Biochemical Engineering and Biotechnology of Medicinal Mushrooms (Advances in Biochemical Engineering/Biotechnology, 184) [1st ed. 2023] 3031369491, 9783031369490

Table of contents :
Preface
Contents
Medicinal Mushrooms: Past, Present and Future
1 Introduction
2 Past
3 Present
3.1 The Pyramid Model of the Mushroom Industry
4 Future Outlook
4.1 Industrial-Scale MM Production
4.2 Mushroom Products: Safety, Standardization, Quality Control
4.3 Other Applications of MM Cultivation
5 Conclusions
6 Closing Remarks
References
Farming of Medicinal Mushrooms
1 Introduction
2 Farming Technology
3 Farming Substrates
4 Farming of Medicinal Mushrooms
4.1 Cultivation of Ganoderma lucidum
4.1.1 Cultivation on Natural Wood Logs
4.1.2 Substrate Cultivation in Bags and Bottles: Synthetic Log Cultivation
4.1.3 Cultivation in Trays or Beds
4.2 Cultivation of Grifola frondosa
4.2.1 Nutrition and Substrate Compositions
4.2.2 Cultivation in Bags
4.2.3 Cultivation in Bottles
4.2.4 Cultivation in Bed Cultures
4.3 Agaricus brasiliensis (Agaricus blazei)
4.3.1 Cultivation in Beds
4.4 Pleurotus Species
4.4.1 Cultivation on Wood Logs
4.4.2 Bag Cultivation
4.4.3 Bottle Cultivation
4.4.4 Cultivation in Beds: Shelf Cultivation
4.4.5 Substrate Improvements and Alternatives for Pleurotus Cultivation
4.5 Lentinus edodes
4.5.1 Production of Lentinus edodes on Wood Logs
4.5.2 Growing Lentinus edodes in Bags (Synthetic Log Production)
4.5.3 Bed Cultures
4.5.4 Substrate Modifications and Improvements
5 Conclusions
References
Mushroom Production in the Southern Cone of South America: Bioeconomy, Sustainable Development and Its Current Bloom
1 Introduction
2 Edible and Medicinal Mushroom Production Traits in the Southern Cone of South America
2.1 Mushroom Cultivation Diversifies the Labour Supply and Encourages the Transfer of Science to the Socio-Cultural Sphere and...
2.2 Mushroom Cultivation Increases the Availability of Healthy Foods
2.3 Mushroom Cultivation Promotes Innovation Aimed at Environmental Sustainability
3 Prospects for the Southern Cone of South America
3.1 1st Bottleneck: The Supply Chain of Substrates
3.2 2nd Bottleneck: Provision of Supplies, Equipment and Infrastructure
3.3 3rd Bottleneck: Authorization, Commercialization and Use of Residual Substrate
4 A Case Study: Bioconversion of Sunflower Seed Hulls and Solid Residues from Olive-Oil Production to Mushrooms of the Ganoder...
5 Sunflower and Olive Lignocellulosic Residues as a Starting Reservoir for the Generation of Nutraceuticals and Energy
6 Conclusion
References
Barriers to the Use of Medicinal Mushrooms for Production of Metabolites
1 Introduction
2 Background to the Story
3 Growing Mushrooms in Liquids
4 Morphology
5 Oxygen Transfer
6 Scale-up
7 Power Input
8 Oxygen Transfer Coefficient
9 Impeller Tip Speed
10 Study Your Data Before You Report Them
11 Conclusion
Reference
Advances in Production of Medicinal Mushrooms Biomass in Solid State and Submerged Bioreactors
1 Introduction
2 Cultivation Technologies
3 Solid-State Cultivation (SSC)
3.1 Substrates for SSC
3.2 Ganoderma lucidum
3.3 Grifola frondosa
3.4 Trametes versicolor
3.5 Hericium erinaceus
3.6 Cordyceps militaris
4 Submerged Cultivation
4.1 Ganoderma lucidum
4.1.1 Inoculum Preparation and Density
4.1.2 Effect of Initial Medium pH
4.1.3 Influence of Aeration and Agitation
4.1.4 Temperature
4.1.5 Influence of Substrate Composition and Carbon Sources
4.1.6 Effect of Nitrogen Sources and Carbon to Nitrogen (C/N) Ratio
4.1.7 Influence of Macro- and Micro-elements
4.1.8 Effect of Plant Oils and Fatty Acids
4.1.9 Effect of Polymer Additives
4.1.10 Batch Cultivation
Batch Cultivation in Airlift Reactor
Batch Cultivation in Stirred Tank Reactor
Batch Cultivation in System of Combined Reactors
4.1.11 Multi-pulse Feeding Integrated Strategy
4.1.12 Fed-Batch Cultivation
4.1.13 Repeated Fed-Batch Cultivation
4.2 Grifola frondosa
4.2.1 Inoculum
4.2.2 Effect of Initial pH
4.2.3 Effect of Carbon and Nitrogen Sources
4.2.4 Effects of Plant Oils and Surfactants
4.2.5 Effects of Oxygen Concentration
4.2.6 Cultivation of G. frondosa in a Stirred Tank Reactor
4.2.7 Cultivation of G. frondosa in Airlift Reactor
Batch Cultivation
Fed-Batch Cultivation
4.3 Trametes versicolor
4.4 Hericium erinaceus
4.5 Cordyceps militaris
4.6 Other Species
5 Conclusions
References
Advances in Pilot-Scale Stirred Bioreactors in Solid-State and Submerged Cultivations of Medicinal Mushrooms
1 Introduction
2 Solid-State Cultivation
2.1 Ganoderma lucidum
2.2 Grifola frondosa
2.3 Trametes versicolor
2.4 Hericium erinaceus
2.5 Cordyceps militaris
3 Submerged Cultivation
3.1 Pleurotus ostreatus
3.2 Ganoderma australe
3.3 Cordyceps militaris
3.4 Ganoderma lucidum
3.5 Ganoderma lucidum EPS Production by Simulation Scale-Up
3.6 Hericium erinaceus
4 Discussion
5 Conclusions
References
Downstream Processing of Medicinal Mushroom Products
1 Introduction
2 Medicinal Mushroom Products
3 General Downstream Procedures
3.1 Sample Drying
3.2 Sample Disruption
3.3 Extraction
3.4 Fractionation
3.5 Purification
4 Examples of Downstream Processing of Typical Medicinal Mushroom Products
4.1 Downstream Processing of Polysaccharides
4.2 Downstream Processing of Triterpenoids
5 Conclusions
References
Bioactive Compounds from Medicinal Mushrooms
1 Introduction
2 Bioactive Compounds
2.1 Polysaccharides
2.1.1 Homopolysaccharides
2.1.2 Heteropolysaccharides
2.2 Proteins
2.3 Triterpenoids
2.4 Meroterpenoids
2.5 Steroids
2.6 Polyphenols
2.7 Nitrogen-Containing Compounds
3 Bioactivity of Polysaccharides of MMs
3.1 Immunomodulatory Activity
3.2 Antitumor Activity
3.3 Hypoglycemic Activity
3.4 Hepatoprotective Activity
3.5 Activity of Regulation of Intestinal Flora
4 Conclusion
References
Research Progress on Edible Fungi Genetic System
1 Elements for Genetic Transformation of Edible Fungi
1.1 Promoter Type
1.2 Selectable Marker
1.2.1 Antibiotic Selectable Marker
1.2.2 Auxotrophic Selectable Markers
1.2.3 Herbicide and Fungicide Resistance Markers
1.2.4 Metabolite Resistance Markers
2 Transformation Method
2.1 ATMT Method
2.2 LMMT Method
2.3 PEG Transformation
2.4 Electroporation Method
2.5 Restriction Enzyme-Mediated Integration
3 Application of Transformation Systems in Edible Fungi
3.1 Gene Knockdown
3.2 Gene Knockout
3.3 Gene Overexpression
4 Conclusions
References
The Health and Clinical Benefits of Medicinal Fungi
1 Introduction
2 High-Molecular-Weight Active Compounds
2.1 The Importance of β-Glucans
2.2 Chitin, The Second Most Abundant Biopolymer on Earth
2.3 Mushrooms, The Most Effective Prebiotic Fiber
3 Low-Molecular-Weight Active Compounds
3.1 Phenolics
3.2 Terpenes
3.3 Indoles, Amines, Non-Protein Amino Acids, Lectins, and Alkaloids (N-Containing Compounds)
3.4 Volatile Compounds and Scents
3.5 Alkaloids
3.6 Nutrition
4 Products for Clinical Use, Prepared Medicines
4.1 Current Products Sold for Medical Practitioners and Consumers: Quality Issues
5 Sourcing Mushrooms and Medicinal Mushroom Products
5.1 Quality and Standardization Considerations
5.2 Cultivation
5.3 Extraction
5.4 Finished Products
6 Bottom of Form
6.1 Individual Mushrooms Species Profiles: Clinical Trials and Observational Studies
7 Conclusions
References

