High value fermentation products. Volume 2, Human welfare 9781119555384, 1119555388, 9781119554837

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High Value Fermentation Products Volume 2

Scrivener Publishing 100 Cummings Center, Suite 541J Beverly, MA 01915-6106 Publishers at Scrivener Martin Scrivener ([email protected]) Phillip Carmical ([email protected])

High Value Fermentation Products Volume 2 Human Welfare

Edited by

Saurabh Saran, Vikash Babu, and Asha Chaubey

This edition first published 2019 by John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA and Scrivener Publishing LLC, 100 Cummings Center, Suite 541J, Beverly, MA 01915, USA © 2019 Scrivener Publishing LLC For more information about Scrivener publications please visit www.scrivenerpublishing.com. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. Wiley Global Headquarters 111 River Street, Hoboken, NJ 07030, USA For details of our global editorial offices, customer services, and more information about Wiley products visit us at www. wiley.com. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials, or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Library of Congress Cataloging-in-Publication Data ISBN 978-1-119-55483-7 Cover image: Capsule counting machine, Garn Phakathunya | Dreamstime.com Cover design Kris Hackerott Set in size of 11pt and Minion Pro by Exeter Premedia Services Private Ltd., Chennai, India Printed in the USA 10 9 8 7 6 5 4 3 2 1

Contents Foreword

xv

Acknowledgements

xvii

Preface

xix

About the Editors

xxi

1

Challenges and Opportunities for the Production of Industrial Enzymes by Fermentation Andrés Illanes 1.1 Introduction 1.1.1 Sources of Enzymes as Process Catalysts 1.1.2 Advantages of Microorganisms as Hosts for Enzyme Production 1.2 Production of Microbial Enzymes by Fermentation: A Process Perspective 1.2.1 Enzyme Synthesis 1.2.2 Enzyme Recovery 1.2.3 Enzyme Purification 1.2.4 Enzyme Formulation 1.2.5 Market Outlook for Industrial Enzymes 1.3 Building Up Enzyme Catalysts for Process Applications 1.4 Conclusions References

2 Biotechnology of Leather: An Alternative to Conventional Leather Processing Saurabh Saran, Shifali Chib and R. K. Saxena 2.1 Introduction 2.2 Structure and Significance of Leather Industry 2.3 History of Leather 2.3.1 The Making of Leather Goods 2.4 Conventional Methods for Leather Processing 2.4.1 Pre-Tanning or Beam House Operations 2.4.1.1 Curing 2.4.1.2 Soaking v

1 1 1 2 5 6 6 7 10 11 12 15 15 23 23 25 26 27 28 29 29 29

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Contents 2.4.1.3 Liming 2.4.1.4 Dehairing 2.4.1.5 Deliming 2.4.1.6 Bating 2.4.1.7 Pickling 2.4.1.8 Degreasing 2.4.2 Tanning 2.4.3 Post-Tanning 2.4.4 Finishing 2.5 Biotechnology in Leather Industry 2.6 A Good Alternative to Conventional Chemicals in Leather Processing 2.7 Enzymes for Leather Processing 2.7.1 Enzymes for Soaking 2.7.2 Enzymes for Dehairing 2.7.3 Enzymes for Liming 2.7.4 Enzymes for Bating 2.7.5 Enzymes for Degreasing 2.8 Importers and Exporters of Top Leather 2.9 Outlook References

3 Enzyme Catalysis: A Workforce to Productivity of Textile Industry Sharrel Rebello, Embalil Mathachan Aneesh, Raveendran Sindhu, Parameswaran Binod, Ashok Pandey and Edgard Gnansounou 3.1 Introduction 3.2 Major Textile Enzymes, Mechanism of Action and Microbial Sources 3.2.1 Amylases 3.2.2 Cellulases 3.2.3 Proteases 3.2.4 Laccases 3.3 Applications in Textile Industry 3.3.1 Desizing 3.3.2 Bioscouring 3.3.3 Biobleaching 3.3.4 Stone Washing 3.3.5 Enzyme-Assisted Dyeing 3.3.6 Effluent Treatment 3.4 Technological Advancements to Enhanced Production of Textile Enzymes 3.5 Conclusion Acknowledgement References

29 29 30 30 30 30 31 31 31 32 34 35 36 37 40 40 41 41 42 42 49

49 51 51 54 54 57 57 57 57 58 58 58 59 59 60 60 60

Contents vii 4 Current Trends in the Production of Ligninolytic Enzymes Susana Rodríguez-Couto 4.1 Introduction 4.2 Ligninolytic Enzymes 4.2.1 Lignin Peroxidase 4.2.2 Manganese-Dependent Peroxidase 4.2.3 Versatile Peroxidase 4.2.4 Laccase 4.3 Sources of Ligninolytic Enzymes 4.3.1 Wood-Degrading Fungi 4.3.2 Bacteria 4.4 Production of Ligninolytic Enzymes 4.5 Purification of Ligninolytic Enzymes 4.6 Potential Applications of Ligninoytic Enzymes 4.7 Outlook References

67

5 Asava-Arishta: A Multi-Advantageous Fermented Product in Ayurveda Varun Kumar Singh, Avinash Narwaria and C. K. Katiyar 5.1 Introduction 5.2 Definition of Asava and Arishta 5.3 Method of Preparation for Asava Arishta 5.3.1 Purvakarma Includes (Pre-Fermentation Process) 5.3.2 Pradhanakarma Includes (Fermentation Process) 5.3.3 Pashchata Karma Includes (Post-Fermentation) 5.3.4 Industry Practice for Preparation of Asava-Arishta 5.3.4.1 Preparation of Raw Materials 5.3.4.2 Fermentation 5.3.4.3 Filtration and Maturation 5.3.4.4 Quality Check, Packaging and Storage 5.4 Role of Ingredients and Process 5.4.1 Role of Extraction 5.4.2 Role of Sugar/Sugar Source 5.4.3 Role of Dhataki Flower and Microbes 5.4.4 Role of Prakshep Dravya/Spices 5.5 Advantages of Asava–Arishta Over Other Dosage Form 5.6 Future Perspective Acknowledgements References

89

6 Production and Applications of Polyunsaturated Fatty Acids Sabeela Beevi Ummalyma, Raveendran Sindhu, Parameswaran Binod, Ashok Pandey and Edgard Gnansounou 6.1 Introduction

67 68 68 71 72 73 75 75 77 77 78 78 80 81

89 90 91 92 92 92 92 92 96 97 98 98 98 100 101 103 104 104 105 106 109

109

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Contents 6.2 Biosynthesis of Polyunsaturated Fatty Acids 6.3 Sources of Polyunsaturated Fatty Acids 6.3.1 Microbial Source 6.3.2 Plant and Microalgal Source 6.4 Different Fermentation Process for PUFA Production 6.5 Application of PUFAs 6.6 Future Perspectives 6.7 Conclusion Acknowledgements References

110 112 112 113 118 119 119 120 120 120

7 Functional Foods and Their Health Benefits Rwivoo Baruah, Krishan Kumar and Arun Goyal 7.1 Introduction 7.2 Fermented Functional Foods 7.2.1 Benefits of Fermented Functional Foods 7.2.2 Synthesis of Nutraceuticals and Bioactive Compounds in Fermented Functional Food 7.2.3 Fermented Functional Foods and Their Health Benefits 7.2.3.1 Yoghurt 7.2.3.2 Cheese 7.2.3.3 Kimchi 7.2.3.4 Soy Sauce 7.2.3.5 Sourdough 7.3 Functional Whole Foods 7.3.1 Cereals 7.3.2 Culinary Herbs and Spices 7.4 Conclusion References

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8 Industrially Important Biomolecules From Cyanobacteria Y. P. Reddy, R. K. Yadav, K. N. Tripathi, A. Borah, P. Jaiswal and G. Abraham 8.1 Introduction 8.2 Cultivation Methods for Cyanobacteria 8.3 Multifaceted Role of Biocompounds Found in Cyanobacteria 8.3.1 Plant Growth Promoting Compounds 8.3.2 Soil Health Improvement 8.3.3 Bioremediation 8.3.4 Compounds for Biocontrol 8.3.5 Bioenergy Applications 8.3.6 Medical Applications 8.3.7 Applications in Food Sector 8.3.8 Other Value-Added Biomolecules 8.4 Industrial-Scale Production and Commercial Status

147

127 128 128 129 129 129 131 132 133 134 135 135 139 140 141

147 148 150 151 151 152 152 153 153 154 154 155

Contents ix 8.5 Future Perspectives Acknowledgements References

155 156 156

9 Augmenting Bioactivity of Plant-Based Foods Using Fermentation Sonam Chouhan, Kanika Sharma and Sanjay Guleria 9.1 Introduction 9.2 Effect of Fermentation on Bioactivity of Plant-Based Foods 9.3 Different Fermentation Procedures 9.3.1 Solid State Fermentation (SSF) 9.3.2 Submerged Fermentation/ Liquid Fermentation (SmF/LF) 9.4 Factors Affecting Fermentation Process 9.5 Bioactive Properties of Fermented Foods 9.5.1 Antioxidant Effect 9.5.2 Antimicrobial Effect 9.5.3 Anti-Inflammatory Effect 9.5.4 Antidiabetic Effect 9.5.5 Other Biological Effects of Fermented Plant Extracts 9.6 Conclusion and Future Perspectives References

165

10 Probiotic Intervention for Human Health and Disease Bilqeesa Bhat and Bijender Kumar Bajaj 10.1 Introduction 10.2 Various Sources of Probiotics 10.3 Commercially Developed Probiotic Products 10.4 Global Market of Probiotics 10.5 Probiotic Production From Fermentation Process 10.6 Health Implications of Gut Microbiota Dynamics 10.6.1 Probiotics and Cardio Vascular Diseases, Blood Pressure, Obesity and Diabetes 10.6.2 Cholesterol Reducing and Total Lipid Profile Influencing Potential of Probiotics 10.6.3 Probiotic Intervention in Neurological Disorders 10.6.4 Health Benefits of Probiotic Exopolysaccharides 10.6.5 Antimicrobial Potential of Probiotics 10.6.6 Probiotics for Bacterial Vaginosis 10.6.7 Probiotics for Treatment of Diarrhea 10.6.8 Probiotics for Urinary Tract Infections 10.7 Conclusions Acknowledgement References

185

165 166 168 168 169 169 172 173 174 175 176 177 177 178

185 188 190 190 194 194 195 196 198 199 199 201 201 202 203 203 204

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Contents

11 Saccharomyces - Eukaryotic Probiotic for Human Applications Alok Malaviya1, Shruti Malviya, Anil Agarwal, Madhvi Mishra and Valina Dalmida 11.1 Introduction 11.2 Advantages of Eukaryotic Probiotics Over Prokaryotic Probiotics 11.3 Probiotic Properties of Approved Yeast Strains 11.3.1 Survival in Gastrointestinal Tract 11.3.2 Stress Tolerance Capability 11.3.3 Ability to Adhere with Gastrointestinal Tract 11.4 Pharmacodynamics of S. Boulardii 11.4.1 Effect on Enteric Pathogens 11.4.2 Neutralization of Bacterial Toxins 11.4.3 Modification of Host Cell Signalling 11.4.4 Trophic Effect on Intestinal Mucosa 11.4.5 Anti-Inflammatory Effect 11.5 Therapeutic Potentials of S.boulardii 11.5.1 Antibiotic-Associated Diarrhoea 11.5.2 Traveller’s Diarrhoea 11.5.3 Acute Diarrhoea in Children 11.5.4 Tube-Feeding-Associated Diarrhoea 11.5.5 Diarrhoea Associated with AIDS 11.5.6 Irritable Bowel Diseases and Irritable Bowel Syndrome 11.6 Fermentative Production of S. Boulardii 11.7 Commercial Impact of Probiotic Products (Eukaryotic Probiotics) 11.8 Safety 11.9 Conclusion and Future Prospects References 12 Bioactive Polysaccharides Produced by Microorganisms: Production and Applications Gilberto V. de Melo Pereiraa, Susan G. Karp, Luiz A. J. Letti, Maria G. B. Pagnoncelli, Ana M. Finco, Maria R. Machado and Carlos R. Soccol 12.1 Introduction 12.2 Classification and Structure of Polysaccharides 12.3 Fungal Polysaccharides 12.4 Bacterial Polysaccharides 12.5 Algal Polysaccharides 12.6 Microbial Polysaccharides Versus Plant Polysaccharides 12.7 Biological Activities of Microbial Polysaccharides 12.8 Microbial Polysaccharides Production 12.9 Microbial Polysaccharides Recovery 12.10 Conclusion References

211

211 213 215 215 217 217 217 218 218 218 219 219 219 219 219 220 220 220 220 220 224 224 225 226 231

231 232 233 238 239 240 241 242 243 244 244

Contents xi 13 Shikimic Acid: A Compound of Industrial Interest with Respect to Swine/Avian Flu P. Tripathi 13.1 Introduction 13.1.1 Swine/Avian Flu Pandemics – A Historical Account 13.1.2 Prevention/Treatment of Swine and Avian Flu 13.2 Shikimic Acid 13.2.1 General Concept 13.2.2 Detection of Shikimic Acid 13.2.3 Current Scenario of Shikimic Acid Production 13.2.3.1 Production of Shikimic Acid From Chemical Synthetic Route 13.2.3.2 Production of Shikimic Acid From Plant Route 13.2.3.3 Production of Shikimic Acid From Microbial Route and Its Extraction 13.2.3.4 Enzymatic Modifications for Shikimic Acid Production 13.3 Applications of Shikimic Acid 13.4 Conclusion References 14 1,3-Propanediol: From Waste to Wardrobe Jasmine Isar, Harshvardhan Joshi and Vidhya Rangaswamy 14.1 Introduction 14.2 Applications of 1,3-PDO 14.3 Microbial Production of 1,3-PDO 14.4 Challenges in Microbial Production of 1,3-PDO 14.5 Different Strategies for the Production of 1,3-PDO 14.5.1 Conversion of Glycerol to 1,3-PDO 14.5.2 Conversion of Glucose to Glycerol 14.5.3 Two-Stage Fermentation 14.5.4 Co-Fermentation 14.5.5 Micro-Aerobic Conditions 14.5.6 Repeated Batch/fed-Batch Fermentation 14.5.7 Non-Sterile Fermentation 14.5.8 Effect of Process Parameters 14.5.9 Genetic Engineering 14.5.10 Other Strategies 14.6 Downstream Processing of 1,3-PDO 14.7 Economic Importance of 1,3-PDO 14.8 Future Prospects and Outlook 14.9 Conclusions References

253 253 255 256 257 257 259 260 260 264 267 269 270 271 272 281 281 283 284 285 286 290 292 293 294 295 296 296 297 298 302 303 306 307 308 308

xii Contents 15 Biomedical and Nutraceutical Applications of Chitin and Chitosan Manish Kumar, Amandeep Brar, V. Vivekanand and Nidhi Pareek 15.1 Introduction 15.1.1 Chitin 15.1.2 Chitosan 15.2 Different Forms of Chitin and CHS 15.3 Biosynthesis of Chitin and CHS 15.4 Biomedical Applications of Chitin and CHS 15.4.1 Tissue Engineering 15.4.1.1 Bone Tissue Engineering 15.4.1.2 Cartilage Tissue Engineering 15.4.1.3 Tendon and Ligament Tissue Engineering 15.4.1.4 Nerve Tissue Engineering 15.4.1.5 Liver Tissue Engineering 15.4.1.6 Skin Tissue Engineering 15.4.2 Drugs and Growth Factors Delivery 15.4.2.1 Drug Delivery 15.4.2.2 Growth Factor Delivery 15.4.3 In Wound Healing 15.5 Nutraceutical Application of Chitin and CHS 15.5.1 Antimicrobial Activity 15.5.2 Anti-Inflammatory Activity 15.5.3 Antioxidant Activity 15.5.4 Anticarcinogenic Activity 15.5.5 Antiulcer Activity 15.5.6 Renal Disease Recovery 15.5.7 Dietary Fibers 15.6 Commercial Products 15.7 Conclusion References

319

16 Microbial Polyhydroxyalkanoates: Current Status and Future Prospects Jyotsana Dalal and Banwari Lal 16.1 Introduction 16.2 PHA Structure and Diversity 16.3 Physical Properties of PHA 16.4 Environment, Phylogeny and Diversity of PHA Producing Microorganisms 16.5 PHA Biosynthesis 16.6 Problems and Challenges in PHA Production on an Industrial Scale 16.6.1 Renewable Sources as Substrate for PHA Production 16.6.1.1 Whey 16.6.1.2 Lignocellulosic Biomass 16.6.1.3 Plant Oils 16.6.1.4 Glycerol 16.6.2 Photosynthetic Bacteria

351

319 320 321 322 322 325 325 325 326 327 328 328 329 330 330 331 332 333 333 333 334 335 335 336 337 337 338 339

351 353 354 357 361 363 363 363 365 366 368 369

Contents 16.6.3 Halophiles 16.6.4 Co-Production of PHA with Other Valuable Bioproducts 16.6.5 Blends of PHAs 16.7 Applications of PHAs in the Medical Industry 16.8 Conclusions and Future Prospects Acknowledgements References

xiii 370 371 372 373 374 375 376

List of Contributors

389

Index

393

Foreword From last two decades we have witnesses unprecedented growth and development in biotechnology positioning the bioeconomy as a major indicator of advancement. Today, the global fermentation-based industry is already worth over 127 billion dollars. Based on the experience and expertise in this filed, we are trying to collect the different technologies advancement and products developed in biotechnology. This book ‘High Value Fermentation Products-Volume II (Human Welfare) is divided into various important sections related to Human Health like antibiotics, sugar & sugar alcohols, enzymes, nutraceuticals, metabolic engineered derived products, this will help the readers to understand the importance of fermentation derived product for the betterment of human health. This book will also help to overcome of various bottle necks of the Industry/ scientific community and shall be useful for the betterment of the society and environment. This book will also shares an insight into the recent research, cutting edge technologies, high value products, industrial demand which bring immense interest among young and brilliant researchers, cultivated scientists, industry personnals and talented student communities. The contents of the book have been designed in such a way that it is providing extensive coverage of new developments, state of the art technologies, current and future trends in biotechnology and fermentation. The reader will be introduced with basic and advanced methodologies on industrial microbiology and fermentation technology. The main goal of this book is to share and enhance the knowledge of each and every individual in this fermentation world. Ram A Vishwakarma Director, CSIR-IIIM

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Acknowledgements The Editors take this opportunity to gratefully acknowledge the assistance and contribution of the people who have faith in us in this undertaking for compiling of the Book “High Value Fermentation Products”-Volume II (Human Welfare). We are in debt of Dr. Ram A. Vishwakarma, Director, CSIR-Indian Institute of Integrative Medicine, Jammu for his valuable and esteemed guidance to carry out this task. His scholarship and authorative knowledge has been a great source of motivation and inspiration. First and foremost, it is not enough to express our gratitude in words to all the contributors for devotion and providing excellent matter of chapters on time. The help and support provided by Mr. Chand Ji Raina, Mr. R.K. Khajuria and Mrs. Urmila Jamwal, was important and we acknowledge all of them with sincere thanks. We are also thankful to the students of Fermentation Technology Division, CSIRIIIM for their sincere efforts, dedication and determination to achieve objectives for the completion of this task in a given time. Where emotions are involved, words cease to mean for our family members for the consistent motivation during the planning and edition of this book. We avail the opportunity to express our heartiest thanks to ‘Almighty’ for pouring His care and blessings throughout and making this work a success. Saurabh Saran Vikash Babu Asha Chaubey

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Preface Fermentation processes are being used for generations to meet the requirements for sustainable production of enzymes, food & dairy products and nutraceutical products etc. Thus, the modern biotechnology offers numerous opportunities for human welfare. Fermentation processes involve the proven scientific and engineering principles by biological agents. It uses variety of microorganisms such as bacteria, yeast and fungi for production of variety of value added products and biomolecules such as enzymes, antibiotics, hormones, organic acids, drug precursors and other metabolites. The book entitled “High Value Fermentation Products” has been divided in two volumes namely, Human Health and Human Welfare. The Volume 1 of the book has 18 chapters focussed on basics to fermentation technology, antibiotics & immunosuppressants, antibodies, peptides & proteins, sugars & sugar alcohols and metabolic engineering derived products. The Volume 2 of the book with the theme ‘Human Welfare” has 16 chapters which primarily focus on enzymes, nutraceuticals, probiotics, biopolymers, and organic acids. The first chapter entitled ‘Challenges and opportunities for the production of industrial enzymes by fermentation’ aims to provide the insights on the industrial enzymes, their bioprocess and associated challenges. The second chapter on ‘Biotechnology of leather: An alternative to conventional leather processing’ provides the techniques and enzymatic methods involved in leather processing, an alternative green approach in leather processing. The third chapter on ‘Enzyme catalysis: a workforce to productivity of textile industry’ compiles the various enzymes involved in the textile industry. The fourth chapter entitled ‘Current trends in the production of ligninolytic enzymes’ highlights the recent biotechnology driven fermentation approaches for production of ligninolytic enzymes. Fifth chapter emphasizes on production of Ayurvedic preparations, i.e. Asava & Arishtas by fermentation. Production and applications of poly unsaturated fatty acids have been elaborated in the sixth chapter. Seventh chapter on ‘Functional foods and their health benefits’ provides information the importance of functions foods. The eighth chapter entitled ‘Industrially important biomolecules from cyanobacteria’ describes various types of biomolecules produced by cyanobacteria. ‘Augmenting bioactivity of plant based foods using fermentation’, the ninth chapter of the book elaborates the bioactivities of plant based fermentation products. The tenth chapter ‘Probiotic intervention for human health and disease’ emphasizes on the probiotics and their importance in human health and protection against diseases. Eleventh chapter of the volume elaborates the Saccharomyces

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xx Preface sp. based probiotics. Chapter twelve entitled ‘Bioactive polysaccharides produced by microorganisms: Production and applications’ elaborates various polysaccharides produced by microorganisms, their bioactivities and applications. Thirteenth chapter ‘Shikimic acid: A compound of industrial interest with respect to swine/avian flu’ is focused on an important compound, shikimik acid with special reference to swine flu. ‘1,3-Propanediol: From Waste to Wardrobe’ is the fourteenth chapter of the volume, which talks about another important compound with innumerable applications. Fifteenth chapter ‘Biomedical and Nutraceutical Applications of Chitin and Chitosan’ explains wide applications of chitin and chitosan. The last chapter of the volume 2 of the book is ‘Microbial polyhydroxyalkanoates: current status and future prospects’ which elaborates the features and importance of polyhydroxyalkanoates with wide industrial applications. In the Volume-2 of the book ‘High Value Fermentation Products’, editors have tried their best to compile contributions that provide applications and recent trends in the area of fermentation based processes for production of enzymes, biopolymers, probiotics and other nutraceuticals.

About the Editors Dr. Saurabh Saran, PhD, is a Fermentation Scientist having experience in Industrial microbiology, Biotechnology and Fermentation Technology for more than 15 years. Dr. Saran has completed his PhD from Delhi University, India. Dr. Saran has got hands-on experience in working both industries and academic. He has worked in the industries like Reliance Industries Ltd., India. Later he was appointed as a Research Professor at Republic of Korea, South Korea. He has also worked as a Coordinator at the Technology Based Incubator, Delhi University South Campus, Delhi, Inida. Presently, he is working as a Senior Scientist, Fermentation technology division, CSIR-IIIM, Jammu, India. He has an expertise on the screening, isolation, production and scale up of Industrial Enzymes, Biochemicals & Biofuels. Expert in process development/engineering, scale up to 5L, 10L, 30L, 100, 300 L & 500L fermentation size, (batch, fed batch and continuation fermentation) strain engineering, downstream processing and applications of industrially important biomolecules. To my credentials, I have 3 patents and more than 25 international publications in peer reviewed international journals on fermentation technology. Dr. Vikash Babu, PhD was born in Bulandshahr district of Uttar Pradesh, India on 1st September 1981. He did his Bachelor’s degree from I.P (PG) College Bulandshahr, India. After qualifying all India combined entrance exam for biotechnology conducted by JNU, New Delhi, India, he did his degree in Biotechnology from Kumaun University, Nainital. After completing his M.Sc degree, he qualified many national level

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xxii About the Editors competitive exams such as DBT-JRF- 2005, CSIR-UGC NET for lecturership- Dec. 2004 & June 2005 and GATE-2005. In Nov. 2005, he joined as a DBT-JRF in the Department of Biotechnology, Indian Institute of Technology, Roorkee under the superivision of Dr. Bijan Choudhury and registered for the Ph.D in the same department and Institute and completed his Ph.D degree in the year 2011. After finishing his Ph.D research work he joined Mangalayatan University, Beswan, Aligarh (India) as a lecturer. He left Manglayatan University in the year 2012 and joined Graphic Era University, Dehradun (India) as an assistant professor where he worked till June 2013. Currently, he is working as a scientist in CSIR-IIIM. Dr. Asha Chaubey, Ph.D is Principal Scientist and Head of Fermentation Technology Division, CSIR-Indian Institute of Integrative Medicine, Jammu, India. She has about 15 years of research experience in the area of enzymology and fermentation technology. She is actively engaged in development of indigenous process development. Her research interests include exploration and exploitation of microorganisms for production of enzymes and bioactives in special reference to industrial applications. She has published research articles in the area of bioactives production, enzyme immobilization, biotransformation, kinetic resolution of racemic drug intermediates. She has also published several review articles and has been actively involved in the development of biosensors for health care and environmental monitoring and has several patents on lactate and cholesterol biosensors.

1 Challenges and Opportunities for the Production of Industrial Enzymes by Fermentation Andrés Illanes School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso Av. Brasil 2085, Valparaíso, P.O. Box 4059, Valparaíso, (Chile)

Abstract Enzymes have been used as industrial catalysts for over a century now. Plant and animal tissues and fluids have been gradually replaced by microorganisms as sources of enzymes, because of the advantages of intensive production by fermentation at high productivity and under controlled conditions. Genetic engineering has allowed the production of enzymes from any origin in microbial hosts, including higher organisms, non-culturable microbes and metagenomic pools. Complementarily, protein engineering tools allow producing enzyme variants with improved features as process catalysts. Enzyme production by fermentation is reviewed from a process perspective, and strategies for building up enzyme catalysts for process applications are presented considering the evolution of biocatalysis from rather simple reactions of hydrolysis to more complex reactions of organic synthesis where stringent conditions impose new demands for enzyme performance. Advances in the field as well as challenges both in the production and utilization of microbial enzymes are discussed. Keywords: Microbial enzymes, biocatalysis, immobilized enzymes, protein engineering

1.1

Introduction

1.1.1

Sources of Enzymes as Process Catalysts

Enzymes are the catalysts of life. The metabolism of all living cell forms depends on enzymes since they allow the biochemical reactions to proceed at a sustained pace at the mild conditions required for cell integrity. Enzymes are complex molecules that have evolved to act with an outstanding molecular precision, being both specific in terms of substrate recognition and selective in terms of the reaction catalyzed [1]. These are outstanding properties that make enzymes attractive catalysts for chemical processes. However, as process catalysts, enzymes should perform efficiently under conditions usually far apart from physiological. This is a major challenge and most efforts in the last 50 years have been devoted to making these metabolic catalysts robust enough to Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (1–22) © 2019 Scrivener Publishing LLC

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High Value Fermentation Products Volume 2

withstand the usually harsh conditions of an industrial process of biotransformation [2]. The industrial use of enzymes dates back to the early years of the last century. At that time, most enzymes used were extracted from plant tissues and from animal organs [3]. Some of these early industrial enzymes from plant and animal origin are still being produced in significant amounts. This is the case of the plant proteases papain and bromelain, which are used in food and beverage processing and also in cosmetic and pharmaceutical products [4, 5], and several lipases and proteases extracted from animal tissues that are used in food and leather processing, and also in some pharmaceutical products [6, 7]. Main industrial enzymes are listed in Table 1.1. Most of the enzymes listed are hydrolases and even though most of these industrial applications are mature technology, technological improvements are still ongoing. Highly resistant enzymes for laundry (proteases, lipases and amylases), intensive use of enzymes in animal feeding (phytases, β-glucanases) and, above all, enzymes used for biofuel production (amylases, cellulases, hemicellulases, lipases), are major areas of development already having a strong impact on the enzyme market. As seen in Table  1.1, most of these industrial enzymes are of microbial origin, mostly from bacteria, yeasts and molds, so its production is tightly bound to fermentation technology. The development of submerged fermentation and its success in the large-scale production of penicillin and other antibiotics triggered the production of enzymes by microbial fermentations that started to displace the former plant and animal enzymes, so that by 1960 30% of the enzymes were already produced from microbial strains, and two decades later the situation had reversed and more than 70% were produced intensively and independent of season and climate in well-controlled industrial fermentation processes. Up to now, living cells are the only source of biocatalysts. The creation of synthetic molecules that mimic the active sites of enzymes (enzyme mimics) has been pursued in organic chemistry as a way of solving some of the restrictions of natural enzymes, like high production cost, narrow substrate specificity and propensity to degradation. Small molecular weight active site constructs (chemzymes) and catalysts based on molecular imprinting in synthetic polymers, and also in some inorganic matrices like silica and zeolites, with products and transition state analogues (abzymes) have been studied and evaluated as enzyme-like catalysts [8]. However, very few of them are able to catalyze reactions at the conditions in which enzymes perform and in such cases activity and stability are very low, but the wide range of catalytic activities that may be obtained and the continuing progress in the field will eventually represent a technological option to natural enzymes in the long term [9].

1.1.2 Advantages of Microorganisms as Hosts for Enzyme Production Microorganisms are ideal hosts for producing enzymes. Reasons underlying are many: microbes are vigorous organisms with high specific growth rates and simple nutritional requirements that are rather easy to manipulate both genetically and environmentally, representing a huge reservoir of genetic material. These features are technologically meaningful, making the production of microbial enzymes more reliable, simpler and cheaper, using readily available raw materials in a controlled environment, so favoring process validation. Production of enzymes in microbial

Challenges and Opportunities for the Production

3

Table 1.1 Commonly used industrial enzymes and their applications. Enzyme

Source

Application

Glycosidases α-amylase

mold

bakery, confectionery, brewery, firstgeneration bioethanol

α-amylase

bacteria

starch liquefaction, detergent, fabrics desizing, first-generation bioethanol

α-arabinofuranosidase

yeast, mold

wine making

β-amylase

plant, bacteria

glucose syrup, brewery

cellulase

mold

juice extraction and clarification, detergent, denim, second-generation bioethanol

β-galactosidase

yeast, mold

delactosed milk and dairy products, whey upgrading

β-glucanase

mold

animal feed supplement, brewery

β-glucosidase

yeast, mold

wine making

glucoamylase

mold

glucose syrup

invertase

yeast, mold

confectionery

naringinase

mold

juice debittering

pectinase

mold

juice clarification and extraction, baby foods, wine making

phytase

bacteria

animal feed supplement

xylanase

mold, bacteria

wood pulping and bleaching, bioethanol

Proteases alkaline protease

bacteria

detergent, leather tanning and dehairing, stickwater treatment

aminopeptidase

mold, bacteria

protein hydrolyzate debittering

bromelain

Ananas comosus stem

anti-inflammatory and burn healing preparations, drug absorption

chymosin

animal, recombinant yeast and mold

cheese-making

neutral protease

mold, bacteria

baking, protein hydrolyzate (Continued)

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High Value Fermentation Products Volume 2

Table 1.1 Cont. Enzyme

Source

Application

papain

Carica papaya latex

yeast and meat extracts, brewery, protein hydrolyzates, meat tenderizing, tanning, digestive aids, skin wound healing preparations

pepsin

animal

cheese-making

Other Hydrolases lipases

animal, yeast, fungi, bacteria

flavor enhancer, detergent, biodiesel

aminoacylase

mold

food and feed fortification

penicillin acylase

mold, bacteria

β-lactam antibiotics

urease

bacteria

alcoholic beverages, urea removal

enzymes

Non-hydrolytic

glucose isomerase

bacteria, actinomycetes

high-fructose syrup

glucose oxidase

mold

food and beverage preservation

catalase

bacteria

food preservation, peroxide removal

nitrile hydratase

bacteria

acrylamide, nicotinamide

aspartate ammonia lyase

bacteria

aspartic acid

hosts is no longer restricted to the microbial genome since the development of genetic engineering that allows the expression into suitable microbial hosts of foreign genetic material, coming from any kind of organism, including higher organisms, non-culturable microbes, and even from metagenomic pools [10]. Cloning genes of extremophiles into suitable mesophilic microbial hosts has a striking importance for producing enzymes able to withstand the harsh conditions that may occur in biocatalytic processes [11, 12]; complementing the above, protein engineering strategies, like site-directed mutagenesis and directed evolution, are powerful tools for producing enzyme variants better suited for performing biocatalysis [13]. On the other hand, impressive advances in fermentation technology and process control allow obtaining high cell concentrations so that the volumetric productivity of enzymes can be greatly increased [14]. Only in the case of eukaryotic glycoenzymes (i.e., urokinase, tissue plasminogen activator) production in established animal cell lines is a better option because of the ability of these cells to perform post-translational glycosylation properly [15]; in this case, high costs associated to production can be absorbed because of the very high unit price of the enzyme [16].

Challenges and Opportunities for the Production

5

Summing up, the advantages of using microorganisms for producing enzymes explain why now more than 90% of the enzymes marketed are from microbial origin and a significant fraction of them are produced with genetically manipulated microorganisms [17]. The case of chymosin clearly illustrates this: chymosin is a very specific aspartic acid protease that hydrolyzes the Phe105-Met106 peptide bond of κ-casein triggering its clotting in the presence of calcium ions to yield the curd [18]. It was traditionally produced by extraction from calf abomasum as a by-product of veal production; shortage prompted its replacement for cheaper and more readily available sources, so that now it has been replaced to a considerable extent by recombinant chymosin produced in suitable microbial hosts, mostly from the genus Kluyveromyces [19] and Aspergillus [20]; in addition, chymosin variants produced by protein engineering have been obtained with increased specificity and better pH profile than the native enzyme [21]. Improvements of the kind have been applied to ton scale industrial enzymes, as illustrated by the case of tailor-made proteases specifically designed to act efficiently under the harsh conditions of laundering [22]. Improving microbial enzymes by the rational modification of their structure and selection of variants with improved features by high throughput screening methods is already making an impact on enzyme biotechnology [23, 24].

1.2

Production of Microbial Enzymes by Fermentation: A Process Perspective

There is a broad spectrum of applications of enzymes as process catalysts, going from large-scale processes for the production of commodities, where enzymes are used mostly as barely purified preparations [25], to highly sophisticated uses in the chemical synthesis of bioactive molecules where higher purity is required [26]. Production process is very much dependent on the level of production and type of application. In fact, industrial enzymes for the detergent and textile industries, and also many of the enzymes used for food and feed applications, are usually produced in large quantities as rather crude preparations [27]. On the other hand, enzymes used in more sophisticated processes of chemical synthesis for the production of drugs and other specialties are usually required in smaller amounts and higher purity. Immobilized enzymes are increasingly being used in both small- and largescale processes and in such cases increasing the purity of the enzyme starting material may be rewarded by the higher specific activity of the biocatalyst obtained [28, 29]. Localization of the enzyme is another key feature determining the production process: exported enzymes will be recovered from the spent fermentation medium, while cell-bound enzymes will be recovered from the biosolids that will be further subjected to extraction by cell disruption or permeabilization [30, 31]. An enzyme production process can be divided into four stages: enzyme synthesis by propagation of the producing microorganism; enzyme recovery involving solidliquid separation, concentration of the spent medium or cell disruption and cell debris removal; enzyme purification, consisting in one or more operations after enzyme

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recovery aiming to remove unwanted contaminants; enzyme product formulation including final polishing operations, stabilization and standardization.

1.2.1

Enzyme Synthesis

Microbial enzymes are mostly produced by submerged fermentations under highly controlled conditions; however, solid-state fermentation is a viable option when the producing microorganism is well conditioned for surface growth and a suitable solid substrate is available [32]. Submerged fermentation can be conducted in batch, fedbatch or continuous operation. Fed-batch is particularly appealing for the production of enzymes because it allows the control of metabolic responses of the producing cells and the operation is rather simple, thus it is often a preferred option at industrial level [33]. Continuous culture is in principle the most productive and controlled operation for producing microbial metabolites; however, industry is rather reluctant to adopt it because the prolonged operation times imply the hazard of washout of the producing strain by contamination or mutation [34], the latter being particularly critical when using recombinant microorganisms [35]. Besides enzyme localization, specific activity of the producing strains (units of enzyme activity per unit mass of microbial cells) is quite important, since it directly affects not only the cost of fermentation but also the cost of downstream operations; therefore, much effort has been spent in increasing it by both environmental manipulations (fermentation medium design, temperature and pH optimization, aeration and agitation rates) and genetic manipulations (genetic engineering and mutagenesis) [36]. Enzymes are subjected to different mechanisms of control, so that the corresponding triggering signals have to be considered for proper medium design. Most industrially relevant microbial enzymes are growth-associated, so increasing cell growth rate means increasing enzyme productivity. However, optimum conditions for growth seldom match those optimal for enzyme synthesis, so inevitably a compromise arises that should be judiciously solved in terms of enzyme production. Safety status of the producing strain is another key issue for selecting the enzyme and its usage at industrial scale. For instance, enzymes intended for food or pharmaceutical applications should be produced by microorganisms with the corresponding safety status. Obtaining such status may be costly and time-consuming so that in occasions it is a better option to clone the enzyme structural gene and express it in a safety host [37]. Regulatory issues with respect to enzymes produced from recombinant microorganisms [38], and genetic stability and safety are of paramount importance when producing recombinant enzymes [39].

1.2.2

Enzyme Recovery

After fermentation, solid-liquid separation is required to either collect the spent medium removing the cells in the case of extracellular enzymes, or the other way around in the case of cell-associated enzymes. Solid-liquid separation is a conventional unit operation that can be done by filtration or centrifugation, depending on the morphology of the producing microorganism [40]. Enzyme release is a highly desirable feature since the cell membrane acts as a powerful purification tool, so that further purification steps will be significantly reduced and

Challenges and Opportunities for the Production

7

in some cases unnecessary. In this case, a rather diluted liquid stream will be obtained; for a 20 g L-1 cell concentration, extracellular protein is not expected to be much higher than 1 g L-1 and even in the case of high cell density fermentations, extracellular protein will not exceed a few grams per liter, so that concentration of the spent medium will be required. It can be estimated that concentration should be increased by no less than one order of magnitude prior to the purification step, being a key determinant of the production cost of extracellular enzymes [41]. In this respect, the production of enzymes by solid-state fermentation is convenient since a much concentrated liquid can be obtained by pressing the fermented solids or recovering them with a reduced amount of extractant [42]. Vacuum evaporation and ultrafiltration are the most used operations for enzyme concentration, the latter being a preferred option for being gentler and readily scalable [43]. In the case of cell-associated enzymes, they should be recovered from the solid cell paste by extraction. The operation of extraction will be mostly determined by the location of the enzyme within the cell structure (periplasmic, cytoplasmic, or membrane bound) and the structure of the cell envelope. Operations for the recovery of cell-bound enzymes can be roughly divided into that producing cell disruption and that producing cell permeabilization by membrane damage. Periplasmic enzymes can be effectively recovered by permeabilization, while intracellular enzymes may require cell disruption. Microbial cells are hard to disrupt, especially bacteria and yeast, because of the resilient nature of their cell envelopes. Most used methods for recovery of cell-bound enzymes are in Table 1.2. Not all of them are amenable for scale-up at production level and the cost of this operation can represent a significant part of the enzyme production cost [44]. Extracts, especially those obtained by cell disruption, are complex mixtures containing most of the intracellular components, so that downstream operations for enzyme purification become complex and costly, this being a major drawback of cell-associated enzymes. In this respect, gentle and more selective methods of enzyme recovery, involving permeabilization rather than disruption, are welcomed whenever possible [45, 46]. Whatever the method, enzyme extraction can be optimized in terms of recovery of active enzyme if sound models for the kinetics of protein extraction and enzyme inactivation at the conditions of extraction are developed [47]. A good method is one which is tough on cells and soft on proteins. After extraction, cell debris should be removed, which implies a second operation of liquid-solid separation. Besides the conventional methods, biphasic partition based on polymer incompatibility has been used successfully for producing particle-free extracts for the subsequent operations of purification [48]; the operation is readily scalable and equipment used is the same as used in liquid-liquid extraction [49].

