression from primordium to mature fruiting
bodies (Akiyama et al., 2002; Sakai and Kajiwara, 2003). A high-energy demand
is necessary for active signal transduction and transcriptional regulation to
regulate the expression and reconstruction of genes and proteins, respectively, for
the morphogenesis of complex fruiting bodies.

2.2.2.4 Genes for Mature Fruiting Bodies Formation

Stipe Elongation. Elongationless3 (ELN3) and cytochrome P450 may be
involved in the stipe elongation of the fruiting bodies in L. edodes. The eln3
mutation in C. cinereus affects stipe elongation during fruiting body formation
(Arima et al., 2004). The gene homologue to eln3 is highly expressed in the
mature fruiting bodies of L. edodes. Complementary DNAs that were derived from
the gill of the fruiting body have been compared with cDNAs from the mycelia
by differential screening to identify six fruiting body -specific genes: fbg03, 08,
13, 14, 16, and 21 (Hirano et al., 2004). The deduced proteins include cytochrome
P450 and the riboflavin aldehyde-forming enzyme (Hirano et al., 2004).

Cytochrome P450 functions in diverse biological pathways among various liv-
ing organisms. It catalyzes the oxidation of xenobiotic and endogenous natural
compounds in different biological pathways. In L. edodes, some cytochrome P450
genes are differentially expressed in the primordium to the mature fruiting bodies,
especially in stipe rather than pileus and gill tissue (Akiyama et al., 2002; Hirano
et al., 2004; Miyazaki et al., 2005).

Gill Development and Basidiospore Formation. Signal transduction for
this process may be started from primordium because certain signal transduction
genes, such as Le.ras and Le.MAPK, are differentially expressed in primordium
(Table 2.1, Figure 2.4).



ISOLATION OF GENES 45



Le.ga/1

Serine/theronine protein kinase, Le.MAPK, Le.ras, Le.nikl

i

mfbAc , priB, Le.recQ
SPS19, Le.paa, Le.-FAD2, Le.-FAD1, exgl, Exg2, uckl, Le.rnr2c

Figure 2.4 Genes that may be related to gill development and basidiospore formation.

During the production of basidiospores, the biosynthesis of nucleic acids, car-
bohydrates, and lipids is active (Kaneko et al., 1998). Uridine diphosphate (UDP),
cytidine diphosphate (CDP), and adenosine diphosphate (ADP) synthesized by
the uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase serve
as precursors for the synthesis of uridine 5'-triphosphate (UTP) and deoxythymi-
dine triphosphate (dTTP), cytidine triphosphate (CTP) and deoxy cytidine triphos-
phate (dCTP), and adenosine triphosphate (ATP) and deoxyadenosine triphosphate
(dATP), and all serve as substrates for RNA and DNA synthesis. UTP and CTP
are also involved in the generation of other biosynthesis intermediates, such as
UDP-glucose and UDP-galactose in carbohydrate synthesis and CDP-acylglycerol
in lipid synthesis (Kaneko et al., 1998).

The mature fruiting body -specific cDNA, mfbAc, encodes a high-molecular-
weight cell adhesion protein that contains an Arg-Gly-Asp motif (Kondoh and
Shishido, 1995).

The Le.rnrlc gene that encodes the protein homologous to the ribonucleotide
reductase (RNR) small subunit is highly expressed in the hymenophores of
the mature fruiting body of L. edodes (Kaneko and Shishido, 2001). In situ
RNA-RNA hybridization analysis shows that transcripts of Le.rnrl and uckl
are highly abundant in the hymenium and in other regions of the trama in the
hymenophore (Kaneko and Shishido, 2001). The hymenium contains basidia in
which two nuclei are fused for meiosis and replication to produce basidiospores
(Kaneko and Shishido, 2001). Thus, Le.rnrl and uckl may play important roles in
the nucleotide biosynthesis that is essential for the production of basidiospores
(Kaneko and Shishido, 2001). Le.paa encoding a regulatory subunit A (PR65)
homologue of protein phosphates 2A is actively transcribed during the late stages
of fruiting body development (Ishizaki et al., 2000). The Le.paa transcript has
higher expression in gill tissue than in pileus or stipe; therefore it may play a role
in gills in which basidiospores are produced (Ishizaki et al., 2000).

The Le-FADl and Le-FAD2 genes that encode proteins similar to delta 9
fatty acid desaturases and delta 12 fatty acid desaturases have been cloned and
sequenced (Sakai and Kajiwara, 2003, 2005). The transcript level of Le-FAD2
increases following a shift from 25 to 18��C and also from primordia to mature fruit-
ing bodies, whereas the transcript level of Le-FADl is higher in the primordium and
fruiting body than in mycelia cultivated at 1 8 or 25��C. Le-FADl and Le-FAD2 tran-
scripts increase during L. edodes fruiting, and this correlates with an increase in the



46 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

unsaturated fatty acid content in total lipids. Therefore, delta 9 and 12 desaturase
may be needed in fruiting body formation (Sakai and Kajiwara, 2003, 2005).

By genomic binding site cloning, three target genes of the developmental reg-
ulator (PRIB) have been identified in L. edodes. These are previously cloned priB
and uckl and a new gene named mfbC (Miyazaki et al., 2004b). The product of
mfbC, MFBC protein, is highly homologous to Saccharomyces cerevisiae YJR070
C/Lial, the protein interacting with a putative translation initiation factor. The
mfbC transcripts have been found only in mature fruiting bodies, which suggest a
need for mfbC in the final stage of fruiting body formation (Miyazaki et al., 2004b).

The Le.recQ gene homologue that encodes RecQ-type DNA helicase has been
isolated (Katsukawa et al., 2004). The expression level of the Le.recQ transcripts
is similar at all of the developmental stages, and the mycelial growth rate increases
with the increase of Le.recQ transcripts. This suggests that it is necessary for
the good growth of mycelial cells (Katsukawa et al., 2004). As shown by in
situ RNA-RNA hybridization, however, the Le.recQ transcript level within the
hymenophore is higher in the hymenium, subhymenium, and outer region of the
trama, which suggests that the Le.recQ gene may be involved in basidiospore
formation (Katsukawa and Shishido, 2005).

An exo-(\ -> 3)-/J-glucanase-encoding gene (exgl) is expressed in fruiting
bodies but not in vegetative mycelia. The expression is higher in the stipe than in
the pileus of young fruiting bodies but is high in the gills of mature fruiting bodies
(Sakamoto et al., 2005a). Thus, exgl may play a role in fruiting body formation,
including the stipe elongation of L. edodes (Sakamoto et al., 2005a). The exgl
gene that encodes exo-(l — > 3)-/3-glucanase (Sakamoto et al., 2005b), unlike the
exgl gene, is low in transcription and translation in the gills of mature fruiting
bodies but increases after harvesting, which suggests that it is a lentinan-degrading
enzyme-encoding gene (Sakamoto et al., 2005b).

Two laccase genes, lacl and lac2, express higher in the caps of fruiting bodies
than in the stipes and primordium (Zhao and Kwan, 1999). Strong laccase activity
is present in the caps of fruiting bodies, which indicates that laccase may catalyze
the formation of extracellular pigments by oxidation polymerization and is there-
fore important for fruiting body morphogenesis (Zhao and Kwan, 1999). Another
study of the transcriptional regulation of laccase and cellulase genes during the
growth and fruiting of L. edodes, however, has shown that laccase activity is high
during colonization and then declines rapidly during fruiting body formation (Ohga
and Royse, 2001). The difference between these studies may be due to the differ-
ent supplemented sawdust that L. edodes was grown on, the different strains of
L. edodes used, or the different substrates used to determine laccase activity (Ohga
and Royse, 2001). No matter what affected the results, laccase activity does change
during fruiting body development.

In summary, the molecular studies so far have advanced our understanding of
the molecular mechanisms of the fruiting initiation and/or the primordium forma-
tion of L. edodes (Table 2.1). The results show that during the fruiting initiation
and/or the primordium formation the signal is transferred to active transcription,



ISOLATION OF GENES 47



energy production, and protein turnover for fruiting body development. From pri-
mordium to young fruiting bodies, the genes expressed are those responsible for
morphological changes such as pileus and stipe formation. In addition, some genes
may be related to responses to stresses such as temperature change. More studies
of the gene expression profiles of dikaryotic mycelium and the mature fruiting
bodies and sporulating fruiting bodies of L. edodes, however, are required to better
understand the growth and developmental processes of L. edodes.

2.2.3 Physiological Processes in Lentinula edodes

2.2.3. 1 Signal Transduction Signal transduction is important in the growth
and development of any organism. Several genes that may be involved in
signal transduction have been isolated from L. edodes: Le.ras, Le.Ga, Le.mfbC,
Le.recQ, Le.MAPK, Le.DRMIP, and Le.nikl. Mitogen-activated protein kinase
(MAPK) is one of the important signal transduction proteins in the cascades
that regulates growth and development in many organisms. It is regulated by the
phosphorylation cascade of upstream MAPK kinase (MEK) and MEK kinase
(MEKK), and MAPK regulates such downstream effectors as transcription
factors and growth regulators. The MAPK homologue Le.MAPK was identified
from the primordium of L. edodes (Leung et al., 2000). The expression profiles
of Le.MAPK and its interacting novel gene that encodes the developmentally
regulated MAPK-interacting protein, Le.DRMIP, suggest their importance in
fruiting body initiation and development (Szeto et al., 2007). Their expressions
are highest in the primordial stage, and their transcripts are located in the very
young fruiting bodies in which future gill development takes place, which
suggests that MAPK and its interacting partner, Le.PRMIP, play some role in
cell differentiation and morphogenesis during the gill development of L. edodes
(Szeto et al., 2007). Fungal histidine kinase plays an essential role in cell wall
assembly, virulence, sporulation, hyphal development, fungicide resistance, and
osmoregulation (Miller et al., 2002; Nagahashi et al., 1998; Posas et al., 1996; Pott
et al., 2000; Yoshimi et al., 2004). The histidine kinase gene in basidiomycetes,
namely Le.nikl, has been isolated from L. edodes by reverse-blot hybridization
screening (Szeto et al., 2008). The transcript expression level of Le.nikl increases
from mycelium to fruiting body, which indicates that Le.nikl has a role in the
initiation and development of the fruiting body (Szeto et al., 2008). As most
of the other signal transduction gene {Le.ras, Le.Ga, Le.mfbC, and Le.recQ)
transcripts are located in the outer region of the hymenophore in L. edodes (Hori
etal., 1991; Katsukawa and Shishido, 2005; Miyazaki etal., 2004b; Tanaka
et al., 2005), Le.nikl may play a vital role in trama cell development and in
transferring the signal (i.e., basidiospore formation and release) from the middle
trama cell to the outer hymenophore (Szeto et al., 2008). Le.nikl may regulate
important downstream developmental and stress-responding genes (Szeto et al.,
2008).

2.2.3.2 Energy Production In the different developmental stages of
L. edodes, there is the expression of various different genes for different



48 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

metabolic pathways to produce energy. To meet energy demand during the
rapid growth of the fruiting bodies, higher mitochondrial activities may be
required. LeMPP is highly expressed during the development of the fruiting
bodies (Zhang et al., 1998). Surprisingly, both aerobic and anaerobic pathways
are used in the primordial stages to produce energy for their active growth
and development. The genes that encode enzymes for the TCA cycle, citrate
synthase and aconitase, and the genes homologous to the enzymes for alcohol
dehydrogenase, alcohol dehydrogenase and pyruvate decarboxylase, are highly
expressed in the primordium (Miyazaki et al., 2005). Less energy seems to be
needed for mature fruiting bodies, and the genes that encode the enzymes for
glycolysis, fructose- 1,6-bisphosphatase dehydrogenase (Leung etal., 2000), and
glucose-6-phosphate (Miyazaki et al., 2005) are more highly expressed.

2.2.3.3 Structural Proteins in Development Hydrophobins are essential
for morphogenesis and pathogenesis in fungi and fruiting body development
in mushrooms (Kershaw and Talbot, 1998). Hydrophobin-encoding genes have
been isolated from many fungi, such as S. commune, Agaricus bisporus, and
Pleurotus ostreatus. Their expressions are developmentally regulated (Ng et al.,
2000). Two hydrophobin-encoding genes (Le.hydl and Le.hyd2) have been
isolated from L. edodes and characterized to be differentially expressed in
different developmental stages (Ng et al., 2000). The transcript level of Le.hydl
is higher in primordium, and that of Le.hydl is higher in dikaryotic mycelium.
The results indicate that these two L. edodes hydrophobins have distinct roles in
the fruiting body development of L. edodes. In mature fruiting bodies, the genes
for structural components, including various hydrophobins, are not abundantly
expressed. Structural protein production appears to be important from mycelium
to primordium but not for mature fruiting bodies.



2.3 MOLECULAR GENETICS

Since the 1930s modern cultivation methods with pure cultured mycelia as
inoculum have been developed for mushroom cultivation, and increasing efforts
have been made to develop new cultivated strains by mating between strains with
desirable characteristics (Hasebe etal., 1991; Terashima etal., 2002a; Tokimoto
and Komatsu, 1995). Molecular markers have been used to develop suitable
strains for breeding and strain improvement (Table 2.2). They are also used in
genetic mapping, identifying and cloning genes, and studying genetic diversity
(Table 2.2). PCRs using arbitrary primers (AP-PCRs), restriction fragment
length polymorphisms (RFLPs), random-amplified polymorphic DNA (RAPD)
markers, amplified fragment length polymorphism (AFLP) analysis, sequence
characterized amplified region (SCAR) markers, and inter- simple sequence
repeat markers (ISSRs) have been used to generate markers or identify strains
with desirable characteristics for mating (Table 2.2).



MOLECULAR GENETICS 49



TABLE 2.2 Approaches Used for Molecular Genetics of L. edodes


lVInlppiilfir AnrvrnnplipQ


Invpcti crfitinn


R f fi p t*p ti c p


Multilocus enzyme


Genotype identification


RoyseandMay, 1987


electrophoresis






Polymerase chain reaction


Strain typing


Chang et al., 1995; Kwan


using arbitrary primer




etal, 1992a


(AP-PCR)






Mitochondrial DNA


Examination of modes of


Fukuda et al., 1995


(mtDNA) restriction


mitochondrial




fragment length


inheritance in sexual




polymorphisms


crosses and protoplast




(RFLPs)


cell fusions




Random-amplified


1. Strain typing


Zhang and Molina, 1995


polymorphic DNA






(RAPD)








2. Construction of genetic


Kwan and Xu, 2002




linkage map




DNA fingerprints


Strain characterization by


Saito et al., 2002




subrepeat regions




Amplified fragment length


Construction of genetic


Terashima et al., 2002b


polymorphism (AFLP)


linkage map




analysis








Genetic diversity and


Terashima et al., 2002a




strain typing




Sequence-characterized


1 . Screen molecular


Tanaka et al., 2004


amplified region


markers linked to




(SCAR) markers


mating factors using






randomly amplified






polymorphic DNA






(RAPD)






2. Strain identification by


Qin et al., 2006




ISSR




Inter simple sequence


Strain typing in China


Zhang et al„ 2007


repeat markers (ISSR)







2.3.1 Generation of Markers

RAPD is based on a PCR with a pair arbitrary primer that has a predominantly
G/C composition (60-80%) and a relatively low temperature (Gostimskii et al.,
2005; Welsh and McClelland, 1990; Williams et al., 1990). By using RAPD, seven
primers have been used to produce polymorphisms in 15 tested strains of L. edo-
des, and it was found that 13 of them had unique DNA fingerprints (Zhang and
Molina, 1995).

ISSR is also a PCR-based method, but it uses oligonucleotide primers with
repetitive units and the so-called anchor at the 3' or 5' end (Gostimskii et al., 2005;



50 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

Zietkiewicz et al., 1994). Seventeen Chinese strains of L. edodes, including 15
cultivated strains and two wild strains, were clustered into two distinct groups — H
(high-temperature) type or B (broad-temperature) type and L (low-temperature)
type or M (medium-temperature) type (Zhang et al., 2007). Subrepeat regions
within the nuclear rDNA intergenic spacers IGS1 and IGS2 have been amplified
to analyze the relationships between 16 commercial cultivars of L. edodes (Saito
et al., 2002). The DNA fingerprinting from the PCR products of the subrepeat
regions (SRI and SR2) of IGS1 and IGS2 are useful to discriminate among L.
edodes cultivars (Saito et al., 2002).

SCAR markers are based on PCR using long, specific primers (Gostimskii et al.,
2005; Kesseli et al., 1993). A pair of SCAR primers can precisely amplify a single
unique fragment from 85 strains of L. edodes (Qin et al., 2006).



2.3.2 Typing/Fingerprinting

AP-PCR is a PCR-based method that has been found to be better than rDNA
internal transcribed spacer region sequence comparison for L. edodes strain typ-
ing (Chang et al., 1995; Kwan et al., 1992a). The method provides almost unique
DNA profiles for each of the 15 L. edodes strains (Kwan et al., 1992a).

AFLP is based on the selective PCR amplification of genomic DNA restriction
fragments to produce polymorphic loci (Vos et al., 1995). To identify some major
cultivated strains in Japan, six pairs of AFLP primers have been used, and they
detected 304 DNA fragments from 13 cultivated strains for wood log cultivation
and two strains for sawdust cultivation (Terashima et al., 2002b).



2.3.3 Genetic Mapping

It is estimated that the complete map size of L. edodes is about 1200 cM (Kwan
and Xu, 2002). More that half of the genome is covered by an RAPD-constructed
genetic linkage map that contains 14 linkage groups (Kwan and Xu, 2002).

AFLP markers have also been used to generate a medium-dense genetic linkage
map consisting of 11 linkage groups that comprise eight large (over 100-cM) and
three small (less then 100-cM) groups, for a total of 1956.7 cM (Terashima et al.,
2002b).



2.4 FUNCTIONAL GENOMIC APPROACHES FOR
GENE EXPRESSION ANALYSIS

Identifying more differentially expressed genes and analyzing their expression pro-
files under various developmental stages allow us to gain a better understanding of
fruiting body development. The methods used have included RAP-PCR, ESTs,
cDNA random sequencing, and SAGE (Table 2.3).



FUNCTIONAL GENOMIC APPROACHES FOR GENE EXPRESSION ANALYSIS 51

TABLE 2.3 Molecular Approaches Used for Gene Expression Studies of L. edodes



Molecular Approaches

RNA fingerprinting by

arbitrarily primed PCR

(RAP-PCR)
cDNA representational

difference analysis

(cDNA-RDA)



Serial analysis of gene
expression (SAGE)



cDNA microarray



Suppression subtractive
hybridization



Expressed sequence tag
(EST)

Yeast two-hybrid system



Sequencing-by-synthesis
approach (454 Life
Science)



Applications

13 cDNA fragments were

differentially expressed

in primordium
105 genes differentially

expressed in

primordium or mature

fruiting body were

isolated
Gene expression profiles

of monokaryotic and

dikaryotic mycelium,

primordium, and

fruiting body before and

during spore formation
Gene expression profiles

of monokaryotic

mycelium, dikaryotic

mycelium, and

primordium
Selection of differentially

expressed genes in

dikaryons against either

monokaryotic mycelium

parent
Gene expression profiles

of primordium
Isolation of interacting

proteins of functional

proteins
Transcriptome analysis of

dikaryotic mycelium

and mature fruiting

bodies



References
Leung et al., 2000

Miyazaki et al, 2005



Chum et al., 2006a, 2008



Shih, 2003



Shih, 2003



Unpublished

Lee et al., 2007; Szeto
et al, 2007

Chum et al., 2006b; Kwok
et al, 2006



2.4.1 Differential Display: RAP-PCR

Some molecular methods, such as AP-PCR and RAP-PCR, have been used to
study the genomes of mushrooms and to identify the genes that are differentially
expressed during fruiting body development. RAP-PCR has been used for this
purpose in L. edodes. More than 100 genes have been isolated and sequenced,
and 15 have been studied further (Leung et al., 2000). Thirteen RAP fragments
were found to be highly homologous to known genes that function in transport
across the plasma membrane (drug efflux pump and sugar transporter); cell



52 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

cycle control (cyclin B); signal transduction and transcriptional regulation
(mitogen-activated protein kinase, Cdc39/Notl, PriA, and Jun-D); intracel-
lular molecular trafficking (ubiquitin, plasma membrane proton ATPase, and
a-adaptin); mitochondrial biogenesis (mitochondrial processing peptidase beta
subunit, mitochondrial glycerol-3-phosphate dehydrogenase); and intermediary
metabolism (fructose- 1,6-bisphosphatase) (Leung et al., 2000).

Six clones identified as fruiting body-specific genes have been isolated by dif-
ferential screening (Hirano et al., 2004). The deduced proteins include cytochrome
P450 and a riboflavin aldehyde-forming enzyme (Hirano et al., 2004).

2.4.2 cDNA Representation Difference Analysis

To study the molecular mechanisms of fruiting in L. edodes, cDNA-RDA between
vegetatively growing mycelium and two developmental substages, primordium and
mature fruiting body, has been used to isolate 105 individual genes (51 in pri-
mordium and 54 in the mature fruiting body) (Miyazaki et al., 2005).

2.4.3 SAGE and LongSAGE

SAGE was first proposed by Velculescu et al. (1995). It allows simultaneous
comparative and quantitative analysis of the level of transcripts (Yamamoto et al.,
2001). SAGE can analyze each transcript without the transcripts, as in microarray
hybridization (Vedoy et al., 1999; Velculescu et al., 2000). When compared
with differential displays and ESTs, SAGE is more effective and can provide
quantitative and comprehensive profiles (Sun et al., 2004; Vedoy et al., 1999;
Velculescu et al., 2000). SAGE is based on three main principles as follows:

1. Nine to 14-bp short sequence tags are obtained from a defined region within
each transcript that contains sufficient information to identify a transcript
uniquely.

2. Ditags (two individual tags ligated randomly) are ligated together to form a
concatemer, which is a long DNA sequence that can be cloned for sequenc-
ing. Sequencing of the concatemer clones results in the identification of
individual tags.

3. SAGE 2000 Software (version 45) (Invitrogen) is used to analyze the expres-
sion level of the transcript by counting the number of copies of a particular
tag.

LongSAGE is the conventional SAGE method modified by using a different
type of the IIS restriction enzyme, Mmel, to generate longer SAGE tags (Saha
et al., 2002). LongSAGE is based on the acquisition of a sequence tag (15-21 bp)
that, theoretically, can be uniquely assigned to a single genomic position (Saha
et al., 2002). A LongSAGE tag contains more information for identification and is
more reliable in the correct identification of the genomic locus that corresponds to
a certain transcript (Wahl et al., 2005).



FUNCTIONAL GENOMIC APPROACHES FOR GENE EXPRESSION ANALYSIS 53

2.4.3.1 SAGE Profiles: Mycelium to Primordium A total of 6363 tags
have been extracted (3278 from dikaryotic mycelium and 3085 from primordium),
and 919 tags (293 unique tags) match to an in-house EST database (Chum and
Kwan, 2005; Chum etal., 2008). The expression profiles of dikaryotic mycelium
and primordium are very different and reveal that the transcriptional expression
of a specific set of genes is required for initial fruiting body development.
One hundred and thirteen tags are more highly expressed in the dikaryotic
mycelium. Some of them match ESTs that encode various putative proteins.
These are mycelial hydrophobins, serine-rich proteins, the reduced form of
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase I, mitochondrial
phosphate carrier, ATP/PDP carrier protein, NADH dehydrogenase I subcomplex,
phosphatidylethanolanine-binding protein, and lectin. One hundred and forty-four
genes are differentially expressed in primordium. Some of them match ESTs
that encode putative proteins, including Ga-binding protein, ADP ribosylation
factor 1, hesp-379, methallothionein, cytochrome p450, ATP-synthase, ubiquitin,
hydrophobin 1, and riboflavin aldehyde-forming enzyme.

Different structural proteins are abundantly expressed in the two stages. At the
primordial stage, the expression of ribosomal proteins is higher than in mycelium,
which indicates that protein synthesis is active.

2.4.3.2 SAGE Profiles: Fruiting Bodies Maturation to Sporulation Two
LongSAGE libraries were generated to obtain 4000 and 7000 LongSAGE tags
from mature fruiting bodies before and during spore formation (Chum et al.,
2006b). These LongSAGE libraries have been compared to identify the genes that
are more highly expressed in fruiting bodies during sporulation. The expressions
of the genes relevant to sporulation were expected to increase, and the products
of some genes were stored in basidiospores for use during germination. The
LongSAGE tags have also been compared with the SAGE tags obtained in other
developmental stages, such as dikaryotic mycelium and primordium, to analyze
the transcription profiles of certain interesting genes at different stages (Chum
et al., 2006b). Most of the LongSAGE tags, however, do not match the L. edodes
gene databases that were generated from dikaryotic mycelium and primordium.

2.4.4 cDNA Microarray

DNA microarray analysis is commonly used to investigate the expression of
several thousand genes simultaneously, such as screening for interesting genes
on a genomic scale. Lentinula edodes DNA microarray slides were constructed
in-house. About 500 ESTs extracted from primordium and thousands of clones
randomly selected from the subtractive library of dikaryotic mycelium were dotted
on slides for microarray hybridization. The gene expression of monokaryotic
mycelium parents and their dikaryotic mycelium were compared by cDNA
microarray hybridization. In addition, 30 strains of L. edodes were selected from
different regions of mainland China and cultivated in the same conditions. Their
mycelial growth rate, fruiting date, number of fruiting bodies, and weight of



54 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

fruiting bodies were recorded. Eleven of these 30 strains with contrasting char-
acteristics in mycelial growth rates and/or fruiting body amounts were analyzed
by cDNA microarray hybridization. The data were analyzed with The Institute for
Genome Research (TIGR) MultiExperiment Viewer (MeV), and genes were clus-
tered based on their expression patterns through the experiments (unpublished).

2.4.5 Expressed Sequence Tag

A total of 447 unique ESTs were obtained from the single-pass sequencing of 670
random cDNA clones (unpublished). By BLASTX, 238 ESTs showed moderately
to highly significant matches to protein sequences in the databases, and 209 ESTs
had weakly significant or no matches. Among the genes with putative identities,
34% are involved in protein synthesis, 21% in metabolism, 7% in RNA synthe-
sis, and 7% in cell defense. Some ESTs are involved in cell signaling (5%), cell
structure (5%), or cell division (1%). Dot-blot hybridization was used to analyze
the mycelium- primordium expression ratio of 235 ESTs and revealed that 154
ESTs were differentially expressed. Among these, 145 ESTs were more highly
expressed in primordium. ESTs are assigned to roles in cell communication, cell
defense, RNA and protein synthesis, and metabolism. Thus, these cellular pro-
cesses may be important in fruiting body initiation. Because the genes involved
in different cellular processes show primordial preferential expression patterns, it
appears that fruiting body initiation is a complicated process that depends on the
integral functions of the genes involved in different cellular roles.

2.4.6 Yeast Two-Hybrid System

Yeast two-hybrid analysis (Fields and Song, 1989) is one of the most powerful
tools for the investigation of entire protein interaction networks. It has three main
applications:

1 . Testing interactions between known proteins

2. Screening libraries for proteins that interact with a known protein

3. Defining the domains and/or amino acids required for an interaction

It has been used to screen proteins that interact with the signal transduction
genes: Le.nikl, Le.MAPK, and Le.DRMIP (Szeto et al., 2007), and the endocytosis
genes Le.Rab5 and Le.RACKl interact with Le. Rab7 (Lee et al., 2007).

2.4.7 Sequencing-by-Synthesis Approach (454 Life Science)

In a typical large-scale sequencing project, for example, whole-genome
sequencing, it is necessary to clone DNA fragments into vectors to amplify and
purify individual templates followed by Sanger sequencing using fluorescent
chain-terminating nucleotide analogs and either slab gel or capillary electrophore-
sis. High-throughput sequencing technologies are being developed to displace



TRANSCRIPTIONAL REGULATION 55



the use of vectors and Sanger sequencing as the main generators of sequencing
information (Margulies et al., 2005). The cost, complexity, and time required to
sequence large amounts of DNA have thus been reduced.

To understand the biological mechanisms of L. edodes, it is important to iden-
tify more genes. A genome sequence of L. edodes, however, is not available, and
few genes have been isolated. Most of the isolated genes are related to the initia-
tion of fruiting bodies/primordium formation, and few genes that relate to mature
fruiting bodies and sporulating fruiting bodies have been isolated. Large-scale
cDNA sequencing can provide more information on the genes that are important in
mature fruiting bodies. 454 Life Science has developed a scalable, highly parallel
sequencing system with raw throughput that is significantly greater than that of
the state-of-the-art capillary electrophoresis instrument. This system uses a novel
fiber-optic slide (PicoTitle Plate) of individual wells and is able to sequence 25
million bases at ^ 99% accuracy in each four-hour run. This is about a 100-fold
increase in throughput over the current Sanger sequencing technology (Margulies
et al., 2005). To achieve high throughput, an emulsion method for DNA amplifica-
tion and an instrument for sequencing (Genome Sequencer 20 system) by synthesis
using a pyrosequencing protocol optimized for solid support and picoliter-scale
volume have been developed. Using this sequencing-by-synthesis approach, more
than 5000 and 7000 cDNA contigs from L. edodes dikaryotic mycelium and fruit-
ing bodies have been generated.

2.5 TRANSCRIPTIONAL REGULATION

2.5.1 Transcriptional Factors

Many biological processes, such as cell growth, environmental adaptation, and
cell development, are regulated at the transcriptional level. The mechanisms of
such regulations are conserved among eukaryotes (Struhl, 1995). One of these
mechanisms is the specific binding of transcriptional factors (TFs) onto a spe-
cific DNA sequence called transcriptional factor binding sites (TFBSs). TFBSs
are 5-25 nucleotides located at the 5' flanking region before the transcription start
site. TFs and their corresponding binding sites are important in gene regulation.
Current information about TFs and TFBSs in L. edodes is very limited. Three tran-
scription factors, LePriA, PriB, and Le-cdc5, have been identified. Transformants
of L. edodes that overexpress the PriA gene decrease in zinc ion acumination,
which indicates that fruiting body development may involve changes in intracellu-
lar metal ion concentration (Ishizaki and Shishido, 2000).

2.5.2 Promoter Analysis

Studies of the 2-kb nucleotide sequence, including the S-flanking region of a
cell adhesion protein-encoding gene (mfbA) that was isolated from L. edodes,
have revealed that the promoter region contains a TATA box, a GC box, a CAAT
box, and a CT-rich sequence element from upstream to downstream (Kondoh



56 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

and Shishido, 1995). The 3' noncoding region of the priB gene contains several
promoter-like motifs — a GC boxlike sequence, two CAAT boxes and two TATA
boxlike sequences, and two CT motifs — and has promoter activity in S. cerevisiae
(Yamazaki et al., 2002). The PriA promoter has been introduced into Coprinopsis
cinereus to drive the expression of the P. ostreatus manganese (II) peroxidase
gene mnp and of the xylanase genes from Aspergillus oryzae and Bacillus subtilis
(Kikuchi et al, 1999, 2004; Ogawa et al, 1998).



2.6 TRANSFORMATION

Cross breeding is the traditional method to improve strains of mushrooms, but it
is inefficient. Transgenic breeding is a new way for the genetic improvement of
mushrooms to increase their yields and quality. Transformation is an important
tool in transgenic breeding and functional genomics studies. Many approaches are
introduced through this technique, such as eliminating, destructing, or silencing a
gene to reach different targets or needs (Table 2.4). It is powerful in the study of
physiology and gene functions. Transformation protocols can be very specific at
the species level and even at the strain level. Much effort is required to develop
different precautions and protocols for transformation (Table 2.4).



2.6.1 Transformation Methods

Some model fungi have been genetically modified using transformation, includ-
ing C. cinereus, P. ostreatus, and A. bisporus. In L. edodes studies, transformation
has been established for some strains such as dikaryotic strain S-l (Hirano et al.,
2000; Sato et al., 1998). Lentinula edodes has been transformed using the restric-
tion enzyme-mediated DNA integration (REMI) method (Hirano et al., 2000; Irie
et al, 2003; Sato et al., 1998) and the polyethylene glycol (PEG)-mediated trans-
formation method (Li et al., 2006; Sun et al., 2001), which are described below.

2.6.1.1 PEG-Mediated Transformation PEG-mediated transformation
uses PEG as a medium to induce the porous cell membrane for DNA to enter the
cell. The PEG-mediated transformation of L. edodes protoplast has been used to
express vector p301-bGl, which contains a gus gene and a bialaphos resistance
gene; both are driven by the glyceraldehyde-3-phosphate dehydrogenase (GPD)
gene promoter isolated from L. edodes (Sun et al., 2001). The efficiency of
PEG-mediated transformation, however, is usually affected by variations in
conditions and is too low for most studies (Meyer et al., 2003). A modified
PEG-mediated transformation method was used to transfer the hph gene into
L. edodes, resulting in 85-100 transformants per microgram of DNA per 10 7
viable protoplasts (Li et al., 2006).



TRANSFORMATION 57



TABLE 2.4 L. edodes Genes for Transformation









PriA gene


Expresses Streptomyces


Yanaietal., 1996


terminator


hygroscopicus bialaphos resistance






gene in P. ostreatus






Expresses E. coli hygromycin B


Sato et al., 1998




phosphotransferase hph gene in






L. edodes by REMI






Expresses Bacillus subtilis endo- /J-l,


Kikuchi et al., 1999




4-D-xylanase xyn in C. cinereus






Expresses E. coli hygromycin B


Hirano et al., 2000




phosphotransferase hph gene in






L. edodes by REMI






Expresses rat cytochrome P450


Orihara et al., 2005




CYP1A1 in Coriolus hirsutus




PriA gene


Expresses P. ostreatus manganese (II)


Ogawa et al., 1998


promoter


peroxidase gene mnp in C. cinereus






Expresses Aspergillus oryzae


Kikuchi et al., 2004




xylanase XynFl in C. cinereus






Expresses laccase gene led in


Kilaru et al., 2006




C. cinereus




GPD promoter


Express E. coli hygromycin B


Hirano et al., 2000


and terminator


phosphotransferase hph gene in






L. edodes by REMI






Express E. coli hygromycin B


Irie et al., 2001




phosphotransferase hph gene in






P. ostreatus by REMI




Ras gene


Expresses S. hygroscopicus bialaphos


Yanaietal., 1996


promoter


resistance gene in P. ostreatus






Expresses P. ostreatus manganese (II)


Ogawa et al., 1998




peroxidase gene mnp in C. cinereus






Expresses E. coli hygromycin B


Sato et al., 1998




phosphotransferase hph gene in






L. edodes by REMI






Expresses B. subtilis endo- B-l,


Kikuchi et al., 1999




4-D-xylanase xyn in C. cinereus





2.6.1.2 Restriction Enzyme-Mediated Integration REMI transforma-
tion uses restriction enzymes to increase efficiency. Different enzymes are added
to the cell and enter the nuclear membrane to cleave chromosomal DNA in
vivo at their particular restriction sites. The free chromosomal DNA ends that
are generated can be ligated to restriction enzyme -linearized plasmid DNA
by the host cell enzymes. This was first applied in S. cerevisiae to introduce
random tagged mutations into the host genome efficiently (Schiestl and Petes,
1991). REMI was used in transformation with pLCl-hph, a recombinant plasmid



58 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM

containing L. edodes transcriptional signals, and an Escherichia coli hygromycin
B phosphotransferase gene into L. edodes (Hirano et al., 2000; Sato et al., 1998).
An iron sulfur protein (Ip) subunit gene from L. edodes was cloned and used to
construct a homologous drug-resistant marker, Cbx^, which was then successfully
introduced into L. edodes by REMI (Irie et al., 2003).

2.6.1.3 Others

Electroporation. Electroporation, a simple, convenient, and effective technique
for transformation, is widely used in many species. The principle of electropora-
tion is the use of an electric field of high intensity that can induce inner membrane
permeabilization. This permeabilization remains for a short period after the pulse.
It is less often used in mushrooms. Only Agrocybe aegerita (Noel and Labarere,
1994) and Flammulina velutipes (Kuo et al., 2004) have been transformed using
electroporation. There are so far no reports of transformating L. edodes with elec-
troporation.

Particle Bombardment Particle bombardment is the introduction of DNA into
intact cells or tissues by using high-velocity microprojectiles via a mechanism
that breaches cell walls and membrane. Heavy particles, such as gold, tungsten,
and platinum, with the adequate momentum to penetrate into the appropriate tis-
sue are used as carriers. This technique has been successfully applied in different
plants, such as tobacco, soybeans, maize, and others (Yin et al., 2004). This method
makes it possible to carry DNA directly into cells or tissue. Similar to electropo-
ration, a nonoptimal particle bombardment procedure will decrease cell viability
and transformation efficiency. After optimization, its efficiency is very high, and it
is convenient for many studies. This method has only been used in transformating
P. ostreatus (Sunagawa and Magae, 2002) and Volvariella volvacea (Guo et al.,
2005), but not L. edodes.

2.6.2 Lentinula edodes Genes Used in Transformation

The GPD gene is a key enzyme of the glycolytic pathway. It is strongly and con-
stitutively expressed in most tissues of L. edodes (Hirano et al., 1999). Because
only one copy of the GPD gene was detected in L. edodes, it was suggested that
the GPD promoter is very active and can be a useful component of transforma-
tion vectors (Hirano et al., 1999). A plasmid pLG-hph was constructed with an
L. edodes GPD promoter and terminator and introduced into L. edodes by REMI
transformation (Hirano et al., 2000). Under the regulation of a GPD promoter, the
heterologous gene can be stably expressed in L. edodes (Hirano et al., 2000). In
addition, GPD expression signals show that the stable and integrative transforma-
tion of P. ostreatus to hygromycin B resistance is maintained (Irie et al., 2001) and
that foreign genes introduced in L. edodes by PEG-mediated transformation are
effectively expressed (Sun et al., 2001). The PriA gene was isolated and found
to be highly expressed in the primordia/immature fruiting bodies of L. edodes



PROCESS ANALYSIS 59



(Kajiwara et al., 1992). The PriA promoter and/or terminator has been used in
the transformation of P. ostreatus (Yanai et al., 1996), C. cinereus (Ogawa et al.,
1998), L. edodes (Hirano et al., 2000; Sato et al., 1998), and Coriolus hirsutus (Ori-
hara et al., 2005). The Ras gene is highly and constitutively expressed in L. edodes
(Hori et al., 1991), and the Ras gene promoter has been used in the transformation
of P. ostreatus (Yanai et al., 1996), C. cinereus (Ogawa et al., 1998), and L. edo-
des (Sato et al., 1998). The Ras gene promoter has been used with the PriA gene
promoter to effectively regulate heterologous gene expression in different fungi.

2.7 PROCESS ANALYSIS

2.7.1 Postharvest Studies

Studies of lentinan degradation and fruiting body senescence during the posthar-
vest preservation of L. edodes have shown an increase in glucanase activity and
isolated three genes, tlgl, exgl, and exg2 (Sakamoto et al., 2005a, 2005b; 2006).
The tlgl gene encodes a thaumatin-like protein that may be involved in lentinan
and cell wall degradation during senescence following harvest and spore diffusion
(Sakamoto et al., 2006). The exgl gene encodes exo-(l 3)-/J-glucanase, and the
exg2 gene encodes exo-(l -> 3)-/3 -glucanase.

2.7.2 Stress Responses

2. 7.2. 1 Studies of Temperature Stress in Mushrooms Although temper-
ature stress is commonly employed in forced fruiting and appears to be a crucial
factor in fruiting body induction, it has not been studied at the molecular level in
L. edodes. Some studies have been carried out, however, in another popular edible
mushroom, the phoenix tail mushroom Pleurotus sajor-caju (Jeong et al., 2000;
Lee et al., 2006).

2.7.2.2 Studies of Molecular Chaperones in Fungi Molecular chaper-
ones are the typical target genes in studies of stress responses in fungi. Several
chaperones in L. edodes have been studied.

Role of Molecular Chaperones. Molecular chaperones are proteins that medi-
ate appropriate protein folding by binding to unfolded or unassembled proteins and
protein translocation from cytosol to target organelles during normal growth. They
also protect certain other proteins from misfolding by renaturation under heat and
other physiological stresses (Craig et al., 1993). In addition, some molecular chap-
erones, such as HSP70, have been found to prevent the activation of stress kinase
(Gabai et al., 1997). The 70-kilodalton heat-shock proteins (HSP70) are the most
well characterized due to their highly conserved amino acid sequences (50-98%
similarity) among all species, from bacteria to human (Lindquist and Craig, 1988).
The differential expression of the chaperones, Ssb and TCP1, and the cochaper-
ones, Mgel and Stil, during the fruiting body development of L. edodes suggests



60 MOLECULAR ANALYSIS AND GENOMIC STUDIES OF SHIITAKE MUSHROOM



TABLE 2.5 Lignocellulytic Enzymes Isolated in L. edodes



Biopolymer"


Enzymes


Genes


References


Cellulose


endo- 1 ,4-fi -D-Glucanase


Le.egl


Kwok et al., 2006




Cellulase


Cel7A and 6B


Leeetal., 2001


Xylan


Xylanase


xynllA


Lee et al, 2005


Lignin


Manganese-dependent


Mn(II) peroxidase


NCBI accession




peroxidase 1




no. BAE79199




Laccase


lacl and 2


Zhao and Kwan,








1999



their biological significance to mushroom development (Bian, 2001). Although
in most studies the mechanisms are still unclear, the effect of temperature stress
on development is still worth studying at the molecular level for insights into the
low-temperature-induced forced-fruiting phenomenon.

2.7.3 Lignocellulose Degradation

As it is a white rot fungus, L. edodes secretes extracellular enzymes to degrade
both lignin and cellulose to leave a light, white, and fibrous residue from wood
(Table 2.5). Lignin is a phenylpropanoid polymer that has a complex, heteroge-
neous structure that is difficult to degrade. Up to 30% of plant material, how-
ever, is composed of lignin, which allows plants to have an integrity structure
and protects against pests and pathogens. The lignin-degrading enzyme lignin
peroxidase has been isolated from the white rot fungus Phanerochaete chrysospo-
rium. Lignin is degraded with the presence of hydrogen peroxide, and the haem
enzyme releases highly reactive, transient free radicals of oxygen that break the
covalent bonds of lignin to release the phenolic compounds that are characteris-
tic of lignin breakdown (Carlile and Watkinson, 1997b; Kirk and Fenn, 1982). To
use lignin, L. edodes has to generate specific enzymes to degrade lignin. Only a
few genes of ligninocellulytic enzymes have been isolated in L. edodes. There-
fore, the molecular aspects of biodegradation by L. edodes are still unclear. Using
the sequencing-by-synthesis approach (454 Life Science), Le.egl has been isolated
from the lignin grown mycelium of L. edodes. Cel7A and 65, xynllA, and Le.egl
for cellulose and xylan degradation have also been isolated (Kwok et al., 2006; Lee
et al., 2001, 2005).. Also, lad and lac2, encoding laccase proteins, were isolated
from L. edodes (Zhao and Kwan, 1999). The gene expressions of lacl and lac2
in different substrates and various developmental stages have been analyzed and
shown to be very variable (Zhao and Kwan 1999).

2.7.4 Meiosis

Ribonucleotide reductase small subunit cDNA, Le.rnrlc, is most actively tran-
scribed in the hymenophores of the mature fruiting bodies of L. edodes (Kaneko



REFERENCES 61



and Shishido, 2001), and in situ hybridization analysis has shown this to be the
case. This enzyme plays a role in the nucleotide biosynthesis that would be essen-
tial both for the production of basidiospores and for the divergence of trama cells
into subhymenium cells in the hymenophore (Kaneko and Shishido, 2001).



2.8 CONCLUSION

Our understanding of the growth and development of L. edodes is still fragmented.
More studies are required to unravel the mechanisms of growth and development
in this popular edible mushroom. In the past, molecular genetic studies using
RAPD, AP-PCR, and other PCR-based strain-typing methods have been carried
out to identify strains with desirable characteristics for breeding and to generate a
genetic linkage map of L. edodes. More recently, gene cloning and transformation
techniques have been developed to identify the functions of the genes and to
study the physiological processes of the mushroom. Recently, gene expression
profiles under different conditions or developmental stages have been studied
by using high-throughput technologies such as cDNA-RDA, SAGE, microarray,
and sequencing by synthesis. The transformation efficiency of L. edodes,
however, is too low to perform functional tests on the novel genes isolated with
high-throughput technologies. Current knowledge obtained from the molecular
studies of L. edodes reveals that gene expression profiles are very different in
dikaryotic mycelium to primordium and eventually in mature fruiting bodies.
The genes differentially expressed at the initiation of the fruiting body can be
categorized into (1) the initiation- stress response and specific signal transduction;
(2) the reconstruction of proteome- protein degradation, modification, and
biosynthesis; and (3) the switching of biochemical pathways and structural
components (Chum et al., 2006a). In particular, certain biological processes, such
as postharvest degradation, lignindegradation, and endocytosis, have been studied.
Molecular studies of L. edodes advance our understanding of its growth and
developmental processes and of those of edible mushrooms in general.

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CHAPTER 3



Nutritional Value and Health
Benefits of Mushrooms

Peter C. K. Cheung

Food and Nutritional Sciences Programme, Department of Biology, The Chinese University of
Hong Kong, Hong Kong, China

CONTENTS

3.1 Introduction

3.2 Wild and Cultivated Edible Mushrooms

3.3 Production of Cultivated Mushrooms

3.4 Nutritional Composition

3.5 Newly Cultivated/Nonconventional Mushrooms

3.6 Nutritional Evaluation

3.7 Health Benefits of Edible Mushrooms

3.8 Conclusion
References



3.1 INTRODUCTION

Since the work of Crisan and Sands in the 1970s (Crisan and Sands, 1978), only
a few comprehensive reviews have been produced on the nutritional composition
of wild and edible mushrooms (Chang and Miles, 2004; de Roman et al., 2006).
Although the number of cultivated mushroom species (and especially those grow-
ing in Asia) has grown recently, there is a lack of knowledge of their chemical
composition and nutritional value. This chapter reviews the recent findings on
newly developed species of cultivated mushroom and discusses current investi-
gations into the health benefits of edible mushrooms to humans, with emphasis on
the prevention of chronic conditions such as cardiovascular disease and diabetes.



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



71



72 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS



3.2 WILD AND CULTIVATED EDIBLE MUSHROOMS

Wild edible mushrooms have been part of the human diet for thousands of years
due to their nutritional, organoleptic characteristics and medicinal properties (de
Roman et al., 2006). An overview of the uses and importance of wild edible mush-
rooms was published recently in which more than a thousand species of wild mush-
rooms consumed in 85 countries were reported (Boa, 2004). Among these species,
the black truffle (Tuber melanosporum), boletes (Boletus spp.), and chanterelles
(Cantharellus cibarius) from Europe and the matsutake (Tricholoma matsutake)
from China and Japan have the highest economic value.

Fewer than 25 edible mushroom species are widely cultivated and accepted as
a food of economic importance (Smith, 1972). In more affluent countries, mush-
rooms are considered a somewhat expensive type of vegetable and are eaten almost
exclusively for their culinary properties and to provide flavor or as a garnish for
other foods (Flegg and Maw, 1977). Currently, the possibility of using mushrooms
as a source of protein in the human diet in developing countries is being consid-
ered (Boras, 1996), as mushrooms are a healthy food that is low in calories and fat
but rich in protein, dietary fiber, vitamins, and minerals (Crisan and Sands, 1978).
Several studies have been carried out on the chemical composition and nutritional
quality of edible mushrooms grown in different parts of the world, including those
produced in tropical areas (Aletor, 1995; Sanmee et al., 2003), India (Longvah
and Deosthale, 1998), North America (Leichter and Bandoni, 1980), and Europe
(Senatore, 1992; Dfez and Alvarez, 2001; Manzi et al., 2001; Caglarirmak et al.,
2002).

The fruiting bodies of mushrooms are the most common edible form in the
human diet (Gray, 1973), but the sclerotia of some mushroom species can also
be consumed. The mycelia are less commonly utilized as a human food despite
their shorter production time and comparable nutritional value to fruiting bodies
(Litchfield, 1967; El-Kattan et al., 1991; Cheung, 1997b).

3.3 PRODUCTION OF CULTIVATED MUSHROOMS

The world production of cultivated mushrooms was estimated to be 6.1 million
tons in 1997 and 12.2 million tons in 2002 (Chang, 2006), which represents a dou-
bling in just five years. The world production of Lentinula edodes increased from
14.3 to 25.4% (in terms of output, from 180,000 to 1.5 million tons), and that
of Pleurotus spp. from 2.8 to 14.2% (in terms of output, from 35,000 to 876,000
tons). More than 10 new mushroom species, including Agaricus blazei, Pleurotus
eryngii, and Agrocybe aegerita, have been cultivated in recent years on a small
commercial scale, and the potential for expansion is great. However, the output
yield of the 10 main cultivated mushrooms makes up 91.6% of the total world
production (Table 3.1), with the 6 most important, namely, Agaricus (31.8%),
Lentinula (25.4%), Pleurotus (14.2%), Auricularia (7.90%), Flammulina (4.60%),
and Volvariella (3.0%), contributing up to 86.9%. In China, Tremella is mainly cul-
tivated, whereas in Japan Hypsizygus marmoreus and Grifola frondosa are the most



NUTRITIONAL COMPOSITION 73



TABLE 3.1 World Production of Cultivated Edible Mushrooms in 1997



Production (Fresh wt x 1000 metric tons)



Rest of North Latin



Species


China


Japan


Asia


America


America


Europe


Africa


Agaricus bisporus


330


NP


68.4


425.3


51.6


990.2


36.0


Lentinula edodes


1397


115.3


47.4


3.60


0.30


0.80


NP


Pleurotus spp.


760


13.3


88.4


1.50


0.20


12.0


0.20


Auricularia spp.


480


NP


5.30


NP


NP


NP


NP


Volvariella volvacea


120


NP


60.8


NP


NP


NP


NP


Flammulina spp.


150


109


25.7


NP


NP


NP


NP


Tremella spp.


130


NP


0.50


NP


NP


NP


NP


Hypsizygus marmoreus


2.10


72.0


0.10


NP


NP


NP


NP


Pholiota nameko


31.0


24.5


NP


NP


NP


NP


NP


Grifola frondosa


2.00


31.0


NP


NP


NP


NP


NP


Hericium erinaceus


0.80


NP


NP


NP


NP


NP


NP


Coprinus comatus


0.50


NP


NP


NP


NP


NP


NP


Others"


514.9


2.90


0.80


0.40


NP


0.30


NP


Total


3918.3


368


297.4


430.8


52.1


1003.3


36.2


Percent of total world


63.6


6.00


4.80


7.00


0.80


16.3


0.60



production



"Mushrooms with emerging commercial potential, e.g., A. blazei, Lepista nuda, P. eryngii, A. aegerita,
Tricholoma giganteum, Auricularia fuscosuccinea, Tremella cinnabarina, etc.
Note: NP, no production.
Source: From Chang (1999).

common cultivated species. The total mushroom production in China in 1997 was
3.9 million tons, which accounted for 63.6% of the total world output, demonstrat-
ing that China has become a leading producer and consumer of cultivated edible
mushrooms. Lentinula edodes is the leading cultivated mushroom in China, with
a production of 1.4 million tons in 1997, which was equal to 35.6% of the total
mushroom production in that year.



3.4 NUTRITIONAL COMPOSITION
3.4.1 Conventional Edible Mushrooms

3.4. 1. 1 Moisture In general, the moisture content of mushrooms ranges from
85 to 95% of their fresh weight. The specific moisture content of some fleshy
edible mushrooms is listed as follows: Grifola frondosa (86.1%), Pleurotus ostrea-
tus (85.2-94.7%), P. eryngii ferulae (88.1%), Pleurotus plumonarius (87.7%),
L. edodes (81.8-90%), Flammulina velutipes (87.2-89.1%), Pleurotus cystidi-
ous (86.7%), and Agaricus bisporus (92.8-94.8%) (Manzi et al, 1999, 2001; Mau
et al., 2001b, c; Yang et al., 2000). However, it must be noted that the moisture
content of mushrooms is affected by the time of cropping, watering conditions



74 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

during cultivation, postharvest period, and temperature and relative humidity dur-
ing growth (Bano and Rajarathnan, 1988). The moisture content of dry mushrooms
is generally less than 10% (w/w), with figures of 9.05% for Dictypphora indusiata
and 5.30% for Schizophyllum commune being previously reported (Longvah and
Deosthale, 1998; Mau et al, 2001b, c).

3.4.1.2 Protein and Amino Acids The crude protein content of edible
mushrooms is usually high, but varies greatly and is affected by factors such as
species and stage of development (Longvah and Deosthale, 1998). The crude
protein content [percent dry weight (%DW)] of some common edible mushrooms
is as follows: L. edodes (15.2-23.0%), Schizophyllum commune (16.0-27.0%),
Tricholoma giganteum (16.1%), P. ostreatus (19.9-34.7%), P. eryngii ferulae
(23.2%), and Tricholoma terreum (15.0%) (Longvah and Deosthale, 1998; Manzi
et al, 1999; Dfez and Alvarez, 2001).

The essential amino acid content (g/100 g protein DW) of mushrooms ranges
from 34 to 47%, although one previous report showed that essential amino acid
levels can be as high as 61.8% (Tricholoma portentosum) and 63.3% (T. terreum)
(Manzi et al., 1999). The protein of edible mushrooms is comparatively rich in glu-
tamic acid (12.6-24.0%), aspartic acid (9.10-12.1%), and arginine (3.70-13.9%).
The essential amino acid profiles of eight mushrooms, together with the Food
and Agricultural Organization/World Health Organization (FAOAVHO) require-
ment patterns (FAO, 1991), are shown in Table 3.2 and reveal that the proteins
are deficient in sulfur-containing amino acids, including methionine (1.22-21.6
mg/g protein) and cysteine (15.7-19.1 mg/g protein). However, these edible mush-
rooms are comparatively rich in threonine (41.2-94.5 mg/g protein) and valine
(35.8-88.7 mg/g protein). It has been reported that lysine, leucine, isoleucine,
and tryptophan are the limiting amino acids in some edible mushroom proteins
(Cheung, 1997b; Manzi etal., 1999; Dfez and Alvarez, 2001). The amino acid
composition of other wild and cultivated mushrooms was reported previously by
Crisan and Sands (1978) and Mdachi et al. (2003).

The free amino acid level in mushrooms is low, ranging from 7.14 to 12.3
mg/g in dry edible mushrooms, with glutamic acid (21.7-23.7%) and alanine
(17.7-17.9%) predominating (Manzi etal., 1999). Free amino acids, and
especially highly basic amino acids, contribute the main flavor properties of
mushrooms (Sugahara etal., 1975; Maga, 1981), but a low level of other free
amino acids, such as /-amino butyric acid (GABA) (10.2-281 mg/100 g DW)
and ornithine (88.7-392 mg/100 g DW), has been reported in edible Pleurotus
mushrooms (Manzi et al., 1999). The profiles of free amino acids in mushrooms
are considerably different. Aspartic acid and glutamic acids are monosodium
glutamate (MSG) -like components that give the most typical mushroom taste,
that is, the umami taste or palatable taste that is the characteristic taste of MSG and
5'-nucleotides (Mau et al., 2001b, c). The content of these MSG-like components
in common mushrooms is relatively low, ranging from 22.7 to 47.1 mg/g DW
(Tseng and Mau, 1999), 11.2-26.2 mg/g DW in V. volvacea (Mau etal., 1997),
10.9-11.9 mg/g DW in Agrocybe cylindracea (Mau and Tseng, 1998), and



NUTRITIONAL COMPOSITION 75



TABLE 3.2 Essential Amino Acid Profiles (mg/g protein) of Edible Mushrooms



Mushrooms


lie


Leu


Lys


Met


Cys


Phe


Tyr


The


Val


Trp a


Volvariella bombycina


54.1


50.1


54.1


1.22


19.1


60.2


45.8


46.5


35.8


ND


L. ulmarius


58.9


101


46.1


19.1


18.2


51.1


72.1


41.2


56.1


ND


Pleurotus citrinopileatus


35.1


71.2


56.3


25.4


15.8


39.7


32.2


49.2


60.7


ND


T. portentosum


37.2


93.7


86.3


29.6


16.2


43.6


32.1


94.5


77.6


9.60


T. terreum


35.8


81.5


76.3


34.6


17.0


66.1


30.0


90.7


88.7


10.6


L. edodes


33.0


63.8


49.8


21.6


34.0


38.1


26.0


55.5


381


19.2


P. eryngii female


41.1


65.6


67.1


16.9


15.7


40.4


34.2


50.4


45.1


12.2


P. ostreatus


44.5


72.8


61.1


20.1


16.8


46.9


40.6


51.6


48.8


40.6


FAO (1991)


28


66


58


25*


63


c


34


35


11



requirement pattern



"ND, not determined.
*Value includes Met and Cys.
c Value includes Tyr and Phe.

Source: From FAO, 1991; Cheung, 1997b; Manzi et al., 1999; Dfez and Alvarez, 2001; Dabbour and
Takruri, 2002a.