Citation preview

Advances in Biochemical Engineering/Biotechnology 184 Series Editor: Roland Ulber

Marin Berovic Jian-Jiang Zhong   Editors

Biochemical Engineering and Biotechnology of Medicinal Mushrooms

184 Advances in Biochemical Engineering/Biotechnology Series Editor Roland Ulber, Kaiserslautern, Germany Editorial Board Members Thomas Scheper, Hannover, Germany Shimshon Belkin, Jerusalem, Israel Thomas Bley, Dresden, Germany Jörg Bohlmann, Vancouver, Canada Man Bock Gu, Seoul, Korea (Republic of) Wei Shou Hu, Minneapolis, MN, USA Bo Mattiasson, Lund, Sweden Lisbeth Olsson, Göteborg, Sweden Harald Seitz, Potsdam, Germany Ana Catarina Silva, Porto, Portugal An-Ping Zeng, Hamburg, Germany Jian-Jiang Zhong, Shanghai, Minhang, China Weichang Zhou, Shanghai, China

Aims and Scope This book series reviews current trends in modern biotechnology and biochemical engineering. Its aim is to cover all aspects of these interdisciplinary disciplines, where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, molecular biology, chemical engineering and computer science. Volumes are organized topically and provide a comprehensive discussion of developments in the field over the past 3–5 years. The series also discusses new discoveries and applications. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. In general, volumes are edited by well-known guest editors. The series editor and publisher will, however, always be pleased to receive suggestions and supplementary information. Manuscripts are accepted in English. In references, Advances in Biochemical Engineering/Biotechnology is abbreviated as Adv. Biochem. Engin./Biotechnol. and cited as a journal.

Marin Berovic • Jian-Jiang Zhong Editors

Biochemical Engineering and Biotechnology of Medicinal Mushrooms With contributions by M. Berovic
M. Bidegain
J. Buswell
D. Caprile
S. Chang
J. Feng
N. Feng
D. Figlas
R. González Matute
C. Hobbs
B. Kristiansen
Y. Li
L. Liu
R. Liu
Y. Liu
H. Luo
M. Nikšić
B. Boh Podgornik
P. Postemsky
A. Ren
V. Salazar-Vidal
M. Saparrat
L. Shi
Y. Yang
H. Zhang
J. Zhang
M. Zhao
J.-J. Zhong
J. Zhu

Editors Marin Berovic Department of Chemical and Biochemical Engineering University of Ljubljana Ljubljana, Slovenia

Jian-Jiang Zhong School of Life Sciences and Biotechnology Shanghai Jiao Tong University Shanghai, China

ISSN 0724-6145 ISSN 1616-8542 (electronic) Advances in Biochemical Engineering/Biotechnology ISBN 978-3-031-36949-0 ISBN 978-3-031-36950-6 (eBook) https://doi.org/10.1007/978-3-031-36950-6 Graphic Art and All Graphic Abstract (besides Chapter 3) by Marin Berovic © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Mushrooms have the potential to serve as a healthy food source. Pure refined products can be incorporated into one’s diet as a form of medicine to address compromised health. Crude extract products, on the other hand, are primarily used as dietary supplements (nutraceuticals) for individuals in healthy, sub-healthy, and ill states. Human history bears witness to thousands of years of knowledge and longterm utilization of medicinal mushrooms in treating various diseases. This is evident in Europe through the discovery of Ötzi, the Iceman, frozen 5000 years ago, as well as medical records in China and Ayurveda in East Asia, which showcases the development of this knowledge over the past 2000 years and its clinical use as folk medicine. Continuous research and the use of many new types of medicinal mushrooms have been prevalent in East Asia. Fungal polysaccharides, proteins, proteinoglucans, terpenoids, sterols, and steroids, among others, represent a remarkable collection of naturally produced molecules with highly proven efficacy. The diverse bioactivities of polysaccharides from medicinal mushrooms, such as immunomodulatory, antitumor, hypoglycemic, and hepatoprotective activities, are already widely recognized. Surprisingly, these results and research have not been sufficient to encourage greater production of medicinal mushrooms and their products in the Western world, where the Western Pharmaceutical Industry did not perceive significant profitability. Even with the fast and comprehensive submerged cultivation of highly active medicinal mushroom biomass and their products, there has been a lack of strong interest and investment from the industry. One of the most common objections and non-recommendations regarding the use of medicinal mushroom derivatives in modern medicine is the lack of knowledge and recognition of their effectiveness and rich medical heritage. Most people, including doctors, are not adequately informed about the health benefits, clinical effectiveness, and safety of using medicinal mushrooms. In this regard, the clinical experience primarily gained in Chinese hospitals serves as proof of the effectiveness of such treatment with drugs. Patients with various types of cancers who received chemotherapy alongside mushroom v