1.2.3

Enzyme Purification

After recovery, the clarified concentrated broth containing the extracellular enzyme or the crude extract containing the intracellular enzyme is subjected to purification. Purification implies a series of operations aimed at removing the contaminants and producing an enzyme product of the desired purity. As said before, purification is much tougher for intracellular enzyme extracts. Leaving aside contaminants that can be rather easily removed, purification is focused mostly on removing contaminant proteins, so

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Table 1.2 Operations for the recovery of cell-bound enzymes [44]. Operation

Principle

Applicability at large scale

Pressure

compression, shear stress

moderate

Homogenization

shear stress, cavitation

feasible

Milling

compression, shear stress

highly feasible

Sonication

cavitation

moderate

Decompression

de compressive explosion

moderate

Freezing-thawing

shear stress

unlikely

Dispersion in water

osmotic shock

unlikely

Thermolysis

cell wall rupture

moderate

Alkali treatment

cell wall digestion

unlikely

Solvent treatment

membrane digestion

moderate

Enzymatic lysis

cell wall digestion and osmotic rupture

feasible

Autolysis

cell wall digestion and osmotic rupture

in some cases

Cell rupture

Cell permeabilization

purification is essentially a protein fractionation stage. Purification is evaluated in terms of purification factor (PF) and yield of recovery (Y). Values for each purification operation (i) and for the whole purification stage composed by “n” consecutive operations are then defined by Eqs. 1.1 to 1.4





(1.1)



(1.2) (1.3)



(1.4)

Where a is the specific activity of the enzyme (units of activity per unit mass of protein) and E is the total units of enzyme activity; subindex 0 denotes the initial values before purification.

Challenges and Opportunities for the Production

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Since (PF)i will be higher than 1, while Yi will be lower than 1, it is apparent that PF will increase while Y will decrease with the number of steps, so purity and recovery run into opposite directions; this means that the higher the purity required, the lower amount of enzyme recovered. Then, the criterion for establishing the final purity of the enzyme preparation is critical. For the case of so-called technical enzymes, which are produced at a large scale as commodities, the criterion of purification is the minimum compatible with its intended use. Purification at large scale is complex and costly and generally not justified by the benefit of producing a purer protein; in such cases Y rather than PF is the objective function and in practice it means few to none purification steps, in the case of extracellular enzymes. The situation is quite different in the case of specialty enzymes, where purity cannot be sacrificed in favor of yield. In the case of enzymes that will be further immobilized, there is an incentive in purification even for bulk enzymes, since higher mass activities can be obtained. Methods for protein fractionation have been thoroughly developed at laboratory scale aiming to obtain pure proteins for structural and functional studies. Some, but not all of them, are applicable for the production of enzymes, and reported data on protocols for enzyme purification at laboratory scale are quite poor in terms of yield having little meaning for production purposes [50]. Besides PF and Y, throughput and rate of the operation have to be considered. Those methods that can be scaled up for production purposes can be divided into those based on differential solubility and those based on differential retention by interaction with a stationary phase, usually referred to as chromatography [47]. The former are based on the differential precipitation under non-denaturing conditions by the action of precipitating agents (salts, organic co-solvents, polymers). PF values attainable by fractional precipitation are rather modest, well below ten in most cases [51], and these operations are used rather as an initial concentration step since precipitates formed can be redissolved in a reduced volume of liquid so that further purification steps are conducted over a significantly lower volume. Chromatographic fractionation is a very powerful technique for protein purification, initially developed for analytical purposes, but preparative chromatography has been extensively used for laboratory scale enzyme purification. Several types of chromatography exist according to the principle of fractionation that have been applied for enzyme purification: size-exclusion chromatography (gel permeation) is based on the molecular size [52], ionic-exchange is based on ionic interactions among charged groups [53], hydrophobic chromatography is based on apolar interactions [54], and affinity chromatography is based on functional properties of the protein [55]; affinity chromatography is in principle the most selective but also the more complex and expensive method. Chromatography is quite powerful in terms of purification at laboratory scale; however, scale up to production level is not an easy task and a compromise exits between resolution and throughput. Despite the very high resolution of chromatography, because of hydrodynamic considerations, chromatographic columns cannot be scaled up by geometric congruence so that column diameter to bed height ratio increases with size and dispersion is increased reducing resolution [56]. Since enzyme purification is envisioned as a series of sequential operations, some guidelines have been proposed for the design of the purification stage. Operations

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should be selected to fully exploit the difference in physicochemical or functional properties of the enzyme with respect to the contaminant proteins, each operation should be based on a different property and simple though not very selective operations should be conducted first to early reduce the processing volume, using more complex and selective operations for a later stage to act on a reduced volume of product stream [57]. Rational design and optimization of protein purification processes have been developed and optimal operation sequences determined by using expert system [58, 59].

1.2.4 Enzyme Formulation After purification, the preparation has to be formulated as a product to be stored and delivered to the customer in a format adequate to its intended use. This is a rather neglected aspect of enzyme production that is seldom reported in the open literature. However, this is important both for bulk industrial enzymes and specialty enzymes that should meet strict regulations, and represents a competitive edge for the enzyme producer. Health and safety aspects regarding enzyme production are a major concern, and regulations are determined by the end use of the enzyme and may vary considerably according to it; regulations may also vary from country to country [60]. Enzyme formulation involves final polishing operations, stabilization and standardization. Polishing operations include the removal of trace contaminants (i.e., pyrogens, endotoxins, viruses), and in the case of bulk industrial enzymes may include salt removal by diafiltration or size-exclusion chromatography, pH adjustment if produced in a liquid format and drying (usually spray-drying) if produced as a solid preparation. Additional polishing operations may be required in the case of enzymes for special uses. Production of the enzyme as a liquid or solid preparation is mostly dictated by the end use of the enzyme, but decision is not trivial. Concentrated liquid preparations present some advantages: containment is simpler, drying cost is avoided and dosage is simpler. Solid preparations are intended mostly when enzymes are included in solid products, like detergents, and have the advantage of easier handling and transportation, and in many cases extended storage stability. Stabilization is a key issue in enzyme formulation, since the enzyme product should withstand long periods of storage and transportation maintaining its catalytic integrity. Microbial degradation should be avoided and structural conformation preserved. The first goal may be achieved either by adding authorized preservatives or by sterilization using absolute filtration. Preservation of the enzyme conformation, avoiding aggregation, unfolding or any deleterious alteration of its three-dimensional conformation, is mostly important; proteins are more stable in concentrated solutions, so concentration also plays that role in the case of liquid preparations, but enzymes are usually formulated in the presence of structural stabilizers like neutral salts and polyols and sometimes more specific conformational protectants are used, like the enzyme cofactor, the enzyme substrate, a substrate analogue or even an inhibitor [61]. Enzyme products should be standardized to ensure a product of uniform quality to the customer or end user. Catalytic potency of the enzyme in terms of its specific activity should be warranted and since batch to batch variations are unavoidable, standardization is done by diluting the enzyme activity to a certified value using varying amounts of excipients to that purpose that may be the same substances used for preservation [62]. Enzyme producers have their own way of measuring

Challenges and Opportunities for the Production

11

activity which may or may not be relevant to the user, but this method has to be clearly described by the producer to be checked by the user. Storage stability is also important and should be clearly specified in the product sheet provided by the producer. Product sheets usually contain additional information about physical characteristics of the products, but information about excipients and other components is seldom reported. In some cases, additional information like pH and temperature profiles, and even values of kinetic parameters are also provided, which is welcomed. An enzyme product must comply with all requirements of quality and compatibility with intended used before being launched and be produced according to good manufacturing practices. Most enzyme producing companies have the corresponding ISO (International Organization for Standardization) 9001–2008 certificates warranting that the production process complies with the requirements for standardization and quality assurance. Enzymes are sometimes produced in immobilized format and in this sense immobilization could be considered as a final step of product formulation; however, immobilization has a much more significant meaning since it is oriented to improve the enzyme catalyst performance, so it will be presented in the next section.

1.2.5 Market Outlook for Industrial Enzymes Enzymes are increasingly being used as industrial catalysts not only for conventional degradation processes but also for more sophisticated processes of organic synthesis [63]. Global industrial enzyme market was estimated close to US$ 4.5 billion by 2015 and might well be over US$ 5 billion by now. So-called technical enzymes represent 34%, enzymes for food and beverages represent 27%, and enzymes for other uses represent 39% [64]. The first two categories are mostly hydrolases acting on carbohydrates (glycosidases), on proteins (proteases) and on lipids (lipases). The last category includes the specialty enzymes being used in high-added value processes of organic synthesis, and represent the most dynamic sector. Lipases are outstanding catalysts for organic synthesis, being well suited for performing in low-water environments, as usually required for organic synthesis; its molecular structure is well conditioned to such purpose, many lipases being activated at water-oil interfaces and in hydrophobic solvents where they can catalyze esterification, transesterification and interesterification reactions [65]. Other hydrolases have been also used in their synthetic capacity; worthwhile mentioning is the use of proteases in peptide synthesis (i.e., synthesis of the dipeptide aspartame with thermolysin) [66] and glycosidases in oligosaccharide synthesis (i.e., synthesis of prebiotic galacto-oligosaccharides with β-galactosidase) [67]. Market figures should be taken with caution since a significant fraction of enzymes are produced by their own users or by joint ventures with supplying companies, so that the real commercial value of enzymes is definitely larger that the figures above given. Hydrolases still represent the major share of the enzyme market (about 45%); lyases represent more than 15% due to the massive use of nitrile hydratase in the bulk production of acrylamide; isomerases represent over 5% but this is referred solely to the use of glucose (xylose) isomerase in the production of high-fructose syrups for the food industry; oxidoreductases are co-enzyme requiring specialty enzymes used mostly for the synthesis of bioactive molecules for the pharmaceutical and fine-chemicals sectors

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Table 1.3 Main industrial enzymes producers. Company

Country

Main industrial areas of application

Novozymes

Denmark

Food and feed, biofuels, pulp and paper, leather, detergents

DuPont (Genencor)

U.S.A.

Food and feed, biofuels, detergents, textiles

DSM

The Netherlands

Food and feed, biofuels, detergents, textiles

BASF (Verenium)

Germany (U.S.A.)

Biofuels, animal health, pulp and paper, textiles

Amano

Japan

Food, pharmaceuticals

and have experienced the most significant increase in recent years representing now close to 30% of the global market [26, 68]. Enzyme market is covered by a rather small number of highly specialized companies with outstanding skills in screening for new and improved enzymes, in highly controlled fermentation, in enzyme purification at large-scale, and in enzyme product formulation [69]. A list of most relevant industrial enzyme producers is presented in Table 1.3. Novozymes and DuPont (Genencor) together represent about two-thirds of the enzyme market, the remaining one third being covered mostly by DSM, BASF and several Japanese companies. In quantitative terms, food and feed, detergents, pulp and paper and leather are the most enzyme-demanding areas, where hydrolases are still prevalent. Emerging areas like biofuel and several applications in organic synthesis for the production of bioactive compounds are rapidly gaining a prominent place within the enzyme market, where not only conventional hydrolases are being used, but also coenzyme-requiring more complex enzymes like oxidoreductases, lyases and transferases that are progressively coming into play [70]. On the other hand, China, India and South Korea are increasingly relevant countries for industrial enzyme production.

1.3

Building Up Enzyme Catalysts for Process Applications

Enzymes are physiological catalysts that are not necessarily well suited to perform under process conditions. Converting these physiological catalysts into robust process catalysts is undoubtedly the major challenge for enzyme biocatalysis. This is even more so now, when enzymes are increasingly being used in reactions of organic synthesis, which are usually conducted under rather harsh conditions. Several strategies to tackle this problem are available, considering biocatalyst design, medium engineering and also bioreactor design [71].

Challenges and Opportunities for the Production

13

Nature is an endless source of bioactive molecules and new enzymes with adequate properties for process applications can be obtained by bioprospecting [72] and the metagenomic approach is gaining increasing importance for the discovery of novel enzyme functions [73–75]. However, wild-type enzymes are frequently not active or stable enough for process applications so that improving them by molecular redesign and other protein engineering techniques already mentioned are of paramount importance for building-up industrial enzyme biocatalysts, both for conventional degradation processes and for organic synthesis [76–79]. Enzyme stabilization has received considerable attention, being a key issue for developing industrially useful enzyme biocatalysts [80]. Enzyme immobilization is probably the most powerful tool for producing robust process biocatalysts, salient features of immobilized enzymes being their increased stability, reusability and flexibility for reactor operation [81, 82]. Many strategies for enzyme immobilization are envisaged considering both carrier-bound and carrier-free systems [83]. The former allows high flexibility in biocatalyst design, produce very robust catalysts with increased stability and susceptibility to reactivation, but the considerable amount of inert material implies additional costs and low specific activities [84]. On the other hand, carrier-free biocatalysts, especially cross-linked enzyme aggregates (CLEAs) have very high specific activities because a substantial portion of the catalyst mass is active protein, and are simple to produce [85]; however, their operational stability and handling at process conditions are drawbacks to be overcome. Optimization of enzyme immobilization is a complex task, since it is a multivariable process and a sound, but not obvious, objective function is required for evaluation, so that no rational guidelines have been developed and the best system and conditions have to be determined in each case [86]. Despite this, immobilized enzymes have had a profound impact on enzyme biocatalysis at industrial level [87, 88] and are being increasingly important as catalysts for organic synthesis where operation conditions can be harmful for maintaining the enzyme active configuration [89]. Advances in several fields, like molecular biology, bioinformatics and material sciences are allowing an increasingly rational approach to enzyme immobilization [90–92], and sometimes the concept of immobilization engineering has been used to refer to this more rational approach to enzyme immobilization [93, 94]. In the last two decades, enzyme biocatalysis has experienced a change in paradigm: enzymes were traditionally considered as catalysts for aqueous media and, in fact, most of the early enzymes used were hydrolases acting in high water activity environments, but a gradual but sustained trend of biocatalysis as an alternative for organic synthesis has moved the attention to non-hydrolytic enzymes and hydrolases acting in reverse. The former are complex, labile and coenzyme-requiring proteins, so technological development has been complex; however, the high added value of some processes of synthesis [95] and the compliance of biocatalysis with the principles of green chemistry [96], have been the driving forces for development. Despite their complexities, oxidoreductases [97], lyases [98] and transferases [99] are being increasingly evaluated as catalysts for the synthesis of sophisticated bioactive molecules, where the molecular precision of the reaction is a key feature [100]. Hydrolases, that are robust and readily available enzymes, over which biocatalysis was developed, can under certain conditions act in reverse catalyzing bond formation instead of hydrolysis. This is of paramount significance since hydrolases can act with very high molecular precision to

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catalyze reactions of bond formation [101]: lipases may catalyze esterification, interesterification and transesterification reactions [102–104], proteases and acylases may catalyze the formation of peptide bonds [105–107], and glycosidases may catalyze the formation of glycosidic linkages [108–110]. To do so, reaction medium has to be engineered in order to depress the hydrolytic potential of these hydrolytic enzyme and reactions should be conducted at reduced water activity. This can be done by partial or (almost) total replacement of water by an organic solvent or a neoteric solvent, or using high solids concentrations. Organic solvents have been the most used reaction media for organic synthesis [111], despite their obvious economic and environmental burdens, so that neoteric solvents have been evaluated like supercritical fluids [112, 113], ionic liquids [114] and, more recently, deep eutectic solvents [115]. Neoteric solvents are in more compliance with the principles of green chemistry but for the most part there are still technological and economic constraints precluding their extensive use as reaction media for biocatalysis. The use of high solids concentration is a strategy to depress water activity when substrates are poorly soluble in non-aqueous media that have been successfully applied to the synthesis of β-lactam antibiotics with penicillin acylase [116] and to oligosaccharide synthesis with β-galactosidases [117]. Among hydrolases, lipases clearly outstand as catalysts for organic synthesis and have been profusely used in a large number of chemo, regio and stereoselective transformations, many of them of technological relevance [7]. This is because lipases are structurally well conditioned for performing in low water media and at water-oil interfaces. Many of them have a peculiar structure in which a polypeptide chain, called lid, can bend over the active site impeding catalysis (closed conformation); in the presence of an interface or a hydrophobic surrounding the lid moves away leaving the active site available for substrate binding and conversion (open configuration) [118]. Even so, lipases are conveniently used in immobilized form for giving a more robust configuration in non-aqueous media [119]. Activity and stability are major concerns when using enzymes in non-conventional media, and this is more critical for other hydrolases, like proteases and glycosidases, and even more so for non-hydrolytic enzymes, so that enzymes are usually used in immobilized format and mutant enzymes selected by improved traits related to performance in such media are actively searched for [120]. It is worthwhile mentioning that commercial immobilized hydrolases have been usually designed for hydrolytic reactions so that they are not necessarily well suited to perform in reactions of synthesis. For instance, diffusional restrictions are quite limiting for fast hydrolytic reactions, but may be not so for slower reactions of synthesis [121]; this is to highlight that the current trend is designing the biocatalyst according to the process in which it will be used and not the other way around. Medium engineering implies a judicious choice of the one in which the compromise between solubility of substrates, enzyme activity and stability and substrates and product stability, are optimally balanced. For the moment, this is to be experimentally determined for each case. Reactor design is another factor to be taken into consideration. High yields and productivities are required for making biocatalytic processes competitive, so reactors should be designed according to a process intensification strategy to meet such purpose [122]. Membrane reactors could be a good strategy when product size is smaller than the substrate. Since most data on biocatalytic processes comes from laboratory

Challenges and Opportunities for the Production

15

scale operations it is important to design those experiments in a scale-down concept so that results obtained at such scale are more predictive to production scale.

1.4

Conclusions

Enzymes have played a significant role in the industrial production of commodities as well as specialty products. Enzyme applications have evolved from rather simple hydrolytic processes where they were mostly used in dissolved form in aqueous media to more complex processes of organic synthesis usually conducted in non-conventional reaction media. Enzyme biocatalysis is now considered a strategic technology, having a noticeable social impact on health and in food, fuel and chemicals supply under sustainable conditions. Enzymes are now produced mostly by fermentation and this will continue to be so in the foreseeable future. Impressive advances in molecular biology, fermentation technology, bioinformatics, nanotechnology, material sciences and process control are the cornerstones over which biocatalysis development is sustained.

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High Value Fermentation Products Volume 2 of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris. Protein Expr. Purif., 60(2), 140–146, 2008. Altamirano, C., Illanes, A., Canessa, R., Becerra, S., Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production. Electron. J. Biotechnol., 9(1), 61–68, 2006. Chu, L., Robinson, D.K., Industrial choices for protein production by large-scale cell culture. Curr. Opin. Biotechnol., 12(2), 180–187, 2001. Illanes, A., Cauerhff, A., Wilson, L., Castro, G.R., Recent trends in biocatalysis engineering. Bioresour. Technol., 115, 48–57, 2012. Visser, S., van Rooijen, P.J., Schattenkerk, C., Kerling, K.E.T., Peptide substrates for chymosin (Rennin) Kinetic studies with bovine κ-casein-(103–108)-hexapeptide analogues. Biochimica et Biophysica Acta (BBA) - Enzymology, 481(1), 171–176, 1977. van den Berg, J.A., van der Laken, K.J., van Ooyen, A.J., Renniers, T.C., Rietveld, K., Schaap, A., et al., Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Biotechnology. (N.Y.)., 8(2), 135, 1990. Ward, M., Wilson, L.J., Kodama, K.H., Rey, M.W., Berka, R.M., Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Biotechnology (N.Y.)., 8(5), 435–440, 1990. Mantafounis, D., Pitts, J., Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis. Protein. Eng. Des. Sel., 3(7), 605–609, 1990. Maurer, K.H., Detergent proteases. Curr. Opin. Biotechnol., 15(4), 330–334, 2004. Luetz, S., Giver, L., Lalonde, J., Engineered enzymes for chemical production. Biotechnol. Bioeng., 101(4), 647–653, 2008. Woodley, J.M., Protein engineering of enzymes for process applications. Curr. Opin. Chem. Biol., 17(2), 310–316, 2013. Hodgson, J., The changing bulk biocatalyst market. Biotechnology (N.Y.)., 12(8), 789–790, 1994. Muñoz Solano, D., Hoyos, P., Hernáiz, M.J., Alcántara, A.R., Sánchez-Montero, J.M., Industrial biotransformations in the synthesis of building blocks leading to enantiopure drugs. Bioresour. Technol., 115, 196–207, 2012. Headon, D.R., Walsh, G., The industrial production of enzymes. Biotechnol. Adv., 12(4), 635–646, 1994. Cao, L., Langen, L., Sheldon, R.A., Immobilised enzymes: carrier-bound or carrier-free? Curr. Opin. Biotechnol., 14(4), 387–394, 2003. Garcia-Galan, C., Berenguer-Murcia, Ángel., Fernandez-Lafuente, R., Rodrigues, R.C., Potential of different enzyme immobilization strategies to improve enzyme performance. Adv. Synth. Catal., 353(16), 2885–2904, 2011. Middelberg, A.P., Process-scale disruption of microorganisms. Biotechnol. Adv., 13(3), 491–551, 1995. Salazar, O., Asenjo, J.A., Enzymatic lysis of microbial cells. Biotechnol. Lett., 29(7), 985– 994, 2007. Singhania, R.R., Sukumaran, R.K., Patel, A.K., Larroche, C., Pandey, A., Advancement and comparative profiles in the production technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Microb. Technol., 46(7), 541–549, 2010. Singh, J., Vohra, R.M., Sahoo, D.K., Enhanced production of alkaline proteases by Bacillus sphaericus using fed-batch culture. Process Biochemistry, 39(9), 1093–1101, 2004. Acevedo, F., Gentina, J.C., Cinética de fermentaciones. In: Acevedo F, Gentina J. C, eds. Ediciones Universitarias de Valparaíso, 2002.

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35. Cereghino, G.P., Cereghino, J.L., Ilgen, C., Cregg, J.M., Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol., 13(4), 329–332, 2002. 36. Parekh, S., Vinci, V.A., Strobel, R.J., Improvement of microbial strains and fermentation processes. Appl. Microbiol. Biotechnol., 54(3), 287–301, 2000. 37. Ahmad, M., Hirz, M., Pichler, H., Schwab, H., Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol., 98(12), 5301–5317, 2014. 38. Olempska-Beer, Z.S., Merker, R.I., Ditto, M.D., DiNovi, M.J., Food-processing enzymes from recombinant microorganisms-a review. Regul. Toxicol. Pharmacol., 45(2), 144–158, 2006. 39. Demain, A.L., Vaishnav, P., Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv., 27(3), 297–306, 2009. 40. Medronho, R., Solid-liquid separations. In: Hatti-Kaul R, Mattiasson B, eds. Isolation and Purification of Proteins. CRC Press. pp. 117–174, 2003. 41. Liu, Y.C., Liao, L.C., Wu, W.T., Proceedings of the national scientific council republic of china B, 24, 156, 2000. 42. Singh, S.K., Sczakas, G., Soccol, C.R., Pandey, A., Production of enzymes by solid-state fermentation. In: Pandey A, Soccol C. R, Larroche C, eds. Current Developments in Solid-State Fermentation. Springer. pp. 183–204, 2008. 43. Ulber, R., Plate, K., Reif, O.W., Melzner, D., Membranes for protein isolation and purification. In: Hatti-Kaul R, Mattiasson B, eds. Isolation and Purification of Proteins. CRC Press. pp. 175–207, 2003. 44. Fonseca, L.P., Cabral, J.M.S., Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization. J. Chem. Technol. Biotechnol., 77(2), 159– 167, 2002. 45. Kula, M.-R., Schütte, H., Purification of proteins and the disruption of microbial cells. Biotechnol. Prog., 3(1), 31–42, 1987. 46. Illanes, A., Gorgollón, Y., Kinetics of extraction of invertase from autolysed bakers' yeast cells. Enzyme Microb. Technol., 8(2), 81–84, 1986. 47. Illanes, A., Enzyme purification. In: Illanes A, ed. Enzyme Biocatalysis: Principles and Applications. Springer. pp. 57–106, 2008. 48. Kula, M.R., Kroner, K.H., Hustedt, H., Schütte, H., Technical aspects of extractive enzyme purification. Ann. N. Y. Acad. Sci., 369(1 Biochemical E), 341–354, 1981. 49. Cunha, T., Aires-Barros, R., Large-scale extraction of proteins. Mol. Biotechnol., 20(1), 029–040, 2002. 50. Patel, R.K., Dodia, M.S., Joshi, R.H., Singh, S.P., Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochem., 41(9), 2002–2009, 2006. 51. Farag, A.M., Hassan, M.A., Purification, characterization and immobilization of a keratinase from Aspergillus oryzae. Enzyme Microb. Technol., 34(2), 85–93, 2004. 52. Singh, R.S., Dhaliwal, R., Puri, M., Partial purification and characterization of exoinulinase from Kluyveromyces marxianus YS-1 for preparation of high-fructose syrup. J. Microbiol. Biotechnol., 17(5), 733, 2007. 53. Aguilar, O., Albiter, V., Serrano-Carreón, L., Rito-Palomares, M., Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli. J. Chromatogr. B., 835(1-2), 77–83, 2006.

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54. Lee, K.-W., Bae, H.-A., Shin, G.-S., Lee, Y.-H., Purification and catalytic properties of novel enantioselective lipase from Acinetobacter sp. ES-1 for hydrolysis of (S)-ketoprofen ethyl ester. Enzyme Microb. Technol., 38(3-4), 443–448, 2006. 55. Mendu, D.R., Ratnam, B.V.V., Purnima, A., Ayyanna, C., Affinity chromatography of α-amylase from Bacillus licheniformis. Enzyme Microb. Technol., 37(7), 712–717, 2005. 56. Tallarek, U., Bayer, E., Guiochon, G., Study of dispersion in packed chromatographic columns by pulsed field gradient nuclear magnetic resonance. J. Am. Chem. Soc., 120(7), 1494–1505, 1998. 57. Watanabe, E., Tsoka, S., Asenjo, J.A., Selection of chromatographic protein purification operations based on physicochemical properties. Ann. N. Y. Acad. Sci., 721(1 Recombinant D), 348–364, 1994. 58. Vásquez-Alvarez, E., Lienqueo, M.E., Pinto, J.M., Optimal synthesis of protein purification processes. Biotechnol. Prog., 17(4), 685–696, 2001. 59. Simeonidis, E., Pinto, J.M., Lienqueo, M.E., Tsoka, S., Papageorgiou, L.G., MINLP models for the synthesis of optimal peptide tags and downstream protein processing. Biotechnol. Prog., 21(3), 875–884, 2005. 60. Spök, A., Safety regulations of food enzymes. Food Technol. Biotechnol., 44, 197–303, 2006. 61. Illanes, A., Stability of biocatalysts. Electron. J. Biotechnol., 2(1), 1999. 62. Schoemaker, H.E., Mink, D., Wubbolts, M.G., Dispelling the myths--biocatalysis in industrial synthesis. Science, 299(5613), 1694–1697, 2003. 63. Adrio, J.L., Demain, A.L., Microbial enzymes: tools for biotechnological processes. Biomolecules, 4(1), 117–139, 2014. 64. Binod, P., Palkhiwala, P., Gaikaiwari, R., Madhavan Nampoothiri, K., Duggal, A., Dey, K., J. Sci. & Ind. Res. India, 72, 271, 2013. 65. Gotor-Fernández, V., Brieva, R., Gotor, V., Lipases: Useful biocatalysts for the preparation of pharmaceuticals. J. Mol. Catal. B- Enzym., 40(3-4), 111–120, 2006. 66. Leuchtenberger, W., Huthmacher, K., Drauz, K., Biotechnological production of amino acids and derivatives: current status and prospects. Appl. Microbiol. Biotechnol., 69(1), 1–8, 2005. 67. Vera, C., Córdova, A., Aburto, C., Guerrero, C., Suárez, S., Illanes, A., Synthesis and purification of galacto-oligosaccharides: state of the art. World J. Microbiol. Biotechnol., 32(12), 197, 2016. 68. Wells, A.S., Finch, G.L., Michels, P.C., Wong, J.W., Use of enzymes in the manufacture of active pharmaceutical ingredients—A science and safety-based approach to ensure patient safety and drug quality. Org. Process Res. Dev., 16(12), 1986–1993, 2012. 69. Sarrouh, B., Up-to-date insight on industrial enzymes applications and global market. J. Bioprocess. Biotech. s4: 002, 2012. 70. Singh, R., Kumar, M., Mittal, A., Mehta, P.K., Kumar Mehta, P., Microbial enzymes: industrial progress in 21st century. 3 Biotech, 6(2), 174, 2016. 71. Illanes, A., Fernández-Lafuente, R., Guisán, J.M., Wilson, L., “Heterogeneous enzyme kinetics”. In: Illanes A, ed. Enzyme Biocatalysis: Principles and Applications. Springer. pp. 155–203, 2008. 72. Banerjee, G., Scott-Craig, J.S., Walton, J.D., Improving enzymes for biomass conversion: A basic research perspective. Bioenerg. Res., 3(1), 82–92, 2010. 73. Davids, T., Schmidt, M., Böttcher, D., Bornscheuer, U.T., Strategies for the discovery and engineering of enzymes for biocatalysis. Curr. Opin. Chem. Biol., 17(2), 215–220, 2013. 74. Ferrer, M., Martínez-Martínez, M., Bargiela, R., Streit, W.R., Golyshina, O.V., Golyshin, P.N., Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. Microb. Biotechnol., 9(1), 22–34, 2016.

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75. Jemli, S., Ayadi-Zouari, D., Hlima, H.B., Bejar, S., Biocatalysts: application and engineering for industrial purposes. Crit. Rev. Biotechnol., 36(2), 246–258, 2016. 76. Cherry, J.R., Fidantsef, A.L., Directed evolution of industrial enzymes: an update. Curr. Opin. Biotechnol., 14(4), 438–443, 2003. 77. Kim, D.Y., Han, M.K., Oh, H.W., Bae, K.S., Jeong, T.S., Kim, S.U., et al., Novel intracellular GH10 xylanase from Cohnella laeviribosi HY-21: Biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues. Bioresour. Technol., 101(22), 8814–8821, 2010. 78. Bommarius, A.S., Blum, J.K., Abrahamson, M.J., Status of protein engineering for biocatalysts: how to design an industrially useful biocatalyst. Curr. Opin. Chem. Biol., 15(2), 194–200, 2011. 79. Bornscheuer, U.T., Huisman, G.W., Kazlauskas, R.J., Lutz, S., Moore, J.C., Robins, K., Engineering the third wave of biocatalysis. Nature, 485(7397), 185–194, 2012. 80. Silva, C., Martins, M., Jing, S., Fu, J., Cavaco-Paulo, A., Practical insights on enzyme stabilization. Crit. Rev. Biotechnol., 38(3), 335–350, 2018. 81. Mateo, C., Palomo, J.M., Fernández-Lorente, G., Guisán, J.M., Fernández-Lafuente, R., Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme Microb. Technol., 40(6), 1451–1463, 2007. 82. Fernández-Lorente, G., Lopez-Gallego, F., Bolivar, J., Rocha-Martin, J., Moreno-Perez, S., Guisan, J., Immobilization of proteins on highly activated glyoxyl supports: Dramatic increase of the enzyme stability via multipoint immobilization on pre-existing carriers. Curr. Org. Chem., 19(17), 1719–1731, 2015. 83. Tischer, W., Kasche, V., Immobilized enzymes: crystals or carriers? Trends Biotechnol., 17(8), 326–335, 1999. 84. Romero, O., Guisán, J.M., Illanes, A., Wilson, L., Reactivation of penicillin acylase biocatalysts: Effect of the intensity of enzyme–support attachment and enzyme load. J. Mol. Catal. B- Enzym., 74(3-4), 224–229, 2012. 85. Sheldon, R.A., Cross-linked enzyme aggregates as industrial biocatalysts. Org. Process Res. Dev., 15(1), 213–223, 2011. 86. Illanes, A., Immobilized biocatalysts. In: Moo-Young M, ed. Comprehensive Biotechnology (Second Edition) Volume 1: Scientific Fundamentals of Biotechnology. Elsevier. pp. 25–39, 2011. 87. DiCosimo, R., McAuliffe, J., Poulose, A.J., Bohlmann, G., Industrial use of immobilized enzymes. Chem. Soc. Rev., 42(15), 6437, 2013. 88. Liese, A., Hilterhaus, L., Evaluation of immobilized enzymes for industrial applications. Chem. Soc. Rev., 42(15), 6236, 2013. 89. Rodrigues, R.C., Ortiz, C., Berenguer-Murcia, Á., Torres, R., Fernández-Lafuente, R., Modifying enzyme activity and selectivity by immobilization. Chem. Soc. Rev., 42(15), 6290–6307, 2013. 90. Nestl, B.M., Nebel, B.A., Hauer, B., Recent progress in industrial biocatalysis. Curr. Opin. Chem. Biol., 15(2), 187–193, 2011. 91. Bolivar, J.M., Eisl, I., Nidetzky, B., Advanced characterization of immobilized enzymes as heterogeneous biocatalysts. Catal. Today, 259, 66–80, 2016. 92. Wang, M., Qi, W., Su, R., He, Z., Advances in carrier-bound and carrier-free immobilized nanobiocatalysts. Chem. Eng. Sci., 135, 21–32, 2015. 93. Jackson, E., López-Gallego, F., Guisán, J.M., Betancor, L., Enhanced stability of L -lactate dehydrogenase through immobilization engineering. Process Biochem., 51(9), 1248–1255, 2016.

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94. Weiser, D., Nagy, F., Bánóczi, G., Oláh, M., Farkas, A., Szilágyi, A., et al., Immobilization engineering – How to design advanced sol–gel systems for biocatalysis? Green Chem., 19(16), 3927–3937, 2017. 95. Hummel, W., Kuzu, M., Geueke, B., An efficient and selective enzymatic oxidation system for the synthesis of enantiomerically pure D-tert-leucine. Org. Lett., 5(20), 3649–3650, 2003. 96. Woodley, J.M., New opportunities for biocatalysis: making pharmaceutical processes greener. Trends Biotechnol., 26(6), 321–327, 2008. 97. Berenguer-Murcia, A., Fernandez-Lafuente, R., New trends in the recycling of NAD(P)H for the design of sustainable asymmetric reductions catalyzed by dehydrogenases. Curr. Org. Chem., 14(10), 1000–1021, 2010. 98. Ardao, I., Alvaro, G., Benaiges, M.D., Dolors Benaiges, M., Reversible immobilization of rhamnulose-L-phosphate aldolase for biocatalysis: Enzyme loading optimization and aldol addition kinetic modeling. Biochem. Eng. J., 56(3), 190–197, 2011. 99. Guo, F., Berglund, P., Transaminase biocatalysis: optimization and application. Green Chem., 19(2), 333–360, 2017. 100. Nestl, B.M., Hammer, S.C., Nebel, B.A., Hauer, B., New generation of biocatalysts for organic synthesis. Angew. Chem. Int. Ed. Engl., 53(12), 3070–3095, 2014. 101. Méndez-Sánchez, D., López-Iglesias, M., Gotor-Fernández, V., Hydrolases in organic chemisry. Recent achievements in the synthesis of pharmaceuticals.. Curr. Org. Chem., 20(11), 1186–1203, 2016. 102. Toledo, M.V., José, C., Collins, S.E., Ferreira, M.L., Briand, L.E., Towards a green enantiomeric esterification of R/S-ketoprofen: A theoretical and experimental investigation. J. Mol. Catal. B- Enzym., 118, 52–61, 2015. 103. Sankaran, R., Show, P.L., Chang, J.-S., Biodiesel production using immobilized lipase: feasibility and challenges. Biofuels Bioprod. Bioref., 10(6), 896–916, 2016. 104. Chávez-Flores, L., Beltran, H., Arrieta-Baez, D., Reyes-Duarte, D., Regioselective synthesis of lactulose esters by Candida antarctica and Thermomyces lanuginosus lipases. Catalysts, 7(9), 263, 2017. 105. Illanes, A., Wilson, L., Kinetically-controlled synthesis of B-lactam antibiotics. Chim. Oggi., 24(5), 27–30, 2006. 106. Yagasaki, M., Hashimoto, S., Synthesis and application of dipeptides; current status and perspectives. Appl. Microbiol. Biotechnol., 81(1), 13–22, 2008. 107. Bahamondes, C., Wilson, L., Bernal, C., Illanes, A., Álvaro, G., Guzmán, F., Synthesis of the kyotorphin precursor benzoyl-L-tyrosine-L-argininamide with immobilized α-chymotrypsin in sequential batch with enzyme reactivation. Biotechnol. Prog., 32(1), 54–59, 2016. 108. Linde, D., Rodríguez-Colinas, B., Estévez, M., Poveda, A., Plou, F.J., Fernández Lobato, M., Analysis of neofructooligosaccharides production mediated by the extracellular β-fructofuranosidase from Xanthophyllomyces dendrorhous. Bioresour. Technol., 109, 123– 130, 2012. 109. Vera, C., Córdova, A., Aburto, C., Guerrero, C., Suárez, S., Illanes, A., Synthesis and purification of galacto-oligosaccharides: state of the art. World J. Microbiol. Biotechnol., 32(12), 197, 2016. 110. Vera, C., Guerrero, C., Wilson, L., Illanes, A., Synthesis of propyl-β-D-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae. Process Biochem., 53, 162–171, 2017. 111. Koskinen, A.M.P., Klibanov, A.M., Enzymatic Reactions in Organic Media, London. Blackie Academic & Professional, 1996.

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112. Matsuda, T., Harada, T., Nakamura, K., Organic synthesis using enzymes in supercritical carbon dioxide. Green Chem., 6(9), 440, 2004. 113. Lozano, P., Garcia-Verdugo, E., Luis, S.V., Pucheault, M., Vaultier, M., (Bio)Catalytic continuous flow processes in scCO2 and/or ILS: Towards sustainable (bio)catalytic synthetic platforms. Curr. Org. Synth., 8, 810, 2011. 114. Potdar, M.K., Kelso, G.F., Schwarz, L., Zhang, C., Hearn, M.T., Recent developments in chemical synthesis with biocatalysts in ionic liquids. Molecules, 20(9), 16788–16816, 2015. 115. Xu, P., Zheng, G.W., Zong, M.H., Li, N., Lou, W.Y., Recent progress on deep eutectic solvents in biocatalysis. Bioresour. Bioprocess., 4(1), 34, 2017. 116. Illanes, A., Wilson, L., Corrotea, O., Tavernini, L., Zamorano, F., Aguirre, C., Synthesis of cephalexin with immobilized penicillin acylase at very high substrate concentrations in fully aqueous medium. J. Mol. Catal B- Enzym., 47(1-2), 72–78, 2007. 117. Huerta, L.M., Vera, C., Guerrero, C., Wilson, L., Illanes, A., Synthesis of galacto-oligosaccharides at very high lactose concentrations with immobilized β-galactosidases from Aspergillus oryzae. Process Biochem., 46(1), 245–252, 2011. 118. Nardini, M., Lang, D.A., Liebeton, K., Jaeger, K.E., Dijkstra, B.W., Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases. J. Biol. Chem., 275(40), 31219, 2000. 119. Adlercreutz, P., Immobilisation and application of lipases in organic media. Chem. Soc. Rev., 42(15), 6406–6436, 2013. 120. Choi, J.M., Han, S.S., Kim, H.S., Industrial applications of enzyme biocatalysis: Current status and future aspects. Biotechnol. Adv., 33(7), 1443–1454, 2015. 121. Valencia, P., Flores, S., Wilson, L., Illanes, A., Batch reactor performance for the enzymatic synthesis of cephalexin: influence of catalyst enzyme loading and particle size. New-. Biotechnol., 29(2), 218–226, 2012. 122. Satyawali, Y., Vanbroekhoven, K., Dejonghe, W., Process intensification: The future for enzymatic processes? Biochem. Eng. J., 121, 196–223, 2017.