3.75-9.06 mg/g DW in L. edodes (Lin, 1988). The levels of sweet components,
which are mainly soluble sugars (Ala + Gly + Ser + Thr), are also low, ranging
from 0.36 to 8.71 mg/g DW. The levels of bitter components (Arg + His + He +
Leu + Met + Phe + Trp + Val) are high, ranging from 2.37 to 6.46 mg/g DW.
However, bitterness from the bitter components can be unequivocally masked by
the sweet components (Mau et al., 2001b, c; Yang et al., 2002).

3.4.1.3 Fat Edible mushrooms generally have a low lipid level of less than
10% DW but nevertheless are a source of unsaturated fatty acids, and especially
oleic and linoleic acids. In some species, the lipid content may be as low as 2.0%
(L. edodes, S. commune, and P. ostreatus) (Longvah and Deosthale, 1998). The
lipid content (%DW) of some other mushrooms are as follows: Dictyophora
indusiata (2.98%), T. giganteum (4.28%), G. frondosa (3.10%), L. edodes
(5.71-6.34%), Lyophyllum ulmarius (2.09%), V. bombycina (2.75%), and
T. terreum (6.60%) (Mau et al., 2001b, c; J. H. Yang etal., 2000). The levels of
polyunsaturated fatty acids in mushrooms are high, constituting more than 75%
of the total fatty acids, of which palmitic (19.2%), oleic (8.3%), and linoleic
(68.8-84.0%) acids are the most significant (Cheung, 1997b; Longvah and
Deosthale, 1998; Dfez and Alvarez, 2001; J. H. Yang et al., 2002). Linolenic acid
levels are generally low in mushrooms (Yilmaz et al., 2006), but despite its small
quantity, this compound is strongly related to flavor in certain mushrooms, as it
is the precursor to l-octen-3-ol, or the "alcohol" of fungi, and is the principal
aromatic compound in most mushrooms (Maga, 1981).

3.4. 1.4 Ash and Minerals The ash content in mild edible mushrooms ranges
from 6 to 10.9% DW and represents a wide variety of minerals (Zakhary et al.,



76 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

1983). Recently, the metal content of wild edible mushroom species in the Mediter-
ranean was reported (Ouzouni et al., 2007). The ash content (%DW) of some
edible mushrooms is as follows: P. ostreatus (6.90%), P. eryngii ferulae (8.60%),
L. edodes (5.27-5.85%), and Hericium erinaceus (9.35%) (Manzi et al., 1999;
Mau et al., 2001b, c). Cultivated mushrooms are a good source of minerals, con-
taining macroelements such as calcium, magnesium, sodium, potassium, and phos-
phorus and microelements such as copper, iron, manganese, and zinc. Some com-
mon cultivated edible mushrooms, including P. ostreatus, L. edodes, and A. bis-
porus, are rich in potassium (2670-4730 mg/100 g DW) and are a good source
of phosphorus (493-1390 mg/100 g DW), magnesium (20-200 mg/100 g DW),
zinc (4.70-9.20 mg/100 g DW), and copper (0.52-3.50 mg/100 g DW) but are
low in sodium (130-420 mg/100 g DW), calcium (1-25.0 mg/100 g DW), iron
(2.80-12.30 mg/100 g DW), and manganese (0.51-2.1 mg/100 g DW) (Zakhary
et al., 1983; Verma et al., 1987; Vetter, 1990; Shah et al., 1997).

Mushrooms are known to accumulate heavy metals, but the concentration
of these elements is generally assumed to be species dependent, with substrate
composition also being an important factor (Kalac and Svoboda, 2000; Demirba��,
2001; Stijve etal, 2004; Svoboda etal., 2000, 2006). The levels of cadmium
(3.60-120 M-g/100 g DW) and selenium (3.90-320 |xg/100 g DW) vary (Bano
and Rajarathnam, 1988; Vetter, 1994a, b, 1995; Mattila etal., 2001), but low
levels of lead (2.0-18.0 |xg/100 g DW) and mercury (0.09-0.13 mg/100 g DW)
have generally been reported (Piepponen et al., 1983; Svoboda et al., 2002).
Mushrooms in general and species in the Boletus genus in particular are rich in
selenium (Cocchi et al., 2006).

3.4.1.5 Vitamins Cultivated mushrooms are a good source of several vita-
mins, such as riboflavin (vitamin B2), niacin, and folates, with concentrations that
vary within the range of 1.8-5.1, 31-65, and 0.30-0.64 mg/100 g DW, respec-
tively, depending on the species. The vitamin B2 content in mushrooms is higher
than that generally found in vegetables, and some varieties of A. bisporus even
have a level of vitamin B2 as high as that found in egg and cheese (Mattila et al.,
2001). The vitamin B2 content of other cultivated edible mushrooms is as fol-
lows: P. ostreatus (2.27-8.97 mg/100 g DW), A. bisporus (3.70-5.10 mg/100 g
DW), andL. edodes (0.90-1.80 mg/100 g DW) (Crisan and Sand, 1978; Bano and
Rajaratham, 1986, 1988; Miles and Chang, 1997; Mattila et al., 2001). Cultivated
mushrooms are rich in niacin, but again the content varies, from 33.8-109 mg/100
g DW for P. ostreatus, 11.9-98.5 mg/100 g DW for L. edodes, and 36.2-57.0
mg/100 g DW for A. bisporus (Crisan and Sand, 1978; Bano and Rajaratham, 1986,
1988; Miles and Chang, 1997). Mushrooms contain moderately high amounts of
folates at concentrations that are of the same magnitude as is generally found
in vegetables. Furthermore, the bioavailability of folates is as good as that for
folic acids (Clifford et al., 1991), with a content of 640-1412 Ltg/100 g DW for
P. ostreatus, 590-933 ^g/i00 g DW for A. bisporus, and 300 (xg/100 g DW for
L. edodes (Bano and Rajaratham, 1986, 1988; Mattila et al., 2001). In addition to
riboflavin, niacin, and folates, cultivated mushrooms also contain small amounts



NUTRITIONAL COMPOSITION 77



of vitamin C and vitamin Bl and traces of vitamins B12 and D2. The variation
in the vitamin C content of mushrooms is wide, ranging from 17.0 in A. bisporus
and 25.0 mg/100 g DW in L. edodes to 36.4-144 mg/100 g DW in P. ostreatus
and 40.4-59.9 mg/100 g DW in L. edodes. (Li and Chang, 1985; Beelman and
Edwards, 1989; Kurzman, 1997). The vitamin Bl content in mushrooms is quite
low, ranging from 0.60 to 0.90 mg/100 g DW but is of the same magnitude as is
generally found in vegetables. Traces of vitamin B12 (0.60-0.80 |xg/100 g DW)
have also been found in cultivated mushrooms (Walker, 1996). Vitamin D is almost
absent in cultivated mushrooms, although the level present depends on the culti-
vation conditions (Mattila et al., 2001). It has been reported that the vitamin D2
content in A. bisporus (0.21 (j,g/100 g DW) cultivated in the dark is lower than that
of L. edodes (22.0-110 |xg/100 g DW) cultivated in natural climatic conditions
(Takamura et al., 1991), which is mainly due to the influence of illumination on
the conversion of ergocalciferol (provitamin D) to vitamin D (Mattila et al., 2001).

3.4.1.6 Dietary Fiber All fungal cell walls contain a mixture of fibrillar
components and amorphous or matrix components. In Basidiomycetes, the main
fibrillar components include chitin, which is a straight-chain (1 — > 4)-/Minked
polymer of N-acetyl-glucosamine, and matrix components, which include various
polysaccharides such as (1 -> 3)-a- and (1 3)-j6-D-glucans and mannans
(Bartnicki-Garcia, 1968). All of these cell wall components are nonstarch
polysaccharides that can be classified as dietary fiber [American Association of
Cereal Chemists (AACC), 2001].

There is a large variation in the total dietary fiber (TDF) content of edible
mushrooms, which depends on their morphological form and species. The fruit-
ing bodies of some mushroom species have a low level of TDF (4.50% DW in
T. giganteum, 9.26% DW in D. indusiata, and 8.74% DW in P. cystidious), whereas
others have a high TDF level, such as T. portentosum (45.0%), T. terreum (50.0%),
Auricularia auricula (49.7%), Tremellafuciformis (54.5%), V. bombycina (25.7%),
L. ulmarius (33.2%), and Pleurotus citrinopileatus (35.6%) (Cheung, 1997b; Dfez
and Alvarez, 2001). In general, mushrooms are a good source of dietary fiber, with
100 g of fresh mushrooms providing between 10 and 40.0% of the recommended
dietary intake of fiber (Manzi et al., 2001). The TDF in mushrooms is predomi-
nantly composed of insoluble dietary fiber (IDF) and a low level of soluble dietary
fiber (SDF) of less than 10%. This may be partly due to the differing amounts of
chitin found in different mushrooms (Manzi et al., 1999), which in Pleurotus varies
from 4.70 to 4.90% DW (Yoshida et al., 1986). The /3-glucan content ranges from
0.21 to 0.53 g/100 g DW in mushrooms, of which 16.8-46.0% is found in SDF
and 53.9-83.2% in IDF (Manzi and Pizzoferrato, 2000). A previous study showed
that neutral and amino sugars are the main components in the TDF of A. bisporus
(84.8%), Auricularia auricula (81.4%), Auricularia fuciformis (81.4%), Pleurotus
sajor-caju (77.8%), andL. edodes (92.8-92.9%) (Cheung, 1996a, 1997a; Cheung
and Lee, 1998), with a small amount of uronic acid (2.10-12.6% of the TDF) and
Klason lignin (2.40-12.9%) also being reported (Cheung, 1997a, 1998). Glucose



78 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS



(43.1-82.8% of the TDF) is the predominant sugar in mushrooms, but the pres-
ence of other sugars, such as mannose, xylose, rhamnose, galactose, and uronic
acids, in the TDF reflects that the main cell wall polysaccharides in most fungi are
hemicelluloses, such as /3-glucan, glucuronoxylomannan, pectic substances, and
chitin (Cheung, 1996a, 1997a). It is worth noting that mushroom sclerotia, which
are the dry compact biomass of fungal hyphae, contain over 80% DW of TDF that
is composed mainly of /3-glucans (Wong et al., 2003). The structure and function
of sclerotial TDF is discussed in more detail in Chapter 4.

3.4.1.7 Carbohydrates The carbohydrate content of edible mushrooms
varies with species and ranges from 35 to 70% DW. The carbohydrate content
of some edible mushrooms is as follows: P. indusiata (67% DW), G. frondosa
(58.8% DW), T. giganteum (70% DW), L. edodes (62.3-64.4% DW), P. cystidious
(63.1% DW), S. commune (61.1% DW), and P. ostreatus (61.1% DW) (Longvah
and Deosthale, 1998; Dfez and Alvarez, 2001; Mau etal., 2001b, c). Edible
mushrooms are believed to contain a high level of oligosaccharides and only a low
level of total soluble sugars (182 mg/100 g DW for T. portentosun and 55 mg/100
g DW for T. terreum) and glycogen (15.6 mg/100 g DW for T. portentosun and
10.6 mg/100 g DW for T. terreum) (Dfez and Alvarez, 2001). The profiles of
soluble sugars differ across species. Arabitol (127 mg/g DW), glucose (4.91-39.4
mg/g DW), mannitol (9.36-50.9 mg/g DW), trehalose (9.71-341 mg/g DW), and
inositol (1.43-3.20 mg/g DW) are found in edible mushrooms, with mannitol
and trehalose being the two major components in V. volvocea and P. ostreatus,
respectively (Bano and Rajarathnam, 1988; Mau et al., 1997).

3.4.1.8 Energy The energy content of edible mushrooms is generally low,
which allows them to be used in low-energy diets. Previous reports have shown
that the energy content in Tricholoma robustus is 3.02 kcal/g DW, in Psathyrella
antroumbonata is 2.50 kcal/g DW, in A. bisporus is 4.17-4.20 kcal/g DW, in
P. ostreatus is 4.16-4.23 kcal/g DW, and in the Boletus group is 4.20-4.27 kcal/g
DW (Aletor, 1995; Manzi et al., 2001). A serving of 100 g of fresh edible mush-
rooms provides only 1.4-4.4% of the daily energy requirement for a 70-kg adult
male who engages in moderate physical activity (LARN, 1996).

3.4. 1.9 Other Components Mushrooms contain a very low level of phenolic
compounds, with flavonoid and lignan contents that are usually below the limits of
detection (Mattila et al., 2001). The content of total 5' -nucleotides is high, ranging
from 7.43 to 31.9 mg/g DW. Flavor 5'-nucleotides are found to be 5'-guanosine
monophosphate (5'-GMP), 5'-inosine monophosphate (5'-IMP), and 5'-xanthosine
monophosphate (5'-XMP) (Chen, 1989). The flavor 5'-nucleotide content in some
common mushrooms is particularly high, between 0.62 and 13.6 mg/g DW (Lin,
1988; Mau etal., 2001b, c; Tseng and Mau, 1999). 5'-GMP gives a meaty fla-
vor and is a stronger flavor enhancer than MSG (Litchfield, 1967). The synergistic
effects of flavor 5' -nucleotides with MSG-like components can greatly increase the
umamic taste of mushrooms (Yamaguchi et al., 1971). The DNA level (0.21-0.26



NEWLY CULTIVATED/NONCONVENTIONAL MUSHROOMS 79



g/100 g DW) in mushrooms is much lower than the level of RNA (Li and Chang,
1982; Cheung, 1997b). The total nucleic acid content of mushrooms, which ranges
from 3.51 to 4.15% (Cheung, 1997b), can be considered safe, as the maximum
daily dietary intake of nucleic acid suggested by the Protein Advisory Group of
the United Nations System is 4 g (PAG, 1970). The phytic acid (160-360 mg/100
g DW), phytic acid-phosphorus (50-600 mg/100 g DW), and oxalate (80-220
mg/100 g DW) levels of mushrooms are generally not higher than those reported
for cowpeas and soybeans (Aletor, 1995). Phytic acids can chelate certain mineral
elements, such as calcium, magnesium, iron, and zinc, and render them metaboli-
cally unavailable, which interferes with the basic proteins to inhibit certain activ-
ities of digestive enzymes (a-amylase, pepsin, and pancreatin) (Huisman, 1991).
However, the level of oxalate in edible mushrooms is quite low (80-220 mg/100 g)
compared with the values of 2.30-5.80 g/100 g reported earlier for some varieties
of guinea corn (Aletor, 1995).



3.5 NEWLY CULTIVATED/NONCONVENTIONAL MUSHROOMS

Some wild-grown edible mushrooms with great nutritional and medicinal potential
have been artificially cultivated in mass production by modern agricultural tech-
nology. More than 10 new species of mushrooms have been cultivated in recent
years on a small scale, and these have a great potential for expansion in the future
(Chang, 1999). As the consumption of mushrooms is increasing and the develop-
ment in cultivation techniques is rapid, new data on the chemical composition and
nutritional values of mushrooms are urgently needed (Mattila, et al. 2001).

Several species of edible Pleurotus mushrooms and other lesser known edible
mushrooms newly developed and cultivated by the Sanming Mycological Institute
in Sanming, Fujian, China, were studied to determine their chemical composition
and nutritional values (Figure 3.1) (Wong, 2003). Tables 3.3 and 3.4 summarize
the nutritional composition of 11 newly cultivated Pleurotus mushroom and 12
lesser known cultivated mushroom species, respectively, and Figures 3.2a -3.2z
show pictures of dry specimens of these edible mushrooms. The moisture content
of the dried mushrooms was less than 10%. The crude protein content of both
groups was reasonably high (14.9-28.8 and 9.40-27.3% DW, respectively), and
all of the mushrooms were low in lipids (less than 6.0% DW) and ash (4.55-7.71
and 3.78-9.47% DW, respectively). The TDF of the edible Pleurotus mushrooms
was 26.7-43.6% DW, and that of the lesser known edible species was 26.7-44.0%
DW. The TDF predominantly consisted of IDF (92.6-98.4 and 87.9-98.6% DW
for the two groups, respectively) and a small amount of SDF (1.60-7.40 and
1.40-12.1% DW, respectively). Potassium was the major element found in the
mushrooms, together with low levels of sodium, magnesium, and calcium in the
ash (Wong, 2003). The levels of heavy metal were found to be very low in both
groups (Wong, 2003), and all were low in energy (less than 300 kcal/100 g DW)
(Wong, 2003).



80



NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS



Basidiomycota (phyla)
Hymenomycetes (class)



Ascomycota



Zygomycota



Agaricales (order)



i

Aphyllophorales





, 1 ,

Hydnaceae Polyporaceae

Hericium erinaceus Grifola frondosa
Hericium ramosum


1

Pleurotaceae (family)

Pleurotus abalones
Pleurotus citrinopileatus
Pleurotus cornucopiae
Pleurotus djamor


i

Pluteaceae

Volvariella volvacea


i

Agaricaceae

Agahcus bisporus
Agaricus blazei


i

Strophariaceae

Stropharia rugoso-annulata
Pholiota adipose
Pholiota nameko



Pleurotus eryngii Bolbitiaceae

Pleurotus eryngii var female Agrocybe aegerita

Pleurotus eryngii var nebrodensis Agrocybe chaxinggu
Pleurotus nebrodensis
Pleurotus ostreatus
Pleurotus plumonarius
Pleurotus sapidus
Lentinula edodes
Lentinula giganteus



Tricholomataceae

Hypsizigus marmoreus
Flammulina velutipes



Coprinaceae

Coprinus comatus



Figure 3.1 The classification of the edible mushrooms being investigated.



3.6 NUTRITIONAL EVALUATION
3.6.1 General Aspects

Mushrooms are rich in high-quality protein, contain a high level of dietary fiber
and a high proportion of unsaturated fatty acids, are rich in various vitamins and
minerals, and have an acceptably low level of nucleic acids, all of which suggests
the suitability of their daily use as a vegetable (Chang, 1999). Edible mushrooms
or fungi that are a good and inexpensive source of protein are therefore good can-
didates for the relief of the problem of chronic protein deficiency. The idea of
the development of ribonucleic acid-reduced biomass from fungal hyphae culti-
vated in submerged fermentation (mycoprotein) originated in the early 1960s in
response to severe dietary protein shortages (Litchfield, 1967; PAG, 1970), and
today mycoprotein is still being considered as an alternative to meat (Rodger,
2001).



3.6.2 Biological Methods for Nutritional Evaluation

In addition to comparing the amino acid content of a food with the amino
acid requirements of humans, extensive evaluation of existing in vitro and in
vivo methods for food has been conducted that indicates that the rat balance







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Figure 3.2 Photos of dried samples of newly developed cultivated mushrooms. (See insert
for color representation.)



84



NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS





NUTRITIONAL EVALUATION




86 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS




NUTRITIONAL EVALUATION 87




(y) Pholiota nameko (Pn) (z) Stropharia rugoso-annulata (Sra)

Figure 3.2 (Continued)



method is the most practical for predicting protein digestibility in humans (FAO,
1991; Boutrif, 1991). Indeed, the protein efficiency ratio (PER) and net protein
ratio (NPR) methods have been used to measure the ability of a protein to sup-
port the growth in young and rapidly growing rats in many countries (Boutrif,
1991). However, as the PER method cannot properly account for the protein used
for maintenance purposes, the Codex Committee on Vegetable Proteins (CCVP)
proposed the protein-digestibility-corrected amino acid score (PDCAAS), which
is deemed to be the most suitable routine method for assessing the protein quality
of most vegetable protein products and other food products (Codex Alimentarius
Commission, 1989). This method has since been extensively used for evaluating
the protein quality of various foods sources (Boutrif, 1991; Dabbour and Takruri,
2002b). However, it has also been suggested that the PDCAAS method overes-
timates the protein quality of certain foods that may contain naturally occurring
antinutritional factors and of poorly digestible proteins supplemented with limiting
amino acids in rats (Sarwar, 1996).

In vitro methods are relatively rapid, and involve only small amounts of raw
materials (Ezquerra et al., 1998). Various tests, such as multienzymatic digestion
and measurement of the ensuing pH change and enzymatic pH-stat assays, have
been developed and extensively used to measure the in vitro protein digestibility
(IVPD) of different protein sources and to determine the protein quality of different
foods. Although in vitro enzymatic procedures that involve samples of plant and
animal proteins yield only an approximate estimate of protein digestibility, they
are still extensively used (Pedersen and Eggum, 1983).



3.6.3 Mushroom Protein Quality

There are only a few publications on the biological quality of protein in edible
mushrooms (Crisan and Sands, 1978; Khana and Garcha, 1986; Longvah and
Deosthale, 1998).



88 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

Pleurotus is one of the main genera of edible mushroom with commercial value,
and the biological value of the protein in some of the new strains and cultivated
species of this edible mushroom has recently been reported (Wong, 2003; Valencia
del Toro et al., 2006).

The IVPD values of T. portentosum and T. terreum are 72.9 and 73.2%, respec-
tively (Dfez and Alvarez, 2001), which are similar to those of legumes but lower
than those of protein from animal sources.

The in vivo true protein digestibility (TPD) values for Terfezia claveryi,
P. ostreatus, T. terrum, and Agaricus macrosporus are 61.4, 73.4, 52.6, and
80.5%, respectively (Dabbour and Takruri, 2002a), and similar findings have been
observed for S. commune (53.2%) and L. edodes (76.3%) (Adewusi et al., 1993).
These values are again comparable to those of certain legumes, such as Phaseolus
annularis (70.1%), Phaseolus calcaratus (75.8%), Dolichos lablab (75.8%)
(Wong and Cheung, 1998), Phaseolus vulgaris (72.4%) (Carbonaro et al., 2000),
and the pinto bean (72.6%) but lower than those of the animal protein sources
casein (98.6%), tuna fish (96.6%), cheese (94.4%), nonfat dry milk (NFDM)
(87.9%), and rolled oats (98.8%) (McDonough et al., 1990). Based on the FAO
(1991) requirement pattern for children, the chemical scores for lysine (the
limiting amino acid) in T. claveryi and T. terrum are 0.71 and 0.67, respectively
(Dabbour and Takruri, 2002a), which agrees with previous findings for T. claveryi
(0.75) (Sawaya et al., 1985). The chemical scores for sulfur-containing amino
acids in P. ostreatus and A. macrosporus are 0.61 and 0.50, respectively (Dabbour
and Takruri, 2002a), which are consistent with those reported for P. ostreaus and
A. bisporus (Alofe, 1991; Dannel and Eaker, 1992). Other protein sources, such
as legumes, are also found to have a low content of essential sulfur amino acids
(methionine and cysteine) (Carbonaro et al., 2000).

A recent study has shown that low intakes of edible mushrooms in rats con-
tribute to small gains in body weight (Dabbour and Takruri, 2002a). Although a
meager weight gain (0. 1 1 g/rat/day) of rats fed with S. commune was observed,
rats fed with L. edodes experienced a significantly greater body weight gain (0.97
g/rat/day) (Adewusi et al, 1993).

Nevertheless, the NPR of the mushrooms T. robustus (0.80) and L. edodes
(1.70) (Adewusi et al., 1993) is significantly lower than that of legumes such as
P. angularis (6.38) and D. lablab {6.1 A) (Wong and Cheung, 1998). It has also been
found that the PER and net protein utilization (NPU) values of casein-fed rats (3.55
and 78.5) were significantly higher than those fed a mushroom diet consisting
of T. claveryi (—1.76 and 32.6), P. ostreatus (—0.23 and 38.5), T. terrum (—0.98
and 29.1), and A. macrosporus (—0.41 and 31.5). A previous report also showed
similar PER results in rats fed with dried mushrooms (Adewusi et al., 1993), and
similar NPU values for L. edodes (45.8) and S. commune (23.7) have been reported
(Adewusi et al., 1993). The negative PER values indicate that the mushrooms
failed to support the growth of rats, although their crude protein contents were
high (Dabbour and Takruri, 2002a). The presence of alkaloids and tannins in
mushrooms has been reported to cause liver necrosis, and tannins are also known
to retard growth by reducing digestion and the absorption of amino acids and



HEALTH BENEFITS OF EDIBLE MUSHROOMS



89



minerals (Adewusi et al., 1993). Phenolic compounds that complex with protein
decrease amino acid availability (DaDamio and Thompson, 1992). In addition, the
unpalatability of a mushroom diet may have had a negative effect on rat growth
and on the protein quality of the test food (Sarwar and McDonough, 1990). It has
also been suggested that a higher requirement for sulfur-containing amino acids
(methionine and cystine), histidine, isoleucine, threonine, and valine in rats leads to
the underestimation of the protein quality of mushrooms as a human food (Boutrif,
1991; FAO, 1991). Although previous reports have suggested that mushroom
protein is nutritionally incomplete (Bano and Rajarathnam, 1988; Tshinyangu,
1996) and may not be able to support growth as effectively as animal proteins
(Dabbour and Takruri, 2002a), a combination of cereal protein, which supplies
an adequate level of methionine, and mushroom protein could provide a good
balance of amino acids and a good source of dietary protein for human beings.



3.7 HEALTH BENEFITS OF EDIBLE MUSHROOMS

3.7.1 General Aspects

The increasing number of cultivated edible mushrooms being introduced into the
market has led to greater attention in the food and nutritional sciences being paid
to their potential health benefits to humans. This has resulted in many scientific
publications, to the extent that there is now a body of scientific evidence about the
specific health effects of mushrooms and their bioactive molecules. In this section,
the health benefits of edible mushrooms are explored, with an emphasis on the
potential contribution of mushrooms as functional foods and ingredients.

3.7.2 Antioxidants in Mushrooms

3.7.2.1 Bioactive Components and Their Antioxidative Activities

Recently, many studies have found that edible mushrooms possess potent antiox-
idants. The following sections review the antioxidant properties of mushrooms
and give details of the characteristics and biosynthesis of mushroom phenolic
antioxidants.

Mushrooms possess many antioxidant properties. Research conducted in Japan
that studied the antioxidant activity of the crude ethanol extract of 150 Japanese
mushrooms using the peroxide value in the methyl linoleate system showed that
many mushrooms, especially those belonging to the Suillus genus, had a perox-
ide value some 80% lower than the control (Kasuga et al., 1993). The same study
proposed that there may be an intragenus relationship with antioxidant activity
and found that both polar (diethyl ether) and nonpolar (petroleum ether) extracts
of oogitake, kugitake, and maitake mushrooms showed high antioxidant activity
in assay, which suggests the presence of both polar and nonpolar antioxidants
(Kasuga et al., 1993).

It has been reported that polysaccharide extracts isolated from several mush-
rooms are potent scavengers of hydroxyl and superoxide free radicals but that none



90 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

of the species tested showed antioxidant activity as measured by the malondialde-
hyde content of liver microsomes (Liu et al., 1997). Recent research has focused
on the determination of the total phenolic content and antioxidant properties of
several commercial mushrooms. A study of methanolic extracts from black, red,
and snow ear mushrooms found that they had an inhibitory effect on lipid perox-
idation, l,l-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, and hydroxyl
radical scavenging and a strong reducing power and ability to chelate ferrous ions
(Mau et al., 2001a). Similar studies on other mushrooms, including D. indusiata,
G. frondosa, H. erinaceus, T. giganteum, F. velutipes, L. edodes, P. cystidiosus, and
P. ostreatus, showed that these mushrooms also possess the aforementioned antiox-
idant properties (J. H. Yang, et al., 2002; Mau et al., 2002). It is therefore likely that
most mushrooms possess hydroxyl and DPPH radical scavenging effects, inhibit
lipid peroxidation, chelate metals, and have a strong reducing effect.

Methanolic extracts of D. indusiata, G. frondosa, H. erinaceus, and T. gigan-
teum show polyphenolics to be the major naturally occurring antioxidant compo-
nents, which have excellent reducing power, scavenging effect, and chelating effect
on ferrous ions (Mau et al., 2002). Similar antioxidant properties have also been
reported for other edible mushrooms, including Agrocybe cylindracea (Tsai et al.,
1972) and H. marmoreus, both of which belong to the Tricholomataceae family
(Lee et al., 2007).

Potent antioxidant activity was found in crude methanol and water extracts of
the three common Chinese edible mushrooms L. edodes (shiitake mushroom),
Pleurotus tuber-regium, and V. volvacea (straw mushroom). The activity was
evaluated by the /^-carotene bleaching method, DPPH radical scavenging activity,
and erythrocyte hemolysis assay (Cheung, 2001; Cheung et al., 2003). Fractiona-
tion of the crude methanol and water extracts of L. edodes, P. tuber-regium, and
V. volvacea further indicated that the dichloromethane and ethyl acetate fractions
of these mushrooms have the strongest antioxidant activity, as they showed the
lowest median effective concentration (EC50) values (Cheung, 2001).

The antioxidant potential of a lesser known edible mushroom, A. aegerita,
which belongs to the family Bolbitiaceae, was recently studied (Lo and Cheung,
2005) and was shown to exhibit strong in vitro antioxidant activity as expressed
in the inhibition of /J-carotene bleaching, DPPH radical scavenging, and the
inhibition of erythrocyte hemolysis in crude water and methanol extracts (Lo and
Cheung, 2005). In the fractionation of the extracts, the high antioxidant activity
was demonstrated by radical scavenging ability and low-density lipoprotein (LDL)
oxidation in the ethyl acetate and butanol subfractions to be positively correlated
with the total phenolic content (Lo and Cheung, 2005).

A recent report on antioxidant activity and antioxidant compounds in seven
wild edible mushrooms (Elmastas et al., 2007) determined the potential antioxi-
dant compounds, including phenolics, ce-tocopherol, and /3-carotene in methanolic
extracts, and their in vitro antioxidant systems, including their reducing power,
free-radical scavenging, superoxide anion radical scavenging, total antioxidant
activity, and metal-chelating activities (Elmastas et al., 2007).



HEALTH BENEFITS OF EDIBLE MUSHROOMS



91




(c) Ornatipolide (d) Flavoglaucin

Figure 3.3 The structures of some phenolic compounds isolated from fungi.



Phenolics with antioxidant ability have also been found in other mushroom
species. Flavoglaucin, which is a phenolic compound isolated from the mycelial
mat of Eurotium chevalieri, is an excellent antioxidant in vegetable oil at a con-
centration of 0.05% (Ishikawa et al., 1984).

The inhibition of lipid peroxidation by mushrooms has also been reported
recently, with the finding that L. edodes and V. volvacea display antioxidant
behavior by scavenging the free radicals, such as peroxyl radicals, that are
generated in lipid peroxidation (Cheung et al., 2003, 2005). A positive correlation
was also found between the total phenolic content in the mushroom extracts and
their antioxidative properties. This further confirms that edible mushrooms have a
potential as natural antioxidants due to the ability of their phenolics to inhibit lipid
oxidation.

3.7.2.2 Characterization of Mushroom Phenolic Antioxidants

Research on the characterization of mushroom antioxidants is scant. In one of the
few studies in this area, 750 strains of filamentous fungi from soil were screened
for their antioxidant activity (Aoyama et al., 1982). Among them, two strains were
studied for their microbial products, during which curvulic acid, procatechuic
acid, and citrinin were found to have antioxidant activity, with citrinin appearing



92 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

to be as effective as a -tocopherol and curvulic acid and procatechuic acid showing
a slightly less effective activity than a-tocopherol (Aoyama et al., 1982). However,
despite its abundance, citrinin is a known myotoxin and has a limited potential in
foods, whereas procatechuic acid is a known antioxidant, and curvulic acid has
proved to have a low toxicity at 150 mg/kg body weight in rats (Aoyama et al.,
1982).

Figure 3. 3d shows flavoglaucin, which is a phenolic compound, isolated
from the mycelial mat of E. chevalieri. The compound was found to possess
an excellent antioxidant activity in vegetable oils (Ishikawa et al., 1984),
stabilizing lard when used in combination with a -tocopherol at a concentration
of 0.04%. However, this fungal antioxidative compound did not show any
mutagenic activity on Salmonella typhimurium and would thus seem to have
limited potential for use in food (Ishikawa et al., 1984). Babitskaya et al. (1996)
were probably the earliest investigators to characterize phenolics in the edible
mushroom P. ostreatus. Using thin-layer chromatography (TLC) and chemical
detection methods, they identified the presence of simple phenols and flavones
in the mushroom, although they did not carry out the structural identification of
compounds exerting an antioxidant effect. Wada et al. (1996) successfully isolated
and fully characterized two novel prenylated phenolics from the fruiting body of
Boletinus asiaticus using both chemical and spectroscopic methods, including
infrared (IR) spectrometry, mass spectrometry (MS), and nuclear magnetic
resonance (NMR). Two compounds were found that were structural isomers
of each other and were named asiaticusin A and asiaticusin B. The molecular
formulas of both were found to be C27H 36 0s, and their structure is as shown in
Figures 3.3a, b. Later, Shibata etal. (1998) isolated and characterized another
novel macrolide phenolic compound — ornatipolide — from the fruiting body of
another fungus, Boletus ornatipes. The structure of this phenolic metabolite is
shown in Figure 3.3c. In addition to the fruiting body, other structural units of
the fungus were also found to contain phenolic compounds. More recently, liquid
chromatography-mass spectrometry (LC-MS) analysis identified a polyphenolic
compound — epigallocatechin 3-gallate [molecular weight (MW) 458.38] — in
the ethyl acetate subfraction of P. tuber-regium that showed potential antioxidant
activity (Cheung, 2001). In addition, the gas chromatography-mass spectrometry
(GC-MS) method has revealed the presence of several phenolic acids, including
frans-cinnamic acid, hydroxybenzoic acid, protocatechuic acid, and caffeic acid,
in A. bisporus and L. edodes (Mattila et al, 2001).

Other types of phenolic antioxidants, such as sterols, are also present in abun-
dance in mushrooms. Although its antioxidant activity is generally weaker than
that of phenolic antioxidants, ergosterol is also found in abundance in mushrooms
(Mattila et al., 2002). In some cultivated mushrooms, such as A. bisporus, P. ostrea-
tus, and L. edodes, over 600 mg of ergosterol can be found in 100 g of mushrooms.
Other sterol antioxidants, such as fungisterol, are also present, but in lower quan-
tities (Mattila et al., 2002).



HEALTH BENEFITS OF EDIBLE MUSHROOMS



93



These results indicate that mushrooms can be used as a potential dietary source
of phenolic antioxidants to enrich the endogenous antioxidant status of the human
body.

3.7.2.3 Biosynthesis of Phenolic Compounds from Mushrooms or
Fungi The phenolic compounds in fungi are secondary metabolites derived
from intermediates of the shikimic acid pathway, the primary role of which
is to provide the essential aromatic amino acids phenylalanine, tyrosine, and
tryptophan (Turner, 1971). The intermediates of the shikimic acid pathway are
precursors of aromatic compounds, including phenolic compounds. One group of
compounds so derived in fungi are simple phenolic compounds, which are usually
classified as C6-C3, C6-C2, and C6-C1 depending on the length of the carbon
side chain. The C6-C3 compounds include cinnamic acid and its derivatives,
the C6-C2 compounds are phenylacetic acid and its derivatives, and the Cg-Ci
compounds include benzoic acid and its derivatives.

The biosynthesis of the C6-C3 compounds in the Basidiomycetes, especially
Lentinula lepideus, has demonstrated that they possess enzymes such as
ammonia-lyases that convert phenylalanine and tyrosine to cinnamic acids (Power
et al., 1965). There are three possible pathways for the biosynthesis of Cg-Ci
compounds. In Basidiomycetes the C6-C3 compounds are converted into Cg-Ci
compounds. In Sporobolomyces roseus, this was found to involve the conversion
of C6-C3 compounds, such as cinnamic, p-coumaric, and caffeic acids, into a
Cg-Ci compound — protocatechuic acid — by oxidation in the washed cells,
probably via benzoic and p-hydroxybenzoic acids (Moore et al., 1968). The
second pathway is the formation of protocatechuic acid and gallic acid from
dehydroshikimic acid. The third pathway involves the stepwise degradation of
C6-C3 compounds into C��-C\ compounds via C6-C2 compounds, as identified
in Polyporus tumulosus (Turner, 1971). The C6-C3 compounds also serve as
intermediates in the biosynthesis of flavonoids (C6-C3-C6 compounds), which
are another type of phenolic antioxidant. This pathway plays a smaller role
in fungi, as only two flavonoid compounds have been isolated so far, namely,
chlorflavonin and dihydrochalcone, which were isolated from Aspergillus



OH O




(a) Chlorflavonin (b) Dihydrochalcone

Figure 3.4 Structure of two flavonoids isolated from fungi.



94 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

candidus and Phallus impudicus, respectively (Bu'Lock, 1967; List and Freund,
1968) (Figure 3.4).

3.7.3 Hypocholesterolemic Effect of Mushrooms

Cardiovascular disease is associated with atherosclerosis, LDL oxidation, and
hypercholesterolemia, and thus the regulation of the cholesterol level is important
for the prevention and treatment of this disease. Edible mushrooms are an
ideal food for the dietetic prevention of atherosclerosis due to their high fiber
and low fat content. Indeed, the inclusion of edible mushrooms in a natural
hypocholesterolemic and antisclerotic diet is often prescribed in Oriental medicine
(Sun et al., 1984). The hypolipidemic effects of some edible mushrooms are
summarized in Table 3.5

Initial research on the cholesterol-lowering effects of mushrooms was con-
ducted in Japan in the 1960s (Kaneda and Tokuda, 1966), and it was demonstrated
that when rats were fed with a high-fat and high-cholesterol diet supplemented
with 5% DW of the fruiting bodies of L. edodes (shiitake mushroom) for 10
weeks, the plasma cholesterol levels of the animals decreased significantly
(Kaneda and Tokuda, 1966). The adenosine derivative lentinacin or lentysine
(currently known as eritadenine) [2(R), 3(7?)-dihydroxy-4-(9-adenyl)-butyric
acid] (Figure 3.5) was subsequently isolated and identified to be one of the active
hypocholesterolemic components in the shiitake mushroom (Tokita et al., 1972).
Eritadenine has also been found to reduce the serum cholesterol level in mice, not
only by the inhibition of cholesterol biosynthesis but also by the acceleration of
the excretion of ingested cholesterol and its metabolic decomposition (Suzuki and
Ohshima, 1976). Various studies have shown that Lentinula mushrooms can lower
both the blood pressure and the free cholesterol level in plasma and can accelerate
the accumulation of lipids in the liver by removing them from circulation (Kabir
and Kumura, 1989). Eritadenine affects the metabolism not only of cholesterol
but also of phospholipids and fatty acids in rats (Sugiyama et al., 1995; Shimada
et al., 2003). The dietary supplementation of eritadenine may therefore decrease
phosphatidylcholine biosynthesis by altering the phosphatidylethanolamine
concentration (Sugiyama et al., 1995). Similar to soybean protein, eritadenine
lowers cholesterol by decreasing the ratio of phosphatidylcholine (PC) to
phosphatidylethanolamine (PE) in liver microsomes and altering the composition
of PC (Sugiyama and Yamakawa, 1996). Eritadenine can also suppress the
metabolism of lipids (linoleic acid) by suppressing A 6— desaturase activity
(Sugiyama et al., 1997; Shimada et al., 2003). Several other studies on Lentinula
extracts have shown them to cause a significant decrease in serum cholesterol in
young women and people older than 60 years of age in Japan (Hobbs, 1995).

Recently, it has been reported that eritadenine may elicit its effect by the sup-
pression of the hyperhomocysteinic effect of guanidinoacetic acid, which leads to
the decreased production of homocysteine and increased cystathionine formation
(Fukada et al., 2006). In addition to eritadenine, nucleic acid compounds extracted
from L. edodes also show an inhibition in platelet agglutination (Wasser and Weis,
1999).



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Lovastatin (mevinolin) and its analogues (Figure 3.6) are powerful inhibitors
of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and as such are
well-known cholesterol-lowering agents (Endo, 1988). They can be isolated
from the fruiting bodies of various types of oyster mushroom {Pleurotus spp.),
including P. eryngii, P. sapidus, P. ostreatus, and P. cornucopiae (Cimerman
and Cimerman, 1995). It has also been found that the addition of 2 and 4% of
P. ostreatus to a hyperlipidemic diet can prevent the accumulation of cholesterol
and triglyceride in both the sera and livers of rats with exogenous, endogenous,
or genetically induced hyperlipidemia (Bobek et al., 1991, 1993). A reduction of
the serum cholesterol level of up to 80% also resulted from the feeding of the
whole mushroom, water, and 30% ethanol extract of P. ostreatus to rats. This
led to the proposal that the hypolipidemic effect may be attributable to the fiber
complex of the mushroom, which limits the reabsorption of cholesterol in the
gastrointestinal tract. An undefined substance that influences the metabolism out-
side of the phase of reabsorption may also contribute to the cholesterol-lowering
effect (Bobek etal., 1991, 1993). In another study, dietary fiber extracted from
P. cornucopiae had a marked antiatherosclerotic effect in vitro (Ryong et al.,
1989), and patients with coronary disease showed a decreased atherogenic
activity (20-40%) in their sera after the consumption of this mushroom, which
confirms that it has a natural cholesterol-lowering agent that is responsible for
this hypocholesterolemic effect (Ryong et al., 1989). It has also been found that
the addition of 1-5% of oyster mushroom to a hyperlipidemic diet efficiently
prevents the accumulation of cholesterol (and especially LDL cholesterol) and
triglycerides in both the blood and liver of rats with hyperlipidemia (Bobek et al.,




Figure 3.6 Chemical structure of lovastatin.



HEALTH BENEFITS OF EDIBLE MUSHROOMS



97



1998) and also reduces cholesterol biosynthesis by suppressing the activity of
hepatic HMG-CoA reductase (Bobek et al., 1995) and accelerated cholesterol
catabolism by up-regulating hepatic cholesterol la -hydroxylase (Bobek et al.,
1994). It has been suggested that the fruiting bodies of oyster mushrooms could
be recommended for consumption as a natural cholesterol-lowering agent in the
human diet (Cimerman, 1999).

In addition to lovastatin and eritadenine, dietary fiber (nonstarch polysac-
charides, mainly /6-glucans) has also been suggested to be an important
hypocholesterolemic component in mushrooms, and dietary fiber isolated from
Auricularia auricula-judae (Jew's ear) and Tremella fuciformis (white jelly-leaf)
can significantly decrease the serum total cholesterol (TC) and LDL cholesterol
levels (Cheung, 1996b). Furthermore, exopolysaccharides produced by the
submerged fermentation of the mycelium of V. volvacea can reduce the levels of
serum TC, LDL cholesterol, and liver TC in alimentarially induced hypercholes-
terolemic rats (Cheung, 1996c). Fibers from G. frondosa (maitake mushroom)
can greatly increase the fecal sterol excretion, which reduces the total body
"sterol pools" (Kubo and Nanba, 1997), and fibers from F. velutipes (enokitake
mushroom) and A. bisporus (button mushroom) can dramatically enhance the
hepatic LDL receptor messenger RNA (mRNA), causing the diminution of the
serum TC (Fukushima et al., 2000, 2001).

Other mushroom species, such as A. auricula-judae, display anticoagulation,
antiaggregatory activity in the blood platelets of mice and rats, thus serving
to lower their TC, total triglyceride, and lipid levels (Chen, 1989; Sheng and
Chen, 1990). The supplementation of 5% DW of V. volvacea (straw mushroom)
to hamsters fed a hypercholesterolemic diet (0.1% cholesterol and 10% fat)
significantly lowered their levels of plasma and hepatic cholesterol and increased
the fecal excretion of neutral sterols (Cheung, 1998). Another mushroom,
G. frondosa, reduced blood pressure in rats without changing the plasma
high-density lipoprotein (HDL) level or serum cholesterol level (Mizuno, 1995).
It has also been reported that dried A. aegerita can significantly reduce the serum
TC, triglyceride, atherogenic index, hepatic TC, and total triglyceride levels in
rats fed a semisynthetic high-cholesterol diet compared with the control group
(Yeung and Cheung, 2002).

The hypocholesterolemic effect of A. aegerita has been suggested to be linked
with its antioxidant activity (Ng, 2005). Hot water and ethanolic extracts obtained
from A. aegerita have shown the in vitro inhibition of LDL oxidation, as expressed
through thiobarbituric acid reactive substances (Ng, 2005). Apart from having
potent antioxidant activity and a high total phenolic content, A. aegerita also
exhibits an in vivo hypocholesterolemic effect and has potential as a natural
source of phenolic antioxidants and a hypocholesterolemic agent.

3.7.4 Hypoglycemic Effect of Mushrooms

An extensive search for traditional plant treatments for diabetes has been con-
ducted (Alarcon-Aguilara etal., 1998) that recognized edible mushrooms as an
ideal food for the dietetic prevention of hyperglycemia because of their high dietary



98 NUTRITIONAL VALUE AND HEALTH BENEFITS OF MUSHROOMS

fiber and protein and low fat content. Many studies have been conducted on the
hypoglycemic activity of whole mushrooms and their fruiting bodies (Horio and
Ohtsuru, 2001) and on mushroom bioactive components, including polysaccha-
rides (Kiho et al., 1994a, b, 2000, 2002; Mori et al., 1998; Kurushima et al., 2000)
and lectins (Ewart et al., 1975) isolated from the fruiting bodies. Moreover, endo-
and exopolymers produced in submerged mycelial cultures have also been found
to have a hypoglycemic effect (Kim et al., 1997, 2001). The most common animal
models used for the study of the hypoglycemic effects of mushrooms are rats and
mice with insulin-dependent diabetes mellitus (IDDM) induced by streptozotocin
(STZ) and genetically diabetic mice with non-insulin-dependent diabetes mellitus
(NIDDM) (Beattie et al., 1980; Swanston-Flatt et al., 1989; Kiho et al., 2002).

The administration of G. frondosa to IDDM STZ diabetic albino Wistar
rats at 20% DW in a semipurified diet for 100 days resulted in an increase
in insulin excretion and a decrease in the blood glucose level in the animals
(Horio and Ohtsuru, 2001). It has also been demonstrated that G. frondosa has
an antidiabetic effect in NIDDM KK-A- V mice, which is produced by reducing
the blood glucose level (Kubo et al., 1994; Kubo and Nanba, 1997). Mush-
room polysaccharides, including /J-glucan isolated from the fruiting bodies of
A. cylindracea (Kiho et al., 1994a), H. erinaceus (Xue et al., 1989; Mori et al.,
1998), and G. frondosa (Kurushima et al., 2000), have been investigated for
their hypoglycemic effect. The /2-glucans isolated from A. cylindracea showed
remarkable hypoglycemic activity in both normal and STZ-induced diabetic mice
when administered intraperitoneally (Kiho et al., 1994a). The (1 4)-linked
or (1 — > 6)-linked residues in the (1 — > 6)-/?-branched (1 — > 3)-/2-D-glucan of
the isolated polysaccharides seemed to be necessary for their hypoglycemic
effect (Kiho et al., 1994a). The hypoglycemic /J-glucans isolated from
H. erinaceus differed from those of A. cylindracea in having a backbone of
(1 -> 6)-j6-linked-D-glucose with (1 -> 6)-y3-linked residue (Mori etal., 1998).
The polysaccharide fraction with blood-glucose-depressing effect isolated from
G. frondosa also has (1 — > 6)-j6-linked glucose as the main chain, which is similar
to that in H. erinaceus but has a (1 -> 4)-a-linked glucopyranosyl residue as a
branch chain (X fraction) (Kurushima et al., 2000). This X fraction promotes the
responsiveness of the insulin receptors and can lead to some recovery from the
NIDDM (Kubo et al., 1994).

Acidic polysaccharides isolated from the fruiting bodies of A. auricula-judae
(Yuan etal., 1998a, b), Tremella aurantia (Kiho etal., 1995, 2002), and
T. fuciformis (Kiho et al., 1994b) have also been studied for their antidiabetic
effects. The antidiabetic acidic polysaccharide isolated from T. aurantia has a
branched structure with a (1 — > 4)-a-linked-D-mannopyranosyl backbone and side
chains of /J-D-xylopyranosyl residues, with /J-D-glucuropyranosyluronic residues
linked to the terminal a-D-mannopyranose (Kiho et al., 2000). Tremella aurantia
has been shown to exert its hypoglycemic effect in diabetic mouse models of
both IDDM and NIDDM following intraperitoneal administration (Kiho et al.,
1995). The antidiabetic activity of this mushroom may also be mediated by an



REFERENCES 99



increase in the activities of glucokinase, hexokinase, and glucose-6-phosphate
dehydrogenase and a decrease in the activity of glucose-6-phosphatase in normal
and IDDM diabetic mouse livers after intraperitoneal administration (Kiho et al.,
2000). Tremella aurantia has also been shown to be effective in lowering the
plasma glucose level when administered orally in KK-A } mice (Kiho et al., 2002).
Another acidic polysaccharide from the fruiting bodies of T. fuciformis was found
to be effective in STZ-induced diabetic mice when administered orally (Kiho
et al., 1994b).

A significant reduction in the level of plasma glucose was also observed
in STZ-induced diabetic Sprague-Dawley rats fed with exopolymers (mainly
polysaccharides) isolated from the submerged mycelial cultures of five types of
common edible mushrooms (Kim et al., 2001).

Lectins isolated from mushrooms (Agaricus campestris and A. bisporus) have
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and Flatt, 1998). Guanidine, which is a known hypoglycemic substance related
to the biguanide class of oral antidiabetic drugs, has been found in edible mush-
rooms (Windholz, 1983), but the detailed principles of these active components in
mushrooms remain to be elucidated.



3.8 CONCLUSION

The detailed mechanisms of the various health benefits of mushrooms to
humans still require intensive investigation, especially given the emergence of
new evidence of their health benefits, such as their prebiotic, hypotensive, and
hepatoprotective effects. The exploration of newly cultivated mushrooms and their
active ingredients with potential therapeutic value therefore remains a challenge.

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CHAPTER 4



Sclerotia: Emerging Functional Food
Derived from Mushrooms

Ka-Hing Wong

Department of Biology, The Chinese University of Hong Kong, Hong Kong, China
Peter C. K. Cheung

Food and Nutritional Sciences Programme, Department of Biology, The Chinese University of
Hong Kong, Hong Kong, China

CONTENTS

4. 1 Introduction

4.2 Concepts of Mushroom Sclerotia

4.3 Ontogeny of Sclerotia

4.4 Structure of Sclerotia

4.5 Cultivation of Mushroom Sclerotia

4.6 Biochemical, Nutritional, and Technological Characteristics of Mushroom
Sclerotia

4.7 Biopharmacological Values of Mushroom Sclerotia of P. tuber-regium,
P. rhinocerus, and W. cocos

4.8 Conclusion
References

4.1 INTRODUCTION

Unlike plants and animals, filamentous fungi do not form three-dimensional
complex tissues via mitotic activity (Elliott, 1994; Moore, 2003). Instead, the
complex structures (e.g., strands, rhizomorphs, synnemata, stromata, sclerotia) of
fungi develop by the aggregation of the specialized hyphae that result from the
differentiations that are governed by tight genetic control (Elliott, 1994; Moore,
2003). These multihyphal structures can be further classified into elongated (e.g.,
strands) and rounded (e.g., sclerotia) structures. The elongated organs not only



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



111



112 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

enable the connection of one fungus resource to another (e.g., rhizomorph) but
also allow the fungal reproductive organs to reach a location that is favorable
for the dispersal of spores (e.g., synnemata). The main functions of the rounded
structures (i.e., sclerotia) are to allow the fungus to survive under unfavorable
environmental conditions and to provide reserves for the fungus to germinate.

In addition to enzymatic preparation (Wong and Cheung, 2004, 2005a), frac-
tionation (Cheung and Lee, 1999, 2000; Zhang et al., 2003a), and biochemical,
microstructural, physicochemical, and functional characterizations (Cheung,
1997; Cheung and Lee, 1998; Wong et al., 2003; Wong and Cheung, 2005a;
L. N. Zhang etal., 2001; M. Zhang etal., 2003b), our research team has for
the past decade been actively studying the physiological functions (Chau et al.,
2007; Cheung and Wong, 2004; Lai and Cheung, 2004; Lai, 2005; Lai et al.,
2005; Tao et al., 2006; Wong and Cheung, 2005b; Wong et al., 2005, 2006, 2007;
M. Zhang etal., 2001, 2004a, b, 2006a, b) of the nonstarch polysaccharides
(NSPs) isolated from three Chinese edible and medicinal mushroom sclerotia,
namely Pleurotus tuber-regium (Fr.) Sing., Polyporus rhinocerus Cooke, and
Wolfiporia cocos (Schw.) Ryv. et Gilbn. In this chapter, in addition to the concepts,
ontogeny, and cultivation of mushroom sclerotia, we share our previous research
on the NSPs of the aforementioned sclerotia and their potential to be developed as
novel functional foods or nutraceuticals.

4.2 CONCEPTS OF MUSHROOM SCLEROTIA

According to Willetts and Bullock (1992), the sclerotium can be described,
from a functional point of view, as a morphologically variable, nutrient-rich,
multihyphal aggregated structure that can remain dormant or quiescent when
the environment is unfavorable. However, when the conditions improve, the
sclerotium can germinate again to produce the fungus. The size of a sclerotium is
species dependent and ranges from microscopic (only a few cells, e.g., Verticillium
dahliae) to enormous (more than 30 cm in diameter, e.g., Polyporus mylittae),
whereas its shape is usually spherical to oval (Carlisle et al., 2001; Moore, 2003).
Sclerotia occur sporadically in the Ascomycotina (e.g., Claviceps purpurea;
Luttrell, 1980), Basidiomycotina (e.g., Sclerotium rolfsii; Chet and Henis, 1975),
and Deuteromycotina (e.g., V. dahliae). Among these three subdivisions, the most
studied sclerotium-forming fungi are those of economic importance, such as the
Sclerotiniaceae (e.g., Monilinia, Botryotinia, and Sclerotinia; Willetts, 1997) and
the Typhula spp. (Willetts et al., 1990).

4.3 ONTOGENY OF SCLEROTIA
4.3.1 Morphological Aspects

The ontogeny study of sclerotia was reported as early as the nineteenth century by
Brefeld (1877) and De Bary (1887). In general, sclerotial ontogeny can be divided



ONTOGENY OF SCLEROTIA



113



into three overlapping stages: (i) initiation (when the hyphae begin to aggregate
to form small, discrete initials); (ii) development (when the initials grow to full
size, accumulating nutritional reserves from the parent mycelia and droplet excre-
tion); and (iii) maturation (surface delimitation, pigmentation of the peripheral
hyphae, conversion of the reserve nutrients into a suitable form for long-term stor-
age, and internal consolidation). These three developmental stages are complicated
processes that are accompanied by both morphological and biochemical differen-
tiations under tight genetic control (Carlisle etal., 2001; Chet and Henis, 1975;
Cooke, 1970; Townsend and Willetts, 1954).

Although numerous endogenous and exogenous factors are reported to be
involved in sclerotial initiation, the sclerotium is basically initiated by the onset of
starvation conditions or other circumstances that are unfavorable for continuous
mycelial growth (Carlisle et al., 2001; Willetts and Bullock, 1992). The environ-
mental (e.g., light, temperature, pH), mechanical (e.g., when mycelia are cut, torn,
grow against the side of the culture vessel), biochemical (e.g., when mycelia grow
in contact with staling products, other microorganisms, antibiotics, phenolics, and
polyphenoloxidases), nutritional (e.g., C/N ratio, minerals, vitamins), and internal
morphogenetic factors that affect sclerotial initiation have been comprehensively
reviewed in the literature (Cooke, 1983; Chet and Henis, 1975; Willetts, 1978;
Willetts and Bullock, 1992). Thus, these factors will not be discussed here.

There are three main types of sclerotial development — loose, terminal, and
lateral — all of which have been discussed in detail by Willetts (1972). When
hyphae aggregate to form multihyphal structures such as sclerotia, their repulsion,
which occurs in normal hyphal growth, must disappear or even be substituted
by attraction for hyphal association. Apart from a few classic studies reviewed
by Moore (1984), information on the surface chemistry of hyphae is scarce.
Autotrophic agents and the mucilage matrix, which accumulate over the surface
of vegetative hyphae, are believed to play an important role in specific cell-to-cell
adhesion in the development of multihyphal structures such as sclerotia (Reijnders
and Moore, 1985; Willetts, 1972, 1978).

Sclerotial initials develop from one or several knot(s) of aggregated hyphae
(e.g., strands in the case of S. rolfsii; Townsend and Willetts, 1954) within a
mycelial mass. As the sclerotial initials grow and increase in size, the central
hyphae exhibit remarkable dichotomous branching, and their cells become
swollen and vacuolated. This facilitates their main role of accumulating nutritional
reserves (including glycogen, polyphosphate, proteins, and lipids) from the
parental mycelia. For the outer hyphae of the developing sclerotia, the cells
differentiate to become shortened, followed by wall thickening and melanizing
to form a protective layer over the sclerotial surface (rind). The tightly packed
structure of the rind together with the deposits between the outer hyphal cells
is likely to prevent the apoplastic transfer of the solutes (Carlisle et al., 2001).
During sclerotial development, considerable nutrients are absorbed from the
substrate, followed by degradation by the various enzymes (e.g., arylesterase
and acid phosphatase; Wong and Willetts, 1974), to provide energy and nutrients
for the developing sclerotium. Thus, the nutrient status of the substrate directly



114 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

determines the number and size of the sclerotia produced (Willetts and Wong,
1971). Furthermore, the substratum may be partially enclosed in the sclerotium
and surrounded by the melanized rind cells.