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Preface

extracts significantly improved their survival rates and experienced a better quality of life by reducing chemotherapy side effects such as fatigue, depression, and nausea. The notable surge in interest in products derived from mushroom extracts in the market and media can also be attributed to the fact that modern medicine and pharmacy have not yet produced drugs that are nearly as non-toxic and proven safe as mushroom-based medications. These medications simultaneously exhibit beneficial, regulating, and activating effects on our immune function and the functioning of other body systems. It is worth mentioning that many medicinal products are inexpensive compared to numerous pharmaceutical drugs, and they have been clinically confirmed to be entirely safe or with minimal side effects. The healing power of mushrooms has a rich tradition in the Eastern world and a proven effectiveness surpassing that of existing modern pharmaceutical drugs. In comparison, the Western world is only just beginning to explore this field. This volume is the result of collaborative work by selected groups engaged in medicinal mushroom research from both the West and the East. It combines the collective knowledge in this book, serving as a reference for comprehensive information and progress in all areas of research, ranging from farming, solid-state, and submerged cultivation engineering of medicinal mushrooms to downstream processing of their products. It also covers the latest aspects and findings in modern biochemistry, genetic engineering, and clinical experiences. In this format, it serves as a guidebook for postgraduate students, young researchers, and engineers who are interested and involved in the development of this exceptional field. The publication aims to showcase the potential of naturally occurring active substances that have been present for thousands of years and their relevance and application in our everyday lives. Ljubljana, Slovenia Shanghai, China

Marin Berovic Jian Jiang Zhong

Contents

Medicinal Mushrooms: Past, Present and Future . . . . . . . . . . . . . . . . . . Shuting Chang and John Buswell

1

Farming of Medicinal Mushrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miomir Nikšić, Bojana Boh Podgornik, and Marin Berovic

29

Mushroom Production in the Southern Cone of South America: Bioeconomy, Sustainable Development and Its Current Bloom . . . . . . . . Pablo Postemsky, Maximiliano Bidegain, Ramiro González Matute, Débora Figlas, Daniela Caprile, Viviana Salazar-Vidal, and Mario Saparrat

77

Barriers to the Use of Medicinal Mushrooms for Production of Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Bjørn Kristiansen and M. Berovic Advances in Production of Medicinal Mushrooms Biomass in Solid State and Submerged Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Marin Berovic and Jian-Jiang Zhong Advances in Pilot-Scale Stirred Bioreactors in Solid-State and Submerged Cultivations of Medicinal Mushrooms . . . . . . . . . . . . . . . . . 163 Marin Berovic and Jian-Jiang Zhong Downstream Processing of Medicinal Mushroom Products . . . . . . . . . . . 187 Haiyan Luo and Yingbo Li Bioactive Compounds from Medicinal Mushrooms . . . . . . . . . . . . . . . . 219 Jingsong Zhang, Na Feng, Yangfang Liu, Henan Zhang, Yan Yang, Liping Liu, and Jie Feng

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Contents

Research Progress on Edible Fungi Genetic System . . . . . . . . . . . . . . . . 269 Liang Shi, Ang Ren, Jing Zhu, Rui Liu, and Mingwen Zhao The Health and Clinical Benefits of Medicinal Fungi . . . . . . . . . . . . . . . 285 Christopher Hobbs

Adv Biochem Eng Biotechnol (2023) 184: 1–28 https://doi.org/10.1007/10_2021_197 © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 Published online: 27 February 2022

Medicinal Mushrooms: Past, Present and Future Shuting Chang and John Buswell

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Past . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 The Pyramid Model of the Mushroom Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Industrial-Scale MM Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Mushroom Products: Safety, Standardization, Quality Control . . . . . . . . . . . . . . . . . . . . . . . 4.3 Other Applications of MM Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 5 7 9 15 16 19 21 22 23 24

Abstract The survival of Homo sapiens is continually under threat from agencies capable of inflicting calamitous damage to the overall health and well-being of humankind. One strategy aimed at combatting this threat is focused on medicinal mushrooms and derivatives thereof. Mushrooms themselves have been consumed as part of the human diet for centuries, whereas ‘mushroom nutriceuticals’ is a more recently adopted term describing mushroom-derived products taken as dietary supplements to enhance general health and fitness. Among the most extensively studied pharmacologically active components of mushrooms are polysaccharides and polysaccharide-protein complexes, triterpenes, lectins, and fungal immunomodulatory proteins. Medicinal mushrooms have been credited with a wide range of therapeutic properties including antitumour/anti-cancer, antioxidant, hepatoprotective, anti-diabetic, antimicrobial, cholesterol-lowering and

S. Chang (*) Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, China J. Buswell Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China

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S. Chang and J. Buswell

genoprotective activities as well as protection against atherosclerosis, cardiovascular, chronic inflammatory and autoimmune diseases, and neurodegenerative conditions. This review examines the past, present and future of medicinal mushroom development including the two legs concept for the mushroom industry and the pyramid model summarizing the various human applications of mushrooms. It considers numerous issues the industry needs to address to exploit fully the opportunities presented by the continued increasing demand for medicinal mushrooms, and by the future overall expansion of the medicinal mushroom movement. Graphical Abstract

Keywords Climate change, Dietary supplements, Functional foods, Medicinal mushrooms, Mushroom nutriceuticals, Mushroom spawn, Nutraceutical, Polysaccharides

Abbreviations AIDS COVID-19 DS EM HIV IJMM IMMC IPCC

Acquired immunodeficiency syndrome Coronavirus disease 2019 Dietary supplement Ectomycorrhizal Human immunodeficiency virus International Journal of Medicinal Mushrooms International Medicinal Mushroom Conference Intergovernmental Panel on Climate Change

Medicinal Mushrooms: Past, Present and Future

ISMM ISMS ITS MIRCEN MM PBDEs PCBs RAPD RFLP RIPs SARS TCM UNESCO WSMBMP

3

International Society for Medicinal Mushrooms International Society for Mushroom Science Internal transcribed spacer Microbial Resource Centre Medicinal mushroom Polybrominated diphenyl ethers Polychlorinated biphenyls Random amplified polymorphism DNA Restriction fragment length polymorphism Ribosome-inactivating proteins Severe acute respiratory syndrome Traditional Chinese Medicine United Nations Educational, Scientific and Cultural Organization World Society for Mushroom Biology and Mushroom Products