2 Biotechnology of Leather: An Alternative to Conventional Leather Processing Saurabh Saran1,*, Shifali Chib1 and R. K. Saxena2 1

Fermentation Technology Group, CSIR-Indian Institute of Integrative Medicine (IIIM), Canal Road, Jammu Tawi-180001 , (India) 2 Department of Microbiology, University of Delhi South Campus, New Delhi-110021, (India)

Abstract The leather industry is an important contributor to the world economy as annual global trade reached up to US$100 billion per annum in 2016. Leather has its own significance, market strategy, economy, and market growth in terms of its existence. Leather finds applications in garments, footwear, bags, carpets, tents, rugs, etc. Conventional chemical-based leather processing involves several unit operations classified in four groups: pre-tanning, tanning, post-tanning and finishing, of which pre-tanning and tanning processes are known to contribute more than 90% of the total pollution load. Due to this pollution, the global leather industry is undergoing a paradigm shift from chemical- to bio-based leather making to meet the growing environmental challenges. In this respect, use of hydrolytic enzymes can be a boon for the leather industry as they have very little impact on the environment and are more specific, and their activity often works at a milder condition. Enzyme-mediated leather processing presents a breakthrough approach in making the pertaining operations of the leather industry chemical free. Keywords: Leather, biotechnology, production, value addition, enzyme, finance, economy

2.1

Introduction

The making of leather had its origin in the antiquity of man as an empirical development. It is generally conceded to have been the first manufacturing process by man. The leather industry has always emerged as an important economic activity in several developing countries of Southern Asia, mainly those dependent on an agricultural economy [1, 2], Nowadays, leather processing has become one of the important industries, closely related to everyday life. The leather industry has a prominent place in the Indian economy in terms of employment, export, finance, and growth. The Indian leather industry has evolved over nearly two centuries now. There has been a planned development in the leather industry in terms of its production utilizing the available

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (23–48) © 2019 Scrivener Publishing LLC

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raw materials minimizing the waste and maximizing the products or value added products for exports. There has been a tremendous transformation of technology in the past two decades which has been observed in terms of exportation of value added finished products in comparison to raw materials supply in the 1960 s. Leather is a unique commodity that links the rural farmer to the fashion world. It finds applications in garments, footwear, bags, gloves, carpets, clothes, furniture, tents, rugs or interiors in cars [3, 4, 5, 6]. Historically, about 65% of leather went into footwear, but this proportion has been decreasing lately toward 55%. The annual world production of leather is estimated to be 1.85 billion square meter. Earlier, in 2001, it was estimated that about 1.67 × 109 m2 of leather was annually being made in the world [7]. India has exported leather articles to around 100 countries of the world including Hong Kong, Italy, China, Vietnam, Korea, Malaysia, Spain, Indonesia, Germany, Portugal, Turkey, etc. India exports leather to its four biggest partners (Hong Kong, Italy, China and Vietnam) and recorded 61.17% of the total leather exports. In terms of value, the exports to these four countries was worth USD 211 million, USD 102 million, USD 96 million and USD 93 million respectively in 2017 [8]. The Indian leather industry today has established itself as a prominent industry both in the international as well as in the domestic market. Apart from being the ninth-largest exporter of leather and leather products, it is also the second-largest producer of footwear and leather garments [8]. India is one of the major players in the global leather trade. India is the secondlargest producer of footwear and leather garments in the world. Leather has assumed importance as an opportunity sector for social development, employment generation and export realization. The Indian leather industry has evolved over nearly two centuries. In 2001 it was reported that, there were about 2,100 tanneries located in different parts of India, with a processing capacity of 0.9 × 106 tons of rawhides and skins [9], making this a thrust area in national planning for the development of India. Major tannery clusters in India are located in the states of Tamil Nadu, West Bengal, Uttar Pradesh and Punjab. India’s share in global market observed in 2000 was 2.1 percent but had declined in 2009 with the rate of 1.9 percent. Although India possesses the world’s largest livestock production, which puts India in the driving seat for leather production for its availability of raw materials, its high end value products is still debatable and limited [10]. This untapped potential provides the sector significant opportunities for expansion and diversification. According to the latest census, India ranks first among the major livestock holding countries in the world. There exists a large raw material base. This is on account of a population of 194 million cattle, 70 million buffaloes, 95 million goats. In respect of sheep, with 48 million sheep, it claims the sixth position. These four species provide the basic raw material for the leather industry. The annual availability of 166 million pieces of hides and skins is the main strength of the industry. Some of the goat/calf/sheep skins available in India are regarded as speciality products commanding a good market. Abundance of traditional skills in training, finishing and manufacturing downstream products and relatively low wage rates are the two other factors of comparative advantage for India. The world scenario shows that the leather industry aims at higher productivity, own design, enhanced cost competitiveness and capacity to cater to all price segments. The domestic market is expected to double in the next five years from the present level

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Table 2.1 Report of working group on leather & leather products twelfth five year plan period (2012–2017) [12]. Product

Capacity

Leather Hides

65 million pieces

Skins

170 million pieces

Footwear & Footwear Components Shoes

100 million pairs

Leather shoe uppers

100 million pairs

Non-leather shoes/chappals etc

1056 million pairs

Leather Garments

16 million pieces

Leather Products

63 million pieces

Industrial Gloves

52 million pairs

Saddlery

12.50 million pieces

and reach USD 18 billion by 2020. Exports are projected to reach USD 9.0 billion by 2020, from the present level of USD 5.85 billion. A conservative estimate shows that additional employment of 3 lakh people in organized manufacturing and 5 lakh in unorganized manufacturing and feeder areas and enhanced wages to nearly 3 lakh existing workers will be the outcome. The leather Industry in India is planned well to grow rapidly in the next five years with a growth target of 50% in exports from 2016 to 20. Domestic consumption of leather-based footwear in India is expected to reach 5 billion pairs in the next 2–3 years, meaning that the per capita consumption of footwear has increased rapidly. This would help employ nearly 3 million people, among which 30 percent are women. Every 1 crore investment in the leather industry can generate more than 250 jobs [11].

2.2

Structure and Significance of Leather Industry

The leather industry is spread in different segments, namely, tanning & finishing, footwear & footwear components, leather garments, leather goods including saddlery & harness, etc. The estimated production capacity in different segments is presented in Table 2.1. The Indian leather industry holds a significant position in the Indian economy in terms of high potential shown for employment, growth and exports. It is estimated that the industry employs a workforce of around 2.5 million directly or indirectly, and 30% of the workforce are women. The major production centers for leather and leather

26

High Value Fermentation Products Volume 2 Cattle

Hides

Heavy leather (bovine)

Goats

Goats skins

Light leather (bovine)

Sheep

Sheep skins

Light leather (sheep & goats)

Leather products ¥ Footwear ¥ Upholstery ¥ Garments ¥ Others

Trade

Marketing Local market

Figure 2.1 Value Chain of Leather Industry [12].

products are located at Chennai, Ambur, Ranipet, Vaniyambadi, Trichi, Dindigul in Tamil Nadu, Calcutta in West Bengal, Kanpur in Uttar Pradesh, Jalandhar in Punjab, Bangalore in Karnataka, Delhi and Hyderabad in Andhra Pradesh. It is among one of the industries in which India has abundance of raw material (India is endowed with 21% of world cattle & buffalo and 11% of the world goat & sheep population), adequately skilled manpower and technology, apart from compliance with all international environmental standards and support from associated industries. Since the last three decades the Indian leather industry has undergone a significant structural change, from being merely an exporter for raw materials during the 1960s to a preferred supplier of value added products, the industry is now one of the top ten foreign exchange earners for the country. The Indian industries comprises of tanneries (where hide and skins are transformed into leather) and manufacturing units (where leather footwear, garments and outerwear, and assorted leather goods are manufactured) [12].

2.3

History of Leather

Primitive people, who lived during the Ice Age, were likely the first to use the skins of animals to protect their bodies from the elements. The Egyptian civilization is the one that has brought the most information on leather tanning, in the form of engraving in papyruses or in wall paintings and objects in Egyptian tombs; sandals, clothes, gloves, buckets, bottles, shrouds for burying the dead and military equipment, due to their belief that the dead should be buried with all of their possessions to enjoy them in the next life [13]. Just as leather today is a byproduct, our ancient ancestors hunted animals primarily for food, but once they had eaten the meat, they would clean the skin by scraping off the flesh and then sling it over their shoulders as a crude form of coat. The main problem that primitive men encountered was that after a relatively short time the skins decayed and rotted away. With their limited knowledge and experience, primitive men had no idea how to preserve these hides. As centuries passed, it was noticed that several things could slow down the decay of leather. If the skins were stretched out and allowed to dry in the sun, it made them stiff and hard but they lasted much longer. Subsequently, oily substances were then rubbed into the skins to soften them. As time passed, it was eventually discovered that the bark of certain trees contained ‘tannin’ or tannic acid which could be used to convert raw skins into what we recognize today as ‘leather’.

Biotechnology of Leather: An Alternative to Conventional Leather

(a)

Americ ans Indians

(b)

Roman legend

(c)

27

Arabian craftsmen

Figure: 2.2 Making of leather goods (adapted from Acron - The Olde Hide House (http://www. leathertown.com).

Somewhat later, techniques used by the American Indian are very similar to those used in this early period. These Indians took the ashes from their campfires, put water on them and soaked the skins in this solution. In a few weeks the hair and bits of flesh came off, leaving only the raw hide. This tanning method, which used a solution of hemlock and oak bark, took about three months to complete after which the leather was worked by hand to make the hide soft and pliable (Figure 2.1).

2.3.1 The Making of Leather Goods The tanning of leather was used by mankind in numerous geographical areas throughout the early periods of human civilization. It was discovered that water would keep fresh and cool in a leather bag. Leather was also found suitable for such other items as tents, beds, rugs, carpet, armor and harnesses. Ancient Egypt, one of the most developed civilizations in the early period, valued leather as an important item of trade. The Egyptians made leather sandals, belts, bags, shields, harness, cushions and chair seats from tanned skins. Many of these items are in fact still made from leather today. Similarly, the Greeks and Romans used leather to make many different styles of sandals, boot and shoes. When the Roman legions marched in conquest across Europe, they were well attired in leather armor and leather capes (Figure 2.2). In fact, right up until the early 18th century, the shield carried by the ordinary soldier was more likely to be made of leather than metal. As we move into the Middle Ages, leather continued to increase in popularity. By far the cleverest craftsmen with leather in medieval times were the Arabs (Figure 2.2). In medieval England, most industries were carried out by master craftsmen aided by apprentices under the supervision of the appropriate Craft Guilds.

28

High Value Fermentation Products Volume 2 Pre- tanning

Curing Soaking Liming Dehairing Deliming Bating Pickling Degreasing

Tanning

Post- tanning

Chrome splitting Shaving Retaining, dyeing, Fatiliquoring drying

Finishing

Leather

Figure 2.3 Inflowand out flow diagram for leather processing [1].

Until the later part of the 19th century, there were relatively few changes in the methods used to produce leather. In fact, the process had changed very little in over 200 years. However, the Industrial Revolution did not bypass tanning – one of the oldest and most basic forms of manufacturing. Science was quickly introduced to the art and craft of leather making. A wider range of dyestuffs, synthetic tanning agents and oils were introduced. Together with precision machinery, these changes and continued innovations to the present day have combined to make tanning into a viable, modern manufacturing industry.

2.4

Conventional Methods for Leather Processing

The rawhide and skin has to undergo a series of chemical treatments before it turns into flattering leather. The term ‘hide’ is used for the skin of large animals (e.g., cows, buffalos or ehorses), while ‘skin’ is used for that of small animals (e.g., sheeps, goats). Hides and skins are mostly by-products of slaughterhouses, although they may also come from animals that have died naturally or been hunted or trapped. The conventional method for leather processing is a multi-step process that includes a series of unit operations, as shown in (Figure 2.3). The operations involved in leather processing may be classified in four groups: (i) pre-tanning or beam-house operation, (ii) tanning, (iii) post-tanning and (iv) finishing. It is important to note that the conventional method of leather processing discharges enormous amount of pollutants [14, 15, 16]. For all the steps, the chemicals used are quite toxic. Major pollution arises from beam house operations. Several other major pollutants, chemical-based unhairing methods, include the use of chlorine dioxide [17], dimethyl sulfates (DMAS) [18], hydrogen peroxide [19], and carbonates [20]. The beam house process involves ‘do-undo operations’, such as curing (dehydration) - soaking (rehydration), liming (swelling) - deliming (deswelling), pickling (acidification) depickling (basification), etc [5]. Thus, due to the pre-tanning operations, the leather processing industry is one of the worst offenders of the environment [21].

Biotechnology of Leather: An Alternative to Conventional Leather

29

2.4.1 Pre-Tanning or Beam House Operations 2.4.1.1

Curing

Animal skins and hides are usually salted to preserve. Skin is stretched out on board from the flesh side for drying. A good amount of salt is poured on the skin usually ‘two kilogram of salt per kilogram of the skin’. Every part of the skin must be covered with salt. The skin is folded to flesh side to flesh side and rolled. In a day or so the skin is unrolled, the old salt is shaken out and this process is repeated. After drying, skins and hides are piled on one to the other and kept in a cool dry place at 1–4 °C for 2–6 months to prevent putrefaction [22].

2.4.1.2 Soaking Soaking aims at reversal of cured hides and skins to near the status of the freshly flayed condition, in terms of dimension and composition. For soaking, preservatives/wetting agents are used with large amounts of water. The common salt, when removed from the skin during soaking, constitutes a major source of pollution also called soak liquor from tanneries [23]. This soak liquor is characterized by high Total Dissolved Solids (TDS) and chloride contents [24]. An obnoxious smell and ammoniacal odor emanating from degraded protein are also associated with spent soak liquor. In addition to the wastewater it also leads to significant increase of salinity to the soil [25].

2.4.1.3

Liming

In this operation, lime and sodium sulfide are used along with substantial qualities of water [26]. Lime causes the ionic imbalance in the matrix due to the increase in the skin pH, which further causes osmotic swelling [27]. Swelling causes change in dimension and charge characteristics of the skin and hide. Liming operations lead to not only wastewater containing significant amounts of Biological Oxygen Demand (BOD), TDS and S2- but also substantial quantities of solid wastes containing lime sludge, fleshing and hair. Sodium sulfide, a good reducing agent, interferes in the oxidation of organic wastes and contributes significantly to the BOD and Chemical Oxygen Demand (COD) concentrations in wastewater [28].

2.4.1.4 Dehairing Dehairing is one of the main operations in the beam house. Dehairing used to be followed by opening up of fiber structure in ‘liming’. Four methods of dehairing are generally adopted, viz. (i) clipping process, (ii) scalding process, (iii) chemical process, and (iv) sweating process. Of these, the most commonly practiced method of dehairing of hides and skins are the chemical process using lime and sodium sulphide at soaking stage. However, the use of high concentrations of lime and sodium sulphide creates an extremely alkaline environment resulting in the pulping of hair and its subsequent removal.

30

High Value Fermentation Products Volume 2

It contributes in no small measure to the pollution load [29]. Out of all pre-tanning processes, dehairing contributes maximally to the pollution load. About 75% of the organic waste from a tannery is from the pre-tanning and 70% of this waste is from hair after dehairing, which is rich in nitrogen. If left untreated, it can cause major problems in the sewers [22, 25].

2.4.1.5 Deliming After mechanical removal of the subcutaneous tissue, deliming is performed in order to remove the adsorbed lime from the hide and to eliminate the lime swell. Deliming agents based on weakly acidic salts such as ammonium chloride and ammonium sulfate are used to neutralize lime. It is now realized that the use of nitrogen-bearing salts could affect the N:P:K ratio of soil [30]. The resultant salts of this operation would in turn increase the amounts of COD and TDS [31].

2.4.1.6

Bating

Bating is a complete removal of unwanted interfibrillary materials and short hairs by using enzymatic applications [24]. The liquid wastes from this operation contain usually small quantities of proteineous matter and other debris, which contribute to BOD and COD significantly.

2.4.1.7 Pickling Pickling is a process in which the partially anionic matrix is temporarily converted into a cationic matrix in order to prepare the stock for the subsequent chrome tanning operation [27]. Lowering of the pH value to the acidic region. This must be done in the presence of salts. Pickling is normally done to help with the penetration of certain tanning agents, e.g., chromium (and other metals), aldehydic and some polymeric tanning agents. Pickling acidifies the skin and kills bacterial growth. Pickling can also be used to hold the skin safely for storage. Pickling is a temporary preservative [32].

2.4.1.8

Degreasing

Excess natural grease in raw animal skins, leading to various defects in the finished leather, are traditionally removed by solvent extraction or by use of surfactants. Biocatalysts offer innovative, environment-friendly methods for the fat removal, potentially leading to improved quality of finished leather. The traditional fats removal methods was based on solvent extraction. In the last decades, solvent extraction methods were progressively substituted by emulsification in the presence of surface-active agents in aqueous medium. Nevertheless, the pollution problems arising out of the discharge of degreasing effluents remain critical [33].

Biotechnology of Leather: An Alternative to Conventional Leather

2.4.2

31

Tanning

Tanning is a process in which the leather-making protein is permanently stabilized against heat, enzymatic biodegradation, and thermo-chemical stress [24]. The tanning process consists in strengthening the hide’s protein structure by creating a bond between the peptide chains. The hide is composed of three layers: epidermis, dermis and subcutaneous layer. The dermis consists of about 30% to 35% protein, which is mostly collagen, with the remainder being water and fat. The dermis is used to make leather after the other layers have been removed using chemical and mechanical means. The acidity of hides once they have finished pickling will typically be between pH of 2.8–3.2. At this point the hides are loaded in a drum and immersed in a float containing the tanning liquor. The hides are allowed to soak (while the drum slowly rotates about its axle) and the tanning liquor slowly penetrates through the full substance of the hide. Regular checks will be made to see the penetration by cutting the cross section of a hide and observing the degree of penetration. Once an even degree of penetration is observed, the pH of the float is slowly raised in a process called basification. This basification process fixes the tanning material to the leather, and the more tanning material fixed, the higher the hydrothermal stability and increased shrinkage temperature resistance of the leather. The pH of the leather when chrome tanned would typically finish somewhere between 3.8–4.2 [32]. In commercial practice, vegetable and chrome tanning methods are widely used. Vegetable tanning is known to be hard to biodegrade [34], and hence wastes bearing vegetables tannins degrade slowly. Amongst the various tanning system, chrome tanning is the most commonly used tanning system in commercial practice. In chrome tanning, the cationic matrix (pickled kin) is treated with is a mixture of many molecular species such as octaaqua-μ-dihydroxochromium(III), octaaqua-μ-dioxochromium (III), hexaaqua-μ-dihydroxo-μ-sulfactochromium(III), and tetraaquahydroxosulfactochromium -(III) [35, 36]. When the uptake level of this mixture are low, higher amounts of chromium along with neutral salts are discharged, which increase the COD, TDS, and SO2-4 content in the spent chrome liquor [37]. The spent chrome tanning solutions are sources of both TDS, COD, SO2-4 and chromium pollution.

2.4.3 Post-Tanning Post-tanning operations, in general, attempt the addition of aesthetic values and improvement of intrinsic properties of leather [31]. Post-tanning processes contribute to neutral salts, COD, TDS, and heavy metal pollution like chromium [38]. Additionally, azodyes and biocides adds to the toxic load of waste water streams [39].

2.4.4

Finishing

The pollution loads generated in finishing operations are not significant. This waste stream is characterized by the presence of polymeric binders, heavy-metal based pigments, solvents, nitrocellulose and other topcoat materials, as described by [14]. In addition to that, use of formaldehyde, un-reacted acrylic monomers, toxic-metalbased pigment formulation, and solvent-based top coats causes gaseous wastes. The

32

High Value Fermentation Products Volume 2

Salted Salted hides hides / skins / skins

Water Water Lime Lime Sulfide Sulfide Enzymes Enzymes Saltand andacid acid Salt

Salt Salt Protein Protein Lime Lime BEAM BEAM HOUSE HOUSE OPERATIONS OPERATIONS

Dusted Dusted salt salt Trimming Trimming Fleshing Fleshing Hair Hair

Sulfide Sulfide

Limed Limedhides hides/ /skins skins

Tanned Tanned leather leather

CrustCrust leather leather

Water Water Chrome Chrome Veg .. tannina tannina

Water Water Chrome Chrome Syntans Syntans Dyes Dyes Fatliquors liquors Fat

Binders Binders Pigments Pigments Organic solvents Organic solvents Formaldehyde Formaldehyde Lacquers Lacquers

TANNING TANNING OPERATIONS OPERATIONS Salt Salt Chromium Chromium Tannins Tannins

POST-TANNING POST -TANNING OPERATIONS OPERATIONS

Dyes,grease Dyes, Grease Syntans Syntans Chrome Chrome

Tanned Tanned wastes wastes Buffering Buffering dustdust

FINISHING FINISHING

FINISHED LEATHER LEATHER FINISHED

Splits Splits Shaving Shaving Veg Veg.. bark bark

Org. Solvents Org. Solvents Buffering Buffering dust dust Formaldehyde Formaldehyde

Figure 2.4 Inflow and out flowdiagram for leather processing [1].

various chemical inputs and outputs into leather processing are given in Figure 2.4 and Table 2.2. Now we can easily say that leather and the environment can be described as two sides of the coin. On the one hand, the industry has gained a negative image in the society with respect to pollution. On the other hand, the leather industry had made traceable and visible impacts in the socioeconomic area through both employment generation and export earning. Thus, leather processing activity is therefore facing a serious challenge and there is public outcry against the industry.

2.5

Biotechnology in Leather Industry

Conventional leather processing involves several unit operations that cause negative impact on the environment, as already discussed above. Despite making significant contributions to the economy, the leather industry causes environmental concerns owing to the use and discharge of various chemicals (Cleaner processing: a sulphide—free approach for depilation of skins [29]. The use of chemicals in different stages of leather processing specially in beam-house operation brings about almost 80–90% of the pollution [4, 40, 41], and generates noxious gases as well as solid wastes, e.g., hydrogen sulfide and lime [22]. All these made leather processing a resource intensive process producing large amounts of liquid, solid and hazardous wastes [42]. This results in a net increase in TDS comprising sludge from tannery, chrome sludge and other minerals in tannery wastewater [43, 44]. Liming operation for the removal of hair and unwanted adhering subcutaneous layer causes nearly 40% of BOD and 50% of COD [45, 46]. The

Biotechnology of Leather: An Alternative to Conventional Leather

33

Table 2.2 (Manual for oxazolidine leather tanning LIFE08 ENV/E/000140) [13]. RAW HIDES OR SKIN BEAM HOUSE

Sorting – Trimming Soaking – Liming Fleshing – Pelt splitting

TANNING

Deliming – Bating Degreasing Pickling Tanning Sammying

POST TANNING

Splitting Shaving Retanning Neutralisation Dyeing Fatliquoring Sammying Drying Staking

FINISHING

Finishing Mechanical operations Sorting – Packing – Dispatching FINISHED LEATHER

BOD is mainly contributed by hair as pulp and subcutaneous layer and COD is due to large amount of chemicals used for dehairing [47]. The effluent of leather tanneries was associated to a huge foaming problem on surface waters too [48]. The high concentrations of pollutants with low biodegradability in tannery wastewater represent a serious and actual technological and environmental challenge [49]. Major pollutants from pre-tanning may have significant environmental impact include lime, sulphide and chromium [50]. It has been reported that the conventional dehairing process with sodium sulfide and lime is responsible for 84% of biochemical oxygen demand (BOD), 75% of the COD and 92% of the suspended solids from a tannery

34

High Value Fermentation Products Volume 2

[51]. In fact, one-third of the pollution caused by the leather industries results from the wastes generated during dehairing operations. The wastes from the tanneries are let out into the drains which in turn empty into the main sewerage causing hazard to those who use this water. Many tanneries have been forced to close down because of their noncompliance with the standards laid down. In a short span of time, the Indian leather industry has faced serious challenges such as Germany’s ban on pentachlorophenate, certain azo dyes, formaldehyde, etc., on one hand, and court order for compliance with environmental regulations on the other. Besides that environmental pollution caused by the effluents generated during the processing/manufacture of leather is one of the serious problems our country is facing today. Therefore, despite traceable and visible impact of the leather industry on the socio-economic through employment and export earning, the industry has gained a negative image in society owing to the resulting pollution [22]. Today, growing global concern about environmental health is forcing all the processing industries to adopt greener and cleaner manufacturing practices. In recent years, questions have already been raised about the negative consequence of pollution from wastewaters, careless disposal of solid wastes and gaseous emissions [52]. In India, the attention of tanners is moved towards revamping the processing methods, recovery systems, and effluent treatment techniques to make leather processing ecofriendly. Intensive efforts are being directed towards using a viable alternative technology for beam-house processes using enzymes [40]. This could be one of the ways of solving the industrial pollution problems resulting from tannery effluents. Hence, the leather industries are seeking alternative strategies to replace the conventional chemical processes with enzyme-based processes [46, 53]. As an alternative to chemical unhairing, enzyme-based unhairing processes using alkaline proteases are being developed to gain environmental benefits. Proteolytic enzymes are of great commercial importance, contributing to more than 40% of the world’s commercially produced enzymes. Microbial proteases are more environmentally friendly than chemical processes and have tremendous potential application in the leather industry and other industries [54, 55] (Figure 2.5).

2.6

A Good Alternative to Conventional Chemicals in Leather Processing

An environmentally benign approach demands that the discharge from any process should be minimized and valuable materials recovered. The ideal strategy should be aimed at zero or near-zero discharge of waste liquours [56]. An approach has been made to reduce the pollution by replacing the toxic chemicals and also by altering the individual steps. This will not only help in the reduction of pollutants, but also reduce the water consumption substantially. Enzyme-mediated processes are rapidly gaining interest because of reduced process time, intake of low energy input, cost effective, nontoxic and eco-friendly characteristics [53, 54]. In this direction, enzymes seem to be quite promising as they can be

Biotechnology of Leather: An Alternative to Conventional Leather

(a) Chemical treatment

35

(b) Enzyme treatment

Figure 2.5 Comparative analysisin appearance of dehaired skin after chemical and enzymatic treatment.

used in the beam house operations in leather manufacturing. Development of enzymatic processes for dehairing is being encouraged because they not only yield quality improved products but also reduce or totally remove the use of hazardous and polluting chemicals [1, 4, 22, 42–45, 57]. The important factor in choosing an enzyme as a dehairing agent depends on the specificity of the enzyme used, which should not attack the collagenous matter. A wide range of enzymes are used in leather processing, viz., neutral proteases in soaking, alkaline proteases in dehairing and acid proteases in bating [58]. Their specificity offers much finer product control, while their efficiency, requiring low energy inputs and mild conditions, has distinct environmental advantages. They can be used to treat biological waste from industries, and are themselves biodegradable, being readily absorbed back into nature. The benefits from enzymatic process would be reduction in the use of chemicals, water and power. Elimination of several processes, viz, liming, reliming, deliming, pickling and basifying is possible with subsequent reduction in process time, TDS, COD, sludge volumes (total elimination of lime sludge) and also better exhaustion of chromium in tanning. They are exemplary agents of green technology [52]. However, at present, biological methods are being used with relative success in soaking (cleaning and rehydration), dehairing (removal of hair), liming and bating.

2.7

Enzymes for Leather Processing

An important enzyme used in pre-tanning processes belongs to the group of proteolytic enzymes, proteases. Microbial proteases are more environmentally friendly than chemical processes and have tremendous potential application in the leather industry and other industries [54, 55]. Obtained by microbial fermentation, the proteases are meant for use in the leather industry for soaking, dehairing, liming, bating and degreasing processes [56]. Animal proteases and microbial proteases from bacteria and

36

High Value Fermentation Products Volume 2 HIDE PRESERVATION

SOAKING

Proteases, Lipases

DEHAIRING

Proteases,Keratinases

BATING

Proteases

TANNING

Figure 2.6 An overview of the leather production process, emphasizing steps that are at fully or least partially dependent upon enzymatic activity.

fungi are used in the pre-tanning processes of leather manufacture. The animal proteases are mixtures of trypsin, chymotrypsin and various peptidases which may contain amylase or lipase as secondary enzymes [21]. Mainly for economic reasons, enzymes from microorganisms have come to play a significant role in recent years and enzyme products of microbial origin are already being produced on a wide scale. Apart from bacterial and fungal proteases, specific proteases like keratinases are known. Keratinase can be used for dehairing [59]. Some of the lipase-producing microorganisms are also used in degreasing. Andrioli [60] reported on the enzymatic–oxidative unhairing process using protease produced by SMF with a concentration of 100–300 U/g of hide in combination with hydrogen peroxide. Therefore, we aim to produce highly potential alkaline protease by SSF from a novel bacterium for chemical free enzymatic unhairing process to reduce the pollution load which causes deleterious effect on the environment. Below is the flow chart of the steps involved in the beam house operations of tannery and the possible usage of enzyme in the steps involved (Figure 2.6).

2.7.1

Enzymes for Soaking

The soaking stage is the most polluting stage of the process since it contributes to 50–55% of the total pollution [61] due to chemical products used for the treatment of hide, such as lime, sodium carbonate, sodium hydroxide, etc. Soaking is the cleaning operation of the hides and skins with water. Proteases with an optimum pH of around 9–10 are now widely used to clean the stock and facilitate the water uptake of the hide or skin. These enzymes break down soluble proteins inside the matrix, thus improving the removal of salt and hyaluronic acid. This makes room for the water. Enzymes such as Aspergilus parasiticus, A. flavus, A. oryzae, and Bacillus subtilis have been used alone or in mixtures [62]. demonstrated that the use of enzyme preparation in soaking of rabbit skins improves the softness and elasticity and increases the area yield of the fur

Biotechnology of Leather: An Alternative to Conventional Leather

37

by 3.3% while reducing the processing time by 10–20 hr. Alkaline proteases of bacterial and fungal origin have been used for soaking which reduces the need for the liming chemicals [21]. Besides proteases, microbial amylases have also been reported for soaking dried wool lamb skins [63].

2.7.2 Enzymes for Dehairing Dehairing is one of the main operations in the beam house. Enzymatic dehairing is suggested as an environmental friendly alternative to the conventional chemical process. The enzyme digests the basal cells of the hair bulb and the cells of the malphigian layer. This is followed by loosening of hair with an attack on the outermost sheath and subsequent swelling and breakdown of the inner root sheath and parts of the hair that are not keratinized. Proteolytic enzymes are of great commercial importance in dehairing process. Microbial proteases are derived from a wide variety of yeast, molds and bacteria. Yeast proteases are mainly intracellular in nature and therefore these enzymes have not gained significant commercial interest. The protease from A. flavus was earlier being used for dehairing, and later it was reported that simultaneous dehairing and bating is possible with the protease of A. flavus [64]. Earlier studies using commercial enzymes for sheep skin dehairing revealed a high correlation between dehairing activity and proteolytic activity [65]. Some microorganisms producing extracellular enzyme with dehairing activity have been described, e.g., Streptomyces sp [66]. Proteolytic enzymes used for dehairing of hides and skins are majorly derived from a large number of Bacillus sp. and Streptomyces sp [66, 67]. Strains of B. subtilis and B. amylolequifaciens have also been characterized for dehairing purposes [67]. Alkaline protease produced by different bacterial strains can also used to dehair animal-skin [66–69]. Varela [67] reported the use of B. subtilis IIQDB32 alkaline protease for dehairing sheepskin [64]. used B. amyloliquefaciens alkaline protease for dehairing of hides and skins. Hameed [66] used B. subtilis K2 protease in bating and leather processing. Several different proteases can be used in the individual stages of the leather manufacturing process. Table 2.3 gives a brief account of protease producing microorganism used for dehairing. Four methods of application are commonly used in the enzymatic dehairing process: (i) paste method, (ii) paint method, (iii) dip method, and (iv) spray method. In the paste method, the enzyme solution is applied on the flesh side of hides and skins, piled flesh to flesh, covered with polythene sheets and kept till dehairing takes place. In the paint method, the enzyme solution is mixed with an inert material like kaolin, made into a thin paste, adjusted to the required pH, applied on the flesh side of hides and skins, covered with polythene sheets and kept till dehairing takes place. In the dip method of enzymatic unhairing, the hides or skins are kept immersed in the enzyme solution at the required pH in a pit or tub. The disadvantage encountered in this method is the unavoidable dilution of the enzyme solution. Even though enzyme penetration is observed to be uniform, dehairing at backbone and neck is not up to the mark. A novel spraying technique has been adopted for the application of multienzyme concentrate in depilation. The advantages of this method over the painting and dip methods are that (i) even concentrated solutions can be sprayed, (ii) when the enzyme

11

8.0

9–11

4.0 -

Bacillus subtilis

A. terreus

A. flavus

Aspergillus flavus

B. cereus

8.0.

9.0.

Brevibacterium luteolum (MTCC 5982)

-

Bacillus cereus VITSN04

B. cereus

13.0

7

Bacillus cereus MCM B-326

Bacillus subtilis 168

9.6

9–11

Aspergillus tamarii

Bacillus pumilus

9.6

Bacillus pumilus

8

8.0

Bacillus subtilis

Pseudomonas aeruginosa

pH

Source

30 to 39 °C,

30 °C

37

35

40

30 °C

45–60 °C

45–60 °C

45 °C

28

37

50

30–37

37

30

Temp °C

96 hr

16 hr

72 hr

-

48 hr

7 day

72 hr

72 hr

7 hr

21

24

2.3–3

18

4

4

Incubation Period (h)

Cow

goat

Cow dung

-

Red gram husk

-

goat

goat

goat

Buffalo

Pig

Cow

Goat

Skin

Sheep

Skin/hide type

Table 2.3 Sources and properties of some of the alkaline proteases used in dehairing.

1193.77 U/g

200.1 ± 0.68 U/ml

4813.0

170.32 ± 1.5 IU/ml

258.0

(49.3 U mL-1

(2.048 U/mL)/ (6699 U)

(1.006 U/mL)/ (1023 U).

548 U/ml

1% enz (126 U/ml)

400

2 U/ml

1% enz w/w

300 μg

900

Enzyme conc. (U)

[79]

[78]

[77]

[76]

[75]

[74]

[73]

[73]

[72]

[40, 57]

[71]

[70]

[46]

[41]

[67]

References

38 High Value Fermentation Products Volume 2

Biotechnology of Leather: An Alternative to Conventional Leather

(a) Goat skin

(b) Sheep skin

39

(C) Buffalo skin

Figure 2.7 Dehaired skins and hides using proteolytic enzymes.

solution is sprayed on the flesh side with force, entry becomes easier, (iii) backbone and neck can be sprayed with more amount of enzyme, thereby making the process quicker, (iv) there is no effluent arising out of this method, and (v) after depilation hair will be almost free from all the adhering skin tissues. Of late, dehairing by drumming is being practiced, and industrially this should be feasible [80]. This enzymatic process is safer and more suitable than traditional methods using sodium sulphide treatment [81, 82]. As the alkaline conditions enable the swelling of hair roots and subsequent attack of proteases on the hair follicle protein aid in the easy removal of hair [83]. The results obtained after dehairing studies on goat skin, sheep skin and buffalo hides are presented in Figure 2.7. The skin was much smoother in appearance and the texture showed that the quality of skin was much better as compared to chemical processed skin. Further, when the washouts of the dehairing experiments were analyzed, it was found that COD and BOD of the washout of the enzyme mediated dehairing was much less as compared to chemical or enzyme assisted dehairing. This all forms a part of making the tannery go green. In general, the overall performance of the enzymatically processed leather is either comparable or even superior to the conventionally processed leather. Kandasamy [84] also reported enzyme-dehaired leathers exhibit similar or improved characteristics. Advantages of enzymatic dehairing are: 1. Significant reduction or even complete elimination of the use of sodium sulphide. 2. Recovery of hair of good quality and strength with a good saleable value. 3. Creation of an ecologically conducive atmosphere for the workers.

40

High Value Fermentation Products Volume 2 4. Enzymatically dehaired leathers have shown better strength properties and greater surface area. 5. Simplification of pre-tanning processes by cutting down one step, i.e., bating. 6. A significant nature of the enzymatic dehairing process is the time factor involved. The lime-sulphide process takes about 16 hr, whereas the enzymatic dehairing could be completed between 12 and 20 hr.

2.7.3 Enzymes for Liming In the liming stage, hair, skin and an emulsion fats are removed from the hides and are released in the effluent thus increasing the total pollutant load [85]. Alkaline proteases and lipases are used in this process as liming agents in order to speed up the reactions of the chemicals normally used. For example, the enzymes combine to break down fat and proteinaceous matter such as dermatan sulphate. This facilitates the opening up of the structure and the removal of hair. The result is a clean and relaxed pelt that is ready for the next step [21].

2.7.4 Enzymes for Bating Bating process involves removing the non-leather forming proteinous materials like albumin, globulin, and mucoids from hides and/or skins. This gives leather desired characteristic properties including softness and pliability [86]. To make leather pliable, the hides and skins require an enzymatic treatment before tanning known as bating. During bating, scud is loosened and other unwanted proteins are removed. Bating deswells swollen pelts and prepares leather for tanning. It makes the grain surface of the finished leather clean, smooth, pliable and fine [86]. Bating with enzymes is an indispensable operation of leather processing to obtain best quality of leather and cannot be substituted with a chemical process. Bio-technical developments in science have now completely replaced these methods with use of industrial enzymes. Deliming and bating, the subsequent steps in the processing of the pelts after liming, are basically two separate operations although they are usually carried out in one step and often overlap each other. The principle materials which a bate contains are a proteolytic enzyme, a carrier for the enzyme like wood flour, and a suitable deliming agent like ammonium chloride or sulphate or both. Trypsin (EC 3.4.21.4), a serine protease, has been utilized in many fields, for instance the leather bating [87, 88, 89]. The deliming agents are used for the removal of lime salts which are used during the dehairing process [21]. Bating by enzymatic process was practiced for more than a thousand years, at first by pancreatic enzymes, later by plant enzymes, and finally by enzymes produced by microorganisms. Innumerable variations of the enzymatic bating process have been proposed. The proteolytic enzymes in the pancreas are present in inactive forms; chynotrypsin as chymotrypsinogen, trypsin as trypsinogen and carboxypeptidases as procarboxypeptidases. Their processing for the recovery of technically useful products is not inconsiderably limited by the value of these glands for the production of insulin. Also, many countries, for example Australia, have enacted strong supervisory

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regulations to protect against virus infections which can be transmitted by pancreatic products. In order to overcome the drawbacks related to pancreatic enzymes, microbial enzyme are now be used in bating. Underkofler and Hickey [90] have described a process for the manufacture of enzyme bate from mold source. A procedure has been developed for bating pig skins, using an enzyme preparation from Bacillus subtilis, and bated skins exhibit good physiological properties [62]. Despite knowing all the shortcomings of pancreatic enzymes, still some content of pancreatic trypsin could heretofore not be omitted from any first-rate enzymatic bating agent. Bacterial preparation from S. rimosus and B. licheniforms have been tested for their bating action and it is found that solubilization of collagen has been less pronounced under the influence of microbial proteases than under the influence of pancreatic protease [91]. A combination of both mold and pancreatic enzymes in suitable proportions will be an ideal bate for different types of leather.