Dormant sclerotia may survive for several years, and the pattern of sclero-
tial germination is species dependent (Willetts and Bullock, 1992). During dor-
mancy, considerable accumulated endogenous reserves, together with the inter-
hyphal materials, are used collectively to sustain the sclerotium during the peri-
ods of survival and germination (e.g., Sclerotinia minor; Bullock and Willetts,
1996) when conditions are favorable. The germination of sclerotia can be car-
ried out by means of asexual spores (sporogenic), fruiting bodies (carpogenic),
mycelia (myceliogenic), or a combination of these. However, only sporogenic and
carpogenic germinations are the general characteristics of larger sclerotia, which
are produced by airborne fungal pathogens such as mushrooms (Garrett, 1970).
Sclerotia germinate as a result of various factors, including exposure to nutrients,
appropriate light and temperature conditions (e.g., P. mylittae, Carlisle et al., 2001 ;
S. minor, Imolehin etal., 1980; Sclerotinia sclerotiorum, Huang, 1991), or even
stimulation by substances emitted by a host plant (e.g., Sclerotinia, Carlisle et al.,
2001; S. minor, Hall et al., 1982). Obviously, complex and genetic-controlled pro-
cesses are likely be involved in sclerotial germination (Elliott, 1994; Moore, 2003).
As suggested by Carlisle and his co-investigators (2001), the formation of sclero-
tia, which remain at the site of production, is an effective strategy for the survival
of fungi. This is because the area that has been suitable for the growth of the host
plants naturally allows the seeds of the host plant to remain, thus allowing the next
generation to grow. This then increases the chance of fungal infection in the plants.

4.3.2 Physiological Aspects

4.3.2.1 Translocation As reported by Jennings (1987), during sclerotial
development, the insoluble carbohydrates (e.g., glycogen) that accumulate in
the mycelium during vegetative growth are converted to soluble forms (e.g.,
trehalose and mannitol). These chemical changes lead to the development of a
turgor gradient between the vegetative mycelium (nutrient base) and the sclerotial
initials (nutrient sinks). As a result, the movement of water and nutrients to
the developing sclerotia is carried out by a turgor-driven mass flow via a few
specialized conducting hyphae (Wilcoxson and Sudia, 1968). As the translocated
nutrients are utilized by the developing sclerotia, the turgor gradient is maintained.
As suggested by Jennings (1987), the maintenance of metabolic gradients during
translocation may even be tightly controlled by evaporation at the surface of the
multihyphal structures and by the trehalose in the mycelium, which fine tunes the
sugar concentration in both the nutrient base and the sink. In contrast to the early
stage of sclerotial development, the nutrient requirement for growth is diminished
when the sclerotium is mature because of its large size and compactness. To
avoid the accumulation of soluble nutrients, which could affect translocation and
other metabolic activities, soluble nutrients are converted to insoluble forms and
accumulate as intra- and extracellular reserves, with the greatest deposition in



STRUCTURE OF SCLEROTIA 115



the rind and cortex regions (at the periphery) of the sclerotia (at the end of the
translocatory stream) for subsequent use by the fungus, such as in germination
under favorable conditions.

4.3.2.2 Exudation Exudation is a common phenomenon during the early
stages of sclerotial development (Townsend and Willetts, 1954) and has been
extensively reviewed by Colotelo (1978). In early sclerotial development, small
droplets begin to form on the surface of the sclerotia, and these coalesce to form
a few large droplets when the sclerotia become mature and pigmented. In the
case of Cristulariella spp., the mature sclerotia are even found to bathe in liquid
(Willetts and Bullock, 1992). The droplets may remain on the sclerotia from
several days (under evaporation) to several weeks, with some of the constituents
being reabsorbed and probably utilized by the sclerotial tissue (Colotelo, 1978).
The color of the droplets, which is probably due to the accumulation of oxidized
phenolics, varies from clear to pale or dark brown (mature sclerotia only), even
on the same sclerotium (Willetts and Bullock, 1982). The composition of the
droplets is complex. In addition to carbohydrates (such as trehalose, mannitol,
inositol, and glucose) and enzymes (including polyphenoloxidase, peroxidase,
glucosidase, and cellulose), amino acids and fatty acids have been reported
previously (Cooke, 1969; Jones, 1970). Cooke (1969, 1970) and Jennings (1987)
reported that the permeability of the hyphal tip is significantly different from that
of the rest of the hypha and exudation from the hyphal tips is probably an active
and selective process that assists in dissipating the excessive hydrostatic pressure
that is generated during the aforementioned translocation.



4.4 STRUCTURE OF SCLEROTIA

A sclerotium commonly includes a pseudoparenchymatous and melanized "rind"
that encases a broad "medulla" of interwoven hyphae. In some sclerotia (e.g.,
S. minor), a narrow layer of close-fitting hyphae, namely the "cortex," is dis-
cernible between the rind and the medulla (Willetts and Bullock, 1992). The rind
is a continuous layer of tightly packed hyphal tips that become thick walled and
pigmented to form an impervious outer surface layer. The medulla constitutes the
main part of the sclerotium, the hyphae of which (together with those of the cortex
if present) are the main storage area for the intracellular reserves (Carlisle et al.,
2001; Moore, 1995; Willetts and Bullock, 1992), whereas the interhyphal space
is usually filled with an extracellular matrix (continuous or containing lacunae)
(Moore, 1995; Willetts and Bullock, 1992).

4.4.1 Rind

The rind is formed when the hyphal tips at the periphery of the sclerotium become
closely packed to form a continuous layer, with the septa lying close to the apices
and the terminal cells becoming swollen and rounded (Bullock et al., 1980). In the



116 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

terminal cells of a young differentiating rind, such organelles as nuclei, mitochon-
dria, small amounts of endoplasmic recticulum (ER), and vacuoles are observed
(Willetts and Bullock, 1992). At the early stage of rind development, the cell walls
begin to thicken, and many multivesicular bodies that are closely associated with
the plasmalemma are observed (S. minor, Bullock et al., 1980), which suggests
their possible role in the synthesis of the hyphal cell walls of sclerotia (Marchant
et al., 1967; Khan and Aldrich, 1973). In mature sclerotia, the cell wall of the rind
is the thickest (an eight fold increase in the case of S. minor; Bullock et al., 1980).
As the rind develops, vacuolation occurs rapidly through an increase in the number
of vacuoles, which then coalesce to form one large central vacuole surrounded by
a thin layer of cytoplasm. Although mature rind cells are usually devoid of content,
large phenol-rich bodies have been observed in the rind cells of the mature scle-
rotia of Sclerotiniaceae (Kohn and Grenville, 1989a, b) and Typhula incarrnata
(Willetts et al., 1990). They were probably involved in resistance against antago-
nistic microorganisms. The factors that control rind differentiation are not clear. In
S. minor, the sclerotium grows to almost full size before the rind begins to differen-
tiate (Bullock et al., 1980), whereas in Sclerotinia trifoliorum and S. sclerotiorum,
there is considerable enlargement after the rind starts to develop (Cook, 1971). The
enlargement of preexisting rind cells and/or the incorporation of new tips into the
rind layer from underlying hyphae (e.g., S. minor; Bullock et al., 1980) allow the
rind to expand to accommodate an increase in the surface area of the sclerotium.
These rind cells may also serve as specialized passage cells that allow the sclerotial
reserves to pass through the impermeable rind and be utilized for hyphal growth
outside of the sclerotium (i.e., for sclerotial germination and secondary sclerotium
formation). A characteristic feature of sclerotial rind formation is a change of color
from white to buff to dark brown or black, which is caused by an accumulation of
melanin (Chet et al., 1967; Chet and Henis, 1968; Jones, 1970). At maturity, most
of the sclerotium rind cells have collapsed and are dead; thus, the symplastic trans-
port of water and nutrients across the rind would be unlikely in a normal situation.
The barrier role of the pigmented rind is partially supported by the findings of
Young and Ashfold (1992), who reported that, as the rind cells differentiate, there
is a reduction in the permeability of the sclerotia to the apoplastic tracer, sulforho-
damine, which corresponds with the wall thickening and pigmentation of the rind
cells.

4.4.2 Cortex

In some sclerotia, as the rind begins to differentiate and the sclerotium grows to
almost its full size, a cortex of close-fitting rounded cells becomes discernible.
The width of this cortex varies between and even within species (ranging from
imperceptible to six cells wide; Townsend and Willetts, 1954; Willetts and Bul-
lock, 1982; Kohn and Grenville, 1989a, b). There is no clear definition of the outer
and inner boundaries of the cortex. The outermost cortical cells have some rindlike
features, such as pigmentation and vacuolation (although with a thinner cell wall),
whereas the innermost cortical cells grade into the medulla (but possess greater



CULTIVATION OF MUSHROOM SCLEROTIA 117



density and a rounded shape; Willetts and Bullock, 1992). The laying down of
abundant storage bodies within the cortical hyphae is a major feature of corti-
cal development, as the cortex is the region for the accumulation and storage of
reserve materials. Similarly to the genus Sclerotinia, the cortex in the sclerotia of
T. incarrnata cannot be distinguished (Willetts et al., 1990).

4.4.3 Medulla

Most of the sclerotium consists of a medulla of prosenchymatous tissue that is
formed by the interweaving of the hyphae with a few septa and branches (Willetts
et al., 1990; Willetts and Bullock, 1992). The greatest hyphal density is usually
in the outer region of the medulla (e.g., T. incarrnata; Willetts et al., 1990). The
most conspicuous differentiation feature of the medulla is the accumulation of an
extracellular matrix in the interhyphal spaces (e.g., T. incarrnata; Willetts et al.,
1990). In addition to the shrinkage of the extracellular matrix (the air drying of
the sclerotia), the presence of lacunae in the extracellular matrix is attributed to
the density of the medulla hyphae, which determines whether sufficient materi-
als are produced to fill all of the interhyphal spaces (Willetts and Bullock, 1992).
Compared with cortical hyphae, medullary hyphae share similar kinds of storage
bodies, the deposition of reserves, and wall thickening.



4.5 CULTIVATION OF MUSHROOM SCLEROTIA

With the enormous improvement in mushroom cultivation technologies, the world
production of cultivated edible mushrooms increased more than 12% annually
between 1981 and 1997 (to 6.158 million metric tons; Chang, 1999), and their
commercial value in 1998 was estimated to be about U.S. $18 billion, which is sim-
ilar to that of coffee production (Wasser et al., 2000). In China, the production of
cultivated edible mushrooms increased drastically from 60,000 metric tons in 1978
to 4.35 million metric tons in 1998 (more that 40 species and accounting for about
half of the world's total output), and it is expected to reach more than 6 million
metric tons by 2010 (Huang, 1999b, 2000). Thus, China is becoming a significant
international edible mushroom producer (Chang, 1999; Huang, 2000). As reported
by Huang (1999a), the sclerotium-forming, edible, and/or medicinal mushrooms
in China are mainly Grifola umbelata (Pers.), Omphalia lapidenscens Schroet,
Xylaria nigripes (Kl.) Sacc, W. cocos (Schw.) Ryv. et Gilbn., P. tuber-regium
(Fr.) Sing., and P. rhinocerus Cooke. In addition to W. cocos, P. tuber-regium and
P. rhinocerus are two of the most economically important sclerotium-forming fungi
to gain popularity in China recently (Huang, 2000). Although the main source
for edible and medicinal use at present is still wild sclerotia, their natural habitats
have been gradually destroyed in recent years due to insufficient protection from
the rapid development of agricultural and urban areas. For the conservation and
exploration of mushroom sclerotia as a functional food, research on the cultivation
of mushroom sclerotia under well-controlled artificial conditions needs to be



118 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

comprehensively conducted, so that their scale and efficiency of production can
be improved and their commercialization facilitated.

4.5.1 Sclerotia of Pleurotus tuber-regium (Fries) Singer

Pleurotus tuber-regium is an edible and medicinal mushroom from the Basidiomy-
cotina, which are mainly distributed in tropical and subtropical regions such as
China, Australia, and Africa (especially Nigeria) (Oso, 1975, 1977; Singer, 1961;
Zadrazil, 1996; Zoberi, 1973). Pleurotus tuber-regium is the only Pleurotus species
in which the fruiting bodies arise from a sclerotium (Isikhuemhen and Nerud,
1999). The sclerotium of P. tuber-regium is large and subterranean and is spheri-
cal to oval in shape (about 10-25 cm in diameter toward the end of the growing
season, which is from April to September), and new fruiting bodies (that are light
brown in color, up to 10 cm in size, and depressed in the center) are formed on it
in the subsequent growing season (Oso, 1977; Zoberi, 1972, 1973). The rind of the
sclerotia is shiny and dark brown, whereas the internal structure is hard, powdery,
and white (Oso, 1977).

Although it is quite expensive, the sclerotium of P. tuber-regium is popularly
consumed in Nigeria (Okhuoya and Etugo, 1993; Oso, 1977) and considered to
be a delicacy (Okhuoya and Okogbo, 1990). The sclerotium is chopped or ground
into powder before being put into soup, egusi, or melon seed ball preparations
(Akobundu andEluchie, 1992; Nwokolo, 1987; Oso, 1977; Zadrazil, 1996; Zoberi,
1973). In addition to using P. tuber-regium sclerotial powder as a tablet disintegrant
(Iwuagwu and Onyekweli, 2002), its successful incorporation into pork sausage
has also been reported by Akobundu and Eluchie (1992). In addition, the sclerotia
of P. tuber-regium can be kept for years without losing their nutritional quality as a
foodstuff or even their ability to produce fruiting bodies (Nwokolo, 1987; Zadrazil,
1996). Furthermore, the sclerotium of P. tuber-regium is also used for medicinal
purposes by medical practitioners in Nigeria (Oso, 1977; Zoberi, 1972, 1973) to
cure headaches, stomach ailments, colds, constipation, fever, asthma, smallpox,
nervous disorders, and high blood pressure (Oso, 1977; Fasidi and Olorunmaiye,
1994; Zadrazil, 1996). In China, P. tuber-regium is commonly known as the "tiger
milk mushroom" and is mainly found in the southern region, particularly in Yun-
nan Province (Deng et al., 2000). Recently, because of the nutraceutical benefits
of its various polysaccharide fractions, such as antitumor and immunopotentiating
effects (Cheung and Lee, 2000; M. Zhang etal., 2001), the consumption of
P. tuber-regium sclerotia is growing in popularity and economic importance
(Huang, 2000).

Pleurotus tuber-regium is a classic, wood-rotting fungus that can utilize a
wide range of broad-leaf and needle-leaf trees. The successful cultivation of
P. tuber-regium sclerotia has been reported by numerous studies (Fasidi and
Ekuere, 1993; Jiang et al, 2000; Okhuoya and Okogbo, 1990). In China, in addi-
tion to the logs of broad-leaf trees, P. tuber-regium sclerotia have been cultivated
using substrate bags. The two general formulations of compost used were (1)
saw dust (78%), wheat bran (20%), white sugar (1%), CaC0 3 (1%), and water



CULTIVATION OF MUSHROOM SCLEROTIA



119



(1 : 1.1-1.3) and (2) saw dust (39%), wheat bran (49%), sugar cane (1%), CaC0 3
(1%), and water (1 : 1.1-1.3) (He etal., 2000). Similarly to other Pleurotus
spp., P. tuber-regium sclerotia have also been found to grow (five to six weeks
after spawning) on a variety of cellulosic waste materials, such as cotton waste
(biological efficiency 30.11%), rice straw (29.51%), corn cobs (22.85%), and
banana leaves (13.58%) (Fasidi and Ekuere, 1993; Garcha etal, 1984; Jandaik,
1974) and on the moist drill dust from the wood of Daniella oliveri and Elaeis
guineensis trees after 65 and 71 days inoculation, respectively (Okhuoya and
Okogbo, 1990). As suggested by Fasidi and Ekuere (1993), the high saprophytic
ability of P. tuber-regium can most likely be attributed to its capability of secreting
a wide range of hydrolyzing and oxidizing enzymes, as other members of the
genus do (Isikhuemhen and Nerud, 1999; Kadiri and Fasidi, 1990; Toyama and
Ogawa, 1974; Ulezlo et al., 1975;). Furthermore, recent studies (Jiang et al.,
2000) have reported that, in addition to a sufficient oxygen supply, the optimum
conditions for the cultivation of P. tuber-regium sclerotia are a temperature of
23-28��C, a pH of 7.5-8, a culture medium-to-water ratio of 1 : 2.2, and corn
starch, wheat bran, and cow dung carbon and nitrogen sources.

4.5.2 Sclerotia of Polyporus rhinocerus Cooke

Polyporus rhinocerus (Chinese common name, hurulingzhi) is a type of white-rot
fungus that is mainly distributed in China, Malaysia, Sri Lanka, the Philippines,
Australia, and East Africa (Huang, 1999a). Very limited information on this mush-
room has been reported in the literature.

The sclerotium of P. rhinocerus is subterranean with a spherical, oval, or even
irregular shape (about 4-5 cm in diameter). The rough and wrinkly surface (rind)
of the sclerotia (which is white to pale brown in color), on which oval- shaped fruit-
ing bodies (that are tea brown in color, ciliated, and depressed in the center) are
grown, is thin, and the internal structure is white and powdery (Huang, 1999b). The
P. rhinocerus sclerotium is an expensive folk medicine used by Chinese physicians
to treat liver cancer, chronic hepatitis, and gastric ulcers. Because of its success-
ful cultivation, the scientific name and taxonomy of this mushroom have recently
been further identified and confirmed by conventional morphology characteriza-
tion as P. rhinocerus Cooke or Lignosus rhinocerus Cooke Ryv., belonging to
Eumycota, Basidiomycotina, Hymenomycetes, Aphllophorales, and Polyporaceae
(Huang, 1999a, b).

In contrast to P. tuber-regium, information concerning the cultivation of
P. rhinocerus sclerotia is very limited. Nevertheless, the successful cultivation
of P. rhinocerus sclerotia using substrate bags was reported by Huang (1999b).
The ingredients of the compost used were saw dust (80%), wheat bran (18%),
sugar cane (1%), CaCC>3 (1%), and water (1 : 1-1.4). Briefly, all of the compost
ingredients were well mixed and filled into transparent polypropylene plastic bags
(170 x 350 x 380 x 0.05mm) to two-thirds height. After putting them on plastic
rings, the open ends of the plastic bags were plugged with cotton plugs, wrapped
in paper, and tied up with rubber bands prior to sterilization with an autoclave.



1 20 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

It is interesting to note that loosely packed compost was found to cause wrinkles
on the surface of the resulting sclerotia (Huang, 1999b). When the plastic bags
had cooled down to about 30��C, each substrate bag was aseptically inoculated
with spawn (young P. rhinocerus mycelia with sclerotium-forming ability, grown
on a sawdust-wheat bran medium for 30-40 days at 20-25��C), followed by
incubation at 20-26��C in incubation rooms. After about one and a half months,
the mycelia had fully colonized the substrates in the plastic bags, and sclerotia had
begun to form. At this stage, the sclerotia could either have been kept growing in
the substrate bags or buried in soil (without the plastic bags) under broad-leaf trees
and covered with loose soil and withered leaves. When the sclerotia maintained
with the compost in the substrate bags had shrunk drastically, become softened,
and undergone exudation (at about six months), they were mature enough for
harvesting. All of the harvested sclerotia were washed clean and sun or oven dried
prior to further processing for edible or medicinal use.

4.5.3 Sclerotia of Wolfiporia cocos (Schw.) Ryv. Et Gilbn
[Poria cocos (Schw.) Wolf]

Wolfiporia cocos is also known as Fu Ling or Hoelen (the Chinese names for its
sclerotium) and is mainly distributed in the southern provinces of China such as
Yunnan and Fujian (Bi et al., 1993). The sclerotium of W. cocos is subterranean
with a spherical, oval, or even irregular shape and ranges in diameter from 10 to 30
cm (Keys, 1976; Ooi, 2000). When it is fresh, the sclerotium of W. cocos is slightly
soft, but it becomes very hard when it is dry (Bi et al., 1993; Liu and Bau, 1980).
Wolfiporia cocos sclerotia can be collected all year round, especially in August and
September (Liu and Bau, 1980).

The sclerotium of W. cocos is one of the earliest and most commonly used
fungi in Chinese medicine. The rough and wrinkly surface (rind) of the sclerotia
(which are brownish yellow to dark brown in color) is mainly used as a diuretic
(that regulates the K and Na balance) (Keys, 1976; Xu and Wang, 2002) and as
a decoction for coughs. Umbrella-shaped fruiting bodies (that are white or pale
yellow when fresh, with nearly no stem) are resupinately grown on the sclerotia
and form a thin layer (Bi et al., 1993; Ooi, 2000). Their white or pink interiors
are powdery and used as a cardiotonic agent and to relieve the uneasiness that
arises from pregnancy. The sliced or whole sclerotium is often applied to treat
jaundice and to induce menstruation (Ooi, 2000). The polysaccharides extracted
from the sclerotium of W. cocos, such as debranched pachyman, exhibit strong
antitumor and immunomodulatory effects (Chihara et al., 1970; Ding et al., 1998),
whereas its low-molecular-weight tetracyclic triterpenes have been found to have
immunostimulating, antiviral, tumor inhibitory, and cytotoxic properties (Hobbs,
1995; Kaminaga et al., 1996; Ukiya et al., 2002; Wang et al., 1995). Other folk
medicinal functions of W. cocos sclerotia include the treatment of diarrhea, spleen
dampness, and insomnia. They are also used as a sedative to tranquilize the mind
and refresh the spirit (Keys, 1976; Xu and Wang, 2002).

Wolfiporia cocos is a brown rot fungus that mainly grows in association with
the roots of various conifers, especially the Chinese red pine and the Taiwanese



BIOCHEMICAL, NUTRITIONAL, AND TECHNOLOGICAL CHARACTERISTICS 1 21

pine, and oaks (Keys, 1976; Ooi, 2000). At present, W. cocos sclerotia for medic-
inal use are primarily obtained from cultivation. In China, W. cocos sclerotia are
mainly cultivated on the surface of pine logs that are buried in caverns after the
spawn (young mycelia with sclerotium-forming ability) are inoculated. As the pro-
cedures for and information on this cultivation technique (including the season
for cultivation, preparation of the spawn, type and method of inoculation, man-
agement after inoculation, and harvesting method) have been comprehensively
reviewed in the recent literature [Central Agricultural Broadcasting and Televi-
sion School and National Farmer's Science and Technology Education Training
Centre (CABTS/NFSTTC), 2006], they will not be described in detail here. Igari
et al. (2000) reported that 10 strains of W. cocos sclerotia (about 5 cm in diam-
eter; 62 kg/m 3 pine log) were successfully cultivated on the surface of pine logs
buried in a field after spawn inoculation for 21 months and that their quality (in
terms of ash content, TLC patterns, and amount of pachymic and debydropachymic
acids in the diluted ethanol extracts) was highly comparable to that of commer-
cial strains. Recently, an indoor cultivation technique (without soil) for W. cocos
sclerotia was developed and reported by Kubo et al. (2006). Briefly, W. cocos scle-
rotia were cultivated in mushroom culture bottles that contained three pine logs
(Pinus densiflora SIEB. Et Zucc; 5 cm in diameter and 10 cm in length), and
the caps of the bottles were equipped with commercially available cloth air filters.
After spawn [W. cocos mycelia with proven sclerotium-forming ability, grown on a
sawdust-rice bran medium (3 : 1 v/v) with a moisture content of 70% at 30��C for
one month] inoculation on the pine logs, all of the culture bottles were incubated
in the dark at 25��C for 24 weeks to facilitate sclerotia formation. Compared with
the efficiency of field cultivation (21 kg/m 3 DW, 21 months), the indoor cultiva-
tion of W. cocos sclerotia exhibited a remarkably faster growth rate (14 weeks) and
higher productivity (110 kg/m 3 DW, about 15 cm in length). In addition, both culti-
vated and commercial W. cocos sclerotia share similar TLC patterns and pachymic
and debydropachymic acid content in their methanol extracts. Furthermore, the
progressive decay (mainly hemicellulose and cellulose) of the pine logs by the
W. cocos also markedly increased its alkaline solubility in 1% aqueous NaOH
(based on the weight of the decayed wood and the percentage weight loss due to
wood decay), which is consistent with the main characteristic of brown rot fungi.
As this indoor technique successfully cultivated W. cocos sclerotia with a high
yield in a short period of time, further study on its applicability to other mushroom
sclerotia, such as those of P. tuber-regium and P. rhinocerus, would be worthwhile.



4.6 BIOCHEMICAL, NUTRITIONAL, AND TECHNOLOGICAL
CHARACTERISTICS OF MUSHROOM SCLEROTIA

4.6.1 Biochemical Components of Mushroom Sclerotia

4.6.1.1 Cell Walls Similarly to the fungal cell wall of most Basidiomycetes,
the cell wall of sclerotial hyphae usually contains chitin and /3-glucans (with 1,3



1 22 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

and 1,6 linkages at different degrees) as its major structural and matrix compo-
nents (Backhouse and Willetts, 1984; Bullock et al., 1980; Jones et al., 1972; Kohn
and Grenville, 1989a, b; Willetts et al., 1990). Protein has also been detected in
the hyphal walls of some sclerotia (Chet et al., 1967; Kohn and Grenville, 1989a,
b). Melanins, or oxidized phenolic pigments, are found to be deposited in large
amounts in the rind walls and sometimes in the walls of the outer cortical cells
(Bullock et al., 1980; Kohn and Grenville, 1989a, b). In addition to reducing the
permeability of the cell wall of the peripheral hyphae, melanins also contribute to
its resistance to radiation and biological degradation (Willetts, 1971). When form-
ing a complex with the chitin of the fungal wall, melanins also act as inhibitors
of the polysaccharide-hydrolytic enzymes of the fungus itself and its antagonistic
microorganisms (Bull, 1970). This finding could well explain why the rind remains
intact even when the medullary hyphae have been completely lysed by the activ-
ities of the antagonistic microorganisms (Coley-Smith, 1980) and even after the
sclerotia have been degraded during germination (Backhouse and Willetts, 1985;
Bullock et al., 1983).

4.6. 1.2 Extracellular Matrix In various mushroom sclerotia, the major chem-
ical composition of their extracellular matrix, which consists of highly hydrated
materials expanding and filling the interhyphal spaces within the sclerotia, is found
to be similar to a structure that is composed mainly of /?-l,3-glucan backbone with
/5-1,6-linked side branches (Backhouse and Willetts, 1984; Bullock and Willetts;
1996; Bullock et al., 1980; Dubourdieu et al., 1981). In addition to morphogenesis
and the storage and supply of water to withstand unfavorable environmental condi-
tions (e.g., drought), one of the most important functions of the extracellular matrix
is to provide a large energy reserve of carbohydrates during sclerotial germination
(Backhouse and Willetts, 1985; Bullock et al., 1983; Ueno et al., 1980).

4.6.1.3 Cytoplasmic Reserves The identification of sclerotial cytoplasmic
reserves by histochemistry has been reported in numerous studies (Backhouse and
Willetts, 1984; Bullock et al., 1980; Moore et al., 1991; Willetts et al., 1990), and
the main cytoplasmic reserves detected in mushroom sclerotia include glycogen,
protein polyphosphate, and lipids (Moore, 1995; Willetts and Bullock, 1992).

Glycogen. Glycogen is present throughout the cytoplasm of cortical and
medullary hyphae at all stages of sclerotial differentiation, whereas the hyphal
cells in the rind region contain a much lower quantity of glycogen than do those
of other regions, even at the beginning of rind differentiation. As reported by
Bullock etal. (1980), granular glycogen deposits, which fill the spaces between
other storage bodies and organelles, were observed in the medulla hyphae of the
sclerotia of S. minor by a transmission electron microscopy. Glycogen decreases
in fully grown sclerotia and is probably utilized in the synthesis of other reserves,
such as proteins.



BIOCHEMICAL, NUTRITIONAL, AND TECHNOLOGICAL CHARACTERISTICS 123

Polyphosphates. Polyphosphate granules are present in vegetative hyphae and
sclerotial initials, and their quantity increases during the growth of sclerotia, par-
ticularly in the outer medulla and cortex (the main storage region at maturity)
(Kohn and Grenville, 1989a, b; Willetts et al., 1990). The presence of phosphates
in the metachromatic granules and tissue of the sclerotia of Paxillus involutus was
confirmed by Moore etal. (1991) using energy-dispersive X-ray microanalysis.
Polyphosphates play several possible roles in sclerotia, including energy storage,
the regulation of soluble phosphate levels, and phosphorus storage (Harold, 1966;
Kulaev, 1975).

Protein. The size and number of protein bodies increase throughout sclerotial
development, particularly at the stage at which the sclerotium has grown to
almost its full size and is not yet fully pigmented. Under a transmission electron
microscope, protein bodies are round or elongated membrane-bound structures
and are completely filled with moderately electron-dense materials (Willetts and
Bullock, 1992; Willetts et al., 1990). ER, in the form of long and parallel cisternae,
becomes much more abundant in sclerotial hyphae when the protein bodies are
being formed, which suggests its possible role in protein synthesis, as occurs in
other animal and plant systems (Gunning and Steer, 1975; Jorgensen et al., 1977).
In the sclerotia of P. involutus (Moore etal., 1991) and T. incarrncita (Willetts
et al., 1990), polyphosphates embedded in the matrix of the protein bodies were
observed. In mature sclerotia, protein bodies are the major cytoplasmic storage
reserve.

Lipids. Rather than special storage bodies, Backhouse and Willetts (1984) sug-
gested that the lipid bodies in the sclerotia of Botrytis cinerea and Botrytis fabae
are normal constituents of the hyphae, as they resemble both the sclerotia and veg-
etative hyphae of the Botrytis spp. However, remarkable amounts of lipids were
reported in the sclerotia of Monilinia fructicola (Kohn and Grenville, 1989a, b)
and V. dahliae (Willetts and Bullock, 1992). In addition to being species depen-
dent, the lipid content of mushroom sclerotia may also be partially attributed to the
culture medium and the particular isolate used (Kohn and Grenville, 1987).

4.6.2 Nutritional Evaluation of Mushroom Sclerotia

4.6.2.1 Proximate Composition Our previous studies (Wong etal., 2003)
have found that the sclerotia of P. tuber-regium, P. rhinocerus, and W. cocos exhibit
similar patterns of proximate composition, with a substantial amount of carbohy-
drates [ranging from 90.5 to 98.1% dry matter (DM)] and an extremely low lipid
content (ranging from 0.02 to 0.14% DM). This indicates that all three sclerotia
may belong to the carbohydrate-rich sclerotia type, as was previously suggested
by Coley-Smith and Cooke (1971). Similar results were reported previously by
Cheung (1997), Fasidi and Ekuere (1993), Nwokolo (1987), and Ude et al. (2001).
The crude protein (0.67-6.71% DM) and ash contents (ranging from 1.09 to 2.78%
DM) of the three sclerotia were low and were significantly different from one



124 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

another (P. tuber-regium was the highest and W. cocos the lowest). The crude
protein content (6.71% DM) of the sclerotia of P. tuber-regium was similar to
that of cultivated specimens grown on different cellulosic waste materials (rang-
ing from 6.32 to 8.24% DM; Fasidi and Ekuere, 1993) but was much lower than
that (10.8% DM) reported by Ude et al. (2001). Nevertheless, these data would be
comparable if a protein conversion factor of 4.38, rather than 6.25, was used in
the latter case (7.58% DM). In fact, Basidiomycetes such as P. tuber-regium pos-
sess an appreciable amount of nonprotein N (mainly from chitin/chitosan), which
must be corrected for before the crude protein content can be estimated from the N
content and the protein conversion factor (Nwokolo, 1987; Kurasawa et al., 1991).
The ash content of P. tuber-regium (2.78% DM) lies within the range for cultivated
P. tuber-regium sclerotia (ranging from 0.54 to 4.00 DM) in the previous literature
(Fasidi and Ekuere, 1993; Nwokolo, 1987; Ude et al., 2001). All of the air-dried
sclerotia possessed a notably high level of moisture, with that of P. tuber-regium
being significantly the lowest. Although the moisture content of the P. tuber-regium
sclerotium (12.9% DW) was consistent with that of specimens grown in Nigeria
(14.9% DW) (Ude et al, 2001), a much higher moisture content level (23.7% DW)
for this air-dried sclerotium was reported by Nwokolo (1987). Except for the mois-
ture content, all of the proximate compositions of W. cocos were in agreement with
those of a previous study (Cheung, 1997).



4.6.2.2 Sclerotial Dietary Fiber Extensive research over the past three
decades has demonstrated that the intake of sufficient dietary fiber (DF) has
benefits for health maintenance and disease prevention [American Diabetic
Association (ADA), 2002]. In general, DF can be divided into soluble (SDF)
and insoluble (IDF) fractions based on its solubility in an aqueous medium.
The viscosity of SDF is responsible for slower digestion and the absorption
of nutrients, which helps to attenuate blood cholesterol and glucose levels. In
contrast, IDF is characterized by its ability to increase fecal bulk (nonfermentable
or partially fermentable in colonic microflora) and decrease intestinal transit
time, thus promoting laxation (Potty, 1996). The increased awareness of the
potential health benefits of DF among consumers has undoubtedly encouraged
food manufacturers to explore new DF sources and develop fiber-enriched or
fiber-fortified food products such as snack foods, beverages, cookies, and canned
meats (McKee and Latner, 2000; Sloan, 2001). Today, most fiber supplements are
obtained from the by-products that result from the processing (e.g., milling) of
cereals, fruits, vegetables, and legumes (McKee and Latner, 2000). Because of
the highly competitive market for fiber-enriched food products, there is an urgent
need to explore new sources of DF. Because chitin and /5-linked glucose-based
polysaccharides cannot be digested or absorbed in the human intestine, mushroom
sclerotia obviously contain an abundant amount of cell wall and extracellular
matrix materials that can be classified as DF and may thus serve as an alternative
source of DF in the food industry (Cheung, 1997; Wasser and Weis, 1999).



BIOCHEMICAL, NUTRITIONAL, AND TECHNOLOGICAL CHARACTERISTICS 125



Our previous studies (Wong et al., 2003; Wong and Cheung, 2005a) have shown
that the sclerotia of P. tuber-regium, P. rhinoceros, and W. cocos possess remark-
ably high levels of IDF content (ranging from 77.4 to 94.6% DM) and excep-
tionally small levels of SDF content (ranging from 1.45 to 2.50% DM), whereas
their TDF content is (ranging from 81.7 to 96.3% sample DM) comparable to
that of some commercial DF-rich supplements [HUMAMIL (glucomannan) 82.9%
DM; FYBOGEL (Ispaghula husk) 88.5% DM; FIBRAPLAN (soluble fiber from
algae, seeds, flour, and nonspecified plants) 86.6% DM] (Goni and Martin-Carron,
1998). This finding suggests that the DF of all three sclerotia has great potential
to act as an alternative source of high fiber in the food industry. In addition, the
three types of sclerotial DF have notably high levels of NSP (86.6-94.3% scle-
rotial TDF DM), in which the predominant glucose residues (89.7-94.5% NSPs
DM), together with the glucosamine content (1.83-6.28% NSP DM), collectively
ensure that /3-glucans and chitin are the main matrix and fibrillar components of
the fungal cell wall polysaccharide in the three sclerotia (Wong et al., 2003; Wil-
letts and Bullock, 1992). Other minor sugar residues found in the three types of
sclerotial DF include mannose, galactose, rhamnose, and uronic acids, which may
indicate the presence of small amounts of mannan, galactan, and polyuronides. The
three sclerotia may possess glucuronic acids, as the presence of glucuronic acids
in other edible fungi such as Tremella aurantia and Tremella fuciformis has also
been reported (Gao et al., 1996; Kiho et al., 2000). Furthermore, scanning electron
micrographs have shown fragments of interwoven hyphae and insoluble materials
in the three sclerotial IDF fractions, but only the amorphous structure of soluble
materials was observed in the SDF fractions (Wong et al., 2003).

All three types of sclerotial DF exhibited very low levels (<30.0 p.g/g of
sclerotial DF) of five nutritionally important divalent minerals — calcium (Ca),
magnesium (Mg), copper (Cu), iron (Fe), and zinc (Zn) — when compared
with those of common DF sources such as cereals (wheat bran, rice bran, and
oats: Ca 701-1904 \x,glg; Mg 771-8825 u,g/g), fruits (apples and oranges: Mg
519-879 |xg/g; Zn 9-16 M-g/gX legumes (butter beans, broad beans, lentils: Ca
977-1730 (ig/g; Mg 286-424 |xg/g; Fe 180-390 |xg/g; Cu 11.1-30 |xg/g; Zn
29-62 |xg/g), vegetables (tomato and sugar beet fiber: Mg 1530-3475 |ig/g; Zn
13-41 |xg/g) (Elhardallou and Walker, 1999; Idouraine et al., 1995; Thibault
and Ralet, 2001), and some commercial DF supplements (e.g., Citrucel, Fiber
One, All-Bran, Metamucil: Ca 333-6340 |ig/g) (Luccia and Kunkel, 2002). This
finding indicates that a considerable amount of these five minerals was most likely
lost during the enzymatic preparation of sclerotial DF, as considerably higher
amounts (Ca 6200-21000 u,g/g; Mg 5800-15100 |ig/g; Fe 100-500 |xg/g;
Cu 100-500 M-g/g; Zn 100-500 M-g/g) were previously reported in cultivated
P. tuber-regium sclerotia, in addition to an abundant amount of potassium
(24,500-95, 600 |xg/g) and small amounts of phosphorus (8200-18, 300 u,g/g)
and manganese (100-1000 |xg/g) (Fasidi and Ekuere, 1993). Furthermore, by
using an energy-dispersive X-ray microanalyzer, trace amounts of Ca-oxalate,
Si, and Al were detected in the crystalline substances of W. cocos sclerotia



1 26 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

(Tanaka, 1990). As suggested by Fasidi and Ekuere (1993), the mineral con-
tent in mushroom sclerotia is highly dependent on their cultivated or natural
environments.

4.6.3 Physicochemical and Functional Properties of
Mushroom Sclerotial DF

In addition to its physiological benefits, DF has desirable functional properties,
such as providing texture, gelling, thickening, emulsification, and stabilization
in DF-enriched foods (Nelson, 2001; Dreher, 1987). Therefore, DF research,
particularly in the growing nutraceutical industry, has gained a lot of attention
recently (Jalili etal., 2000; Thebaudin etal., 1997). DF of different origins
possesses different structures, chemical compositions, and physicochemical
properties that exhibit different nutritional, technological, and physiological
benefits (Blackwood etal., 2000; Guillon and Champ, 2000; Nelson, 2001;
Thebaudin et al., 1997). The preparation of sclerotial DF from P. tuber-regium,
P. rhinocerus, and W. cocos using industrial food-grade glycolytic and proteolytic
enzymes has recently been carried out in our laboratory (Wong and Cheung,
2004). To evaluate their potential for developing fiber-enriched products with a
high level of consumer acceptance, some of their physicochemical and functional
properties [such as color, pH, water-binding capacity (WBC), oil-holding capacity
(OHC), emulsifying activities (EA), and emulsion stability (ES)] were investigated
and compared with those of a commercial fiber-rich barley ingredient (Wong and
Cheung, 2005a). We found that the pH of the suspension of all three types of
sclerotial DF was slightly acidic (ranging from 5.59 to 6.1 1), which was consistent
with their low levels of uronic acids (0.51-2.14% DM). In addition, their pH
values were significantly lower (p < 0.025) than those of the barley DF and
higher than those of the DF concentrates prepared from peaches (ranging from
3.63 to 3.86) (Grigelmo-Miguel and Martin-Belloso, 1997) and oranges (ranging
from 3.85 to 3.93) (Grigelmo-Miguel and Martin-Belloso, 1999).

Compared with the color of the barley DF control, both the P. tuber-regium
and P. rhinocerus DF possessed significantly higher (p < 0.025) values of light-
ness (L*) but smaller increments of redness (a*) and yellowness (b*). Such a high
degree of whiteness is a technological advantage for these two types of sclerotial
DF when they are added to such bakery products as white bread and sugar-type
cookies because their incorporation is not likely to produce an off color (darker
than desired) (Good, 2002; Nelson, 2001). The color of the W. cocos DF was char-
acterized by similar values of L* and a*, but a significantly lower (p < 0.025)
value of b* when compared with the color of the barley control. Among the three
types of sclerotial DF, the value of the total color difference (AZs *) between the W.
cocos DF and the barley DF control was the lowest (9.84; p < 0.025), indicating
their relatively high similarity in color compared to the other two types of sclerotial
DF. The pinkish brown color of the W. cocos DF suggests that its incorporation into
a food system may affect the color of the final product. Color is influenced by many
factors, including species variety, the maturity of the sample, and the processing
method (e.g., drying) (Grigelmo-Miguel and Martin-Belloso, 1999).



BIOCHEMICAL, NUTRITIONAL, AND TECHNOLOGICAL CHARACTERISTICS 1 27

The WBC of a fiber measures the amount of water that it retains after it is sub-
ject to a stress such as centrifugation (Nelson, 2001). This hydration property of
a DF ingredient is crucial to its successful application in food that will be subject
to physical stress (e.g., the extrusion of cereals). The W. cocos DF had the highest
(p < 0.05) value of WBC (6.26 g/g DW), which was highly comparable to that
of some DF derived from cereal processing by-products [wheat bran 6.4-6.6 g/g
DW (Adams et al., 1986; Ralet al, 1990); oat bran 5.5 g/g DW (Cadden, 1987)],
fruits [apple DF 6.3-6.9 g/g DW and pear DF 6.8 g/g DW (Grigelmo-Miguel
and Martin-Belloso, 1997)], and some commercial DF-rich supplements [AGIO-
LAX (Ispaghula seed and husk, cassia fruit) 6.6 g/g DW (Goni and Martin-Carron,
1998); FIBREX (sugar beet) 4.56 g/g DW (Abdul-Hamid and Luan, 2000)]. The
WBC of P. tuber-regium (2.78 g/g DW) and P. rhinocerus DF (2.72 g/g DW) did
not differ significantly (p < 0.025) from that of the barley DF (2.54 g/g DW),
but their WBC levels were also consistent with those of various high-fiber ingre-
dients from apple pulp (2.3 g/g DW), wheat bran (2.6 g/g DW), corn bran (2.5 g/g
DW), and soy bran (2.4 g/g DW) (Dreher, 1987). The remarkably high WBC of the
W. cocos DF suggests that this material could be used as a functional ingredient to
avoid syneresis (weeping) and to improve the rubbery texture of formulated prod-
ucts such as cheese (Nelson, 2001), in addition to reducing calories by the total or
partial substitution of high-energy ingredients. The methods of measurement and
food system environments (such as pH, ionic strength, concentration, and pres-
ence of other water binding materials) are crucially important factors that affect
the WBC of high-fiber ingredients (Auffret et al., 1994; Fleury and Lahaye, 1991;
Nelson, 2001).

Only the OHC of P. rhinocerus DF (1.87 g/g DW) was comparable to that of
barley DF (1.88 g/g DW) and wheat DF (2.3 g/g DW) (Thebaudin et al., 1997).
Although the OHC values of the DF obtained from P. tuber-regium and W. cocos
were significantly lower (p < 0.025) (1.36-1.37 g/g DW), they were compa-
rable to those of orange DF concentrate (0.86-1.28 g/g) (Grigelmo-Miguel and
Martin-Belloso, 1999) and the commercial DF-rich supplement FIBREX (1.29 g/g
DW) (Abdul-Hamid and Luan, 2000).

The ability of a fiber to bind oil is more a function of the porosity of the fiber
structure than the affinity of the fiber molecule for oil (Nelson, 2001). Other fac-
tors, such as the number of lipophilic sites, overall hydrophobicity, and capillary
attraction (Kinsella, 1976), may also contribute to the variations of OHC in scle-
rotial DF. A high-fiber ingredient with a high OHC allows the stabilization of
high fat content and emulsion in formulated food products such as comminuted
or emulsified meat by retaining the fat. In low-fat meat applications, the OHC of
the high-fiber ingredients can also retain the low amount of fat present, which aids
in the flavor, texture, and juiciness of the final cooked product.

The emulsion formed by all of the DF samples was generally good, as their EA
values (56.7-71.9%) were >50% (Wang and Kinsella, 1976; Yasumatsu etal.,
1972). In addition, the EA of all of the sclerotial DF was notably higher (p <
0.0.5) than that of the barley DF control (56.7%), rice bran DF (14.4%), and the
commercial fiber-rich supplement FIBREX (3.46%) (Fleury and Lahaye, 1991),



1 28 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

thus suggesting their great potential to act as an emulsifier in formulated food.
Furthermore, the emulsions formed by all of the DF samples were very stable, as
evidenced by their similarly high percentage of ES after incubation at 80�� C for 30
minutes.

The three novel types of sclerotial DF appear to be versatile low-calorie food
ingredients with several technological advantages (natural origin, high DF content,
and good physicochemical functional properties) that are of interest to the food
ingredient market, and they could thus be incorporated into a wide range of formu-
lated foods such as bakery products, noodles, and snacks. Further investigation of
their potential role as a functional food fiber or nutraceutical via the assessment of
some of their physiological benefits would be interesting.

4.7 BIOPHARMACOLOGICAL VALUES OF MUSHROOM SCLEROTIA

OF P. tuber-regium, P. rhinocerus, AND W. cocos

4.7.1 In Vitro Mineral Binding Capacity

The recommendation for an increase in DF intake has raised questions about
the possible negative effects on mineral bioavailability, particularly in high-risk
population groups such as the elderly, infants, and pregnant women (Idouraine
et al., 1995, 1996). Because the electrostatistic binding and/or trapping of minerals
within DF particles is one of the main factors that determines the undesirable
effects of DF on mineral bioavailability, the in vitro mineral binding capacity
of DF is believed to be a crucial parameter for predicting its effect on mineral
bioavailability in humans (Laszlo, 1989). To predict the possible effects of
sclerotial DF prepared from P. tuber-regium, P. rhinocerus, and W. cocos on
mineral bioavailability in the human gastrointestinal tract, their in vitro mineral
binding capacity on five nutritionally important divalent minerals — calcium
(Ca), magnesium (Mg), copper (Cu), iron (Fe), and zinc (Zn) — under sequential
simulated physiological conditions of the human stomach, small intestine, and
colon was investigated and compared (Wong and Cheung, 2005b). In addition to
releasing most of their endogenous Ca (ranging from 96.9 to 97.9% removal) and
Mg (ranging from 95.9 to 96.7% removal), the simulated physiological conditions
of the stomach also attenuated the possible adverse binding effects of the three
types of sclerotial DF to the exogenous minerals by lowering their cation exchange
capacity (ranging from 20.8 to 32.3%) and removing a substantial amount of
their potential mineral chelators, including protein (ranging from 16.2 to 37.8%)
and phytate (ranging from 58.5 to 64.2%). This finding suggests that when the
three sclerotial DF types reach the stomach, most of their endogenous Ca and Mg
will be readily released, and about half of their endogenous Cu, Fe, and Zn will
remain bound. The in vitro mineral binding capacity of the three sclerotial types
of DF under the simulated physiological conditions of the small intestine was
found to be low, especially for Ca (ranging from 4.79 to 5.91% binding) and Mg
(ranging from 3.16 to 4.18% binding) and was highly correlated (r > 0.97) with



BIOPHARMACOLOGICAL VALUES OF MUSHROOM SCLEROTIA 129

their residual protein content. The three types of partially demineralized sclerotial
DF from the stomach could only rebind a limited amount of the five nutritionally
important minerals in the small intestine and may not have a detrimental effect
on mineral bioavailability compared with other fibers (Harland, 1989; Kelsay,
1986; Munoz and Harland, 1993; Reinhold et al, 1976). Under the simulated
physiological conditions of the colon with a slightly acidic pH (5.80), only
bound Ca was readily released (ranging from 34.2 to 72.3% releasing) from the
three types of sclerotial DF. This finding indicates the potential physiological
benefits of the three sclerotial DF types on Ca bioavailability. On reaching the
human colon, part of the small intestinal condition-treated sclerotial DF would be
fermented by the anaerobic microflora in the large intestine, thus releasing some
of their bound minerals. If the fermentability of the three types of small intestinal
condition-treated sclerotial DF was high enough to create an acidic colonic
environment (pH < 5.80), then this not only would release an appreciable amount
(34.2-72.3%) of bound Ca from the three types of nonfermented sclerotial DF
but also might promote their ionization together with the already released and
unabsorbed minerals. As a result, passive mineral absorption, especially Ca, in the
large intestine might be enhanced and the overall Ca bioavailability might then
be improved. The additional absorption of Ca in the colon is especially important
in elderly people and postmenopausal women who have insufficient Ca intake
or insufficiently active Ca absorption from the small intestine. Nevertheless, the
main criterion in determining the potential enhancing effect of the three types
of sclerotial DF on passive Ca absorption in the human large intestine is their
fermentability.

4.7.2 In Vitro Fermentability

DF escapes digestion and absorption in the human small intestine and constitutes
the main substrate for colonic fermentation (Cummings, 1982). The fermentative
breakdown of DF in the human colon by anaerobic saccharolytic microflora leads
to the production of certain gases (CO2, CH4, and H2), microbial biomass, and
short-chain fatty acids (SCFAs), which considerably influences the physiological
functions of humans (Topping and Clifton, 2001; Tungland and Meyer, 2002).
The rate and extent of fiber fermentation depend on two main categories of fac-
tors: (1) host-specific factors, such as the activities and composition of the colonic
microflora and gastrointestinal tract transit time, and (2) substrate-specific factors,
including the physicochemical properties (e.g., particle size, solubility, and cell
wall architecture) of the fiber source and the chemical composition (monosaccha-
ride profile) and structural arrangement (degree of branching and linkages between
monosaccharide) of the fiber constituents (Auffret et al., 1993; McBurney and
Thompson, 1989; Titgemeyer et al., 1991). The SCFAs produced during fermen-
tation are rapidly absorbed by the colonic mucosa, stimulating water and sodium
absorption and peristalsis, which, in turn, aid the bowel function (Cummings et al.,
1987; Ruppin et al., 1980). An appreciable amount of SCFAs resulting from highly
fermentable DF would also lower the colonic pH, thus modulating the composition



130 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

of the colonic microflora by inhibiting the growth of pathogenic bacteria (non-acid
tolerant) but stimulating the growth of those, such as Lactobacillus, that are ben-
eficial (Cummings et al., 2001; Gibson and Roberfroid, 1995). To predict the fate
of the sclerotial DF prepared from P. tuber-regium, P. rhinocerus, and W. cocos in
the human colon after consumption as a food ingredient, their fermentability was
evaluated in vitro by using human fecal microflora and comparing it to a cellulose
control (Wong et al., 2005).

Briefly, all of the DF samples (0.5 g each) were fermented in vitro with a human
fecal homogenate (10 mL) in a batch system (total volume 50 mL) under strictly
anaerobic conditions (using an oxygen-reducing enzyme under argon atmosphere)
at 37��C for 24 h. All three types of novel sclerotial DF exhibited notably higher
DM disappearance (P. tuber-regium 8.56%; P. rhinocerus 13.5%; W. cocos 53.4%)
and organic matter disappearance (P. tuber-regium 9.82%; P. rhinocerus 14.6%;
W. cocos 57.4%) when compared with the cellulose control. Nevertheless, only
the W. cocos DF was remarkably degraded to produce a considerable amount of
total SCFAs (5.23 mmol/g DF on an organic matter basis, with a relatively higher
molar ratio of propionate) that lowered the pH of the nonfermented residue to a
slightly acidic level (5.89). These findings suggest that during the fermentation
of the W. cocos DF by the human colonic bacteria, the high fermentability may
result in a sufficient amount of SCFAs to acidify the colonic pH, which may in
turn promote the ionization of the unabsorbed minerals and enhance their pas-
sive absorption in the colon, as in other highly fermented DF reported previously
(Coudray etal., 1997; Morohashi etal., 1998; Tahiri etal., 2001; Younes etal.,
2001). From a physiological point of view, the relatively higher level of propionate
produced by the readily fermented W. cocos DF implies that it may have hypo-
glycemic (probably by increasing hepatic glucose utilization or maximizing the
insulin response) and/or hypocholesterolemic effect(s) on humans (probably by
suppressing the synthesis of hepatic cholesterol or redistributing cholesterol from
the plasma to the liver), as has been proposed in previous human (Todesco et al.,
1991) and animal (Chen et al., 1984) studies. The P. tuber-regium and P. rhinocerus
DF remains nonfermented in the human colon, which, in turn, contributes to the
fecal bulking capacity and the bacterial biomass. In addition to diluting the car-
cinogenic and toxic substances by providing a bulkier stool, the nonfermented
P. tuber-regium and P. rhinocerus DF may also decrease the transit time of the stool
through the colon and lower the chance of exposure to carcinogens similar to other
cereal polysaccharides (Karppinen et al., 2000). Interestingly, the fermentation of
the /M,4-glucan-rich samples, the P. tuber-regium DF, and the cellulose control
exhibited similar SCFA profiles with a relatively higher molar ratio of butyrate
(acetate-propionate-butyrate in P. tuber-regium DF 2.1 : 1 : 1.4; cellulose 1.86 :
1 : 1.14). This finding is also comparable to that of the /J-1,4 linkage-rich glucan
from cereals with a relative molar ratio of 2.09 : 1 : 1.84 (Botham et al., 1998). In
the case of the DF of both P. rhinocerus and W. cocos, which was rich in /J -1,3 link-
ages, it had a relatively higher molar ratio of propionate (P. rhinocerus DF 4.75 :
2.25 : 1; W. cocos DF 2.82 : 1.46 : 1) after 24 hours in vitro fermentation, and
the SCFA profiles of both were also similar. The production of a relatively higher



BIOPHARMACOLOGICAL VALUES OF MUSHROOM SCLEROTIA 131

molar ratio of propionate has also been reported in the fermentation of curdlan
(3.72 : 1.41 : 1) (Shimizu etal., 2001) and laminarian (1.7 : 1.25 : 1) (Michel
et al., 1996). An obvious structure -function relationship between the three types
of sclerotial DF and their in vitro fermentability was present, and the variations
in their in vitro fermentability may mainly be attributed to the different amounts
of interwoven hyphae present (different amounts of the enzyme-inaccessible cell
wall component) and to the possibly different structural arrangements (linkage and
degree of branching) of their /6-glucan components. Thus, further investigation of
the possible enhancing effect of the sclerotial DF of W. cocos on passive mineral
absorption in the large intestine by using an animal model would be interesting.

4.7.3 In Vivo Ca and Mg Absorption

Previous studies have shown that DF, especially its insoluble fraction, can bind
strongly to Ca and form unabsorbable complexes owing to its anionic nature
(Piatt and Clydesdale, 1987). As a result, it has been proposed that DF may
impair Ca absorption. However, during the past decade, there has been substantial
evidence to indicate that Ca absorption is not affected by the fiber component
per se (Harrington etal., 2001; Kennefick and Cashman, 2000). Many recent
studies have also shown that fermentable DF, including oligosaccharides (e.g.,
fructo-oligosaccharides and inulin) and polysaccharides (e.g., resistant starch),
even improves overall Ca absorption in both humans (Coudray et al., 1997; Tahiri
et al., 2001) and rats (Morohashi et al., 1998; Younes et al., 2001).

According to Campbell. (1997), the beneficial effect of DF on overall Ca
absorption depends on its fermentability, the dosage used, and the duration of the
animal experiments. Although the detailed mechanisms of the enhancing effect
of fermentable DF on overall Ca absorption remain unclear, it is widely accepted
that the microbial degradation of fermentable DF in the large intestine is the
most important factor (Ohta etal., 1995; Shiga etal., 1998). The fermentation
by-products, SCFAs, are also believed to be the major contributor (Kishi et al.,
1999; Lutz, 1991) to increasing the concentration of ionized Ca and promoting its
absorption in the large intestine (Mineo et al., 2001; Younes et al., 1996).

Compared with cellulose control, the effects of the sclerotial DF prepared from
P. tuber-regium, P. rhinocerus, and W. cocos on apparent Ca and Mg absorption
were evaluated in ovariectomized (OVX) rats fed sclerotial DF-based and low-Ca
(0.3%) diets for 14 days (Wong et al., 2006). All three of the sclerotial DF-based
diet groups possessed significantly higher (p < 0.025) molar concentrations of
total SCFAs (ranging from 80.3 to 204 |xmol/g of cecal content) in their cecums,
with the W. cocos DF group being the highest (p < 0.025). However, only the
cecal pH (about 5.88) and cecal content (1.15 g) of the W. cocos DF group were
significantly lower (p < 0.025) than those of the cellulose control group. These
findings suggest that the ingestion of W. cocos DF leads to greater cecal fermen-
tation and produces significantly higher (p < 0.025) amounts of total SCFAs
that lower cecal pH to a slightly acidic level. The slightly acidic environment
established in the W. cocos DF group also led to the remarkable (p < 0.025) aug-
mentation of the cecal-soluble Ca (2.56-fold) and Mg (1.22-fold) concentrations.