1 Introduction First of all, it may be interesting to have a charming mushroom poem as a beginning for this review article: ‘WITHOUT LEAVES, WITHOUT BUDS, WITHOUT FLOWERS, YET, THEY FORM FRUIT; AS A FOOD, AS A TONIC, AS A MEDICINE, THE ENTIRE CREATION IS PRECIOUS’ [1]. The first sentence describes the morphological and physiological characteristics of mushrooms while the second sentence recounts their nutritional and medicinal properties. We are living in an age of human health crises remindful of the Black Death (bubonic plague), caused by the bacterium Yersinia pestis, that killed 50 million people in Europe and Asia during the fourteenth century. More recently, huge numbers of people worldwide have died due to HIV/AIDS, SARS, and now COVID-19. On January 13, 2021, the Inquirer section in the Weekend Australian newspaper reported that the seven-day average of new daily global cases of COVID19 was 733,000. In addition to infectious diseases, there have been worldwide upsurges of hypertension, cardiovascular disorders, cancer, dementia and neurodegenerative conditions. To combat these threats, humankind is focusing more and more attention on mushrooms and mushroom products. Mushrooms themselves are consumed regularly as part of the human diet and are treated as healthy or functional foods. On the other hand, the term mushroom nutriceuticals or dietary supplements has been applied to products derived from medicinal mushrooms that are taken to enhance general health and fitness but are not a regular food item. These have been used in China for over 2000 years and became the focus of considerable attention in the late 1980s due to the beneficial effects they had on various biological functions. In some cases, they have also been used in the prevention and treatment of several human diseases and have the advantage of lacking the troublesome side effects that

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frequently accompany synthetic drug counterparts. The main components of these supplements are polysaccharides, triterpenes and immunomodulatory proteins. Polysaccharide components, in particular, have been widely investigated as a source of antitumour and immunostimulating agents. They are widely distributed in mushrooms, with over 660 species from 183 genera reported to contain pharmacologically active polysaccharides. About 77% of all medicinal mushroom products are derived from the fruiting bodies, which have been either commercially farmed or collected from the wild, 21% from culture mycelium and 2% from culture broths. Precisely how these products work is still a matter of conjecture, but numerous laboratory animal tests as well as human clinical trials have shown them to be effective. In some cases, attention has focused on a single bioactive mushroom component and its effectiveness in treating specific disease conditions, much like a pharmaceutical. In the case of nutriceuticals/dietary supplements, emphasis has been placed on a combination of components that collectively impact on an individual’s overall health and quality of life. Many such products are currently available, and their market value worldwide increased from USD1.2 billion in 1991 to USD3.6 billion in 1994. The combined market value of medicinal mushrooms, mushroom extracts and derived products in 1999 was estimated to be USD 6.0 billion. That year, the United States nutraceutical market alone was valued at USD35 million. Since then, demand has increased between 20% and 40% annually depending on the species, with Ganoderma-based dietary supplements alone valued at USD1.6 billion. The MM industry has grown from small-scale (cottage-based) operations aimed at supplementing household incomes, to medium and mega-sized industrial ventures. This review examines the past, present and future of MM development and includes a pyramid model addressing key issues. For the purpose of this review, the term ‘medicinal’ is used to describe mushrooms that improve the well-being of the human condition. For several reasons, the continuing increase in the global demand for such mushrooms shows no sign of abating. To begin with, mushrooms have maintained their status as an important constituent of the human diet due to the consumer’s long-standing appreciation of their nutritional and organoleptic qualities [2]. Recent decades have also seen a huge expansion in mushrooms as a source of ‘mushroom nutriceuticals’, mushroomderived concoctions that exhibit tonic and therapeutic properties and which are marketed as dietary supplements (DSs) [3]. The application of modern scientific methods has since led to the isolation and identification of individual mushroomderived drugs called ‘mushroom pharmaceuticals’. MMs are also a source of flavour compounds used in the food service industry, components with cosmetic properties (cosmeceuticals), antibiotics and antiviral agents and natural bio-control agents for agricultural use [4, 5]. Mushrooms are the fruiting bodies of macrofungi. They include both edible/ medicinal and poisonous species. A 440 million-year-old fossilized mushroom reported in the Oxford Research Encyclopedia of Environmental Science [6] may be the oldest organism to have lived on dry land [4]. The consumption of mushrooms by humankind probably predates recorded history, and the historical record is, indeed, an ancient one. Considered as a special food, their matchless flavour has

Medicinal Mushrooms: Past, Present and Future

5

always been popular with gastronomes. The Greeks believed that mushrooms provided strength for warriors, the Pharaohs prized them as a delicacy, and the Romans regarded mushrooms as the ‘Food of the Gods’, to be served only on festive occasions. In Mexico, the indigenous population used mushrooms as hallucinogens in religious ceremonies and in witchcraft, as well as for therapeutic purposes [1]. In China, mushrooms have always been considered as a health food, the ‘elixir of life’. This is illustrated in a well-known Chinese legend which depicts the high regard people had for Ganoderma; ‘There are these two White and Black Snake-Spirit sisters, who steal Ganoderma from a remote mountain garden. The garden belongs to a heavenly god, and is very well protected. After a severe fight with the guards of the garden, the two Snake-Spirit sisters finally succeed, and get away with the mushroom. What followed? The mushroom brings Mr. Hsu, White Snake-Spirit’s husband, back to life . . .!’ There is little doubt that early humans were familiar with mushrooms and, by trial and error, discovered which types were worth collecting and which should be avoided. Traditionally, Ganoderma could only be found in the wild and, since the fruit bodies were often difficult to find, they were highly cherished and expensive. Even in the 1950s, it was customary for Ganoderma picked in the wild to be presented to high ranked officials in China and Taiwan. It was not until the 1970s that Ganoderma was first cultivated artificially, and since then production of the mushroom has increased rapidly. Ganoderma is cultivated mainly using either wood logs or, more commonly, on a lignocellulosic substrate (e.g. sawdust) in bottles or plastic bags [7]. Although classified nutritionally as a vegetable, mushrooms are actually fungi, which in the past were considered to be plants. However, unlike plants, mushrooms possess cell walls that contain chitin rather than cellulose, and they lack chloroplasts [8, 9]. Mushrooms are rich in protein, low in lipids, but high in unsaturated fatty acids. They are also low in cholesterol, but high in fibre, minerals and vitamins. Mushrooms are, indeed, superb health foods, and some of them have been scientifically proven to display strong medicinal potency. Therefore, they can be used to improve the health of individuals who have been affected by some of the most horrendous and psychologically devastating diseases of our time, including cancer. They have also been used traditionally in many different cultures for the maintenance of health and for the prevention and treatment of conditions such as blood platelet aggregation and hypertension [10]. Some medicinal mushroom extracts exhibit prophylactic or therapeutic effects against pneumonic superinfections and severe lung inflammation that often complicate COVID-19 infections and could prove effective in the battle against this virus [11].