2.7.5 Enzymes for Degreasing Enzymatic degreasing is suggested as a viable alternative to combat the pollution problems caused by the use of solvents and detergents. The advantages of using enzymes for degreasing are the elimination of solvents, reduction in surfactants, and possible recovery of valuable by-products. Lipases which are projected as an excellent alternative for solvents and detergents, catalyze the breakdown of fats and can be obtained from animal, microbial and plant sources [92–94]. Lipases are a type of enzyme that specifically degrades fat and so cannot damage the leather itself. Lipases hydrolyse not just the fat on the outside of the hides and skins, but also the fat inside the skin structure. Once most of the natural fat has been removed, subsequent chemical treatments such as tanning, re-tanning and dyeing have a better effect. The main advantages of using lipases are a more uniform colour and a cleaner appearance. For sheepskins, which contain up to 20–30% fat, the use of solvents is very common and these can also be replaced with lipases and surfactants [95]. Solvents tend to dry out the skin and give it a pale colour. If surfactants are used for sheep skins, they are usually not as effective and may be harmful to the environment. Stronger surfactants such as nonyl phenol ethoxylate have a better effect but they are more detrimental to the environment. When using lipases, the original surfactant dosage can be reduced by at least 50% in the case of both sheep and pigskins. Zang and Zang [96] reported use of alkaline lipase in combination with the proteinase and pancreatin in softening pig skin to improve the degreasing effect. Pirozzi and Greco [33] reported highest hexocellular lipase activity as well as god protease activity from Candida rugosa.

2.8

Importers and Exporters of Top Leather

In 2016, world leather exports and imports totaled around US$2 billion. The largest exporter and importer of leather goods were Italy and China respectively.

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In a global context, leather is one of the most widely traded commodities in the world. The leather industry plays a prominent role in the world’s economy with an estimated global trade value of approximately US$100 billion per year on average. In the last half-decade, Ethiopia, Belgium, Cambodia, Albania and Philippines were the top 5 fastest-growing leather importing countries while Kenya, Zambia, Albania, Serbia and Turkey were the top 5 fastest-growing countries with regard to leather exports. Given the current dynamics of the leather industry, both leather imports and exports declined globally in 2016; imported leather declined by approximately 29% while exported leather dropped by 44% from 2012.

2.9

Outlook

Today, science touches all edges in various spheres of life and has successfully developed techniques and processes to fulfill the demands of consumers in terms of ‘natural’ or ‘green’ products. The driving forces for developing environmentally benign technology or green technology are the current need to decrease pollution caused by conventional leather processing and to replace the use of hazardous chemicals with environmentally friendly technology. In this direction, the use of enzymes are found to be very promising in leather processing as enzymes can reduce the use of chemicals significantly. They are also responsible for major reductions in the amount of water used since the replacement of chemicals by enzymes lightens the rinsing and cleaning processes. Ultimately, using state-of-the-art enzyme solutions can make the leather-making process quicker, cheaper and more efficient, while at the same time easing the load on the environment. Beside this, the use of enzymes has several other advantages: (i) Cost-efficient leather production, (ii) Reduced processing costs through speeding up of the soaking process, (iii) Reduced use of traditional chemicals, (iv) Increased yield by expanding the leather area, (v) Improved physical qualities of finished leather that are not achievable with traditional chemicals, (vi) Tailor-made solutions to specific technical problems that are not possible using conventional chemistry, and (vii) Natural and environment-friendly technology based on biodegradable products. In the above context, the enzymes used in the leather industry have also proved their potentiality in the “beam house operations” of the leather industry as this can do chemical free, enzymatic dehairing of animal skins and hides. This protease can be therefore be useful for the leather industry and will certainly help in lowering the environmental threats to near zero level. In this regard, the future may witness eco-labeled leather/leather products emerging as niche products, and the experience gained by the Indian leather industry in this area might greatly help India to emerge as a global leader in the leather industry. Thus, it is envisaged that success in this direction will certainly result in an environmentally benign technology and subsequently an ‘industrial revolution’.

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2. Yamamoto, G.T., Şekeroğlu, Ö., Bayramoğlu, E.E., Marketing activities in the leather industry: comparative country analysis. International Journal of Economics and Management Sciences, 1(3), 37, 2011. 3. Covington, A.D., Tanning chemistry: the science of leather. Royal Society of Chemistry, 2009. 4. Rao, R.J., Thanikaivelan, P., Nair, B., An eco-friendly option for less-chrome and dye-free leather processing: in situ generation of natural colours in leathers tanned with Cr–Fe complex. Clean Techn. Environ. Policy, 4(2), 115–121, 2002. 5. Black, M., Canova, M., Rydin, S., Scalet, B.M., Roudier, S., Sancho, L.D., Best available techniques (BAT) reference document for the tanning of hides and skins. European Commission Database, 46, 2013. 6. Laurenti, R., Redwood, M., Puig, R., Frostell, B., Measuring the environmental footprint of leather processing technologies. Journal of Industrial Ecology, 21(5), 1180–1187, 2017. 7. FAO, Rome: Food and agriculture organization of the united nations, 1982, 2001. 8. Export Genius: Leather export from India, 2017. Available from: http://www.exportgenius. in/blog/leather-export-from-india-in-2017-list-of-leather-suppliers-in-india-211.php. 9. Velappan, K.C., Muralidharan, C., Chennai Proceedings of the 35th LERIG. P, 15, 2001. 10. UNIDO, project number: TE/ETH/08/008, 2011. 11. Department of industrial policy and promotions. 2016. 12. Report of working group on leather & leather products twelfth five year plan period 2012‐17 13. Manual for oxazolidine leather tanning LIFE08 ENV/E/000140. 14. Greif, M., Journal of American Leather Chemical Association,, 85, 36, 1990. 15. Ludvik, J., Study on the scope for decrease of pollution load in leather processing, UNIDO Report, 1997. 16. Rao, J.R., Environmental Science and Technology, 41(23), 8084, 2005. 17. Scroggie, J.G., A comparative assessment of leather produced by'chlorine dioxide'and lime-sulfide unhairing. The Journal of the American Leather Chemists Association, 64, 628, 1969. 18. Frendrup, W., UNIDO manual, 20, 2000. 19. Morera, J.M., Bartolí, E., Gavilanes, R.M., Hide unhairing: achieving lower pollution loads, decreased wastewater toxicity and solid waste reduction. J. Clean. Prod., 112, 3040–3047, 2016. 20. Kanagaraj, J., Panda, R.C., Senthilvelan, T., Process safety and environmental protection, 100, 36, 2016. 21. Kamini, N.R., Hemachander, C., Geraldine, S.M.J., Puvanakrishnan, R., Curr. Sci., 7, 80, 1999. 22. Thanikaivelan, P., Rao, J.R., Nair, B.U., Ramasami, T., Progress and recent trends in biotechnological methods for leather processing. Trends Biotechnol., 22(4), 181–188, 2004. 23. Ludvik, J., Orlita, A., Kozarstvi, 36, 83, 1986. 24. Ramasami, T., Sreeram, K.J., Gayatri, UNIDO, R., Chennai, 20, 1999b. 25. Daniels, R., World Leather, 10, 41, 1997. 26. Money, C.A., Journal of the Society of Leather Technologists and Chemists, 80, 175, 1996. 27. Bienkiewicz, K., Malabar FL: Robert E. Krieger Publishing Company, 149, 1983. 28. Steven, W., The treatment of beamhouse and fellmongering waste waters. Journal of the Society of Leather Technologists and Chemists, 67, 127, 1983. 29. Ranjithkumar, A., Durga, J., Ramesh, R., Rose, C., Muralidharan, C., Cleaner processing: a sulphide-free approach for depilation of skins.. Environ. Sci. Pollut. Res. Int., 24(1), 180–188, 2017. 30. Dix, J.P., Part 1: Beam house and chrome tanning operations. World Leather, 42–45, 2000.

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31. Ramasami, T., Rao, J.R., Chandrababu, N.K., Parthasarathi, K., Rao, P.G., Saravanan, P., et al., Journal of the Society of Leather Technologists and Chemists, 83, 39, 1999a. 32. Örnberg, J., Oy, K.N., Managing Director, Interview, Kokkola, Finland, 2017. 33. Pirozzi, D., Greco, G., Activity and stability of lipases in the synthesis of butyl lactate. Enzyme Microb. Technol., 34(2), 94–100, 2004. 34. Healy, J.B., Young, L.Y., Catechol and phenol degradation by a methanogenic population of bacteria. Appl. Environ. Microbiol., 35(1), 216, 1978. 35. Rao, J.R., Nair, B.U., Ramasami, T., Isolation and characterisation of a low affinity chromium (III) complex in chrome tanning solutions. Journal of the Society of Leather Technologists and Chemists, 81, 234, 1997. 36. Darbra.Guill´en, D.A., Ginebreda, M., Petrovic, D., Barcel´o, R., Rydin, S., Additives in the Leather Industry, 2012. 37. Chandrasekaran, B., Rao, J.R., Prasad, B.G.S., Nair, B.U., Journal of Industrial Association Environmental Management, 16, 168, 1989. 38. Simoncini, A., Sammarco, U., Germany Proceedings of the XXIII IULTCS Congress, 1995. 39. BASF, Information on legislation and consumer protection. World Leather, 65, 1998. 40. Zambare, V.P., Nilegaonkar, S.S., Kanekar, P.P., Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent. World J. Microbiol. Biotechnol., 23(11), 1569–1574, 2007. 41. Huang, Q., Peng, Y., Li, X., Wang, H., Zhang, Y., Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol., 46(3), 169–173, 2003. 42. Saravanabhavan, S., Aravindhan, R., Thanikaivelan, P., Rao, J.R., Nair, B.U., Ramasami, T., A source reduction approach: Integrated bio-based tanning methods and the role of enzymes in dehairing and fibre opening. Clean Techn. Environ. Policy, 7(1), 3–14, 2004. 43. Saravanbhavan, S., Aravindham, R., Thanikaivelan, P., Chandrasekaran, B., Rao, J.R., Nair, B.U., Journal of the Society of Leather Technologists and Chemists,, 87, 2003a. 44. Saravanabhavan, S., Aravindhan, R., Thanikaivelan, P., Raghava Rao, J., Nair, B.U., Green solution for tannery pollution: effect of enzyme based lime-free unhairing and fibre opening in combination with pickle-free chrome tanning. Green Chem., 5(6), 707, 2003b. 45. Taylor, M.M., Bailey, D.G., Feairheller, S.H., Journal of the Society of Leather Technologists and Chemist,, 82, 153, 1987. 46. Dayanandan, A., Kanagaraj, J., Sounderraj, L., Govindaraju, R., Rajkumar, G.S., Application of an alkaline protease in leather processing: an ecofriendly approach. J. Clean. Prod., 11(5), 533–536, 2003. 47. Balasubramanian, S., Pugalenthi, V., Water Resources, 34, 4201, 2000. 48. Schilling, K., Bletterie, U., Kroiss, H., Zessner, M., Environ. Sci. Policy, 1(23), 68, 2012. 49. Schrank, S.G., José, H.J., Moreira, R.F.P.M., Simultaneous photocatalytic Cr(VI) reduction and dye oxidation in a TiO2 slurry reactor. Journal of Photochemistry and Photobiology A Chemistry, 147(1), 71–76, 2002. 50. Alessandro, R., Silvia, O., Adriano, B., Journal of Chemical Technology and Biotechnology, 78, 855, 2003. 51. Marsal, A., Cot, J., Boza, E.G., Celma, P.J., Manich, A.M., Journal of the Society of Leather Technologists and Chemists, 83, 310, 1999. 52. Aravindhan, R., Saravanabhavan, S., Thanikaivelan, P., Rao, J.R., Nair, B.U., A chemo-enzymatic pathway leads towards zero discharge tanning. J. Clean. Prod., 15(13-14), 1217–1227, 2007. 53. Ogino, H., Otsubo, T., Ishikawa, H., Biochem. Eng. J., 2007; in press.

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54. Zhu, Y., Huang, W., Ni, J., A promising clean process for production of diosgenin from Dioscorea zingiberensis C. H. Wright. J. Clean. Prod., 18(3), 242–247, 2010. 55. Mahmoodi, N.M., Arami, M., Mazaheri, F., Rahimi, S., Degradation of sericin (degumming) of Persian silk by ultrasound and enzymes as a cleaner and environmentally friendly process. J. Clean. Prod., 18(2), 146–151, 2010. 56. Sykes, G., Leather board manufacture. World Leather, 10(7), 60, 1997. 57. Nilegaonkar, S.S., Zambare, V.P., Kanekar, P.P., Dhakephalkar, P.K., Sarnaik, S.S., Production and partial characterization of dehairing protease from Bacillus cereus MCM B-326. Bioresour. Technol., 98(6), 1238–1245, 2007. 58. Valeika, V., Beleška, K., Valeikienė, V., Kolodzeiskis, V., An approach to cleaner production: from hair burning to hair saving using a lime-free unhairing system. J. Clean. Prod., 17(2), 214–221, 2009. 59. Macedo, A.J., da Silva, W.O., Gava, R., Driemeier, D., Henriques, J.A., Termignoni, C, Alexandre, J.M., Walter, O., Beys, D.S., Renata, G., David, D., Joao, A., et al., Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilities. Appl. Environ. Microbiol., 71(1), 594, 2005. 60. Andrioli, E., Petry, L., Gutterres, M., Process safety and environmental protection, 1(93), 9, 2015. 61. Chowdhury, M., Mostafa, M.G., Biswas, T.K., Mandal, A., Saha, A.K., Characterization of the Effluents from Leather Processing Industries. Environ. Process., 2(1), 173–187, 2015. 62. Jareckas, G., Shibakovskaya, N.M., Maceiviciene, O., Strumskiene, A., Kozh Obuvn. Promst, 6, 44, 1985. 63. Botev, I., Esaulenko, L., Toshev, T., Kozh-Obuvn. Prom-st., 17, 13, 1976. 64. Rohm, O., German Patent No, 88, 740, 1953. 65. Huang, Q., Peng, Y., Li, X., Wang, H., Zhang, Y, Qing, H., Yong, P., Xin, L., Haifeng, W., Yizheng, Z., Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol., 46(3), 169, 2003. 66. Mukhopadhyay, R.P., Chandra, A., Indian Journal Experimental Biology,, 1, 557, 1993. 67. Varela, H., Daniel Ferrari, M., Belobrajdic, L., Vazquez, A., Lyliam Loperena, M., Ferrai, M.D., Biotechnol. Lett., 19(8), 755–758, 1997. 68. George, S., Raju, V., Krishnan, M.R.V., Subramanian, T.V., Jayaraman, K., Production of protease by Bacillus amyloliquefaciens in solid-state fermentation and its application in the unhairing of hides and skins. Process Biochemistry, 30(5), 457–462, 1995. 69. Hameed, A., Keshavaraz, T., Evans, C.S., Effect of dissolved oxygen tension and ph on the production of extracellular protease from a new isolate of bacillus subtilis K2, for use in leather processing. Journal of chemical Technology and Biotechnology, 47, 5, 1999. 70. Najafi, M.F., Deobagkar, D., Deobagkar, D., Potential application of protease isolated from Pseudomonas aeruginosa PD100. Electron. J. Biotechnol., 8(2), 197–203, 2005. 71. Wang, C.T., Ji, B.P., Li, B., Nout, R., Li, P.L., Ji, H., et al., Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J. Ind. Microbiol. Biotechnol., 33(9), 750–758, 2006. 72. Arunachalam, C., Saritha, K., Protease enzyme: an eco-friendly alternative for leather industry. Indian J. Sci. Technol., 2(12), 29, 2009. 73. Chellapandi, P., Production and preliminary characterization of alkaline protease from Aspergillus flavus and Aspergillus terreus. Journal of Chemistry, 7(2), 479, 2010. 74. Muthulakshmi, C., Gomathi, D., Kumar, D.G., Ravikumar, G., Kalaiselvi, M., Uma, C., Protease-based cross-linked enzyme aggregates with improved catalytic stability, silver removal, and dehairing potentials. Biological. 75. Rathakrishnan, P., Nagarajan, P., Journal of Science Inventor Tod, 1, 114, 2012.

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76. Vanitha, N., Rajan, S., Murugesan, A.G., Optimization and production of alkaline protease enzyme from Bacillus subtilis 168 isolated from food industry waste. Int. J. Curr. Microbiol. Appl. Sci., 3(6), 36, 2014. 77. Vijayaraghavan, P., Lazarus, S., Vincent, S.G., De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties. Saudi J. Biol. Sci., 21(1), 27–34, 2014. 78. Sundararajan, S., Kannan, C.N., Chittibabu, S., Alkaline protease from Bacillus cereus VITSN04: Potential application as a dehairing agent. J. Biosci. Bioeng., 111(2), 128–133, 2011. 79. Renganath Rao, R., Vimudha, M., Kamini, N.R., Gowthaman, M.K., Chandrasekran, B., Saravanan, P., Alkaline protease production from Brevibacterium luteolum (MTCC 5982) under solid-state fermentation and its application for sulfide-free unhairing of cowhides. Appl. Biochem. Biotechnol., 182(2), 511–528, 2017. 80. Chandrasekaran, S., Dhar, S.C., Studies on the development of a multiple proteinase concentrate and its application in the depilation of skin. Leather Sciencep, 32, 297, 1985. 81. Kumar, C.G., Takagi, H., Microbial alkaline proteases. Biotechnol. Adv., 17(7), 561–594, 1999. 82. Thangam, E.B., Rajkumar, G.S., Purification and characterization of alkaline protease from Alcaligenes faecalis. Biotechnol. Appl. Biochem., 35(2), 149, 2002. 83. Tang, X.M., Lakay, F.M., Shen, W., Shao, W.L., Fang, H.Y., Prior, B.A., et al., Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis. Biotechnol. Lett., 26(18), 1421–1424, 2004. 84. Kandasamy, N., Velmurugan, P., Sundarvel, A., Raghava, R.J., Bangaru, C., Palanisamy, T., Eco-benign enzymatic dehairing of goatskins utilizing a protease from a Pseudomonas fluorescens species isolated from fish visceral waste. J. Clean. Prod., 1, 25, 2012. 85. Chowdhury, M., Mostafa, M.G., Biswas, T.K., Saha, A.K., Treatment of leather industrial effluents by filtration and coagulation processes. Water Resources and Industry, 3(3), 11–22, 2013. 86. Puvanakrishnan, R., Dhar, S.C., NICLAI Publication. Madras, 178, 1988. 87. Braia, M., Loureiro, D., Tubio, G., Lienqueo, M.E., Romanini, D., Interaction between trypsin and alginate: an ITC and DLS approach to the formation of insoluble complexes. Colloids and Surfaces B: Biointerfaces, 155, 507–511, 2017. 88. Ketnawa, S., Benjakul, S., Martínez-Alvarez, O., Rawdkuen, S., Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability. Food Chem., 215, 383–390, 2017. 89. Vijayaraghavan, P., Arun, A., Al-Dhabi, N.A., Vincent, S.G.P., Arasu, M.V., Choi, K.C., Novel Bacillus subtilis IND19 cell factory for the simultaneous production of carboxy methyl cellulase and protease using cow dung substrate in solid-substrate fermentation. Biotechnology and Biofuels, 9(1), 73, 2016. 90. Underkofler, L.A., Hickey, R.J., Industrial fermentations. Chemical Publishing Co., 2, 109, 1954. 91. Pokorny, M., Restek, A., Petravic, J.. Kozarstv, 33, 224, 1983. 92. Muthukumaran, N., Dhar, S.C., Comparative studies on the degreasing of skins using acid lipase and solvent with reference to the quality of finished leathers. Leather Science, 29, 417, 1982. 93. Addy, V.L., Covington, A.D., Langridge, D.A., Watts, A., Microscopy methods to study lipase Degreasing, part 2: a study of the interaction of ovine cutaneous adipocytes with lipase enzymes using microscopy. Journal of the Society of Leather Technologies and Chemist, 85, 52, 2001.

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94. Palop, R., Marshal, A., Cot, J., Optimization of the aqueous degreasing process with enzymes and its influence on reducing the contaminant load. Journal of the Society of Leather Technologies and Chemists, 84, 170, 2000. 95. Simoncini, A., Biotechnology in the tanning industry. Journal of American Leather Chemical Association, 82, 226, 1987. 96. Zhang, X., Zhang, Y., Test use of alkalinelipase in degreasing of pigskin. Pige Keji, 40, 16–21, 1982.

3 Enzyme Catalysis: A Workforce to Productivity of Textile Industry Sharrel Rebello1, Embalil Mathachan Aneesh1, Raveendran Sindhu2,*, Parameswaran Binod2, Ashok Pandey3 and Edgard Gnansounou4 1

Communicable Disease Research Laboratory, St. Joseph’s College, Irinjalakuda, (India) Microbial Processes and Technology Division, CSIR-National Institute of Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum – 695 019, (India) 3 CSIR- Indian Institute for Toxicology Research (CSIR-IITR), 31 MG Marg, Lucknow-226 001, (India) 4 Ecole Polytechnique Federale de Lausanne, ENAC GR-GN, GC A3, Station 18, CH-1015, Lausanne, (Switzerland) 2

Abstract Enzyme guided catalysis of biological reactions and their industrial synthesis is of great interest, accounting for less energy consumption, eco-friendly solutions and high substrate specificity provided by these proteins. The textile industry accounts for a major portion of the industrial enzymes in various steps of the desizing, bioscouring, biobleaching, stone washing and dyeing process. The current review focuses on providing the role of various enzymes utilised in the textile industry, their mechanism of action and microbial sources. Finally, the key strategies adapted to optimised production and increased yield of enzymes are also discussed. Keywords: Enzymes, cellulase, protease, amylase, microbes

3.1

Introduction

Enzyme-based catalysis by microbes such as yeasts can be traced back to early alcoholic fermentation, even before much scientific knowledge was obtained on it. Traced back from the use of industrially produced protease additives in detergents and hydrolases in the food industry, enzymes have played their role in different industries. Microbial enzymes can be considered as horsepower workforce in various industries as these catalysts speed the catalysis rate of various industrial processes [1]. Apart from lowering energy consumption, the release of environment-friendly end products compatible with the ecosystem makes enzymes the candidates of choice for various processes.

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (49–66) © 2019 Scrivener Publishing LLC

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Food industry Detergents

Biofuel production

Cosmetics

R&D

Diagnostics

Pharmaceuticals Enzymes

Miscellaneous

Biocatalysis Feed industry

Figure 3.1 Role of enzymes in different fields of life.

Enzymes can be used in crude, pure, composite mixtures, immobilized forms or as viable cell products based on the process that is used. A close look at various industries indicates that multiple enzymes contribute to its effective functioning. The pharma industry for instance greatly relies on anticancerous enzymes (glutaminases, asparaginases), digestion enhancers (trypsin, pepsin), fibrinolysis (nattokinase), anticoagulants (streptokinase, urokinase), antiviral agents (ribonuclease), anti-inflammatory agents (superoxide dismutase), disease diagnostic markers (glucose oxidase for diabetics, creatinase for kidney diseases), etc [2, 3]. A big network of enzymes including amylase for starch digestion [4], transglutaminase for flour quality enhancement [5], lipases as flavour enhancers [6], rennet for cheese production [7], esterases, lactase, aminopeptidase, lysozyme, lactoperoxidase in dairy products [8], pectinases in juice clarification [9] etc., are some examples of enzymes utilised in the food industry [10]. Figure 3.1 depicts the areas where enzymes find their application. The global industrial enzyme market has increased steadily in consecutive years and is expected to rise to $6.3 billion in 2022 at a compound annual growth rate (CAGR) of 5.8% from 2017 [11]. Based on their extent of use and utility in different sectors, enzymes can be grouped as technical, food associated and feed associated enzymes. The entire industrial enzymes can be grouped into 56% of technical enzymes, followed by food enzymes constituting 29% and feed enzymes representing the remaining 15% [12]. The vast majority of sales and utility of industrial enzymes is accounted to protease, amylase and cellulase due to their utility in textile, paper, leather, detergent and personal care products. The second prevalent group of enzymes is used in the food industry including lipases, pectinases etc., in various baking, brewing and fermentative

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techniques [13]. Finally, the feed enzymes viz. phytase [14], xylanase and glucanase [15] represent the final group which are used in animal and poultry feed to increase the nutritive absorption by the hosts. The textile industry accounts for almost 20% of the water pollution with release of toxic chemicals, heavy metals, dyes, bleaches, detergents which persist in the environment causing harm [16]. Studies indicate that such detergents and chemicals released by conventional methods of textile processing often result in high levels of energy requirement and the pollutants in turn greatly increase the extent of global warming [17]. The incorporation of enzymes in the textile industry effectively reduces the energy-water consumption for textile processing, reduces the negative impact of effluent pollutants in the environment, adds more eco-friendliness to clothes we wear, lowers greenhouse gas release and reduces global warming [18, 19]. Moreover, the substrate specificity, biodegradability, rapid action and catalysis at milder conditions of enzymes increase their effective utilization [20]. The above review lays out the various enzymes used in the textile industry, their sources and the mode of their application in industry. A brief outline of the enzymes is generally provided along with their precise role in different stages of textile processing.

3.2

Major Textile Enzymes, Mechanism of Action and Microbial Sources

The textile industry utilizes enzymes at different stages of desizing, scouring, bleaching, stone washing, dyeing and in effluent remediation. Various enzymes are used at different stages to make the processes more effective and nature-friendly, as listed in Table 3.1. The textile industry represents the second highest polluting industry causing drastic effects to the environment. The rationale for use of enzymes in fabric processing is to find biological solutions to harsh treatments of textiles which could generate toxic waste products into the environment. The enzymes used mostly include oxidoreductases and hydrolases in various processes such as desizing, stone washing, degumming, fluff removal, etc [21]. The production of cotton clothes involve steps such as weaving, sizing of yarn with starch to prevent its breaking, desizing after weaving, bioscouring to remove the non-cellulosic elements of the textiles, bleaching, dyeing and polishing. The current section outlines these major enzymes, mode of action and sources of production.

3.2.1 Amylases Being the first enzyme discovered in 1833, amylase has been well studied and utilized in different sectors. Amylases catalyze the breakdown of starch to glucose units and finds applications in almost all industries [22]. Technically amylases are of four types viz. α amylase, β amylase, glucoamylase and pullulanase based on the mode of action on starch and their end products. Alpha-amylases are endoenzymes with a molecular weight between 10 kDa to 210 kDa, breaking internal α −1,4-glycosidic bonds of linear starch molecules to yield glucose, maltose, linear oligosaccharides,

Fusarium oxysporium, Fusarium polycaprolactone, Streptomyces, Colletotrichum, Thermobifida fusca Bacillus sp., Aspergillus fumigatus, Aspergillus niger Bacillus sp., Cyanobacteria Propionibacterium,Candida,Mycoderma, Debaryomyces kloeckeri

Cutinases

Proteases

Xylanases

Lipases

(Continued)

Penicillium notatum, Bacillus sp., Penicillium notatum, Aspergillus fumigatus, Aspergillus kawachii, Aspergillus niger

Aerobacter aerogenes, Bacillus acidopullulyticus, Bacillus flavocaldarius, Bacillus thermoleovorans Klebsiella pneumonia, Streptomyces sp., Clostridium sp. Thermoscaldophilus, Pyrococcus woesei, Pyrococcus furiosus

Pullulanase

Pectinases

Aspergillus niger, Aspergillus oryzae

Amyloglucosidase

Bioscouring

Bacillus sp.

Amylase

Desizing

Microbial sources

Enzyme used

Textile processing step

Table 3.1 Major enzymes used in the processing of textiles.

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Effluent treatment

Pseudomonas, Enterobacter

Azoreductase,

Aspergillus sp., Penicillium sp., Trametes versicolor

Laccase

Laccase,

Bacillus, Aspergillus niger, Penicillium funiculosum

Cellulase

As noted earlier

Simultaneous use of lipase, protease, cellulase, pectinase

Biobased Stonewashing

Aspergillus sp., Penicillium sp.

glucose oxidase

Biobleaching

Microbial sources

Enzyme used

Cont.

Textile processing step

Table 3.1

Enzyme Catalysis: A Workforce to Productivity 53

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α -limit dextrin [23]. β -amylases are exoenzymes that cleave α −1, 4-glycosidic linkages of starch yielding maltose and β limit dextrin. Glucoamylases are exoenzymes breaking the α −1,4-glycosidic linkages of amylose and amylopectin to glucose subunits, enabling complete starch hydrolysis [24]. Pullulanase enzymes can be produced both as exo- as well as endoenzyme capable of cleaving both the α −1,4-glycosidic linkages and β−1,6- glycosidic linkages of partly digested amylopectin and are frequently applied in combination with glucoamylases in industries [25]. Amylases are produced from a variety of plant and animal sources; however, bacterial and fungal amylases have gained momentum in the industrial sector [26]. The ease of synthesis of enzymes from microbes, their easy availability and plasticity are some factors contributing to this preference. The amylases in textile industries are available in different forms to be used in mild, high and psychrophilic temperatures and thus microbes from different niches are explored to obtain such enzymes as depicted in Table 3.2. Amylases are mostly produced by Bacillus sp., Clostridium thermosulfurogenes, Geobacillus etc., providing variants of alkali stable and heat tolerant enzymes [27–30]. Most of the amylases from Bacillus are found to be thermostable, yet halotolerant amylases are also reported from various marine bacteria [31]. The fungal sources include Aspergillus species, Penicillium, Aureobasidium pullulans[32]. Most of the industrial amylases are produced by Bacillus sp. owing to its thermo-tolerance, high yield and generally regarded as safe nature [33].

3.2.2 Cellulases Cellulases catalyze the hydrolytic cleavage of cellulose into smaller sugar components like glucose units by cleaving the β−1, 4-glucosidic bonds of the parent molecule. The degradation of cellulose is usually accomplished by the action of three types of cellulases viz. endoglucanase (E.C. 3.2.1.4), exoglucanase (E.C. 3.2.1.176) and (E.C. 3.2.1.91) and β-glucosidase (E.C. 3.2.1.21) [34]. Endoglucanases mostly provide random cuts on the internal bonds of glycan chain to generate shorter cello-oligosaccharides which can be further cleaved by exoglucanase to yield cellobiose. The β-glucosidases thus act on the cellobiose to release glucose to be utilized by microbes [35]. Cellulose degradation is brought about mainly by aerobic bacteria, fungi and yeast [36]. However, anaerobic bacteria are not that effective in cellulase enzyme production [31]. Bacterial cellulases are found to be attached to their cell wall whereas fungal cellulases are excreted outside, thereby increasing the ease of extraction [34]. Production of cellulase is documented also in plants and in a number of invertebrate taxa that includes insects, crustaceans, annelids, molluscs, mussels, and nematodes. Fungal cellulases are found to be better tolerant to harsh conditions and are industrially produced mostly by Trichoderma species compared to Aspergillus and Humicola species [37].

3.2.3 Proteases As per the global market analysis proteases represented the largest share of industrial enzymes and would exceed US$3 billion by 2024 [38]. Proteases are proteolytic enzymes produced by a variety of bacteria and fungi and they play a critical role in many industries. Bacterial proteases are noteworthy for their thermo-tolerance than the fungal

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Table 3.2 Companies involved in commercial synthesis of textile enzymes and their products. Company’s name

Textile enzymes provided

Products name & use

AB Enzymes

Cellulases

BIOTOUCH® - Innovative Textile Finishing ECOSTONE® - Efficiency And Value For Denim Treatment

Novozymes

Blend of catalase & neutral cellulase

Novozymes Cellusoft®-neutral and acid biopolishing

Recombinant heat-stable α-amylase by Bacillus licheniformis.

Novozymes Termamyl®-desizing

Pectate lyase from Bacillus

Novozymes BioPrep® - bioscouring, for combined biopolishing & desizing

Amylase

Amylazyme -

Cellulase

Cellazyme C

Amylase from Bacillus sp (Psychrophilic, medium and thermophilic)

Desizyme –L, M,H for desizing

Cellulase from Trichoderma

Stonenzyme – denim washing

Cellulase from Trichoderma

Polishzyme- biopolishing

mixture of various enzymes from Bacillus

Scourzyme- for bioscouring of textiles

catalase enzymes from fungal Aspergillus sp

Peroxzyme – for Bleach cleanup to remove residual H2O2

alkaline protease enzymes from Bacillus sp

Biosilkzyme- degumming of silk.

Megazyme

Aumenzymes

protease enzymes from Bacillus sp Woolzyme- finishing of wool. (Continued)

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Table 3.2 Cont. Company’s name

Textile enzymes provided

Products name & use

Maps Enzymes Limited

Alpha amylase with low, medium and psychrophilic variants

Palkozyme and its variants- desizing at different temperatures

Cellulase

Palkofeel, Palkofeel C- for biopolishing cotton and blended fabric and garment

Cellulase

Palkowash, Palkocel Palkostone- for denim finishing

Catalase

Palkoperox- bleach cleanup to remove residual H2O2

Multienzyme mixture

Palkoscour- bioscouring

counterparts. They are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. Based on the nature of the functional group in their active site, they are classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases [39]. Generally, serine proteases are mostly alkaline and neutral proteases of low molecular weight (18.5–35 kDa) produced by microbes as well as animals and are widely used as detergent additives [40]. A detail on the mechanism of its action and catalytic role is reviewed [41]. Bacteria such as Bacillus sp., Clostridium, Pseudomonas contribute to the major level of production of this enzyme [42]. Cysteine proteases are members of the papain family and are characterized by Cys–His–Asn triad at the active site. The cysteine residue attacks to the carbon of the reactive peptide bond and His serves as a proton donor, which results in the cleavage of thioester bond to produce carboxylic moiety from the parent protein [43]. Aspartic proteases represent the acidic proteases and are produced by viruses, bacteria, fungi, plants, and animals [44]. The high stability and activity of aspartic proteases account for their use in food processing industry such as protein haze of wine, manufacture of cheese, flavour enhancement of food, etc [45]. The metalloproteases require metallic ions for their activity and thus are classified so. Apart from its industrial significance these serine proteases play a critical role in physiological and patho-physiological processes as signalling molecules in humans and are prospective drug targets [46, 47]. Some examples of proteases physiological role are in blood coagulation, fibrinolysis, complement activation, hormone production, metamorphosis and proper digestion [48]. Cysteine proteases also play a major role in immunomodulation and antigen presentation, haemoglobin hydrolysis, digestion, parasitic invasion and processing surface proteins [49]. Proteases are generally produced by microbes such as Bacillus, Aspergillus, Penicillium, Pseudomonas, Streptomyces, etc.

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3.2.4

57

Laccases

Laccases are multi-copper enzymes unique in their ability to oxidize pesticides, steroids, phenolic as well as nonphenolics pollutants, etc [50]. They are basically oxidoreductases and have a wide range of substrates including polyphenols, methoxy-substituted monophenols, aliphatic and aromatic amines [51, 52]. They are capable of ring cleavage, breakage of polymers mediated by oxidation of substrate electron along with generation of water. Laccase mediated remediation of industrial effluents from textiles, dairy, pharmaceutical industries is widely practised [53, 54]. Laccases are produced by plants, insects and bacteria, but fungi such as Deuteromycetes, Ascomycetes as well as Basidiomycetes are prominent laccase producers [55, 56]. Studies also indicate that bacterial sources of laccases are found to be promising in dye degradation studies [57]. Quite often laccases produced in multiple isoforms than single isoforms and provide 95% degradation of dyes, as noted in a recent study [58].

3.3

Applications in Textile Industry

Commercial synthesis and use of microbial derived enzymes in the textile industry has advanced to a great level today. The key players in the textile industry include Novozymes, Sigma Chemical Industries, Lumis Enzymes, AB Enzymes, and Refnol. Table 3.2 describes various companies that successfully market these enzymes under different names to provide more eco-friendly solutions to the textile industry.

3.3.1 Desizing The process of desizing or removal of starch from textiles using amylases has been widely practised [59]. Amylases can be produced from a wide variety of bacteria and fungi and these desizing agents selectively act against the starch not affecting the cellulose fibres [60]. The use of gellan-immobilized amylases are found to be effective in removing the glue attached to archaeological textile samples without damaging the cellulosic fibres [61].Yet the traditionally amylases generate glucose units and shorter oligosaccharide units thereby increasing the COD and BOD of the resultant effluents. Studies suggest the use of amyloglucosidase/pullulanase mixture instead of traditional amylases for desizing and this in combination with glucose oxidase to bring about bleaching action due to hydrogen peroxide, catalase based removal of H2O2 followed by dyeing generation in a single bath [62]. Combined desizing, scouring and depilling using a triple enzymatic mixture of amylase, pectinase and cellulase in a single bath could be a eco-friendly, water-saving approach to traditional techniques [63].

3.3.2 Bioscouring Scouring involves the process of removing all non-cellulosic materials such as waxes, pectins, gums and oils from the textile materials. This can be achieved by using chemicals such as alkaline sodium hydroxide or using biological enzymes. Some of the enzymes used in bioscouring include pectinases [64], cutinases [65, 66], proteases [67],

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xylanases [68] and lipases. The process of bioscouring was found to be more advantageous than alkaline scouring technique, imparting less damage to cotton terry fabrics after 20 washings in a domestic washing machine, imparting greater resistance to abrasion, reduced degree of polymerisation and a higher fabric hand feeling [69]. Bioscouring usually increases the wettability of the textile, reduces water consumption for processing, and it takes up the dye effectively. Moreover, the amount of total dissolved solids in textile effluents is greatly reduced on adoption of bioscouring techniques [70].

3.3.3 Biobleaching The process of bleaching of textiles conventionally involves treatment with hydrogen peroxide, sodium hydroxide, sodium silicate, sodium carbonate and magnesium sulphate at boiling temperatures [71]. This method is disadvantageous on account of the damage it causes to cotton fibrils and the large amount of water needed for the entire process. Thus the inclusion of biogenically derived hydrogen peroxide using enzymes such as glucose oxidase has been suggested as an alternative [72] and the method was found to significantly decrease the chemical oxygen demand when combined with ultrasonic waves [73]. Further efforts in this direction also developed a bio-bleaching recipe of in situ formed peracetic acid and enzymes (lipase, protease, cellulase, pectinase) involving lesser temperatures of 60 °C, less batch times and alkali consumption than conventional counterparts [74]. Peracetic acid could be used instead of hydrogen peroxide when medium degree of whiteness is required [75].

3.3.4 Stone Washing The most popular shades of denim shirts or jeans obtain their stone washed or faded appearance due to the treatment with microbial cellulases [76]. Enzymatic treatment of such fabrics is a greener means of updated fashion, proving less of a burden to the environment [77]. The difficulties associated with older methods of rubbing with pumice stones, clogging with broken pumice pieces and their disposal is fully overcome with the aid of biological cellulase treatment. This causes fading of dyes at the point of application of the enzyme to get shades as our interest [78]. The use of cellulases along with laccases co-immobilized onto the reversibly soluble polymers (Eudragit S-100 or Eudragit L-100) was found to be more advantageous than cellulase alone, giving the denim better tensile strength post-treatment [79]. More benefits of UV protection and antibacterial role of denims treated with enzyme (Cellzyme SPL H/C) would surely gain more interest in the present scenario [80].

3.3.5

Enzyme-Assisted Dyeing

The use of microbial enzymes as an alternative to metal mordents has been suggested in textile industry to make the process more eco-friendly. Enzyme pretreatment of fibres contributes to better and faster uptake of dye along with better absorbency and adherence. The combinatorial use of physical techniques such as ultrasonication also can avoid the use of high temperatures as required in conventional dyeing [81].

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Though not commercially feasible, microwave-assisted dyeing of enzyme treated textile fibres were found to give good results in another study [82]. Dyeing of silk with commercial dye Rubia has good results when they were treated with enzymes such as protease, phytase, xylanase, amylase and pectinase in a one-step process even at room temperature [83]. Further modification of textiles to gain properties such as UV protection can be obtained by laccase-assisted polyphenol coating of textile fibres [84].