132 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

Compared with the cellulose control group, the apparent Ca and Mg absorption of
the W. cocos DF group was also notably (p < 0.025) enhanced (Ca 16.5%; Mg
15.3%), together with a significant (p < 0.025) elevation of their serum Ca (3.61
mmol/L) and Mg (1.07 mmol/L) concentrations but suppression of their serum
PTH levels (57.1 pg/mL). These findings illustrate that the detrimental effects
induced by both ovariectomy and a low-Ca diet can be alleviated by the ingestion
of W. cocos DF. Its enhancing effect on Ca and Mg absorption in the cecum of OVX
rats is of particular interest for populations with inefficiently active Ca absorption,
such as elderly people and postmenopausal women. Fermentability is the main
factor that determines the enhancing effect of nondigestible carbohydrates on min-
eral absorption. Therefore, to explore the three types of novel sclerotial DF as
functional food ingredients for the enhancement of overall Ca and Mg absorption,
their fermentability should be improved (Cashman, 2003), probably by isolating
their main DF component, /J-glucan-rich polysaccharides, or even preparing some
novel fi -glucose-based oligosaccharides from the three types of sclerotial DF using
partial acid or enzymatic hydrolysis.

4.7.4 Antitumor and Immunomodulatory Activities

/6-Glucans, the major structural component of fungal cell walls (Bartnicki-Garcia,
1970), have been found to stimulate both the innate and adaptive immunity of the
host, followed by a wide range of immunopharmacological activities, particularly
antitumor activities, via their cytokine production and signaling cascade (Bohn
and BeMiller, 1995; Moradali et al., 2007). Mushroom sclerotia (a dried compact
biomass of fungal hyphae) have been found to possess substantial amounts of
/6-glucans (>80% on a DM basis; Wong et al., 2003), which have exhibited
remarkable immunomodulatory and antitumor activities in numerous previous
studies (Wong et al., 2007; Zhang et al., 2006a). Our research team has been
actively studying the immunomodulatory and antitumor activities of both native
and chemically modified sclerotial /J-glucans for the past six years (Chau et al.,
2007; Lai and Cheung, 2004; Lai, 2005; Lai et al., 2005; Tao et al., 2006; Wong
etal, 2007; M. Zhang etal., 2001, 2004a, b, 2006a, b). We have found that
sclerotial /J-glucan fractions isolated from P. tuber-regium (hot alkaline soluble,
hot water soluble, ultrasonic, sulfated, and carboxymethylated fractions) and
P. rhinocerus (hot water soluble and ultrasonic fractions) possess remarkable
immunomodulatory and antitumor activities when they are administered intraperi-
toneally on BALB/c mice bearing sarcoma 180 (allogeneic solid tumor cells) (M.
Zhang et al., 2001, 2004a, b; Lai, 2005; Lai et al., 2005; Tao et al., 2006). These
sclerotial /2-glucan fractions also exhibited a direct cytotoxic effect on various
mammalian cancer cell lines (such as HL-60, MCF-7, and HepG2) but were
noncytotoxic to normal kidney cells from monkey (VERO) cells (M. Zhang et al.,
2001, 2004a, b; Lai, 2005; Tao et al., 2006). Furthermore, flow cytometric analysis
showed that the sclerotial /6-glucan fractions isolated from P. tuber-regium
(carboxymethylated fractions) and P. rhinocerus (hot water-soluble fractions)
not only arrested the cell cycle progression of the MCF-7 (with down regulation



BIOPHARMACOLOGICAL VALUES OF MUSHROOM SCLEROTIA 133

of cyclin Dl and cyclin E expressions) and HL-60 cells, respectively, at the Gi
phase but also induced their apoptosis with decreased expression of Bcl-2 and
increased expression of the Bax/Bcl-2 ratio (Lai, 2005; Zhang et al., 2006a).
In the case of W. cocos, its sclerotial /2-glucan fractions (1% sodium carbonate
soluble) not only significantly induced nitric oxide (NO) production by the
peritoneal macrophages of B6 C3F1 mice but also inducible NO synthase (iNOS)
transcription of the murine macrophage-like cell line, RAW 264.7, via activation
of the transcription factor, namely nuclear factor-/cB/Rel (NF-/cB/Rel) (Lee and
Jeon, 2003). Its methanol extract and isolated triterpene acids were also found
to inhibit 12-0-tetradecanoylphorbol-13-acetate-induced ear edema and tumor
promotion in mouse skin (Kaminaga et al., 1996).

Despite the fact that sclerotial /2-glucans exhibited the aforementioned remark-
able immunomodulatory and antitumor activities, their underlying in vivo mecha-
nisms are still not fully understood, even though this issue has been of interest to
many scientists over the past five decades (Ooi and Liu, 2000). The most controver-
sial issue is how these macromolecules, fungal /J-glucans, act on or are recognized
by the innate immunity prior to triggering the acquired immune response and exert-
ing their antitumor effects.

Recently, a novel cell surface receptor, Dectin-1, that recognizes the yeast
/6-glucan (zymosan) and mediates its immunomodulatory and antitumor
effects, has been found on the surface of various innate immune cells [such as
macrophages, natural killer (NK) cells, and dendritic cells] in both humans and
mice (Brown and Gordon, 2001; Brown, 2006; Heinsbroek et al., 2006; Taylor
et al., 2002). Although the discovery of /6-glucan receptors on the surface of innate
immune cells would undoubtedly provide a stepping stone for an investigation
of the underlying mechanisms of the immunomodulatory and antitumor effects
of fungal /3-glucans, nearly all related studies have been limited to yeast-derived
/?-glucans, zymosan (Goodridge et al., 2007; Olsson and Sundler, 2007), but not
in the case of other important fungi such as mushroom sclerotia. Thus, it would
be valuable to determine whether there is a unique cell surface receptor(s) for the
/2-glucans of other important fungi, especially mushroom sclerotia.

Our latest in vivo immunophenotyping studies on the peritoneal exudate cells
and hepatic mononuclear cells of healthy BALB/c mice have discovered that scle-
rotial /3-glucan fractions isolated from P. tuber-regium (hot water-soluble fraction)
and P. rhinocerus (hot water-soluble and ultrasonic fractions) exhibit a remarkable
stimulatory effect on both the NK cells (CD56 + ) and macrophages (Mac— 1 + ) of
the innate immunity (unpublished data). In addition to a significant increase in
the weight of their spleens, the levels of various cytokines (including interleukins
IL-12 and IL-13) and macrophage inflammatory proteins were also notably ele-
vated in the serum of the mice pretreated with the hot water-soluble /J-glucans that
were isolated from P. rhinocerus (Lai et al., 2005).

Using this research as background, the underlying mechanisms of the innate
antitumor immunity that is mediated by these previously proven immunopotenti-
ating mushroom sclerotial /J-glucan fractions could be further investigated using
athymic nude mice with human tumor xenografts, as this animal model is T-cell



134 SCLEROTIA: EMERGING FUNCTIONAL FOOD DERIVED FROM MUSHROOMS

deficient to support the growth of human tumors without immune rejection. Innate
immune cells pretreated with these mushroom sclerotial /3-glucan fractions could
also be isolated from healthy athymic nude mice followed by an assessment of
their functional activities. Furthermore, the /J-glucan receptor(s) that are specific
to these mushroom sclerotial /2-glucan fractions could be identified on the surface
of innate human and murine primary cells (isolated from human peripheral blood
and athymic nude mice, respectively) by a molecular technique termed "phage
display." These findings would be a major breakthrough in antitumor studies of
mushroom sclerotia, as they would provide a new perspective to explain their
immunomodulatory and antitumor effects in terms of the kind of innate immune
cells involved, the type of cytokines induced, and the possible cell surface /J-glucan
receptor(s) identified. By increasing our knowledge of the interaction between
mushroom sclerotial /3-glucans and innate immunity, a more effective utilization
of these macromolecules as antitumor agents can be made possible.

4.8 CONCLUSION

The detailed mechanistic actions of the bioactive components in mushroom sclero-
tia are still not completely understood. Mushroom sclerotia thus remain underuti-
lized at the moment. It is anticipated that, with advances in molecular biology and
biotechnology, the content and the structure of the bioactive components of sclero-
tia, especially /2-glucans, can be manipulated to produce "tailor-made" functional
food products in the future.

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CHAPTER 5



Antitumor and Immunomodulatory
Activities of Mushroom
Polysaccharides

Vincent E. C. Ooi

Department of Biology and Institute of Chinese Medicine, The Chinese University of
Hong Kong, Hong Kong, China

CONTENTS

5.1 Introduction

5.2 Antitumor Polysaccharides from Mushrooms (Higher Fungi)

5.3 Mechanisms of Antitumor Action of Mushroom Polysaccharides

5.4 Structure and Antitumor Activity Relationship of Polysaccharides

5.5 Conclusions
References



5.1 INTRODUCTION

Mushrooms have a long history of medicinal application in addition to their nutri-
tional value. They have been used as food and medicinal materials in many Oriental
countries such as China, Japan, and Korea. In the past three to four decades, there
has been an upsurge of interest in research on the medicinal value of mushrooms
and their products. Chief among the most promising biopharmacological activities
of mushrooms are their immunomodulation and antitumor effects. Polysaccha-
rides are the best known mushroom-derived substances with potent antitumor and
immunomodulatory properties (Ooi and Liu, 2000; Wasser, 2002; Moradali et al.,
2007; Zhang et al., 2007). The beneficial uses of many mushroom polysaccha-
rides as therapeutic adjuvants or dietary supplements have been extensively studied
and their new wider usages and trials are further explored (Borchers et al., 2004;



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



147



148 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

Zekovic et al., 2005; Sullivan et al., 2006). Polysaccharides represent a structurally
diverse class of biological macromolecules of relatively widespread occurrence in
nature. Unlike proteins and nucleic acids, they contain repetitive structural fea-
tures which are polymers of monosaccharide residues joined to each other by
glycosidic linkages and can interconnect at several points to form a wide vari-
ety of branched or linear structures. Among these macromolecules, polysaccha-
rides offer the highest capacity for carrying biological information because they
have the greatest potential for structural variability. This enormous variability in
polysaccharide structures gives the necessary flexibility for the precise regulatory
mechanisms of various cell-cell interactions in higher organisms (Sharon and
Lis, 1993). Polysaccharides or polysaccharide-protein complexes derived from
mushrooms have attracted much attention of research because they are gener-
ally believed to be able to suppress the tumor growth of the host by restoring or
enhancing the immune defense system, which is vitally important for the mainte-
nance of homeostasis. They are often considered as host defense potentiators or
biological response modifiers (BRMs) (Bohn and BeMiller, 1995; Ooi and Liu,
2000; Leung et al, 2006; Wasser, 2002; Moradali et al, 2007). In addition, these
biomacromolecules have also been acclaimed to prevent carcinogenesis and tumor
metastasis (Kim et al., 1999; Baek et al, 2002; Guterres et al., 2005; Lee etal.,
2005). Although the mechanisms of antitumor action of polysaccharides are not
completely clear, they can potentiate cell-mediated immune responses through
the activation of specific immune cells to enhance a variety of cellular functions
such as cytotoxic and phagocytic responses against tumor cells. They are consid-
ered as multicytokine inducers that are able to induce gene expression of vari-
ous immunomodulatory cytokines by immunocompetent cells in the innate immu-
nity (Ooi and Liu, 2000; Wasser, 2002; Moradali et al., 2007). Nonetheless, many
of these macromolecules have also been documented to have direct cytotoxicity
against cancer cell lines in vitro (Chen and Chang, 2004; Zaidman et al., 2005;
M. Zhang et al., 2006a, b, 2007; Hui et al., 2005). The mechanistic action of anti-
tumor polysaccharides in the regulation of cell cycle and in the activation of the
cell death program (apoptosis) has recently received more attention of research
(Fullerton et al., 2000; M. Zhang et al., 2006a, b; Fang et al., 2006; Lavi et al.,
2006; Wong et al., 2007).

In the 1970s and 1980s, several antitumor polysaccharides, such as lentinan,
schizophyllan, and polysaccharide-protein complexes (PSK, PSP), were isolated
from Lentinus edodes, Schizophyllum commune, and Coriolus (Trametes) versi-
color, respectively, and have since become very popular in Japan, China, and other
Oriental regions (Mizuno et al., 1995b; Ooi and Liu, 1999, 2000). ,6-D-Glucans
from Grifola frondosa, Sparassis crispa, Agaricus blazei, Phellinus linteus, and
many others have also been widely used as dietary supplements or therapeutic
adjuvants for cancer treatment. Several excellent review articles about the iso-
lation, biological activity, chemical structure and modification, and drug devel-
opment of antitumor polysaccharides have been published in the last few years
(Ooi and Liu, 2000; Wasser, 2002; Zhang et al., 2007; Moradali et al., 2007). This
present review focuses on the recent findings of the antitumor polysaccharides of



ANTITUMOR POLYSACCHARIDES FROM MUSHROOMS (HIGHER FUNGI) 149

mushrooms with an emphasis on the relationship between their structure and anti-
tumor activity, elucidation of their antitumor and immunomodulatory mechanisms
of action at the molecular and cellular level, and improvement of their various
biological activities by chemical modifications.

5.2 ANTITUMOR POLYSACCHARIDES FROM MUSHROOMS
(HIGHER FUNGI)

Mushrooms are considered as macrofungi with a distinctive fruiting body which
is large enough to be seen with the naked eyes. Most macrofungi belong to
the class Basidiomycetes, but there are also others from the class Ascomycetes
(Chang and Miles, 1989). The life cycles of mushroom-like fungi are complex
and may involve a number of different morphological forms, including mycelium,
fruiting body, and sclerotium. The number of large filamentous fungi in the
sense of this definition is at least 14,000 species and perhaps as many as 22,000
species (Hawks worth, 2001). It is estimated that more than 2000 are safe as
edible mushrooms, and about 700 species are known to possess significant
pharmacological properties (Wasser, 2002). Thus, mushrooms comprise a vast and
yet largely untapped source of powerful new pharmaceutical products. For modern
medicine, in particular, they represent an unlimited source of polysaccharides
with antitumor and immunomodulatory properties. Many, if not all, mushrooms
contain biologically active polysaccharides in fruiting bodies, cultured mycelia,
sclerotia, and culture filtrates. Wasser (2002) reported that at least 651 species and
7 infraspecific taxa representing 182 genera of Basidiomycetes (mushroom-like
fungi) contain antitumor and immunostimulatory polysaccharides. These polysac-
charides may vary in their chemical composition, structure, and antitumor activity
(Ooi and Liu, 2000; Wasser, 2002; Zhang et al., 2007). Many polysaccharides
purified from mushroom fungi are mainly present as glucans with different types
of glycosidic linkages, but some are true heteroglycans, while others mostly bind
to protein residues as polysaccharide-protein complexes (Ooi and Liu, 2000).
The main source of mushroom antitumor polysaccharides appears to be related to
the cell walls, which consist of polysaccharides such as chitin, cellulose, (1 — > 3,
1 6)-y6-glucans, and (1 -> 3)-a-glucans or polysaccharide-protein complexes
such as galactomannan-protein, glucuromannan-protein (Zhang et al., 2007).
However, chitin and chitosan (fungal chitin) have not been reported to have any
antitumor activity (Mizuno et al., 1995b).

Modern research with mushroom polysaccharides can be traced to the 1960s
in Japan to the work of the Ikegawa group on the host-mediated antitumor activ-
ity of hot- water extracts of several edible mushrooms using sarcoma 180-bearing
mice (Ikekawa etal., 1969) and that of Chihara etal. (1970b) on the antitumor
polysaccharides isolated from L. edodes. Since then, numerous polysaccharides
and polysaccharide-protein complexes have been isolated and characterized from
mushrooms and used as a source of therapeutic agents for cancers.

In the last three to four decades, numerous polysaccharides and polysac-
charide-protein complexes have been isolated from mushrooms and used as



150



ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES



a source of nutraceuticals and therapeutic agents. Three antitumor mushroom
polysaccharides, that is, lentinan, schizophyllan, and polysaccharide-protein
complexes (PSK, PSP), have become very popular nutraceuticals in the Oriental
countries. Lentinan and schizophyllan are pure /3-glucans (Komatsu et al., 1969;
Chihara etal., 1970b, 1989), whereas PSK (Krestin) is a /3-glucan-protein
complex containing 25-38% protein residues (Tsukagoshi etal., 1984). It is a
(1 -> 4)-j6-glucan with (1 6)-y6-glucopyranosidic side chains for every fourth
glucose unit and has a structure with branches at the 3 and 6 positions in a
proportion of one per every several residual groups of 1 -> 4 bonds and associated
with peptide moiety (Tsukagoshi et al., 1984). PSP (polysaccharopeptide) isolated
from a strain of C. (T. ) versicolor in China (Yang et al., 1992) is found to be quite
similar in glucan structure to PSK in Japan. Lentinan from the fruiting body of
L. edodes is a representative mushroom (1 3)-/6-glucan with effective antitu-
mor and immunopotentiating activity. Its primary structure is a (1 — > 3)-/3-glucan
consisting of five (1 -> 3)-/6-glucose residues in a linear linkage and two
(1 6)-/J-glucopyranoside branches in side chains which result in a right-handed
triple-helical structure (Chihara etal., 1989)]. Another highly potent antitumor
polysaccharide, schizophyllan from S. commune, is also a (1 3)-/6-glucan
having a /J-glucopyranosyl group linked 1 6 to every third or fourth residue of
the main chain. It is similar to lentinan in its triple-helix structure and biological
activity but physicochemically unlike lentinan (Komatsu et al., 1969; Ohno et al.,
1995).

More recent additions to the list of antitumor polysaccharides that are widely
used include /6-D-glucans isolated from G.frondosa, A. blazei, S. crispa, P. linteus,
and many others. A bioactive /3-glucan extracted from the maitake mushroom has
a cytotoxic effect on prostatic cancer cells in vitro, leading to apoptosis (Fuller-
ton etal., 2000). A D-fraction, a (1 — > 3)-branched (1 — > 6)-/J-glucan extracted
from the fruiting bodies of G. frondosa, has strong antiproliferative activity against
human prostatic cancer PC-3 cells in vitro and shows a synergistic potentiation
when coadministered with vitamin C or an anticancer agent carmustine (BCNU)
(Konno et al., 2002). It can activate macrophages, dendritic cells, and T cells and
inhibit the growth of tumor cells. The D-fraction enhances the cytotoxicity of natu-
ral killer (NK) cells through the production of interleukin (IL) 12 by macrophages
activated by D-fraction (Kodama et al., 2005a). A 21-kDa heteropolysaccharide,
coded as GFPS 1 b, obtained from the cultured mycelia of G. frondosa exhibits more
potent antiproliferative activity on MCF-7 cells than other polysaccharide frac-
tions. GFPS lb was an acidic polysaccharide with a backbone consisting of (1 — >
4)-a-linked D-galacopyranosyl and (1 — > 3)-a-linked D-glucopyranosyl residues
(Cui et al., 2007).

Agaricus blazei is an edible mushroom that is widely considered to be medically
important. Polysaccharide fractions prepared from cultured A. blazei mainly show
antitumor activity against the solid form of sarcoma 180 in ICR mice. The highly
branched (1 — > 3)-/3-glucan segment forms the active center of the antitumor activ-
ity (Ohno et al., 2001; Chung et al, 2005). A (1 -> 6)-/6-D-glucan extracted from



ANTITUMOR POLYSACCHARIDES FROM MUSHROOMS (HIGHER FUNGI) 151

A. blazei has cytotoxic effect against human ovarian cancer HRA cells in vitro,
promoting p38 MAPK activity for suppressing HRA cell proliferation and ampli-
fying the apoptosis cascade. In mice, oral supplementation with /2-glucan reduces
pulmonary metastasis of 3LL cells and peritoneal disseminated metastasis of HRA
cells and inhibits the growth of these metastatic tumors in lung or peritoneal cav-
ity (Kobayashi et al., 2005). The mycelium polysaccharide and exopolysaccharide
(EPS) of Agaricus brasiliensis also demonstrate a strong antitumor action against
the solid form of sarcoma 180 in ICR mice (Fan et al., 2007).

Sparassis crispa Fr., an edible mushroom recently cultivable in Japan, contains
a remarkably high content of 6-branched (1 — > 3)-/J-D-glucan (SCG) showing
antitumor activity (Ohno et al., 2000). The addition of recombinant murine
granulocyte-macrophage colony-stimulating foctor (rMuGM-CSF) to spleen cell
cultures from various strains of mice synergistically enhanced interferon gamma
(IFN-y), tumor necrosis factor alpha (TNF-a), and IL-12p70 in the presence
of SCG (Harada et al., 2002, 2004). Acidic polysaccharide (PL) isolated from
P. linteus exhibits antitumor activity against B16 melanoma cells in a
dose-dependent manner through the up-regulation of nitric oxide (NO) and
TNF-a production, suggesting that PL acts as an effective immunomodulator that
enhances the antitumor activity (Kim et al., 2004a). Protein-bound polysaccharide
from P. linteus induces G2/M phase arrest and apoptosis in SW480 human colon
cancer cells (Li et al., 2004).

Ganoderma lucidum and other related Ganoderma species, including
G. lucidum, G. tsugae, G. capense, and G. applanatum, are the most well known
medicinal fungi in the Orient. The extract of G. lucidum (LZE) has antitumor activ-
ity with proapoptotic and anti-inflammatory functions, as well as inhibitory effects
on cytokine expression during early inflammation in colonic carcinoma cells
(Hong et al., 2004). It exerts its effect on cancer cells by multiple mechanisms and
suppresses the growth of breast cancer cells through the inhibition of Akt/nuclear
factor kappa B (NF-/cB) signaling pathway (Jiang et al., 2004a). Various antitumor
polysaccharide components such as /3-glucan, glucuronoglycan, mannoglucan,
and other active heteroglycans as well as polysaccharide-protein complexes have
been isolated and purified from Ganoderma species for medicinal use (Mizuno
etal., 1995b; Ooi and Liu, 2000; Gao etal., 2004). GLP, a polysaccharide
fraction isolated from fruiting bodies of G. lucidum, significantly suppresses in
vivo the growth of sarcoma 180 solid tumor. GLP induces a marked increase
in the expression levels of IL-la (two fold), IL-1/3 (3-fold), TNF-a (2-fold),
IL-12 p35 (up to 6-fold), and IL-12 p40. In the macrophages, GLP promotes a
remarkable increase in the expression levels of IL— 1/3 (2.5-to 3-fold), TNF-a (up
to 6-fold), and macrophage colony-stimulating factor (M-CSF) (up to 2-fold) (Ooi
et al., 2002). Ganopoly, the refined polysaccharide extracted from G. lucidum,
exhibits antitumor potential with a broad spectrum of immunomodulating
activities, including significantly increased cytotoxic T-lymphocyte cytotoxicity
and NK cell activity in mice (Gao etal., 2005). One /3-glucan, named Lzps-1
(MW 8000), obtained from G. lucidum spore has antitumor activity against
sarcoma 180 and Lewis lung cancer in mice and enhances the NK cell activity



152 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

(Jiang et al., 2005). Ganoderan (MW 20,000), an immunomodulatory /3-glucan of
G. lucidum, induces potent antitumor immunity in tumour-bearing mice (Han
et al., 1995). Several /3-glucans, heteroglycans, and glycan-protein complexes
having high antitumor activity have been isolated from the fruiting body and
mycelium of G. applanatum, with the molecular weight of the primary polysac-
charides ranging from 30 x 10 3 to 1000 x 10 3 , and the basic chemical structure
is (1 — > 3)-/6-glucopyranan having 1-15 (1 — > 6)-monoglucosyl side chains. It
seems that the greater the molecular weight and the higher the water solubility of
these polysaccharids, the higher the antitumor activity (Mizuno, 1995). Among
the seven strong antitumor polysaccharide-protein complexes from Ganoderma
tsugae, two are identified as protein-containing glucogalactans associated with
mannose and fucose, and five are protein-containing (1 3)-/6-glucans. It is
noteworthy that antitumor polysaccharides having high activity from fruiting
bodies are mostly heteropolysaccharides with molecular weight of about 10,000
and consisting of galactose, glucose, mannose, and fucose, whereas the highly
active polysaccharides from mycelia are mainly protein-containing glucans with
molecular weight of 10,000 (Zhang et al., 1994a; Mizuno et al., 1995c; Gao et al.,
2004; Peng et al., 2005). The active polysaccharides isolated from the fruiting
bodies and mycelia of these three Ganoderma species are markedly different in
their component monosaccharides, their protein moiety content, and their average
molecular weight.

A highly branched (1 — > 3)-/6-glucan with a pentasaccharide segment
consisting of one nonreducing terminus on 3,6-O-substituted and three
3-mono-O-substituted /3-glucopyranosyl side chains isolated from Pleurotus
ostreatus was reported to have strong antitumor activity (Yoshioka et al., 1985).
A newly identified low-molecular-weight a-glucan from the same mushroom
exhibits antiproliferative and proapoptotic activities against HT-29 colon cancer
cells in vitro via the induction of programmed cell death (Lavi et al., 2006). Sev-
eral potent antitumor polysaccharide-protein complexes have been purified from
fruiting bodies of a Chinese edible mushroom, Pleurotus sajor-caju, including
(a) protein-containing xyloglucan (MW 280,000) with polysaccharide-protein
ratio 76 : 24 (w/w); (b) protein-containing mannogalactan (MW 120,000) with
polysaccharide-protein ratio 76 : 16; (c) protein-containing xylan (MW 200,00)
with polysacchairde-protein ratio 62 : 21; (d) protein-containing glucoxylan (MW
90,000) with polysaccharide-protein ratio 71 : 15; and (e) protein-containing
xyloglucan (MW 70,000) with polysaccharide-protein ratio 69 : 3 (Zhuang et al.,
1993). Another interesting edible mushroom, Pleurotus tuber-regium (PTR), exists
as a stage of mycelium, fruiting body, or sclerotium. The water-soluble nonstarch
polysaccharides (NSPs) extracted from the fruiting body, mycelium, and culture
medium (coded as HWE, EDP, and CEP, respectively) of P. tuber-regium have
been shown to exert antiproliferative activity through the induction of apoptosis
in HL-60 cells with an increase in the ratio of Bax/Bcl-2 (Wong et al., 2007).
The mechanism for the antitumor activity of a water-soluble carboxymethylated
/3-glucan (CMPTR), partially synthesized from an insoluble native glucan
isolated from the sclerotia of P. tuber-regium, is related to cell cycle arrest



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 153

and apoptosis induction in human breast carcinoma MCF-7 breast cancer cells
in vitro (Zhang et al., 2006a). An immunomodulating polysaccharide isolated
from the aqueous extract of Pleurotus florida fruiting bodies exhibits significant
macrophage activity through the release of nitric oxide (Rout et al., 2004).
Three polysaccharides, namely, (1 — > 3)-/?-D-glucan, (1 — > 6)-/J-D-glucan, and
(1 — > 3, 1 — > 6)-/3-D-glucan, purified from fruiting bodies of Lyophyllum decastes
Sing., a newly cultivated mushroom in Japan, show marked antitumor activity
against sarcoma 180 (Ukawa et al., 2000). EA6 (a protein-bound polysaccharide)
isolated from the culinary-medicinal mushroom Flammulina velutipes augments
the antitumor immunity in combination with surgical excision (SE) in Meth-A
fibrosarcoma-bearing mice, and the effect is mediated by CD4-positive T cells
(Maruyama and Ikekawa, 2005).

The complete list of the antitumor polysaccharides and polysaccharide-protein
complexes from mushroom fungi are presented separately in Tables 5.1 and 5.2.



5.3 MECHANISMS OF ANTITUMOR ACTION OF
MUSHROOM POLYSACCHARIDES

Numerous polysaccharides or polysaccharide-protein complexes from mush-
rooms have been identified and shown to have antitumor activities (Ooi and Liu,
2000b; Wasser, 2002; Zhang et al, 2007). Although a complete answer to the
mechanisms of antitumor action of polysaccharides or polysaccharide-protein
complexes is not yet available, they are generally known as biological response
modifiers which are able to restore or enhance various immune responses in vivo
and in vitro. Their actions are predominantly considered to be host mediated.
However, many of these macromolecules have been documented to also possess
direct cytotoxic effects on cancer cells. It is possible that, in some instances,
these two types of inhibitory action may be interwoven. Therefore, the possible
modes of anticancer action may include both (1) direct cytotoxicity to cancer cells
as shown in many in vitro studies and (2) indirect antitumor inhibition through
immunomodulation of the body defense system.

5.3.1 Anti proliferation of Cancer Cells and Induction of Apoptosis

Various glucans such as lentinan (yS-glucan of L. edodes) show no direct
growth-inhibitory effects on tumor cell lines in vitro (Ooi and Liu, 2000b; Wasser,
2002). However, the indirect cytotoxicity of lentinan is observed to enhance the
antitumor cytotoxic activity of peritoneal macrophages against human melanoma
target cells in vitro (Ladanyi et al., 1993). The activity of lymphokine-activated
killer cells (LAK) stimulated by IL-2 and lentinan against autologous tumour cells
and K562 human erythroleukemia cells is greater than that stimulated by IL-2
alone in vitro showing the augmentation of cytotoxicity of LAK by lentinan (Tani
etal., 1993). When the water-insoluble a-(l — > 3)-D-glucan (L-FV-II) isolated
from fruiting bodies of L. edodes is chemically modified to the water-soluble



154



ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES



TABLE 5.1 Antitumor Polysaccharides from Mushrooms



Mushroom
Species



Polysaccharide
Component



References



Agaricus blazei



Agaricus brasiliensis

Agrocybe

cylindracea
Amanita muscaria

Armillariella
tabescens
Auricularia auricula

Collybia dryophila

Cordyceps sinensis
Cryptoporus volvatus
Dictyophora

indusiata
Flammulina

velutipes
Ganoderma lucidum

Ganoderma tsugae

Grifora frondosa



Grifora umbellata

Hericium erinaceus
Hypsizigus

marmoreus
Lyophyllum decastes
Lentinus edodes

Omphalia

lapidescens
Phellinus linteus



Homoglucans
(1 4)-a- and (1 6)- ,6-Glucan

1 — >�� 6)- a- and (1 -»• 4)-a-Glucan
1 -> 6)- 0- and (1 -> 3)- / 6-Glucan

1 6)- P- and (1 3)- / 6-Glucan

1 -»• 3)-/S-Glucan (fruiting body)

1 ->• 3)-a-Glucan

1 3)-/S-Glucan (fruiting body)

1 ->• 3)-a-Glucan

1 -»• 3)-/S-Glucan (fruiting body)

1 -»• 3)-/S-Glucan (fruiting body)

Cordyglucan (1 — >• 3)-/S-glucan
1 ->• 3)-/S-Glucan (fruiting body)
1 ->• 3)- /i-Glucan (fruiting body)



3)-/S-Glucan (fruiting body)



Ganoderan [(1 -> 3)-/S-glucan]
1 ->• 3)-/S-Glucan (spore)
1 ->• 4)-a- and (1 ->• 3)-/S-Glucan

(mycelium)
Grifola (1 ->• 3)- ( 6-D-Glucan
1 3)-/3-Glucan (fruiting body,

mycelium, medium product)
1 ->• 3)-j8-Glucan (sclerotium)

1 ->�� 3)-,S-Glucan

1 ->• 3)-/S-Glucan (fruiting body)

1 -s- 6)- (6-Glucan

Lentinan, (1 -> 3)-/6-glucan (fruiting

body, mycelium)
1 ->• 3)-/S-Glucan (fruiting body)

1 3)-j8-Glucan (fruiting body)



Fujimiya et al., 1998;
Kobayashi et al., 2005
Mizuno et al., 1998
Mizuno et al., 1990a;
Chung et al., 2005
Camelini et al., 2005;
Angeli et al., 2006
Kihoetal., 1989;
Yoshida et al., 1996
Kihoetal., 1994
Kiho etal., 1992a
Kihoetal., 1992b

Misaki and Kakuta,
1995

Pacheco-Sanchez et al,

2006
Yalin et al., 2005
Kitamura et al, 1994
Haraetal., 1991

Smiderle et al., 2006;
Leung and Fung, 1997
Han etal., 1995
Bao et al., 2001
Peng et al., 2005

Zhuang et al., 1994b
Kodama et al, 2002

Ogawa and Kaburagi,

1982
Dong et al., 2006
Ikekawa et al., 1992

Ukawa et al., 2000
Chihara et al, 1970b;
Surenjav et al., 2005
Ohno et al., 1993;
Saito, etal., 1992
Kim et al., 1996



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 155



TABLE 5.1 (Continued)



\A 1 1 q Vi mom


Po 1 v q ?i r* c h t\ ri H p

_F Ulj ftLlCCll£lllLlC






Pnninfitipnf
1 1 1 yj \j i iwi 1 1


R P fi P T*P TI P P Q


Pleurotus eryngii


(1 -> 3)-/S-Glucan


Carbonero et al., 2006


Pleurotus florida


(1 -»• 3)-/S-Glucan


Rout et al., 2005


Pleurotus


(1 -s- 3)-j6-Glucan


Carbonero et al., 2006


ostreatoroseus






Pleurotus


(1 3)-^-Glucan


Gutierrez et al., 1996


pulmonarius






Pleurotus


(1 ->�� 3)-^-D-Glucan (Sclerotium)


Zhang etal., 2003;


tuber-regium




Tao and Zhang, 2006


Polyporus confluens


(1 — »• 3)-/3-D-Glucan (fruiting body)


Mizunoetal., 1992


Poria cocos


Pachyman, (1 — »• 3)- j 6-D-glucan


Kanayama et al., 1986


Porodisculus


/i-Glucan (medium products)


Ogawa and Kaburagi,


pendulus




1982


Sclerotinia libertiana


/J-Glucan (medium products)


Ogawa and Kaburagi,






1982


Sclerotium sclerotia


Scleroglucan, (1 -»• 3)- / S-glucan


Palleschi et al., 2005


Schizophyllum


Schizophyllan, (1 -»• 3)-/J-glucan


Komatsu et al, 1969;


commune




VJgaWa dllLl JVaDUiagl,






1982


Sparassis crispa


(1 -> 3)-£-Glucan


Ohno et al., 2000;






Harada et al 2006


lei flLlLUfftyCCCt


^J. ?" .Jy'-L'-vJILlCd.ll


PhaVrnhnrtv pt nl 9006

V^llajvlaUUl Ly CL al., Z.UVJU








TvfitYiPtPK vihho'ifl

J 1 LI 1 1 1 C- l C l> Cil/(/l/J l*


/3-G1ucan ffniitinp bodv^

j_y v j i ui w cm \ii ui 1 1 1 1 > — . uuu y j


("Varnpcki and Givvbpk

V^ZjCU. UvvlVI till Vj V J 1 ' - V I ' ^ IV .






1995


Trichn In m n

�� i n,i us us it in


(\ — v 'W-R-CWwcnxx (Triiitiiicr hnH\A


TVIi7iinn pt al 1 005a


gigQyiteutn






Tylopilus f elicits


jf3-Glucan (fruiting body)


Grzybek et al.,1990;






l^/^hl tyi iin 7ai" /if q1 1 QQ(i
JVUI11111U11Z,C1 CL d.1., 177U


Visl vlli IcllLl VULVClCcti


-VJlUCall ^llUlllllg UUUj 1 )


KkhiHa Pt al 1 080

JVlMllUd CL d.1., 1707








Agaricus blazei


Mannogalactoglucan


Cho et al., 1999




Riboglucan


Cho et al., 1999


Flammulina


Galactomannoglucan


Ikekawa et al., 1982


velutipes






Fomitella fraxinea


Mannogalactoglucan


Cho et al., 1998


Ganoderma lucidum


(1 3)-/S-Glucuronoglucan


Saito et al., 1989


Ganoderma tsugae


Arabinoglucan


Zhang etal., 1994a


Grifola frondosa


Mannofucoxyloglucan


Zhuanget al., 1994b;






Mizuno and Zhuang,






1995




Xyloglucan


Mizuno et al., 1986


Hericium erinaceus


Galactoxyloglucan


Mizunoetal., 1992b;



Wang et al., 2004



156 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES



TABLE 5.1 (Continued)



Mushroom
Species


Polysaccharide
Component


References


Hohenbuehelia


Galactomannoglucan


Ma etal., 1991


serotina






Inonotus obliquus


Xylogalactoglucan


T. Mizuno et al., 1999


Leucopaxillus


Galactomannoglucan


Mizuno et al., 1995


giganteus






Pleurotus


Mannogalactoglucan


Gutierrez et al., 1996


cornucopiae






Pleurotus


Mannogalactoglucan


Gutierrez et al., 1996


pulmonarius


Xyloglucan


Gutierrez et al., 1996


Polyporus confluens


Xyloglucan


Mizuno et al., 1992


Tremella fuciformis


(1 3)-,S-Mannoglucan


Gaoetal., 1996




Heteroglycans




Agariscu bisporus


Heterogalactan


Shida and Sakai, 2004


Agaricus blazei


(1 ->• 2)-p- and


M. Mizuno et al., 1999




(1 3)-/J-Glucomannan






Glucoxylan (fruiting body)


Mizuno et al., 1990b




Heterogalactan


Shida and Sakai, 2004


Collybia maculata


Galactomann


Lim et al., 2005


Dictyophora


Fucomannogalactan


Haraetal., 1991


indusiata






Flammulina


Xylomannan


Smiderle et al., 2006


velutipes


Heterogalactan


Shida and Sakai, 2004


Fomitella fraxinea


(1 -*�� 6)-a-Mannofucogalactan


Cho et al., 1998


Ganoderma tsugae


Glucogalactan


Wang et al., 1993


Grifola frondosa


Mannogalactofucan


Zhuang et al., 1994a




Heterogalactan


Shida and Sakai, 2004


Hericium erinaceus


Xylan


Mizuno et al., 1992b




Rhamnoglucogalactan


Jia et al, 2004




Glucogalactan


Wang et al., 2004




Mannoglucoxylan


Mizuno et al., 1992b




Fucogalactan


A. Zhang et al., 2006;






Shida and Sakai, 2004


Hypsizigus


Heterogalactan


Shida and Sakai, 2004


marmoreus






Inonotus obliquus


a-Linked fucoglucomannan


Kim et al., 2006


Lampteromycer


Mannan (fruiting body)


Fukuda et al., 1975


japonicus






Lentinus edodes


Galactoglucomannan


Fujii et al., 1978


Pleurotus


Arabinogalactan


Zhang et al., 1994b


citrinopileatus






Pleurotus eryngii


Heterogalactan


Shida and Sakai, 2004



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 157



TABLE 5.1 (Continued)



Mushroom


Pol vsaccharide




Species


Component


References


Pleurotus ostreatus


Heterogalactan


Shida and Sakai, 2004


Pleurotus sajor-caju


Galactoglucomannan


Pramanik et al., 2005


Polyporus confluens


,6-Glucopyranan (mycelium)


Mizunoetal., 1992a


Sarcodon aspratus


Fucogalactan


Mizuno et al., 2000


Tremella fuciformis


jS-Glucuronoxylomannan (fruiting


Misaki and Kakuta,




body)


1995;






Yuietal., 1995;






Gao et al., 1996


Tremella mesenterica


/i-glucuronoxylomannan


Wasser et al., 2002;






Vinogradov et al, 2004


Tricholoma


Xyloglucomannan (fruiting body)


Mizuno et al., 1995a


giganteum







sulfated a-(l -> 3)-D-glucan (SL-FV-II), it has potent antiproliferation action
(52%) on human MCF-7 breast carcinoma cells (Zhang and Cheung, 2002).

Moreover, a (1 — ► 3)-/6-glucan extracted from the maitake mushroom (G.fron-
dosa), known as D-fraction, demonstrates a direct cytotoxicity (at a dosage of
480 mg/mL) to prostatic cancer PC-3 cells, inducing nearly complete cell death
(>95%) in 24 hours, and has a synergistic potentiation when it is coadministered
with vitamic C or an anticancer agent carmustine (BCNU), resulting in a drastic
reduction in cell viability (Fullerton et al., 2000; Konno et al., 2002). GFPSlb, a
21-kDa heteropoly saccharide obtained from the cultured mycelia of G. frondosa,
exhibits more potent antiproliferative activity on MCF-7 cells than other polysac-
charide fractions (Cui et al., 2007). Phellinus linteus polysaccharides (PLs) render
murine or human lung cancer cells susceptible to apoptosis, and in the process
of PL-induced apoptosis, caspase 2 is induced in LNCaP cells, which express the
androgen receptor (AR), but not in PC-3 cells, which lack AR, demonstrating the
AR-dependent and independent apoptotic pathways (Zhu et al., 2007). PL has a
synergistic effect with doxorubicin (Dox) to activate caspases in prostate cancer
LNCaP cells, suggesting that PL has therapeutic potential to augment the magni-
tude of apoptosis induced by antiprostate cancer drugs (Collins et al., 2006).

A newly identified low-molecular- weight a-glucan of P. ostreatus has
promising antitumorigenic properties and demonstrates its direct effect on HT-29
colon cancer cell proliferation via the induction of programmed cell death (Lavi
et al., 2006). A comparative study shows that the water-soluble NSPs extracted
from the fruiting body, mycelium, and culture medium (coded as HWE, EDP,
and CEP, respectively) of a novel edible mushroom PTR can induce apoptosis
in HL-60 cells with an increase in the ratio of Bax/Bcl-2 (Wong et al., 2007).
Among all PTR NSPs, HWE (a heteropolysaccharide-protein complex from
the fruiting body, MW1.86 xlO 6 ) has the strongest antiproliferative activity in



158



ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES



TABLE 5.2 Antitumor Polysaccharide-Protein Complexes from Mushrooms



Mushroom Species



Active Component



References



Agaricus blazei



Armillariella
tabescens
Collybia confluens

Cordyceps

ophioglossoides
Coriolus (Trametes)

versicolor



Flammulina
velutipes
Fomes fomentarius

Fomitella fraxinea



Ganoderma lucidum
Ganoderma tsugae



Grifola frondosa
Hebeloma

crustuliniforme
Lentinus edodes
Laetiporus

sulphureus
Phellinus linteus

Pleurotus

citrinopileatus
Pleurotus ostreatus
Pleurotus sajor-caju



(1 -»• 6)- / 6-D-Glucan-protein
complex



Polysaccharide-protein complex

(ATOM)
Xyloglucan-protein
Protein-containing heteroglycan

(fruiting body)
Protein-bound polysaccharide

(mycelium)
Protein-bound polysaccharide

(medium product)
PSK, protein-bound

polysaccharide (mycelium)
PSP, polysaccharide-peptide

complex (mycelium)

Protein-bound glucan (fruiting

body, mycelium)
Protein-containing polysaccharide

(medium product)
Protein-containing

galactomannoglucan (fruiting

body)
Proteoglycan
Proteoglycan

(1 — > 3)- / 8-Glucan-protein

complex (mycelium)
Glucogalactan-protein complex

(mycelium)
Heteroglycan-protein complex
Polysaccharide-protein complex

Polysaccharide-peptide complex
Protein-polysaccharide (fruiting
body)

Polysaccharide-protein complex

Protein-containing heteroglycan

(fruiting body)
Proteoglycan

Heteroglycan-protein complex
(fruiting body)



Mizuno et al., 1990b;
Kawagishi et al., 1990;
Gonzaga et al, 2005;
Hong and Choi, 2007
Ito et al., 1997

Mizuno etal., 1990b
Kihoetal., 1992b

Kim et al., 1993

Ohmori et al., 1988a,

Tsukagoshi et al., 1984;
Kanazawa et al., 2005
Yang et al., 1992;
Wang et al., 1996a;
Ho et al., 2004
Ikekawa et al., 1982;
Ohkumaetal., 1982
Ito et al., 1976

Choetal., 1995



J. Zhang et al, 2002
Baek et al., 2002
Wang et al., 1993

Zhang etal, 1994a;
Peng et al., 2005
Zhuang et al., 1994a
Cho and Chung, 1999

Liu etal., 1998
Gasiorowski et al, 1993

Songet al., 1995;
Kim et al, 2006
Zhang etal., 1994b

Sarangi et al., 2006
Zhuang et al, 1993



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 159



TABLE 5.2 (Continued)



Mushroom Species


Active Component


References


Polyporus confluens


Xyloglucan-protein (fruiting


Mizuno et al., 1992a




body)




Tremella fuciformis


Heteroglycan-protein (mycelium)


Cho et al., 2006


Tricholoma


Polysaccharide-protein complex


Mizuno et al., 1995a


giganteum


(fruiting body)




Tricholoma


Polysaccharide-protein complex


Liu and Ooi, 1995;


lobayense


(PSPC) (medium product)


Liu et al., 1996a;






Liu et al., 1996b


Tricholoma


a-Glucona-protein complex


Hoshi et al., 2005


matsutake






Tricholoma


Polysaccharide-peptide complex


Wangetal., 1996b


mongolicum


(mycelium)





vitro against human acute promyelocytic leukemia cells (HL-60) whereas CEP
(a mannose-rich polysaccharide, MW 44,000) has the least cytotoxicity. EDP (a
glucose-rich polysaccharide from the mycelium of PTR, MW 509,000) can cause
G2/M arrest in HL-60 cells by lowering the Cdkl expression. HWE exerts S-phase
arrest in the HL-60 cells by a depletion of Cdk2 and an increase in cyclin E
expression (Wong et al., 2007). CMPTR, partially synthesized from an insoluble
native glucan isolated from the sclerotia of PTR, can inhibit the cell proliferation
of MCF-7 by arresting the G phase of its cell cycle, which is associated with the
down-regulation of cyclin D- 1 and cyclin E expressions in breast cancer cells. In
addition, CMPTR-treated MCF-7 cancer cells exhibit a decreased expression of
antiapoptotic Bcl-2 protein and an increased expression of Bax/Bcl-2 ratio (Zhang
et al., 2006a).

A neutral polysaccharide fraction isolated from Poria cocos (PC) shows a potent
activity in suppressing the proliferation of human leukemic cells, U937 and HL-60
cells, and induces more than 50% of U937 cells and HL-60 cells to differen-
tiate into mature monocytes/macrophages, which also markedly express surface
antigens of CDllb, CD14, and CD68 (Chen and Chang, 2004). A recent study
provides the preliminary insights into the mode of direct antitumor activity in vitro
of a yS -glucan from the mycelium of PC on human breast carcinoma MCF-7 cells
via cell cycle arrest and apoptosis induction (Zhang et al., 2006).

Unlike most glucans, polysaccharide-protein complexes (PSK or PSP) from
C. (T. ) versicolor and other mushrooms usually have both direct and indirect cyto-
toxic effects on tumor cell lines. Coriolus versicolor (CV) extract is able to selec-
tively and dose dependently inhibit the proliferation of lymphoma and leukemic
cells possibly via an apoptosis-dependent pathway (Lau et al., 2004). The CV
extract exerts antiproliferative activity through the induction of apoptosis, dif-
ferentially dependent of p53 and Bcl-2 expression in human breast cancer cells
(Ho et al., 2005). The protein-bound polysaccharide of C. versicolor (PSK) could



160 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

suppress cell proliferation and induce subsequent cellular apoptosis in the Burkitt
lymphoma cell line (Namalwa), out of 33 hematological malignant cell lines tested,
indicating the initial evidence of the direct cytotoxic activity of PSK in a can-
cer cell line (Hattori et al., 2004). PSK could also inhibit cell growth and DNA
synthesis in various cell lines such as L1210 leukemia, P388 leukemia, Ehrlich
carcinoma, Yoshida sarcoma, AH- 13, human hepatoma CHC-20, human chorio-
carcinoma GCH-1 and GCH-2, and human breast cancer cell MCF-7 (Tsukagoshi
et al., 1984). Similarly, the polysaccharide peptide of C. versicolor (PSP) extracted
from a strain of C. versicolor has a wide range of antitumor activities in vitro,
inhibiting Ehrlich ascites tumor, leukemia P388, sarcoma 180, and four types of
human cancer cell lines, namely human gastric cancer cells, human lung cancer
cells, mononuclear leukemia cells, and human skin histiocytic lymphoma cells
(Yang et al., 1992; Cui and Chisti, 2003). PSP is also effective in inhibiting cell
proliferation through apoptosis. Cells treated with PSP show a significant reduc-
tion in cell proliferation with the induction of apoptosis via the up-regulation of
p21 and down-regulation of cyclin D-l (Chow et al., 2003). PSP, used in com-
bination therapy, has the ability to lower the cytotoxicity of certain antileukemic
drugs through their interaction with cell cycle -dependent and apoptotic pathways.
Induction of S-phase cell arrest and caspase activation by PSP of C. versicolor
enhances the cell cycle-dependent activity and apoptotic cell death of doxoru-
bicin and etoposide but not cytarabine in HL-60 cells (Hui et al., 2005). PSK, PSP,
and most /J-D-glucans such as lentinan show different modes of antitumor action
in vitro (Sakagami et al., 1991; Ooi and Liu, 2000).

Protein-bound polysaccharide from P. linteus induces G2/M phase arrest and
apoptosis in SW480 human colon cancer cells (Li et al., 2004). The antitumor
polysaccharide-protein complex (PSPC) purified and characterized from the
culture filtrates of Tricholoma lobayense exhibits strong antitumor activity in
both ICR and BALB/c mice. PSPC has the ability to restore the phagocytic
function of the peritoneal exudate cells (macrophages) and the mitogenic activity
of T cells of tumor-bearing mice (Liu and Ooi, 1995; Liu et al., 1996b). PSPC
also exerts indirect cytotoxic activity against P815 mastocytoma cells and L929
mouse fibroblast cells by activating macrophages to release reactive nitrogen
intermediates (RNIs) and TNF-a, which are shown to increase significantly after
PSPC treatment in the tumor-bearing mice (Liu and Ooi, 1995). PSPC both
induces the various immune responses in vivo and exhibits cytotoxicity against
tumor cell lines in the presence of PSPC in vitro (Liu et al., 1996b). Similarly,
the antitumor activity of ATOM, a polysaccharide-protein complex prepared
from the cultured mycelia of A. blazei, is highly effective against four kinds
of established tumors in mice, namely subcutaneously implanted sarcoma 180,
Ehrlich ascites carcinoma, Shionogi carcinoma 42, and Meth A fibrosarcoma
(Itoh et al., 1994, 1997). Heteroglycan-protein complexes from G. frondosa
are shown to depress tumor growth by activating the immune system as a
biological response modifier (Cun et al., 1994). The heteropolysaccharide-protein
complexes (mainly (1 — > 3)-a-D-glucan-bound protein with the presence of
mannose and galactose) from P. cocos mycelia exhibit significant cytotoxic



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 161

effect on the proliferation of HL-60 cells in vitro and the antitumor activity
against sarcoma 180 in vivo (Jin et al, 2003c). Among all PTR NSPs, HWE (a
heteropolysaccharide-protein complex, MW 1.86 xlO 6 ) exerts the strongest
anti-proliferative activity in vitro against human acute promyelocytic leukemia
cells (HL-60) and causes S-phase arrest in the HL-60 cells by a depletion of
Cdk2 and an increase in cyclin E expression (Wong et al., 2007). It is thus
suggested that polysaccharide-protein/polysaccharide-peptide complexes of
many mushrooms may have some unique structural features, possibly generated
from the involvement of protein portions and/or unique structural configurations,
including sugar to sugar linkages, that contribute to their immunomodulatory and
cell cycle -dependent antitumor actions as well as direct cytotoxicity (Ooi and
Liu, 2000b).

5.3.2 Immunomodulation

Immunomodulators are substances from a variety of sources which have the
ability to augment the immune system in multiple ways. They are often considered
pharmacologically as BRMs and have been reported to have antitumor activity
among many biopharmacological properties. The most prominent BRMs are
polysaccharide BRMs, especially (1 — > 3)-/3-D-glucans from mushrooms (higher
fungi), which occur widely in nature (Bohn and BeMiller, 1995; Ooi and Liu,
2000b; Wasser, 2002; Lull et al, 2005).

Many mushroom polysaccharides or polysaccharide-protein complexes have
distinct antitumor activities in murine allogeneic, syngeneic, and autochthonous
hosts (Tsukagoshi et al, 1984; Bohn and BeMiller, 1995). The preliminary
determination of antitumor activity has often relied on a bioassay system normally
based on an allogeneic tumor in mice. There are a number of factors such as
strain of mice, type of tumor, suitable dosage, and strictly planned timing of drug
administration that are essential to achieve the antitumor effect of polysaccharides
(Chihara, 1992; Kerekgyarto et al., 1996). Hundreds of polysaccharides or
polysaccharide-protein complexes have been screened for their antitumor
activity, and three of them, namely schizophyllan, lentinan, and protein-bound
polysacharides (PSK and PSP), have been used widely for more than 30 years
(Chihara, 1992; Yang et al., 1992).

Several polysaccharides such as lentinan (/6-glucan) and PSK (/J-glucan-
protein) have been shown to have effective antitumor action against a variety of
transplantable experimental animal tumors and have been successfully used in
clinical treatments (Chihara, 1992; Kobayashi et al., 1993). The enhancement
or potentiation of host defense mechanisms has been recognized as a possible
means of inhibiting tumor growth without harming the host. The body's defense
against spontaneously arising malignant tumors involves a concerted interplay
of innate and acquired immune responses. Innate immunity comprises cellular
elements such as macrophages, cytotoxic lymphocytes, NK cells, and dendritic
cells (DCs). Both cell-mediated immune response against the target cells initiated
by macrophage -lymphocyte interactions and cytotoxicity induced by antibodies



162 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

to target cells are believed to contribute to the elimination of target tumor cells
(Chihara et al., 1989). This immune system is regulated by chemical mediators
or cytokines and by activation of inflammatory responses. Mushroom polysac-
charides can activate macrophages or NK cells to produce various cytokines such
as interleukins, interferons, and other mediators so that they are targeted toward
destroying tumor cells (Chihara, 1992). NK cells have two relevant functions
related to the natural immune response against pathogens or tumor cells: (1)
cytotoxicity that is mediated by the recognition and lysis of target cells, such
as virus- and bacteria-infected or tumor cells, and (2) production of cytokines
that can modulate natural and specific immune responses. Furthermore, DCs
and macrophages activated by D-fraction produce cytokines such as IL-12 that
stimulate NK cells to rapidly produce other cytokines (including IFN-y, TNF-a,
and GM-CSF) and enhance the cytotoxicity of NK cells (Kodama et al., 2002,
2005a).

The detailed mechanisms of action of polysaccharide immunomodulators or
BRMs are not yet fully known, but it is generally accepted that they act on differ-
ent immunocompetent cells which may initiate a cascade of signal transduction
pathways that are responsible for the immune responses. The first step of the
polysaccharide BRM in the modulation of cellular activity is the recognition of
BRM and the binding of it to the specific immune cell receptors. Some evidence
shows that there are pattern recognition receptors (PRRs) available for the molecu-
lar reception of polysaccharide BRMs (Lowe et al., 2001). The binding of ligands
to PRRs may initiate Rel/NF-/cB -mediated signaling events, which leads to the
induction of gene expression and specific cellular functions of the innate immu-
nity (Leung et al., 2006). It has been reported that some groups of PPRs can
recognize the polysaccharide BRMs, for example, complement receptor 3 (CR3 or
CDllb/CD18) (Ross et al., 1987), dectin-1 (Zimmerman et al, 1998; Taylor et al.,
2002), and toll-like receptors (TLR-2 and TLR-4) (Shao et al., 2004). CR3 plays
an important role as both membrane /6-glucan receptor and adhesion molecule
and occurs on cell membranes of monocytes, macrophages, neutrophils, NK cells,
and DCs. CR3 may mediate a variety of cellular functions as it has the abil-
ity to bind various ligands such as intercellular adhesion molecule- 1 (ICAM-1),
iC3b, /6-glucan, and others (Thornton et al., 1996; Lowe et al., 2001). ,6-Glucans
can thus mediate cytotoxic and phagocytic responses through CR3 by binding
them to these sites of the immune cells (Moradali et al., 2007). Mueller et al.
(2000) suggests that the triple-helical conformation, molecular weight, and charge
of the glucan polymer may be important determinants in CR3-ligand interaction.
Dectin-1 is the /J-glucan receptor and is mainly expressed on the surface of the
monocytes, macrophages, neutrophils, and DCs (Taylor et al., 2002). The biologi-
cal response transduced by dectin-1 depends on the TLR pathway, for example,
dectin-1 is required to cooperate with TLR-2 for the activation of NF-/cB and
induction of TNF-a (Moradali et al., 2007). A proteoglycan isolated from P. lin-
teus (PL) is shown to induce the phenotypic and functional maturation of DCs
through the TLR-2- and/or TLR-4- mediated NF-/cB signal pathways (Kim et al.,
2004b). TLR-4 is also involved in G. lucidum polysaccharide (GLPS)- mediated



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 163

macrophage activation (Shao et al., 2004). It is noteworthy that a thorough under-
standing of the recognition of mushroom polysaccharides (particularly /J-glucan)
by certain receptors on the immune cells and activation of signal transduction path-
ways appears to be a major challenge of future immunomodulation research.