2 Past Our attitudes towards natural phenomena are seldom based on simple observations. Nevertheless, throughout history certain life forms have given rise to a wealth of illusions and mythologies. Many have inspired fear and loathing simply because

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they are seen to be ugly, peculiar, and even evil. For example, snakes, spiders, toads, and owls have, in some communities, been associated with the devil or regarded as harbingers of disease and death. This is one reason why poisonous mushrooms are sometimes called ‘toadstools’ even though there is no scientific basis for the term. Nevertheless, toads have been observed sitting alongside, or even on top of a mushroom, catching insects that are attracted to fruiting bodies [12]. Certainly, some mushrooms (4), (1-->3) or (1-->4), (1->6) – ß-D-glucan [52], lentinan [53], a high-molecular-weight (1-->3)-ß-D-glucan from Lentinus edodes fruiting bodies, and schizophyllan, a high-molecular-weight (1-->3), (1-->6)-ß-D-glucan prepared from Schizophyllum commune culture filtrates [54, 55]. Polysaccharides and a low molecular weight protein-bound polysaccharide (EA6) with high antitumour activity were isolated from another popular edible mushroom, Flammulina velutipes [47]. The biochemical mechanisms underlying the biological activity of these components are still not fully understood. However, numerous studies have shown that regular consumption of certain medicinal mushroom species either as a food or as extracted compounds (nutriceuticals) is effective in both preventing and treating specific diseases, mainly through immunopotentiation and antioxidant activity.

4 Future Outlook Climate change is arguably the most compelling threat facing humankind. A recent report published by the United Nation’s Intergovernmental Panel on Climate Change (IPCC) concluded that eating less meat could help save the planet by preserving vast areas of land from farming and cutting greenhouse gases [56]. It emphasizes the high emission-intensive impact both of deforestation undertaken to create pasture for cattle rearing and of the huge quantities of methane produced by the cattle themselves as they digest their food. Attention was also drawn to the increased soil erosion and desertification, and the reduction in the amount of organic material stored in the ground brought about by intensive farming practices. Furthermore, associated climate change will inevitably cause disruption to global food chains, thereby increasing the risk of food insecurity and famine. Based on these assertions, major reductions in meat consumption worldwide would undoubtedly benefit both the climate and human health. However, gaining general approval of policies aimed at this outcome would, at the very least, require an alternative source of protein acceptable to the consumer. In this context, MMs represent the ideal candidate. Not only are they a source of high-quality protein that can be produced with greater biological efficiency than animal protein, they are rich in fibre, minerals and vitamins, have a low total fat content but a high proportion of polyunsaturated fatty acids relative to total fatty acids, and do not contain cholesterol [57]. MMs can be cultivated in large, industrial-scale production facilities as well as in situations where capital-intensive operations are inappropriate. In the latter case, mushroom production as a ‘cottage industry’ can (a) enrich the diet of the local population, especially in areas of protein deficiency, (b) address shortages of arable land, (c) provide employment particularly for women (thereby raising their socioeconomic status) and (d) generate additional income for farmers in the form of a cash

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crop. They also reduce environmental pollution by utilizing lignocellulosic wastes, disposal of which can often contribute to climate change.

4.1

Industrial-Scale MM Production

The past three decades has witnessed an enormous increase in global mushroom production, driven by consumer pressure and supported by technological advances within the industry. In China, the three most popular MMs are Ganoderma spp., Grifola frondosa and Hericium erinaceus (Table 2). Continued increasing demand for MM-based food and other products is a clear indication that there is abundant scope for further expansion. However, if it is to exploit fully the opportunities presented, there are numerous issues that the industry must address. For example: • A relatively small number of commercial MM strains are used in industrial-scale enterprises. Over time, the genetic material of existing commercial strains may undergo degenerative changes resulting in poorer growth, reduced climatic tolerance and lower yields, outcomes that would have a devastating effect on the industry.

Table 2 Most popular MMs in China (annual production volumes in tons 2001–2019) Year 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019

Ganoderma spp. 21,783 36,708 49,153 60,478 86,731 89,119 116,542 131,906 114,437 91,222 110,027 109,171 83,449 102,611 117,084 123,742 137,292 167,680 185,469

Grifola frondosa 14,605 36,623 24,962 4,937 4,675 5,985 7,115 10,833 6,423 10,778 5,258 14,882 20,957 61,834 21,326 24,044 26,362 28,721 36,334

Source: China Edible Fungi Association 2019 annual report

Hericium erinaceus 9,547 12,591 30,521 15,160 45,348 21,456 57,231 46,368 127,069 127,033 150,631 197,088 123,448 238,896 133,734 170,523 88,689 60,784 63,212

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• While many MMs are grown year-round in sophisticated in-door cultivation facilities, crops are still susceptible to various diseases. For example, Agaricus bisporus is highly vulnerable to a variety of bacterial (Brown Blotch-Pseudomonas tolaasii), fungal (Dry Bubble-Lecanicillium fungicola, Trichoderma spp.) and viral (La France Disease-AbV1 virus) diseases. Although various chemical agents are currently employed to control some of these diseases, increasing public concern over food safety issues may restrict their use in the future. • Loss of genetic diversity due to climate change, urbanization and alternative land use leading to the destruction of habitats, and over-harvesting (see below). • Consumer pressure has also led to a greater emphasis on food ‘freshness’. The industry will need to develop methods for extending the shelf-life and maintaining the quality of their products, which often require transportation over long distances before reaching the market. • Highly prized mushrooms such as truffles (Tuber spp.) and matsutake (Tricholoma matsutake) are among the most economically important edible ectomycorrhizal (EM) mushrooms worldwide. However, attempts at artificial cultivation have so far been unsuccessful. Commercial demand is therefore met by harvesting the fruiting bodies that occur naturally in forests of ectomycorrhizal coniferous trees and, in the case of truffle fruiting bodies, by semi-artificial cultivation. However, the latter is time-consuming, usually taking 4 to 12 years before harvesting of the fruiting body [58]. In the early 1940s, the annual harvest of matsutake in Japan was about 12,000 tons. This has since dropped to 15 cm in diameter), and a length of 25–50 cm. Alternatively, logs can be bundled tightly into a bamboo loop and fit into a larger bag, 40–60 cm in diameter. Massive loading makes it difficult to sterilize effectively. Sterilize bags at high pressure (1.5 kg/cm2) for 1.5 h, or at normal air pressure 100
C for 10 h. Heat-sealed polypropylene or polyethylene bags with microfilter windows can be used. Most producers in USA use “"Unicorn” bags

then buried in a containerized mixture of sawdust and rice bran or sawdust [9]. Subsequently, these logs were topped with soil. Large plastic pots containing treated logs were closely packed, with sides touching each other in a single layer on pebblelined dirt floors in the loop-framed greenhouses. Longer natural logs with multiple drilled-hole inoculation have also been used in the cultivation of Ganoderma mushrooms in North America. These logs are embedded either horizontally or at a slant [9]. Some growers leave the logs above ground. Horizontal implantation of logs outdoors may require an extended period of time, 2 years, for fruiting bodies to develop. These logs may continue to produce mushrooms for 4–5 years.