3.3.6

Effluent Treatment

Effluent treatment from the dyeing industry greatly relies on laccases, proteases, azoreductases, etc. The use of enzymes such as glucose oxidase in azo dye treatment are found to increase the dye degradation level to 5-fold than the control on repeated cycles [85]. Recombinant laccases expressed in Saccharomyces cerevisiae are recently reported to be effective in the remediation of industrial effluents of containing synthetic dyes [86]. Laccases from Phoma immobilized in membrane filters aided in removing 85% of pharmaceuticals from industrial waste [87]. The use of tyrosinases in the complete removal and breakdown of phenolic compounds from effluents has also been suggested as a cheaper alternative compared to laccases and peroxidises [88]. Thus enzymes are found to be a boon providing fruitful solutions to waste water treatment, decolourisation, soil remediation and detoxification of textile industry effluents [89].

3.4

Technological Advancements to Enhanced Production of Textile Enzymes

Media engineering of production media using statistically relevant approaches such as response surface methodology has helped to optimise industrial production of various enzymes cost-effectively [90]. But the production of enzymes has boomed from its primary stages of industrial production, with the advent of genetic engineering and protein engineering technologies. Site-directed mutagenesis to improve enzyme thermo-stability, alkaline stability, better catalytic efficiency also serves as tools to manipulate and improve enzymes in various sectors [9, 58]. The recombinant expression of enzymes in promoter controlled expression systems such as Escherichia coli [91], Saccharomyces cerevisiae [92], Pichia pastoris [93], Kluyveromyces [94], Baculovirus [95, 96] and mammalian cell lines [97] have enabled the enhancing of the production rate of various enzymes. The advantages and drawbacks of various expression hosts used for microbial enzyme production is also well reviewed [92]. The use of metagenomic approaches to identify promising enzymes from the vast genome resources of the environment has also found to be successful [98]. Mutagenesis [99] and metabolic engineering [100] of enzymes to increase their yield, improve or optimise essential properties of enzymes is also widely practised.

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Conclusion

Microbial enzymes individually as well as in combination are widely useful in various industries. The increased production and use of these biocatalysts would greatly contribute to the success in concerned sectors of use. However, with the increasing demands for value-added products with better quality the demand of constituent enzymes is increasing. The utilization of microbial enzymes with better yield, catalytic efficiency and unique properties would help to find a solution to the demand. Textile industry being an indispensable element of man, the addition of enzyme workforce in the industry has greatly reduced the loss of our natural resources such as water and energy. The major advantage of microbial enzymes in the textile industry is the ecological benefits and environmental safety they confer to this industry.

Acknowledgement One of the authors, Sharrel Rebello, acknowledges SERB for NPDF (File no PDF/2015/000472). Raveendran Sindhu acknowledges DST for sanctioning a project under DST-WOS-B scheme. Parameswaran Binod and Raveendran Sindhu acknowledge EPFL, Lausanne, Switzerland, for a visiting fellowship.

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35. Jørgensen, H., Kristensen, J.B., Felby, C., Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod. Bioref., 1(2), 119–134, 2007. 36. Payne, C.M., Knott, B.C., Mayes, H.B., Hansson, H., Himmel, M.E., Sandgren, M., et al., Fungal cellulases. Chem. Rev., 115(3), 1308–1448, 2015. 37. Muhammad, I., Zahid, A., Muhammad, I., Muhammad, J.A., Hassan, A., Advances in Enzyme Research, 4, 44–55, 2016. 38. Proteases market size, industry analysis report, regional outlook, application development potential, price trends, competitive market share & forecast, 2016 – 2024. Available. Available from: https://www.gminsights.com/industry-analysis/proteases-market. 39. Rao, M.B., Tanksale, A.M., Ghatge, M.S., Deshpande, V.V., Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev., 62(3), 597, 1998. 40. Ellaiah, P., Srinivasulu, B., Adinarayana, K., Journal of Scientific & Industrial Research, 61, 690, 2002. 41. Hedstrom, L., Serine protease mechanism and specificity. Chem. Rev., 102(12), 4501–4524, 2002. 42. Gupta, R., Beg, Q.K., Lorenz, P., Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol., 59(1), 15, 2002. 43. Coulombe, R., Grochulski, P., Sivaraman, J., Ménard, R., Mort, J.S., Cygler, M., Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.Embo J., 15(20), 5492–5503, 1996. 44. Hsiao, N.-W., Chen, Y., Kuan, Y.-C., Lee, Y.-C., Lee, S.-K., Chan, H.-H., et al., Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R. Electronic Journal of Biotechnology, 17(2), 89–94, 2014. 45. Theron, L.W., Divol, B., Microbial aspartic proteases: current and potential applications in industry. Appl. Microbiol. Biotechnol., 98(21), 8853–8868, 2014. 46. Turk, B., Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discov., 5(9), 785–799, 2006. 47. Patel, S., A critical review on serine protease: Key immune manipulator and pathology mediator. Allergol. Immunopathol. (Madr)., 45(6), 579–591, 2017. 48. Neurath, H., Walsh, K.A., Role of proteolytic enzymes in biological regulation (a review). Proceedings of the National Academy of Sciences, 73(11), 3825–3832, 1976. 49. Verma, S., Dixit, R., Pandey, K.C., Cysteine proteases: Modes of activation and future prospects as pharmacological targets. Front. Pharmacol., 7(125), 107, 2016. 50. Gianfreda, L., Xu, F., Bollag, J.-M., Laccases: A useful group of oxidoreductive enzymes. Bioremediat. J., 3(1), 1–26, 1999. 51. Karaki, N., Aljawish, A., Humeau, C., Muniglia, L., Jasniewski, J., Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review. Enzyme Microb. Technol., 90, 1–18, 2016. 52. de Freitas, E.N., Bubna, G.A., Brugnari, T., Kato, C.G., Nolli, M., Rauen, T.G., et al., Removal of bisphenol A by laccases from Pleurotus ostreatus and Pleurotus pulmonarius and evaluation of ecotoxicity of degradation products. Chemical Engineering Journal, 330, 1361–1369, 2017. 53. Becker, D., Rodriguez-Mozaz, S., Insa, S., Schoevaart, R., Barceló, D., de Cazes, M., et al., Removal of endocrine disrupting chemicals in wastewater by enzymatic treatment with fungal laccases. Org. Process Res. Dev., 21(4), 480–491, 2017. 54. Asif, M.B., Hai, F.I., Kang, J., van de Merwe, J.P., Leusch, F.D.L., Price, W.E., et  al., Biocatalytic degradation of pharmaceuticals, personal care products, industrial chemicals, steroid hormones and pesticides in a membrane distillation-enzymatic bioreactor. Bioresour. Technol., 247, 528–536, 2018.

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55. Abd El Monssef, R.A., Hassan, E.A., Ramadan, E.M., Production of laccase enzyme for their potential application to decolorize fungal pigments on aging paper and parchment. Annals of Agricultural Sciences, 61(1), 145–154, 2016. 56. Myasoedova, N.M., Renfeld, Z.V., Podieiablonskaia, E.V., Samoilova, A.S., Chernykh, A.M., Classen, T., et al., Novel laccase—producing ascomycetes. Microbiology, 86(4), 503– 511, 2017. 57. Chauhan, P.S., Goradia, B., Saxena, A., Bacterial laccase: recent update on production, properties and industrial applications. 3 Biotech, 7(5), 323, 2017. 58. Luo, Q., Chen, Y., Xia, J., Wang, K.Q., Cai, Y.J., Liao, X.R., et  al., Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis. Enzyme Microb. Technol., 109, 11–19, 2018. 59. Opwis, K., Knittel, D., Kele, A., Schollmeyer, E., Enzymatic recycling of starch-containing desizing liquors. Starch/Stärke, 51(10), 348–353, 1999. 60. Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K., Chauhan, B., Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38(11), 1599–1616, 2003. 61. Ferrari, M., Mazzoli, R., Morales, S., Fedi, M., Liccioli, L., Piccirillo, A., et al., Enzymatic laundry for old clothes: immobilized alpha-amylase from Bacillus sp. for the biocleaning of an ancient Coptic tunic. Appl. Microbiol. Biotechnol., 101(18), 7041–7052, 2017. 62. Eren, H.A., Anis, P., Davulcu, A., Enzymatic one-bath desizing — bleaching — dyeing process for cotton fabrics. Textile Research Journal, 79(12), 1091–1098, 2009. 63. Toprak, T., Anis, P., Combined one-bath desizing–scouring–depilling enzymatic process and effect of some process parameters. Cellulose, 24(1), 383–394, 2017. 64. Niaz, A., Malik, Q.J., Muhammad, S., Shamim, T., Asghar, S., Bioscouring of cellulosic textiles. Coloration Technology, 127(4), 211–216, 2011. 65. Degani, O., Gepstein, S., Dosoretz, C.G., Potential use of cutinase in enzymatic scouring of cotton fiber cuticle. Appl. Biochem. Biotechnol., 102-103(1–6), 277–290, 2002. 66. Zhang, Y., Chen, S., Xu, M., Cavaco-Paulo, A., Cavoco-Paulo, A., Wu, J., Chen, J., et al., Characterization of Thermobifida fusca cutinase-carbohydrate-binding module fusion proteins and their potential application in bioscouring. Appl. Environ. Microbiol., 76(20), 6870–6876, 2010. 67. Lin, C.-H., Hsieh, Y.-L., Direct scouring of greige cotton fabrics with proteases. Textile Research Journal, 71(5), 425–434, 2001. 68. Battan, B., Dhiman, S.S., Ahlawat, S., Mahajan, R., Sharma, J., Application of thermostable xylanase of Bacillus pumilus in textile processing. Indian J. Microbiol., 52(2), 222–229, 2012. 69. Mojsov, K., Enzymatic scouring and bleaching of cotton terry fabrics – opportunity of the improvement on some physicochemical and mechanical properties of the fabrics. Journal of Natural Fibers, 1, 2017. 70. Vigneswaran, C., Journal of Textile and Apparel, Technology and Management, 7, 2011. 71. Charles, T., Department of Textile Engineering, Chemistry and Science College of Textiles North Carolina State University Raleigh. North Carolina, 1992. 72. Buschle-Diller, G., Yang, X.D., Yamamoto, R., Enzymatic bleaching of cotton fabric with glucose oxidase. Textile Research Journal, 71(5), 388–394, 2001. 73. Davulcu, A., Eren, H.A., Avinc, O., Erişmiş, B., Ultrasound assisted biobleaching of cotton. Cellulose, 21(4), 2973–2981, 2014. 74. Usluoglu, A., Arabaci, G., Bleaching of cotton/polyamide fabrics with enzymes and peracetic acid. Asia-Pac. J. Chem. Eng., 9(3), 364–367, 2014. 75. Mojsov, K., Bioscouring and bleaching process of cotton fabrics – an opportunity of saving water and energy. The Journal of The Textile Institute, 107(7), 905–911, 2016.

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76. Gusakov, A.V., Berlin, A.G., Popova, N.N., Okunev, O.N., Sinitsyna, O.A., Sinitsyn, A.P., A comparative study of different cellulase preparations in the enzymatic treatment of cotton fabrics. Appl. Biochem. Biotechnol., 88(1-3), 119–126, 2000. 77. Shahid, M., Zhou, Y., Tang, R.-C., Chen, G., In: Muthu S. S, ed, Textiles and Clothing Sustainability: Sustainable Technologies. Springer, Singapore. p. 67, 2017. 78. Olson, L.A., Treatment of denim with cellulase to produce a stonewashed appearance, ed: Google Patents, 1990. 79. Yu, Y., Wang, Q., Yuan, J., Fan, X., Wang, P., Co-immobilization of cellulase and laccase onto the reversibly soluble polymers for decolorization of denim fabrics. Fibers Polym., 18(5), 993–999, 2017. 80. Pervez, M.N., Rahman, M.A., Yu, L., Cai, Y., A novel study on UV protection and antibacterial properties of washed denim garment. MATEC Web Conf., 108, 03004, 2017. 81. Vankar, P.S., Shanker, R., Ecofriendly ultrasonic natural dyeing of cotton fabric with enzyme pretreatments. Desalination, 230(1-3), 62–69, 2008. 82. Mohamed, N.A., Wan Ahmad, W.Y., Ngalib, K., Ahmad, M.R., Ab Kadir, M.I., Ismail, A. In: Ahmad M. R, Yahya M. F, eds, Proceedings of the International Colloquium in Textile Engineering, Fashion, Apparel and Design. 89. Springer Singapore, ed Singapore, 2014. 83. Vankar, P.S., Shukla, D., Wijayapala, S., Samanta, A.K., Vankar, P.S., Shukla, D., Innovative silk dyeing using enzyme and Rubia cordifolia extract at room temperature. Pigment & Resin Technology, 46(4), 296–302, 2017. 84. Aixue, D., Yuanyuan, Y., Xuerong, F., Qiang, W., Artur, C.-P., Journal of Industrial Textiles, 46, 160, 2015. 85. Ansari, Z., Karimi, A., Sedghi, S., Razzaghi, M., Ebrahimi, S., Glucose oxidase effect on treatment of textile effluent containing reactive azo dyes by Phanerochaete chrysosporium. J. Chem. Technol. Biotechnol., 92(7), 1721–1726, 2017. 86. Antošová, Z., Herkommerová, K., Pichová, I., Sychrová, H., Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent-A comparative study. Biotechnol. Prog., 34(1), 69-80, 2018. 87. Jahangiri, E., Thomas, I., Schulze, A., Seiwert, B., Cabana, H., Schlosser, D., Characterisation of electron beam irradiation-immobilised laccase for application in wastewater treatment. Sci. Total Environ., 624, 309–322, 2018. 88. Agarwal, P., Gupta, R., Agarwal, N., A review on enzymatic treatment of phenols in wastewater. J. Biotechnol. Biomater., 06(04), 2, 2016. 89. Jajpura, L., Detox fashion: Sustainable chemistry and wet processing. In: Muthu S. S, ed. Singapore, Springer. p. 95, 2018. 90. Dinarvand, M., Rezaee, M., Foroughi, M., Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM). Braz. J. Microbiol., 48(3), 427–441, 2017. 91. Chen, H., Huang, R., Zhang, Y.-H.P., Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3). Appl. Microbiol. Biotechnol., 101(11), 4481–4493, 2017. 92. Liu, L., Yang, H., Shin, H.-dong., Chen, R.R., Li, J., Du, G., et al., How to achieve high-level expression of microbial enzymes. Bioengineered, 4(4), 212–223, 2013. 93. Berezina, O.V., Herlet, J., Rykov, S.V., Kornberger, P., Zavyalov, A., Kozlov, D., Thermostable multifunctional GH74 xyloglucanase from Myceliophthora thermophila: high-level expression in Pichia pastoris and characterization of the recombinant protein. Appl. Microbiol. Biotechnol., 1, 2017.

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94. Rocha, S.N., Abrahão-Neto, J., Cerdán, M.E., González-Siso, M.I., Gombert, A.K., Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus. Microb. Cell Fact., 9(1), 4, 2010. 95. Kost, T.A., Condreay, J.P., Jarvis, D.L., Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol., 23(5), 567–575, 2005. 96. Contreras-Gómez, A., Sánchez-Mirón, A., García-Camacho, F., Molina-Grima, E., Chisti, Y., Protein production using the baculovirus-insect cell expression system. Biotechnol. Prog., 30(1), 1–18, 2014. 97. Dumont, J., Euwart, D., Mei, B., Estes, S., Kshirsagar, R., Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Crit. Rev. Biotechnol., 36(6), 1110–1122, 2016. 98. Uchiyama, T., Miyazaki, K., Functional metagenomics for enzyme discovery: challenges to efficient screening. Curr. Opin. Biotechnol., 20(6), 616–622, 2009. 99. Reetz, M.T., Directed evolution of selective enzymes: Catalysts for organic chemistry and biotechnology, 115, 2017. 100. Lee, H., DeLoache, W.C., Dueber, J.E., Spatial organization of enzymes for metabolic engineering. Metab. Eng., 14(3), 242–251, 2012.

4 Current Trends in the Production of Ligninolytic Enzymes Susana Rodríguez-Couto 1

Ceit, Paseo Manuel de Lardizábal 15, 20018, Donostia-San Sebastian, (Spain) Universidad de Navarra, Tecnun, Paseo Manuel de Lardizábal 13, 20018, Donostia-San Sebastian, (Spain) 3 IKERBASQUE, Basque Foundation for Science, Maria Diaz de Haro 3, 48013, Bilbao, (Spain) 2

Abstract Ligninolytic enzymes are involved in the degradation of the complex, heterogeneous and recalcitrant aromatic biopolymer lignin, thus, taking part in the global carbon cycle. In addition to modify or degrade lignin, these enzymes can break down xenobiotic and toxic compounds. Ligninolytic enzymes mainly include peroxidases and laccases. Due to their high versatility, they find application in a wide variety of industries such as food, textile, paper, etc. Consequently, the demand for these enzymes has increased in recent years, which has driven the search for cost-efficient production systems. In this sense, the use of wastes from the agricultural, food and forestry industries as feedstock for ligninolytic enzyme production is seen as an economical and environmentally friendly alternative. Keywords: Ligninolytic enzymes, lignin, white-rot fungi, biocatalysis, fermentation

4.1

Introduction

Lignin can comprise up to 30% of plant biomass and plays an important role in providing mechanical support, waterproofing and pathogen resistance to plant cells [1]. Lignin is, after cellulose, the second most abundant renewable biopolymer on Earth and forms part of the global carbon cycle. It has a heterogeneous, complex and stable structure composed of different aromatic heteropolymers in which its predominant structural components (i.e., phenylopropanoid aryl–C3 units) are linked by a variety of C-O (ether) and C-C bonds [2]. The basic structural units and representative linkages of lignin are illustrated in Figure 4.1 [3]. Lignin is highly recalcitrant and more difficult to degrade than cellulose and hemicellulose due to its complex structure and non-hydrolysable bonds. It has a high molecular weight of about 100 kDa or more which hinders its absorption by the microbial

Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (67–88) © 2019 Scrivener Publishing LLC

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Figure 4.1 Representative linkages present in lignin and basic structural units found in lignin. Reprinted from Renewable and Sustainable Energy Reviews 77, Asina FNU, Brzonova I, Kozliak E, Kubátováb A, Jia Y, Microbial treatment of industrial lignin: Successes, problems and challenges, 1179–1205, Copyright 2017, with permission from Elsevier Ltd., UK.

cell [4]. Thus, the biological degradation of lignin must occur through the activity of extracellular enzymes. The physiological importance of lignin biodegradation is the destruction of the matrix it forms, so that microorganisms can gain access to the real substrates hemicellulose and cellulose [5], from where they obtain energy. Lignin degradation in nature is caused by wood-degrading fungi which can be divided, depending on the type of decay they cause, into three groups: white-rot, brown-rot and soft-rot fungi. The former are the only microorganisms able to degrade the whole wood components and by far the most efficient lignin-degrading microorganisms described to date. Their ability to degrade lignin is due to the secretion of different extracellular oxidative and hydrolytic enzymes together with low-molecular mass effectors [6]. The main extracellular enzymes secreted by the white-rot fungi are lignin peroxidase (LiP, E.C. 1.11.1.14), manganese-dependent peroxidase (MnP, E.C. 1.11.1.13) [7], versatile peroxidase (EC 1.11.1.16) [8] and laccase (E.C. 1.10.3.2) [9].

4.2

Ligninolytic Enzymes

In Table  4.1 [10–13] and Table  4.2 [14–16] the main reactions and characteristics, respectively, of the ligninolytic enzymes are summarised.

4.2.1

Lignin Peroxidase

Lignin peroxidase (EC 1.11.1.14, 1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol:hydrogenperoxide oxidoreductase, family 2 at http://www.cazy.org, LiP) is a glycosylated heme peroxidase. It was first detected in the extracellular culture broth of the white-rot fungus Phanerochaete chrysosporium in the 1980s [17, 18]. It is considered as a true ligninase, since it directly catalyses lignin oxidation in the presence of hydrogen peroxide [18, 19].

O2

Laccase (EC 1.10.3.2) Phenols, mediators, e.g., hydroxybenzotriazole or ABTS

Mn, veratryl alcohol, compounds similar to LiP and MnP

H2O2

Versatile peroxidase (EC 1.11.1.16), VP

Phenols are oxidised to phenoxyl radicals; other reactions in the presence of mediators

Mn(II) oxidised to Mn(III), oxidation of phenolic and non-phenolic compounds and dyes

Mn, organic acids as Mn(II) oxidised to Mn(III); chelated chelators, thiols, Mn(III) oxidises phenolic unsaturated fatty acids compounds to phenoxyl radicals; other reactions in the presence of additional compounds

H2O2

Aromatic ring oxidised to cation radical

Reaction

Manganese peroxidase (EC 1.11.1.13), MnP

Veratryl alcohol

Substrate, mediator

H2O2

Cofactor

Lignin peroxidase (EC 1.11.1.4), LiP

Enzyme and abbreviation

Table 4.1 Ligninolytic enzymes and their main reactions [10-13].

Basidiomycota and Ascomycota, in most white-rot fungi and litterdegrading fungi

Basidiomycota, only in Pleurotus sp., Bjerkandera sp. and Trametes versicolor

Basidiomycota, common in white-rot fungi and litter-degrading fungi

Basidiomycota, only in few white-rot fungi

Occurrence in fungi

Current Trends in the Production of Ligninolytic Enzymes 69

35–48

38–62.5

40–45

54–80

LiP

MnP

VP

Laccase

*in some cases they can reach up to 49%

Molecular mass (kDa)

Enzyme

3–4

3.4–3.9

2.9–7.1

3.1–4.7

Isolectric point (pI)

Yes (10–20%)* N-glycosylated

Yes

Yes (4–18%) N-glycosylated

Yes (up to 20–30%) N-glycosylated

Glycosylation

Table 4.2 Characteristics of the main ligninolytic enzymes [14–16].

0.4–0.8

>1

0.8 (at pH 4.5)

1.2 (at pH 3.0)

Redox potential (eV)

Mostly extracellular

Extracellular

Extracellular

Extracellular

Localization

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Native LiP

71

VA LiP III (FeIIIO2+)

III

(Fe

VA VA+

H2O2

Inactivated` H2O

VA Excess H2O2

LiP II

LiP I (FeIV=O2[P] ±)

VA

VA+

(FeIV=O)

Figure 4.2 The catalytic cycle of lignin peroxidase (LiP); VA·+: veratryl alcohol cation radical. Reprinted from FEBS Letters 243, Wariishi, H. and Gold M. H., Lignin peroxidase Compound III: formation, inactivation and conversion to the native enzyme, 165–168, 1989, with permission from John Wiley & Sons, USA.

LiP has a redox potential higher than MnP and laccase (Table 4.2) and, thus, it can catalyse the non-phenolic units of lignin. During its catalytic cycle (Figure  4.2) [20], LiP is oxidised by hydrogen peroxide forming a two-electron oxidised intermediate (compound I). Next, this intermediate (compound I) oxidises substrates by removing one electron leading to a more reduced enzyme intermediate (compound II) and a substrate radical. Then, this intermediate (compound II) can oxidise substrates by one electron, returning the enzyme to its initial state. However, this compound II is highly reactive with hydrogen peroxide and, thus, in the presence of a poor substrate and an excess of hydrogen peroxide is converted into an inactive form of the enzyme (compound III) [20, 21]. Veratryl alcohol enhances the action of LiP on many substrates, including lignin [19], by acting as a mediator [22] or by protecting the enzyme against inactivation by H2O2 [21].

4.2.2 Manganese-Dependent Peroxidase Manganese-dependent peroxidase (EC 1.11.1.13, Mn (II)-hydrogen-peroxide oxidoreductase, family 2 at http://www.cazy.org, MnP) is an extracellular heme-containing peroxidase. It was first discovered in the ligninolytic fungus P. chrysosporium [23] and its expression and production are regulated by the presence of Mn(II) in the culture medium [24]. It is considered as the most common ligninolytic peroxidase, produced by nearly all white-rot basidiomycetes [25]. MnP catalyses the oxidation of Mn2+ to the highly reactive Mn3+ in the presence of hydrogen peroxide which is stabilised by fungal chelators, such as oxalic acid, and oxidises the phenolic units of lignin forming free radicals.

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[R-OOH] H2O2 Fe3 +

H2O[R-OH]

3+

Mn

Ferric MnP Fe4 +

O

RH MnP-Compound I

H++ R∙ 2+

Mn

Mn2 + 4+

O

Fe

R∙ + H+

MnP-Compound II Mn3 +

RH

Figure 4.3 The catalytic cycle of manganese-dependent peroxidase (MnP). Reprinted from Enzyme and Microbial Technology 30, Hofrichter M., Review: lignin conversion by manganese peroxidase (MnP), 454–466, Copyright 2002, with permission from Elsevier Ltd., UK.

The catalytic cycle of MnP (Figure 4.3) is essentially the same as the one of the LiP save Mn(II) is necessary to complete the cycle. It includes the native ferric enzyme as well as the reactive intermediates (compound I and compound II) [26]. In contrast to other peroxidases, MnP uses Mn2+ as the preferred substrate. The cycle begins with the binding of hydrogen peroxide to the native ferric enzyme and the formation of an ironperoxide complex [26]. The subsequent cleavage of the peroxide oxygen-oxygen bond requires a two-electron transfer from the heme resulting in the formation of the MnPcompound I and the release of a water molecule. A subsequent reduction continues through the MnP-compound II. A mono-chelated Mn2+ ion acts as the one-electron donor for this enzyme intermediate and is oxidised to Mn3+. The reduction of the MnPcompound II proceeds in a similar way and another Mn3+ is formed from Mn2+ leading to the generation of the native enzyme and the release of a second water molecule [26]. MnP-compound I resembles that of the LiP and can, in addition to Mn2+, be reduced by other electron donors (ferrocyanide, phenolics). However, MnP-compound II is reduced by other substrates very slowly and requires Mn2+ to complete the catalytic cycle. The Mn3+ formed during the cycle is stabilised by carboxylic acids (oxalate, malonate, malate, tartrate or lactate). The chelates of Mn3+ cause one-electron oxidations of various substrates. As LiP, MnP is sensitive to high concentrations of hydrogen peroxide which cause reversible inactivation of the enzyme by forming the MnP-compound III.

4.2.3

Versatile Peroxidase

The enzyme versatile peroxidase (EC 1.11.1.16, hybrid peroxidases, polyvalent peroxidases, family 2 at http://www.cazy.org, VP) shares the catalytic properties of both LiP

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−

VA VP

VA

[Fe3+]

C-lB

H2O2 Mn H2O

3+

[Fe3+Trp.]

Mn2+

C-IlA

C-IA [Fe =0 P.] 4+

Mn2+

Mn3+

[Fe4+=0] VA−

VA C-lB

[Fe4+=0 Trp.]

Figure 4.4 The catalytic cycle of versatile peroxidase (VP). Reprinted from Journal of Molecular Biology 354, Pérez-Boada M, Ruiz-Dueñas J, Pogni R, Basosi R, Choinowski T, Martínez MJ, Piontek K, Martínez AT, Versatile peroxidase oxidation of high redox potential aromatic compounds: Site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways, 385–402, Copyright 2005, with permission from Elsevier Ltd., UK.

and MnP [27, 28]. Thus as MnP, it has high affinity for Mn(II) and catalyses the oxidation of Mn(II) to Mn(III) and as LiP, it oxidises both phenolic and non-phenolic substrates in the absence of Mn(II). It was first described in the white-rot fungus Pleurotus eryngii [29] and seems to be produced by fungi from the genera Pleurotus, Bjerkandera and Lepista and maybe by Panus and Trametes species [30]. VP is considered a hybrid between MnP and LiP [25], since it can oxidise not only Mn2+ but also veratryl alcohol and phenolic aromatic compounds with high molecular weight by manganese-independent reactions [31]. In Figure 4.4 the catalytic cycle of VP is shown [30]. It resembles the one reported for LiP (Figure 4.2): the enzyme catalyses the electron transfer from the substrate to form the intermediate compounds I and II.

4.2.4

Laccase

Laccase (EC 1.10.3.2, p-diphenol:oxygen oxidoreductases, lignin oxidases family 1, http://www.cazy.org/Auxiliary-Activities.html) is a multi-copper-containing oxidase. It was first discovered by Yoshida [32] in the exudates of the Japanese lacquer tree Toxicodendron vernicifluum (formerly Rhus vernicifera), from which the name laccase was taken, and characterised as a metal-containing oxidase by Bertrand [33]. Fungal laccases were also discovered during the 19th century [34, 35], white-rot fungi being the most efficient laccase producers [36]. Laccases are widely distributed in nature and, thus, are found in plants, fungi, bacteria [37] and a few insects [38].

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Cu H O +

4R ++

T1

Cu

4H

+

+

Cu

Cu

4RH

O2

+

+

Cu

Cu

H O

Fully reduced enyme ++

++

T3 Cu

++

Cu

++

Cu

Cu 2− O2

T2

++

++

Cu

Cu

+

2H Resting (fully oxidized) enzyme

H O

H2O +

2H

++

++

Cu

Cu

H2O

+

Cu

Peroxide intermediate

O ++

Cu

Oxy radical intermediate

Figure 4.5 The catalytic cycle of laccase. Reprinted from Applied Biochemistry and Biotechnology, Wong D.W.S., Structure and action mechanism of ligninolytic enzymes, 174–209, 2009, reproduced with kind permission of Springer Science and Business Media, Germany.

Besides lignin degradation, fungal laccases are also involved in other processes such as the formation of fruiting bodies, synthesis of melanin and other pigments, sporulation, conidiation and plant pathogenesis [39, 40]. Typical fungal laccases contain four copper atoms in different sites which, based on their spectroscopic characteristics, are classified into three categories [41]: one Type 1 (T1) copper, one Type 2 (T2) copper and two Type 3 (T3) coppers [9]. The T1 site contains the mononuclear “blue” copper and the T2 and T3 sites form a tri-nuclear copper cluster. Some “non-blue” fungal laccases (Pleurotus ostreatus POXA1w, Phellinus ribis laccase) are reported to harness other metals (Zn, Fe, Mn) instead of some of the above-mentioned copper atoms [42, 43]. Laccase catalyses the oxidation of phenols, anilines and aromatic thiols [9] with the simultaneous four electron reduction of one oxygen molecule to water [44]. It is assumed that four electrons are transferred from the T1 reducing substrate-binding site to the T2/T3-copper site during redox reactions [45]. The electron migration between the substrate and the enzyme is regulated by the T1 site [45], and, thus, depends on the redox potential (Table 4.2). The catalytic cycle of laccase enzyme is depicted in Figure 4.5 [31]. The first step is the reduction of the reducing substrate at the T1 site [46–49]. The electrons extracted from the reducing substrate are transferred to the T2/T3 tri-nuclear site, resulting in the conversion of the resting form (fully oxidised) of the enzyme to a fully reduced state. A successive four-electron oxidation (from four substrate molecules) is required to fully reduce the enzyme. Laccase cannot directly oxidise all substrates either because of their large size, which hinders their introduction into the active site of the enzyme, or because of their

Current Trends in the Production of Ligninolytic Enzymes Non-phenolic substrate

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Oxidised non-phenolic substrate

Oxidised mediator

Mediator

O2 Laccase H2O2 Mediator

Figure 4.6 Laccase mediator system (LMS) [49].

particular high redox potential. However, it was shown that in the presence of lowmolecular weight organic compounds acting as electron transfer mediators, laccase was also able to oxidise non-phenolic structures [44, 49]. The first step of the laccase mediator system (LMS) is the oxidation of the mediator by the laccase enzyme forming highly active radical cations capable to oxidise the bulky or high redox potential substrate that laccase alone cannot oxidise (Figure 4.6) [49]. Thus, the mediator acts as an electron shuttle between the substrate and the enzyme [50, 51]. More than 100 redox mediators have been described but the most commonly used are 2,2’ - azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1-hydroxybenzotriazole (HBT). However, these synthetic mediators are toxic and expensive, which has driven the search for natural ones. In this sense, Camarero et al., [52] reported that several ligninderived phenols, such as syringaldehyde and acetosyringone, could be low-cost and environmentally friendly alternatives to synthetic mediators.

4.3

Sources of Ligninolytic Enzymes

4.3.1 Wood-Degrading Fungi Wood-degrading or ligninolytic fungi can be divided into white-rot, brown-rot and soft-rot depending on their ability to degrade or modify different components of wood. White-rot fungi and some related litter-decomposing fungi are the only organisms able to mineralise lignin efficiently [10, 53]. Most white-rot fungi belong to the Basidiomycota phylum (Polyporales and Agaricales orders). Their growth substrates are cellulose and hemicellulose but lignin degradation occurs at the end of the primary growth by secondary metabolism in deficiency of nutrients, such as nitrogen, carbon or sulphur [10, 54]. Fungal attack is an oxidative and non-specific process, which decreases the methoxy, phenolic and aliphatic content of lignin, cleaves the aromatic rings and creates new carbonyl groups [10, 54]. These changes in the lignin molecule result in depolymerisation and carbon dioxide production [54]. The fungus P. chrysosporium (order Thelephorales) was the first white-rot fungus studied and has become a model fungus for lignin biodegradation studies. The name white-rot derives from the white appearance of the wood attacked by these fungi due to the removal of the dark coloured lignin. Most white-rot fungi degrade simultaneously all the main wood components (i.e., lignin, cellulose and hemicellulose) and are called

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non-selective lignin degraders (e.g., Trametes versicolor, Fomes fomentarius). However, some of them degrade lignin and hemicellulose more selectively than cellulose [10, 55] and are called selective lignin degraders (e.g., Ceriporiopsis subvermispora, Dichomitus squalens, P. chrysosporium, Phlebia radiata). The selective white-rot degraders are very interesting from a biotechnological point of view, since they remove lignin leaving the valuable cellulose intact [56]. Simultaneous white-rot occurs mainly on hardwoods (e.g., birch and aspen) whereas selective white-rot occurs both on hardwoods and on softwoods (e.g., spruce and pine). Brown-rot fungi degrade cellulose and hemicellulose from wood while partially modifying lignin [6], resulting in a dark brown shrunken wood broken into cubical fragments. This brown residue may remain in the forest for a long time without further degradation, although it is more reactive than native lignin [10, 54, 55]. Brown-rot fungi comprise only about 7% of the lignin-degrading basidiomycetes [4, 6]. In addition to the degradation of wood, brown-rot fungi together with litter-decomposing fungi are involved in the formation of humic substances, lignin being the most important precursor [57]. Soft-rot fungi are ascomycetes and deuteromycetes that usually attack wet wood, leaving a soft, brown residue [6, 58]. They are predominant in environments that are too harsh for basidomycetous ligninolytic fungi [10, 59, 60]. Thus, they tolerate wider ranges of temperature, pH and oxygen limitation than white- or brown-rot fungi [10, 59, 60]. Soft-rot fungi degrade all wood components, but lignin removal is slow and partial [61, 62]. Most ligninolytic fungi are basidomycetous white-rot fungi. They secrete an extracellular and non-specific enzymatic complex, mainly composed of the ligninolytic enzymes LiP, MnP, VP and laccase, which generate free radicals that randomly attack the lignin molecule, breaking the covalent bonds and releasing a range of mainly phenolic compounds. Almost all white-rot fungi produce MnP and laccase but only some of them produce LiP, the only enzyme able to degrade the non-phenolic lignin units. In addition, other enzymes, such as glyoxal oxidase (E.C. 1.2.3.5), superoxide dismutase (E.C. 1.15.1.1), glucose oxidase (E.C. 1.1.3.4), aryl alcohol oxidase (E.C. 1.1.3.7) and cellobiose dehydrogenase (E.C. 1.1.99.18), are associated with the ligninolytic enzymes in lignin breakdown but they are unable to degrade lignin on their own. They produce the hydrogen peroxide required by peroxidases (LiP, MnP and VP) or serve to link lignocellulose degradation pathways [63]. Besides their extracellular ligninolytic system, white-rot fungi also have an endocellular system, involving the cytochrome P-450 monooxigenase-epoxide hydrolase which is supposed to cooperate with the ligninolytic system in the general mechanism of lignin degradation [64, 65]. Moreover, whiterot fungi secrete low molecular weight mediators that increase the range of compounds that they can degrade [66]. Ligninolytic enzyme production by white-rot fungi occurs during their secondary metabolism triggered by nutrient limitation, particularly nitrogen. However, some taxa, produce LiP, MnP and laccase under conditions of nitrogen sufficiency [67]. Also, growth conditions such as temperature and agitation of fungal cultures significantly affect the production and levels of activity of ligninolytic enzymes in white-rot fungi [68–70]. Additionally, other authors have reported that ligninolytic enzyme production by white-rot fungi is affected by mediator compounds, various other chemicals and required-metal (Mn2+, Cu2+) concentrations [71–73].

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The first studies on lignin degradation by white-rot-fungi were performed with the white-rot fungus P. chrysosporium which has become the model fungus for lignin degradation studies [74]. Since then, other species of white-rot-fungi with promising ability to degrade recalcitrant pollutants have been described such as those belonging to the genera Pleurotus, Bjerkandera, Coriolopsis, Phlebia and Trametes [75] and many more continue being described and discovered. In fact, there is a recent growing interest in discovering new and alternative fungal strains able to produce ligninolytic enzymes with improved properties and/or under extreme environmental conditions.

4.3.2 Bacteria Several genera of bacteria, especially soil bacteria, have shown ability to react with lignin and likely produce small aromatic compounds [1]. However, homologous of the most common fungal ligninolytic peroxidases (i.e., LiPs MnPs and VPs) have not been found in any studies on ligninolytic bacteria [76]. This is likely due to the fact that bacteria lacks the machinery to produce complex enzymes with glycosylation and disulphide bonds. Nevertheless, it has been found that bacteria can produce another type of peroxidase, the so-called dye-decolourising peroxidase (DyP, EC 1.11.1.19) [77]. DyP represents a newly discovered family of heme-containing peroxidases which has recently received attention due to their ability to degrade lignin and other compounds [78–81]. The first discovered member of this enzyme family, DyP from the white-rot fungus Bjerkandera adusta, was isolated and characterised in 1999 [82]. Studies of the activity of this enzyme on synthetic anthraquinone and azo dyes have served to name this family of peroxidases [83]. In recent years, many bacterial DyPs have been described in the literature [84]. Most of the known, studied and applied laccases are from fungal origin. However, in the last few years bacterial laccases have aroused considerable interest concerning their possible role in lignin degradation and other biotechnological applications. Recent advances in genome analysis and other approaches have made possible the identification of numerous laccases in bacteria [85–87], particularly from Streptomyces and Bacillus species [88]. They have the advantage of tolerating high temperatures and alkaline conditions which makes them very attractive for industrial applications.

4.4

Production of Ligninolytic Enzymes

The versatility of ligninolytic enzymes makes them very interesting for a wide variety of industrial and biotechnological applications such as pulp bleaching, delignification of lignocellulosic materials, organic synthesis, bioremediation, wastewater treatment, soil treatment, etc [89–91]. This has led to an increased demand of these enzymes in recent years. The production of enzymes from microbial sources is a costly process, so research in this area is oriented towards the search for efficient production systems. A good strategy to minimise the production cost is the use of low-cost raw materials, such as the wastes from the agricultural, food and forestry industries, under solid-state fermentation (SSF) conditions which is the preferred cultivation method for ligninolytic fungi. Biological wastes may contain significant concentrations of soluble carbohydrates and

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inducers of enzyme synthesis ensuring an efficient production of ligninolytic enzymes. In addition, the use of these wastes helps to solve the environmental problems caused by their disposal, thus, contributing to a sustainable economy which is one of the current initiatives of the European Union (EU). SSF is generally defined as the growth of microorganisms on solid materials in the absence or near absence of free-flowing water [92]. In SSF, the solid material functions both as a nutrient supplier and as an anchorage place for the microorganism [93]. The selection of a suitable support to perform SSF is essential, since the success of the process depends on it [94]. The substrate must be locally available in large quantities and of low-cost to make the entire process cost-effective [95]. Ligninolytic enzymes have been produced using a great variety of biological wastes as support-substrates under SSF conditions by different strains of ligninolytic fungi [94]. Kojani and Rajput [96] have reviewed some recent production of ligninolytic enzymes under SSF conditions using different lignocellulosic wastes as support-substrates. In Table 4.3 recent studies on ligninolytic enzyme production using biological wastes as support-substrate under SSF conditions are presented.