5.3.2.1 Effects of Mushroom Polysaccharides on Macrophages and
Spleen Cells Mushroom polysaccharides can enhance the natural immune
system through the activation of monocytes/macrophages, splenocytes (including
lymphocytes), NK cells, and DCs. The effects of mushroom polysaccharides on
macrophages have been extensively studied in vitro and in vivo. Macrophages
and spleen cells can be induced by mushroom polysaccharides to release several
cytokines, such as IL-1, IL-1/3, IL-6, IL-8, IL-10, IL-12, IL-18, TNF-a, and
GM-CSF. Some of these cytokines are able to directly promote cytotoxicity
of macrophages. The production of cytokines from the immune cells can be
considered as a key event in the initiation and regulation of an immune response
(Lull et al., 2005).

Ganoderma lucidum polysaccharide (GLP) significantly suppresses in vivo
the growth of sarcoma 180 solid tumor, exhibiting antitumor activity, although
GLP shows no direct antiproliferative effect as evaluated in vitro using several
cancer cell lines, such as breast cancer MCF-7, lung cancer SPC-A, and hepatoma
SMMC-7721 cells. A study on the immunomodulatory action of GLP as eluci-
dated through analyzing the induced expression profile of cytokines in the treated
mice using primers of specific cytokines, total RNA, and reverse-transcription
polymerase chain reaction (RT-PCR) in the male inbred BALB/c mice showed
that 7 out of 17 cytokine mRNAs were detected in the splenocytes and peritoneal
exudate cells (macrophages) from the control and treated mice (Ooi et al., 2002).
Among the seven detectable cytokine genes in the splenocytes, GLP induced
a marked increase in the expression levels of IL— la (2-fold), IL— \fi (3-fold),
TNF-a (2-fold), IL-12 p35 (up to 6-fold), and IL-12 p40. In the macrophages,
GLP promoted a remarkable increase in the expression levels of IL— 1/3 (2.5- to
3-fold), TNF-a (up to 6-fold), and M-CSF (up to 2-fold). These results indicate
that antitumor GLP can induce a cascade of immunomodulatory cytokines, but the
potentiation of their gene expression and interaction seems quite complicated. The
potency of TNF-a induction in macrophage is up-regulated after the challenge of
GLPO (MW< 12,000) more than that of GLPI (MW> 12,000), suggesting that
molecular size might be one of the factors in determining the structure-function
relationship of this polysaccharide (Ooi et al., 2002). Lee et al. (2003) reported
that polysaccharide purified from the mycelium of G. lucidum GLP(AI) is the
major component to show the in vivo antitumor effect on fibrosarcoma growth
in C3H mice. It could stimulate blood mononuclear cells to secrete cytokines,
such as TNF-a and IFN-y, which might induce apoptosis and differentiation in
the treated leukemic U937 and HL-60 cells in vitro. The polysaccharide from
G. lucidum (PS-G) is also reported to enhance phagocytic activity of human
primary neutrophils and neutrophilic phenotype cells differentiated from all-trans
retinoic acidy-treated HL-60 cells. Moreover, chemotactic action of PS-G



164



ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES



requires the activities of phosphatidylinositol 3-kinase (PI3K), p38 MAPK, Src
tyrosine kinases, and protein kinase C (PKC), demonstrating the abilities of PS-G
to enhance neutrophil function in phagocytosis and chemotaxis (Hsu et al., 2003).
It was reported that after the treatment of macrophages with a polysaccharide
from fruiting bodies of G. lucidum, the levels of IL-l/J, TNF-a, and IL-6 were,
respectively, 5.1-, 9.8-, and 29-fold higher than in cultures of untreated cells,
and the release of INF-)/ from T lymphocytes was also greatly enhanced in the
presence of this polysaccharide (Wang et al., 1997). The data suggest that the
immunomodulating effects of G. lucidum polysaccharides include the activation
of macrophages, splenocytes, NK cells, and DCs as well as the production of
cytokines promoting antitumor activity (Lin, 2005).

SCG is a major 6-branched (1 -> 3)-j6-D-glucan isolated from S. crispa Fr. with
strong antitumor activities. The splenocytes from the naive DBA/1 and DBA/2
mice strongly react with SCG to produce IFN-y. The addition of GM-CSF to
spleen cell cultures in the presence of SCG synergistically enhances the produc-
tion of IFN-y, TNF-a, and IL-12p70, which would be significantly inhibited by
neutralizing GM-CSF with anti-GM-CSF monoclonal antibody (mAb). It is con-
cluded that GM-CSF plays a key role in regulating cytokine induction by (1 ->
3)-y6-D-glucan SCG in DBA/2 mice in vitro (Harada et al., 2004). A bioactive (1 ->
3, 1 — > 4)-j6-D-glucan from Collybia dryophila polysaccharide (CDP) strongly
inhibits NO production in activated macrophages in a dose-dependent manner
without affecting cell viability (Pacheco-Sanchez et al., 2006). The inhibition of
NO by CDP is consistent with the decreases in both inducible nitric oxide synthase
(iNOS) protein and mRNA expression, suggesting that CDP exerts its effect by
inhibiting iNOS gene expression. CDP also significantly increases prostaglandin E
[PGE(2)] production in LPS- and IFN-y -induced macrophages when compared to
the control (Pacheco-Sanchez et al., 2007). Three polysaccharides, namely, (1 — >
3)-/6-D-glucan, (1 -> 6)-/6-D-glucan, and (1 3, 1 6)-/3-D-glucan or a mixture
of both polysaccharides, purified from the newly cultivated mushroom Lyophyl-
lum decastes Sing, show marked antitumor activities against sarcoma 180 and
increased number of peritoneal macrophages with the complement (C3) -positive
fluorescent cells in mice treated with (1 3)-/6-D-glucan (Ukawa et al., 2000).

Similarly, a purified polysaccharide fraction extracted from the mycelial culture
and fruiting bodies of A. blazei Murill (ABM) could induce bigger increases in NO
secretion than the others, mainly due to an increase in cytokine mRNAs or NO syn-
thase mRNA (Sorimachi et al., 2001). ABM could also promote TNF-a and IL-8
secretion by macrophages derived from rat bone marrow. Thus ABM contains cer-
tain components which activate macrophages contributing to the immune response
in vitro. Different doses of the polysaccharides of ABM synergically enhance the
antitumor effect of cyclophosphamide (CP) in S-180-treated mice, and its mech-
anism is associated with the induced apoptosis of tumor cells and the increased
expressions of IL-2 by macrophages and suppressive gene P27 (Liu et al., 2006).

Lentinan can restore and augment responsiveness of host cells but has no direct
cytotoxicity against tumor cells. Interestingly, the antitumor activity of lentinan



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 165

is also inhibited by pretreatment with antimacrophage agents. Thus, the various
effects of lentinan are thought to be due to potentiation of the response of precursor
Tcells and macrophages to cytokines produced by certain classes of lymphocytes
after specific recognition of tumor cells. In addition, the induction of a marked
increase in the amounts of cerebrospinal fluid (CSF), IL-1, and IL-3 by lentinan
results in maturation, differentiation, and proliferation of the immunocompetent
cells for host defense mechanisms (Chihara et al., 1989). Lentinan is able to restore
the suppressed activity of helper T cells in the tumor-bearing host to their normal
state, leading to the complete restoration of humoral immune responses (Maeda
et al., 1988). The oral administration of lentinan to AKR mice exerts strong anti-
tumor activity, resulting in the raised level of lymphocyte secretion of cytokines
such as IFN-y, TNF-a, IL-2, and IL-la (Yap and Ng, 2003). Lentinan has been
clinically applied as an antitumor and antimetastatic drug and has been reported to
prevent both chemical and viral carcinogenesis. It is known that lentinan affects the
tumorous vascular system resulting in the induction of hemorrhagic necrosis which
is dependent on T cells in the tumor, (Mitamura et al., 2000). The polysaccharide
L-II consisting of D-glucopyranose purified from the fruiting body of L. edodes
demonstrates the antitumor activity in sarcoma 180-bearing mice mediated by
the induction of T cells and macrophage-dependent immune system responses. It
causes also a significant increase in TNF-a and IFN-y but not in IL-2. It can also
raise NO production and catalase activity in macrophages (Ruan et al., 2005).

Granulocytes/macrophages seem to be the major target cell type responsive to
PG101 (a water-soluble extract from Lentinus lepideus) in irradiated mice. PG101
interacts with macrophages or related cells resulting in the activation of the tran-
scription factor NF-/cB, which sets off a series of reactions producing a variety of
proinflammatory and anti-inflammatory cytokines (TNF-a, IL-l/i, IL-10, IL-12,
GM-CSF, IL-1 8) in a sequential manner. Despite its significant biological effect
on various cytokines, PG101 remains nontoxic in both rats and human peripheral
blood mononuclear cells (hPBMCs) even at a biological concentration approxi-
mately 20 times greater (Jin et al., 2003b).

Schizophyllan is similar to lentinan in the composition and biological activity,
and its mechanism of antitumor action appears to be quite similar (Jong et al.,
1991). The antitumor effect of schizophyllan is diminished in mice neonatally
thymectomized and treated with antithymus globulin. Schizophyllan restores and
enhances cellular immunity in the tumor-bearing host by functioning as a T-cell
adjuvant and macrophage activator (Okazaki et al., 1995). Mechanistic study of
the antitumor activity of schizophyllan suggests that macrophages may incorpo-
rate j6-glucans through certain (1 — > 3)-/6-D-glucan-specific mechanisms and/or
other endocytosis pathways and that the glucan-mediated immunopharmacological
activities are dependent on the helical conformation (Okazaki et al., 1995).

Grifolan, a (1 — >• 3)-/6-glucan isolated from G. frondosa, induces the
release of IL-1, IL-6, and TNF-a from macrophages but appears to require a
high-molecular-mass soluble form of grifolan for TNF-a production (Adachi
etal., 1994; Okazaki etal., 1995; Ishibashi et al., 2001). D-fraction, another
(1 3)-y3-glucan purified from G. frondosa, could induce a Th-2 dominant



166 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

response via the activation of macrophages, resulting in the enhancement of
humoral immunity rather than cell-mediated immunity. In addition, it could pro-
mote an increase in the percentage ratio of CD69 and CD89 expression on major
histocompatibility complex 11+ cells, which revealed activation of APCs 4 hours
after D-fraction administration (Kodama et al., 2004). D-fraction is suggested as a
novel inducer for iNOS which contributes at least in part to the antitumor activity
of D-fraction through iNOS-mediated NO production in RAW264.7 macrophages
(Sanzen et al., 2001). These results suggest that D-fraction can decrease the effec-
tive dosage in tumor-bearing mice by increasing the proliferation, differentiation,
and activation of immunocompetent cells and thus provide a potential clinical
benefit for patients with cancer (Kodama et al., 2005a).

A galactomannan isolated from a polar extract of Morchella esculenta, a
highly prized mushroom in the world, exhibits immunostimulatory activity by
enhancing NF-/cB -directed luciferase expression in THP-1 human monocytic
cells (macrophages) (Duncan et al., 2002). Similarly, a fucogalactan isolated
from Sarcodon aspratus elicits the immunomodulating activity by the release
of TNF-a and NO in macrophages of mice in vitro (Mizuno et al., 2000). The
endopolysaccharide extracted from the mycelium of Inonotus obliquus is also an
immunostimulating agent which can specifically activate B cells and macrophages.
It enhances nitrite production and expression of IL— 1/6, IL-6, TNF-a, and iNOS
in macrophages (Kim et al., 2005, 2006). An immunomodulating polysaccharide
isolated from P. florida fruiting bodies exhibits significant macrophage activity
through the release of No (Rout et al., 2004). The tumoricidal activity of peri-
toneal macrophages (PMs) cultured with acidic polysaccharide (PL) isolated from
Phellinus linteus against B16 melanoma cells was enhanced in a dose-dependent
manner (Kim et al., 2003). PL exerts cytotoxicity against Yac-1 cells through
the up-regulation of NO and TNF-a production and enhances the expression of
costimulatory molecules, CD80 and CD86, and major histocompatibility complex
(MHC) molecules II in PM. Such properties of PL may be related to its ability
to induce the production of the tumoricidal effector molecule NO mediated
via the activity of protein tyrosine kinase (PTK) and protein kinase C (PKC)
(Kim et al., 2003, 2004a) and through the enhancement of IFN-y secretion by
T lymphocytes (Oh et al., 2006). A novel polysaccharide-protein complex (PPC)
extracted from P. linteus is able to increase the production of cytokines and NO
from macrophages and enhance the lytic death of NO-sensitive tumor cells, B16
melanoma. PPC is a potent immunomodulator which can stimulate the tumoricidal
activities of macrophages and NK cells and induce the proliferation of B cells in
vitro (Kim et al., 2006).

PSK (a protein bound-polysaccharide K) prepared from C. (T.) versicolor in
Japan has the ability to restore immune potential to the normal level after the
host has been depressed by tumor burden or anticancer chemotherapeutic agents
(Tsukagoshi et al., 1984; Kobayashi et al., 1993). The oral administration of PSK
can improve the impaired antitumor CD4+ T-cell response in gut-associated lym-
phoid tissue of specific -pathogen-free mice (Harada et al., 1997). PSK enhances
the cytotoxic activity of peripheral blood lymphocytes (PBLs) in vivo and in vitro.



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 167

It may accelerate interaction of PBL with tumor cells such as T24 human uri-
nary bladder tumors when both effector cells and target cells are exposed to PSK
simultaneously. It is reported that PSK induces gene expression of some cytokines
such as TNF-a, IL-1, IL-8, and IL-6 in vivo or in vitro (Kato etal., 1995; Liu
et al., 1996a). These cytokines produced by monocytes, macrophage, and various
other immune cell types mediate multiple biological effects by direct stimula-
tion of cytotoxic T cells against tumors, enhancement of antibody production by
B lymphocytes, and induction of IL-2 receptor expression on T lymphocytes. The
induction of TNF-a by PSK would contribute, in part, to potent tumoricidal effects
of this agent since the administration of neutralizing antibody against TNF-a sig-
nificantly attenuates the antitumor activity of PSK in the murine model (Kato et al.,
1995). Similarly, PSP prepared from C. versicolor in China may activate peritoneal
macrophages to produce TNF-a and other cytokines. It is also able to suppress the
growth of various human cancer cell lines and reverse tumor-induced immunode-
ficiencies in mice by increasing immunoglobulin (Ig)G and C3 complement levels
(Yang et al., 1992; Liu et al., 1993; Cui and Chisti, 2003).

The antitumor PSPC purified and characterized from the culture filtrates of
T. lobayense exhibits strong antitumor activity in both ICR and BALB/c mice (Liu
et al., 1996b). PSPC has the ability to restore the phagocytic function of the peri-
toneal exudate cells (PEC) and the mitogenic activity of T cells of tumor-bearing
mice. PSPC also exhibits indirect cytotoxic activity against P815 mastocytoma
cells and L929 mouse fibroblast cells by activating PEC to release reactive nitro-
gen intermediates (RNIs) and TNF-a, which are shown to increase significantly
after PSPC treatment in the tumor-bearing mice (Liu et al., 1996b). Similarly, the
antitumor activity of ATOM, a polysaccharide-protein complex prepared from the
cultured mycelia of A. blazei, is highly effective against four kinds of established
tumors, that is, subcutaneously implanted sarcoma 180 in mice, Ehrlich ascites
carcinoma, Shionogi carcinoma 42, and Meth A fibrosarcoma (Itoh et al., 1994;
Ito et al., 1997). A heteroglycan-protein complex from G. frondosa is shown to
depress tumor growth by activating the immune system as a biological response
modifier (Cun et al., 1994).

5.3.2.2 Effects of Mushroom Polysaccharides on NK Cells Natural
killer cells are a form of cytotoxic lymphocytes which constitutes a major
component of the innate immune system. NK cells can be activated in response to
interferons or macrophage-derived cytokines. Their activity is tightly regulated.
NK cells can recognize the surface changes that occur on a variety of tumor cells
and virally infected cells (Miller, 2002). They express a cascade of activating cell
surface receptors that can trigger cytolytic programs and produce cytokines such
as IFN-y, TNF-a, and GM-CSF, which modulate natural and specific immune
responses (Yokoyama et al., 2004; Vivier et al., 2004).

Maitake D-fraction, a (1 -> 3)-/?-glucan extrated from G. frondosa, exerts its
antitumor effect in tumor-bearing mice by enhancing the immune system through
the activation of macrophages, T cells, and NK cells. Kodama et al. (Kodama et al.
(2002) monitored the level of NK cell cytotoxic activity in cancer patients receiving



168 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

D-fraction and found that the elevated levels of cytotoxic activity were main-
tained for one year. D-fraction is capable of enhancing and maintaining peripheral
blood NK cell activity in patients with lung and breast cancer. It stimulates the
natural immunity related to the activation of NK cells indirectly through IL-12
produced by macrophages and DCs in normal mice (Kodama et al., 2003). Thus,
NK cells are responsible not only for the early effects of D-fraction on tumor
growth but also for the long-term tumor-suppressive effects of D-fraction through
the increased release of IL-12 by macrophages. It appears to repress cancer pro-
gression primarily through the stimulation of NK activity (Kodama et al., 2005a).
Thus immunomodulation effected by the binding of a (1 — >• 3)-/3-glucan molecule
or particle probably includes activation of cytotoxic macrophages, helper T cells,
and NK cells and promotion of T cell differentiation.

AC-PS, a unique polysaccharide component purified from the mycelium of
Antrodia camphorata, has pronounced antitumor effects on both in vitro and in
vivo model. AC-PS alone does not show any direct cytotoxic effect to human
leukemic U937 cells, even at high concentrations (200 mg/mL). However, AC-PS
could inhibit the proliferation of U937 cells via the activation of mononuclear cells
and elicits its antitumor effect by promoting a Thl -dominant state and NK cell
activity (Liu et al., 2004). Ganopoly, the polysaccharide fraction of G. lucidum, is
reported to enhance the host immune functions (e.g., enhanced NK cell activity)
in patients with advanced lung cancer. Administration of Ganopoly for 12 weeks
results in a significant increase in the mitogenic reactivity of lymphocytes to con-
canavalin A, CD3 percentage, and NK cell activity; a marginal increase in the
CD4 percentage and CD4/CD8 ratio; but a marginal reduction of CD8 (Gao et al.,
2003). A well-characterized glycoprotein fraction containing fucose residues in the
extract of G. lucidum polysaccharide (EORP) is reported to enhance CD14 endocy-
tosis of LPS and promote TLR4 signal transduction of cytokine expression. EORP
increases the surface expression of CD14 and TLR4 within murine macrophages
J774A.1 cells in vitro (Hua et al, 2007).

FWE, the water extract containing mainly polysaccharides from five medici-
nal mushrooms, C. versicolor, Cordyceps sinensis, L. edodes, A. blazei, and G.
lucidum in equal amounts, has the activity to enhance phagocytosis of peritoneal
macrophages and NK activity in mice and suppress the growth of B-16 melanoma.
FWE is able to activate NK cells to directly kill tumor cells and to secrete the
cytotoxic agents to elicit the apoptotic pathway of tumor cells or other signaling
pathways (W. Y. Zhang et al., 2004).

5.3.2.3 Effects of Mushroom Polysaccharides on DCs DCs are

antigen-presenting cells (APCs) with a unique ability to induce primary immune
responses. DCs not only induce the activation and polarization of T cells but also
directly activate naive and memory B cells (Banchereau et al., 2000). Mushroom
polysaccharides as BRMs may promote the cytotoxic activity of various effector
cells by the induced production of multiple cytokines and suppression of
immunosuppressive factors. DCs at different stages of differentiation can regulate



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 169

effector cells of the innate immunity, such as NK cells and cytotoxic T cells, and
initiate the induction of tumor immunity (Ogihara et al., 2004; Lull et al., 2005).

PS-G, the polysaccharide component with a branched (16)-/6-D-glucan moiety
of G. lucidum, exerts antitumor activity and can effectively promote the activation
and maturation of immature human monocyte-derived DCs. The treatment of
DCs with PS-G results in the enhanced expression of membrane molecules
such as CD80, CD86, CD83, CD40, CD54, and IL-12p70, p40, and IL-10 and
also IL-12p35, p40, and IL-10 mRNA. PS-G promotes the inhibitor of kappa
B (I-kB) kinase and NF-/cB activity and also LcBa and p38 mitogen-activated
protein kinase (MAPK) phosphorylation. The data demonstrate that PS-G can
effectively promote the activation and maturation of immature DCs, suggesting
it has a potential in regulating immune responses (Lin et al., 2005). In examining
the effects of PS-G on human monocyte-derived DCs with microarray analysis
by Human Genome U133 Plus 2.0 GeneChip, the results also reveal that
PS-G induces changes of gene expression of various immunomodulatory and
proinflammatory cytokines in human DCs and promotes the immune response
of Th-1 in BALB/c mice (Lin etal., 2006). The data from microarray analysis
could be correlated with the in vivo effect of the immune-enhancing compound.
In another study, Cao and Lin (2002) reported that Gl-PS, a fraction of G. lucidum
polysaccharides, could promote not only the maturation of cultured murine bone
marrow -derived DCs but also the initiation of immune response induced by DCs.
GL-M, the extract of G. lucidum (GL) mycelia, could activate the proliferation of
PBMCs and monocytes and stimulate Th-1 and Th-2 cytokine mRNA expression.
GL-M enhances the maturation of DCs in terms of up-regulation of CD40, CD80,
and CD86, and reduces endocytosis (Chan et al., 2005).

PG, a proteoglycan isolated from Phellinus linteus, strongly inhibits the
MCA- 102 tumor growth and proliferation in vivo through a mechanism leading
to a Th-l-dominant immune state and the activation of CDllc + CD8 + DCs. It
induces the phenotypic and functional maturation of bone marrow -derived DCs
in vitro via TLR-2- and TLR-4-mediated NF-kB, ERK, and p38 MAPK signal
pathways. PG enhances the production of IL-12 and IFN-y and surface molecules,
including CD80 and CD86 in MCA- 102 tumor-bearing mice, as well as the
proliferation of CD4(+) and CD8(+) T cells (Kim et al., 2004c). Furthermore,
the acidic PL isolated from P. linteus can also promote the maturation of DCs. PL
significantly increases the membrane surface molecules, including MHC classes I
and II, CD80, and CD86, and IL-12 in DCs. PL markedly reduces the endocytic
activity of DCs and augments their capacity to promote the proliferation of naive
allogeneic T cells (Park et al., 2003).

PSK (a protein-bound polysaccharide) from the cultured mycelium of
C. (T.) versicolor is reported to improve the immunosuppressed state and might
be associated with DC maturation directly. PSK promotes both the phenotypic
and functional maturation of DCs derived from human CD 14+ mononuclear cells
(Kanazawa et al., 2004). It can overcome the defective maturation of DCs exposed
to tumor-derived factors in vitro (Okuzawa et al., 2002). In a clinical trial involving
6 normal adults and 14 patients with gastric or colorectal cancers, the Thl/Th2



170 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

and DC1/DC2 balance becomes Th2 and DC2 dominant in the cancer-bearing
state. PSK therapy results in a shift of the Thl/Th2 and DC1/DC2 balance toward
Thl and DC1 dominance (Kanazawa et al., 2005). It might be possible to combine
DC vaccination therapy with oral PSK to promote the induction of T cell and DC
differentiation in cancer patients (Kanazawa et al., 2005).

5.3.2.4 Effects of Mushroom Polysaccharides on Hematopoietic Stem
Cells MBG (a /6-glucan isolated from the fruiting body of G. frondosd) can
enhance the blood-forming process (hematopoiesis) of mouse bone marrow cells
(BMCs) in vitro and protects BMCs from doxorubicin (DOX) toxicity on fresh
human umbilical cord blood (CB) cells. MBG treatment significantly raises the
response of the colony-formation unit of granulocytes-macrophages (CFU-GM)
over the whole dose range of 12.5-100 mg/mL (P < 0.05). The addition of MBG
to DOX-treated CB cells significantly protectes granulocyte-macrophage colony
formation from the toxicity of DOX, which otherwise produces strong hematopoi-
etic repression. The data show that MBG induces granulocyte colony-stimulating
factor (G-CSF) production in CB CD33 + monocytes, but adult peripheral blood
monocytes did not produce a significant G-CSF response to MBG. They have
the potential to reduce hematopoietic suppression induced by chemotherapy (Lin
et al., 2007). Recently, Lin et al. reported that maitake MD-fraction (obtained by
further purification of D-fraction from the fruiting body of G. frondosa) causes
direct enhancement of the response of CFU-GM of BMC progenitors and enhances
the recovery of the CFU-GM response after DOX-induced hematopoietic suppres-
sion (H. Lin et al., 2004). These studies demonstrate that MBG or MD-fraction
may induce the proliferation of hematopoietic stem cells and differentiation of
CFU-GM in umbilical CB cells and act directly to induce the production of G-CSF.

PG101, a water-soluble extract containing protein-bound polysaccharides
isolated from cultured mycelia of L. lepideus, is a potential biological response
modifier that activates selective cytokines in vitro, mainly by controlling cellular
transcription factor NF-kB (Jin et al., 2003b). It was reported that in irradiated
mice given PG101 by gavage daily for 24 days the number of colony-forming
cells, including CFU-GM) and erythroid burst-forming units (BFU-E), increased
to almost the levels seen in the nonirradiated control as early as 8 days after irradi-
ation. PG101 increases the number of granulocytes (ER-MP12~20 + ) and myeloid
progenitors (ER-MP12 + 20 + ) in the bone marrow cell population and raises the
serum levels of GM-CSF, IL-6, and IL-l/J. The level of TNF-a, elevated by irradi-
ation in the control mice, maintains at the background level in the PG101 -treated
mice. The results suggest that PG101 might induce differentiation of progenitor
cells to granulocytes and/or proliferation of the committed cells and might
effectively suppress TNF-a -related pathological conditions (Jin et al., 2003a).

A branched /3-glucan isolated from fruiting bodies of S. crispa (SCG) is able
to promote the hematopoietic response in cyclophosphamide (CY) -induced
leukopenic mice by prior or postadministration. Monocytes and granulocytes in
the peritoneal cavity, liver, spleen, and bone marrow recover faster than in the
control group. The ratio of NK cells and T cells in the liver, spleen, and peritoneal



MECHANISMS OF ANTITUMOR ACTION OF MUSHROOM POLYSACCHARIDES 171

cavity is also increased. The cotreatment of CY+SCG in the culture of peritoneal
exudate cells (macrophages), spleen cells, and bone marrow cells induces the
production of high amounts of IL-6 than that of the CY-treated group. IL-6 might
be a key cytokine for the enhanced hematopoietic response by SCG (Harada et al.,
2002).

5.3.3 Antimetastasis

Many polysaccharides or polysaccharide-protein complexes from mushrooms
also exhibit antimetastatic effects. Lentinan, a purified (1 -> 3)-y3-D-glucan from
L. edodes, not only markedly prevents chemical and viral carcinogenesis (Suga
et al., 1984), but also suppresses cancer metastasis and recurrence in animal
models (Suga et al., 1989). The administration of lentinan prominently inhibits
colony formation of metastasis in the lung after surgical resection of the tumors
implanted subcutaneously into the mouse footpad, with the inhibition ratios of
95% in DBA/2.MC.CS-T sarcoma and 83% in Lewis lung carcinoma. The results
suggest that lentinan may be effective in the prevention of tumor recurrence and/or
metastasis (Suga et al., 1989).

PSK (a protein-bound polysaccharide from C. versicolor) has also been shown,
that once the progression of carcinogenesis is initiated, to exhibit significant
preventive action on cancer metastasis such as the suppression of pulmonary
metastasis of methylcholanthrene-induced sarcomas, human prostate cancer
DU145M, and lymphatic metastasis of mouse leukemia P388 in the spontaneous
metastatic models. PSK also inhibits the metastasis of rat hepatoma AH60C,
mouse colon cancer 26, and mouse leukemia RL male 1 in artificial metastatic
models (Kobayashi et al., 1995). PSK could prevent distant metastases and
improve the survival rates by 10-20% in colorectal cancer (Yoshikawa et al.,
2004). Kobayashi et al (1995) suggested that PSK is able to influence the
following steps of cancer metastasis: (a) through the inhibition of tumor invasion,
adhesion and production of cell matrix -degrading enzymes; (b) by suppression of
tumor cell attachment to endothelial cells; (c) by suppression of tumor cell motility
and thus cell migration after extravasation; (d) through the inhibition of tumor
angiogenesis and growth; (e) through the modulation of cytokine production and
the augmentation of effector cell functions; and (f) by suppression of malignant
progression of tumor cells through superoxide trapping. PSK may thus suppress
cancer metastasis at any one step, and its primary mechanism of action can be
ascribed to direct action on the tumor cell as well as to immunomodulation
(Kobayashi et al., 1995).

In a study on the effects of natural polysaccharides isolated from Phellinus
gilvus (PG) on human gastric cancer, Bae et al. (2006) showed that PG reduces cell
proliferation and increased cell apoptosis in a dose-dependent manner in vitro and
markedly suppresses tumor growth (peritoneal carcinomatosis) in a nude mouse
model. The data show that PG significantly inhibits tumor growth and metasta-
sis in an orthotopic model of human gastric adenocarcinoma without detectable
adverse effects (Bae et al., 2006).



172 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

An exopolysaccharide fraction (EPSF) prepared from cultivated C sinensis
(Chinese caterpillar fungus) has immunomodulatory and antitumor activity. EPSF
enhances phagocytosis capacity of peritoneal macrophages and proliferation of
spleen lymphocytes in the treated B16-bearing mice. EPSF significantly inhibits
the metastasis of B16 melanoma cells to the lung and liver (Zhang et al., 2005).

M. Zhang et al. (2004) reported that FWE (the water extract composed mainly
of polysaccharides from five medicinal mushrooms, i.e., C. versicolor, C. sinensis,
L. edodes, A. blazei, and G. lucidum) significantly promoted the NK cell activ-
ity of treated mice and depressed the levels of bcl-2 and P53 protein in the liver
and lung cells. The minimum inhibition rates (MIRs) of lung metastasis of B16
melanoma cells by low dose and high dose of FWE were 15.5% and 72.7%, respec-
tively. These results indicate that FWE not only promotes the mouse host-mediated
immunity but also inhibits tumor metastasis (M. Zhang et al., 2004).

5.3.4 Antiangiogenesis

Angiogenesis is the process of new blood vessel formation from the preexisting
microvascular networks (Folkman, 1995). It is crucial to the progressive growth
and metastasis of solid tumors, and thus antiangiogenesis is considered an
important strategy for therapeutic intervention of tumor proliferation (Chen et al.,
1995). The formation of new blood vessels involves the angiogenic switch in
the balance of angiogenic and antiangiogenic factors and the interactions of
tumor cells, endothelial cells, and extracellular matrix, leading to endothelial
proliferation, migration, and tube formation (Hanahan and Folkman, 1996).
Cancer cells can produce several angiogenic factors such as vascular endothelial
growth factor (VEGF) and few inflammatory cytokines.

PSK, a protein-bound polysaccharide immunomodulating agent from C. ver-
sicolor, was reported to have an antiangiogenic effect (Wada et al., 2002). PSK
inhibits the proliferation of human umbilical vein endothelial cells (HUVECs)
in the presence of basic fibroblast growth factor (bFGF) more effectively. At
a high concentration PSK suppresses tube formation of HUVECs and their
adhesion to extracellular matrix proteins. Administration of PSK could reduce
the bFGF-induced angiogenesis in an in vivo rat cornea assay. These data suggest
that PSK binds to bFGF and interferes with its signal transduction to inhibit
the proliferation of HUVECs, resulting in the suppression of angiogenesis
(Wada et al., 2002). Quantitative analysis of microcorrosion casting of the tumor
tissue in the study using the SI 80 tumor-bearing mouse model shows that the
polysaccharopeptide, PSP, isolated from the edible mushroom C. versicolor
has antiangiogenesis properties. The expression of VEGF in these tumors is
suppressed (Ho et al., 2004). Moreover, the EPSF of a cultivated C. sinensis (Cs)
inhibits tumor growth in the mice (C57BL/6). The levels of expression of c-Myc,
c-Fos, and VEGF in the lungs and livers of the EPSF-treated mice are significantly
lower than those of the untreated mice (Yang et al., 2005).

Polysaccharide peptide (Gl-PP) isolated from G. lucidum has antitumor effects
in mice and potential antiangiogenesis. Gl-PP treatment of HUVECs could induce



STRUCTURE AND ANTITUMOR ACTIVITY RELATIONSHIP OF POLYSACCHARIDES 173

cell apoptosis directly and lead to a reduction of Bcl-2 antiapoptotic protein expres-
sion and an increase of Bax proapoptotic protein expression of HUVECs. It is thus
suggested that Gl-PP may directly inhibit vascular endothelial cell proliferation or
indirectly decrease VEGF expression of tumor cells (Cao and Lin, 2006).

In an investigation on antiangiogenic activities of several medicinal fungi,
including Antrodia cinnamomea, Antrodia malicola, Antrodia xantha, Antrodiella
liebmannii, Agaricus murrill, and Rigidoporus ulmarius, on VEGF-induced
tube formation in endothelial cells (ECs), Chen et al. (2005) reported that
polysaccharides isolated from A. xantha and R. ulmarius produce greater
inhibition of endothelial tube formation compared to those from other fungi and
polysaccharides isolated from A. xantha and R. ulmarius provide greater antian-
giogenesis than those from commercialized A. murrill (Brazilian mushroom) and
A. cinnamomea. Polysaccharides isolated from A. cinnamomea inhibit cyclin
Dl expression through inhibition of VEGF receptor signaling, leading to the
suppression of angiogenesis. Antrodia cinnamomea polysaccharides also block
VEGF-induced migration and capillary-like tube formation of ECs on Matrigel
(Cheng et al., 2005).

5.4 STRUCTURE AND ANTITUMOR ACTIVITY
RELATIONSHIP OF POLYSACCHARIDES

Polysaccharides having antitumor action differ greatly in their chemical com-
position, branching configuration, helical conformation, and other physical
properties. Antitumor activity is exhibited in a wide range of glycans extending
from homopolymers to highly complex heteropolymers. Although it is difficult
to correlate the structure and antitumor activity of complex polysaccharides,
some possible relationships can be inferred. It has been reported that most
of the antitumor polysaccharides such as lentinan and schizophyllan show
the same basic /?-glucan structure with different types of glycosidic linkages.
Therefore it is obvious that some structural features such as (1 3)-fi linkages
in the main chain of the glucan and further /J-1,6 branch points are needed for
antitumor action (Franz, 1989; Bohn and BeMiller, 1995). It has been suggested
that the /3-glucans containing mainly (1 -> 6) linkages have less activities, and
polysaccharides with high molecular weight appear to be more effective than those
with low molecular weight (Jong etal., 1991; Sakagami etal., 1991; Surenjav
et al., 2006). However, obvious variations of antitumor polysaccharides are also
noted. There are antitumor polysaccharides with other chemical structures, such
as hetero-j6-glucan (Mizuno et al., 1995a), heteroglycan (Zhuang and Mizuno,

1995) , /J-glucan-protein (Kawagishi et al., 1990a), a-manno-j6-glucan (Mizuno
et al., 1995a), a-glucan-protein (Mizuno et al., 1995a), and heteroglycan-protein
complexes (Zhuang etal., 1993; Liu etal., 1996b; Mizuno etal., 1996). For
example, PSK and PSP are a /J-glucan-protein whereas PSPC from Tricholoma
species are a heteroglycan-protein complex (Liu et al., 1996b; Mizuno et al.,

1996) . Therefore, it is difficult to identify which polysaccharide structure is
essential for antitumor action. It has been shown that the molecular mass, the



174 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

degree of branching, conformation, and chemical modification of antitumor
polysaccharides significantly affect their antitumor and immunomodulatory activ-
ities (Bohn and BeMiller, 1995; Okazaki et al., 1995; Ohno et al., 1995; Surenjav
et al, 2006).

5.4.1 Effect of Molecular Mass

A (1 — > 3)-/6-glucan extracted from the cultured mycelium of G. frondosa with
various molecular masses obtained by heat treatment for varying times at 150��C
manifests changes of biological activities with different molecular masses (Adachi
et al., 1990a). The highest molecular mass fraction (800 kDa) always exhibits the
strongest antitumor and immunomodulatory activity (Adachi et al., 1990a; Kim
et al., 1990). When PSK is separated into four fractions with different molecular
masses (Fl: <50 kDa; F2: 50-100 kDa; F3: 100-200 kDa; F4: >200 kDa) by
successive filtration, the highest molecular mass fraction (>200 kDa) has the most
potent immunomodulatory activity (Kim et al., 1990; Sakagami et al., 1990). The
antitumor activity of the native triple helical (1 — > 3)-/J-D-glucan with high molec-
ular weight and bound protein isolated from L. edodes is higher than that of the
modified (1 — > 3)-y3-D-glucan having only a single flexible chain and low molecu-
lar weight (Surenjav et al., 2006). These investigations suggest that a high molec-
ular mass is required for extensive enhancement of immunological and antitumor
activities. However, the study by Peng et al. (2005) indicates that the water-soluble
polysaccharide with relatively lower weight-averaged molecular mass from the
mycelium of G. tsugae, which contains a moderate content of galactose and bound
protein, is important in the improvement of antitumor activity of polysaccharides.
The chemically modified (1 — > 3)-/6-D-glucans, such as schizophyllan and lenti-
nan, having a linear wormlike, triple-helical structure with weight-averaged molec-
ular mass less than 50x 10 4 g/mol or larger than 1 lOx 10 4 g/mol might stimulate
the monocytes in vitro more efficiently to secrete TNF-a than the samples with
molecular mass in the range 67 — 10 4 — 110 x 10 4 g/mol (Falch etal., 2000).
However, lentinan and schizophyllan with low molecular weight have the same
antitumor activity against sarcoma 180 as those with higher molecular weight
(Sasaki etal., 1976; Tabata etal., 1981; Ogawa and Kaburagi, 1982). Therefore,
the discrepancy about the effect of molecular mass of polysaccharides on antitumor
activity and immunomodulation remains to be clarified.

5.4.2 Impact of Branching Configuration

In general, /6-glucans are active antitumor agents if they have mainly linear struc-
ture, possessing not excessively long branches. Some data indicate that it is the
distribution of the branch units along the backbone chain of (1 — > 3)-/?-D-glucan
that is responsible for the antitumor activity. For example, pachyman, a branched
(1 3)-/2-D-glucan obtained from P. cocos, is inactive, whereas pachymaran,
obtained by debranching pachyman using periodate oxidation and mild hydrolysis,
exhibits pronounced antitumor activity (Chihara et al., 1970a). Lentinan (2/5)



STRUCTURE AND ANTITUMOR ACTIVITY RELATIONSHIP OF POLYSACCHARIDES 175

is a (1 — > 3)-y3-D-glucan with two branches for every five D-glucopyranosyl
residues (Chihara, 1992). Schizophyllan (1/3) is also a (1 -> 3)-/J-D-glucan
with one branch for every three D-glucopyranosyl residues (Tabata et al., 1981).
The debranched lentinan preparations are more effective against sarcoma 180
than the native lentinan at a dose of 2.0 mg/kg for five days in mice (Sasaki
et al., 1976). In addition, when the highly branched (1 -»• 3)-/J-D-glucan (2/3,
two branches for every three D-glucopyranosyl residues), called OL-2, extracted
from Omphalia lapidescens is modified to Smith-type degradation product with
approximately one branch at every 24 main-chain glucosyl units at each C-6
position (number of all main-chain glucosyl units is on average), it still exhibits
potent antitumor activity on the solid form of sarcoma 180 in mice and increases
the life span of mice treated with the ascites form of sarcoma 180 and MH 134
hepatoma (Saito et al., 1992). The alkali-insoluble, branched (1 -> 3)-y6-D-glucan
(glucan II), which is a major constituent of the fruiting body of Auricularia
auricula-judae, shows essentially no inhibitory activity against implanted
sarcoma 180 solid tumor in mice. When glucan II, having numerous branches
attached, is modified by controlled, periodate oxidation, borohydride reduction,
and mild acid hydrolysis, the resulting water-soluble, degraded glucan, having
covalently linked polyhydroxy groups attached at the 0-6 of the (1 — > 3)-linked
D-glucosyl residues, shows potent antitumor activity (Misaki etal., 1981). At
the molecular level, it is found that the patterns of cytokine expression between
schizophyllan (1/3) from P. cocos and OL-2 (2/3) from O. lapidescens are also
different. The mice administered with OL-2 strongly express IL— la, IL— \fi,
and IL-lra genes in peritoneal exudate cells (macrophages), while schizophyllan
only induces the expression of IL— la. In the genes related to hematopoiesis,
OL-2 induces the expression of G-CSF and GM-CSF, but schizophyllan induces
the expression of IL-3 (Nemoto et al., 1993, 1994). Therefore, the relationship
between the antitumor activity and the branch ratios of ^-glucan appear to be
quite complicated. The data indicate that the (1 3)-/6-D-glucan backbone is
essential, and the most active polymers have degrees of branching (DB) between
0.20 and 0.33.

5.4.3 Relationship of Antitumor Activity and Conformation

Conformations of antitumor polysaccharides include single-helix, triple-helix, and
random-coiled conformers. A triple-helix conformer is usually more stable than
the single-helix conformer, since a certain part of the single-helix conformer is
gradually changed to the triple one. Lentinan, schizophyllan, and glucan moieties
of PSK all have triple-helix structure (Tsukagoshi et al., 1984; Chihara 1992;
Ohno etal., 1995). Lentinan, a (1 3)-/?-D-glucan isolated from L. edodes,
exists as triple-helical conformation in aqueous NaCl and as single flexible
chains in dimethyl sulfoxide (DMSO) (Zhang et al., 2002). The antitumor activity
of the native triple-helical (1 -> 3)-j6-D-glucan with high molecular weight
and bound protein isolated from L. edodes is higher than that of the modified
(1 3)-/6-D-glucan having only a single flexible chain and low molecular weight



176 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

(Surenjav et al., 2006). Pachyman from P. cocos which is a (1 -> 3)-/?-D-glucan
having a single-helix conformer is biologically inactive against tumor growth.
However, when pachyman is treated with periodate oxidation and mild hydrolysis,
the newly formed conformer, pachymaran or carboxyl-methyl-pachymaran,
exhibits pronounced antitumor activity (Chihara et al., 1970a; Kanayama et al.,
1986).

Schizophyllan-OH, which has a single-helix structure derived from the
alkaline-treated schizophyllan, shows a reduced ability to inhibit tumor growth as
compared to the native schizophyllan (Chihara, 1984). The cytokine stimulating
activity of (1 — > 3)-/2-D-glucans is also found related to the triple-helix conforma-
tion (Falch et al., 2000). Therefore, the antitumor and immunopharmacological
activities of polysaccharides are dependent on the helical conformation, and
the conformation dependency may vary according to the assays and analytical
methods used (Ohno et al., 1995). The relationship of conformation and antitumor
activity of polysaccharides or polysaccharide-protein complexes suggests the
existence of a biological system within the host body that recognizes the con-
figurational structure of polysaccharides. The antitumor and immunomodulating
properties of branched (1 3)-/3-D-glucans may depend on their branching
pattern, molecular weight, and conformation or higher order structure.

5.4.4 Improvement of Antitumor Activity by Chemical Modifications

To improve the biological activity of antitumor polysaccharides by chemical
modification, carboxymethylated (CM), hydroxylated, formylmethylated,
aminethylated, and sulfated products have been designed. The linear
(1 — > 3)-a-glucans from Amanita muscaria and Agrocybe cylindracea have
little antitumor activity, but their CM derivatives show potent antitumor activity
against sarcoma 180 in mice (Kiho etal, 1989, 1994; Yoshida etal., 1996).
Debranched pachymaran and CM pachynmaran are more effective antitumor
(1 3)-/2-D-glucans, as compared with the natural glucan, pachyman (Chihara
et al., 1970a; Kanayama et al., 1986). The administration of hydroxylated schizo-
phyllan could induce the production of higher concentrations of NO and TNF-a
by peritoneal exudate cells (macrophages) than that by the native schizophyllan
in vivo (Ohno et al., 1995). The antitumor activity of the formylmethylated and
aminoethylated derivatives of schizophyllan against sarcoma 180 solid tumor in
mice is increased more effectively than that of the native schizophyllan (Usui
et al., 1995). A chemically sulfated polysaccharide (S-GAP-P), derived from the
water-insoluble glucan of G. frondosa mycelia, could inhibit in vitro the prolifer-
ation of SGC-7901 tumor cells and induce apoptosis in a dose-dependent manner.
In vivo experiments demonstrate that S-GAP-P significantly inhibits the tumor
growth and enhances the phagocytosis of peritoneal macrophages in S180-bearing
mice (Nie et al., 2006). The sulfated (1 -> 3)-a-D-glucan modified from the native
glucan of L. edodes exhibits potent antiproliferation action on human MCF-7
breast carcinoma cells, whereas its native water-insoluble (1 — > 3)-a-D-glucan
has only moderate antitumor activity (Zhang and Cheung, 2002). The native



STRUCTURE AND ANTITUMOR ACTIVITY RELATIONSHIP OF POLYSACCHARIDES 177

water-soluble (1 — > 3)-/6-D-glucan from L. edodes, which contains bound protein
and exists as triple-helical conformation with high molecular weight, shows
prominent antitumor activity, whereas the modified (1 -> 3)-j6-D-glucan having
only a single flexible chain has significantly decreased effect. The data suggest that
the antitumor activity of the polysaccharides may be related to their conformation,
molecular weight, and content of the bound protein (Surenjav et al., , 2006).
Furthermore, all of the water-insoluble (1 — >• 3)-a-D-glucans from L. edodes,
which were O-sulfonated to obtain derivatives, exhibit higher antitumor activity
than those of the native glucan. The triple-helical conformation and the effect
of O-sulfonation of the polysaccharides seem to play an important role in the
improvement of their antitumor activity (Unursaikhan et al., 2006). The antitumor
activity of the sulfated derivatives of water-insoluble (1 -> 3)-a-D-glucans isolated
from the mycelium of P. cocos against sarcoma 180 tumor both in vitro and in vivo
is significantly higher than that of the native a-D-glucan (Y. L. Lin et al., 2004).
It is also worth mentioning that the native (1 -> 3)-/6-D-glucan isolated from
the fresh sclerotium of P. cocos has no antitumor activity, whereas the sulfated
and carboxymethylated derivatives exhibit significant antitumor activities against
S-180 and gastric carcinoma tumor cells (Y. Wang etal., 2004). The data show
that good water solubility, relatively high chain stiffness, and moderate molecular
mass of the chemically modified derivatives in aqueous solution contribute
favorably to the enhancement of antitumor activity (Y. Wang et al., 2004).

In a series of investigations on chemical modifications of glucan fractions iso-
lated from the sclerotia of P. tuber-regium, M. Zhang et al. (2003, 2004) reported
that seven water-insoluble (1 — > 3)-/6-D-glucan fractions (TM8-1 to TM8-7) and
six hot alkali extracts (HAE-1 to HAE-6) of mushroom (1 — > 3)-/6-glucans having
different molecular mass were carboxymethylated to produce their correspond-
ing water-soluble derivatives (CTM8-1 to CTM8-7 and CMHAE-1 to CMHAE-6,
respectively). On the whole, all the carboxymethylated /6-glucans have higher
water solubility and CMHAE show higher antitumor activity in vivo (sarcoma 180
solid tumor implanted on BALB/c mice) as well as in vitro (HL-60 tumor cell
culture) than that of the native HAE /J-glucans. The results suggest that the anti-
tumor activity of the carboxymethylated /2-glucans may be correlated to its water
solubility and relatively extended chain (M. Zhang et al., 2003, 2004). In another
study, a water-soluble hyperbranched /6-glucan, coded as TM3b, extracted from the
same mushroom was fractionated by treatment with chlorosulfonic acid at 35��C
to synthesize sulfated yS-glucan derivatives. It reveals that both the native TM3b
and its sulfated derivatives exist in a spherical chain conformation in NaCl. Fur-
thermore, the native and sulfated TM3b fractions show potent antitumor activities
in vivo and in vitro, but the sulfated derivatives exhibit relatively higher in vitro
antitumor activity against human hepatic cancer cell line HepG2 than the native
TM3b. Water solubility and introduction of sulfate groups appear to be the main
factors in enhancing the antitumor activities (Tao et al., 2006). These studies show
that the chemical modification of polysaccharides might be an effective approach
for the improvement of the biological activities of polysaccharides.



178 ANTITUMOR AND IMMUNOMODULATORY ACTIVITIES OF MUSHROOM POLYSACCHARIDES

5.5 CONCLUSIONS

In the last few decades, a large number of macrofungi (mushrooms) have been
extensively used as a source of medicinal agents and therapeutic adjuvants or
health food supplements. There is an increasing interest in identifying effective
and safe constituents from medicinal mushrooms for cancer prevention and
treatment. Most mushrooms contain biologically active polysaccharides in
fruiting bodies, cultured mycelia, sclerotia, and culture filtrates. One of the most
promising activities of the polysaccharides or polysaccharide-protein complexes
derived from mushrooms is their immunomodulation and anticancer effects.
However, their mechanisms of antitumor and immunomodulating actions are not
clearly understood. It is widely accepted that antitumor polysaccharides from
higher fungi enhance various immune responses in vivo and in vitro and act
as BRMs. Their actions are predominantly host mediated. The cell-mediated
immunity plays a critical role in the antitumor activity of polysaccharides or
polysaccharide-protein complexes which are able to induce production of various
immunomodulatory cytokines. The major immunomodulatory effects of these
BRMs involve the activation of immunocompetent cells such as monocytes,
macrophages, DCs, NK cells, Th cells, Tc cells, and B cells and hematopoietic
stem cells and the activation of alternative signaling pathways. Macrophages and
DCs may be activated by mushroom polysaccharides to produce various cytokines
and NO which affect the response pathways of cell-mediated immunity. Activated
DCs and macrophages produce cytokines (e.g. IL-12), that stimulate NK cells
to rapidly secrete other cytokines (including IFN-y, TNF-a, and GM-CSF) and
enhance the cytotoxicity of NK cells. Various data support that their mode of
action is due to the enhancement and potentiation of cell-mediated immunity
through the regulation of immunomodulatory cytokines and the activation of a
complement system. Polysaccharides or polysaccharide-protein complexes are
considered multicytokine inducers able to induce gene expression of various
cytokines and cytokine receptors.

The detailed mechanisms of action of polysaccharide immunomodulators or
biological response modifiers are not fully known, but it is generally accepted that
they act on different immunocompetent cells which may initiate a cascade of sig-
nal transduction pathways that are responsible for the immune responses. The first
step of the polysaccharide BRM in the modulation of cellular activity is the recog-
nition of BRM and the binding to immune cell receptors. Some evidence shows
that there are PRRs available for the molecular reception of polysaccharide BRMs.
It has been suggested that some groups of PRRs can recognize the polysaccha-
ride BRMs, such as complement receptor 3 (CR3 or CDllb/CD18), dectin-1, and
toll-like receptors (TLR-2 and TLR-4). These PRRs play an important role as the
membrane /J-glucan receptors, which are mainly expressed on cell membranes of
monocytes, macrophages, neutrophils, NK cells, and DCs. The binding of BRMs
to PRRs may initiate a signaling cascade for the immune responses. PRRs can
mediate a variety of cellular functions such as cytotoxic and phagocytic responses



REFERENCES 179



as they have the ability to bind various ligands, for example, ICAM-1, /3-glucan,
and others, through various specific signal transduction pathways.

Although the mechanisms of antitumor action of polysaccharides or
polysaccharide-protein complexes are generally thought to be due to the enhance-
ment and modulation of the immune system, many of these macromolecules
have also been documented to possess direct cytotoxic effects on cancer cells.
The polysaccharides or polysaccharide-protein complexes may exert anticancer
activity by antiproliferative effect on tumor cells and induction of cell cycle arrest
and apoptosis. It is possible that, in some instances, these two types of inhibitory
action may be interwoven. Therefore, the possible modes of anticancer action
may include both (1) direct cytotoxicity to cancer cells as shown in many in
vitro studies and (2) indirect antitumor inhibition through immunomodulation
of the body defense system. Other mechanisms of antitumor action include
antiangiogenesis, antimetastasis, and antigenotoxicity.

Mushroom polysaccharides with antitumor and immunomodulatory properties
have been extensively documented. However, the relationship of structure and anti-
cancer activity of polysaccharides at the cellular and molecular levels is still not
well characterized. Future studies should therefore focus on investigation of the
relationship between their structure and antitumor activity, elucidation of their anti-
tumor mechanism at the molecular level, improvement of their various biological
activities by chemical modifications, and clinical trials on therapeutic efficacy of
mushroom polysaccharides. It is noteworthy that a thorough understanding of the
recognition of mushroom polysaccharides (particularly /?-glucan) by certain recep-
tors on the immune cells and activation of signal transduction pathways will also
be a challenge of future immunomodulation research.

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CHAPTER 6



Regulatory Issues of Mushrooms
as Functional Foods and Dietary
Supplements: Safety and Efficacy

Solomon P. Wasser and Eden Akavia

International Center of Biotechnology and Biodiversity of Fungi, Institute of Evolution,
University of Haifa, Haifa, Israel

CONTENTS

6. 1 Introduction

6.2 Legal and Regulatory Issues of Introducing and Controlling Dietary
Supplements from Medicinal Mushrooms in Different Countries

6.3 Safety and Diversity of Dietary Supplement Types from Culinary-Medicinal
Mushrooms

6.4 Submerged Culturing as Best Technique for Obtaining Consistent and Safe
Mushroom Products

6.5 Experiences of Seven Countries in Consolidating Their Food Safety
Systems

6.6 Summary
References



6.1 INTRODUCTION

In the second half of the twentieth century, mushroom-producing technologies
grew enormously. In 2004, the value of world mushroom production was esti-
mated at about U.S. $40 billion, which is almost the same as the value of coffee
production (Wasser et al., 2004; Chang and Miles, 2004; Chang, 2006).

Many pharmaceutical substances with potent and unique valuable properties
are used worldwide. Unique anticancer preparations have been developed based
on certain components such as the polysaccharides lentinan [from Lentinus edodes



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



199



200 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

(Berk.) Singer], krestin [from Trametes versicolor (L.:Fr.) Lloyd], maitake
D-fraction [from Grifola frondosa (Dicks.: Fr.) S. F. Gray], schizophyllan (from
Schizophyllum commune Fr.: Fr.), and befungin [from Inonotus obliquus (Pers.:
Fr.) Pilat] (Mizuno, 1999; Chang, 1999, 2001; Wasser and Weis, 1999a, b;
Stamets, 2000, 2002; Wasser et al., 2000a, b, 2001, 2004; Wasser, 2002; Chang
and Buswell, 2003; Chang and Miles, 2004).

Most mushroom-derived preparations and substances find use not as pharma-
ceuticals ("real" medicines) but rather as a novel class of products by a variety
of names: dietary supplements (DSs), functional foods, nutraceuticals, nutriceuti-
cals, phytochemicals, mycochemicals, biochemopreventives, and designer foods
(Chang and Buswell, 1999, 2003; Zeisel, 1999; Wasser et al., 2000a, b, 2001,
2004; Chang and Miles, 2004; Chang, 2006). These terms vary in meaning from
country to country, as does the regulation of these products. In the United States,
the formal definition under the Dietary Supplements Health and Education Act
(DSHEA) (1994) exists only for DSs. "Functional foods" has no legal status or
general acceptance but is used as a definition in the dietetic profession. "Nutraceu-
ticals" and "nutriceuticals" are also accepted definitions in the nutrition/science
community but are not embodied in law or regulation. "Medical foods" are regu-
lated by the U.S. Food and Drug Administration (FDA) Office of Special Nutri-
tionals on a case-by-case basis as enteral foods or foods for sick infants (Smith
et al., 1996, 2000).

DSs are ingredients obtained from foods, plants, and mushrooms (fungi) that
are taken, without further modification, separately from foods for their presumed
health-enhancing benefits (McNamara, 1999). The term DS includes one or more
of certain dietary ingredients — a vitamin, mineral, amino acid, herb, or other
botanical — or it is a dietary substance used to supplement the diet by increasing
the total dietary intake and is intended for ingestion in the form of a capsule,
powder, softgel, or gelcap and is not represented as a conventional food or as a
sole item of a meal or a diet (DSHEA, Public Law 103-417, 1994). Supplements
are easy to add to the daily diet and are often the first steps consumers undertake
toward greater nutritional awareness and the adoption of a healthy lifestyle. The
DS pyramid has been developed recently, which, just like the food pyramid, has
its own rules.

At the same time, the increased interest in traditional remedies for various
physiological disorders and the recognition of numerous biological activity of
mushroom products have led to the coining of the term "mushroom nutriceuticals"
(Chang and Buswell, 1996-2003; Chang, 1999, 2006; Wasser et al., 2000a, b,
2001, 2004; Chang and Miles, 2004), not to be confused with nutraceuticals, func-
tional foods, and pharmaceuticals. A mushroom nutriceutical is a refined, or par-
tially refined, extract or dried biomass from either mycelium or the fruiting body
of a mushroom, which is consumed in the form of capsules or tablets as a DS (not a
food) and has potentially therapeutic applications. Regular intake may enhance the
immune response of the human body, thereby increasing resistance to disease and
in some cases causing regression of the disease state. Thus, acting as immunopo-
tentiators, mushroom preparations modify host biological responses (Hobbs, 1995;



INTRODUCTION 201



Wasser and Weis, 1999a, b; Wasser, 2002; Wasser et al., 2004; Chang, 2006) and
are also known as biological response modifiers (BRMs).