4.1.2

Substrate Cultivation in Bags and Bottles: Synthetic Log Cultivation

Substrate cultivation technology is defined as the use of hardwood sawdust, cottonseed husks, or different agricultural wastes as raw materials to cultivate mushrooms.

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Table 2 Summarized recommendations for Ganoderma spp. spawning and cultivation, adapted from [7] Procedure Spawning

Spawn run and mycelial penetration

Primordia initiation

Embedding in soil

Growth parameters

Description Apply spawn on the cut surface, 3–5 cm thick, usually 5–10 g for each log. When using freshly cut logs, as in traditional log cultivation in Japan, inoculation is applied immediately to avoid contamination (the interior of a healthy tree is sterile). Alternatively, using an inoculation gun, liquid mycelial spawn can be dispensed into the drilled holes Avoid having superficial mycelial growth on the log surface only, as a tough leathery mycelial layer. Due to low oxygen and moisture content, it results in poor mycelial growth and slow growth rate. (In contrast, in cultivation of Shiitake where a mycelial coat on the surface of the colonized log is desirable). Spawn run tolerates high CO2 concentration and is carried out in the absence of light Brief exposure to very little light triggers primordia initiation. Oxygen is also conducive to primordia formation. In contrast, spawn run is carried out in darkness, and less oxygen is required. Primordia are usually formed 50–60 days after. After primordia formation, embed the short logs vertically, with the cut surface where spawning is applied facing upwards, leaving it above the ground level. Sandy soil should be used. Cover soil with chopped straw to retain moisture. During fruiting, the colonized logs become resistant to microbial contamination in the nonsterile soil. An example: embed only 16–21 cm or 9/10 of the log in soil, leaving well-formed primordia above ground. Yields from soil-buried short logs are superior to logs without soil, as moisture can be better conserved, and mycelia can absorb nutrients, particularly minerals and trace elements from the soil. Within the mushroom house, low loop frames with covers usually in two rows are routinely set up. Alternatively, logs can be carried out in the open air in the wild High T near 30
C supports a rapid mycelial growth and shortens the spawn run. Spawn run in the absence of light promotes the formation and accumulation of fungal food reserves, such as glycogen and lipids. These energy reserves are essential for producing mushrooms. The most crucial factor during primordia initiation is to have high humidity, preferably 90–95% R.H., while the most crucial factor during pileus differentiation in fruiting is an increase in ventilation to reduce CO2 build-up. Differentiation of fruiting is highly sensitive to CO2 concentration, which determines whether antler-shaped fruiting bodies (CO2 > 0.1%) or fruiting bodies with a well-formed pileus (CO2 < 0.1%) will be produced. CO2 concentration at 0.04–0.05%, as close to fresh air as possible (0.03% CO2) should be maintained for production of mushrooms with caps. Air humidity can be supplied by a fine mist (1–2, or 3–4 times/day)

Ganoderma mycelium growth, fruiting body yield (Fig. 5), and various biologically active components are all affected by the substrate-related ingredients and culture conditions [10, 11]. Substrate raw materials are divided into main components and supplements. The main ingredients are traditional sawdust, cottonseed hulls, corn

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Table 3 Instructions for G. lucidum cultivation on natural logs in North America, adapted from [8] Procedure Preparation of natural logs

Inoculation (spawn and spawning)

Incubation of inoculated logs and formation of fruiting bodies

Description Natural logs are harvested during the dormant season before the new buds are formed. Broad-leaf hardwoods, oak, pecan, elder, and choke cherry are usually used, depending on the availability. Conifers should be avoided as the bark may contain undesirable resin Log-end sandwich inoculation is the approach used traditionally in Japan. Spawn, 3–5 cm thick, is applied onto the log. In the USA, many models appeared by drilling holes (ca. 3/800  1 1/200 ), 400 apart, around the log with bark. Inoculation is made through the drilled holes with liquid spawn (with an inoculation gun), grain spawn, sawdust-bran spawn, or colonized dowels, then sealed with cheese wax. The spawn plugs are sealed. When colonized dowels are used, holes are slightly larger. Grains such as millet can be used to facilitate mycelial colonization of the wooden dowel In log-end inoculation, often used in Japan and China, two logs are stacked together vertically as a unit at the junction of the inoculum. These “sandwich” log units are stacked together and covered with a plastic sheet. In a shady and naturally moist place spawn run occurred at R.H. 70–75%. Well-colonized logs, firmly adhering to each other in a unit at the junction of the inoculum, are transferred to the mushroom shed for fruiting. These logs are then covered directly with soil in the ground. Wellcolonized logs are more resistant to mold contamination in nonsterile carvings, especially in nutrient-poor sand or soil

cobs, and bagasse. Supplements include wheat bran, rice bran, corn flour, and soybean flour. Generally speaking, when sawdust is used, the fruiting body is of good quality and the surface is hard. When cottonseed hulls are used, high yields can be obtained, but the quality is poor [12]. Substrate cultivation methods are divided into bottle cultivation and bag cultivation, sometimes also called synthetic logs. Bag cultivation has more advantages, such as the use of more substrate, a strong body, and convenient manipulation, so it is more widely used. Both production processes include the following main steps: raw material preparation, mixing, bagging (bottling) and sterilization, inoculation, spawning, embedding in soil (or transfer to mushroom house), fruiting body development, management, and harvesting. Most cultivations of Ganoderma spp. on supplemented sawdust are performed in heat-resistant polypropylene bottles or bags [13]. Sawdust can be supplemented with rice bran (10%) and CaCO3 (3%), moistened with water and filled (700 g) into plastic bags. A plastic collar is then fitted onto each bag and stoppered with a cotton plug. After 5 h of heat treatment (95–100
C) and cooling, substrate is inoculated with grain or sawdust spawn, and incubated for 3–4 weeks (or until the spawn fully