4.5

Purification of Ligninolytic Enzymes

After the fermentation process, several post-fermentation operation units can be needed to get the final enzyme product. As most of the ligninolytic enzymes are extracellular, the first operation is usually the separation of the cells and support from the broth. The final use of the ligninolytic enzyme will dictate the grade of purity needed. Thus, in many industrial applications of the ligninolytic enzymes, such as dye decolouration and biobleaching, the crude enzyme is used. However, some applications prefer the use of purified enzymes [107]. Precipitation (e.g., ammonium sulphate precipitation, acetone precipitation) followed by chromatographic techniques (e.g., ion exchange, affinity, gel filtration) are the methods commonly used to purify ligninolytic enzymes. The number of purification steps required depends on the substrate and the fermentation system used to produce the enzyme.

4.6

Potential Applications of Ligninoytic Enzymes

Due to their low substrate specificity, ligninolytic enzymes can degrade a wide variety of organic compounds, thus having potential applications in numerous fields.

Food industry Laccase can be applied to remove undesirable phenolic compounds from fruit juice, beer and wine, ascorbic acid determination, sugar beet pectin gelation and baking [108–110].

Corn stover

Potato peel waste

Rice husk and rice straw Sunflower seed hulls

Water hyacinth and sawdust Laccase

Rice straw

Paddy straw and corn husk

Rice straw

Orange waste

Pine sawdust, rice straw and MnP soybean powder

Pleurotus eryngii Pleurotus ostreatus Trametes versicolor

P. ostreatus

Ganoderma lucidum

Pycnoporus sanguineus

Schizophyllum commune

P. ostreatus

Pyrenophora phaeocomes

Pleurotus pulmonarius

Irpex lacteus

Laccase

Laccase

Laccase

MnP LiP Laccase

Laccase Laccase

Laccase MnP

Laccase MnP LiP

Laccase

Wheat bran

Cyathus bulleri

Ligninolytic enzyme

Support-substrate

Microorganism

950 U/L (84 h)

12.2 U/mL (day 20)

10859 U/g (day 4)

2.54 U/g dry substrate (day 14)

1846.7 U/g (144 h) 1347.2 U/g (144 h) 316.3 U/g (144 h)

32.02 U/g dry substrate (day 9)

10927 U/kg (day 10) 16442 U/kg (day 5)

6708.3 ± 75 U/L (day 17) 2503.6 ± 50 U/L (day 17)

189.7 ± 4.4 U/L (day 12) 50.4 ± 7.1 U/L (day 9) 369.0 ± 1.8 U/L (day 18)

94.4 U/mg (day 12)

Maximum activity

[106]

[105]

[104]

[103]

[102]

[101]

[100]

[99]

[98]

[97]

Reference

Table 4.3 Recent studies on ligninolytic enzyme production using different substrates under solid-state fermentation (SSF) conditions. References are arranged in chronological order.

Current Trends in the Production of Ligninolytic Enzymes 79

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Pulp and paper industry Ligninolytic enzymes can replace the chemical methods of pulping and bleaching in the pulp and paper industry making these processes environmentally friendly. Thus, one of the most studied applications in the industry is the laccase-mediator bleaching of kraft pulp whose efficiency has been proven in mill-scale trials [111]. Also, LiP was proved to be efficient in biobleaching [112] and MnP in biopulping [113, 114].

Textile industry Ligninolytic enzymes catalyse the transformation of a wide variety of synthetic dyes including azo, triphenylmethane, anthraquinone, phthalocyanine and heterocyclic dyes [115–117]. They are also applied in cotton bleaching [118] and in denim bleaching. Thus, in 1996 Novozyme (Novo Nordisk, Denmark) launched the first commercial laccase for bleaching purposes without requiring a redox mediator under the trade name of DeniliteI™. Later, different enzyme companies commercialised different laccase formulations for specific textile processes [119].

Removal of pollutants Industrial activities result in the release of hazardous pollutants into the environment causing a serious negative impact in the ecosystem and human health. Conventional treatment techniques are not able to remove these pollutants effectively. In this sense, the treatment with ligninolytic enzymes has shown to be a promising technology. Thus, they have been shown to remove a wide range of recalcitrant xenobiotic compounds such as bisphenol A (BPA) [120], polycyclic aromatic hydrocarbons (PAHs) [121], herbicides [122], pesticides [123], pharmaceuticals compounds [124] and synthetic dyes [117].

Other applications Laccase enzymes can be used in organic synthesis due to their ability to generate polymers that are impossible to be produced via chemical synthesis [125]. Also, MnP was used for acrylamide polymerization [126] and LiP showed to be applicable to produce dermatological preparations [127]. Ligninolytic enzymes can also be used for the development of biosensors [128]. Recently, ligninolytic enzymes have been applied in the production of second-generation biofuels [129].

4.7

Outlook

The use of ligninolytic enzymes for industrial and biotechnological applications holds great potential. However, the use of these enzymes in industrial processes has been limited by several factors, mainly their high cost, instability, availability in small amounts, susceptibility to be attacked by proteases and intermediate inhibition processes. The increasing advances in protein engineering and enzyme immobilisation technologies

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are powerful tools to provide enzymes with improved functional properties. This will make possible the development of stable and recoverable ligninolytic enzymes for multipurpose biotechnological and industrial applications.

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37. Dwivedi, U.N., Singh, P., Pandey, V.P., Kumar, A., Structure–function relationship among bacterial, fungal and plant laccases. Journal of Molecular Catalysis B Enzymatic, 68(2), 117–128, 2011. 38. Xu, F., Recent Progress in Laccase Study: Properties, Enzymology, Production, and Applications: The Encyclopedia of Bioprocessing Technology: Fermentation, Biocatalysis, and Bioseparation. New York, John Wiley & Sons, 1999. 39. Alexandre, G., Zhulin, I.B., Laccases are widespread in bacteria. Trends Biotechnol., 18(2), 41–42, 2000. 40. Mayer, A.M., Staples, R.C., Laccase: new functions for an old enzyme. Phytochemistry, 60(6), 551–565, 2002. 41. Malmström, B.G., Enzymology of oxygen. Annu. Rev. Biochem., 51(1), 21–59, 1982. 42. Palmieri, G., Giardina, P., Bianco, C., Scaloni, A., Capasso, A., Sannia, G., A novel white laccase from Pleurotus ostreatus. J. Biol. Chem., 272(50), 31301–31307, 1997. 43. Min, K.L., Kim, Y.H., Kim, Y.W., Jung, H.S., Hah, Y.C., Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis. Arch. Biochem. Biophys., 392(2), 279–286, 2001. 44. Bourbonnais, R., Paice, M.G., Oxidation of non-phenolic substrates. FEBS Lett., 267(1), 99–102, 1990. 45. Solomon, E.I., Sundaram, U.M., Machonkin, T.E., Multicopper oxidases and oxygenases. Chem. Rev., 96(7), 2563–2606, 1996. 46. Messerschmidt, A., Ladenstein, R., Huber, R., Bolognesi, M., Avigliano, L., Petruzzelli, R., et al., Refined crystal structure of ascorbate oxidase at 1.9 A resolution. J. Mol. Biol., 224(1), 179–205, 1992. 47. Zoppellaro, G., Sakurai, T., Huang, H., A novel mixed valence form of Rhus vernicifera laccase and its reaction with dioxygen to give a peroxide intermediate bound to the trinuclear center. J. Biochem., 129(6), 949–953, 2001. 48. Huang, H., Zoppellaro, G., Sakurai, T., Spectroscopic and kinetic studies on the oxygencentered radical formed during the four-electron reduction process of dioxygen by Rhus vernicifera laccase. J. Biol. Chem., 274(46), 32718–32724, 1999. 49. Call, H.P., Mücke, I., History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym®-process). J. Biotechnol., 53(2-3), 163–202, 1997. 50. Galli, C., Gentili, P., Chemical messengers: mediated oxidations with the enzyme laccase. J. Phys. Org. Chem., 17(11), 973–977, 2004. 51. Widsten, P., Kandelbauer, A., Laccase applications in the forest products industry: A review. Enzyme Microb. Technol., 42(4), 293–307, 2008. 52. Camarero, S., Ibarra, D., Martínez, M.J., Martínez, A.T., Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ. Microbiol., 71(4), 1775–1784, 2005. 53. Kirk, T.K., Cullen, D., Enzymology and molecular genetics of wood degradation by whiterot fungi. In: Young R. A, Akhtar M, eds. Environmentally Friendly Technologies for the Pulp and Paper Industry. New York, Wiley. pp. 273–307, 1998. 54. Kirk, T.K., Farrell, R.L., Enzymatic "combustion": the microbial degradation of lignin. Annu. Rev. Microbiol., 41(1), 465–501, 1987. 55. Blanchette, R.A., Degradation of the lignocellulose complex in wood. Can. J. Bot., 73(S1), 999–1010, 1995. 56. Dashtban, M., Schraft, H., Syed, T.A., Qin, W., Fungal biodegradation and enzymatic modification of lignin. Int. J. Biochem. Mol. Biol., 1(1), 36, 2010.

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57. Steffen, K., Tuomela, “Karikkeenhajottajat, M., Litter-decomposing fungi. In: Timonen S, Valkonen J, eds. Sienten Biologia (Biology of Fungi. Tallinn, Gaudeamus Oy. pp. 279–289, 2013. 58. Carlile, M.J., Watkinson, S.C., Gooday, G.W., The Fungi. London, Academic Press, 2001. 59. Blanchette, R.A., Delignification by wood-decay fungi. Annu. Rev. Phytopathol., 29(1), 381–403, 1991. 60. Daniel, G., Nilsson, T., Developments in the study of soft rot and bacterial decay. In: Bruce A, Palfreyman J. W, eds. Forest Products Biotechnology. Great Britain, Taylor & Francis. pp. 37–62, 1998. 61. Rayner, A.D.M., Boddy, L., Fungal Decomposition of Wood: Its Biology and Ecology. New York, John Wiley & Sons, 1988. 62. Eaton, R.A., Hale, M.D.C., Wood Decay, Pests, and Protection. Cambridge, Chapman & Hall, 1993. 63. Leonowicz, A., Cho, N.S., Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., et  al., Fungal laccase: properties and activity on lignin. J. Basic Microbiol., 41(3-4), 185–227, 2001. 64. Bezalel, L., Hadar, Y., Cerniglia, C.E., Enzymatic mechanisms involved in phenanthrene degradation by the white rot fungus pleurotus ostreatus. Appl. Environ. Microbiol., 63(7), 2495, 1997. 65. van den Brink, H.M., van Gorcom, R.F., van den Hondel, C.A., Punt, P.J., Hondel, C.A.M.J.J.Vden., Cytochrome P450 enzyme systems in fungi. Fungal Genet. Biol., 23(1), 1–17, 1998. 66. Pointing, S.B., Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol., 57(1-2), 20, 2001. 67. Buswell, J.A., Mollet, B., Odier, E., Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiol. Lett., 25(2-3), 295–299, 1984. 68. Vyas, B.R.M., Volc, J., Šašek, V., Ligninolytic enzymes of selected white rot fungi cultivated on wheat straw. Folia Microbiol. (Praha)., 39(3), 235–240, 1994. 69. Darah, I., Ibrahim, C.O., Asia Pacific Journal of Molecular Biology and Biotechnology, 4, 174, 1996. 70. Podgornik, H., Podgornik, A., Milavec, P., Perdih, A., The effect of agitation and nitrogen concentration on lignin peroxidase (LiP) isoform composition during fermentation of Phanerochaete chrysosporium. J. Biotechnol., 88(2), 173–176, 2001. 71. Dittmer, J.K., Patel, N.J., Dhawale, S.W., Dhawale, S.S., Production of multiple laccase isoforms by Phanerochaete chrysosporium grown under nutrient sufficiency. FEMS Microbiol. Lett., 149(1), 65–70, 1997. 72. Scheel, T., Höfer, M., Ludwig, S., Hölker, U., Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds. Appl. Microbiol. Biotechnol., 54(5), 686–691, 2000. 73. Galhaup, C., Wagner, H., Hinterstoisser, B., Haltrich, D., Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb. Technol., 30(4), 529–536, 2002. 74. Bumpus, J.A., Tien, M., Wright, D., Aust, S.D., Oxidation of persistent environmental pollutants by a white rot fungus. Science, 228(4706), 1434–1436, 1985. 75. Rodrıguez, E., Nuero, O., Guillén, F., Martınez, A.T., Martınez, M.J., Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase. Soil Biology and Biochemistry, 36(6), 909–916, 2004.

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76. de Gonzalo, G., Colpa, D.I., Habib, M.H., Fraaije, M.W., Bacterial enzymes involved in lignin degradation. J. Biotechnol., 236, 110–119, 2016. 77. van Bloois, E., Torres Pazmiño, D.E., Winter, R.T., Fraaije, M.W., A robust and extracellular heme-containing peroxidase from Thermobifida fusca as prototype of a bacterial peroxidase superfamily. Appl. Microbiol. Biotechnol., 86(5), 1419–1430, 2010. 78. Sugano, Y., DyP-type peroxidases comprise a novel heme peroxidase family. Cell. Mol. Life Sci., 66(8), 1387–1403, 2009. 79. Colpa, D.I., Fraaije, M.W., van Bloois, E., DyP-type peroxidases: a promising and versatile class of enzymes. J. Ind. Microbiol. Biotechnol., 41(1), 1–7, 2014. 80. Singh, R., Eltis, L.D., The multihued palette of dye-decolorizing peroxidases. Arch. Biochem. Biophys., 574, 56–65, 2015. 81. Yoshida, T., Sugano, Y., A structural and functional perspective of DyP-type peroxidase family. Arch. Biochem. Biophys., 574, 49–55, 2015. 82. Kim, S.J., Shoda, M., Purification and characterization of a novel peroxidase from Geotrichum candidum dec 1 involved in decolorization of dyes. Appl. Environ. Microbiol., 65(3), 1029, 1999. 83. Sugano, Y., Muramatsu, R., Ichiyanagi, A., Sato, T., Shoda, M., DyP, a Unique Dyedecolorizing peroxidase, represents a novel heme peroxidase family. J. Biol. Chem., 282(50), 36652–36658, 2007. 84. Lambertz, C., Ece, S., Fischer, R., Commandeur, U., Progress and obstacles in the production and application of recombinant lignin-degrading peroxidases. Bioengineered, 7(3), 145–154, 2016. 85. Alexandre, G., Zhulin, I.B., Laccases are widespread in bacteria. Trends Biotechnol., 18(2), 41–42, 2000. 86. Claus, H., Laccases and their occurrence in prokaryotes. Arch. Microbiol., 179(3), 145–150, 2003. 87. Santhanam, N., Vivanco, J.M., Decker, S.R., Reardon, K.F., Expression of industrially relevant laccases: prokaryotic style. Trends Biotechnol., 29(10), 480–489, 2011. 88. Martins, L.O., Durão, P., Brissos, V., Lindley, P.F., Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology. Cell. Mol. Life Sci., 72(5), 911– 922, 2015. 89. Hamid, M., Khalil-ur-Rehman, Rehman, K.-ur., Potential applications of peroxidases. Food Chem., 115(4), 1177–1186, 2009. 90. Maciel, M.J.M., Siva, A.C., Ribeiro, H.C.T., Electronic Journal of Biotechnology, 13, 2010. 91. Yadav, M., Yadav, H.S., Applications of ligninolytic enzymes to pollutants, wastewater, dyes, soil, coal, paper and polymers. Environ. Chem. Lett., 13(3), 309–318, 2015. 92. Pandey, A., Soccol, C.R., Mitchell, D., New developments in solid state fermentation: I-bioprocesses and products. Process Biochemistry, 35(10), 1153–1169, 2000. 93. Krishna, C., Solid-state fermentation systems-an overview. Crit. Rev. Biotechnol., 25(1-2), 1–30, 2005. 94. Rodríguez Couto, S., Sanromán, M.A., Application of solid-state fermentation to ligninolytic enzyme production. Biochem. Eng. J., 22(3), 211–219, 2005. 95. European Commission, Towards a circular economy: A zero waste programme for europe, European Commission: Brussels. Belgium, 2014. 96. Kojani, R.D., Rajput, K.S., Journal of Bioprocessing and Biotechniques, 5, 2015. 97. Vats, A., Mishra, S., Identification and evaluation of bioremediation potential of laccase isoforms produced by Cyathus bulleri on wheat bran. J. Hazard. Mater., 344, 466–479, 2018.

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98. Wyman, V., Henríquez, J., Palma, C., Carvajal, A., Lignocellulosic waste valorisation strategy through enzyme and biogas production. Bioresour. Technol., 247, 402–411, 2018. 99. Ozcirak Ergun, S., Ozturk Urek, R., Ergun, S.O., Urek, R.O., Production of ligninolytic enzymes by solid state fermentation using Pleurotus ostreatus. Annals of Agrarian Science, 15(2), 273–277, 2017. 100. Postemsky, P.D., Bidegain, M.A., González-Matute, R., Figlas, N.D., Cubitto, M.A., Pilotscale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum. Bioresour. Technol., 231, 85–93, 2017. 101. Wang, Z., Liu, J., Ning, Y., Liao, X., Jia, Y., Eichhornia crassipes: Agro-waster for a novel thermostable laccase production by Pycnoporus sanguineus SYBC-L1. J. Biosci. Bioeng., 123(2), 163–169, 2017. 102. Asgher, M., Wahab, A., Bilal, M., Nasir Iqbal, H.M., Iqbal, H.M.N., Lignocellulose degradation and production of lignin modifying enzymes by Schizophyllum commune IBL-06 in solid-state fermentation. Biocatal. Agric. Biotechnol., 6, 195–201, 2016. 103. Das, A., Bhattacharya, S., Panchanan, G., Navya, B.S., Nambiar, P., Production, characterization and Congo red dye decolourizing efficiency of a laccase from Pleurotus ostreatus MTCC 142 cultivated on co-substrates of paddy straw and corn husk. Journal of Genetic Engineering and Biotechnology, 14(2), 281–288, 2016. 104. Rastogi, S., Soni, R., Kaur, J., Soni, S.K., Unravelling the capability of Pyrenophora phaeocomes S-1 for the production of ligno-hemicellulolytic enzyme cocktail and simultaneous bio-delignification of rice straw for enhanced enzymatic saccharification. Bioresour. Technol., 222, 458–469, 2016. 105. Inácio, F.D., Ferreira, R.O., Araujo, C.A.Vde., Peralta, R.M., Souza, C.G.Mde., Vaz de Araujo, C.A., Production of enzymes and biotransformation of orange waste by Oyster mushroom, pleurotus pulmonarius (Fr.) Quél. Adv. Microbiol., 05(01), 1–8, 2015. 106. Zhao, X., Huang, X., Yao, J., Zhou, Y., Jia, R., Fungal growth and manganese peroxidase production in a deep tray solid-state bioreactor, and in vitro decolorization of Poly R-478 by MnP. J. Microbiol. Biotechnol., 25(6), 803–813, 2015. 107. Kokol, V., Doliška, A., Eichlerová, I., Baldrian, P., Nerud, F., Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase. Enzyme Microb. Technol., 40(7), 1673–1677, 2007. 108. Minussi, R.C., Pastore, G.M., Durán, N., Potential applications of laccase in the food industry. Trends in Food Science & Technology, 13(6-7), 205–216, 2002. 109. Selinheimo, E., Kruus, K., Buchert, J., Hopia, A., Autio, K., Effects of laccase, xylanase and their combination on the rheological properties of wheat doughs. J. Cereal Sci., 43(2), 152–159, 2006. 110. Morozova, O.V., Shumakovich, G.P., Gorbacheva, M.A., Shleev, S.V., Yaropolov, A.I., J. Biochem., 72, 1136, 2007. 111. Srebotnik, E., Hammel, K.E., Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system. J. Biotechnol., 81(2–3), 179–188, 2000. 112. Sigoillot, C., Camarero, S., Vidal, T., Record, E., Asther, M., Pérez-Boada, M., et  al., Comparison of different fungal enzymes for bleaching high-quality paper pulps. J. Biotechnol., 115(4), 333–343, 2005. 113. Kurek, B., Petit-Conil, M., Sigoillot, J.C., Herpoel, I., Ruel, K., Moukha, S., Treatment of high yield pulp with fungal peroxidases from laboratory to pilot scale study. In: Argyropoulos D, ed. Oxidative Delignification Chemistry, Fundamental and Catalysis. Washington D.C, ACS Symposium Series 785, American Chemical Society. pp. 474–486, 2001.

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114. Maijala, P., Mettalo, A., Kleen, M., Westin, C., Poppius-Levin, K., Herranen, K., Treatment of softwood chips with enzymes may reduce refining energy consumption and increase surface charge of fibers, 65, 2007. 115. Ollikka, P., Alhonmäki, K., Leppänen, V.M., Glumoff, T., Raijola, T., Suominen, I., Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium. Appl. Environ. Microbiol., 59(12), 4010, 1993. 116. Tavcar, M., Svobodová, K., Kuplenk, J., Novotny, C., Pavko, A., Acta Chim. Slov., 53, 338, 2006. 117. Bilal, M., Asgher, M., Parra-Saldivar, R., Hu, H., Wang, W., Zhang, X., et al., Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants - A review. Sci. Total Environ., 576, 646–659, 2017. 118. Tzanov, T., Basto, C., Gübitz, G.M., Cavaco-Paulo, A., Laccases to improve the whiteness in a conventional bleaching of cotton. Macromol. Mater. Eng., 288(10), 807–810, 2003. 119. Rodriguez-Couto, S., Laccases for denim bleaching: An eco-friendly alternative. TOTEXTILEJ, 5(1), 1–7, 2012. 120. Gassara, F., Brar, S.K., Verma, M., Tyagi, R.D., Bisphenol A degradation in water by ligninolytic enzymes. Chemosphere, 92(10), 1356–1360, 2013. 121. Kadri, T., Rouissi, T., Kaur Brar, S., Cledon, M., Sarma, S., Verma, M., Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci. (China)., 51, 52–74, 2017. 122. Camacho-Morales, R.L., Gerardo-Gerardo, J.L., Guillén Navarro, K., Sánchez, J.E., Producción de enzimas ligninolíticas durante la degradación del herbicida paraquat por hongos de la pudrición blanca. Revista Argentina de Microbiología, 49(2), 189–196, 2017. 123. Hirai, H., Nakanishi, S., Nishida, T., Oxidative dechlorination of methoxychlor by ligninolytic enzymes from white-rot fungi. Chemosphere, 55(4), 641–645, 2004. 124. Nagdhi, M., Taheran, M., Brar, S.K., Kermanshahi-pour, A., Verma, M., Surampalli, R.Y., Environnemental Pollution, 234, 190, 2018. 125. Aktaş, N., Tanyolaç, A., Reaction conditions for laccase catalyzed polymerization of catechol. Bioresour. Technol., 87(3), 209–214, 2003. 126. Iwahara, K., Hirata, M., Honda, Y., Watanabe, T., Kuwahara, M., Masaaki-Kuwahara, L., Biotechnol. Lett., 22(17), 1355–1361, 2000. 127. Belinky, P., Lasser, H., Dosoretz, C., Rakuto Bio Technologies Ltd, 2005. 128. Christenson, A., Dimcheva, N., Ferapontova, E. E., Gorton, L., Ruzgas, T., Stoica, L., et al., Direct electron transfer between ligninolytic redox enzymes and electrodes. Electroanalysis, 16(1314), 1074–1092, 2004. 129. Plácido, J., Capareda, S., Ligninolytic enzymes: a biotechnological alternative for bioethanol production. Bioresour. Bioprocess., 2(1), 23, 2015.

5 Asava-Arishta: A Multi-Advantageous Fermented Product in Ayurveda Varun Kumar Singh, Avinash Narwaria* and C. K. Katiyar Research & Development Ayurveda- Health Care Division, Emami Limited, 13 BT Road, Kolkata-700056 (India)

Abstract An overview of fermentation as per Ayurvedic literature and its application in modern-day medicines with various aspects of manufacturing at industrial scale is presented. Fermented products (Asava-Arishta) are unique multi-advantageous dosage form in Ayurveda with additional benefit of longer shelf life. They have been used for thousands of years as a drink, diet and health supplement, and Ayurveda specially speaks of every aspect of fermented product; among them Asava-Arishta is interesting to mention. Adoption of technological advancements to prepare an equally potent product at large scale is a challenge but this can be achieved by understanding basic principles of fermentation. Fermentation science also opens up a new window to microbial diversity and their activities impacting fermenting environment to produce therapeutically better derivatives of existing compounds in the self-generated alcohol-aqueous milieu. This milieu is also creating a special delivery pathway for drug uptake at a faster rate and improved bioavailability. Keywords: Sandhan, asava, arishta, fermentation, ayurveda, microbial diversity

5.1

Introduction

Ayurveda is a treasure of various types of dosage forms of medicine to treat health problems. Among them Asava-arishta is a unique formulation with multiple advantages. Arishta and asava are probably the oldest documented knowledge on the science of fermentation used for preparing medicines under the term ‘Sandhan Kalpana’. Sandhan Kalpana (fermentation process) is not new to the 21st century since it is an age-old technique to prepare hydro alcoholic liquid dosage form in order to get enhanced potency, increased shelf life and ease of administration. References to the process are well documented in classical texts from the Vedic age to the modern age. In the arena of the Vedic age, the preparation of ‘Soma-rasa and Sura’ (alcoholic preparation) are described in Rigveda (Rigveda 2/14/1 & 6/66/10), whereas arishta is

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (89–108) © 2019 Scrivener Publishing LLC

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described as madya in Atharva Veda for treatment purposes (Atharva Veda 2/25/14). In Yajurveda too, some references to Sandhan Kalpana were found (Yajurveda 2/10/34), which was used for medicinal purposes. For dietary use consumption of acidic fermented product like curd and vinegar was well known to human beings [1]. In the arena of Ayurvedic wisdom, Charaka Samhita described 9 yonis (source of fermentation), viz. Phala (fruits), Dhanya (cereals), Mula (roots), Pushpa (flowers), Twak (bark), Sara (exudate), Kanda (branches), Patra (leaves), and Sharkara (sugar)) for fermented medicine, and four asava-arishta are specially mentioned for Virechana karma in Kalpasthana [2]. In Sushruta Samhita, use of madya prior to surgery was well mentioned to overcome the pain of surgery. Sushruta Samhita speaks of over 21 Asavaarishta and 46 madyavarga products [3]. During Sangraha period, use of Dhataki flower (as fermentation initiator) was first mentioned in Astanga Hrdya. A total of 17 asavaarishta are mentioned in Astangasangraha and eight asava-arishta are mentioned in Astanga Hrdya [4]. In Sharangdhar Samhita, description of fermentation with indepth process of preparation and its use in disease are mentioned. Here dose, shelf life, proportion of ingredients, preparation and formulation development has been mentioned elaborately. Later on it has been classified into Madya (alcoholic fermentation) and Shukta Kalpana (Acidic fermentation) [5]. In Bhaisajya Ratnawali, a total of 50 fermented products are quoted, among them 15 are Asava and 29 are Arishta [6]. The Ayurvedic Pharmacopoeia of India, Part-II, Vol-II mentions 24 Asava-arishta with their composition, methods of preparation and physico-chemical testing parameters.A total of 57 Asava-Arishta are described in Parts I (37 Nos), II (3 Nos) and III (17 No.s) of Ayurvedic Formulary of India with complete detail of their pharmaceutics and therapeutics. Historically, alcoholic preparations were used for enjoyment due to their effect on the central nervous system (soma-rasa and sura) to produce hallucination and phantom effect on mind and emotion. This knowledge later developed as a systematic method of making medicines by introducing specific herbal ingredients to treat various ailments. In ancient times other liquid dosage forms (self-expressed liquid, decoction, etc.) were used, which were not stable for a longer period of time, thus did not fulfil the demand of medicine to be used by larger group of population over a longer period of time. At the same time sura was observed to be stable over a very long time. So ancient scholars utilized this technique to produce a medicinal product by biomedical fermentation and found a miraculous effect. Ancient scholars also optimized the production of alcohol and constituents in formulation, thus making it specific to ailments and patients.

5.2

Definition of Asava and Arishta

Since fermentation (Sandhana) is a core process that yields a different kind of liquid dosage form depending upon raw material used and process variation. As per Yadav Ji Tikramji, Sandhana has been classified under two main categories1. Madya Sandhana (Alcoholic fermentation) 2. Shukta Sandhana (Acidic fermentation)

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Alcoholic fermentation is further classified into Sura, Sidhu, Varuni, Asava and Arishta on the basis of process and yield but in this chapter we are focusing only on Asava and Arishta aspect of fermentation. According to ancient Acharyas (i.e., Charaka, Sushruta, etc.) there is not much difference between Asava and Arishta. In Charaka Samhita some of the Asavas are mentioned, which are supposed to be prepared by fermentation of different decoction, e.g., Madhavasava and in some of the Arishtas without preparing decoction, e.g., Takrarishta. But with time more clarification was seen in formulation classification and asava was defined as formulations that are prepared by sandhana of drugs without making decoction of raw material, whereas arishta kalpana was made with decoction of raw materials. This process difference with involvement of heat creates a lot of pharmacological variation in between these two process, thus altering the chemical changes occuring within liquid media. As per the Ayurvedic Formulary of India, Asava and Arishta are medicinal preparations made by soaking the drugs, either in powder form or in the form of decoction, in a solution of sugar or jaggery, as the case may be, for a specified period of time, during which it undergoes a process of fermentation generating alcohol, thus facilitating the extraction of the active principles contained in the drugs. The alcohol, so generated, also serves as a preservative [7].

5.3

Method of Preparation for Asava Arishta

Initially, Asava-Arishta not used for commercial purpose as they were used for enjoyment and fun, and later on as medicine. Nowadays in the arena of the global commercial market, the demand for fermented products is growing. Currently we need a larger amount of Asava arishta to fulfill public demand with a reasonable price point. For such purposes, we have to use new techniques to meet the requirement of human healthcare with the same principle and potency as mentioned in classical texts. To cater the emerging needs new techniques has been applied for the production, storage and packaging of asava arishta. New equipments are replacing older ones like larger vessel for decoction, digital controlled fermenter technology, updated filtration technology, controlled storage, and new packaging technology for increasing shelf life. Ayurveda, a classical science, has details about preparation of fermented products in a systematic manner. Preparation of Asava is carried out by Hima (Cold decoction)/ Jala, Swarasa (expressed juice) process. In the preparation of Asava, the drug is coarsely powdered and added to water, to which the prescribed quantities of honey, jaggery /sugar are added. It contains dilute solutions of the readily soluble constituents of crude drugs [8]. Arishta is prepared by soaking the drugs in water for a period of time (8 hr) before decoction, which facilitates the better extraction of active principles into Kashaya (decoction) and thereby increases in potency [9]. Age-old techniques involve earthen pot/wooden pot, husk for maintaining temperature, iron pot for making decoctions etc. The processes utilized in different steps while preparation of AsavaArishta may be divided into three phases [10], namely, Purva Karma, Pradhana Karma and Pashchata Karma.

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5.3.1 Purvakarma Includes (Pre-Fermentation Process) 1. Selection of Sandhana Patra (Fermentation vessel) 2. Dhoopana (Technique used or sterilization) 3. Collection of drugs (Raw material collection and authentication)

5.3.2 Pradhanakarma Includes (Fermentation Process) 1. 2. 3. 4. 5.

5.3.3 1. 2. 3. 4.

Drava Dravya (liquid part preparation cold process or decoction) MadhuraDravya (sweetener) SandhanaDravya (Fermentation initiator/ fermenting agent) Prakshepa Dravya (volatile oil containing drugs) Filling and sealing of Patra (Packaging)

Pashchata Karma Includes (Post-Fermentation) Observations (Quality control) Filtration Maturation Storage

5.3.4 Industry Practice for Preparation of Asava-Arishta Today, industry has practiced vigorous production of Asava arishta, for such efficiency high-end technology is used for manufacturing. The process of fermentation has been divided into the following steps and demonstrated as given in flow chart (Figure 5.1). Industries make asava arishta for commercial purpose in larger quantity and thus use high-end technology other than traditional equipments. Contemporary intervention in processing of asava arishta (Table 5.1) has an impact on the way of processing and ensuring reproducibility of formulation. 1. 2. 3. 4.

Preparation of raw material Fermentation Filtration and maturation Packaging and storage

5.3.4.1 Preparation of Raw Materials For the preparation of alcoholic products (Asava-arishta), raw materials needed are: 1. Decoction or Infusion prepared from medicinal plant drug 2. Source of sugar (predominantly sugar or plant material rich in sugar or carbohydrates) 3. Inoculum (Fermentation initiator/agent like Brewer’s yeast or plant material) 4. Prakshep (volatile oil containing drugs) 5. Water

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Dhataki flower

Sugar source Raw material/ Herbs

Soak in water for 24 hours

For Asava Coarse powder Swarasa,cold infusion etc.

FERMENTOR Incubate/activate for 24 hours

Decoction/ Extraction

For Arishta Liquid decoction

Maintain the temperature (28-32) Cº

Check alcohol content

In case of usingYeast

(desirable between 6-10 % v/v)

Add prakshepdravya 24 -48 Hrs

Stopping fermentation by adding approved preservatives and additional sugar (optional to achieve desired sweetness)

Addition of some excipients like flavour etc.

Filtration and packing

Figure 5.1 Steps of fermentation - traditional vs industrial process.

Preparation of liquid drug is carried out by cold decoction, swarasa (expressed juice) or hot decoction process of ingredients drugs. In these preparations the drug is coarsely powdered and added to water for extraction. After extraction by cold or hot process, the liquids are collected in vessel. For asava preparation, raw drugs may be added with water at the time of mixing of ingredients/initiation of fermentation. In this case, extraction and fermentation occur simultaneously. Madhura dravya are most important for microorganism as food and nutrition and also responsible for the production of alcohol. Madhura dravya (sweetening substance) may be Guda (jaggery), Madhu (honey), Sharkara (sugar), Sita (sugar) or drug itself as in the case of Madhukasava and Kharjurasava. Out of these most commonly used is Guda (Jaggery) but now sugar is used instead. General ratio of water and Guda (to prepare Asava-Arishta) is 39.06% [11]. Sandhana dravya is needed to initiate the fermentation. Ancient Acharya used the following drugs for the augmentation of Sandhana Prakriya. 1. 2. 3. 4.

Dhataki pushpa Madhuka pushpa Surabeeja / Kinwa Brewer’s Yeast

Collection of drugs were done according to season and time. Fresh and mature drugs were preferred.

Collection of drugs (Raw material collection and authentication)

(Continued)

Nowadays quality checks being done by macroscopic, microscopic and physio-chemical testing along with marker based fingerprinting as per Standards set by regulatory authorities.

Not done as cleaning procedure is robust.

Smoke of some plant was given for sterilization and smearing of oil or ghee was done to block pores of earthen pot/wood and prevent cross-contamination and leakage through pore.

Dhoopana (smoke technique used or sterilization) and smearing of oil/ghee

Industrial practice Stainless steel vessels used. Same vessel can be used repeatedly after proper washing.

Sandhanapatra (Fermentation vessel)

Purva Karma

Traditional method Earthen pot, wooden pot. These pots have limitation of breakage and cannot be reused for different formulation.

Key steps

Phase

Table 5.1 Contemporary intervention in fermentation processing.

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Onset of completion of fermentation checked by putting burning stick over mouth and it should be burning. If flame gets off, indicate incomplete fermentation. Done by double layer cotton cloth Mature with time after filtration Stored in large earthen pot, wooden container and china clay pot.

Filtration

Maturation

Storage

Added at start of fermentation along with other ingredient used.

PrakshepDravya (volatile oil containing drugs)

Observations of completion of fermentation (Quality control)

Dhataki flower, Jaggery, Madhuka were used as source of microbes for fermentation. When slow bubbling sound comes from pot and flame of matchstick stop burning when it is placed on mouth of pot indicating fermentation is continuing.

SandhanaDravya (Fermentation initiator/ fermenting agent)

Pashchata Karma

Jaggery, Madhuka, Draksha and other sugar containing fruits were used.

MadhuraDravya (sugar source)

Nowadays larger stainless steel vessels are used for storage and glass bottle for packing.

Maturation done with Prakshepdravya for 24–72.

Filtration done by either high-speed centrifugal separation or by sparkler filter.

Completion being checked by alcohol content.

Many volatile oil containing ingredients have antimicrobial properties. To avoid impediment of fermentation Prakshepdravya being added after desired level of alcohol generation.

Dhataki flower and yeast culture being used for fermentation. Fermentation being checked by visual observation and checking alcohol content.

Majorly sugar and jaggery being used.

In Arishta, decoction is done by traditional method on In practice aqueous extraction being done and mild heat and 16 times of water added and reduced to uniformity of decoction being measured by total one-fourth. Fresh swaras or cold infusion also being solid content to with respect to volume for definite used for asava. amount of raw drugs.

Drava Dravya (liquid part used in fermentation)

Industrial practice

Pradhana Karma

Traditional method

Key steps

Phase

Table 5.1 Cont.

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Fermentation

Sugar

Herbs/ingredients

Yeast/microbes

(Alcohol + Metabolite)

Figure 5.2 Fermentation by yeast.

Rarely Puga, Badara twaka, Babbula twaka were also used. Jaggery also acts as minor source of microorganisms for fermentation. Prakshep dravya is used to increase palatability and specialty of the formulation. This gives good colour and aroma to the medicine and also the medicinal properties get easily assimilated in Asava-Arishta. Prakshepa Dravya is of two types, i.e., Aushadhi Varga (have medicinal value) and Sugandhit dravya Varga (for aroma and taste). Prakshep dravya add medicinal property as well as enhance palatability, taste, aroma and also act as bioavailability enhancer for micro constituents.

5.3.4.2

Fermentation

Fermentation is the process of conversion of carbohydrate/ sugar into alcohol and CO2 by the reaction of enzyme produced by yeast (Figure 5.2). For proper fermentation, we need to control the following conditions of manufacturing. 1. Environmental condition for fermentation, 2. Microorganism diversity and population and 3. Duration Fermentation is a simpler process than oxidation but about 10 times as much energy is liberated as by oxidation. If oxidation is suppressed by lack of oxygen, fermentation continues. If oxidation is permitted to take place again, fermentation discontinues. This phenomenon is called “Pasteur reaction”. This is also the main difference in the process that decides acidic and alcoholic fermentation in Sandhana Kalpana. Thus, fermentation by yeast needed anaerobic condition, and for that purpose pots were sealed in ancient time and kept in husk/cereals. During fermentation there must be controlled temperature to ferment as Yeast cells will be destroyed if formulation were hot or will remain inactive if it is cold. Thus optimum temperature in the range of 20–35 °C is suitable for initiation and continuation of fermentation. An adequate environment is needed around 37 °C for the optimal growth of yeast and development of process [12, 13]. Time duration of fermentation varies according to different seasons when earthen pot/ancient tools are used. Literature revealed that fermentation takes place in 6 days during autumn and summer seasons, 10 days in winter and 8 days in rainy and spring seasons. Generally, in hot tropical climate 7–10 days are enough and 30 days in cool temperature climate [14]. In a broader sense fermentation totally depends on the environment maintained during fermentation. Nowadays we use a digitally controlled

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fermenter where we control temperature, light and oxygen/air and thus create an artificial environment for better fermentation. This helps in reducing the time required for fermentation and increasing process efficiency as well. Microbial diversity is very essential for fermentation as this process is carried out by enzymes produced by yeast. In ancient time, sources of yeast and other microorganisms were Dhataki flower, jaggery and menstruum of previously fermented product that got proliferated in liquid media and performed fermentation. But nowadays the scenario is completely different, and there are many strains of yeast spores available that perform the same action with more accuracy at a faster rate, providing better control on the process. The yeast spore was used by industry to generate the required amount of alcohol. When liberation of CO2 stops, fermentation is considered as complete. In industry, after it reaches the required level of alcohol, the process is stopped by some chemical agents, osmotic agent and pasteurization. After completion of the process, liquid is transferred for filtration.