The term DS (nutriceutical) is broader than BRM and better describes the
qualities of this class of substances. Another shortcoming of the term "biological
response modifiers" in this context is that many of the mushroom preparations
possess particular physiological effects, such as lowering blood cholesterol or
hepatoprotectivie activity (Hobbs, 1995; Chang and Buswell, 1999, 2003; Wasser
and Weis, 1999a, b; Stamets, 2000, 2002).

Several types of culinary -medicinal products are available on the market today:

1. Artificially cultivated fruit body powders, hot- water or alcohol extracts of
these, or the same extract concentrates and their mixtures

2. Dried and pulverized preparations of the combined substrate, mycelium, and
mushroom primordia after inoculation of edible semisolid medium (usually
grains)

3 . Biomass or extracts from mycelium harvested from submerged liquid culture
grown in a fermentation tank

4. Naturally growing, dried mushroom fruiting bodies in the form of capsules
or tablets

There is no doubt that medicinal mushroom-based products can serve as supe-
rior DSs. The problem is that mushroom-based DSs are so diverse, and there are
currently no standard protocols for guaranteeing their product quality and critical
testing. There is a serious need for improved quality and legal control, which are
essential both to increase and maintain consumers' confidence and to meet current
and future standards set by regulatory authorities (Chang and Buswell, 1999, 2003;
Wasser et al., 2000a, b, 2001, 2004; Chang and Miles, 2004; Chang, 2006).

It should be mentioned that most suppliers and distributors of mushroom DSs
provide very little and highly variable information on the source of their materials,
ways of preparation, and composition. The field of mushroom DSs today is very
far from unification and standardization (Wasser et al., 2000a, b; Chang and
Buswell, 2003).

One of the difficulties facing this area is the lack of international consistency
in the regulatory management of DSs per se. In some countries regulation is
effectively based on a three-category system, that is, foods, medicines, and DSs
(or alternate term). In other countries, regulations pertaining to DSs (or other)
generally sit under a broader (two-category) legislative system for either foods
or medicines. By way of example of the potential confusion inherent in some
of these systems, the New Zealand Dietary Supplements Regulations (NZDSR)
regulate products that are predominantly therapeutic products in Australia, yet the
NZDSR place them under the Food Act (1981) in New Zealand. Countries that
have instituted a three-category approach include New Zealand, the United States,
Canada (proposed system), Europe, India, and Japan whereas countries such as
Australia and the United Kingdom use a two-category system, that is, foods and
medicines (under therapeutic products) [Australia New Zealand Food Authority
(ANZFA), 2002].



202 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

6.2 LEGAL AND REGULATORY ISSUES OF INTRODUCING AND
CONTROLLING DIETARY SUPPLEMENTS FROM MEDICINAL
MUSHROOMS IN DIFFERENT COUNTRIES

6.2.1 World Health Organization Guidelines

In 1991, the World Health Organization (WHO) published its Guidelines for the
Assessment of Herbal Medicines. This document was created over a period of five
years (1986-1991) at several international conferences of drug regulatory author-
ities and is based on the WHO's recognition that 80% of the world's population in
developing countries relies on traditional medicine; "a major part of the traditional
therapies involves the use of active constituents of plant extracts"; and "consider-
able growth has occurred in popular, official, and commercial interest in the use of
natural products."

The objective of the WHO guidelines "is to define basic criteria for the evalu-
ation of quality, safety, and efficacy" of all herbal (as usual, including mushrooms
among the herbs) medicines. "As a general rule in this assessment, traditional expe-
rience means that long-term use as well as the medical, historical, and ethnological
background of those products shall be taken into account" (WHO, 1991). Depend-
ing on each country's situation, "the definition of long-term use may vary, but
would be at least several decades. . . . Prolonged and apparently uneventful use of
a substance usually offers testimony of its safety."

According to Alkerele (1992), "safety should be overriding criterion in the
selection of herbal medicines for use in health service systems." The WHO guide-
lines call for various assessments of quality, safety, efficacy, and intended use of
herbal medicines. The guidelines call for reference to pharmacopeia monographs,
if they exist. If none exist, a manufacturer applying for marketing licenses or
registration should supply a monograph with the same components as an official
pharmacopeia. All procedures should correspond to good manufacturing practices
(GMPs), and there needs to be standard testing of the product in its final pack-
aging form. The assessment should also make a distinction between old and new
combination products (Wasser et al., 2000a, b, 2004).

With regard to safety, "a guiding principle should be that if the product has been
traditionally used without demonstrated harm, no specific restrictive regulatory
action should be undertaken unless new evidence demands a revised risk-benefit
assessment" (Alkerele, 1992).

Finally, consumer product information is recommended, including a quantita-
tive list of active ingredients, dosage, dosage form, indications, mode of admin-
istration, duration of use, any major adverse effects, contraindications, warnings,
and so on (Wasser et al., 2000a, b, 2004; Chang and Buswell, 2003; Chang, 2006).

6.2.2 Codex Alimentarius

The Codex Alimentarius Commission implements the joint Food and Agriculture
Organization (FAO, 2005) and WHO Food Standards Programme, the purpose of
which is to protect the health of consumers and to ensure fair practices in the food



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 203

trade. The Codex Alimentarius (Latin, meaning food law or code) is a collection
of internationally adopted food standards presented in a uniform manner. It also
includes provisions of an advisory nature in the form of codes of practice, guide-
lines, and other recommended measures to assist in achieving the purposes of the
Codex Alimentarius. The commission has expressed the view that codes of prac-
tice might provide useful checklists of requirements for national food control or
enforcement authorities. The publication of the Codex Alimentarius is intended to
guide and promote the elaboration and establishment of definitions and require-
ments for foods, to assist in their harmonization, and, in doing so, to facilitate
international trade (FAO, 2005).

The twenty-eighth session of the Codex Alimentarius Commission was held
during July 2005. This event has been the subject of considerable controversy, in
part because many member countries regulate substances as therapeutic goods or
pharmaceuticals and not as foods (if they were not foods, they would be excluded
from the Codex Alimentarius). The Codex seeks not to ban supplements but to
subject them to labeling and composition requirements. Some groups have pointed
to greater concerns related to restrictions on dietary supplement ingredients in
Europe via the European Food Supplements Directive (which utilizes approved
lists of ingredients and ingredient forms) and potentially restrictive dosage limits
to be based on a Codex model via the FAO and WHO Nutrient Risk Assessment
Project. The Codex regulations have been rejected by the FDA in the United States
but accepted by the European Union (EU) trading bloc.

6.2.3 United States

Nearly all Americans have benefited from natural nutritional supplements of some
kind in their lives. Whether taking a multivitamin or eating breakfast cereal forti-
fied with nutrients, people all over the world live healthier lives by supplementing
their diets. Surveys estimate that more than half the U.S. population, or more than
100 million Americans, use DSs such as vitamins, minerals, and herbs as a safe
and natural way to maintain good health and supplement inadequate diets (Natural
Products Association, 2007).

There are approximately 10,000 natural product stores in the United States. In
2004, The U.S. nutrition industry reached sales of $68.6 billion. The industry is
broken down into the following categories (in billions): functional foods $24.5,
supplements $20.3, natural/organic foods $18.4, and natural personal care $5.5.

Natural/organic foods and natural personal care products are the fastest grow-
ing segment of the industry, at a rate of 10% per year, taking 35% of the market.
In the United States, virtually all facets of DS manufacturing, labeling, and adver-
tising are covered by extensive regulations issued and enforced by the Center for
Food Safety and Applied Nutrition (CFSAN, 2005a) and the U.S. Federal Trade
Commission (FTC, 2001).

For decades the FDA regulated DSs as foods to ensure that they were safe and
wholesome and that their labeling was truthful and not misleading. An impor-
tant facet of ensuring safety was the FDA's evaluation of the safety of all new



204 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

ingredients, including those used in DSs, under the 1958 Food Additive Amend-
ments to the Federal Food, Drug, and Cosmetic Act (Zeisel, 1999). GRAS is an
acronym for the phrase "generally recognized as safe." Under Sections 201(s) and
409 of the Federal Food, Drug, and Cosmetic Act, any substance that is inten-
tionally added to food is a food additive, that is, subject to premarket review
and approval by FDA, unless the substance is generally recognized, among qual-
ified experts, as having been adequately shown to be safe under the conditions
of its intended use or unless the use of the substance is otherwise excluded from
the definition of a food additive. For example, substances whose use meets the
definition of a pesticide, a dietary ingredient of a dietary supplement, a color addi-
tive, a new animal drug, or a substance approved for such use prior to September
6, 1958, are excluded from the definition of food additive. Sections 201(s) and
409 were enacted in 1958 as part of the Food Additives Amendment to the act.
While it is impracticable to list all ingredients whose use is GRAS, the FDA pub-
lished a partial list of food ingredients whose use is GRAS to aid the industry's
understanding of what did not require approval. The use of a food substance may
be GRAS either through scientific procedures or, for a substance used in food
before 1958, through experience based on common use in food. Interestingly, the
4-hydroxymethylbenzenediazonium (HMBD) ion has been detected in the com-
monly cultivated and consumed mushroom Agaricus bisporus at levels near 0.6
ppm (Ross et al., 1982). HMBD is a fungal metabolite of agaritine, a naturally
occurring substance found in Agaricus species. HMBD has been implicated in
causing DNA strand breaks at 1.2 mg per 70-kg person (Hiramoto et al., 1995).
The calculated safe dose is less than 4 g mushroom per day or one meal every 100
days. However, since mushrooms are GRAS, HMBD is exempt in this food.

However, with passage of the DSHEA, Congress amended the Food, Drug, and
Cosmetic Act to include several provisions that apply only to DSs and dietary
ingredients of DSs. As a result of these provisions, dietary ingredients used in DSs
are no longer subject to the premarket safety evaluations required for other new
food ingredients or for new uses of old food ingredients. However, they must sat-
isfy several other safety provisions. The Council for Responsible Nutrition (CRN,
2002) outlines different regulations as they apply to foods, DSs, and drugs when
reviewing products.

The DSHEA acknowledges that millions of consumers believe that DSs may
help to augment daily diets and provide health benefits. The intent of Congress in
enacting the DSHEA was to meet the concerns of consumers and manufacturers
to help ensure that safe and appropriately labeled products are available to those
who want to use them. In the findings associated with the DSHEA, Congress stated
that there may be a positive relationship between sound dietary practice and good
health and that, although further scientific research is needed, there may be a con-
nection between dietary supplement use, reduced health care expenses, and disease
prevention.

One of the most important matters of regulation, in which the FDA has
invested much effort in recent years, is the labeling of DS products. The new
nutrition-labeling format for DSs includes a supplement facts box, which lists



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 205

vitamins and minerals always in the same order. The new labels show the amount
of each nutrient in terms of metric quantities (e.g., 60 mg of vitamin C) and in
terms of percentages of the daily value (DV), if one has been established. All
ingredients in the product must be listed, either within the fact box or in a separate
list of ingredients below the fact box. For extracts, additional information may be
provided including the solvent used and the concentration of the extract (Wasser
et al., 2000a, b, 2004).

The final rules for structure-function (SF) claims for DSs are given under the
DSHEA. In April 1988, the FDA published proposed regulations that included a
highly controversial redefinition of the word "disease." Many industry and con-
sumer groups viewed this as an attempt to restrict the scope of the SF claims under
the DSHEA. Since then, the FDA has received 235,000 comments from the pub-
lic about this measure: 213,000 as form letters circulated by consumer and trade
groups and 22,000 as individual letters from consumers, members of industry, and
other interested parties (Wasser et al., 2000a, b, 2004; Blumenthal, 2001).

Formerly, the definition of disease was "any deviation from, impairment of, or
interruption of the normal structure of function." Now, the FDA uses the definition
"damage to an organ, structure, or system. . ." By this alternation of the definition,
the FDA automatically reduces the range or number of health claims for DSs.

On January 6, 2000, the FDA issued its final regulations on SF claims for DSs
under the DSHEA of 1994 (65 FR 1000-1050). The full text is available on the
Internet (FDA, 2000). In these rules, a significant shift is made in relation to SF
claims for over-the-counter (OTC) drugs including DSs. In particular, the FDA has
enlarged the range of SF claims by agreeing with the comments by the American
Herbal Products Association (AHPA) that some claims are not disease claims but,
instead, are claims that deal with the SF of the body. Thus, DSs will be able to make
claims for antacid, digestive aid, short-term laxative, and other uses previously
prohibited to DSs (Blumenthal, 2001).

Many examples of conditions and statements for which SF claims are allowed
under the DSHEA are worked out by these regulations. They include many that are
important for mushroom-derived DSs, such as "immune system function," "main-
tenance of cholesterol levels that are already within the normal range," "tonic," and
"as part of the diet to maintain blood sugar levels." Many other examples remain
disease claims, such as "anti-inflammatory," "lowers cholesterol," and "controls
blood sugar in persons with insufficient insulin." Another important regulation
states that "the name of a product should not contain the name or recognizable
portion of the name of the disease" (Wasser et al., 2000a, b, 2004).

Thus, the FDA regulates all key steps of DS development and application in
the United States. The seemingly important drawback of the FDA empowerment
is that consumers have to be injured prior to banning the product on the shelf.
However, if the FDA has indications that a DS is unsafe, it can issue a warning that
might have an impact on the consumer.

In April 2005, the CFSAN/Office of Nutritional Products, Labeling, and Dietary
Supplements released the Dietary Supplement Labeling Guide. The guide applies
to both dietary supplement ingredients and finished dietary supplement products



206 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

manufactured or produced in the United States as well as those produced in other
countries. The guide covers all aspects of dietary supplement labeling, including
requirements related to statements of identity, net quantity listing supplement facts
panels, ingredient listing, health and SF claims, and premarket notifications for
new dietary ingredients. There are several types of claims (CFSAN, 2005a, b):

(a) An authorized health claim is an "explicit or implied characterization of a
relationship between a substance and a disease or a health-related condition."
According to the FDA, a health claim "describes the effect a substance has
on reducing the risk of or preventing a disease, e.g., 'calcium may reduce
the risk of osteoporosis.'" This type of claim requires significant scientific
agreement and must be authorized by the FDA.

(b) A qualified health claim is supported by less scientific evidence than an
authorized health claim. The FDA requires that qualified claims be accom-
panied by a disclaimer that explains the level of the scientific evidence sup-
porting the relationship.

(c) A SF claim describes the role of a substance intended to maintain the struc-
ture or function of the body. Structure -function claims do not require preap-
proval by the FDA. Structure-function claims are permitted if such state-
ment is truthful and not misleading, including a required disclaimer, and the
marketer notifies the FDA of the claim no later than 30 days after the first
marketing of the product.

When the FDA learns of a supplement claim it believes violates the provisions
of the Food, Drug and Cosmetic Act or regulations promulgated by the act, it sends
a letter to the supplement manufacturer or marketer advising them of the question-
able claims, the potential violations raised by those claims, and a request that the
claims be withdrawn. The FDA posts these letters in a database on the Internet that
is searchable by date, month, and type of violation. Responses to these letters are
also posted when received.

The guide is intended to address questions about dietary supplement labeling
issues and reflects the FDA's current position on these issues. However, it is not
legally binding or precedent setting but rather is merely an interpretation of the
applicable statutes and regulations to educate the industry on how the FDA believes
compliance can be met (CFSAN, 2005b).

Several examples of actions the FDA has taken against violators of the health
claims regulations are available. For example, in December 2001, the FDA's New
York district office recommended detention without physical examination for
Essence of Mushrooms capsules, 400 mg. The product, manufactured by Windsor
Health Products, Kowloon, Hong Kong, was shipped as vitamins via Federal
Express. However, FDA examination found accompanying labeling promoting
the product for treatment of cancer. In addition, the labeling also identified
the manufacturer's website, which was found to be promoting the Essence of
Mushrooms as an alternative therapy for cancer. The FDA refused entry of the
product.



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 207

In 2005, a company named Vitapurity, headquartered in the United States, made
therapeutic claims on its website regarding Miracle Mushroom Blend. The com-
pany established that this product was a drug because it was intended for use in
the cure, mitigation, treatment, or prevention of diseases. The marketing of this
product with these claims violates the Federal Food, Drug, and Cosmetic Act. The
claims were as follows:

• "Reishi Mushroom was comparable to hydrocortisone and aspirin in its ability
to reduce inflammation."

• "When more than 2,000 patients with chronic bronchitis were given Reishi
Mushroom Extract, 60 to 90 percent of these patients showed a marked
improvement in health."

• "Effective in treating conditions such as stomach ulcers and high blood pres-
sure."

• "The Shiitake Mushroom is ... an aid in the prevention of cerebral hemor-
rhagic strokes."

• "Shiitake Mushrooms are promoted to fight the development and progression
of cancer and AIDS. . . . These mushrooms are also said to help prevent heart
disease by lowering cholesterol levels in the blood and treating infections."

• "Research in Japan shows that the mushroom itself can lower blood pressure
in those with hypertension."

• "Giving Shiitake to patients with probable pre AIDS improved the patients
[sic] general conditions and improved their immune status. . . . This agent
may prove to be effective in the suppression of the AIDS condition."

• "The compounds contained in Maitake have the capacity to . . . inhibit tumor
growth."

• "People with Type 2 Diabetes may also benefit from Maitake."

• "Maitake Mushrooms may have the ability to . . . induce apoptosis (cell death)
in cancer cells."

• "Mushroom extracts may improve overall survival and quality of life for can-
cer patients."

The FDA warned Vitapurity the product was not GRAS and effective for the
above-referenced conditions and, therefore, the product is a "new drug" and may
not be legally marketed in the United States.

A similar warning was issued to Health Food Emporium in Idaho, which made
similar claims regarding Garden of Life RM-10. The product contains a combi-
nation of 10 organic medicinal mushrooms which "aid the immune system to
fight Viral Disorders, Allergies, Asthma, Inflammatory Bowel Disease, Hepati-
tis, Cancer, Candida Yeast Overgrowth, Diabetes, Parasitic Infections, Multiple
Sclerosis, Psoriasis, Eczema, Aids, Rheumatoid Arthritis, Lupus, etc." Today the
company lists the following statement on its website: "Statements on this website
have not been evaluated by the Food and Drug Administration. These products are



208 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

not intended to diagnose, treat, cure, or prevent any disease, but rather are dietary
supplements intended solely for nutritional use."

In October 2006 the FDA announced a public hearing on the regulation of cer-
tain conventional foods that companies are marketing as "functional foods." The
purpose of the hearing was for the agency to share its current regulatory framework
and rationale regarding the safety evaluation and labeling of these foods and to
solicit information and comments from interested persons on how the FDA should
regulate these foods under the agency's existing legal authority [U.S. Department
of Health and Human Services (DHHS), 2006] .

In December 2006, the Senate passed the Dietary Supplement and Nonpre-
scription Drug Consumer Protection Act, dubbed the AER bill (S.3546.ES). The
legislation will amend the Federal Food, Drug and Cosmetic Act to require the
reporting of "serious" adverse events for both OTC drugs and dietary supplements
to the FDA. President Bush signed the act on December 22, 2006. The bipartisan
legislation includes several provisions that were instrumental in earning the Natu-
ral Products Association's support. Under this bill, companies would be required
to include contact information on their products' labels for consumers to use in
reporting adverse events. They would further be required to notify the FDA of any
serious adverse event reports received within 15 business days. According to the
bill, the term "adverse event" means any health-related event associated with the
use of a nonprescription drug that is adverse, including:

(a) An event occurring from an overdose of the drug, whether accidental or
intentional

(b) An event occurring from abuse of the drug

(c) An event occurring from withdrawal from the drug

(d) Any failure of expected pharmacological action of the drug

The term "serious adverse event" is an adverse event that results in death, a
life-threatening experience, in-patient hospitalization, a persistent or significant
disability or incapacity, or a congenital anomaly or birth defect or requires, based
on reasonable medical judgment, a medical or surgical intervention to prevent one
of the above outcomes. Among these key provisions are those requiring that only
serious adverse events, not just any complaint, be reported, exempting retail stores
from reporting directly to the FDA and preempting a potential patchwork of state
laws on the issue.

6.2.4 European Union

In Europe, the DSs are known mostly as "food supplements." "Health food" is
a marketing term rather than a legal term, normally denoting a product sold in
specialty stores (Smith et al., 1996; Wasser et al., 2004). In addition, the term DS
is used by the EU for products regulated as Foods for Particular Nutritional Uses
(PARNUTS).



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 209

Consumer safety is Europe's primary concern. A good example of the state-
ment is that individual countries have already banned several supplements avail-
able in the United States. European countries follow the precautionary princi-
ple, which means that when they suspect a product may cause harm, they do not
wait for proof before they take action against it. For example, the FDA warned
American consumers in March 2002 that supplements containing kava, promoted
as a relaxant, may cause liver injury — sometimes so severe that a transplant is
required, sometimes even fatal, based on the case of a 45-year-old woman using
kava who suddenly required a liver transplant as well as reports of 25 similar cases
in Europe. However, while the FDA issued only a warning, several European coun-
tries, including Germany and France, banned the sale of kava (Hecht, 2003).

As the European countries vary in the ways they regulate DSs, the European
Commission (EC) aims to provide a uniform standard of quality and safety for all
member states. The EC divided DSs into two groups for the purpose of regulation:
vitamins/minerals and herbals. The groups are treated separately.

Directive 2002/46/EC, which harmonized EC legislation on vitamins and miner-
als as food supplements, was published in the Official Journal of the EC (L183/51)
on June 10, 2002 (Directive 2002/46/EC). The directive defined the term "food
supplements" and set out labeling requirements. The directive did not immediately
outlaw any products already on the market.

The set standards enable a product approved in one country to be sold in other
member countries. Also, everything in the EC directive follows from the basic prin-
ciple that, under normal circumstances, a balanced diet can provide the necessary
nutrients for development and health. For instance, the directive forbids manufac-
turers to state or even suggest the contrary; a manufacturer may not state that a
supplement is a substitute for a varied diet or that it can prevent, treat, or cure a
disease. Furthermore, rather than setting only general guidelines, the directive lists
exactly which vitamins and minerals may be sold in member countries and in what
form (Hecht, 2003).

Despite the directive's apparent advantages for the consumer, there are some
disadvantages. It will tighten regulation in some countries but loosen it in others
that previously had stricter laws. It also allows each country to decide whether it
will require government notification from manufacturers when a new supplement
comes on the market.

The EC is developing a list of alternative herbal substances. It includes details
about each substance: what it is to be used for, the strength and daily dose, how
it is to be administered, and possible drug interactions or adverse effects. During
2005, the Committee on Herbal Medicinal Products (HMPC) adopted regulatory
guidance documents, which include guidelines on the documentation to be submit-
ted for inclusion in the list of herbal substances, preparations, and combinations
thereof (EMEA/HMPC/107399/2005). The guideline is in effect now, and the var-
ious documents are updated from time to time (HMPC 2006).

The EU relates to herbal DSs in the same way it does to conventional med-
ications. European countries vary in the degree to which they use herbals and
how carefully they regulate them. Germany, Austria, Switzerland, and France lean



210 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

toward a stricter approach, while the Netherlands and the United Kingdom have
traditionally been more lenient. The same products sold as medicine in Germany
are sold as food supplements in the Netherlands (Hecht, 2003).

Germany was in the vanguard of the move toward stricter herbal regulation for
all EU countries, resulting in Directive 2001/83/EC. Although the directive does
not relate to homeopathic medicines, it does cover all herbals used in the treat-
ment or prevention of disease or as modifiers of physiological functions, including
inducing relaxation. Therefore, under the directive, "natural" will no longer auto-
matically mean "good." Manufacturers are not allowed to claim that a product is
safe and effective simply because it is natural (Hecht, 2003). EC legislation does
not refer to mushroom products directly.

The dietary supplements industry in Europe strongly opposed the food supple-
ments directive (2002/46/EC). A large number of consumers throughout Europe,
including over one million in the United Kingdom, and many doctors and scien-
tists have signed petitions against what are viewed by the petitioners as unjustified
restrictions of consumer choice. The European Court of Justice ruled on July 12,
2005, that the directive is valid although the court's own advocate general advised
that the declaration was invalid under EU law [Court of Justice of the European
Communities (CURIA), 2005].

At the end of 2006, Regulation (EC) No 1924/2006 on the use of nutrition and
health claims for foods was adopted by the Council and Parliament. This regulation
made standard rules for the use of health or nutritional claims (such as "low fat,"
"high fibre," and "helps lower cholesterol") on foodstuffs based on nutrient pro-
files. The Health Claims Regulation ensures that any claim made on a food label
in the EU is clear, accurate, and substantiated. In doing so, it enables consumers
to make informed and meaningful choices when it comes to food and drinks. This
also contributes to a higher level of human health protection, as it ties in with the
EC campaign for healthier lifestyle choices by allowing citizens to know exactly
what they are consuming. The regulation also aims to ensure fair competition and
promote and protect innovation in the area of food. Only products offering gen-
uine health or nutritional benefits will be allowed to refer to them on their labels
(European Commission, 2006).

6.2.5 Canada

In Canada, the regulatory framework governing foods, as in other major jurisdic-
tions, is evolving. Food research, product and process innovation, and change in
consumer behavior are all outpacing the adaptation of regulation to new market
realities. Among these realities is growing consumer awareness of nutrition and
interest in health promotion. Increasingly, this awareness is manifested through
consumption of particular foods and dietary supplements believed to contribute to
good health and, in some cases, to hold therapeutic value in the treatment or pre-
vention of specific afflictions or diseases (Smith et al., 1996; Wasser et al., 2004).

Many of these food products are becoming commonly known as nutraceuticals,
or "functional foods." The regulatory environment governing functional foods is
so restrictive that the development of a functional foods industry or even functional



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 21 1

food products in Canada will be severely impaired, if not entirely precluded (Smith
et al., 1996; Wasser et al., 2004).

The Food and Drugs Act (F&DA) is the primary piece of legislation governing
the safety and quality of food sold in Canada. Its scope includes: food labeling,
advertising and claims; food standards and compositional requirements; fortifica-
tion; foods for special dietary uses; food additives; chemical and microbial hazards;
veterinary drug residues; packaging material and pesticides. Created in 1953, the
founding premise of the F&DA is to protect the public from adulterated food,
drink, and drugs and their associated health effects. That same orientation contin-
ues today, and any amendments to the Act must be aligned with this premise.

Health Canada is an agency similar to the U.S. Food and Drug Administration.
It is Health Canada's responsibility to establish the detailed set of regulations and
guidelines that accompany the F&DA to protect the consumer from being misled
about the characteristics of food products. However, the Canadian Food Inspection
Agency (CFIA) is responsible for enforcing the F&DA regulations.

The F&DA specifically forbids the labeling or advertising of food in a manner
that is false, misleading, deceptive, or likely to create an erroneous impression
regarding its character, value, quantity, composition, merit, or safety. Section 3
makes it an offence to advertise or sell a food to the general public as a treatment,
preventative, or cure for any disease referred to in Schedule A of the FD&A. Heart
disease, hypertension, and cancer are examples of diseases listed in Schedule A.
Section 3 and Schedule A of the F&DA prohibits diet-related health messages from
being used on packaging and in advertising.

In an effort to ensure that consumers are provided with consistent information
on the foods they buy, regulations introduced on January 1, 2003 require
mandatory nutrition labeling. The nutrition labeling requirements to the Food and
Drug Regulations provided a three year transition period, making the Nutrition
Facts table mandatory for most prepackaged foods since December 12, 2005
(CFIA, 2003)

The framework for the authorization of health claims for foods in Canada dis-
tinguishes between generic claims and product-specific claims. Generic claims are
associated with nutrients, other food components, foods or food groups that con-
tribute to a dietary pattern of eating associated with a reduction in risk for a disease.
Product-specific claims are associated with a specific food which has demonstrated
a measurable health benefit beyond normal body function, growth, development,
or maintenance of good health. The five authorized health claims are considered
generic claims (Kennedy 2006).

Currently there are three types of generic claims that are associated with nutri-
ents, other food components, foods, or food groups that contribute to a dietary pat-
tern of eating associated with a reduction in risk for a disease. The types are: nutri-
ent content claims, biological role claims, and diet-related health claims. Nutri-
tion claims typically highlight one nutrient found in a food. To ensure nutrition
claims comply with the F&DA requirements (i.e., truthful and not misleading)
Health Canada has established specific rules regarding the language, format, and
compositional criteria for each of these three types of claims. Based on these



212 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

generic types, Health Canada authorized five health claims, all of them applicable
to mushrooms:

1 . "A healthy diet containing foods high in potassium and low in sodium may
reduce the risk of high blood pressure, a risk factor for stroke and heart
disease."

2. "A healthy diet with adequate calcium and vitamin D, and regular physical
activity, help to achieve strong bones and may reduce the risk of osteopo-
rosis."

3. "A healthy diet low in saturated and trans fats may reduce the risk of heart
disease."

4. "A healthy diet rich in a variety of vegetables and fruit may help reduce the
risk of some types of cancer."

5. "Won't cause cavities" or "Does not promote tooth decay" or "Does not
promote dental caries" or "Noncariogenic."

Currently Health Canada is embarking on a major renewal of its health protec-
tion legislation and has proposed the creation of a new Canada Health Protection
Act to replace a number of pieces of legislation, including the Food and Drugs Act.
The goal is to integrate them into a single piece of legislation that has an overall
general policy direction. Until new legislation replaces the F&DA, diet and health
claims for foods must be made within the existing legislative framework (Kennedy
2006).

6.2.6 Australia and New Zealand

The use of natural products in Australia has been widespread for many years, as
evidenced by the presence of an active organization of herbalists formed in 1920
(Blumenthal, 1999). In 1985, the Australian Parliament established the Working
Party on Natural and Nutrition Supplements to review the quality, safety, efficacy,
and labeling of herbs and related products, including mushrooms, for appropri-
ate regulation under the Therapeutic Goods Act of 1990 (Wasser et al., 2000a, b,
2004).

The 1990 South Australian Working Party on Natural and Nutritional Supple-
ments report dealt with approximately 1 144 herbs and "therapeutic substances,"
dividing them into three groups, each having a recommended level of control and
labeling, "appropriate to their level of toxicity."

As in the EU and United States, food regulations between New Zealand and
Australia were awaiting harmonization and standardization. The basic body
responsible for this was the ANZFA. The ANZFA's role is to protect the health
and safety of people in Australia and New Zealand by maintaining a safe food
supply. The ANZFA is a partnership between the Commonwealth government,
Australian States and Territories governments, and the New Zealand government.
As an independent expert body, the ANZFA is responsible for developing and
reviewing food standards for both Australia and New Zealand. The ANZFA makes



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 213

recommendations to change the food standards to the Australia New Zealand
Food Standards Council, a ministerial council made up of commonwealth, state
and territory, and New Zealand health ministers. If the council approves the
recommendations made by the ANZFA, the food standards are automatically
adopted as regulations into the food laws of the Australian States and Territories
and New Zealand (ANZFA, 2002).

Specific regulations were applied to novel and special-purpose foods. DSs
derived from fungi are included (along with herbs and species) into the category
of novel foods. "Novel food" means a nontraditional food (a food that does not
have a history of significant human consumption by the broad community in
Australia or New Zealand) for which there is insufficient knowledge in the broad
community to enable safe use in the form or context in which it is presented,
taking into account:

(a) Composition or structure of product

(b) Levels of undesirable substances in product

(c) Known potential for adverse effects in humans

(d) Traditional preparation and cooking methods

(e) Patterns and levels of consumption of product

Traditional foods of a particular community may be considered novel if they
are made available to a new or wider community without adequate information
regarding applicable presentation and use. Novel foods can take on many forms,
and not all are natural substances. There is an increase in the number and vari-
ety of novel foods on the market as a result of technological developments, trade
opportunities, scientific advances, and increasing ethnic diversity of the population
(ANZFA, 2002)

The New Zealand Food Safety Authority (NZFSA) was established in July
2002 to improve the effectiveness of New Zealand's food safety system by coordi-
nating and harmonizing food safety efforts. Specifically, New Zealand wanted to
address inconsistencies between the methods used in the Ministry of Agriculture
and Forestry's export food safety program and the Ministry of Health's domes-
tic food safety program. The NZFSA has farm-to-table responsibilities — from
primary production through processing to retailers, importing, and exporting as
well as responsibility for consumer education [U.S. General Accountability Office
(GAO), 2005].

The NZFSA has two main areas of focus: to protect and promote public health
and safety through the administration of food-related legislation and to facilitate
access to markets for New Zealand food products and related products (State Ser-
vices Commission, 2006).

6.2.7 Japan

In Japan, food with health claims (FHC) refers to foods that comply with the spec-
ifications and standards established by the Minister of Health, Labor and Welfare



214 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

(MHLW) and are labeled with certain nutritional or health functions. These foods
are categorized into two groups, according to differences in purpose and function:
(a) foods for specified health uses (FOSHU), that is, foods officially approved to
claim their physiological effects on the human body, and (b) foods with nutri-
ent function claims (FNFC), that is, foods that are labeled with the functions of
nutritional ingredients (vitamins and minerals).

Under Japan's Nutrition Improvement Act of 1992 the regulatory system of
FOSHU was developed to delineate functional foods from the pharmaceutical reg-
ulations. The Japanese government defines FOSHU as "foods that are expected to
have certain health benefits, and have been licensed to bear a label claiming that
a person using them for a specified health use may expect to obtain the health
use through the consumption thereof (Shimizu, 2003). The classification or list
has no status outside Japan. As of January 8, 2004, FOSHU totaled 402 items.
Among them, 91 products have been approved since the beginning of 2003. As of
2005, FOSHU account for over $6 billion with over 500 approved products. The
total market value including non-FOSHU functional foods and DSs is three times
higher. FOSHU refer to foods containing ingredients with functions for health and
officially approved to claim its physiological effects on the human body. FOSHU
are intended to be consumed for the maintenance/promotion of health or special
health uses by people who wish to control health conditions, including blood pres-
sure or blood cholesterol. In order to sell a food as FOSHU, the assessment for the
safety of the food and effectiveness of the functions for health is required, and the
claim must be approved by the MHLW.

Requirements for FOSHU approval include the following:

Effectiveness on the human body is clearly proven.

Absence of any safety issues (animal toxicity tests, confirmation of effects in
the cases of excess intake, etc.).

Use of nutritionally appropriate ingredients (e.g., no excessive use of salt, etc.).

Guarantee of compatibility with product specifications by the time of consump-
tion.

Established quality control methods, such as specifications of products and
ingredients, processes, and methods of analysis.

In addition to "regular" FOSHU, new types of FOSHU were introduced to facil-
itate applicants for FOSHU approvals:

• Qualified FOSHU Food with health function which is not substantiated on
scientific evidence that meets the level of FOSHU or food with certain effec-
tiveness but without established mechanism of the effective element for the
function will be approved as qualified FOSHU.

• Standardized FOSHU Standards and specifications are established for foods
with sufficient FOSHU approvals and accumulation of scientific evidence.
Standardized FOSHU are approved when they meet the standards and speci-
fications.



REGULATORY ISSUES OF INTRODUCING AND CONTROLLING DIETARY SUPPLEMENTS 215

• Reduction of Disease Risk FOSHU This claim is permitted when reduction
of disease risk is clinically and nutritionally established in an ingredient.
FOSHU regulated products are typically novel products and not value-added
general-purpose foods. The majority of products are specifically focused on
prevention of disease and maintenance of health status rather than the direct
treatment of disease states. Health agencies of the Japanese government
develop and maintain the regulatory framework that covers products that are
to be regulated under the FOSHU system.

• Foods for Special Dietary Uses (FOSDU) These refer to foods that are
approved/permitted to display that the food is appropriate for specified
dietary use. There are five categories of FOSDU: (1) formulas for pregnant or
lactating women; (2) infant formulas; (3) foods for the elderly with difficulty
in masticating or swallowing; (4) medical foods for the ill; and (5) FOSHU.

• Prohibition of Exaggerated and Misleading Claims ( under Health Promo-
tion Law) Any claims related to health or function made on functional foods
must be relevant and substantiated by scientific evidence. Advertisements are
used in different media to promote food sales. Some are advertised to convey
positive effects on health maintenance and promotion not necessarily having
scientific evidence on the claim. When these advertisements are not regulated
and uncontrolled, consumers who believed the claim might miss an oppor-
tunity for an adequate medical consultation, resulting in adverse affects on
health. Under Paragraph 2, Article 32 of the Food Promotion Law, exagger-
ated and misleading claims are prohibited.

6.2.8 Israel

Hand in hand with the public's growing interest in health care, there has been
an increasing demand for natural health products considered both safe and med-
ically effective. However, on the one hand, many such products have not been
shown to meet efficacy and safety criteria and, therefore, cannot be registered
as pharmaceuticals. On the other hand, it is quite clear that some products do
have pharmacological activity and are being used for therapeutic or preventive
effects. In Israel, the marketing rules for food or DSs prevent their manufactur-
ers from claiming medicinal/healing properties that the product might have and
allow only limited health statements. As great demand for these products has cre-
ated tremendous notoriety, medicinal indications have been attributed to products
whose quality, efficacy, and safety have not been examined and proven according
to accepted medical criteria (Lavy et al., 2000; Wasser et al., 2004).

In October 2006 the Ministry of Health, Department of Food and Dietary Ser-
vices updated the list of mushroom species allowed as food and as DSs in Israel.
The change was necessary because the ministry wanted to align its policy with
those of other countries for products for which knowledge regarding safety was
accumulated. The list includes mushrooms on the FAO list, but only those labeled
as food or edible. Mushrooms on the list labeled as medicinal are not permit-
ted. Nineteen additional mushrooms are permitted and listed in a separate file.
An importer or cultivator may request the use of new species in writing.



216 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

The FAO list of mushrooms is annexed in the book Wild Edible Fungi: A
Global Overview of Their Use and Importance to People (Boa, 2004). Wild edible
fungi are collected for food and to earn money in more than 80 countries. There
is a huge diversity of different types, from truffles to milk-caps, chanterelles to
termite mushrooms, with more than 1100 species recorded during the preparation
of this book. A small group of species are of economic importance in terms of
exports, but the wider significance of wild edible fungi lies with their extensive
subsistence uses in developing countries. They provide a notable contribution to
diet in central and southern Africa during the months of the year when the supply
of food is often perilously low. Elsewhere they are a valued and valuable addition
to diets of rural people.



6.3 SAFETY AND DIVERSITY OF DIETARY SUPPLEMENT TYPES
FROM CULINARY-MEDICINAL MUSHROOMS

Safety of the substances considered is a central feature of any regulation mea-
sure. The substances might have obvious or hidden beneficial actions or exploit
a placebo effect as long as the ideas of health are attached to them in the pub-
lic's mind. However, their safety should be verified and proven as thoroughly as
possible.

Drugs that affect body functions such as immune response, blood pressure,
diuresis, and others are called pharmacodynamic agents. This is the way DSs are
evaluated. Pharmacodynamics is the spectrum of biological responses produced
by an intervention at a given time. The presence of therapeutic pharmacodynamic
activity implies a lack of safety at a sufficiently high dose. This means that a
mushroom preparation, like any other pharmacodynamic agent, cannot have phar-
macological action without toxicological action. A completely safe agent would be
without any activity whatsoever (Huxtable, 1999; Chang and Buswell, 1999, 2003;
Wasser et al., 2000a, b, 2004; Chang and Miles, 2004; Chang, 2006).

It is commonly believed that many botanicals and mushrooms can be con-
sidered safe because of their long history of usage. The shiitake mushroom, for
instance, was described in classical Chinese medicine (Shen Nong Ben Cao Jin,
Compendium of Material Medica of the East Han dynasty) as long as 2000 years
ago (Mizuno, 1999).

However, safety to a pharmacologist is a relative concept which is very differ-
ent from the public notion of safety as an absolute concept. Here, we outline the
reasons why we cannot take the safety of all mushroom-derived DSs for granted
simply because they were used for many centuries in traditional human cultures:

1. Considering the historical perspective, "safety" in traditional terms is very
different from that in modern times. First, mortality patterns of developed
societies today are very different from those of traditional ones. Life
expectancy in the United States today is 76 years or more (Fries and Crapo,
1981), but in medieval China it was 20 years less (these figures do not reflect



SAFETY AND DIVERSITY OF DIETARY SUPPLEMENT TYPES 21 7

a maximum life span, but only a median age at death). Death itself (the
likelihood of dying at a certain age) had a different kinetics in society. Sec-
ond, traditional users rarely had the means to evaluate long-term or chronic
toxicity of the agents. However, we do have cautionary instances of plants
that have been used medicinally for centuries and recently proved to carry
delayed toxic effects. One such example is comfrey {Symphytum officinale
L.), containing pyrrolizidine, alkaloids, which are acutely hepatoxic in high
doses or chronically hepatoxic in low doses (Huxtable, 1992). Nevertheless,
comfrey has been used since classic times as a blood purifier, for enlarged
glands, for female debility, and many more symptoms (Santillo, 1993). An
example of an ill effect is the mushroom Paxillus involutus (Batsch.: Fr.)
Fr. It was widely used in Europe, and it is still taken as a food in many
regions there. Paxillus syndrome was described only in 1971 (Schmidt et al.,
1971); in fact, this syndrome is an immunohemolytic anemia. A patient
who has eaten Paxillus over a long period (sometimes years) on occasion
may develop Paxillus syndrome in a short time period of 1 or 2 hours after
consumption. An antigen of still-unknown structure stimulates a severe
immune shock resulting in such symptoms as diarrhea, subicterus, oliguria,
anuria, hemoglobinuria, and renal pain (Flammer, 1980, 1983; Lefevre,
1982; Bresinsky and Besl, 1990; Wasser et al, 2000a, b, 2004).

2. Many supposedly traditional mushroom products are now marketed in ways
markedly different from those in the past. Today, larger amounts may typ-
ically be taken, or the material is used more frequently, or it is consumed
in the form of enriched extracts, and it may be taken simultaneously with
synthetic drugs. The user of shiitake in old China, for instance, could not
ingest as much active polysaccharide (lentinan) as a modern user taking it in
pure form extracted from shiitake as a DS. Notably, 200 kg of fresh mush-
rooms is needed for extraction of 31 g of lentinan (Chihara et al., 1970). This
heightens the possibility of ill effects from traditionally "safe" mushrooms.

3. Also, many mushrooms or mushroom preparations traditionally taken as
treatments for specific conditions are now often marketed for use as pro-
phylactic agents. The idea of DSs, in many cases, implies that they are taken
in the absence of any indicated conditions to prevent disturbances of health.

4. Finally, reliance on traditional use as an indication of safety involves a dan-
ger, namely, the poor information available to us from antiquity. Huxtable
(1999) recently carried out an analysis of historical sources of such informa-
tion that are in many cases contradictory and vague. Information on acute
toxicity may be located on the same page as the recommendations for use.
The identities of the plants are sometimes dubious. Herbalists copied exten-
sively from each other over thousands of years. The descriptions of symp-
toms are often vague and general (e.g., bloodstream disturbances or liver
malfunction) (Wasser et al., 2000a, b, 2004).

Besides the problems of direct toxicity of some individual nutrients, nutrient
supplementation can cause problems related to nutrient imbalances or adverse



218 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

interactions with medications. Many problems associated with high doses of a
single nutrient may reflect interactions resulting in a relative deficiency of another
nutrient. Many examples are available from studies of DSs that have been used
for a long time and are more common on the market then those derived from
mushrooms. High amounts of calcium inhibit absorption of iron (Cook et al.,
1991). Folic acid can mask hematological signs of vitamin B [2 deficiency, which,
if untreated, can result in irreversible neurological damage. Folic acid can also
interact adversely with anticonvulsant medications (Food and Nutrition Board,
1989). Abundant zinc supplementation can reduce copper status, impair immune
responses, and decrease high-density lipoprotein cholesterol levels..

Because we lack many strict data on the safety of many mushroom preparations,
we could only make estimates on the basis of available scientific knowledge. The
clear advantages of using mushroom-based DSs with regard to safety (as opposed
to herbal preparations) are the following:

1. The overwhelming majority of mushrooms used for production of DSs are
cultivated commercially (and not gathered in the wild). This guarantees
proper identification and pure, unadulterated products. In many cases it also
means genetic uniformity.

2. Mushrooms are easily propagated vegetatively and thus keep to one clone.
The mycelium can be stored for a long time, and the genetic and biochemical
consistency may be checked after considerable time.

3. The main advantage might be the fact that many mushrooms are capable of
growing in the form of mycelial biomass in submerged cultures (Buchalo,
1988; Chang and Buswell, 1996, 2003; Pointing et al., 2000; Wasser et al.,
2000a, b, 2004; Wasser and Reshetnikov, 2002a, b, c; Chang and Miles,
2004; Chang, 2006).

Chang (2006) suggests adopting the five "G" guidelines to enhance quality
mushroom products. Although the text was originally oriented toward mushroom
fruit bodies, it is still valid for other forms of mushroom products such as sub-
merged culturing:

1. GLP (Good Laboratory Practice) A known mushroom strain must be used;
the source and nature of the strain culture must be clearly documented and
should be properly maintained and preserved without contamination and
degeneration.

2. GAP (Good Agriculture Practice) Growth conditions must be known; the
substrate should be free of heavy metals and composed of consistent levels
of ingredients; the environmental conditions should include unpolluted air
and a good sanitary growth area. The product should be harvested at optimal
maturity and free of diseases.

3. GMP ( Good Manufacturing Practice) The parameters for process must be
known and maintained. The temperature, duration, and percentage of sol-
vates used in extraction should be constantly monitored.



SAFETY AND DIVERSITY OF DIETARY SUPPLEMENT TYPES



219



4. GPP (Good Production Practice) The following tests must be conducted:
a chemical analysis of the products to determine organic components and
heavy metal contents; a microbial analysis to determine if the type and level
of microorganisms present are within safe limits; and standardization of the
formulation of the products.

5. GCP (Good Clinical Practice) Medical practitioners must conduct
high-quality clinical trials, including double-blind studies, which should
be conducted to ensure standardization and allow appropriate dosage
determinations and product formulation for the effective treatment of a
particular health problem.

The fifth G is somewhat problematic to DS developers. Once a clinical trial on
humans is performed and bioactivity is confirmed, the product acquires the status
of unapproved active drug. Until the product is approved, it is banned from the
market because it contains an unapproved drug. The process of drug approval is
extremely expensive and lasts several years. By the time the product is approved,
the manufacturers may lose their business. This problem requires that only large
pharmaceutical companies run clinical trials and those large companies do not
engage in the research of traditional DSs. Another reason why DS developers do
not wish to transform a healthy product into a medical product is because some
health organizations, such as the FDA, do not approve a mixture of molecules,
as in the case of mycelial or fruit body extracts, but only approve well-defined,
patentable molecules. On the one hand, most DS marketers will be just happy
enough to sell their mushroom products without printed health claims. They will
advertise them in foreign markets that allow health claims on the Internet or with
word of mouth. On the other hand, perhaps a whole new industry, or even indus-
tries, will arise having greater economic value than those currently producing
mushrooms for food.

In 2004, the FDA published Guidance for Industry: Botanical Drug Products
(FDA, 2004). This guidance explains when a botanical drug may be marketed
under an OTC drug monograph and when FDA regulations require approval for
marketing of a new drug application (NDA). In addition, this document provides
sponsors with guidance on submitting investigational new drug (IND) applications
for botanical drug products, including those currently lawfully marketed as
foods (including conventional foods and dietary supplements) in the United
States. Botanical products are finished, labeled products that contain vegetable
matter as ingredients. A botanical product may be a food (including a dietary
supplement) or a drug (including a biological drug). The term "botanical" includes
plant materials, algae, and macroscopic fungi. It does not include fermentation
products such as extracts made from submerged fermentation of mushroom
biomass. Also, a botanical drug substance does not include a highly purified
or chemically modified substance derived from natural sources. The authors
are not aware of any clinical trials in the United States with mushrooms as the
botanical drug.



220 REGULATORY ISSUES OF MUSHROOMS AS FUNCTIONAL FOODS

6.4 SUBMERGED CULTURING AS BEST TECHNIQUE FOR
OBTAINING CONSISTENT AND SAFE MUSHROOM PRODUCTS

Today, approximately 80% of mushroom products are taken from fruit bodies
either collected in the wild or grown commercially. In both cases, the resulting
products are considerably diverse and unpredictable. The quality of mushroom
fruit bodies is strongly dependent on substrate composition and properties of its
ingredients, and usually these are far from constant. This is explained by the fact
that the main components for mushroom production are of available agricultural
and forest origin such as cereal straw, corn stakes, horse or chicken manure, and
wood sawdust.

The production of many biologically active substances is connected with mat-
uration processes of fruit bodies. Lovastatin was found concentrated primarily in
the lamellae and basidiospores, but not in the stipe or cap tissue, and its amount
depends on fruit body size and age (Gunde-Cimerman, 1999).

Variability of mushroom fruit body composition is the reason why processing
for extraction of polysaccharides from fruit bodies is not considered commercially
feasible, as physicochemical properties of the products resulting from these pro-
cesses are not known or regulated (Ohtsuka et al., 1977).

The cultivation of mushrooms for fruit body production is a long-term process,
taking one to several months for the first fruiting bodies to appear, depending on
species and substrate. By contrast, the growth of pure mushroom cultures in sub-
merged conditions in a liquid culture medium allows one to accelerate the speed of
growth and reduces its duration to several days. Optimization of culture medium
composition and the physicochemical conditions of growth allow regulation of
mushroom metabolism to obtain a high yield of biomass and large amounts of spe-
cific substances of consonant composition (Wasser et al., 2000a, b, 2002, 2004;
Wasser and Reshetnikov, 2002a, b, c; Wasser, 2002).

6.5 EXPERIENCES OF SEVEN COUNTRIES IN CONSOLIDATING
THEIR FOOD SAFETY SYSTEMS

The GAO examined seven countries in consolidating their food safety systems:
Canada, Denmark, Germany, Ireland, the Netherlands, New Zealand, and the
United Kingdom. These countries varied in their approaches and the extent to
which they consolidated. However, the countries' approaches were similar in one
respect: Each established a single agency to lead food safety management or
enforcement of food safety legislation. These countries had two primary reasons
for consolidating their food safety systems: public concern about the safety of
the food supply and the need to improve program effectiveness and efficiency.
The above-mentioned countries faced challenges in (1) deciding whether to place
the agency within the existing health or agriculture ministry or establish it as a
stand-alone agency while also determining what responsibilities the new agency
would have and (2) helping employees adjust to the new agency's culture and
support its priorities.



REFERENCES 221



Although none of the countries had analyzed the results of its consolidation,
government officials consistently stated that the net effect of their country's con-
solidation had been or would likely be beneficial. Officials in most countries stated
their new food safety agencies incurred consolidation start-up costs. However, in
each country, government officials believed that consolidation costs had been or
would likely be exceeded by the benefits. These officials and food industry and
consumer stakeholders cited significant qualitative improvements in the effective-
ness or efficiency of their food safety systems. These improvements include less
overlap in inspections, greater clarity in responsibilities, and more consistent or
timely enforcement of food safety laws and regulations. In addition to these qual-
itative benefits, officials from three countries, Canada, Denmark, and the Nether-
lands, identified areas where they believed financial savings may be achieved as a
result of consolidation. For example, in the Netherlands officials said that reduced
duplication in food safety inspections would likely result in decreased food safety
spending, and they anticipated savings from an expected 25% reduction in admin-
istrative and management personnel.

Although the seven reviewed countries are much smaller than the United States,
they are also high-income countries where consumers have very high expectations
for food safety. Consequently, those countries' experiences in consolidating food
safety systems can offer useful information to U.S. policymakers (GAO, 2005).

6.6 SUMMARY

In this chapter, we discussed legal and regulatory issues introducing and control-
ling DSs from medicinal mushrooms in different countries, including the United
States, the European Community, Canada, Australia, New Zealand, Japan, Israel,
and guidelines of the WHO. A lot of attention is drawn to the safety and diversity
of DS types from culinary-medicinal mushrooms. Most of the mushroom DSs
presently in the marketplace are highly diverse and there are currently few standard
protocols to ensure product quality. There must be thorough analysis and improved
quality and legal control which will, in turn, increase and maintain consumer con-
fidence and achieve the current and future standards set by national regulatory
authorities. We hope that these and future regulations will continue to provide and
protect stable medicinal mushroom products of reliable quality and that culturing
biotechnology will continue to be the best and the most progressive technique for
obtaining consistent and safe mushroom products.

REFERENCES

Alkerele, O. (1992). Summary of WHO guidelines for the assessment of herbal medicines.

Fitoterapia, 62, 99-110.
Australia New Zealand Food Authority (ANZFA) (2002). Initial assessment report (prepare

a new proposal — section 21). Proposal P235. Review of food-type dietary supplements.



222



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Available: www.foodstandards.gov.au/_srcfiles/P235_IA_Report_v2.pdf. Accessed Oct.
16,2007.

Blumenthal, M. (1999). Regulatory models for approval of botanicals as traditional
medicines. In Botanical Medicine. Eshkinazi, D., editor. New York: Mary Ann Liebert,
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INDEX



Accessory food factors, in nutrition, 23

Aconitase, in Lentinus edodes, 48

AC-PS polysaccharide, immunomodulatory

effects of, 168
Acquired immunodeficiency syndrome (AIDS),

mushrooms and, 21-22
Active ingredients, WHO guidelines and, 202
Adenocarcinoma, antimetastatic effects on,

171

Adenosine diphosphate (ADP), in Lentinus
edodes, 45

Adenosine triphosphate (ATP), in Lentinus

edodes genetics, 53
AER bill, 208

Aerobic pathways, in Lentinus edodes, 48
Africa, mushroom production in, 73
Agaricaceae, classification of, 80
Agaricales, 35

classification of, 80
Agaricus

cultivation of, 14

in developing countries, 22

US regulation of dietary supplements from,
204

Agaricus bisporus
antioxidants in, 92
antitumor polysaccharides from, 156
ash and mineral content of, 76
classification of, 80
cultivation of, 14, 18
dietary fiber in, 77
edibility of, 4
energy content of, 78
hypocholesterolemic effects of, 97
hypolipidemic effects of, 95
moisture content of, 73
in mushroom industry, 26
mushroom-related hypoglycemia and, 98-99
protein quality of, 88
US regulation of HMBD from, 204



vitamin content of, 76-77

world production of, 73
Agaricus bitorquis, cultivation of, 14
Agaricus blazei, 18, 85. See also Agaricus
brasiliensis

antimetastatic effects of, 172

antitumor polysaccharide-protein complexes
from, 158, 160, 164

antitumor polysaccharides from, 154, 155, 156

chemical composition of, 82

classification of, 80

FWE water extract from, 158, 172

polysaccharide-protein complex from, 167

polysaccharides isolated from, 148, 150-151

world production of, 72, 73
Agaricus blazei Murill (ABM),

immunomodulatory effects of, 164. See
also Agaricus murrill
Agaricus brasiliensis. See also Agaricus blazei

antitumor polysaccharides from, 154

bed preparation for, 18-19

biology and genetics of, 18

cultivation of, 18-19

polysaccharides isolated from, 151
Agaricus campestris

anatomy of, 3

mushroom-related hypoglycemia and, 98-99
Agaricus macrosporus, protein quality of, 88
Agaricus murrill, antiangiogenesis and, 173. See

also Agaricus blazei Murill (ABM)
Agaritine, US regulation of, 204
Agriculture

applied mushroom biology and, 9

biomass waste from, 10-11

mushroom cultivation as, 11-12
Agrocybe aegerita, 58, 84

chemical composition of, 82

classification of, 80

hypocholesterolemic effects of, 97

world production of, 72, 73



Mushrooms as Functional Foods, Edited by Peter C. K. Cheung
Copyright © 2008 John Wiley & Sons, Inc.