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Fig. 5 Synthetic log cultivation of G. lucidum in bags (photo M. Berovic)

colonizes the substrate). Air T is kept at about 28
C with relative humidity of 85–90%. Basidiocarps begin to appear in about 1–2 weeks after initiation. Approximately 2–3 months after the appearance of primordia, mushrooms are ready to harvest. A mushroom is considered mature when the whitish margin around the edge of the basidiocarp has turned red. Additional examples of diverse substrate formulations have been described elsewhere, with main components being sawdust [10, 14, 15], sunflower seed hulls [16], stillage grain from a rice-spirit distillery [17], sawdust from Chilean native red wood trees [18], and waste of tofu manufacturing [19]. Cultivation in Bottles and Pots Some sources describe cultivation of G. lucidum in bottles and pots. For instance, 21 isolates of nine Ganoderma spp. [20] were grown in bottles on solid substrates based on sawdust. The substrate was prepared by mixing oak sawdust and wheat bran (8:2, v/v), and the water was added to 65% of the total volume. Mixed medium was put into 2,000 ml plastic bottles and sterilized

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at 121
C for 60 min and cooled to 20
C afterward. After inoculation, bottles were moved to the cultivation room, and the caps were removed after the mycelia overgrowth. The cultivation room was maintained at 28–31
C and 85% of R.H. After pinheading the cultivation room was ventilated for 10–20 min. When stipes were grown, the R.H. was controlled as 80–85% and the ventilation was performed 2–3 times. The relative humidity was controlled at 75–80%, and the maximum ventilation was done to form the pileus, as stipes were grown to 3–4 cm. After the pileus were formed, ventilations were made 5–6 times a day, and the R.H. was controlled at 80–85%. As the pileus were thickened and the yellow color of the margin of pileus turned to brown color, the relative R.H. was lowered to 60–70%. The process became standard for bottle cultivation. A Japanese patent [21] claimed a simple structure arrangement for cultivating G. lucidum or other mushrooms. The innovation consisted of special bottle containers and application of negative voltage to the container body for activating the fungi and for producing the mushrooms in a short period and of excellent quality. Another Japanese patent [22] described a cultivation method for Ganoderma in pots by protecting a nursery bed against the infiltration of bacteria by a plastic cover. A substrate, mainly composed of sawdust, was put into a vessel, sterilized and inoculated with a fungus.

4.1.3

Cultivation in Trays or Beds

According to Chen et al. [23] wood-chip or sawdust beds are labor saving, if contamination can be avoided. Substrate, 12 cm deep, of wood chips, supplemented sawdust, or a mixture of the two, was spread evenly over cultivation trays or beds. Colonized sawdust, grain or liquid spawn, 0.5 cm thick or more, was seeded on the surface under still air, then covered with a plastic sheet. Primordia were formed after 1–2 months or sooner. Plastic sheet cover was removed. Mushrooms were maintained at one-third diffused light, 85–95% R.H. and 25
C. Air circulation or aeration was provided three to five times a day, 5–10 min each time. For growth parameters, refer to Table 4. The distance between primordia was maintained at 15 cm  15 cm.

4.2

Cultivation of Grifola frondosa

As a saprophyte, G. frondosa (Japanese – Maitake) (Fig. 6) lives on the stumps of hardwood such as oak, chestnut, or beech tree. Its large size and amazing health benefits are why it has come to be called “the king of mushrooms,” and the cultivation skill has developed. Maitake has become an expensive mushroom species on the market [24]. Within the past two or three decades producers have been able to offer cultivated G. frondosa for food and as a dietary supplement

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Table 4 Management of growth parameters for cultivation of G. lucidum on trays, adapted from [23] Relative humidity 60–70%

Light (Lux) Nil

Primordia initiation Stipe development

85–95% 70–80% or higher

100– 200 150– 200

CO2 Tolerate conc. up to 5% 0.1–0.5% Or lower 0.1–1% branching

Pileus formation for firm pileus

85–95% 50–60% (after maturity)

150– 200 On Off

>0.1– 0.3% 12 h air circulation

Stage Spawn run

O2 air (l/min) 0–1

O2 Low or lower O2

Temperature 25–30
C or lower (20
C) 25–30
C or lower (20
C) 25–30
C days or longer 25–30
C or lower required (thicker)

Duration 1–2 month or as required 1–2 weeks 10– 14 weeks 1–2 months (longer)

Fig. 6 Grifola frondosa (photo A. Gregori)

[25, 26]. Since then, Japan has become the major producer accounting for 98% of worldwide production. Three basic methods of cultivation G. frondosa fruiting bodies have been established: bag culture, bottle culture, and outdoor bed culture.

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4.2.1

43

Nutrition and Substrate Compositions

Commercial production of most G. frondosa is on synthetic substrates contained in polypropylene bottles or bags [13]. A common substrate used for production is composed of sawdust supplemented with rice bran or wheat bran in a 5:1 ratio. For bottle production, the containers are filled with moistened substrate and sterilized or pasteurized prior to inoculation. For production in bags, the moistened substrate (2.5 kg) is filled into microfiltered polypropylene bags and sterilized. After cooling (16–20 h), the substrate is inoculated, and the bags are heat sealed and shaken to uniformly distribute the spawn throughout the substrate. Spawn run lasts about 30–60 days depending on strain and substrate formulation. Temperatures then are lowered from about 22 to 14
C to induce fruiting and fruitbody maturation. Hardwood sawdust (oak, beech, larch, poplar, cottonwood, elm, willow, and alder) is generally used by commercial growers. Oak is the most popular choice in the USA and Japan, while beech and larch are also used in Japan. In China, cottonseed hulls were used as a substitute for sawdust and provided an acceptable yield. Bran derived from cereal grains, such as rice, wheat, oat, and corn bran, are widely used as nutrient supplements. Other nutrient supplements are corn meal and soybean cake. Glucose, soluble starch, maltose, mannose, and fructose, respectively, are the most effective carbon sources utilized for mycelial growth by Maitake. Peptone is the best nitrogen source. Absence of Mn2+, Fe2+, and Cu2+ in the basal medium may result in an apparently decreased growth rate of the mycelium [26]. Another composition of supplemented sawdust-bran substrates consisted of hardwood sawdust (fine + coarse), 3:1 75%; wheat bran, coarse, not refined 23%; Sucrose 1%; Calcium compound 1%; lime (CaCO36H2O) or gypsum (CaSO42H2O); water to moisture content 60–63%; pH 5.5–6.5 [27, 28]. Soil, spent substrates (used substrates after harvest of the mushrooms) can also be used. Soil casing (soil-covered cultivation) has been reported to enhance the yield. Various combinations of wheat bran, rye, millet, and corn meal at 20% total supplement level were examined [26]. Different types of nutrient supplements have significant effects on crop cycle time and mushroom yield and quality. Combinations of 2–3 nutrients selected from wheat bran, millet, and rye were the most desirable formulation with short crop cycle, high quality, and high BE. The specific combination of 10% wheat bran, 10% millet, and 10% rye (BE 47.1%) gave crop cycle of 12 weeks and the combination of 10% wheat bran, 20% rye (BE 44%) gave crop cycle of 10 weeks, and both gave the most consistent yields and quality over time. Stott et al. studied the effect of substrate composition and environmental parameters for production on substrates with eucalyptus sawdust as the main component [29]. The nutritional and environmental conditions required for Maitake cultivation differed significantly from those for cultivation of Shiitake. Supplementation of eucalypt sawdust with maize meal, rice bran, or wheat bran at 10–20% was required for successful cultivation. Three substrate formulations containing beech sawdust supplemented with either 10% wheat bran, 20% corn meal, or 10% wheat bran, and 5% corn meal were tested