5.3.4.3 Filtration and Maturation After completion of fermentation, Asava and arishta were filtered through double-layer cotton cloth in ancient time, but now we use high-end filtration technology like centrifuge separator, sparkler filter, etc., for filtration process. After filtration, clear liquid material gets separated which is stored in vessels for use and remnants are discarded. Maturation generally occurs in two phases 1. After addition of Prakshep drugs 2. At the time of final storage After fermentation prakshep drugs are added to liquid and kept for 24–48 hr for maturation to get proper taste, aroma and flavour. Prakshep material also gets extracted in hydro alcoholic medium, thus adding to therapeutic importance to formulation. In Ayurvedic classical method, Prakshep drugs are also added at the time of starting fermentation, but industry practice is varying to suit process flow and end product characteristics owing to in-house process optimization and validation. After maturation with Prakshep, the liquid is filtered by high-pressure sparkler filter or centrifuge separator in order to get clear and transparent liquid devoid of sediments. Maturation of the finished product continues during long storage of Asava and arishta too. Some ancient scholar has the opinion that there is an increase in the potency as the product becomes older [5]. Even Adhamalla states the same thing for potency of Asava-arishta. Acharya Sharangadhara declared that, Asava and Rasa preparations preserve their qualities even if they become old. This clearly indicates their long shelf life. There are some documented evidences which suggest that rearrangements happen at the molecular level which is attributable to organoleptic and efficacy improvements during maturation. For example, 1. Long chain polyphenol and tannins gets converted into shorter ones and add taste to the formulation.

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High Value Fermentation Products Volume 2 2. Smaller tannins and nectars accumulate and got settle down that was removed, and thus imparts less astringency. 3. Esterification of alcohol in presence of acid (e.g., Acetic acid), if present, add new aroma and reduces alcohol and acid content. 4. Hydrolysis of flavor precursor separated from glucose and impart in new flavor. 5. Continuous complex reaction and controlled slow fermentation also yield more efficacious product in formulation.

5.3.4.4 Quality Check, Packaging and Storage After filtration, the clear liquid is subjected to final quality checking. In process checks are being done throughout the process but final testing is done before packing to ensure quality and safety. For testing, various parameters may be selected including mandatory testing as governed by the ministry of AYUSH, Govt. of India (Table 5.2) [15]. Quality approved finished products are packed in amber colour bottles to avoid direct sunlight exposure. Asava arishta can be stored for an indefinite period as it is said that maturation increases its medicinal value with the passage of time. Care should be taken to avoid mould development at any stage of storage, which leads to rejection of the batch. Industries are analyzing the total microbial load at different stages of storage on commercial batches [16]. As per Gazette notification of Govt. of India, Asava arishta has been assigned a shelf life of 10 years from date of manufacturing [17].

5.4

Role of Ingredients and Process

The ingredients used therein for the process of fermentation have their crucial role in progress of fermentation and decide the future of formulation. The process involved behind the scenes phenomenon like extraction, are critical steps that have to be done properly. The role of ingredients is being analyzed as: 1. 2. 3. 4.

Role of extraction Role of sweetener Role of Dhataki flower and microbes Role of Prakshep

5.4.1 Role of Extraction In Asava and arishta, drava dravya is used as an ingredient derived from either extraction of ingredients drugs or powder drugs with water. Herbal drugs are extracted by a different method depending on the thermal-stability of the phyto-constituents. So the mass transfer from the raw material to liquid media is a major accountable step from the therapeutic efficacy and quality point of view. In asava mainly swarasa (expressed juice), and cold infusion (soaking and maceration) liquid media were taken while in arishta there is a decoction process for extraction of active constituents. For asava, swarasa needs fresh raw material and water in appropriate quantity for better extraction of plant constituents. For preparation of decoction, different

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Table 5.2 Testing parameter of Asava–arishta.

Sr. no.

Testing parameters 1.

Description Colour, Odour

2.

pH

3.

Specific gravity at 25 °C

4.

Total solids

5.

Alcohol content & Test for methanol

6.

Reducing sugar

7.

Non-reducing sugar

8.

Identifications, TLC/HPTLC

9.

Total acidity

10.

Test for heavy metals i. Lead ii. Cadmium iii. Mercury iv. Arsenic

11.

Microbial contamination i. Total bacterial count ii. Total fungal count

12.

Test for specific Pathogen i. E. coli ii. Salmonella spp. iii. S.aureus iv. Pseudomonas aeruginosa

13.

Pesticides residue i. Organochlorine pesticides ii. Organophosphorus pesticides iii. Pyrethroids

ratio of water and raw material were given based on hardness, quantity and nature of plant material. General ratio of herb and water is 1:16, which is boiled and reduced to 1/8th but Acharya Sharangadhara [18] have given a different ratio according to the nature of drug and quantity of the drug in order to achieve better extraction. Ratio of drugs to water according to nature of drug Soft drugs - 4 times water Medium Hard drugs - 8 times water Hard drug - 8 times water

100 High Value Fermentation Products Volume 2 Very Hard drug - 16 times water Ratio of drugs to water according to quantity of drug 12-48 g drugs - 16 times water 48– 192 g drugs - 8 times water More than 192 g drugs - 4 times water On the basis of the above ratio, selection of drugs and water were taken for extraction or water used in formulation. While preparing decoctions, it is better to keep pounded drugs soaked in a small quantity of hot water previous night itself. Next day it has to be squeezed and filtered. The filtered water preserves volatile substances of the drug which may be lost if the decoction is prepared directly. Next day decoction is prepared in Mandagni (low heat) to which that filtrate is added. Now days, industries are using successive extraction technology to extract maximum plant constituents which aid more bioactive available for fermentation but it has a chance of losing volatile oils at some level. Since all the processes are thermodynamically driven and energy is being involved in each transformation occur within liquid medium. So Quantum of heat and the duration of heating are of prime concern for Kashaya preparation (decoction). The purpose of energy in decoction is to drive the therapeutically active principles from the source drug up to maximum possible extent without damaging/denaturing any of the useful constituents in the process [19]. The extraction process was validated through time, temperature and quantity of yield with specific measure of total solid (TS) constituents. It provides reproducibility of the same extent of mass transfer throughout different batches. More mass transfer may be aiding more therapeutically active constituents or more secondary derivative by action of microorganisms. Ultimately, these extracted phyto-constituents are the element that plays a role as therapeutic agent along with self-generated alcohol.

5.4.2

Role of Sugar/Sugar Source

In Ayurveda, it is recommended to use jaggery for better fermentation as it also contains some microbes that help in fermentation initiation. General ratio of water and jaggery (to prepare Asava-Arishta) is 39.06% (Sha. Sa. Ma.Kha.10/3). Increased concentration of carbohydrate in liquid upsurges the viscosity of solution. Only a certain group of microorganisms can survive in a higher concentration up to 65–70%. While at above 40% concentration, only a few osmophilic types of yeast can grow. So at the time of fermentation initiation only about 40% sweetener is needed and remaining (if any) may be added after completion of fermentation [20]. Additionally, sugar added at the time when required alcohol level is achieved for ceasing of fermentation. Adding sugar kills the microbes by increasing osmotic pressure of liquid media. In the process, sweetener is used as source of food/ nutrition for yeast and other microbes so proper amount of sweetener helps in proliferation of microbial diversity to achieve fermentation properly. Sweetener acts as seat of microbes and helps in production of byproduct/metabolite by microbes. Sucrose (C12H22O11)/ glucose get converted into ethanol and carbon dioxide in anaerobic condition. The process is

Asava-Arishta: A Multi-Advantageous Fermented Product 101 exothermic and thus maintaining the temperature inside and also provides energy for molecular conversion.

5.4.3

Role of Dhataki Flower and Microbes

Process of fermentation in sandhana kalpana is majorly performed by yeast. In ancient time fermentation was mainly initiated by the addition of Dhataki flowers (Woodfordia fruticosa Kurz.) which act as source of yeast for fermentation [14]. A total of 24 yeast strains were isolated from the flowers of W. fruticosa that show diversity of microorganisms utilized in fermentation. Arishtas and asavas are prepared by permitting the herbal juices or their decoctions to undergo fermentation with the addition of sugars. The inoculum of yeast comes from Woodfordia fruticosa flowers, which contains the wild species of yeast. Additional spices are also added for improving their assimilation. In fact all preparations are biomedical fermentations mediated by microorganisms. They possess better keeping quality which is most likely due to the contribution of ethyl alcohol produced during fermentation. The enhanced therapeutic properties may be due to the microbial biotransformation of initial compounds into more effective therapeutic compounds as end products. The potential of arishta and asava is controlled by the profile of chemical compounds that can be modulated based on the ingredients, type of fermentation and microorganisms involved [21]. Bhondave et al., described the role of W. flowers in biomedical fermentation. Six yeast cultures were isolated from the flowers and identified as Saccharomyces species and Rhoduntonula muciliginosa. These isolates were used to ferment curcuminoids, and fermentation proceeded more quickly with isolated microbes than with Woodfordia flowers. The authors speculated that R. muciliginosa, a non-fermenting yeast, also had a role in the biotransformation of the active constituents [22]. Different Bacillus spp. were isolated from jaggery, which were also considered to be important in alcohol fermentation. However, difference in jaggery source (old and new) has no effect on physicochemical characters, particularly in alcohol percentage [23]. It has also been proved that there is much difference in herbal tincture prepared in hydro alcoholic solvent and fermented Asava arishta. Recent studies show that Saccharomces cereviciae hydrolyzed glycosides and released aglycones such as mono-terphenol and sesquiterpenes that impacts aroma of fermented formulation [24]. In a study, phenylethanol (metabolite of phenylalanine) was found almost 15 times higher in asava than tincture. It reflects that glycosides are hydrolyzed by alcoholic fermentation which helps in absorption of effective aglycones for the aged [25]. In one study, 16S rRNA gene library of samples of Varunadi kwath revealed the presence of consortia of microorganism which were earlier reported in fermentation of plant-based polymers. Faecalibacterium prausnitzii has been reported as commensal ruminal bacteria and are well known as pectin degraders found in plants [26]. Since these preparations are very rich in carbohydrates, larger group of bacteria

102 High Value Fermentation Products Volume 2 belonged to Bacteriodes and Gram positive anaerobes like Faecalibacterium prausnitzi, Ruminococcus sp., which have been implicated in Anaerobic fermentation systems. Independent research on cellulose degradation for alcohol generation have highlighted the role of Ruminococcus sp. and Clostridium sp. These herbal sources are a very rich source of fiber, which subsequently lead to the selection of microbes with saccharolytic capacity for a wide range of substrates. Similar to Varunadi kwath fermentation, Lactobacillus plantarum has been isolated from nearly all the naturally occurring plantbased fermentation processes, or as a starter culture [27]. The presence of less abundant phyla like Acinetobacter sp., Alcaligenes sp., Cuprividus sp. and Methylobacterium sp. at initiation of fermentation (0 day) of Kutajarishta, signify their ubiquitous presence in the environmental sources. On 8th day of fermentation, the presence of Streptococcus sp. and Bacilluss p., which have high pectinlolytic activity [28]. Some Asava Arishta use grape berries decoction carry lactic acid bacteria, besides yeasts, acetic acid bacteria and molds. Lactic acid bacteria ferment residual sugars, hexoses and pentoses left by yeasts and transform numerous wine components [29]. Fructose and glucose were very low in the dongchimi liquids during the early fermentation period. After six days of fermentation, the concentrations of free sugars increased rapidly, despite increases in the concentrations of lactate, mannitol, and acetate, major fermentation products of the free sugars. After 30 days of fermentation, the concentrations of free sugars decreased rapidly without any increases in lactate, acetate, or mannitol, and instead, the rapid production of glycerol and ethanol was observed [30]. In industry, larger scale production created an environment of CO2 bubbling in Asava Arishta. Increases in CO2 resulted in decreases in the cell yield and growth rate of yeast Saccharomyces in baker’s yeast production. On the other hand, the cell yield decreased by the increases in CO2 under glucose supply limitation, but did not change under oxygen supply limitation. This suggests that these changes of the metabolite yield do not link directly with the yield change of the cells [31]. As the fermentation process undergoes a gradient increase of alcohol level, it extracts a wide range of active ingredients from the herb than any other method of extraction. Fermentation not only removes the contamination but also reduces the toxicity of some toxic components in plants. Herb cells are ruptured by fermentation process and exposed openly to the menstruum where the cell walls are broken down by bacterial enzyme which further assists in the leaching process. Fermentation process creates an active transport system which removes the constituents from the herbal material to the menstruum [32]. There are claims that yeast cell walls naturally bind heavy metals and pesticide residues and act as natural cleaning system, making fermentation of herbal products safer than powder or tinctures. Fermentation also creates an active transport system that moves the dissolved constituents from the herbal material to the solvent [33]. There are indirect statements in Ayurvedic classics that fermentation will enhance the therapeutic properties of herbal drugs. So far, little direct evidence has been shown for such oblique statements. However, experimental proof has been provided for the enhancement of enzyme inhibition relevant in therapeutic action by fermentative transformation of the constituents of a herbal tincture. For example, biotransformation of berberine by fermentation produced its hydroxyl derivatives,

Asava-Arishta: A Multi-Advantageous Fermented Product 103 monohydroxy or dihydroxy-berberine [34, 35]. Surface Plasmon Resonance and enzyme kinetic studies showed that these derivatives had a higher inhibitory potential than berberine towards phospholipase A2 (PLA2). The X-ray crystal structures showed that the bio transformed derivative of berberine was bound to PLA2 in an inverted orientation with respect to the binding of berberine. This study revealed the significance of biotransformation in the generation of better enzyme-inhibitory compounds. Fermentation processes help in rupturing the cells of the herbs and exposing their contents to biotransformation. Fermentation also creates an active transport system with dissolved constituents from the herbal material. There are claims that yeast cell walls naturally bind heavy metals and pesticide residues and act as natural cleaning system, making fermentation of herbal products safer than powder or tinctures [33]. Okutsu et al., studied the concentration of phenyl ethanol, the breakdown product of phenylalanine by yeast and found that it was higher in ginger asava than in the tincture of ginger. In ginger asava, only traces of aldehydes such as geranial and neral were found. The percentages of geraniol and nerol were higher in Asava than in tincture [36]. Chemical analysis of Asava Arishta containing ingredient high in gallic acid, revealed that Gallic acid which was found at the initial stage, transformed into ellagic acid and gallolyl derivatives in later stage. Pseudomonas sp. and Klebsiella sp. have been utilized for their potential to degrade phenolic or aromatic compounds, and tannic acid which is ester of Gallic acid or Ellagitannins. Interestingly, independent reports have significantly highlighted the importance of ruminal microbes in degradation of tannin into non-toxic chemicals, which can be easily digested or assimilated [37] and impart in aroma as well. Fermentation of herbal ingredients are brought about by slow decomposition process of organic compounds and liquefaction of mucilaginous substrates like cellulose and pectin. This slow process also helps in extraction of wide range of active ingredients from herbal formulations [38]. Also a study has been reported where vitamin estimation was done on different asava and arishta, that results in good amount of vitamins B complex and others in the Drakshasava, Ashokarishta, Dashmulrishta, Khadirarishta, Lauhasava, Arjunarishta and Ashwagandharishta thereby supporting the usage of these Asavas & Arishtas in the form of general tonics suitable to be taken in routine life without any side effects due to its herbal base [39].

5.4.4 Role of Prakshep Dravya/Spices Prakshep drugs are mainly spices or volatile oil containing drugs. Since they were thermally unstable substances, they are used in the last step to retain its full swing. Ancient scholars used these spices at the time of fermentation but current practice is to use prakshep after fermentation for better aroma and taste. These drugs were used to generate good colour and aroma to the medicine. Volatile oils and phytoconstituents present in these spices also shows medicinal properties as well as enhanced palatability, taste, aroma; and they also act as bio enhancer for micro constituents. These spices are in a small quantity extracted in hydro-alcoholic medium and serve as bio enhancer for active compounds and enhance the bio-absorption as well as bioavailability of therapeutic agents along with alcohol.

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5.5

Advantages of Asava–Arishta Over Other Dosage Form

Asava-Arishta has unique properties as it is a hydro-alcoholic formulation and the phyto-constituents extracted due to hydro-alcoholic medium have a wide range of applicability. It is considered as superior dosage form in comparison to other liquid dosage form because 1. Except fermentation there is no process to get self-generated alcoholic/ acidic extract in formulation 2. Enhanced shelf life in liquid preparation naturally. 3. Excellent therapeutic activity as wide range of compound comes in formulation. 4. Fermented means one step ahead for its assimilation as it is already heated and partially digested by microbes 5. Formulation with additional metabolites like vitamin B and Folate which get synthesized during the process of fermentation only. 6. Instant absorption as generation of alcohol-aqueous milieu which help in better bioavailability of actives. 7. Fermented products show their superior pharmacology through some of their properties as Laghu (lighter to body), Ruksha (create dryness in body to enhance absorption), Teekshna (Sharper in action), Sukshma (Microform so that reach in every part of body), Amla (alcoholic-acidic in nature), Vyavayi (Spread all over the body), Vikasi (Absorb fast and spread in body vary fastly), Ashu (liquid form), Ushna (provide energy) etc. as mentioned in Charak Samhita [40]. 8. Asava is known for improvement of mental status, body energy, digestive power and Abate Anidra (sleeplessness), Shoka (Sad feeling) and improve sensation [40, 41]. It also give confidence, energy and strength [40]. 9. Arishta exhibits improved digestive power and improved sense perceiving and is used in splenomegaly, fever, gastric disease, respiratory disease, etc [40, 41]. 10. Remove undesirable sugar and convert them into instant energy packets as alcohol. 11. Do biotransformation of active constituents and metabolites into more efficacious one. 12. Some study shows that yeast binds with heavy metal and removes it. 13. Addition of good microbes, thus improvement in gut micro flora. It also acts as a probiotic and provides nutrition to gut microflora.

5.6

Future Perspective

Nowadays, we have robust technologies to monitor the chemical changes during the fermentation process. So utilizing these technologies we can alter the required metabolites and actives therein, by altering the microorganisms, environment of fermentation

Asava-Arishta: A Multi-Advantageous Fermented Product 105 and nature of drugs. Like the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is being recognized to enhance the analytical composition of the wines, so as we may follow the same for production of some new metabolite by controlling microorganisms diversity. Certain non-Saccharomyces species have potential to introduce characteristics to the wine that may improve the aroma profile [42, 43], enhance the glycerol content [44] and reduce the ethanol content [45]. All non-Saccharomyces yeasts produced significantly higher content of glycerol than single S. cerevisiae fermentation. It has been stated that ethyl esters of branched short-chain fatty acids are less correlated with pleasant flavour for young white Solaris wine. Terpenes are originally derived from the grape berries. It is generally admitted that acetates exhibit floral and fruity odours. So we can employ these variations to make Asava-arishta more popular and effective. These fermented products are well suited to promoting the positive health image of probiotics for several reasons [46]. The vital role of probiotics in manufacturing of Sandhana Kalpana may be an area of great interest. Since several reactions occur over a period of time by microbes, changing hydro-alcoholic media and fermentation environment, there are possibilities of new compound generation that may also take part in therapeutics. Research to explore possibilities of new compound generation is also an area to work on. If direction of new compound generation will be defined in future then it opens up a new window to address the ailment and treatment purpose. In the arena of changing lifestyle, population paradigm is shifting towards obesity and people are sitting on a diabetic bomb [47]. In such a scenario, serving asava and arishta may impart high sugar content, so the concept of sugar-free asava and arishta is floating in the market. In traditional manufacturing process of Asava and Arishta nutrition to microbes are provided by plant-based sugar sources. The source of nutrition to microbes could be limited so that it can be completely utilized by microbes during fermentation. This method can be used to produce sugar-free fermented products. Simultaneously comparative efficacy of conventional product with sugar-free product can be conducted to check whether it has same efficacy? Increasing use of fermented products also demands a high focus in all dimensions to ascertain its quality, safety and efficacy. It is also a challenge-cum-opportunity for experts of Indian medicine, pharmaceutical technology, biotechnology, biochemistry and microbiology to work together for intra-disciplinary research to extract cream from ancient Ayurvedic wisdom in the language of the 21st century. The time has come to create a fruitful bridge between experiential Ayurvedic wisdom, modern medicine and contemporary science to validate the effectiveness of Asava-Arishta and practices in terms of molecular pharmacology and in the language of contemporary sciences [48, 49].

Acknowledgements We are grateful to CSIR-IIIM, Jammu for providing insight and valuable inputs for this chapter.

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References 1. Mishra, S.N., Abhinava Bhaishjya Kalpana Vigyana. 4th ed. 08. Varanasi, Chaukhambha Surbharati Prakashan, 1993. 2. Acarya Vaidya Jadava Ji Trikamji, Carak samhita with commentary of cakrapanidatta, Sutra Sthana. 49, Vol. 25, New Delhi, Rashtriya Sanskrita Sansthan Chikitsa Sthana verse 14/13843, 16/111-3, 531, 2002. 3. Acarya Vaidya Jadava Ji Trikamji., Sushrutasamhita with commentary of Dalhana. Sutra Sthana. 7. 45. Varanasi, Chaukhambha Orientalia publishers Chikitsa Sthana. p. 433, 2002. 4. Joshi, D., Jha, C.B., Anc. Sci. Life, 9(3), 125, 1989. 5. Tripathi, B., Sharangdhar Samhita. Varanasi, Chaukhambha Surbharati Prakashan, 2010. 6. Chaudhary, A.K., Lochan, K., In: Varanasi P, Sansthana C. S, eds, Bhaishjya Ratnawali of Govinda Dasji. 1st. 100, 2006. 7. Anonymous, The ayurvedic formulary of india, part-II, new delhi, the controller of publication. Edition, 2000. 8. Reddy, K.R.C., Vijnanam, B.K., A Science of Indian Pharmacy. 2nd ed. Varanasi, Chaukhamba Sanskrit Bhawan, 2001. 9. Paksadhara, J., Asavaarishta Vijnana. 3rd ed. Varanasi, Chaukamba Bharati Academy, 1997. 10. Gupta, S., Mitra, S., Prajapati, P.K., Indian Journal of Ancient Medicine and Yoga, 2229(4), 2009. 11. Tripathi, B., Sharangdhar Samhita. Varanasi, Chaukhambha Surbharati Prakashan, 2010. 12. Kulkarni, S., Chaudhary, A.K., Sachitra Ayurveda, 4, 768, 2003. 13. Lakhani, R., Chaudhary, A.K., Sachitra Ayurveda, 8, 145, 2004. 14. Sekar, S., Mariaappan, S., Wollgast, V., Anklam. Indian Journal of Traditional Knowledge, 7, 548–556, 2008. 15. Lohar, D.R., Protocol for testing of ASU Medicine, P L I M Ghaziabad, Department of AYUSH, Government of India. P, 18, 2011. 16. Shastri, M.V., Vaidya yoga ratnawali, IMPCOPS Madras, 6, 1968. 17. Government of India, The Gazette of India, Extraordinary, part-II, sec-3, subsection-(i), GSR-789(E), 2016. 18. Tripathi, B., Sharangdhar Samhita. Varanasi, Chaukhambha Surbharati Prakashan, 2010. 19. Gupta, S., Mitra, S., Prajapati, P.K., Indian Journal of Ancient Medicine and Yoga, 2, 229, 2009. 20. Chaudhary, A., Singh, N., Dalvi, M., Wele, A., A progressive review of Sandhana kalpana (Biomedical fermentation): An advanced innovative dosage form of Ayurveda. Ayu, 32(3), 408, 2011. 21. Remya, P.M., International Conference and Exhibition on Traditional & Alternative Medicine, 2, 10, 2013. 22. Bhondave, P., Burase, R., Takale, S., Paradkar, A., Patil, S., Mahadik, K., International Journal of Pharmaceutical and Biomedical Research, 4, 37, 2013. 23. Alam, M., Venkatakrishna, D.D., Varadarajan, T.V., Sarma, P.S.N., Purushothaman, K.K., Journal of Research in Indian Medicine Yoga and Homoeopathy, 12, 37, 1977. 24. Ugliano, M., Bartowsky, E.J., McCarthy, J., Moio, L., Henschke, P.A., Hydrolysis and transformation of grape glycosidically bound volatile compounds during fermentation with three Saccharomyces yeast strains. J. Agric. Food Chem., 54(17), 6322–6331, 2006. 25. Okutsu, K., Yoshimitsu, M., Kakiuchi, N., Mikage, M., Differences in volatile compounds between tincture and Ayurvedic herbal liquor "Asava" made from ginger or jujube. Journal of Traditional Medicines, 24, 193, 2007.

Asava-Arishta: A Multi-Advantageous Fermented Product 107 26. Lopez-Siles, M., Khan, T.M., Duncan, S.H., Harmsen, H.J., Garcia-Gil, L.J., Flint, H.J., Cultured representatives of two major phylogroups of human colonic Faecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth. Appl. Environ. Microbiol., 78(2), 420–428, 2012. 27. Dongowski, G., Lorenz, A., Anger, H., Degradation of pectins with different degrees of esterification by Bacteroides thetaiotaomicron isolated from human gut flora. Appl. Environ. Microbiol., 66(4), 1321–1327, 2000. 28. Gupta, S., Abu-Ghannam, N., Probiotic fermentation of plant based products: possibilities and opportunities. Crit. Rev. Food Sci. Nutr., 52(2), 183–199, 2012. 29. Lonvaud-Funel, A., Lactic acid bacteria: Genetics. Metabolism and Applications. Netherlands, Springer. p. 317, 1999. 30. Jeong, S.H., Jung, J.Y., Lee, S.H., Jin, H.M., Jeon, C.O., Microbial succession and metabolite changes during fermentation of dongchimi, traditional Korean watery kimchi. Int. J. Food Microbiol., 164(1), 46–53, 2013. 31. Kuriyama, H., Mahakarnchanakul, W., Matsui, S., Kobayashi, H., The effects of pCO2 on yeast growth and metabolism under continuous fermentation. Biotechnol. Lett., 15(2), 189– 194, 1993. 32. Katiyar, C.K., Asian, S.-E., South-eastAsian (SEA) regional workshop on “extraction technologies for medicinal and aromatic plants” PPP. Central Institute of Medicinal and Aromatic Plants. (CIMAP) Lucknow, India, 2006. 33. Sabu, A., Haridas, M., Fermentation in ancient ayurveda: its present implications. Frontiers in Life Science, 8(4), 324–331, 2015. 34. Chandra, D.N., Preethidan, D.S., Sabu, A., Haridas, M., Frontiers in Life Sciences, 8, 160, 2015. 35. Chandra, D.N., Abhilash, J., Prasanth, G.K., Sabu, A., Sadasivan, C., Haridas, M., Inverted binding due to a minor structural change in berberine enhances its phospholipase A2 inhibitory effect. Int. J. Biol. Macromol., 50(3), 578–585, 2012. 36. Okutsu, K., Yoshimitsu, M., Kakiuchi, N., Differences in volatile compounds between tincture and Ayurvedic herbal liquor "Asava" made from ginger or jujube. Journal of Traditional Medicine, 24, 193, 2007. 37. Bhat, T.K., Singh, B., Sharma, O.P., Microbial degradation of tannins--a current perspective. Biodegradation, 9(5), 343–357, 1998. 38. Mulay, S., Khale, A., International Journal of Pharmaceutical Sciences and Research, 2, 1421, 2011. 39. Singh, M.K., Sharma, A., Sharma, M., Singh, R., Katiyar, C.K., Journal of Food and Pharmaceutical Sciences, 1, 81, 2013. 40. Acarya Vaidya Jadava JI Trikamji, Carak Samhita with commentary of Cakrapanidatta Sutra Sthana, New Delhi: Rashtriya Sanskrita Sansthan (Deemed to be University), 2002. 41. Sharma, P.V., Sushruta Samhita. Varanasi, Chaukhamba Orientalia publication, 1997. 42. Ciani, M., Beco, L., Comitini, F., Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations. Int. J. Food Microbiol., 108(2), 239–245, 2006. 43. Medina, K., Boido, E., Fariña, L., Gioia, O., Gomez, M.E., Barquet, M., et  al., Increased flavour diversity of Chardonnay wines by spontaneous fermentation and co-fermentation with Hanseniaspora vineae. Food Chem., 141(3), 2513–2521, 2013. 44. Sadineni, V., Kondapalli, N., Obulam, V.S.R., Effect of co-fermentation with Saccharomyces cerevisiae and Torulaspora delbrueckii or Metschnikowia pulcherrima on the aroma and sensory properties of mango wine. Ann. Microbiol., 62(4), 1353–1360, 2012. 45. Benito, S., Hofmann, T., Laier, M., Lochbühler, B., Schüttler, A., Ebert, K., et  al., Effect on quality and composition of Riesling wines fermented by sequential inoculation with

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46. 47. 48. 49.

non-Saccharomyces and Saccharomyces cerevisiae. Eur. Food Res. Technol., 241(5), 707– 717, 2015. Heller, K.J., Probiotic bacteria in fermented foods: product characteristics and starter organisms. Am. J. Clin. Nutr., 73(2 Suppl), 374s–379, 2001. Singh, V.K., Reddy, K.R.C., International Journal of Ayurvedic Medicine, 6, 305, 2015. Lele, R.D., Indian biotechnology: Challenges and opportunities- a clinician's perspective. Indian J. Biotechnol., 4, 9–20, 2005. Mashelkar, R.A., Building a golden triangle between traditional medicine, modern medicine and modern science, 2003.

6 Production and Applications of Polyunsaturated Fatty Acids Sabeela Beevi Ummalyma1, Raveendran Sindhu2,*, Parameswaran Binod2, Ashok Pandey3 and Edgard Gnansounou4 1

Institute of Bioresources and Sustainable Development (IBSD), Imphal-795001 Manipur (India) 2 Centre for Biofuels, Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, (CSIR-NIIST), Trivandrum 695019 (India) 3 CSIR-Indian Institute of Toxicology Research (CSIR-IITR), 31 MG Marg, Lucknow-226 001, (India) 4 Ecole Polytechnique Federale de Lausanne, ENAC GR-GN, GC A3, Station 18, CH–1015, Lausanne, (Switzerland)

Abstract Polyunsaturated fatty acids (PUFAs) are key molecules for the control of human as well as animal health. Among the PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are vital compounds for infant brain development and other immune regulatory functions. Commercially available omega fatty acids are obtained from fish sources; nowadays fish oils are not preferred due to its unpalatable taste and odors as well as the fact that this resource is declining. Microalgae and other microorganisms based polyunsaturated fatty acids are gaining more attraction because of its fastest growth and lipids content; it is a preferred resource for vegetarians for omega 3/6 oils. Genetically modified organisms such as yeast are utilized for desired fatty acids production commercially. This chapter discusses the importance of polyunsaturated fatty acids for human health, various sources of PUFAs along with its fermentative productions and future perspective. Keywords: Fatty acids, EPA, DHA, microalgae, fermentation, biosynthesis, yeast

6.1

Introduction

Fatty acids are vital molecule of membranes and crucial for maintaining membrane functions and structures. They are involved in the fluidity of phospholipid bilayer and control the movement and function of membrane associated proteins [1–3]. In eukaryotes, PUFAs are related with selective permeability, fluidity, flexibility of the sub cellular membrane. Furthermore, fatty acids are also controlling protein bounded with membrane such as transport proteins, histocompatibility complexes and ATPase [4]. PUFAs are fatty acids having long chain carbon atoms possess more than two carbon–carbon *Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (109–126) © 2019 Scrivener Publishing LLC

109

110 High Value Fermentation Products Volume 2 double bonds and commonly known as long-chain PUFAs. The word omega fatty acids or an n- fatty acid denotes the terminal methyl end of the fatty acid. Then omega-3 designates the location of the end of carbon–carbon double bond from the omega end of fatty acid. Omega-3/6 PUFAs and their derivatives have a vital role in inflammatory processes, and metabolic, immunologic, coagulation processes [5]. Omega-3 PUFAs, such as EPA which is produced from α-linolenic acid, through carbon addition and desaturation reactions. However, these fatty acids can also be obtained through the diet. EPA can also be converted to DHA as well as eicosanoids, which are essential inflammatory signal molecules. In mammals, eicosanoids such as leukotrienes, prostaglandins and thromboxanes mediates inflammations, clotting, pain, fever, vasodilatation, blood pressure, neurotransmission, and modulation of cholesterol metabolism [6]. Thus, unsaturated fatty acids are conditionally essential for human health and are required for the normal function and development of the body. The arachidonic acid is produced as the intermediate components from omega 6 fatty acids through various enzymatic processes. The arachidonic acid (ARA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-3) are fatty acids found in brain tissues and important nutrients needed for newborn babies due to their potential role in development of neural and retinal functions [7–9]. PUFA are essential fatty acids for the body; the unbalance of various types of PUFAs and their derivatives has been associated with different problems and diseases in humans such as metabolic syndrome, cardiovascular diseases, neurological disorders and inflammation [8, 9]. Mammals including humans cannot synthesize essential fatty acid such as linoleic acid (LA, C18:2 n-6) and α-linolenic acid (ALA, C18:3 n-3). The findings suggested that dietary supplementation of PUFAs significantly improves the symptoms of many chronic diseases, and this has attracted great interest from the general public and food manufacturers. Appropriate intake of dietary fatty acids is thus fortified to offer protection against many diseases and elevated improvements in eyes and brain, as well as functioning of immune systems. Escalation of the market for PUFAs globally is determined by rising pervasiveness of ailments such as cancer, stroke, diabetes and cardiovascular diseases. The PUFAs are used as an ideal component in processed food, infant nutritional products and dietary supplements, and the demand for PUFAs is likely to increase in the future. Furthermore, preference of unsaturated fatty acids against the saturated fats is also enhancing the intake of unsaturated fatty acids across the world. Some consumers are recommended for daily intake (RDI) of PUFAs (90–160 mg/day). However, the increasing cost of fish oils, its scarcity, and costs associated with the R&D activities are anticipated to confine the expansion of the PUFAs market. Worldwide, utilization of PUFAs, especially omega 3 fatty acids is expected at 123.8 thousand metric tons, worth US$ 2.3 billion in 2013, which is projected to be 134.7 thousand metric tons valued at US$ 2.5 billion in 2014. Expected demand for PUFAs globally by 2010 would reach 241 thousand metric tons with a value of US$ 4.96 billion.

6.2

Biosynthesis of Polyunsaturated Fatty Acids

The synthesis of polyunsaturated fatty acid involves the introduction of double bonds which are commonly known as desaturation process of fatty acyl chain and chain

Production and Applications of Polyunsaturated Fatty Acids 111 elongation by two carbons addition in the fatty acyl chain. However, the synthesis of fatty acids involves the alternate action of enzymes such as desaturases and elongases [10, 11]. Desaturation steps in fatty acids synthetic process is an aerobic process and reducing equivalents for the process obtained from electron transport chain [12, 13]. The electron donors for the reaction is dependent on the location of the desaturase enzymes present in cytochrome b5 in endoplasmic reticulum and ferredoxin of bacteria and plastids. Desaturase enzymes are a complex group of protein that could be classified into various forms depending on the type of esterification reaction with fatty acid molecule and its solubility, electron donor; cellular localization and selectivity of the reaction. There are two separate pathways involved for biosynthetic process of PUFAs such as the aerobic pathway which involves desaturase/elongase enzymes and anaerobic PUFA synthase pathway. The aerobic pathway is present in microalgae, bacteria and plants. The specific desaturases and elongases are involved in synthesis of PUFA from C18 fatty acids such as oleic acid, catalyzes separate chain elongation and desaturation reaction and finally goes through a retro-conversion process through the β-oxidation step in the peroxisome for two carbon chain shortening, whereas the production of DHA in eukaryotic organisms initiated from stearic acid and ending with the final desaturation step [14]. Meanwhile, anaerobic pathways is found in prokaryotic bacteria and microalgae. Synthesis of the fatty acids starts with the help of fatty acid synthases. Fatty acid synthases are multi-enzyme complexes with multiple catalytic domains. Synthesis of PUFAs starts from acetyl coenzyme A (CoA) and malonyl-CoA and extended the carbon units respectively in a manner identical to polyketide synthases (PKSs) and fatty acid synthases (FASs) [15–17]. Figure 6.1 represents the biosynthetic pathways of poly unsaturated fatty acids. An anaerobic pathway has great advantages for industrial 18:2n-6

20:3

Elongase

Δ6Fads 18:4n-3

18:3n-6 Elongase

20:4n-3

20:3n-6

20:3n-3

(F Δ ad 8F sC ad 1, s C2 )

Δ5, 11, 14,

18:3n-3

ds ) Fa sA Δ5Fad (

20:2n-6 ) ds C2 Fa 1, Δ8dsC a (F

Δ (F 5F ad ad sA s )

Elongase

20:4Δ5, 11, 14, 17

Δ5Fads (FadsA) 20:5n-3

20:4n-6 Elongase 22:4n-6 24:4n-6 Elongase Δ6Fads 24:5n-6 22:5n-6 ß-oxidation

Δ4Fads

24:5n-3 22:5n-3 Elongase Δ6Fads 22:6n-3 24:6n-3 ß-oxidation

Figure 6.1 Biosynthetic pathways of LC-PUFA from C18 PUFA precursors in vertebrates (Reactions catalysed by fatty acyldesaturases are designated as Δx (Δ6, Δ5, Δ4 and Δ8), whereas elongation reactions are indicated as elongase) (Adapted from Kabeya et al., [18]).

112 High Value Fermentation Products Volume 2 production of PUFAs, due to less requirement of reducing equivalents like NADPH and produces low quantity of by-products with undesirable chain length and unsaturation.

6.3

Sources of Polyunsaturated Fatty Acids

Mammals including humans are unable to synthesize essential fatty acid of their own as they lack desaturase, critical for the synthesis of vey long chain fatty acids de novo and hence they are depend on its supplements via diet. The plant seed oils and marine fish are the major sources of PUFAs available in diet. The over exploitation of fish as well as its lipids for the production of essential fats containing EPA, which has been limited nowadays, due to its unpalatable taste and odor and presence of heavy metals [19]. Hence, quality and quantity of traditional resources of PUFA have some problems in meeting market demand. However, searching for alternative sources is required to overcome challenges associated with the conventional sources of PUFA. Different types of PUFAs have been detected in plants, mosses and various microorganisms including, bacteria, fungi and algae.