227



228 INDEX



Agrocybe chaxinggu, 85

chemical composition of, 82

classification of, 80
Agrocybe cylindracea

antioxidants in, 90

antitumor polysaccharides from, 154

chemical improvement of antitumor
polysaccharides from, 176

mushroom-related hypoglycemia and, 98

protein and amino acid content of, 74-75
Air filters, in Wolfiporia cocos cultivation, 121
Akavia, Eden, xxi, 199

Alanine, in conventional edible mushrooms, 74,
75

Alkaloids, in mushrooms, 88
a-glucans

in chemical improvement of antitumor

activity, 176-177
from mushrooms, 157
a -tocopherol, in mushroom antioxidant assays,
90, 92

Alternative herbal substances, European Union

regulation of, 209
Amanita, toxicity of, 6
Amanita muscaria
anatomy of, 3

antitumor polysaccharides from, 154
chemical improvement of antitumor
polysaccharides from, 176

Amanita phalloides, as poisonous, 4

Amatoxin, 6

American Diabetic Association (ADA), on

sclerotial dietary fiber, 124
American Herbal Products Association (AHPA),

205

Amimethylation, in chemical improvement of

antitumor activity, 176
Amino acids

in conventional edible mushrooms, 74-75
in cultivated mushroom dietary fiber, 77
in dietary supplements, 200
during early sclerotial ontogeny, 1 15
in mushrooms, 88-89
Amplified fragment length polymorphisms
(AFLPs), in generating Lentinus edodes
molecular markers, 48, 49, 50
Anaerobic pathways, in Lentinus edodes, 48
Anaerobic saccharolytic microflora, colonic

fermentation and, 129, 130
Androgen receptor (AR), 157
Angiogenesis, 172
Antiangiogenesis, 179

as antitumor mechanism, 172-173
Antiatherosclerotic effects, of mushrooms, 96



Anticancer preparations, regulation of

mushroom, 199-200
Anticonvulsant medications, folic acid and, 217
Antidiabetic drugs, mushroom-related

hypoglycemia and, 99
Antigenotoxicity, 179

Antigen-presenting cells (APCs), dendritic cells

as, 168
Antimetastasis, 179

as antitumor mechanism, 171-172
Antioxidants, in mushrooms, 89-94
Antiproliferation of cancer cells, as antitumor

mechanism, 153-161
Antisclerotic effects, of mushrooms, 94
Antitumor effects

of mushroom polysaccharides, 147-198
of sclerotia, 132-134
of Wolfiporia cocos, 120
Antitumor polysaccharide-protein complexes,

from mushrooms, 158-159
Antitumor polysaccharides, from mushrooms,
154-157

Antrodia camphorata, polysaccharide from, 168
Antrodia cinnamomea, antiangiogenesis and,
173

Antrodia malicola, antiangiogenesis and, 173
Antrodia xantha, antiangiogenesis and, 173
Antrodiella liebmannii, antiangiogenesis and,
173

Aphyllophorales, 119
classification of, 80
Apoptosis, 157, 159, 160, 168, 179
antimetastatic effects on, 171
as antitumor mechanism, 153-161
polysaccharides and, 148, 150, 152
Applied mushroom biology, 6, 7-11, 28-29

impact of, 9 - 1 1
Arabinogalactan, from mushrooms, 156
Arabinoglucan, from mushrooms, 155
Arabitol, in cultivated mushrooms, 78
Arbitrary primer polymeric chain reactions

(AP-PCRs), in generating Lentinus edodes
molecular markers, 48, 49, 50, 51, 61
Arginine, in conventional edible mushrooms, 74
Armillariella mellea, hypolipidemic effects of,
95

Armillariella tubescens

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154
Aromatic compounds

biosynthesis of, 93

in conventional edible mushrooms, 75
Artificial cultivation, of Ganoderma lucidum, 19



INDEX 229



Ascomycetes, 3, 149

Ascomycota, classification of, 80

Ascomycotina, sclerotia of, 1 12

Ash. See also Minerals

in conventional edible mushrooms, 75-76
in nonconventional edible mushrooms, 79, 8 1
in sclerotia, 123-124

Asia

Lentinus edodes from, 35

medicinal mushroom uses in, 25

medicinal uses of mushroom polysaccharides

in, 147, 150
mushroom production in, 22, 23, 73
Asiaticusins A/B, as mushroom antioxidants, 91,

92

Aspartic acid, in conventional edible

mushrooms, 74
Aspergillus candidus, biosynthesis of phenolic

compounds by, 93-94
Aspergillus oryzae, 56, 57
Aspergillus terreus, hypolipidemic effects of, 95
Atherosclerosis, hypercholesterolemia and, 94
Atkins, F. C, 26

ATOM polysaccharide-protein complex, 160,
167

Auricularia, world production of, 72, 73
Auricularia auricula

antitumor polysaccharides from, 154

dietary fiber in, 77

hypolipidemic effects of, 95
Auricularia auricula-judae

branching configurations of polysaccharides
from, 175

hypocholesterolemic effects of, 97

mushroom-related hypoglycemia and, 98
Auricularia fuscosuccinea, world production of,
73

Auricularia polymelia, hypolipidemic effects of,
95

Australia, 220

introducing medicinal-mushroom dietary

supplements in, 211-212
Australia New Zealand Food Authority

(ANZFA), 201,211-212
Australia New Zealand Food Standards Council,

211-212
Australian Parliament, 211
Authorized health claims, 206
Autotrophic agents, sclerotial ontogeny and, 113

Bacillus subtilis, 56, 57

Bakery products, sclerotial dietary fiber for, 128
Barley dietary fiber, sclerotial dietary fiber
versus, 126-127



Barrier, rind as, 116

Basic fibroblast growth factor (bFGF),

antiangiogenesis and, 172
Basidiomycetes, 3, 149

biosynthesis of phenolic compounds by, 93

cell walls of, 121-122

dietary fiber in, 77

life cycles of, 37

proximate composition of, 124
Basidiomycota, 35

classification of, 80
Basidiomycotina, 119

sclerotia of, 112, 118
Basidiospore formation, in Lentinus edodes,
44-47

Basidiospores, of Lentinus edodes, 14, 37, 38

B cells, dendritic cells and, 168

Beds, in Agaricus brasiliensis cultivation, 18-19

Befungin, regulation of, 200

Benzoic acid, biosynthesis of, 93

/i-carotene bleaching, in mushroom antioxidant

assays, 90
/i-glucans, 179

antitumor and immunomodulatory effects of
sclerotial, 132-134

branching configurations of, 174-175

in chemical improvement of antitumor
activity, 176-177

colonic fermentation and, 1 30

conformations of, 175-176

in cultivated mushroom dietary fiber, 78

D-fraction, 167-168

effects on hematopoietic stem cells, 170-171

grifolan and, 165-166

in immune response, 162

in vivo Ca/Mg absorption and, 132

mushroom-related hypoglycemia and, 98

from mushrooms, 150, 151, 152, 153,
154-155, 157

schizophyllan and, 165

in sclerotial dietary fiber, 125
/i-glucopyranosides, from mushrooms, 150
jg-glucosidases, in mushrooms, 10
P oxidation, in biosynthesis of phenolic

compounds, 93
Biochemistry, of sclerotia, 121-123
Biochemopreventives, regulation of, 200
Bioconversion efficiency, of mushrooms, 22
Bioconversion processes, mushrooms in, 1 1
Bioconversion technology, mushrooms in, 9
Biological efficiency, of mushrooms, 22
Biological response modifiers (BRMs)

mushroom products as, 201

polysaccharides and, 148, 161-162, 178



230 INDEX



Biology

of mushrooms, 6-11, 28-29

specialization in, 7
Biomacromolecules, polysaccharides as, 148
Biomass by-products, reuse of, 28
Biomass wastes, applied mushroom biology and,

9, 10-11, 28
Biopharmacology

mushroom polysaccharides in, 147-148

of sclerotia, 128-134
Bioprocessing, mushrooms and, 8, 23, 28
Bioremediation, mushroom, 7, 8, 9, 11, 28
Biosynthesis

by mushrooms, 10-11, 28

of phenolic compounds from fungi, 93-94
Biotechnology, mushroom, 7-8, 9, 23-25
Bitter components, in conventional edible

mushrooms, 75
Black truffle, 72

BLASTX, in Lentinus edodes genetics, 54
Blood vessels, in angiogenesis, 172
Bolbitiaceae

antioxidants in, 90

classification of, 80
Boletes, 72

Boletinus asiaticus, antioxidants in, 92
Boletus, 72

ash and mineral content of, 76

energy content of, 78
Bone marrow cells (BMCs), 170
Botanical products/botanicals

in dietary supplements, 200

regulation of, 218

safety of, 215
Botryotinia, sclerotia of, 112
Botrytis, lipid bodies in, 123
Botrytis cinerea, lipid bodies in, 123
Botrytis fabae, lipid bodies in, 123
Bottles, in Ganoderma lucidum cultivation,
20

Bowel function, in colonic fiber fermentation,
129

Bran, sclerotial dietary fiber versus, 127
Branching configuration, of antitumor

polysaccharides, 174-175
Brazil, Agaricus brasiliensis cultivation in, 1 8
Breast cancer/carcinoma

chemical improvement of antitumor activity

versus, 176
GLP effects on, 163

mushroom polysaccharides versus, 151, 159
Brown rot fungus, Wolfiporia cocos as, 120-121
Burkitt lymphoma, mushroom polysaccharide-
protein complexes versus, 160



CAAT box, in Lentinus edodes transcriptional

regulation, 55-56
Cadmium, in cultivated mushrooms, 76
Caffeic acid

biosynthesis of, 93

as mushroom antioxidant, 92
Calcium

in cultivated mushrooms, 76

in vitro sclerotial binding of, 128-129

in mushrooms, 24

in nonconventional edible mushrooms, 79

in sclerotial dietary fiber, 125
Calcium absorption, by sclerotia, 131-132
Calories, in mushrooms, 23-24
Canada, 220

food safety systems in, 219-220

introducing medicinal-mushroom dietary
supplements in, 210-211

mushroom nutriceutical regulation in, 201
Cancer cell antiproliferation, as antitumor

mechanism, 153-161
Cancer therapy, mushrooms and, 25
Cantharellus cibarius, 72

ecological classification of, 5
Cap, in mushroom identification, 5
Carbohydrate biosynthesis, in Lentinus edodes,
45

Carbohydrates

in conventional edible mushrooms, 78
in mushrooms, 24
in nutrition, 23
in sclerotia, 123

during sclerotial development, 114, 115
sclerotial extracellular matrix and, 122
Carbon dioxide, in Agaricus cultivation, 14
Carboxymethylated /3-glucan (CMPTR), from

mushrooms, 152-153, 159
Carboxymethylation (CM), in chemical

improvement of antitumor activity, 176
Carcinogenesis, effects of lentinan on, 165
Carcinoma, polysaccharide-protein complexes

versus, 160, 167
Cardiotonic agent, Wolfiporia cocos as, 120
Cardiovascular disease
edible mushrooms and, 7 1
hypercholesterolemia and, 94
Carmustine (BCNU), mushroom

polysaccharides and, 150, 157
fi -Carotene bleaching, in mushroom antioxidant

assays, 90
Casein, protein quality of, 88
Cbx R marker, in Lentinus edodes transformation,

58



INDEX 231



cDNA clones, in Lentinus edodes genetics, 54.

See also Complementary DNA (cDNA)
cDNA microarray, in Lentinus edodes genetics,

36,51,53-54
cDNA representational difference analysis

(cDNA-RDA), of Lentinus edodes, 51, 52,

61. See also Representational difference

analysis (cDNA-RDA)
cDNA sequencing, in Lentinus edodes

sequencing by synthesis, 55
Cel genes, in Lentinus edodes, 60
Cell cycle control genes, of Lentinus edodes, 40
e?ttfo-Cellobiohydrolase, Lentinus edodes and,

36

exo-Cellobiohydrolase, Lentinus edodes and, 36
Cellobiohydrolases, in mushrooms, 10
Cellulase

Lentinus edodes and, 36-57

in mushrooms, 10
Cellulase genes, in Lentinus edodes, 46
Cellulose

in biomass waste, 10

Lentinus edodes degradation of, 36, 60

from mushrooms, 149

in Wolfiporia cocos cultivation, 121
Cellulose control, in vivo Ca/Mg absorption and,

131
Cell walls

in mushroom sclerotia, 121-122

as source of antitumor polysaccharides, 149
Center for Food Safety and Applied Nutrition
(CFSAN), 203

Office of Nutritional Products, Labeling, and
Dietary Supplements, 205-206
Cerebrospinal fluid (CSF), effects of lentinan on,
165

Chang, Shu-Ting, xxi, 1 , 27
Chanterelles, 72

Characteristics, for mushroom identification, 5
Cheese, protein quality of, 88
Chemical improvement, of antitumor

polysaccharides, 176-177
Chemical tests, in mushroom identification, 5
Chemistry, in mushroom cultivation, 20
Chemotaxis, GLP effects on, 164
Chemotherapy, mushrooms and, 25
Cheung, Peter C. K., xxi, xvii, 71, 111
China

Agaricus brasiliensis cultivation in, 18
food safety in, 215

Ganoderma lucidum cultivation in, 19
Lentinus edodes from, 35
mushroom production in, 2 1
mushroom sclerotia production in, 117



mushroom species in, 1-2
Pleurotus tuber-regium consumption in, 118
Polyporus rhinocerus consumption in, 119
total mushroom production in, 72-73
Volvariella cultivation in, 17-18
wild and cultivated edible mushrooms from,
72, 73

Wolfiporia cocos consumption in, 120-121
Chinese medicine

mushroom uses in, 25, 147

shiitake mushroom in, 215
Chi tin

in cultivated mushroom dietary fiber, 77, 78

dietary fiber and, 124, 125

from mushrooms, 149

in sclerotia, 124

in sclerotial cell walls, 122
Chitinase, Lentinus edodes and, 36
Chitosan

from mushrooms, 149

in sclerotia, 124
Chlorflavonin, biosynthesis of, 93-94
Chlorophyll, as lacking in mushrooms, 10
Cholesterol biosynthesis, mushrooms as

reducing, 96-97
Chromosomal DNA, in Lentinus edodes
transformation, 57-58. See also
Deoxyribonucleic acid (DNA)
Chum, Winnie W. Y, xxi, 35
Cinnamic acid, biosynthesis of, 93
rrans-Cinnamic acid, as mushroom antioxidant,
92

Citrate cycle, in Lentinus edodes, 48
Citrate synthase, in Lentinus edodes, 48
Citrinin, in mushrooms, 91-92
Classical Chinese medicine, shiitake mushroom
in, 215

Classification, of edible mushrooms, 80
Claviceps purpurea, sclerotia of, 112
Clones, in Lentinus edodes genetics, 52
CMHAE carboxylated /i-glucans, in chemical

improvement of antitumor activity, 177
Codex Alimentarius, on introducing

medicinal-mushroom dietary supplements,

202-203

Codex Alimentarius Commission, 202-203

in nutritional evaluation, 87
Codex Committee on Vegetable Proteins

(CCVP), in nutritional evaluation, 87
Cold shock, in Lentinus edodes primordium

formation, 38
Collybia confluens, antitumor

polysaccharide-protein complexes from,

158



232 INDEX



Collybia dryophila, antitumor polysaccharides
from, 154

Collybia dryophila polysaccharide (CDP), 164
Collybia maculata, antitumor polysaccharides
from, 156

Colonic fermentation, of dietary fiber, 129-131
Colonic pH, in colonic fiber fermentation,
129-130

Colony formation unit of granulocyte

macrophages (CSU-GM), MBG treatment
and, 170

Color. See also Pigmentation; Pigments

in mushroom identification, 5

of Polyporus rhinocerus, 1 19

of sclerotial dietary fiber, 126

of Wolfiporia cocos, 120
Color change

of cortex, 116

of rind, 116
Column packing, in Pleurotus cultivation, 17
Comfrey, safety of, 216
Commercial-scale cultivation, of Lentinus
edodes, 16

Competitive microorganisms, in mushroom

cultivation, 13
Complementary DNA (cDNA), in Lentinus

edodes genetics, 36, 43, 44. See also cDNA

entries

Complement receptor 3 (CR3), in immune

response, 162
Compost

for Pleurotus tuber-regium, 118-119

for Polyporus rhinocerus, 119-120

for Wolfiporia cocos, 121
Compost preparation phase

for Agaricus mushrooms, 14

in mushroom cultivation, 12, 13
Compost technology, mushrooms and, 8
Conducting hyphae, during sclerotial

development, 114
Conformation, of antitumor polysaccharides,

175-176
Congress, DSHEA and, 204
Conservation, in mushroom cultivation, 21
Copper

in cultivated mushrooms, 76

in vitro sclerotial binding of, 128-129

in sclerotial dietary fiber, 125
Coprinaceae, classification of, 80
Coprinopsis cinerea, 44, 56. See also Coprinus
cinereus

Coprinus cinereus, 57, 59. See also Coprinopsis

cinerea
Coprinus comatus, 85



chemical composition of, 82

classification of, 80

world production of, 73
Cord blood (CB) cells, 170
Cordyceps, xvii

Cordyceps ophioglossoides, antitumor

polysaccharide-protein complexes from,
158

Cordyceps sinensis

antiangiogenesis and, 172

antimetastatic effects of, 172

antitumor polysaccharides from, 154

FWE water extract from, 158, 171

hypolipidemic effects of, 95
Cordyglucan, from mushrooms, 154
Coriolus (Trametes) versicolor. See also
Trametes versicolor

antiangiogenesis and, 172

antimetastatic effects of, 171, 172

antitumor polysaccharide-protein complexes
from, 158, 159-160

FWE water extract from, 158, 171

medicines from, 25

polysaccharides isolated from, 148, 150
PSK polysaccharide from, 166-167, 169-170
Coriolus hirsutus, 57, 59
Cortex, structure of, 115, 116-117
Cottage-scale cultivation, of Lentinus edodes, 16
Council for Responsible Nutrition (CRN), 204
Cristulariella, during early sclerotial ontogeny,
115

Crop frequency, in Agaricus cultivation, 14
Cross breeding, of Lentinus edodes, 56, 57
Crude protein

in conventional edible mushrooms, 74
in nonconventional edible mushrooms, 79
in sclerotia, 123-124
CT-rich sequence element, in Lentinus edodes

transcriptional regulation, 55-56
Culinary-medicinal products, types of, 201
Cultivated edible mushrooms, 72, 73. See also
Edible mushrooms; Newly developed
cultivated mushrooms
in mushroom industry, 2
species of, 7 1

world production of, 72-73
Cultivation. See also Mushroom cultivation

of Pleurotus tuber-regium, 118-119

of Polyporus rhinocerus, 119-120

of sclerotia, 117-121

of Wolfiporia cocos, 121
Cultivation substrate. See also Substrate entries

in Agaricus brasiliensis cultivation, 18-19



INDEX 233



in Ganoderma lucidum cultivation, 19-20

in Pleurotus cultivation, 17
Culture filtrates, of mushrooms, 149
Culture media, in Lentinus edodes cultivation, 15
Culturing, submerged, 219
Curvulic acid, in mushrooms, 91, 92
Cyclic adenosine monophosphate (cAMP), in

Lentinus edodes primordium formation, 38
Cyclin B, in Lentinus edodes genetics, 52
Cyclins, from mushrooms, 159, 161
Cyclophosphamide (CP), 170-171

immunomodulatory effects of, 164
Cystathionine, mushroom-related
hypocholesterolemia and, 94
Cysteine

in conventional edible mushrooms, 74, 75
in mushroom protein, 88, 89
Cytarabine, 160

Cytochrome P450, Lentinus edodes and, 44
Cytokine expression, mushroom polysaccharides
and, 151

Cytokine production, antitumor and

immunomodulatory effects and, 132, 133
Cytokines, 178

AC-PS effects on, 168

in angiogenesis, 172

antimetastatic effects on, 171

dendritic cells and, 168-169

effects of lentinan on, 165

effects of PG101 and, 165

effects of PSK and, 167

GLP effects on, 163

in immune response, 162

mushroom polysaccharide effects on,
167-168

polysaccharides and, 148
Cytoplasm

glycogen in hyphal, 122

in rind cells, 116
Cytoplasmic reserves, of sclerotia, 122-123
Cytotoxicity, 178, 179

as antitumor mechanism, 153, 159

of polysaccharide-protein complexes,

160- 161

Cytotoxic lymphocytes, in immune response,

161- 162

Cytotoxic properties, of Wolfiporia cocos, 120
Cytotoxic responses, polysaccharides and, 148,
150

Dampness, mushrooms and, 2-3
Daniella oliveri, as Pleurotus tuber-regium

compost, 119
Databases, in mushroom cultivation, 21



Death, food safety and, 215-216. See also
Apoptosis

Debydropachymic acid, in Wolfiporia cocos

cultivation, 121
Dectin- 1

in immune response, 162
in sclerotial antitumor and

immunomodulatory studies, 133
Dehydrogenases, in Lentinus edodes, 48, 53
Dendritic cells (DCs)

in immune response, 161-162
mushroom polysaccharides and, 150, 163,

168-170
in sclerotial antitumor and

immunomodulatory studies, 133
Denmark, food safety systems in, 219-220
Deoxyribonucleic acid (DNA). See also
Complementary DNA (cDNA); DNA
entries; Mitochondrial DNA (mtDNA)
RFLPs; Recombinant DNA (rDNA)
in Lentinus edodes particle bombardment

transformation, 57-58
in mushroom cultivation, 20
Department of Food and Dietary Services

(Israel), 214
Department of Health and Human Services

(DHHS), 208
Descriptions, in mushroom identification, 6
Desert truffle, 2

Designer foods, regulation of, 200
Deuteromycotina, sclerotia of, 112
Developing countries

mushroom harvests in, 22

mushrooms in, 72
Developmental structural proteins, in Lentinus
edodes, 48

Development stage, of sclerotial ontogeny,

113-114, 114-115
D-fraction

grifolan and, 165, 166

immunomodulatory effects of, 167-168

regulation of, 200
Diabetes, 207

edible mushrooms and, 7 1

mushrooms in treatment of, 97-99
Dictyophora indusiata

antioxidants in, 90

antitumor polysaccharides from, 154, 156
carbohydrate content of, 78
dietary fiber in, 77
fat content of, 75
moisture content of, 74
Dietary enrichment, via mushroom cultivation,
21, 22, 23



234 INDEX



Dietary fiber (DF). See Fiber
Dietary food supplements

mushroom polysaccharides as, 147-148

mushrooms as, 23, 24-25
Dietary protein shortages, mushrooms as

alleviating, 80
Dietary Supplement and Nonprescription Drug

Consumer Protection Act of 2006, 208
Dietary Supplement Guide, 205-206
Dietary supplement pyramid, 200
Dietary supplements (DSs)

in Australia and New Zealand, 21 1-212

in Canada, 210-211

in European Union, 208-210

in Israel, 214-215

in Japan, 212-214

regulation of, 200-201

safety and diversity of, 215-218

US regulation of, 204-208
Dietary Supplements Health and Education Act

(DSHEA), 200, 204, 205
Differential display, in Lentinus edodes genetics,
51-52

Differential gene expression, in Lentinus edodes,
38

Dihydrochalcone, biosynthesis of, 93-94
Dikaryotic mycelia, of Lentinus edodes, 37,
38-44

Dimethyl suulfoxide (DMSO), 175

Direct cytotoxicity, 179. See also Direct toxicity

as antitumor mechanism, 153
Directives, on European dietary supplements,
209,210

Direct toxicity, of nutrients, 216-217. See also

Direct cytotoxicity
Disease, defined, 205
Ditags, in Lentinus edodes genetics, 52
Diversity, 220

of dietary supplement types, 215-218
of polysaccharides, 148
DNA fingerprints, for Lentinus edodes, 49-50.

See also Deoxyribonucleic acid (DNA)
DNA fragments, in Lentinus edodes sequencing

by synthesis, 54-55
DNA helicases, in Lentinus edodes, 46
DNA microarray analysis, in Lentinus edodes

genetics, 36,51,53-54,61
DNA synthesis, in Lentinus edodes, 45
Dolichos lablab, protein quality of, 88
Dormancy, in sclerotial ontogeny, 114
Dosages, WHO guidelines and, 202
Dot-blot hybridization, in Lentinus edodes

genetics, 54
Doxorubicin (DOX), 157, 170



DPPH radical scavenging, in mushroom

antioxidant assays, 90
Drug abuse, US regulations and, 208
Drug approval, 218
Drugs, safety of, 215

Drug withdrawal, US regulations and, 208
Dry matter (DM), in sclerotia, 123-124
Dry mushrooms, moisture content of, 74

Ecological classification, of mushrooms, 4, 5
Ecology, mushrooms in, 9
Economic importance, of mushrooms, 72, 73
Edible mushrooms, 3, 4. See also Cultivated
edible mushrooms; Wild edible entries
classification of, 80
distinguishing from poisonous, 6
health benefits of, 71, 89-99
international movement for, 26, 27
in mushroom industry, 26
nutritional composition of, 73-79, 81-87
wild and cultivated, 72, 73
Edible mycorrhizal mushrooms, international

movement for, 27
Egusi, Pleurotus tuber-regium in, 118
Ehrlich carcinoma, mushroom

polysaccharide-protein complexes versus,
160

Elaeis guineensis, as Pleurotus tuber-regium

compost, 119
Electroporation, for Lentinus edodes

transformation, 58
ELN3 protein, Lentinus edodes and, 44
Emulsification, of sclerotial dietary fiber, 126
Emulsifying activities (EA), of sclerotial dietary

fiber, 126, 127-128
Emulsion stability (ES), of sclerotial dietary

fiber, 126, 128
efttfo-cellobiohydrolase, Lentinus edodes and, 36
Endocytosis genes

of Lentinus edodes, 42

in Lentinus edodes yeast two-hybrid analysis,
54

Endogenous factors, during sclerotial ontogeny,
113

Endoglucanases, in mushrooms, 10
Endoplasmic reticulum (ER), in rind cells, 116
Energy

from conventional edible mushrooms, 78
from nonconventional edible mushrooms, 79
Energy demand, in Lentinus edodes, 44
Energy production, in Lentinus edodes, 47-48
Energy production genes, of Lentinus edodes, 41
Energy sources, in nutrition, 23



INDEX 235



Enokitake mushroom, hypocholesterolemic

effects of, 97
Environment

applied mushroom biology and, 9,11
in mushroom identification, 5
population growth and, 10, 28
Environmental decontamination, via mushroom

cultivation, 21
Environmental shocks, in mushroom cultivation,
12

Environmental technology, mushrooms and, 8
Enzymatic pH-stat assays, in nutritional

evaluation, 87
Enzymes

during early sclerotial ontogeny, 115
of Lentinus edodes, 36—37
in Lentinus edodes energy production, 48
in Lentinus edodes screening, 52
in Lentinus edodes SAGE and LongSAGE,
52-53

in Lentinus edodes transformation, 57-58

in Lentinus edodes wood degradation, 60

in lignin degradation, 60

in mushrooms, 10-11

in sclerotial cell walls, 122

in sclerotial dietary fiber preparation, 126
Epigallocatechin 3-gallate, as mushroom

antioxidant, 92
EPSF (exopolysaccharide fraction)

antiangiogenesis and, 172

antimetastatic effects of, 172
Equipment, in Lentinus edodes cultivation, 16
Ergocalciferol, in cultivated mushrooms, 77
Eritadenine

hypocholesterolemic effects of, 94

molecular structure of, 96
Erythrocyte hemolysis, in mushroom antioxidant
assays, 90

Erythroleukemia, mushroom polysaccharides

versus, 153
Escherichia coli, 57, 58

Essence of Mushrooms capsules, FDA detention
of, 206

Essential amino acids, in conventional edible

mushrooms, 74
Essential nutrients, in mushroom cultivation, 13
Eumycota, 119

Europe. See also European Union

introducing medicinal-mushroom dietary

supplements in, 203
mushroom nutriceutical regulation in, 201
wild and cultivated edible mushrooms from,
72,73



European Commission (EC), quality standards

of, 209
European Community, 220
European Court of Justice, on dietary

supplements, 210
European Food Supplements Directive, 203
European Union, introducing

medicinal-mushroom dietary supplements

in, 208-210
Eurotium chevalieri, antioxidants in, 9 1 , 92
exgl gene, in Lentinus edodes, 46
exg genes, in Lentinus edodes, 59
exo-cellobiohydrolase, Lentinus edodes and, 36
Exogenous factors, during sclerotial ontogeny,

113

Exopolymers, in mushroom-related

hypoglycemia, 98, 99
Exopolysaccharide (EPS), 151
Experts, in mushroom identification, 6
Expressed sequence tags (ESTs), in Lentinus

edodes genetics, 36, 50, 51, 53, 54
Ex situ conservation, in mushroom cultivation,

21

Extracellular enzymes, of Lentinus edodes,

36-37
Extracellular matrix

interhyphal space and, 115
of sclerotia, 122
Extract of Ganoderma lucidum polysaccharide

(EORP), AC-PS effects on, 168
Exudation, during early sclerotial ontogeny, 1 15

Fat(s)

in conventional edible mushrooms, 75, 81

in mushrooms, 23

in nutrition, 23
Fatty acid desaturases, in Lentinus edodes, 45
Fatty acids

in conventional edible mushrooms, 75, 80

during early sclerotial ontogeny, 115

in mushrooms, 24
Federal Food, Drug, and Cosmetic Act, 204,
206, 208

Federal Trade Commission (FTC), 203
Fermentability, of sclerotia, 129-131
Fermentation

in Agaricus cultivation, 14

in mushroom cultivation, 10-11
Fermentation products, regulation of, 218
Fermentation technology, mushrooms and, 8, 23
Fiber

in conventional edible mushrooms, 77-78
hypocholesterolemic effects of, 96, 97
of sclerotia, 124-126



236 INDEX



Fiber-optic slides, in Lentinus edodes

sequencing by synthesis, 55
Fiber supplements, sclerotia and, 124, 125
FIBRAPLAN dietary fiber supplement, sclerotia

and, 125

FIBREX dietary fiber supplement, 127-128
Fibrosarcoma

mushroom polysaccharides versus, 153

polysaccharide-protein complexes versus,
160, 167
Filamentous fungi

medicinal value of, 149

structure of, 111-112
Fingerprinting, of Lentinus edodes, 50
First International Conference on Mushroom

Science, 26
Flammulina, world production of, 72, 73
Flammulina velutipes, 58, 85

antioxidants in, 90

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154, 155, 156

chemical composition of, 82

classification of, 80

hypocholesterolemic effects of, 97

hypolipidemic effects of, 95

moisture content of, 73

polysaccharides isolated from, 153
Flavoglaucin, as mushroom antioxidant, 9 1 , 92
Flavonoids

biosynthesis of, 93

in cultivated mushrooms, 78
Flavor 5'-nucleotides, in cultivated mushrooms,
78-79

Flavorful production genes, of Lentinus edodes,
42

Flow cytometric analysis, in sclerotial antitumor
and immunomodulatory studies, 132

Folates, in cultivated mushrooms, 76

Folic acid, adverse effects of, 217

Fomes fomentarius, antitumor

polysaccharide-protein complexes from,
158

Fomitella fraxinea

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 155, 156
Food. See also Designer foods; Functional
foods; Medical foods; Novel foods
applied mushroom biology and, 8-9
mushrooms as, xvii, 1-34, 72
natural and organic in US, 203
population growth and, 10, 28
sclerotia as, 1 1 1-146



Food Act of 1981 (New Zealand), 201
Food Additive Amendments (1958), 204
Food additives, US regulation of, 204
Food and Agricultural Organization (FAO),

202-204, 214-215
on mushroom protein and amino acid content,

74, 75

Food and Drug Administration (FDA)

European Union and, 209

food regulation by, 203-204, 204-208, 218

medical food regulation by, 200
Food fortification policy, in Canada, 210-211
Food industry, mushrooms in, 10-11
Food processing industry, biomass waste from,
10-11

Food products, desirability of, 24
Food safety systems, 219-220
Foods for Particular Nutritional Uses

(PARNUTS), 208
Foods for Special Dietary Uses (FOSDU), in

Japan, 214

Foods for specified health uses (FOSHU), in

Japan, 212-214
Food supplements

European Union regulation of, 209
mushrooms as, 23, 24-25
Foods with nutrient function claims (FNFC), in
Japan, 213

Food with health claims (FHC), in Japan, 212
Forestry

biomass waste from, 10-11

Ganoderma lucidum cultivation and, 19-20
Forests, applied mushroom biology and, 9
Formylmethylation, in chemical improvement of

antitumor activity, 176
Free amino acids, in conventional edible

mushrooms, 74-75
Free radicals

lignin degradation by, 60

mushrooms and, 24

mushroom scavenging of, 89-90
Fruiting bodies, 178, 219

antitumor polysaccharides from, 152, 153

in human diet, 72

of Lentinus edodes, 36, 38-44, 44-47
medicinal value of, 149
mushroom nutriceuticals from, 24-25
in mushroom-related hypoglycemia, 98, 99
of Pleurotus tuber-regium, 118
SAGE profiles of Lentinus edodes, 53
of Wolfiporia cocos, 120
Fruiting body morphogenesis, in Lentinus
edodes, 46

Fruiting body senescence, in Lentinus edodes, 59



INDEX 237



Fruiting culture

of Lentinus edodes, 14, 15, 16

in mushroom cultivation, 13

preparation of, 13-14
Fruiting cycle, of Lentinus edodes, 37-38
Fruiting growth phase, in mushroom cultivation,
11-12

Fruiting management phase, in mushroom

cultivation, 12, 13
Fucogalactan

antitumor effects of, 166

from mushrooms, 156, 157
Fucoglucomannan, from mushrooms, 156
Fu ling mushroom, 120
Functional foods

in Canada, 210

FDA on, 208

mushrooms as, xvii, 1-34
regulation of, 200
Functional genomics studies, transformation in,
56, 57

Fungal histidine kinase, in Lentinus edodes, 47
Fungal hyphae, 80
Fungal protein, from mushrooms, 1 1
Fungi. See also Wood rot fungus
biology of, 6-7

biosynthesis of phenolic compounds by,
93-94

cell walls of, 121-122

identification of, 4-5

Israeli regulation of, 214-215

molecular chaperones in, 59-60

mushrooms as, 2-3, 3-4, 10-11, 149

phenolic antioxidants in, 91-93

structure of filamentous, 111-112
Fungisterol, as mushroom antioxidant, 92
FWE water extract

AC-PS effects on, 168

antimetastatic effects of, 172
FYBOGEL dietary fiber supplement, sclerotia
and, 125

Galactan, in sclerotial dietary fiber, 125
Galactoglucomannan, from mushrooms, 157
Galactomannan

antitumor effects of, 166

from mushrooms, 156
Galactomannan -protein complexes, 149
Galactomannoglucan, from mushrooms, 155,

156
Galactose

in cultivated mushroom dietary fiber, 78
in sclerotial dietary fiber, 125
Galactoxyloglucan, from mushrooms, 155



Gallic acid, biosynthesis of, 93

y-amino butyric acid (GABA), in conventional

edible mushrooms, 74
Ganoderan, from mushrooms, 154
Ganoderma, xvii

ecological classification of, 5

polysaccharides isolated from, 151-152

as saprophytes, 4
Ganoderma applanatum, polysaccharides

isolated from, 151, 152
Ganoderma capense, polysaccharides isolated

from, 151
Ganoderma lucidum

antiangiogenesis and, 172-173

antimetastatic effects of, 172

antitumor polysaccharide-protein complexes
from, 158, 162-163

antitumor polysaccharides from, 154, 155

cultivation of, 19-19

ecological classification of, 5

FWE water extract from, 158, 172

ganopoly from, 168

hypolipidemic effects of, 95

medicinal applications of, 4

medicinal effects of, 24

medicines from, 25

polysaccharides isolated from, 151, 152, 169
as saprophyte, 4
Ganoderma lucidum polysaccharide (GLP),
163-163
in immune response, 162-163
Ganoderma tsugae

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154, 155, 156
molecular mass of polysaccharides from, 174
polysaccharides isolated from, 151, 152
Ganopoly, 151

immunomodulatory effects of, 168
Garden of Life RM-10, FDA detention of,

207-208
Garnishes, mushrooms as, 72
Gas chromatography-mass spectrometry
(GC-MS), in antioxidant assays, 92
Gaseous exchange, in mushroom cultivation, 1 3
Gastrointestinal tract, in vitro sclerotial binding

of minerals in, 128-129
GC box, in Lentinus edodes transcriptional

regulation, 55-56
Gelling, of sclerotial dietary fiber, 126
Gene combinations, in mushroom cultivation, 20
Gene expression, 178

Gene expression analysis, of Lentinus edodes,
50-55



238 INDEX



General Accounting Office (GAO), on food

safety systems, 219-220
"Generally recognized as safe" (GRAS) criteria,

204, 207
Genes

of Lentinus edodes, 36-48

in Lentinus edodes development, 48

in Lentinus edodes energy production, 47-48

for Lentinus edodes mature fruiting body

formation, 44-47
in Lentinus edodes signal transduction, 47, 61
in Lentinus edodes sequencing by synthesis,

55

in Lentinus edodes transformation, 58-59
Genetic mapping, of Lentinus edodes, 50
Genome Sequencer 20 system, in Lentinus

edodes sequencing by synthesis, 55
Genome sequencing, in Lentinus edodes

sequencing by synthesis, 55
Germany

food safety systems in, 219-220
regulation of dietary supplements in, 210
Germination, in sclerotial ontogeny, 1 14
Germplasm, in mushroom cultivation, 20-21
Germplasm databases, in mushroom cultivation,
21

GFPSlb polysaccharide, isolation of, 150
Gill development, in Lentinus edodes, 44-47
Gills

of Lentinus edodes, 38
in mushroom identification, 5
GK16 mushroom, 85

chemical composition of, 82
Gl-PP (Ganoderma lucidum polysaccharide

peptide), in angiogenesis, 172-173
Glucanase activity, in Lentinus edodes, 59
Glucanase-encoding gene, in Lentinus edodes,
46

Glucan-protein complexes

from mushrooms, 158, 159

structure and antitumor activity of, 173-174
Glucans. See also a-glucans; /j-glucans

antitumor mechanisms of, 153-157

branching configurations of, 175

from mushrooms, 149

sclerotial extracellular matrix and, 122
Glucoamylase, Lentinus edodes and, 36-57
Glucogalactan-protein complex, from

mushrooms, 158
Glucogalactans, from mushrooms, 152, 156
Glucokinase, mushroom-related hypoglycemia

and, 98-99
Glucomannan, from mushrooms, 156
Glucopyranan, from mushrooms, 157



Glucopyranosyl residue, mushroom-related

hypoglycemia and, 98
Glucosamine, in sclerotial dietary fiber, 125
Glucose

colonic fermentation and, 130
in cultivated mushrooms, 78
in cultivated mushroom dietary fiber, 77-78
during early sclerotial ontogeny, 115
mushroom-related hypoglycemia and, 98
Glucose-6-phosphate dehydrogenase,

mushroom-related hypoglycemia and,
98-99

Glucose-based oligosaccharides (GOSs), in vivo

Ca/Mg absorption and, 132
Glucose-based polysaccharides, dietary fiber

and, 124

Glucose residues, in sclerotial dietary fiber, 125
Glucosidases, Lentinus edodes and, 36
/3-Glucosidases, in mushrooms, 10
Glucoxylan, from mushrooms, 152, 156
Glucuromannan-protein complexes, 149
Glucuronic acids, in sclerotial dietary fiber, 125
Glucuronoglycan, 151
Glucuronoxylomannan

in cultivated mushroom dietary fiber, 78
from mushrooms, 157
Glucuropyranosyluronic residues,

mushroom-related hypoglycemia and, 98
Glutamic acid, in conventional edible

mushrooms, 74
Glyceraldehyde-3-phosphate dehydrogenase

(GPD), in PEG-mediated Lentinus edodes

58. See also GPD entries
Glycine, in conventional edible mushrooms, 75
Glycogen

in cultivated mushrooms, 78
in sclerotial cytoplasmic reserves, 122
during sclerotial development, 114
Glycolysis, in Lentinus edodes, 48
Glycosidic linkages, in polysaccharides, 148
GM-CSF (granulocyte macrophage

colony-stimulating factor), 164, 178. See

also Granulocyte colony-stimulating factor

(G-CSF); Macrophage colony-stimulating

factor (M-CSF)
effects of PG101 and, 165
Good agriculture practice (GAP), 217
Good clinical practice (GCP), 218
Good laboratory practice (GLP), 217
Good manufacturing practice (GMP), 217

WHO guidelines and, 202
Good production practice (GPP), 218



INDEX 239



GPD gene, in Lentinus edodes transformation,

58. See also Glyceraldehyde-3-phosphate

dehydrogenase (GPD)
GPD promoter and terminator, in Lentinus

edodes transformation, 57, 58
Granular glycogen deposits, in sclerotial

cytoplasmic reserves, 122
Granulocyte colony-stimulating factor (G-CSF),

170. See also GM-CSF (granulocyte

macrophage colony-stimulating factor)
Granulocytes

PG101 treatment and, 170
SCGand, 170-171
Greenhouse gases, population growth and, 10
Grey oyster mushroom, 17. See also Pleurotus

sajor-caju
Grifola frondosa, 85. See also Maitake

mushroom
antioxidants in, 90

antitumor glucans from, 157, 165-166
antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154, 155, 156
carbohydrate content of, 78
chemical composition of, 82
chemical improvement of antitumor

polysaccharides from, 176
classification of, 80
D-fraction from, 167-168
fat content of, 75

heteroglycan-protein complex from, 167

hypocholesterolemic effects of, 97

hypolipidemic effects of, 95

Japanese production of, 72-73

MBG from, 170

moisture content of, 73

molecular mass of polysaccharides from, 174

mushroom-related hypoglycemia and, 98

polysaccharides isolated from, 148, 150

regulation of polysaccharides from, 199-200

Grifolan, antitumor effects of, 165-166

Grifola umbellata

antitumor polysaccharides from, 154
cultivation of sclerotia of, 117

Growth genes, of Lentinus edodes, 36-37,
39-42

Guanidine, mushroom-related hypoglycemia
and, 99

Guanidinoacetic acid, mushroom-related

hypocholesterolemia and, 94
Guanosine monophosphate (GMP), in cultivated

mushrooms, 78
Guidance for Industry: Botanical Drug Products

(FDA), 218



Guidelines for the Assessment of Herbal

Medicine (WHO), 202
Guidelines for the Safety Assessment of Novel

Foods Derived from Plants and

Microorganisms, 211

Haploid monokaryotic mycelia, of Lentinus
edodes, 38

Harvesting phase, in mushroom cultivation, 12,
13

HDL cholesterol levels, 97

Health, applied mushroom biology and, 8-9

Health benefits

of edible mushrooms, 89-99

of mushrooms, 71-109
Health Canada, 210-211
Health claims, 206

in Japan, 214
Health Food Emporium, FDA detention of,
207-208

Health Promotion Law, in Japan, 214
Heat shock proteins (HSPs), as molecular

chaperones, 59
Heavy metals

in cultivated mushrooms, 76
in nonconventional edible mushrooms, 79
Hebeloma crustuliniforme, antitumor

polysaccharide-protein complexes from,
158

Helicases, in Lentinus edodes, 46
Helper T cells, effects of lentinan on, 165
Hematopoietic stem cells, mushroom

polysaccharide effects on, 170-171
Hemicelluloses

in biomass waste, 10
in cultivated mushroom dietary fiber, 78
Lentinus edodes degradation of, 36
in Wolfiporia cocos cultivation, 121
Hepatic cholesterol, mushroom effects on, 97
Hepatic glucose, colonic fermentation and, 130
Hepatic HMG-CoA reductase, 97
Herbal medicines, WHO guidelines and, 202
Herbal substances, European Union regulation
of, 209

Herbs, in dietary supplements, 200
Hericium erinaceus, 86
antioxidants in, 90

antitumor polysaccharides from, 154, 155, 156
ash and mineral content of, 76
chemical composition of, 82
classification of, 80

mushroom-related hypoglycemia and, 98
world production of, 73



240 INDEX



Hericium ramosum, 86

chemical composition of, 82

classification of, 80
Heterogalactan, from mushrooms, 156, 157
Heteroglucans, from mushrooms, 155-156
Heteroglycan-protein complexes

immunomodulatory effects of, 167

from mushrooms, 158, 159

structure and antitumor activity of, 173-174
Heteroglycans, from mushrooms, 149, 156-157
Heteropolysaccharide-protein complexes, from

mushrooms, 160-161
Heteropoysaccharides, from mushrooms, 152
Hexokinase, mushroom-related hypoglycemia

and, 98-99
High density lipoprotein (HDL), 97
High-throughput sequencing technologies, in
Lentinus edodes sequencing by synthesis,
54-55
Histidine

in conventional edible mushrooms, 75
in mushroom protein, 89
Histidine kinase, in Lentinus edodes, 47
Hoelen mushroom, 120
Hohenbuehelia serotina, antitumor

polysaccharides from, 156
Homeostasis, polysaccharides and, 148
Homobasidiomycetes, 35
Homocysteine, mushroom-related

hypocholesterolemia and, 94
Homoglucans, from mushrooms, 154-155
Hong Kong, 206

mushroom conference in, 27
Host-specific factors, in colonic fiber

fermentation, 129
hph gene, in PEG-mediated Lentinus edodes

transformation, 56
HRA cell proliferation, mushroom

polysaccharides versus, 151
HSP70 protein, as molecular chaperone, 59
HUMAMIL dietary fiber supplement, sclerotia

and, 125

Human fecal microflora, colonic fermentation
and, 130

Human immunodeficiency virus (HIV),

mushrooms and, 21-22
Human peripheral blood mononuclear cells

(hPBMCs), effects of PG101 and, 165
Humans

applied mushroom biology and, 8-9
benefits of mushrooms for, 7 1
feeding, 23

health benefits of mushrooms to, 89-99
mushrooms in diet of, 72



Human tumor xenografts, in sclerotial antitumor

and immunomodulatory studies, 133-134
Human umbilical vein endothelial cells

(HUVECs), antiangiogenesis and, 172-173
Human welfare, mushrooms and, 28-29
Hungary, mushroom production in, 21
Hurulingzhi mushroom, 119
HWE heteropolysaccharide-protein complex,

157-159, 161
Hybridization, in Lentinus edodes genetics, 54
Hybridization analysis, Lentinus edodes meiosis

and, 61

Hydnaceae, classification of, 80
Hydration properties, of sclerotial dietary fiber,
127

Hydrogen peroxide, lignin degradation by, 60
Hydrophobins, of Lentinus edodes, 44, 48, 53
Hydroxybenzoic acid, as mushroom antioxidant,
92

p-Hydroxybenzoic acid, biosynthesis of, 93
Hydroxylation, in chemical improvement of

antitumor activity, 176
Hydroxyl free radicals, mushroom scavenging

of, 89-90

4-Hydroxymethylbenzenediazonium (HMBD),

US regulation of, 204
Hygromycin B phosphotransferase gene, in

Lentinus edodes transformation, 58
Hygromycin B resistance, Lentinus edodes

transformation and, 58
Hymenium, in Lentinus edodes, 45
Hymenomycetes, 119
classification of, 80
Hymenophore, Lentinus edodes meiosis and,

60-61

Hypercholesterolemia, mushrooms versus, 94
Hyperglycemia, mushrooms in treatment of,
97-99

Hyperhomocysteinic effects, mushroom-related

hypocholesterolemia and, 94
Hyphae

cell walls of, 121-122

of cortex, 117

of filamentous fungi, 111-112
of medulla, 117
polyphosphate granules in, 123
of rind, 116

during sclerotial ontogeny, 113-114

structure of, 115
Hyphal cells, of rind, 116
Hyphal tips

during early sclerotial ontogeny, 1 15

rind and, 115-116

structure of, 115



INDEX 241



Hypocholesterolemia

colonic fermentation and, 130

from mushrooms, 94-97
Hypoglycemia, from mushrooms, 97-99
Hypolipidemia, from mushrooms, 94, 95, 96
Hypsizygus marmoreus, 86

antioxidants in, 90

antitumor polysaccharides from, 154, 156
chemical composition of, 82
classification of, 80
Japanese production of, 72-73

Identification, of mushrooms, 4-6
IL-12 interleukin, 162. See also Interleukins
(ILs)

IL-12p35 interleukin, 163

IL-12p70 interleukin, 151

Immune response, cancer and, 161-162

Immunomodulation, as antitumor mechanism,

153, 161-171
Immunomodulators, 161
Immunomodulatory effects

of mushroom polysaccharides, 147-198

of sclerotia, 132-134

of Wolfiporia cocos, 120
Immunomodulatory proteins, in mushrooms, 25
Immunopotentiators, mushroom products as,

200-201
Incubation

in Lentinus edodes cultivation, 16

in Pleurotus cultivation, 17
India

mushroom nutriceutical regulation in, 201

mushroom production in, 2 1
Indoor fermentation, in Agaricus cultivation, 14
Inducible nitric oxide synthase (iNOS) protein,
164

Information, in mushroom cultivation, 20, 2 1
Infrared (IR) spectrometry, in antioxidant assays,
92

Ingredients

labeling of, 204-205

WHO guidelines and, 202
Inhibitor of kappa B (1-kB) kinase, dendritic

cells and, 169
Initials, in sclerotial ontogeny, 113-114
Initiation stage, of sclerotial ontogeny, 113
Innate immunity, cancer and, 161-162
Innovation, in mushroom industry, 22
Inonotus obliquus

antitumor polysaccharides from, 156

endopolysaccharide from, 166

regulation of polysaccharides from, 199-200
Inorganic compounds, in nutrition, 23



Inosine monophosphate (IMP), in cultivated

mushrooms, 78
Inositol

in cultivated mushrooms, 78
during early sclerotial ontogeny, 115
Insects, inside mushrooms, 2
In situ conservation, in mushroom cultivation, 21
Insoluble dietary fiber (IDF)
in cultivated mushrooms, 77, 81
of sclerotia, 124, 125
Insulin-dependent diabetes mellitus (IDDM),
mushroom-related hypoglycemia and,
98-99

Insulin release, mushroom-related hypoglycemia
and, 99

Intercellular adhesion molecule-1 (ICAM-1), in

immune response, 162, 179
Interferon gamma (IFN-y), 151, 162, 163, 164,

178

effects of lentinan on, 165
Interhyphal materials, in sclerotial ontogeny, 1 14
Interhyphal space

of medulla, 1 17

sclerotial extracellular matrix and, 122
structure of, 115
Interleukins (ILs). See also IL-12 entries
effects of lentinan on, 165
effects of PG101 and, 165
effects of PSK and, 167
GLP effects on, 164
grifolan and, 165
Internal morphogenetic factors, during sclerotial

ontogeny, 113
International Commission on Mushroom

Science, formation of, 26
International Conference on Mycorrhizas, 27
International Conference for Mushroom Biology

and Mushroom Products (ICMBMP), 27
International Journal of Medicinal Mushrooms
(IJMM), 27

International Medicinal Mushroom Conference

(IMMC), 27
International Society of Mushroom Science

(ISMS), 21
formation of, 26
International Workshop on Edible Mycorrhizal

Mushrooms (IW-EMM), 21, 27
InterBsimple sequence repeat markers (ISSRs),

for Lentinus edodes, 48, 49-50
Inulin, in vivo Ca/Mg absorption and, 131
Investigational new drug (IND), 218
In vitro fermentability, of sclerotia, 129-131
In vitro methods, in nutritional evaluation, 87



242 INDEX



In vitro mineral binding capacity, of sclerotia,
128-129

In vitro protein digestibility (IVPD), in

nutritional evaluation, 87, 88
In vivo calcium absorption, by sclerotia,

131-132

In vivo magnesium absorption, by sclerotia,
131-132

Ireland, food safety systems in, 219-220
Iron

in cultivated mushrooms, 76

in vitro sclerotial binding of, 128-129

in mushrooms, 23

in sclerotial dietary fiber, 125
Iron sulfur protein (Ip) subunit gene, in Lentinus

edodes transformation, 58
Isoleucine

in conventional edible mushrooms, 74, 75
in mushroom protein, 89
Israel, 220

introducing medicinal-mushroom dietary
supplements in, 214-215

Japan, 207, 220
Agaricus brasiliensis cultivation in, 18
introducing medicinal-mushroom dietary

supplements in, 212-214
Lentinus edodes from, 35
medicinal mushroom uses in, 25
medicinal uses of mushroom polysaccharides

in, 147

mushroom antioxidant studies in, 89
mushroom nutriceutical regulation in, 201
mushroom-related hypocholesterolemia

studies in, 94
wild and cultivated edible mushrooms from,

72, 73

Jaundice, Wolfiporia cocos in, 120

Jew's ear mushroom, hypocholesterolemic

effects of, 97. See also Auricularia

auricula-judae

Karyogamy, of Lentinus edodes, 38
Kava

for mushroom identification, 5, 6

warnings against, 209
Korea, medicinal uses of mushroom

polysaccharides in, 147
Krestin. See also PSK polysaccharide-protein
complex

from mushrooms, 25, 150

regulation of, 200
Kugitake mushrooms, antioxidants in, 89
Kwan, Hoi-Shan, xxi, 35



Labeling

of Canadian dietary supplements, 210
false or misleading, 206-208
US regulation of, 203-204, 204-205
Laccase, in mushrooms, 10
Laccase genes, in Lentinus edodes, 46
lac genes, in Lentinus edodes, 46, 60
Lactobacillus, in colonic fiber fermentation, 130
Laetiporus sulphureus, antitumor

polysaccharide-protein complexes from,
158

Laminarinase, Lentinus edodes and, 36
Lampteromycer japonicus, antitumor

polysaccharides from, 156
Lateral development, of sclerotia, 113
Latin America, mushroom production in, 22, 23,

73

LDL cholesterol levels, mushrooms reducing,

97. See also Low density lipoprotein (LDL)

oxidation
Lead, in cultivated mushrooms, 76
Le-cdc5 gene, in Lentinus edodes transcriptional

regulation, 55
Le.CDC5 protein, of Lentinus edodes, 43
LeClb gene, of Lentinus edodes, 43
Lectins, mushroom-related hypoglycemia and,

99

Le.cyp genes, of Lentinus edodes, 44
Le.DRMlP gene

in Lentinus edodes, 43, 47

in Lentinus edodes yeast two-hybrid analysis,
54

Le.egll gene, in Lentinus edodes, 60
Le. -FAD genes, of Lentinus edodes, 44, 45
Le.-FDAl gene, of Lentinus edodes, 44
Le. Ga gene, in Lentinus edodes, 47
Legal issues, in introducing

medicinal-mushroom dietary supplements,

202-215
Legumes, protein quality of, 88
Le.hyd genes, in Lentinus edodes, 48
Le.MAPK gene

in Lentinus edodes, 43, 44, 47

in Lentinus edodes yeast two-hybrid analysis,

54

Le.mfbC gene, in Lentinus edodes, 47
LeMPP gene, in Lentinus edodes, 48
Le.nikl gene

in Lentinus edodes, Al

in Lentinus edodes yeast two-hybrid analysis,
54

LeNotl gene, of Lentinus edodes, 43
Lentinacin (lentysine), hypocholesterolemic
effects of, 94



INDEX 243



Lentinan

antitumor mechanism of, 153-157

branching configuration of, 174-175

conformation of, 175

immunomodulatory effects of, 164-165

isolation of, 148, 150

from mushrooms, 25, 154, 161-162

regulation of, 199-200
Lentinan degradation, Lentinus edodes and, 59
Lentinan degradation genes, of Lentinus edodes,
40

Lentinula, 35

Lentinula edodes. See Lentinus edodes
Lentinus

hypocholesterolemic effects of, 94

world production of, 72
Lentinus edodes, 35-69, 165. See also Shiitake
mushroom

anatomy of, 3

antimetastatic effects of, 171, 172
antioxidants in, 90, 91, 92
antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154, 156
ash and mineral content of, 76
biology and genetics of, 14, 15
carbohydrate content of, 78
chemical improvement of antitumor

polysaccharides from, 176-177
Chinese production of, 73
classification of, 80
conformation of polysaccharides from,

175-176
cultivation of, 14-16
development of, 37-47
dietary fiber in, 77
economic value of, 35-36
fat content of, 75

FWE water extract from, 158, 171

gene expression analysis of, 50-55

genetically improving, 35-36

growth genes of, 36-37, 39-42

hypocholesterolemic effects of, 94

isolation of genes of, 36-48

life cycle of, 14-15, 37-38

lignocellulolytic enzymes of, 10

medicinal value of, 25, 35

moisture content of, 73

molecular genetics of, 36, 48-50

molecular mass of polysaccharides from, 174

physiological processes in, 47-48, 61

polysaccharides isolated from, 148, 149, 150

process analysis of, 59-61

protein and amino acid content of, 74, 75



protein quality of, 88
provenance of, 35

regulation of polysaccharides from, 199-200
taxonomy of, 35

transcriptional regulation of, 55-56

transformation of, 56-59

vitamin content of, 76-77

as wood rot fungus, 15

world production of, 72, 73
Lentinus giganteus, 86

chemical composition of, 82

classification of, 80
Lentinus lepideus

biosynthesis of phenolic compounds by, 93

polysaccharide extracted from, 165, 170
Lepiota, toxicity of, 6
Lepista nuda, world production of, 73
LePriA gene, in Lentinus edodes transcriptional

regulation, 55
Le.ras gene, in Lentinus edodes, 44, 47
Le.recQ gene, in Lentinus edodes, 46, 47
Le.rnr2e gene

in Lentinus edodes, 45

in Lentinus edodes meiosis, 60-61
Leucine, in conventional edible mushrooms, 74,
75

Leucopaxillus giganteus, antitumor

polysaccharides from, 156
Leukemic cells, mushroom

polysaccharide-protein complexes versus,
159-160, 161
Lewis lung cancer, mushroom polysaccharides

versus, 151-152
Life cycles

of Lentinus edodes, 37-38
of mushrooms, 149
Life expectancy, food safety and, 215
Life-span increases, mushrooms and, 25
Lignin

in biomass waste, 10
in cultivated mushrooms, 77, 78
Lentinus edodes degradation of, 36, 60, 61
Ligninases, in mushrooms, 10
Lignin peroxidase, from Lentinus edodes, 60
Lignocellulolytic enzymes
of Lentinus edodes, 36
in mushrooms, 10
Lignocellulose degradation, by Lentinus edodes,
60

Lignocellulosic biomass waste, applied

mushroom biology and, 9, 10- 1 1, 21, 28
Lignolytic system, of Lentinus edodes, 36
Lignosus rhinoceros, 119. See also Polyporus
rhinocerus



244 INDEX



Linoleic acid

in conventional edible mushrooms, 75
mushroom-related hypocholesterolemia and,
94

Linolenic acid, in conventional edible

mushrooms, 75
Lipid biosynthesis, in Lentinus edodes, 45
Lipid bodies, in sclerotia, 123
Lipid metabolism, mushroom-related

hypocholesterolemia and, 94
Lipid peroxidation inhibition, by mushrooms, 9 1
Lipids

in conventional edible mushrooms, 75
in mushrooms, 24

in nonconventional edible mushrooms, 79
in sclerotia, 123

in sclerotial cytoplasmic reserves, 122, 123
Liquid chromatography-mass spectrometry

(LC-MS), in antioxidant assays, 92
Liver injury, from kava, 209
Log cultivation, of Ganoderma lucidum, 19—20
LongSAGE, in Lentinus edodes genetics, 52-53.