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[30]. Only on the sawdust with 20% corn meal fruitbodies developed, with an average yield of 310 g G. frondosa mushrooms per block. Wheat bran, rye, millet, and corn meal supplements used alone or in various combinations with mixed oak sawdust significantly influenced crop cycle time, biological efficiency, and basidiome quality of Maitake [31]. The combination of 10% wheat bran, 10% millet and 10% rye and the combination of 10% wheat bran, 20% rye gave the highest and the most consistent yields. The shortest crop cycle time achieved (9 weeks) was with 20% wheat bran and 10% rye and 30% wheat bran only, while the best quality mushrooms were produced with 20% millet and 10% rye. An equal mixture of wheat bran (10%), millet (10%), and rye (10%) proved the best formulas for the most widely used commercial isolate (WC828) in the USA. In several Japanese patents, the authors claimed better yield of G. frondosa. In a patent by Kawakami et al. [32], a part of sawdust powder was replaced by blending a crushed vegetable waste. In another patent [33] the substrate was formed by mixing a bean hull with a bean-curd refuse in the preferred ratio 8:1 to 4:4. This was added to one or more culture medium supports selected from: sawdust, corn cob, cottonseed hull, a waste log of the mushroom log cultivation, or a waste mushroom bed of the mushroom bed cultivation. A culture medium for artificial cultivation of G. frondosa, containing a base material (bran, corn starch or sawdust of a Japanese beech or a Japanese oak), and a coral sand as a nutrient source for growth promotion (comprising a powder containing minerals such as zinc, or a reactional liquid prepared by reacting the coral sand with an acid) was patented [34]. A Japanese patent [35] claimed that N-acetylglucosamine (preferably D-isomer) or chitin oligomer (e.g., 2–8-mer of N-acetylglucosamine) as an active component induced the formation of fruiting bodies of several mushrooms and among them was G. frondosa. The inducing agent was added to a medium in a bottle culture or a box culture, using sawdust as the main medium, preferably when the mycelia was fully grown in nourished state. In the case of immersing a bed log in water, the agent was added to the immersion water in an amount of 0.001–0.5 wt.%. The quality and yield of the formed fruiting body was increased, and the production was decreased. In another Japanese patent [36], adenine compound selected from adenine, adenosine, and adenosine monophosphate was used as an active component for growth promotion of mushrooms, applicable to natural cultivation as well as artificial cultivation, and capable of giving fruiting bodies in a short time in high yield. The adenine was used at 0.5–100 ppm based on the medium. The agent was preferably added in the preparation of culture medium or added to the medium after every harvest of the fruiting body. Cultivation of G. frondosa on crop straw (corn cob, corn straw, rice straw, and soybean straw) as a substrate was optimized by Song et al., and the alternative of crop straw as a substitute for sawdust in the substrate composition was determined [37]. There was a significant positive correlation existing between the yield and corn cob. The growth cycle was negatively correlated with sawdust, corn cob, and soybean straw, with sawdust significantly shortening the growth cycle of G. frondosa. The optimized high-yielding formula included 73.12% corn cob,

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1.87% rice straw, 23% wheat bran, and 2% light calcium carbonate (CaCO3) (C/N ¼ 48.40).

4.2.2

Cultivation in Bags

Bag cultures are used most frequently today by commercial producers [26]. The moistened substrate (2.5 kg) is filled into polypropylene or high-density polyethylene bags. After sterilization and cooling, the substrate is inoculated with G. frondosa spawn. A spawn run lasts about 30–60 days, depending on isolate and substrate formulation. Temperatures are lowered from about 22 to 14
C to induce fruiting and fruitbody maturation. Expected yields are in the range of 0.35–0.68 kg per 2.5 kg bag of 58% moisture substrate. Several other authors provided data on Maitake cultivation in Japan and in North America [27, 38, 39], as presented in Table 5. Shen investigated the specific substrate and growth conditions for the cultivation of G. frondosa fruiting bodies in polyethylene bags [26]. The general substrate formulation consisted of 74.8% mixed oak sawdust, 15% white millet, 10%, wheat bran, and 0.2% gypsum (CaSO4) with moisture content of 55–58%. This formula was initially chosen to determine the effects of genotypes on mushroom yield. All ingredients were combined, mixed, pasteurized, cooled, inoculated, and bagged with an autoclaving paddle mixer. Spawn run temperature was 20  1
C. The bags were sealed with a twist tie, the spawned substrate was incubated for 1 week, and 20 slits (5 mm long) were made to provide for gas exchange. A crop cycle of 12 weeks or less was considered; 90–95% R.H. was maintained by water atomizers; 4 h of light was provided daily, and T was maintained at 17  2
C. Sufficient air changes were maintained to hold CO2 concentrations below 700 ppm (μl/l). Among 23 isolates from the wild that the author tested, nine (39%) did not fruit under these conditions. The results clearly indicated that type and quality of nutrient supplements influenced Table 5 . Grifola frondosa cultivation in Japan and in North America, adapted from [27, 38, 39]

Japan

North America

Cultivation stage Spawn run

Temperature (
C) 22–23 air, 24– 25 substrate

Primordia initiation Fruiting body development Spawn run Primordia initiation Fruiting body development

22–23

Relative humidity (%) 70 (60–80)

16–18

70 (60–80) 85–95

21–24 10–15.6

95–100 95

13–16 (18)

85–90

Light (lux) 50

50 200– 500 N/A 100– 500 500– 1,000

Time (days) 30–40

CO2 Tolerates higher CO2 conc. 5,000 ppm

Primordia Initiation Optimum range 22–25
C ž 20–28
C) Under plastic cover, 2 days, 90–95% R.H.

Fruiting-body Development 20–33
C

22–25
C 70–85% R.H.




21–24 C 80–90% R.H. Minimal 100–200 F.C. (foot candle) 400–800 ppm, 5–7 air exchange per hr.

23–28
C (day-night) 24–27
C 75–85% R.H. Minimal 100–200 F.C. (foot candle) 10,000 ppm