See also Serial analysis of gene expression

(SAGE)

Loose development, of sclerotia, 113
Lovastatin

hypocholesterolemic effects of, 96

submerged culturing of, 219
Low density lipoprotein (LDL) oxidation. See
also LDL cholesterol levels

hypercholesterolemia and, 94

hypocholesterolemia and, 97

in mushroom antioxidant assays, 90
LPK15 mushroom, 86

chemical composition of, 82
Luciferase expression, 166
Lung cancer

GLP effects on, 163

mushroom polysaccharides versus, 151-152
Lung cancer cells, antitumor polysaccharides

versus, 157
Lymphocytes

AC-PS effects on, 168

in immune response, 161-162

mushroom polysaccharide effects on, 163
Lymphoma, mushroom polysaccharide-protein

complexes versus, 159-160
Lyophyllum decastes

antitumor polysaccharides from, 154, 164

polysaccharides isolated from, 153
Lyophyllum ulmarius

dietary fiber in, 77

fat content of, 75

protein and amino acid content of, 75



Lysine

in conventional edible mushrooms, 74, 75

in mushroom protein, 88
LZE extract, 151
Lzps-1, 151-152

Macrofungi, mushrooms as, 2, 3, 149
Macromolecules, polysaccharides as, 148
Macrophage colony-stimulating factor (M-CSF),
151. See also GM-CSF (granulocyte
macrophage colony-stimulating factor)
Macrophages, 178

in chemical improvement of antitumor
activity, 176

D-fraction and, 167-168

galactomannan and, 166

grifolan and, 165-166

in immune response, 161-162, 162-163

mushroom polysaccharides and, 150, 151,
159, 160, 163-167

schizophyllan and, 165

in sclerotial antitumor and

immunomodulatory studies, 133
Magnesium

in cultivated mushrooms, 76

in vitro sclerotial binding of, 128-129

in nonconventional edible mushrooms, 79

in sclerotial dietary fiber, 125
Magnesium absorption, by sclerotia, 131-132
Maitake D-fraction. See D-fraction
Maitake mushroom, 207. See also Grifola
frondosa

antioxidants in, 89

antitumor glucans from, 157

antitumor polysaccharides from, 150

hypocholesterolemic effects of, 97
Manganese

in cultivated mushrooms, 76

in Lentinus edodes cellulose degradation, 60
Manganese peroxidase, in mushrooms, 10
Manna, from mushrooms, 2, 156
Mannans

in cultivated mushroom dietary fiber, 77

in sclerotial dietary fiber, 125
Mannitol

in cultivated mushrooms, 78

during sclerotial development, 114, 115
Mannofucogalactan, from mushrooms, 156
Mannofucoxyloglucan, from mushrooms, 155
Mannogalactan, from mushrooms, 152
Mannogalactofucan, from mushrooms, 156
Mannogalactoglucan, from mushrooms, 155,
156

Mannoglucan, from mushrooms, 156



INDEX 245



Mannoglucoxylan, from mushrooms, 156
Mannopyranose, mushroom-related

hypoglycemia and, 98
Mannose

in cultivated mushroom dietary fiber, 78

in sclerotial dietary fiber, 125
MAPK kinase (MEK)

dendritic cells and, 169

in Lentinus edodes, 47
Marketing licenses, WHO guidelines and, 202
Mass spectrometry (MS), in antioxidant assays,
92

Mating factor gene, of Lentinus edodes, 40
Mating-type genes, of Lentinus edodes, 38
Matsutake truffle, 72

as saprophyte, 4
Maturation, of rind, 116
Maturation stage, of sclerotial ontogeny, 113,
114

Mature fruiting body formation, of Lentinus

edodes, 44-47
Maturity

of Lentinus edodes, 14
in mushroom cultivation, 12
MBG /3-glucan, effects on hematopoietic stem
cells, 170

Medical care, population growth and, 10, 28
Medical foods, regulation of, 200
Medications, European Union regulation of, 209
Medicinal mushroom products, 220. See also

Mushroom medicinal products
Medicinal mushrooms

international movement for, 27

Lentinus edodes, 35-36

in mushroom industry, 26
Medicinal uses/value

of mushroom polysaccharides, 147-198

of mushrooms, xvii, 3, 4, 21-22, 24, 28-29

of Pleurotus tuber-regium, 118

of Polyporus rhinocerus, 1 19

of Wolfiporia cocos, 120, 121
Medicines, European Union regulation of, 209
Medulla

cortex and, 116-117

structure of, 115, 117
Meiosis, in Lentinus edodes, 60-61
MEK kinase (MEKK), in Lentinus edodes, 47
Melanins, in sclerotial cell walls, 122
Melanoma cells, 166
Melon seed ball preparations, Pleurotus

tuber-regium in, 118
Memory B cells, dendritic cells and, 168
Menstruation, Wolfiporia cocos in, 120
Mercury, in cultivated mushrooms, 76



Metabolic pathways, in Lentinus edodes, 48
Metal oxalates, in sclerotial dietary fiber, 125
Metals, in cultivated mushrooms, 76. See also

Heavy metals
Methanol extracts

in sclerotial antitumor and

immunomodulatory studies, 133

in Wolfiporia cocos cultivation, 121
Methionine

in conventional edible mushrooms, 74, 75

in mushroom protein, 88, 89
Mevinolin, hypocholesterolemic effects of, 96
mfbC gene, in Lentinus edodes, 46
Mgel gene, molecular chaperones and, 59-60
Mice

antitumor polysaccharide-protein complex

studies in, 160, 161
grifolan studies in, 166
lentinan studies in, 165
mushroom-related hypoglycemia studies in,

98,99

polysaccharide branching configuration
studies using, 175

PS-G studies in, 169

PSK studies in, 171

PSPC studies in, 167

PSP studies in, 167

SCG studies in, 164

schizophyllan studies in, 165

sclerotial antitumor and immunomodulatory
studies in, 132

studies of chemical improvement of antitumor
activity in, 177
Microarray hybridization, in Lentinus edodes

genetics, 36, 51, 53-54
Microfungi, 2
Microorganisms

in Agaricus cultivation, 14

in mushroom cultivation, 13

sclerotial cell walls and, 122

during sclerotial ontogeny, 113
Mineral binding capacity, of sclerotia, 128-129
Minerals. See also Ash

in conventional edible mushrooms, 75-76, 80

in dietary supplements, 200

European Union regulation of, 209

labeling of, 204-205

in mushrooms, 88

in sclerotial dietary fiber, 125
Minimum inhibition rates (MIRs), 172
Ministry of Health (Israel), 214
Minister of Health, Labor and Welfare (MHLW),
in Japan, 212, 213



246 INDEX



Miracle Mushroom Blend, FDA detention of,
207

Mitochondria, in rind cells, 1 16
Mitochondrial DNA (mtDNA) RFLPs, for

Lentinus edodes, 49. See also

Deoxyribonucleic acid (DNA)
Mitogen-activated protein kinase, in Lentinus

edodes genetics, 52
Mitosis, of Lentinus edodes, 37
mnp gene, in Lentinus edodes transcriptional

regulation, 56
Moisture

in conventional edible mushrooms, 73-74

in nonconventional edible mushrooms, 79,81

in sclerotia, 1 24

in Wolfiporia cocos, 121
Molecular chaperones, in fungi, 59-60
Molecular genetics, of Lentinus edodes, 48-50
Molecular markers, for Lentinus edodes, 48-49,
49-50

Molecular mass, of antitumor polysaccharides,
174

Monilinia, sclerotia of, 112
Monilinia fructicola, lipid bodies in, 123
Monoclonal antibody (mAb), 164
Monocytes, 178

mushroom polysaccharides and, 159
Monokaryons, of Lentinus edodes, 37
Monokaryotic mycelia, of Lentinus edodes, 38
Monosaccharide residues, of polysaccharides,
148

Monosodium glutamate (MSG)

in conventional edible mushrooms, 74-75

in cultivated mushrooms, 78-79
Morchella esculenta, galactomannan from, 166
Morphogenesis genes, of Lentinus edodes, 41
Morphology

of sclerotia, 1 12

of sclerotia during ontogeny, 112-114
Mortality patterns, safety and, 215
Mother spawn

of Lentinus edodes, 14, 15

in mushroom cultivation, 13-14
mRNA expression, 164. See also Ribonucleic
acid (RNA)

Mucilage matrix, sclerotial ontogeny and, 1 13
Multicytokine inducers, polysaccharides and,
148

Multienzymatic digestion test, in nutritional

evaluation, 87
MultiExperiment Viewer (MeV), in Lentinus

edodes genetics, 54
Multihyphal structures. See also Hyphae;

Hyphal entries



of filamentous fungi, 111-112

sclerotial ontogeny and, 113
Multilocus enzyme electrophoresis, for Lentinus

edodes, 49
Mushroom biology, 6-11, 23, 28-29

applied, 6,7-11,28-29

as a discipline, 7, 8-9
Mushroom bioremediation, 7, 8, 9, 11, 28
Mushroom biotechnology, 7-8, 9, 23-25

dietary supplements and, 24-25

medicinal value of mushrooms and, 23-24

nutriceuticals and, 24-25

nutritional value of mushrooms and, 23-24
Mushroom cultivation, 1-34. See also
Cultivation entries

Agaricus, 14

Agaricus brasiliensis, 18—19

Ganodenna lucidum, 19—20

Lentinus edodes, 14—16

mushroom germplasm in, 20-21

phases of, 12-13

Pleurotus sajor-caju, 17

stages of, 13-14

Volvariella, 17-18
Mushroom industry, 21-23
Mushroom industry movements, development
of, 25-27

Mushroom medicinal products, regulation of,

200-201. See also Medicinal mushroom

products
Mushroom microbiology, 23
Mushroom nutriceuticals, 3, 9, 10, 11, 24-25,

28-29, 150. See also Nutriceuticals
Mushroom polysaccharides

antitumor mechanisms of, 153-173, 173-177
identifying antitumor, 149-153, 154-157,

158-158

medicinal uses/value of, 147-198, 178-179
structures and antitumor activities of,
173-177

Mushroom preparations, safety of, 216
Mushroom products, submerged culturing of,
219

Mushrooms, 3-6

anatomy/structure of, 3-4

antipollution uses of, 3

bioactive components of, 89-91

biological aspects of, xvii

in biomass waste processing, 28-29

biosynthesis of phenolic compounds by,

93-94
as botanical drugs, 218
chemical aspects of, xvii
cross breeding of, 56, 57



INDEX 247



cultivation of sclerotiaof, 117-121
defined, 3-4
as dietary supplements, 3
domesticated, 2

ecological classification of, 4, 5
economic importance of, 72, 73
edible, 3, 4
fear of, 2
fossilized, 2

health benefits of, 71-109

hypocholesterolemic effects of, 94-97

identification of, 4-6

insects inside, 2

Israeli regulation of, 214-215

life cycles of, 149

lifestyles of, 2-3

literature on, xvii

medicinal value of, xvii, 3, 4, 21-22, 28-29

moisture content of, 23

nutritional evaluation of, 80-89

nutritional value of, xvii, 3-4, 28-29, 71-109

plants versus, 2, 10-11

poetry about, 2

poisonous, 2

poisonous versus edible, 6

propagation of, 217

protein quality of, 87-89

recent interest in, 3

regulation of, 199-224

safety of, 215

sclerotiaof, 111-148

seasonality of, 2-3

symbiotic with plants, 4

temperature stress in, 59, 60

value of, 21-22

widely cultivated, 72, 73

world production of, 21-23, 199
Mushroom science, 7, 8, 9
Mushroom spawn, in mushroom cultivation, 13.

See also Spawn development phase
Mushroom species, number of known, 1-2
Mycelia, 3-4, 179

in Agaricus brasiliensis cultivation, 18

antitumor polysaccharides from, 152, 159,
160-161

applied mushroom biology and, 1 1

in human diet, 72

of Lentinus edodes, 14, 37, 38

in Lentinus edodes cultivation, 15

medicinal value of, 149

mushroom nutriceuticals from, 24-25

in mushroom vegetative growth phase, 11,12

of Polyporus rhinocerus, 120

SAGE profiles of Lentinus edodes, 53



during sclerotial development, 114
during sclerotial ontogeny, 113
of Wolfiporia cocos, 121
Mycelial cultures, medicinal effects of, 24
Mycelial hydrophobins, in Lentinus edodes

genetics, 53
Mycelial running phase, in mushroom

cultivation, 12, 13
Myceteae, 3

Mycochemicals, regulation of, 200
Mycology, 6-7
"Mycomeat," 10-11
Mycophiles, 1
Mycophobes, 1
Mycoprotein, 80
"Mycorestoration," 1 1

Mycorrhiza. See also Mycorrhizal mushrooms

international movement for, 27

mushrooms as, 4, 5
Mycorrhizal mushrooms, harvesting of, 21-22
Myeloid progenitors, PG101 treatment and, 170
Myotoxins, 92

Naive B cells, dendritic cells and, 168
Natural foods, US regulation of, 203
Natural killer (NK) cells, 166, 178
in immune response, 161-162
mushroom polysaccharides and, 150, 163,

167-168
in sclerotial antitumor and

immunomodulatory studies, 133
Natural personal care products, US regulation of,
203

Natural Products Association, 208
Netherlands, food safety systems in, 219-220
Net protein ratio (NPR), in nutritional

evaluation, 87, 88
Net protein utilization (NPU), 88
Neurospora crassa, 37

Neutral sugars, in cultivated mushroom dietary
fiber, 77

Neutrophils, GLP effects on, 163, 164
New drug application (NDA), 218
Newly developed cultivated mushrooms
chemical composition of, 82
health and nutritional benefits of, 79-80,
81-87
New Zealand, 220

food safety systems in, 219-220
introducing medicinal-mushroom dietary
supplements in, 211-212
New Zealand Dietary Supplements Regulations
(NZDSR), 201



248 INDEX



New Zealand Food Safety Authority (NZFSA),
212

Niacin, in cultivated mushrooms, 76
Nicotinamide adenine dinucleotide phosphate

(NADPH), in Lentinus edodes genetics, 53
Nigeria, Pleurotus tuber-regium consumption in,

118

Nitric oxide (NO), 151, 153

Nitric oxide production, in sclerotial antitumor

and immunomodulatory studies, 133
Nonconventional mushrooms, health and

nutritional benefits of, 79-80, 81-87
Nonfat dry milk (NFDM), protein quality of, 88
Nongreen organisms, mushrooms as, 10-11
Nongreen revolution, mushrooms in, 9-11,

28-29

Non-insulin-dependent diabetes mellitus
(NIDDM), mushroom-related
hypoglycemia and, 98
Nonprotein N, in sclerotia, 124
Nonstarch polysaccharides (NSPs)

in cultivated mushroom dietary fiber, 77
from mushrooms, 152
North America

medicinal mushroom uses in, 25
mushroom production in, 22, 23, 73
Novel foods

in Australia and New Zealand, 212
in Japan, 212-214
Novel Foods Regulations (Canada), 2 1 1
Nuclear factor kappa B (NF-kB), 151, 166
dendritic cells and, 169
effects ofPGlOl and, 165
Nuclear magnetic resonance (NMR), in

antioxidant assays, 92
Nucleic acid biosynthesis, in Lentinus edodes, 45
Nucleic acids, in cultivated mushrooms, 78-79.
See also Deoxyribonucleic acid (DNA);
Ribonucleic acid (RNA)
Nucleotide biosynthesis genes, of Lentinus
edodes, 41

Nucleotides, in cultivated mushrooms, 78-79
Nutraceuticals. See also Nutriceuticals

in Canada, 210

regulation of, 200
Nutriceuticals. See also Mushroom nutriceuticals

defined, 200

regulation of, 200-201

sclerotial dietary fiber in, 126
Nutrients

in conventional edible mushrooms, 73-79,
81-87

direct toxicity of, 216-217
labeling of, 204-205



in mushroom cultivation, 13

during sclerotial development, 1 14-115

for sclerotial ontogeny, 113-114
Nutritional evaluation

biological methods for, 80-87

of mushrooms, 80-89

of sclerotia, 123-126
Nutritional supplements, US regulation of,
203-208

Nutritional value, of mushrooms, 24, 71-109
Nutrition Improvement Act of 1992 (Japan), 213
Nutrition industry, US regulation of, 203

Office of Nutritional Products, Labeling, and

Dietary Supplements, 205-206
Office of Special Nutritionals, medical food

regulation by, 200
Official Journal of the EC, 209
Oil-holding capacity (OHC), of sclerotial dietary

fiber, 126, 127
Oleic acid, in conventional edible mushrooms,

75

Oligosaccharides

in cultivated mushrooms, 78
in vivo Ca/Mg absorption and, 131, 132
Omphalia lapidescens

antitumor polysaccharides from, 154
branching configurations of polysaccharides

from, 175
cultivation of sclerotia of, 117
Ontogeny, of sclerotia, 112-115
Oogitake mushrooms, antioxidants in, 89
Ooi, Vincent E. C, xii, 147
Organic foods, US regulation of, 203
Ornatipolide, as mushroom antioxidant, 91, 92
Ornithine, in conventional edible mushrooms, 74
O-sulfonation, in chemical improvement of

antitumor activity, 177
Ovarian cancer, mushroom polysaccharides

versus, 151
Ovariectomized (OVX) rats, in vivo Ca/Mg

absorption studies using, 131-132
Overdoses, US regulations and, 208
Over-the-counter (OTC) drugs, US regulation of,

205
Oxalates

in cultivated mushrooms, 79
in sclerotial dietary fiber, 125
Oxidases, in Lentinus edodes genetics, 53
Oxidation, in biosynthesis of phenolic

compounds, 93
Oyster mushrooms, 17. See also Pleurotus
ostreatus

hypocholesterolemic effects of, 96, 97



INDEX 249



p38 MAPK, GLP effects on, 164
Pachyman

conformation of, 176
from mushrooms, 155
Pachymaran, in chemical improvement of

antitumor activity, 176
Pachymic acid, in Wolfiporia cocos cultivation,
121

Palmitic acid, in conventional edible

mushrooms, 75
Parasites, mushrooms as, 4, 5
Particle bombardment, for Lentinus edodes

transformation, 58
Pasteurization, in mushroom cultivation, 13
Pathogens, mushrooms as, 5
P. atroumborata, energy content of, 78
Pattern recognition receptors (PRRs), in immune

response, 162, 178
Paxillus involutus

polyphosphate granules in, 123
protein bodies in, 123
safety of, 216
Paxillus syndrome, 216
Pectic substances, in cultivated mushroom

dietary fiber, 78
PEG-mediated transformation, for Lentinus

edodes, 56
Perigold black truffle, as saprophyte, 4
Peripheral blood lymphocytes (PBLs),

immunomodulatory responses of, 166-167
Peristalsis, in colonic fiber fermentation, 129
Peritoneal exudate cells (PEC)
GLP effects on, 163
PSPC effects on, 167
Peritoneal macrophages (PMs), 166
Peroxide value, of Japanese mushrooms, 89
Peroxyl radical inhibition, by mushrooms, 91
PG101 polysaccharide, from Lentinus lepideus,

165, 170
Phagocytes, 178
Phagocytic responses, 162

polysaccharides and, 148
Phallus impudicus, biosynthesis of phenolic

compounds by, 94
Phanerochaete chrysosporium, lignin peroxidase

from, 60
Pharmaceuticals

from mushrooms, 10, 11, 28
regulation of mushroom, 199-200
Pharmacodynamic agents, safety of, 215
Pharmacology, of sclerotia, 128-134
Pharmacopeia monographs, WHO guidelines

and, 202

Phaseolus angularis, protein quality of, 88



Phaseolus calcaratus, protein quality of, 88
Phaseolus vulgaris, protein quality of, 88
Phellinus gilvus, antimetastatic effects of, 171
Phellinus linteus

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 154, 157
polysaccharide-protein complexes from, 160,
162, 166

polysaccharides isolated from, 148, 150, 151

proteoglycan from, 169.?
Phenolic acids, as mushroom antioxidants, 92
Phenolic antioxidants, in mushrooms, 91-93
Phenolic compounds

in cultivated mushrooms, 78

fungal biosynthesis of, 93-94

in mushrooms, 88-89, 90-91
Phenolic pigments, in sclerotial cell walls, 122
Phenotypic variation, in mushroom cultivation,
20

Phenylacetic acid, biosynthesis of, 93
Phenylalanine

biosynthesis of, 93

in conventional edible mushrooms, 75
Pholiota adiposa, 86

chemical composition of, 82
classification of, 80
Pholiota nameko, 87

chemical composition of, 82
classification of, 80
world production of, 73
Phosphatidyl biosynthesis, mushroom-related

hypocholesterolemia and, 94
Phosphatidylcholine (PC), mushroom-related

hypocholesterolemia and, 94
Phosphatidylethanolamine (PE),

mushroom-related hypocholesterolemia
and, 94

Phosphatidylinositol 1 -kinase (PI3K), GLP

effects on, 164
Phosphorus

in cultivated mushrooms, 76

in mushrooms, 23

in sclerotial dietary fiber, 125
Photosynthesis, as lacking in mushrooms, 10, 28
pH values, of sclerotial dietary fiber, 126
Physicochemical properties, of sclerotia,
126-128

Physiological processes, in Lentinus edodes,
47-48, 61

Physiology, of sclerotia during ontogeny,
114-115

Phytic acids, in cultivated mushrooms, 79
Phytochemicals, regulation of, 200



250 INDEX



PicoTitle Plate, in Lentinus edodes sequencing

by synthesis, 55
Pigmentation, of cortex, 116. See also Color

entries

Pigments, in sclerotial cell walls, 122
Pinto beans, protein quality of, 88
Pinus densiflora, in Wolfiporia cocos cultivation,
121

Planting spawn

of Lentinus edodes, 14, 15-16

in mushroom cultivation, 13-14
Plants

mushrooms versus, 2, 10-11

symbiotic with mushrooms, 4
Plasma, mushroom effects on, 97
Plasma membrane, in Lentinus edodes genetics,
51-52

Plasmid DNA, in Lentinus edodes

transformation, 57-58
Plastic bags, in Ganoderma lucidum cultivation,

20

Platelets, mushroom effects on, 97
pLCl-hph plasmid, in Lentinus edodes

transformation, 57-58
Pleurotaceae, classification of, 80
Pleurotus, 83-84

biology and genetics of, 17

chemical compositions of, 8 1

health and nutritional benefits of, 79

hypocholesterolemic effects of, 96

protein and amino acid content of, 74

protein quality of, 88

world production of, 72, 73
Pleurotus abalones, 83

chemical composition of, 8 1

classification of, 80
Pleurotus citrinopileatus, 83

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 156

chemical composition of, 81

classification of, 80

dietary fiber in, 77

protein and amino acid content of, 75
Pleurotus cornucopiae, 83

antitumor polysaccharides from, 156

chemical composition of, 8 1

classification of, 80

hypocholesterolemic effects of, 96
Pleurotus cystidiosus

antioxidants in, 90

carbohydrate content of, 78

dietary fiber in, 77

moisture content of, 73



Pleurotus djamor, 83

chemical composition of, 8 1

classification of, 80
Pleurotus eryngii, 83

antitumor polysaccharides from, 155, 156

chemical composition of, 8 1

classification of, 80

hypocholesterolemic effects of, 96

world production of, 72, 73
Pleurotus eryngii var ferulae, 83. See also
Pleurotus ferulae

ash and mineral content of, 76

chemical composition of, 8 1

classification of, 80

moisture content of, 73

protein and amino acid content of, 74, 75
Pleurotus eryngii var nebrodensis, 84. See also
Pleurotus nebrodensis

chemical composition of, 8 1

classification of, 80
Pleurotus ferulae, chemical composition of, 81,

82. See also Pleurotus eryngii var ferulae
Pleurotus florida

antitumor polysaccharides from, 155

immunomodulating polysaccharide from, 166

polysaccharides isolated from, 153
Pleurotus nebrodensis, 84. See also Pleurotus
eryngii var nebrodensis

chemical composition of, 8 1

classification of, 80
Pleurotus ostreatoroseus , antitumor

polysaccharides from, 155
Pleurotus ostreatus, 56, 57, 58, 59, 84

antioxidants in, 90, 92

antitumor glucans from, 157-159

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 157

ash and mineral content of, 76

carbohydrate content of, 78

chemical composition of, 8 1

classification of, 80

energy content of, 78

fat content of, 75

hypocholesterolemic effects of, 96
moisture content of, 73
polysaccharides isolated from, 152
protein and amino acid content of, 74, 75
protein quality of, 88
vitamin content of, 76-77
Pleurotus pulmonarius, 84

antitumor polysaccharides from, 155, 156
chemical composition of, 8 1



INDEX 251



classification of, 80

moisture content of, 73
Pleurotus sajor-caju

antitumor polysaccharide-protein complexes
from, 158

antitumor polysaccharides from, 157

cultivation of, 17

dietary fiber in, 77

lignocellulolytic enzymes of, 10

polysaccharides isolated from, 152

temperature stress in, 59
Pleurotus sapidus, 84

chemical composition of, 8 1

classification of, 80

hypocholesterolemic effects of, 96
Pleurotus tuber-regium, 121

antioxidants in, 90, 92

antitumor effects of, 132-133

antitumor polysaccharides from, 155

biopharmacological value of sclerotia from,
128-134

chemical improvement of antitumor

polysaccharides from, 177
cultivation of sclerotia of, 117
dietary fiber in, 125, 126-128
fermentation of dietary fiber from, 130
in vivo Ca/Mg absorption and, 131
Polyporus rhinocerus versus, 119
polysaccharides isolated from, 152-153
preparation of dietary fiber from, 126
proximate composition of, 123-124
sclerotia of, 118-119

Pluteaceae, classification of, 80

Poisonous mushrooms, distinguishing from
edible, 6

Poland, mushroom production in, 21
Pollution

applied mushroom biology and, 9,11
mushrooms versus, 3
population growth and, 10, 28
Polyethylene glycol (PEG), 56
Polymerase chain reaction (RAP-PCR), in

Lentinus edodes genetics, 36, 50, 51-52
Polymeric chain reactions (PCRs), in generating
Lentinus edodes molecular markers, 48,
49-50, 61
Polymers, polysaccharides as, 148
Polyphosphates, in sclerotial cytoplasmic

reserves, 122, 123
Polyporaceae, 119

classification of, 80
Polyporus confluens

antitumor polysaccharide-protein complexes
from, 159



antitumor polysaccharides from, 155, 156, 157
hypolipidemic effects of, 95
Polyporus mylittae

sclerotial germination of, 1 14
sclerotia of, 112
Polyporus rhinocerus, 121
antitumor effects of, 132-133
biopharmacological value of sclerotia from,

128-134
classification of, 119
cultivation of sclerotia of, 1 17
dietary fiber in, 125, 126-128
fermentation of dietary fiber from, 1 30
in vivo Ca/Mg absorption and, 131
preparation of dietary fiber from, 126
proximate composition of, 123
sclerotia of, 119-120
Polyporus tumulosus, biosynthesis of phenolic

compounds by, 93
Polysaccharide extracts, of mushrooms, 89-90
Polysaccharide from Ganoderma lucidum
(PS-G), 163-164. See also Ganoderma
lucidum polysaccharide (GLP)
dendritic cells and, 169
Polysaccharide-hydrolytic enzymes, in sclerotial

cell walls, 122
Polysaccharide medicines, from mushrooms, 25
Polysaccharide-protein complex (PSPC). See
also PSP polysaccharide-protein complex
mushrooms and, 24, 25, 160, 167
structure and antitumor activity of, 173-174
Polysaccharide-protein complexes, 148,
149-150, 152, 153, 178, 179
from mushrooms, 158-159
Polysaccharides, 178-179. See also Mushroom
polysaccharides
in cultivated mushroom dietary fiber, 77
dietary fiber and, 124
in vivo Ca/Mg absorption and, 131, 132
Lentinus edodes degradation of, 36-37
mushroom-related hypoglycemia and, 98
in mushrooms, 25
in sclerotial dietary fiber, 125
structural features of, 148
of Wolfiporia cocos, 120
Polysaccharopeptide (PSP), from mushrooms,
150. See also PSP polysaccharide-protein
complex

Polyunsaturated fatty acids, in conventional

edible mushrooms, 75, 80
Polyuronides, in sclerotial dietary fiber, 125
Population growth, applied mushroom biology

and, 9-10, 28



252 INDEX



Poria cocos, 120. See also Wolfiporia cocos
antitumor polysaccharides from, 155, 159,
160-161

branching configurations of polysaccharides

from, 174-175
in chemical improvement of antitumor

activity, 177
conformation of polysacchaides from, 176
Pork sausage, Pleurotus tuber-regium in, 118
Porodisculus pendulus, antitumor

polysaccharides from, 155
Postharvest studies, of Lentinus edodes, 59, 61
Potassium

in cultivated mushrooms, 76
in nonconventional edible mushrooms, 79
Potato dextrose agar (PDA), in Lentinus edodes

cultivation, 15
Pregnancy, Wolfiporia cocos in, 120
Preparation, of sclerotial dietary fiber, 126
PriA gene

in Lentinus edodes primordium formation, 38
in Lentinus edodes transcriptional regulation,
55, 56

Lentinus edodes transformation and, 58-59
PriA gene promoter, in Lentinus edodes

transformation, 57, 59
PriA gene terminator, in Lentinus edodes

transformation, 57, 59
PriB gene, in Lentinus edodes transcriptional

regulation, 55, 56
PRIB protein, in Lentinus edodes primordium

formation, 38-43
PRIB regulator, in Lentinus edodes, 46
Primordium, SAGE profiles of Lentinus edodes,

53

Primordium development genes, of Lentinus

edodes, 43, 44-47
Primordium formation, in Lentinus edodes,

38-44

Procatechoic acid, in mushrooms, 91, 92
Process analysis, of Lentinus edodes, 59-61
Prohibition of Exaggerated and Misleading

Claims law, in Japan, 214
Promoter analysis, in Lentinus edodes
transcriptional regulation, 55-56
Propagation, of mushrooms, 217
Propionate, colonic fermentation and, 130
Prosenchymatous tissue, in medulla, 117
Prostatic cancer

antimetastatic effects on, 171

mushroom polysaccharides versus, 150, 157
Protein. See also polysaccharide-protein
complexes; Proteins

in conventional edible mushrooms, 74-75



from mushrooms, 10-11, 23, 72, 80
in nonconventional edible mushrooms, 79, 8 1
in sclerotial cytoplasmic reserves, 122, 123
Protein Advisory Group of United Nations

System, 79
Protein bodies, in sclerotial cytoplasmic

reserves, 123
Protein chelators, in vitro sclerotial binding of

minerals and, 128-129
Protein degradation genes, of Lentinus edodes,
42

Protein digestibility, in nutritional evaluation, 87
Protein-digestibility-corrected amino acid score

(PDCAAS), in nutritional evaluation, 87
Protein efficiency ratio (PER), in nutritional

evaluation, 87, 88
Protein enrichment, via mushroom cultivation,

21, 22

Protein kinase C (PKC), 164, 166
Protein phosphates, in Lentinus edodes, 45
Protein quality, of mushrooms, 87-89
Proteins

Lentinus edodes degradation of, 36

in Lentinus edodes genetics, 53

as molecular chaperones, 59-60

in mushrooms, 25

polysaccharides and, 148
Protein tyrosine kinase (PTK), 166
Proteoglycans

in immune response, 162

immunomodulatory effects of, 169

from mushrooms, 158
Protocatechuic acid

biosynthesis of, 93

as mushroom antioxidant, 92
Proximate composition, of sclerotia, 123-124
PSK polysaccharide-protein complex. See also
Krestin

in angiogenesis, 172

antimetastatic effects of, 171

conformation of, 175

immunomodulatory effects of, 166-167,
169-170

isolation of, 148, 150

molecular masses of fractions of, 174

from mushrooms, 158, 159-160, 161-162

structure and antitumor activity of, 173-174
PSP polysaccharide-protein complex. See also
polysaccharide-protein complex (PSPC);
Polysaccharopeptide (PSP)

isolation of, 148, 150

from mushrooms, 158, 159-160, 167

structure and antitumor activity of, 173-174
Pyruvate decarboxylase, in Lentinus edodes, 48



INDEX 253



Qualified FOSHU, in Japan, 213
Qualified health claims, 206
Quality control, 220

in European Union, 209

of mushroom dietary supplements, 201

Radiation treatment, mushrooms and, 25
Rain forests, mushrooms in, 2
Random-amplified polymorphic DNA (RAPD),

in generating Lentinus edodes molecular

markers, 48, 49, 61
Ras gene promoter, in Lentinus edodes

transformation, 57, 59

Rats

in vivo Ca/Mg absorption studies using,
131-132

mushroom protein intake by, 88, 89
mushroom-related hypocholesterolemia

studies in, 94
mushroom-related hypoglycemia studies in,

98, 99

Reactive nitrogen intermediates (RNIs), 160

PSPC effects on, 167
Recombinant DNA (rDNA), in mushroom

cultivation, 20. See also Deoxyribonucleic

acid (DNA)

RecQ-type DNA helicase, in Lentinus edodes, 46
Redness, of sclerotial dietary fiber, 126
Reduction of Disease Risk FOSHU, in Japan,
213-214

Reference works, in mushroom identification, 6

Regulation

of mushrooms, 199-224
standardization problems with, 20 1

Regulatory mechanisms, polysaccharides in, 148

Reishi mushroom, 207

Representational difference analysis

(cDNA-RDA), in Lentinus edodes genetics,
36. See also cDNA representational
difference analysis (cDNA-RDA)

Reproductive growth phase, in mushroom
cultivation, 11-12

Reproductive organs, of filamentous fungi,
111-112

Restriction enzyme-linearized plasmid DNA, in

Lentinus edodes transformation, 57-58
Restriction enzyme-mediated integration

(REMI), for Lentinus edodes

transformation, 57-58
Restriction enzymes, in Lentinus edodes

transformation, 57-58
Restriction fragment length polymorphisms

(RFLPs), in generating Lentinus edodes

molecular markers, 48, 49



Rhamnoglucogalactan, from mushrooms, 156
Rhamnose

in cultivated mushroom dietary fiber, 78
in sclerotial dietary fiber, 125
Rhizomorphs, of filamentous fungi, 111, 112
Riboflavin, in cultivated mushrooms, 76
Riboglucan, from mushrooms, 155
Ribonucleic acid (RNA), in cultivated

mushrooms, 79. See also mRNA

expression; RNA synthesis
Ribonucleic acid fingerprinting, of Lentinus

edodes, 36

Ribonucleic acid-reduced biomass, from fungal

hyphae, 80
Ribonucleotide reductase (RNR)

in Lentinus edodes, 45

in Lentinus edodes meiosis, 60—61
Rice straw, in Volvariella cultivation, 17-18
Rigidoporus ulmarius, antiangiogenesis and, 173
Rind

cortex and, 116

of Polyporus rhinocerus, 119

in sclerotial ontogeny, 113

structure of, 115-116

of Wolfiporia cocos, 120
RNA synthesis, in Lentinus edodes, 45. See also

Ribonucleic acid (RNA)
Rolled oats, protein quality of, 88

Saccharomyces cerevisiae, 46, 57, 56
Safety, 220. See also Food safety systems
defined, 215-216

of dietary supplement types, 215-218
of functional foods, 208
via submerged culturing, 219
WHO guidelines and, 202
Safety warnings, for US dietary supplements,
205

SAGE 2000 Software, in Lentinus edodes

genetics, 52
Salmonella typhimurium, in antioxidant assays,

92

Sammin Mycological Institute, 79, 81, 82
Sanger sequencing technology, in Lentinus

edodes sequencing by synthesis, 55
Saprophytes, mushrooms as, 4, 5
Sarcodon aspratus

antitumor polysaccharides from, 157

fucogalactan from, 166
Sarcoma, 175

antimetastatic effects on, 171

chemical improvement of antitumor activity
versus, 177



254 INDEX



Sarcoma, (Continued)

mushroom polysaccharide-protein complexes
versus, 160

mushroom polysaccharides versus, 150-151
Sawdust, in Lentinus edodes cultivation, 16
SCG (Sparassis crispa /j-glucan), 151

immunomodulatory effects of, 164, 170-171
Schizophyllan

antitumor effects of, 165

branching configuration of, 175

in chemical improvement of antitumor
activity, 176

conformation of, 175

isolation of, 148, 150

from mushrooms, 155, 161, 165

regulation of, 200
Schizophyllan-OH, conformation of, 176
Schizophyllum commune, 38

antitumor polysaccharides from, 155

carbohydrate content of, 78

fat content of, 75

medicines from, 25

moisture content of, 74

polysaccharides isolated from, 148, 150

protein and amino acid content of, 74

protein quality of, 88

regulation of polysaccharides from, 199-200
Schizosaccharomyces pombe, 43
Science, in mushroom cultivation, 20-21
Scleroglucan, from mushrooms, 155
Sclerotia, xvii, 111-146, 178

antitumor polysaccharides from, 159

biochemical characteristics of, 121-123

biopharmacological values of, 128-134

cell walls of, 121-122

cultivation of, 117-121

cytoplasmic reserves of, 122-123

dietary fiber in, 124-126

extracellular matrix of, 122

in human diet, 72

lipid bodies in, 123

medicinal value of, 149

morphology and size of, 112

nutritional evaluation of, 123-126

ontogeny of, 112-115

physicochemical properties of, 126-128

polyphosphate granules in, 123

protein bodies in, 123

structure of, 115-117

world production of, 117
Sclerotinia

cortex of, 117

sclerotial germination of, 1 14
sclerotia of, 112



Sclerotiniaceae, sclerotia of, 112

Sclerotinia libertiana, antitumor polysaccharides

from, 155
Sclerotinia minor

cortex of, 115

glycogen reserves of, 122

rind of, 116

sclerotial germination of, 114
Sclerotinia sclerotiorum, rind of, 116
Sclerotinia trifoliorum, rind of, 116
Sclerotinium sclerotiorum, sclerotial germination

of, 114
Sclerotium rolfsii

ontogeny of, 113

sclerotia of, 112
Sclerotium sclerotia, antitumor polysaccharides

from, 155
Seasonality, of mushrooms, 2-3
Secretion phase, in mushroom cultivation, 12, 13
Selection phase, in mushroom cultivation, 12-13
Selective breeding, in mushroom cultivation, 20
Selenium, in cultivated mushrooms, 76
Selenium enrichment, of mushrooms, 24
Septa, of rind, 115

Sequence characterized amplified region

(SCAR) markers, for Lentinus edodes, 48,
49, 50

Sequence tags, in Lentinus edodes genetics, 52,
53

Sequencing-by-synthesis approach (454 Life

Science), in Lentinus edodes genetics, 36,

51,54-55, 60
Serial analysis of gene expression (SAGE), in

Lentinus edodes genetics, 36, 50, 51,

52-53, 61
Serine-rich proteins, in Lentinus edodes

genetics, 53
Serious adverse effects, under US regulations,

208

Serum, mushroom effects on, 97

Serum total cholesterol (TC), mushrooms

reducing, 97
S-GAP-P polysaccharide, in chemical

improvement of antitumor activity, 176
Shiitake mushroom, xvii, 14, 35-69, 207. See
also Lentinus edodes
hypocholesterolemic effects of, 94
popularity of, 35
safety of, 215
Shikimic acid pathway, in phenolic compound

biosynthesis, 93
Short-chain fatty acids (SCFAs), colonic
fermentation and, 129-130



INDEX



255



Short-log cultivation, of Ganoderma lucidum,
19-20

Signal transduction, in Lentinus edodes, 47, 61
Signal transduction genes

in Lentinus edodes, 44-45, 54

in Lentinus edodes primordium formation, 38,
39-40

Single-helix conformation, 175, 176

Size, of sclerotia, 1 12

Sodium

in cultivated mushrooms, 76

in nonconventional edible mushrooms, 79
Solid-state fermentation, in mushroom

cultivation, 10-11
Soluble dietary fiber (SDF)

in cultivated mushrooms, 77, 81

of sclerotia, 124, 125
Soluble sugars, in cultivated mushrooms, 78
Soup, Pleurotus tuber-regium in, 118
South Australian Working Party on Natural and

Nutrition Supplements, 211
South East Asia, Volvariella cultivation in,
17-18

Soybean waste, applied mushroom biology and,
11

Sparassis crispa, 164

antitumor polysaccharides from, 155

branched /i-glucan from, 170-171

polysaccharides isolated from, 148, 150, 151
Spawn development phase, in mushroom

cultivation, 12, 13
Spawn inoculation, of Wolfiporia cocos, 121
Spawn running

in Agaricus cultivation, 14

of Lentinus edodes, 14

in mushroom cultivation, 13
Spawn substrates

in Lentinus edodes cultivation, 15-16

in Pleurotus cultivation, 17
Specialists, in mushroom identification, 6
Specialization, in biology, 7
Spleen cells (splenocytes), mushroom

polysaccharide effects on, 163-167
Spore formation genes, of Lentinus edodes, 41
Spore germination, in Lentinus edodes

cultivation, 15
Spore print, in mushroom identification, 5
Spores, in mushroom identification, 5
Sporobolomyces roseus, biosynthesis of phenolic

compounds by, 93
Ssb gene, molecular chaperones and, 59-60
Stabilization, of sclerotial dietary fiber, 126
Stalk, in mushroom identification, 5
Standardization



in Australia and New Zealand, 211-212

of European dietary supplements, 209

of mushroom dietary supplements, 20 1
Standardized FOSHU, in Japan, 213
Starch, Lentinus edodes degradation of, 36-37
Starch utilization genes, of Lentinus edodes, 40
Sterilization, in mushroom cultivation, 13
Sterols, as mushroom antioxidants, 92
Stil gene, molecular chaperones and, 59-60
Stipe elongation, in Lentinus edodes, 44
Stock culture

in mushroom cultivation, 13

preparation of, 13-14
Strains, of Lentinus edodes, 50
Strands

of filamentous fungi, 111

in sclerotial ontogeny, 113
Straw mushroom, 17-18. See also Volvariella

hypocholesterolemic effects of, 97
Streptomyces hygroscopicus, 57
Streptozotocin (STZ), mushroom-related

hypoglycemia and, 98, 99
Stress response genes, of Lentinus edodes, 42
Stress responses, of Lentinus edodes, 59-60, 61
Stromata, of filamentous fungi, 111
Strophariaceae, classification of, 80

Stropharia rugoso-annulata, 87

chemical composition of, 82
classification of, 80

Structural proteins, in Lentinus edodes, 48
Structure, of sclerotia, 115-117
Structure-function (SF) claims, US regulation

of, 205-208
Submerged culturing, safety via, 219
Substrate, in sclerotial ontogeny, 113-114. See

also Cultivation substrate
Substrate preparation phase

in Agaricus brasiliensis cultivation, 18-19
in Ganoderma lucidum cultivation, 19-20
in Lentinus edodes cultivation, 15-16
in mushroom cultivation, 12, 13, 14
in Pleurotus cultivation, 17
Substrate-specific factors, in colonic fiber

fermentation, 129
Substrate-utilizing genes, of Lentinus edodes,
36-37

Sugar residues, in sclerotial dietary fiber, 125

Sugars. See also Carbohydrates

in conventional edible mushrooms, 75

in cultivated mushroom dietary fiber, 77-78

in mushrooms, 25

Suillus, peroxide value of, 89

Sulfation, in chemical improvement of antitumor
activity, 176, 177



256 INDEX



Sulfonation, in chemical improvement of

antitumor activity, 177
Sulfur-containing amino acids, in mushroom

protein, 88, 89
Superoxide free radicals, mushroom scavenging

of, 89-90

Supplement facts box, in labeling, 204-205
Suppression subtractive hybridization, of

Lentinus edodes, 5 1
Sweet components, in conventional edible

mushrooms, 75
Symphytum officinale, safety of, 216
Synnemata, of filamentous fungi, 111, 112

Tags, in Lentinus edodes genetics, 52, 53. See

also Expressed sequence tags (ESTs)
Taiwan, Wolfiporia cocos consumption in,

120-121
Tannins, in mushrooms, 88
TATA box, in Lentinus edodes transcriptional

regulation, 55-56
TCA (tricarboxylic acid; citrate) cycle, in

Lentinus edodes, 48. See also Tricarboxylic
acid (TCA)
T cells

D-fraction and, 167-168
effects of lentinan on, 165
mushroom polysaccharides and, 150
PSK response of, 166-167
TCP1 gene, molecular chaperones and, 59-60
Technological innovation, in mushroom industry,
22

Temperature

in Agaricus brasiliensis cultivation, 18

in Agaricus cultivation, 14

in Lentinus edodes cultivation, 15

in Pleurotus cultivation, 17

in Volvariella cultivation, 17-18
Temperature stress, in mushrooms, 59, 60
Terfezia arnenari, 2
Terfezia claveryi, protein quality of, 88
Terminal cells, of rind, 115-116
Terminal development, of sclerotia, 1 1 3
Termitomyces eurhinus, antitumor

polysaccharides from, 155
Termitomyces robustus

energy content of, 78

protein quality of, 88
Texture, of sclerotial dietary fiber, 126
The Institute for Genome Research (TIGR), in

Lentinus edodes genetics, 54
Therapeutic adjuvants, mushroom
polysaccharides as, 147-148
Therapeutic agents, from mushrooms, 150



Therapeutic pharmacodynamic activity, safety
of, 215

Thickening, of sclerotial dietary fiber, 1 26
Thin-layer chromatography (TLC), in

antioxidant assays, 92
Threonine

in conventional edible mushrooms, 74, 75

in mushroom protein, 89
Tiger milk mushroom, 118
tlgl gene, in Lentinus edodes, 59
TLR pathway, in immune response, 162
Toads, mushrooms and, 2
Toadstools, 2

a-Tocopherol, in mushroom antioxidant assays,
90, 92

Total color difference, of sclerotial dietary fiber,
126

Total dietary fiber (TDF)

in cultivated mushrooms, 77-78
in nonconventional edible mushrooms, 79,81
in sclerotia, 125
Total productivity, of biomass materials, 28
Toxicity

of mushrooms, 6
of nutrients, 216-217
Toxic substances, in mushroom cultivation, 13
Traditional mushroom products, safety of, 216
Trametes, hypolipidemic effects of, 95
Trametes gibbosa, antitumor polysaccharides

from, 155
Trametes versicolor, regulation of

polysaccharides from, 199-200. See also
Coriolus (Trametes) versicolor
Transcriptional factor binding sites (TFBSs), in
Lentinus edodes transcriptional regulation,
55

Transcriptional factors (TFs), in Lentinus edodes

transcriptional regulation, 55
Transcriptional regulation, of Lentinus edodes,

55- 56

Transcription regulation genes, in Lentinus

edodes primordium formation, 38, 39-40
Transformation, of Lentinus edodes, 56-59
Transformation methods, for Lentinus edodes,

56- 58

Transgenic breeding, transformation in, 56, 57
Translocation, of sclerotia during ontogeny,
114-115

Transmission electron microscopy (TEM), in
sclerotial cytoplasm studies, 122

Transport genes, of Lentinus edodes, 38, 41

Trehalose

in cultivated mushrooms, 78
during sclerotial development, 1 14



INDEX 257



Tremella, Chinese production of, 72, 73
Tremella aurantia

dietary fiber in, 125

hypolipidemic effects of, 95

mushroom-related hypoglycemia and, 98-99
Tremella cinnabarina, world production of, 73
Tremella fuciformis

antitumor polysaccharide-protein complexes
from, 159

antitumor polysaccharides from, 156, 157
dietary fiber in, 77, 125
hypocholesterolemic effects of, 97
hypolipidemic effects of, 95
mushroom-related hypoglycemia and, 98-99
Tremella mesenterica, antitumor polysaccharides
from, 157

Triangular model, for mushroom classification, 5
Tricarboxylic acid (TCA), Lentinus edodes and,

44. See also TCA (tricarboxylic acid;

citrate) cycle
Tricholoma, 173
Tricholoma giganteum
antioxidants in, 90

antitumor polysaccharide-protein complexes
from, 159

antitumor polysaccharides from, 155, 157
carbohydrate content of, 78
dietary fiber in, 77
fat content of, 75

protein and amino acid content of, 74
world production of, 73
Tricholoma lobayense

antitumor polysaccharide-protein complexes

from, 159
medicines from, 25

polysaccharide-protein complexes from, 160,
167

Tricholoma matsutake, 72

antitumor polysaccharide-protein complexes

from, 159
ecological classification of, 5
as saprophyte, 4
Tricholoma mongolicum, antitumor

polysaccharide-protein complexes from,
159

Tricholoma portentosum
dietary fiber in, 77

protein and amino acid content of, 74, 75

protein quality of, 88
Tricholomataceae, 35

antioxidants in, 90

classification of, 80
Tricholoma terreum

carbohydrate content of, 78



dietary fiber in, 77
fat content of, 75

protein and amino acid content of, 74, 75
protein quality of, 88
Triggers

in Lentinus edodes primordium formation, 38

in mushroom cultivation, 12
Triglycerides, mushrooms as lowering, 96-97
Triple-helix conformation, 175-176
Triterpenes

in mushrooms, 25

in sclerotial antitumor and

immunomodulatory studies, 133
True protein digestibility (TPD), of mushrooms,
88

Truffles

desert, 2

matsutake, 4, 5

Pengold black, 4
Tryptophan

biosynthesis of, 93

in conventional edible mushrooms, 74, 75
Tuber melanosporum, 72

ecological classification of, 5

as saprophyte, 4
Tumor necrosis factor alpha (TNF-a), 151, 160,
163, 164, 166, 167, 178

effects of lentinan on, 165
Tumor xenografts, in sclerotial antitumor and

immunomodulatory studies, 133-134
Tuna fish, protein quality of, 88
Tylopilus felleus, antitumor polysaccharides

from, 155
Type 2 diabetes, 207
Typhula, sclerotia of, 112
Typhula incarnata

cortex of, 117

medulla of, 117

protein bodies in, 123

rind of, 116
Typing, of Lentinus edodes, 50
Tyrosine

biosynthesis of, 93

in conventional edible mushrooms, 75

Ubiquitins, of Lentinus edodes, 43-44
Umami flavor, of edible mushrooms, 74-75,
78-79

Umbilical cord blood (CB) cells, 170
UMP-CMP kinase, in Lentinus edodes, 45
United Kingdom

food safety systems in, 219-220
mushroom nutriceutical regulation in, 201
regulation of dietary supplements in, 210



258 INDEX



United States, 220. See also American entries;

Federal entries; Food and Drug

Administration (FDA)
drug regulation in, 218
food safety in, 215

introducing medicinal-mushroom dietary

supplements in, 203-208
medicinal mushroom use in, 25
mushroom nutriceutical regulation in, 200,

201

Unsaturated fatty acids, in conventional edible

mushrooms, 75, 80
Uronic acids

in cultivated mushroom dietary fiber, 77, 78

in sclerotial dietary fiber, 125

Vacuolation, of cortex, 116
Vacuoles, in rind cells, 116
Valine

in conventional edible mushrooms, 74, 75

in mushroom protein, 89
Variability, of polysaccharides, 148
Vascular endothelial growth factor (VEGF), in

angiogenesis, 172
Vegetables, mushrooms as, 72
Vegetative growth phase, in mushroom

cultivation, 11, 12
Verticillium dahlae

lipid bodies in, 123

sclerotia of, 112
Vietnam, mushroom production in, 21
Vitamin Bi, in cultivated mushrooms, 77
Vitamin B2, in cultivated mushrooms, 76
Vitamin B12, in cultivated mushrooms, 77
Vitamin B 12 deficiency, 217
Vitamin C

in cultivated mushrooms, 77

mushroom polysaccharides and, 150
Vitamin D2, in cultivated mushrooms, 77
Vitamins

in conventional edible mushrooms, 76-77

in dietary supplements, 200

European Union regulation of, 209

labeling of, 204-205

in mushrooms, 23, 80

in nutrition, 23
Vitapurity, FDA detention of, 207
Volvariella

cultivation of, 17-18

industrial-scale cultivation of, 18

world production of, 72, 73
Volvariella bombycina

dietary fiber in, 77

fat content of, 75



protein and amino acid content of, 75
Volvariella volvacea, 58
anatomy of, 3
antioxidants in, 90, 91
antitumor polysaccharides from, 155
carbohydrate content of, 78
classification of, 80
hypocholesterolemic effects of, 97
hypolipidemic effects of, 95
lignocellulolytic enzymes of, 10
medicinal effects of, 24
protein and amino acid content of, 74
world production of, 73

Wasser, Solomon P., xxi, 27, 199

Wasson, R. Gordon, 1

Wastes, applied mushroom biology and, 9,

10-11, 28
Water

in nutrition, 23

during sclerotial development, 114
Water-binding capacity (WBC), of sclerotial

dietary fiber, 126, 127
White jelly-leaf mushroom, hypocholesterolemic

effects of, 97
Whiteness, of sclerotial dietary fiber, 126
White rot fungus

Lentinus edodes as, 60
Polyporus rhinocerus as, 119
WHO Food Standards Programme, 202-203.

See also World Health Organization (WHO)
WHO Nutrient Risk Project, 203
Wild edible fungi, Israeli regulation of, 214-215
Wild Edible Fungi: A Global Overview of Their

Use and Importance to People (FAO),

214-215

Wild edible mushrooms, health and nutritional

benefits of, 79-80, 81-87
Wild mushrooms
edible, 72, 73

international movement for, 27
in mushroom industry, 26
Wolfiporia cocos. See also Poria cocos
antitumor effects of, 133
biopharmacological value of sclerotia from,

128-134
cultivation of sclerotia of, 117
dietary fiber in, 125-126, 126-128
fermentation of dietary fiber from, 130-131
hypolipidemic effects of, 95
in vivo Ca/Mg absorption and, 131-132
preparation of dietary fiber from, 126
proximate composition of, 123, 124
sclerotia of, 120-121



INDEX 259



Wong, Ka-Hing, xxi, 111
Wood

in biomass waste, 10

in Ganoderma lucidum cultivation, 19-20
in Lentinus edodes cultivation, 15, 16
in Wolfiporia cocos cultivation, 121
Wood rot fungus

Lentinus edodes as, 15
Pleurotus tuber-regium as, 118
Working Party on Natural and Nutrition

Supplements, 211
World Health Organization (WHO). See also
WHO entries
on introducing medicinal-mushroom dietary

supplements, 202, 220
on mushroom protein and amino acid content,
74

World mushroom production, 21-23
World population growth, applied mushroom

biology and, 9-10, 28
World production, of cultivated mushrooms,

72-73, 117
World Society for Mushroom Biology and

Mushroom Products (WSMBMP), 21
formation of, 27

Xanthosine monophosphate (XMP), in cultivated

mushrooms, 78
Xenografts, in sclerotial antitumor and

immunomodulatory studies, 133-134
X fraction, mushroom-related hypoglycemia

and, 98

Xiang gu mushroom, 14, 35. See also Lentinus
edodes

X-ray microanalysis, of polyphosphate granules,
123

X-ray microanalyzer, in sclerotial dietary fiber
studies, 125



Xylan

Lentinus edodes degradation of, 60
from mushrooms, 152, 156
Xylanase genes, in Lentinus edodes

transcriptional regulation, 56
Xylaria nigripes, cultivation of sclerotia of,
117

Xylogalactoglucan, from mushrooms, 156
Xyloglucan, from mushrooms, 152, 155, 156
Xyloglucan-protein complex, from mushrooms,
158, 159

Xyloglucomannan, from mushrooms, 157
Xylomannan, from mushrooms, 156
Xylopyranosyl residues, mushroom-related

hypoglycemia and, 98
Xylose, in cultivated mushroom dietary fiber,

78

xynllA gene, in Lentinus edodes, 60

Yeast, in sclerotial antitumor and
immunomodulatory studies, 133

Yeast two-hybrid system, in Lentinus edodes
genetics, 51, 54

Yellowness, of sclerotial dietary fiber, 126

Yunnan Province, Pleurotus tuber-regium
consumption in, 118

Zero emissions, 28
Zinc

in cultivated mushrooms, 76
in vitro sclerotial binding of, 128-129
in Lentinus edodes primordium formation,
38-43

in sclerotial dietary fiber, 125
Zygomycota, classification of, 80
Zymosan, in sclerotial antitumor and
immunomodulatory studies, 133



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