6.3.1

Microbial Source

Microbes are alternative potential oil producers due to their fast growth rate and fast growth rates, easy to handle and delete because they are easy to handle and manipulate. The microbial diversity can facilitate the selection of potent oleaginous strains with the target fatty acids. There are several microorganisms that have been studied recently as a substitute to agricultural and fish oil product [19]. In order to meet the increased demands for EPA and DHA, alternative clean and sustainable source of the omega-3 fatty acid through fermentation process was developed using metabolic engineering approach with the yeast strains of Yarrowia lipolytica [20]. Some oleaginous yeast has fast growth and possessing 20% of cell weight of lipids. Most of the oil producing yeast can able to accumulate lipids up to 40%, that can be further enhanced to 70% under nutrient limited condition in fermentation. However, lipid as well as fatty acid profile of the species is varied based on the culture conditions and nutrients present in the media. The potent oleaginous yeasts are found in genera Cryptococcus, Candida, Rhodosporidium, Lipomyces, Rhodotorula, Rhizopus, Trichosporon and Yarrowia [21]. A report showed fatty acids variation from yeast R. glutinis with different carbon substrates [22]. The maximum lipid production 34% of TGA was obtained in media having mixture of dextrose and glycerol as carbon source. This result showed that percentage of unsaturated fatty acids in the TAGs was reliant on type of carbon present in the media the high lipid production of 53% obtained from glycerol as substrate and low lipids 25% on xylose. The supplementation of whey permeate in the culture media for enhancing lipid from different yeast strains, L. starkeyi ATCC 12659 was produced maximum accumulation of lipids among others such as Apiotrichon curvatum, Cryptocooccus albidus, L. starkeyi and Rhodosporidium toruloides [23]. Lipomyces starkeyi is transgenic yeast possessing (Δ15 desaturase) gene from flax capable of accumulating 60% of their body weight as lipids. Before modification of L. starkeyi contained 56.03 mgL-1 DHA along with 71.4 mgL-1 EPA and 42.2 mgL-1 ALA. After transformation of genes from

Production and Applications of Polyunsaturated Fatty Acids 113 flax, the modified yeast can able to accumulate 1080 mg/L-1 DHA (17.4 %) along with 74.28 mgL-1 EPA and 126.72 mgL-1 ALA, might be due to alteration in the synthetic pathways of PUFA [24]. Many oleaginous fungal species such as Mortierella alpina, Claviceps purpurea, Mortierella isabellina, Aspergillus terreus and Tolyposporium are able to accumulate lipids. Several of the fungal species are used for the PUFAs production studies. The Mucor rouxii, reported to accumulate a high level of intracellular lipids with gamma linolenic acids (GLA). Eroshin [25] reported that the production ARA as maximum of 4.5 gL−1 by M. alpina with maximum productivity of 19.2 mgL-1 h−1 with the use of nitrogen source as potassium nitrate [26]. They successfully produced 7.1 gL−1 ARA using the fungus Mortierella alliacea in a 50L jar with a working volume of 25L with media having 12% glucose and 3% yeast extract, produced 46.1 gL−1 biomass with 42.3% lipids in 7 days of cultivation. The age of the mycelia should be an important parameter to be considered for the production of poly unsaturated fatty acids [27], observed that lipid production was highest in young mycelia and production decreased in age of the culture increases [27]. Cultivation of T. fermentans in media having nitrogen source as peptone, carbon as glucose and a C/N ratio of 163, enhanced biomass production of 28.1 gL−1 with lipid production 62.4% [28]. Recently fungus Mortierellais abellina was exploited for lipids rich in GLA during fermentation on deproteinized whey permeate as carbon source [29].

6.3.2

Plant and Microalgal Source

Microalgae are known producers of PUFAs essential for human nutrition [30, 31]. It has been reported that dinoflagellates and marine protists such as species of Schizochytrium, Thraustochytrium and Crypthecodinium are the sources of docosahexaenoic acid, while eicosapentanoic acid are produced from microalgae like Phaeodactylum and Monodus. The most well-known DHA producer among the microalgae species from dinoflagellate is called Crypthecodinium cohnii, which is a heterotrophic organism containing 50% (w/w) DHA [32, 33]. PUFAs based from these organisms are commercially available (DHASCOTM). Other important DHA producers from green microalgae are the genus Schizochytrium [34]. The potential of phototrophic microalgae is the capability to synthesize lipids from sunlight and atmospheric CO2 as carbon and energy sources. Algae can be grown as phototrophic, heterotrophic and mixotrophic cultivation. Cultivation mode is chosen based on the availabilities of carbon substrates, CO2 and sunlight for their metabolisms. Microalgae can be cultivated in large-scale operations in open pond raceways systems under natural growth conditions and this system of cultivation requires low cost for manufacture. However, conditions such as less dissolved CO2 and insufficient light intensities makes low cell densities, which further affect the downstream processing which is an energy-intensive and costly process. Moreover, open pond systems are limited to a few numbers of robust species having potential to tolerate high salinity or fast-growth rate and produce biomass against the competitive organisms and other algal feeders [30]. Commonly, open pond systems are used for the production of bulk products such as carotenoids or biomass which is used as a human dietary supplement. However, the production of high-value products like PUFAs for human nutrition are obtained

114 High Value Fermentation Products Volume 2 economically under controlled conditions in photobioreactors. Photobioreactors are used for high cell density cultivation of algae which require significantly less space compared to open pond systems [35]. Some microalgae produce an enormous amount of oils containing long chain fatty acids. Long chain PUFA is important to human nutrition, and a report showed that few of these PUFAs are associated with visual, mental and physical development in infants [36]. Moreover, PUFAs are an element of a healthy food aid in reducing the risks of many ailments such as cancers, arthritis, cardiovascular disease and dementia [37]. Several algal groups are identified as rich in high levels of LC-PUFA, including diatoms, Cryptophytes, Chrysophytes, Dinoflagellates and others [37–39]. Table 6.1 represents the predominant PUFA from different microalgal species. DHA is a predominant fatty acid present in neurological tissues, consists of 20–25% of the total fatty acids in the gray matter of the human brain and 50–60% in retina rod outer segments [4, 42]. Humans are unable to produce DHA de novo and the capability to synthesize DHA from its precursor molecule alpha linolenic acid. Thus, adequate supplies of DHA have to be obtained from food [43]. Microalgae such as Crypthecodinium cohnii (40–50% DHA), Schizochytrium (40% DHA) and Ulkenia sp. are right organism for the production of DHA [40, 44]. The Crypthecodinium cohnii, a dinoflagellate can produce majority of its lipid as DHA [39]. Since 1990 onwards, various organizations for health and nutrition suggested that addition of DHA in infant formula. The infant formula world market is now estimated to be about US$ 10 billion per annum [45]. Martek’s DHA oil (DHASCO; Martek, Columbia, MD, USA) is produced from C. cohnii and contains 40–50% DHA [32, 33, 45]. DHA oil produced from C. cohnii is currently available worldwide [46]. Another commercial source of DHA is produced from Schizochytrium and mainly used for adult dietary supplement and foods for nursing pregnant women [40]. Moreover, Omega Tech (USA) by Martek, exploits Schizochytrium to produce low-cost oil called as DHA Gold [33]. The oil is now used as dietary supplement in health foods, animal feeds, maricultural products and beverages. Finally, Nutrinova (Frankfurt, Germany) uses Ulkenia sp for DHA production in 80 m3 fermenters and this oil is sold under the name of DHA active [33]. Different types of algae has been proposed for EPA production such as Nitzschia sp [47], Nanochloropsis [48], Navicula sp [49], Phaeodactylum [50] and Porphyridium [51]. Alteration of EPA levels can considerably change the individual coronary vascular conditions, because eicosanoids are the metabolic products of EPA having antithrombotic and anti-aggregatory effects [52]. Technology for producing EPA, from Phaeodactylum tricornutum has been developed by the University of Almeria in Spain. An economic analysis of production facility of 430 kg of 96% pure EPA per year, calculated the total cost of production at US$ 4602/kg with 40% from the biomass production and 60% of the cost from harvesting process [53]. They believed that if the cost is reduced by 80%, then the EPA production is economically viable. The residual biomass after extraction of the EPA to be sold as animal feed [53]. The annual worldwide demand of EPA is 300 t [54]. In addition, new algal sources for PUFAs production especially, EPA from Glossomastix chrysoplasta [55] and Thraustochytrium sp. are used for optimization of PUFA production [56].Table 6.2 shows various microbial sources used for PUFA production.

Chemical structure

18:3 ω6, 9,12

20:4 ω6, 9,12,15

20:5 ω3, 6,9,12,15

22:6 ω3, 6,9,12,15,18

PUFA

γ-Linolenic acid(GLA)

Arachidonic acid(AA)

Eicosapentaenoic acid(EPA)

Docosahexaenoicacid (DHA)

Infant formulas for full-term/preterm infants Nutritional supplements, Aquaculture

Nutritional supplements Aquaculture.

Infant formulas for full-term/preterm infants, Nutritional supplements.

Infant formulas for full-term infants, Nutritional supplements.

Potential application

Crypthecodinium, Schizochytrium

Nannochloropsis, Phaeodactylum,Nitzschia

Porphyridium

Arthrospira

Algal source

Table 6.1 Algae as bioresources for PUFA production (adapted from spolaore et al., [40]: brennan and owende [41].

Production and Applications of Polyunsaturated Fatty Acids 115

Microbial sources

Fungus Cunninghamella blakesleeana-JSK2 Cunninghamella echinulata Mucor circinelloides Thamnidium elegans Zygorhynchus moelleri Yeast Yarrowia lipolytica

Fungus Halophytophthora spinosa var. spinosa IMB162 Mortierella alpina D20 Microalgae Khawkinea quartana SAG 1204 Palmodictyon varium SAG 3.92 Parietochloris incisa Rhabdomonas incurva SAG 1271–8

PUFAs

GLA

AA

Table 6.2 Microbial sources of PUFAs.

[65] [65] [66] [67] 34.3 73.8 44 41.3 NA NA 23 NA

(Continued)

[63] [64]

[62] 25 45.4

20.0

52.7

[57] [58] [59] [60] [61]

Reference

NA 52.7

21.1 11–18 14.6–26 13.6 10–18

PUFAs/l (%)

41 30–48 14.7–35.6 6.1 10.6

Dcw (%)

116 High Value Fermentation Products Volume 2

Yeast Yarrowia lipolytica Microalgae Aurantiochytrium limacinum Aurantiochytrium sp. Crypthecodinium cohniia Isochrysis galbana Isochrysis sp. T-iso Phaeodactylum tricornutum Prorocentrum triestinum S2 Schizochytrium limacinum

DHA

Fungi Halophytophthora spinosa Mortierella alpina ST1358(GM) Yarrowia lipolytica (GM) Microalgae Asterionella sp. S2 Nannochloropsis oculata strains Nannochloropsis salina Pavlova lutheri Phaeodactylum tricornutum Porphyridium cruentum Porphyridium purpureum Tetraselmis gracilis Tetraselmis sp.

Microbial sources

Cont.

PUFAs

Table 6.2

[63] [76] [65] [74] [77] [78] [79] [80] [72] [79] [81] [71]

18.4 26.4 56.6 26.4 19 23.6 18.7 57 3.6 15.8 14 9.4

1.8 30 26.6 NA NA 8 2.4 12.7 NA

[64] [69] [70] [71] [72] [73] [74] [75] 48.5 35 25 12.7 4.6 12 20.4 19.1

63.8 57.2 32 NA 20 NA 3.7 75 NA NA 30

[68]

Reference

5.6

PUFAs/l (%)

NA

Dcw (%)

Production and Applications of Polyunsaturated Fatty Acids 117

118 High Value Fermentation Products Volume 2

6.4

Different Fermentation Process for PUFA Production

The oleaginous algae strain Aurantiochytrium limacinum SR21, initially known as Schizochytrium limacinum SR21 [82] showed that this alga can produce intracellular lipid up to 70% cell weight rich in 30–40% of the total fatty acid as DHA. Presently several studies have targeted DHA production from A. Limacinum SR21 through glycerol-based fermentation processes [34, 83]. Research conducted by Chi et al., [34] obtained high biomass of 22.1 gL−1 containing DHA content of 4.91 gL−1 from A. limacinum SR21. Another study by Chi et al., [84] applied a two-stage oxygen control method during fermentation, which elevated biomass and DHA in A. limacinum SR21 to37.9 1 gL−1 and 6.56 1 gL−1, respectively. Ethier et al., [85] adapted continuous fermentation method for the cultivation of A. limacinum SR21, yielding a biomass of 11.8 gL−1and DHA content of 1.74 gL−1. Liang et al., [86] used sweet sorghum juice as culture medium produced biomass of 9.41 gL−1 and DHA production of 0.51 gL−1 day_1. All those reports are discussed on fermentation condition for both biomass and DHA productivity in A. limacinum SR21 must be further improved with better fermentation parameters. The carbon to nitrogen ratio (C: N ratio) is another critical factor affecting microorganism growth and DHA production during fermentation. Chi et al., [84] and Qu et al., [87] reported that during fermentation of A. limacinum SR21, dissolved oxygen levels directly affecting biomass and DHA productivity. In another case, intermittent oxygen feeding to fermentation broth to maintain dissolved oxygen level of 50% with C: N ratio of 1.25 for Schizochytrium limacinum SR21 for obtaining biomass of 61.76 gL−1 with DHA concentration at 20.3 gL−1also been reported [31]. The lipids and fatty acids within oleaginous organism therefore acts as a active storage materials which are produced in huge amount when carbon is plenty or starvation when carbon is scarce in the media [33, 88, 89]. Moreover, the high carbon to nitrogen ratio is required for lipid production, while the DHA concentration varied with supplementation of glucose [90]. In addition to this, it has been proved that production of DHA was favored in low dissolved oxygen (DO) [91] and hence the fermentation approach was conducted by controlling DO for optimization of DHA production [84]. A stepwise aeration control approach was performed for fed-batch fermentation of DHA production in 1500L reactor [92] or sporadic oxygen feeding method to maintaining DO level of 50% [31]. Fermentation of Schizochytrium sp. LU310 in baffled flask for getting higher oxygen supply for improving DHA production showed that significant improvements of DHA fermentation by batch mode. By applying strategy for nitrogen-feeding in 1000 mL baffled flasks, the biomass, DHA productivity and DHA concentration were increased by 110.4, 110.4, and 117.9% respectively. Moreover, DHA content of 21.06 gL−1was obtained by feeding of 15 gL−1 glucose occasionally, which enhance the production of 41.25% over batch mode fermentation. Another innovative strategy was performed by intermittent carbon feeding and simultaneous nitrogen feeding in fermentation broth. The maximum DHA concentration and DHA productivity in the fed-batch cultivation reached to 24.74 gL−1 and 241.5 mg/L-1h-1, respectively(93). Waste water recycling technologies are used for fermentation of fungus and microalgae to produce polyunsaturated fatty acids. Song et al., [93] showed that wastewater obtained after fermentation

Production and Applications of Polyunsaturated Fatty Acids 119 of Aurantiochytrium (DHA) and Mortierella alpina (ARA) is used for further next cycle of fermentation process. The fermentation experiments conducted in 5 L fedbatch mode with cross-recycle technology, DHA and ARA productions are 30.4 and 5.13 gL−1, respectively [93]. Hence, this machinery has great potential for industrial fermentation for low-cost polyunsaturated fatty acid production along with nutrient remediation.

6.5

Application of PUFAs

According to the recommendation of the American Heart Association (AHA), all adults should increase their consumption of food derived from n-3 PUFA. The AHA also suggested that people suffering from coronary heart disease take approximately 1 g of eicosapentaenoic acid and docosahexaenoic acid every day. However, PUFAs are potentially applied for many disorders indirectly or directly connected with cardiovascular disease risk such as mood depression, rheumatological diseases, chronic inflammatory lung diseases, chronic kidney disease and others [94]. The AHA also recommended that n3/n6 fatty acids supplements are beneficial to patients suffering from chronic hypertriglyceridemia (>500 mg of triglycerides per deciliter (5.6 mmol/l), and its effective doses are higher, minimum 2 g to 4 g of EPA/DHA per day is required to decrease the triglyceride levels by 20–40% [95]. Recent evidence suggested that EPA and DHA has anticancer properties therefore, it is applied for anticancer drug targeting in the cells. The n-3 PUFAs could apply their anticancer actions by influencing multiple targets associated with the various stages of cancer development, involving angiogenesis, cell proliferation, cell survival, metastasis as well as inflammation against various stages of cancers [96, 97]. Moreover, n-3 PUFAs mainly imposed excellent actions of anti-inflammation and antioxidation as well as their restorative actions. The PUFAs are used as a carrier molecule for targeting anticancer drug in to the tumor cells. The lipophilic nature of PUFAs is readily incorporated into cell lipid bilayer; this will disrupt the membrane structure and fluidity of the cells, therefore chemo-sensitivity is influenced in cancer cells. Therefore, PUFAs can be used as a carrier molecule to increase the therapeutic efficiency of antitumor drugs [98]. In addition, PUFAs possess anticancer activity against the cell lines of PANC-1, Mia- Pa-Ca-2 pancreatic, CFPAC and HL-60 leukemia cell lines. The inhibition of tumor growth by DHA was mediated through decrease in intracellular cAMP through G-protein-coupled signal transduction pathway which is the unique characteristic and vital requirement for tumor cells will make PUFAs find potent applications in drug development.

6.6

Future Perspectives

Recently poly unsaturated fatty acids are considered as conditionally essential fatty acids. The industrial production of these biomolecules in large-scale cultivation of microalgae can address the global demand for these chemicals. The commercial market for these fatty acids are limited by supply, therefore exploitation as well as isolation of novel strains

120 High Value Fermentation Products Volume 2 with important n-3 PUFA research is still going on worldwide. Fermentative processes for oil production from micro organisms are expensive. Alternative low-cost production process will reduce the price of high-value products like fatty acids for human diet. Genetically modified yeast and fungus are used for production of tuned fatty acids such as DHA and EPA but challenging issues for the recovery of this tailor-made fatty acids from this organisms is difficult. Therefore, focus should be given to extraction of oils from modified organisms. Algal oils are really valued for human health, but production cost needs to be minimized, through using cheap organic carbon and low-cost nutrients for their growth along with cheap harvesting process for biomass recovery and better lipids extraction process, which will address the issue related to high price of algal oils. Bio refinery approaches for PUFAs production along with other routes for complete exploitation of microbial oils will again address the issues associated with production cost of PUFAs.

6.7

Conclusion

PUFAs from fish sources are not sustainable due to declining stocks. Phototrophic, heterotrophic microalgae and other organisms have been exploited for polyunsaturated fatty acids productions. Large-scale production of algae through phototrophic systems are beneficial since these systems do not require costly organic carbon source, hence can avoid the problems faced in heterotrophic cultivation systems such as contamination of culture with other organisms. Bio-refinery concepts are attractive; all PUFAs can be separated from the microbial lipids and other portion can be utilized for biofuel applications; the rest of the biomass can be used for valuable protein rich animal feeds. This approaches carried out in a large scale would address the important areas like transportation energy, health and food security.

Acknowledgements One of the authors, Raveendran Sindhu, acknowledges DST for sanctioning a project under DST-WOS-B scheme. Sabeela Beevi Ummalyma, Parameswaran Binod and Raveendran Sindhu acknowledge EPFL, Lausanne, Switzerland, for providing visiting fellowship.

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7 Functional Foods and Their Health Benefits Rwivoo Baruaha, Krishan Kumara and Arun Goyal* Carbohydrate Enzyme Biotechnology Laboratory, Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, Assam, (India)

Abstract The concept of “functional foods” was developed after the widespread understanding of the impact of certain foods on human health. Functional foods can include whole, fortified, enriched or enhanced foods possessing a beneficial effect on health when consumed on a regular basis as a varied diet. Traditional and familiar foods can also be functional foods where recent research findings have brought forth their new health benefits or dispelled negative perception. Fermented foods are one such class of food which have recently earned interest for their various benefits to human health. Besides the health-promoting nutrients present in these foods, native live microorganisms (probiotics) also play an important role in conferring health benefits and bio-transformation of food material to high-value microbial substances. Keywords: Functional foods, fermented foods, probiotics, lactic acid bacteria

7.1

Introduction

Functional foods provide additional health benefits due to the presence of physiologically-active compound(s) [1]. The term “functional food” was first used in Japan in 1984, when some specific food products were marketed with the addition of special constituents which were advantageous to human health. In 1991, the Japanese Ministry of Health introduced certain rules and approved the specific health-related food category called FOSHU (Food for Specific Health Uses) [2]. The largest market for functional foods in the world is Japan followed by the USA and the European market [3]. Japan, being the birthplace of functional foods, has a wide range of such products. It was reported in 1999 that more than 1,700 functional food products were launched between 1989 and 1999 with an estimated turnover of around US$14 billion [4]. Functional foods are of four types: (a) Fortified foods: foods that are fortified with additional components (e.g., fruit juices fortified with vitamin C, zinc and calcium), (b) Enriched foods: foods with additional components, which benefits health and are a

RB, KK and KK contributed equally.

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (127–146) © 2019 Scrivener Publishing LLC

127

128 High Value Fermentation Products Volume 2 not found in a particular food (e.g., probiotics and prebiotics), (c) Altered foods: foods from which an unwanted component is either removed, reduced or replaced by a beneficial component (e.g., fibers as fat releasers in meat), (d) Enhanced foods: foods in which a beneficial component is naturally enhanced (e.g., eggs with increased omega-3 fatty acids). Fermented foods occupy an important position in human diet. Probiotics are “live microorganisms which when consumed in adequate number confer health benefit on the host” [5]. Prebiotics are non-digestible food ingredients that beneficially affect the host by selectively stimulating growth and/or activity of one or a limited number of beneficial bacteria in the colon, thus improving the host’s health [6]. Fermented foods are the foods where a controlled microbial growth is responsible for the conversion of its components from its original form to its final form through various enzymatic processes [7]. Many microbes found in various fermented foods are probiotic in nature and the foods may also contain prebiotics. The fermented foods containing probiotics and prebiotics are popular as functional foods. Fermented foods can be either traditional such as yoghurt or new fermented food such as cereal-based “Yosa,” which is made from oat bran pudding (water cooked) fermented with lactic acid bacteria (LAB) and bifidobacteria, it is consumed in Finland and other Scandinavian countries [8].

7.2

Fermented Functional Foods

7.2.1

Benefits of Fermented Functional Foods

Humans have been consuming fermented foods for thousands of years, and there are a wide variety of fermented foods throughout the world. Their origin is from diverse sources such as dairy, cereals, legumes, vegetables, fruits, meat and fish. The major groups of microorganisms associated with fermented foods are lactic acid bacteria (Lactobacillus, Leuconostoc and Streptococcus), Acetobacter, Propionibacterium, yeast and moulds [7]. Fermented foods have gained popularity in recent years owing to their health-promoting characteristics that are not found in their initial food material. There are several health benefits of fermented foods such as improvement in glucose metabolism or anti-obesity and anti-diabetic effects [9, 10]. The probiotic microbiota associated with these health-promoting fermented functional foods plays a pivotal role. The probiotic bacteria capable of producing bile salt hydrolase (BSH) enzyme have gained interest owing to their role in improving heart health [11]. BSH producing strain Lactobacillus reuteri NCIMB 30242 when supplied to humans through yoghurt or capsule, helps in reduction of total cholesterol by 5–9%, low-density lipoprotein cholesterol (LDL) by 9–12% and the ratio of apolipoprotein B to A (apoB/apoA) by up to 13%, all positively affecting the human heart health [12]. Lactobacillus reuteri NCIMB 30242 was shown to reduce both atherogenic C-reactive protein (CRP) and fibrinogen, the two major factors of atherogenesis along with the increased levels of 25-hydroxyvitamin D, which is a modest cardioprotectant [13].

Functional Foods and Their Health Benefits 129

7.2.2 Synthesis of Nutraceuticals and Bioactive Compounds in Fermented Functional Food An array of health promoting compounds are available in fermented functional food that are consumed by humans (Table 7.1). Lactic acid (or lactate) is produced by lactic acid bacteria through sugar fermentation which can be over 1%. Lactic acid is associated with the reduction of pro-inflammatory cytokine secretion in immune cells in a dose dependent manner [29]. Lactate is also responsible for the reduction of reactive oxygen species in intestinal enterocytes [30]. Lactic acid in lower GI tract helps in maintaining a low pH environment, inhibiting the growth of pathogenic bacteria. β-galactosidase is one of the enzymes which hydrolyzes lactose to galactose and glucose, which is produced by probiotic bacteria associated with the fermented dairy products. It is able to retain activity even after passing through acidic conditions in the stomach, as it is physically protected within the bacterial cells. It is helpful to lactoseintolerant individuals, as it improves their digestion capacity for lactose-containing milk products [31]. LAB produces low molecular weight peptides called bacteriocins that show antibacterial activity towards other Gram positive bacteria. These bacteriocin producing LABs are used for food preservation and show activity against foodborne pathogens such as Clostridium botulinum, Staphylococcus aureus and Listeria monocytogenes [32]. Most of the other microbial products are strain dependent, which include vitamin B (riboflavin, folate and B12), amino acids and their derivatives (γ-aminobutyric acid, GABA) and microbial exopolysaccharides and various prebiotic oligosaccharides. Exopolysacchrides are complex polysaccharides with either same or different monosaccharide units [33]. LAB are the producers of exopolysaccharides in fermented foods and are well studied. Exopolysaccharides are used for their various functional applications such as for their hypocholesterolemic activities, as antioxidants, for prevention of pathogenic microbes from adhering the intestinal mucosa [7], and as a prebiotic food additive [34, 35].

7.2.3 Fermented Functional Foods and Their Health Benefits 7.2.3.1

Yoghurt

Yoghurt is a fermented product produced by culturing the mixture of milk and cream with the lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus). Yoghurt may also be supplemented with additional bacteria that contribute beneficial factors [36]. Yoghurt can also be produced by changing the composition (low fat, or without fat milk) or by adding extra flavors, and additives such as fruits, natural sweeteners or by adding extra supplements like vitamin D. Yoghurt is rich in dietary minerals as it was reported that 100 g of yoghurt contains 234 mg potassium, 183 mg calcium, 144 mg phosphorous, 17 mg magnesium, and 0.9 mg zinc. In yoghurt fermentation, LAB create an acidic environment which maintains the minerals in their ionic forms that increases the mineral absorption in the intestine [14, 37]. Yoghurt is also a rich source of vitamins: a 100 g yoghurt contains 0.21 mg riboflavin, 0.11 mg niacin, 0.05 mg vitamin B6, and 0.56 mg vitamin B12 [38]. Yoghurt is good source of

Organisms involved

Lactobacillus delbrueckii subsp. bulgaricus Streptococcus thermophiles

Lactobacillus, Lactococcus, Leuconostoc and Streptococcus (Starter culture) Lactobacillus, Pediococcus, Enterococcus and Leuconostoc (Non-starter culture)

Leuconostoc, Lactococcus, Lactobacillus and Weissella

Aspergillus oryzae Aspergillus sojae Pediococcus halophilus Zygosaccharomyces rouxii and Candida species

Lactobacillus, Pediococcus, Leuconostoc and Streptococcus Saccharomyces,Candida and Pichia

Fermented food

Yogurt

Cheese

Kimchi

Soy sauce

Sourdough

Table 7.1 Fermented functional foods.

Organic acids (lactic acid and acetic acid) Exopolysaccharides Bacteriocins

Ethanol HEMF, Shoyuflavones A, B and C

Vitamins A and C, -Cartenoids Polyphenols, Anthocyanins Thiosulfates

Vitamin A, Vitamin B Proteins and free amino acids

Proteins and free amino acids, Vitamin B, Zinc Calcium

Products formed/rich in

Regulates blood glucose levels Improves mineral bioavailability

Antimicrobial Antioxidative Enhancement of gastric juice secretion Mediates inflammation

Decrease Cholesterol level Regulates Blood pressure Antibacterial in nature

Delivery of probiotic bacteria Bioactive peptides γ-aminobutyric acid (GABA)

Delivery of probiotic bacteria Improves bone density and muscle health Increases HDL

Functional application

[27, 28]

[23–26]

[21, 22]

[17–20]

[14–16]

References

130 High Value Fermentation Products Volume 2

Functional Foods and Their Health Benefits 131 essential amino acids and contains high protein content. The proteolytic activity of bacteria in yoghurt predigests the milk proteins and releases free amino acids that result in better protein digestibility [14]. Yoghurt, being a good source of protein and calcium, enhances bone density and muscle health [15]. Consumption of yoghurt by postmenopausal women resulted in the reduction of N-telopeptide, which is a urinary marker of bone resorption [39]. A study indicated that yoghurt consumers have better metabolic profile compared to non-consumers such as lower bone mass index, reduction in triglycerides, fasting glucose and insulin and increase in High Density Lipoprotein (HDL) cholesterol [16]. Most of the elderly people suffer from protein-energy malnutrition and have deficiencies in zinc and vitamin B6 [40]. Pneumonia is prevalent among them because of their deficiency in zinc [41]. The intestinal microflora of the eldery is also affected as a result of use of antibiotics, reduced intestinal motility and changes in gastrointestinal architecture. Therefore, yoghurt may be the best option as it contains zinc, protein, vitamin B6 and probiotic bacteria and can improve gut-associated problems.

7.2.3.2

Cheese

Cheese is produced from the ripening of coagulated milk solids after separating from the whey. The coagulated milk solids are obtained by acidification of milk by starter cultures or acids or addition of enzyme rennet. The coagulated solids are pressed and ripened. The ripening process can range from 2 weeks to 2 years, during which several processes such as proteolysis, lipolysis, aroma production and colour production occur [42]. Cheese contains a host of micro nutrients like calcium, phosphorus, magnesium, zinc and vitamins like vitamin A, vitamin B2 (riboflavin) and vitamin B12 [43]. The major protein present in cheese is casein along with trace amounts of alpha-lactalbumin and beta lactoglobulin [7]. Cheese protein is among the high-quality proteins from foods for its unique amino acid composition, having a relatively higher amount of essential amino acid lysine. Lactose is the major carbohydrate found in milk; during cheese production the majority of the lactose is removed with the whey and the remaining small amount is later fermented to lactic acid during ripening. Due to the low amount of lactose present in cheese, it can be consumed by lactose intolerant individuals [7]. The major fat content of cheese undergoes hydrolysis during ripening, resulting in the release of volatile fatty acids such as butyric, caproic and caprylic acids, which also contribute to the flavor and texture of the cheese [43]. Most cheeses are fermented twice during its production, once by starter culture for milk coagulation and finally by non-starter culture during the ripening process. Starter cultures were introduced in cheese making, in the 19th century, to standardize the rate of acidification for milk coagulation. The major starter cultures used for cheese making are lactic acid bacteria (SLAB) from the mesophilic or themophilic species of Lactobacillus, Lactococcus, Leuconostoc and Streptococcus. Non-starter lactic acid bacteria (NSLAB) dominate cheese microbiota during ripening. The genus Lactobacillus contains both homo and hetero-fermentative species and is dominant among NSLAB. Non lactobacilli cultures among NSLAB also include Pediococcus, Enterococcus and Leuconostoc, which can be of the same species as the starter cultures [44]. During

132 High Value Fermentation Products Volume 2

Figure 7.1 Kimchi (Source: https://i.ytimg.com/vi/0sX_wDCbeuU/maxresdefault.jpg).

ripening the NSLAB utilize the residual lactose and other carbohydrates or metabolites left from SLAB fermentation. The NSLAB degrade the casein and peptides to form free amino acids (FAA) which act as precursors to volatile aroma compounds and contribute to flavor of the cheese [17]. Cheese is a suitable matrix for the delivery of probiotic bacteria due to its higher pH, buffering capacity and fat content. It can protect probiotic bacteria more efficiently than a fluid environment, during transit through the human GI tract [18]. Bioactive peptides produced by the NSLAB fermentation of milk proteins are reported to show hypotensive and/or Angiotensin-I-converting enzyme (ACE)-inhibitory activity [19]. Some NSLAB also biosynthesize γ-aminobutyric acid (GABA) from L-glutamate, GABA has beneficial effects on human health such as blood pressure regulation [20].

7.2.3.3

Kimchi

Kimchi (Figure 7.1) is the most popular traditional dish of Korea, which is obtained by the fermentation of simple vegetables such as cabbage, pumpkin, radish, sweet potato and cucumber with seasoning like salts, red and black pepper, garlic, ginger and leek. The ingredients used for the preparation of Kimchi are vegetables, spices, seasonings and other additional materials [45]. The most commonly used vegetables are Chinese cabbage, radish, ponytail radish, young oriental radish, sweet potato and cucumber [46]. The spices that are commonly used for Kimchi preparation are red and black pepper, ginger, cinnamon, mustard, garlic and onion. The seasonings used in Kimchi are salt and salt-pickled seafood, sesame seed, corn syrup, and soybean sauce. Some additional ingredients used are carrot, rice, barley, apple, leek, peas, meat like pork and beef. The microorganisms involved for the fermentation of Kimchi belong to Lactic acid bacteria such as Leuconostoc mesenteroides, Ln. carnosum, Ln. gelidum, Ln citreum,

Functional Foods and Their Health Benefits 133 Ln kimchi, Ln gasicomitatum, Lactococcus lactis, Lactobacillus plantarum, Lb sakei, Lb spicheri, Lb brevis, Lb parabrevis, Lb curvatus, Weissella confusa, W. cibaria and W. koreensis [47, 48]. In 2006 Health magazine listed Kimchi as one of the world’s top five healthiest foods [49]. This was counted among the top healthy foods because it possesses high nutritional values of the raw materials like Chinese cabbage which contains vitamin A and C, minerals (Ca, Mg, K and Na), Cartenoids (lutein and α-carotene) [50]. Radish and onion are the sources of phytochemicals such as polyphenols, anthocyanins and thiosulfates [21, 51]. Onion also contains flavonoids (quercetin glucosides) with reported anti-inflammatory [22, 52] and anti-carcinogenic [53] action. Various studies showed that consumption of Kimchi daily decreases the cholesterol level in the body and increases the immune system of the body [54]. Consumption of fermented Kimchi has been shown to be more effective on factors associated with systolic and diastolic blood pressures, fasting glucose, percent body fat, and total cholesterol as compared with the fresh Kimchi, which suggests that fermentation of Kimchi adds favourable effects on metabolic parameters. Kimchi also has antibacterial potential because of the presence of sulphur containing compounds and LAB [55].

7.2.3.4

Soy Sauce

Soy sauce is the most popular oriental condiment and colouring agent in food preparation or for regular table use. It is widely consumed in Asian counties especially China, Japan and Korea. It is a dark brown liquid, which can be kept at room temperature without the need for refrigeration due to its high salt content and low water. Traditionally, soy sauce is produced by fermentation of soybean and wheat flour, which are inoculated with mold (Aspergillus oryzae). After the growth of mold, it is placed in brine for a secondary fermentation by bacteria and yeast to finally obtain soy sauce. Inferior soy sauce is produced by acid hydrolysis of soybean and wheat mixtures; as a result, it lacks the rich flavor of the fermented product. There are two major kinds of fermented soy sauce: Chinese and Japanese. The Chinese variety is made by using either soybeans alone or by a mixture of soybeans and wheat, having a higher percentage of soybean in the mix. Japanese soy sauce is primarily made with equal portions of soybean and wheat. Both varieties are different in aroma, taste and viscosity [56]. In general soy sauce contains 2–5% (w/v) reducing sugars, 1.0–1.65% (w/v) nitrogen, 1–2% (w/v) organic acids, 2.0–2.5% (w/v) ethanol and 17–19% (w/v) sodium chloride. A total of 18 amino acids are found in soy sauce of which glutamic and aspartic acids are the major components. Among sugars glucose, arabinose, mannose, galactose and xylose along with a few unidentified sugars have been reported. Organic acids present in soy sauce are acetic, citric, succinic, formic and lactic acids [57]. Soy sauce also contains varieties of other components contributing to its distinctive flavor and texture. Traditionally in fermented soy sauce, the mixture of soybean and wheat is initially inoculated with “Koji”. Koji is a pre-inoculum of mold such as Aspergillus oryzae or Aspergillus sojae which are grown on steamed rice or other cereals [56]. Koji acts as an enzymatic preparation having high proteolytic and amylolytic activity which breaks down the protein and starch respectively to simpler compounds for the secondary fermentation [57]. The secondary fermentation takes place under high salt concentration

134 High Value Fermentation Products Volume 2

(a)

(b)

Figure 7.2 Sourdough (a) Liquid (Source: https://breadtopia.com/wp-content/uploads/2016/08/starter. jpg) (b) Bread (Source: https://cms.splendidtable.org /sites/default/files/styles/w2000/public/7115933903 _a5cdff7c77 _z.jpg?itok = DiRxD1 eP).

or brine, which is inoculated with halotolerant lactic acid bacteria Pediococcus halophilus and halotolerant yeasts such as Zygosaccharomyces rouxii and Candida species. The high salt concentration of 20% (w/v) prevents the growth of undesirable microbes. The yeasts are inoculated after the pH has reached 5.0 from 6.5 and fermentation temperature is raised from 15°C to 28°C and is kept for 4 months [57]. Soy sauce possesses several health promoting properties, which makes it functional food [58]. Some of these properties include the enhancement of gastric juice secretion [23]. Soy sauce has been reported to have antimicrobial properties against pathogenic bacteria due to its salt and ethanol content as well as its low pH [24]. Japanese soy sauce contains a flavor component, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2 hr)-furanone (HEMF) which is a potent antioxidant and active anticarcinogen [25]. Soy sauce also contains 3 tartaric isoflavone derivatives, named shoyuflavones A, B and C [26]. Shoyuflavones are exclusive to fermented soy sauce and shown to possess histidine decarboxylase (HDC) inhibitory activities, as a result of which histamine is produced from L-histidine. Histamine is responsible for mediating inflammation, allergies, neurotransmission and gastric acid secretion [26].

7.2.3.5

Sourdough

Sourdough is a mixture of one of the cereal flours (wheat, buckwheat, oat, maize and rye) and water (Figure 7.2 a & b), that is fermented with the help of yeasts and lactic acid bacteria [59]. Lactic acid bacteria found from sourdoughs are Lactobacillus, Pediococcus, Leuconostoc and Streptococcus. Many species of yeasts are also identified in sourdoughs such as Saccharomyces cerevisiae, Torulopsis holmii, S. exiguous, Candida krusei, Hansenula anomala and Pichia norvegensis [60]. In wheat sourdoughs, Lb. sanfranciscencis, Lb. brevis linderi, Lb. plantarum and Lb. brevis increase the levels of free amino acids [60, 61] whereas the yeasts S. cerevisiae and S. exiguus decrease the amount of amino acid [62]. Therefore, combination of lactic acid bacteria and yeast shows intermediate levels of amino acid. Lactic acid bacteria in sourdough produce exopolysaccharides which act as hydrocolloids and beneficial to the sourdough texture and shelf life [35, 63].

Functional Foods and Their Health Benefits 135 White wheat bread, potato, rice products and many cereals are known as starchy foods having high glycaemic response. These high glycaemic foods show immediate increased level of blood glucose [27]. The lactic acid produced in Sourdough fermentation creates the interaction between starch and gluten and reduce the digestibility of starch [64]. Whole meal cereals are source of minerals such as K, Mg, P, or Zn, but utilization of minerals is limited due the presence of phytic acid in cereals [28]. Phytic acid forms insoluble complexes with cations hindering the mineral’s bioavailability [28]. Phytic acid degrading enzymes require the pH value of 4.5 for efficient hydrolysis of phytic acid [65]. In sourdoughs fermentation pH can be adjusted to increase phytic acid hydrolysis and to improve mineral bioavailability [66]. Sourdough also has antimicrobial activity due to the production of organic acids (lactic acid and acetic acid), ethanol, carbon dioxide, hydrogen peroxide and diacetyl by Lactic acid bacteria [67]. The inhibition activities also produce a low molecularmass peptide or proteins called bacteriocins, having bactericidal mode of action [68]. Therefore, use of bacteriocin producing Lactic acid bacteria in the fermentation of cereals is currently under investigation.

7.3

Functional Whole Foods

7.3.1

Cereals

Cereals are the most cost-effective food in the world and they are considered as nutraceuticals or functional food because they are the source of dietary fibres, proteins, vitamins, energy, minerals and antioxidants. Wheat, rice, barley, millets, oat, corn, sorghum, flaxseed, psyllium and their products are the most common cereals functional foods containing nutraceuticals (Table 7.2). Wheat (Triticum aestivum) contains the free and esterified phenolic acid that possesses strong antioxidant activity. It has been shown that the solubility and antioxidant activity of the phenolic acid increases at gastrointestinal pH [69]. Wheat contains magnesium in abundance which acts as co-factor for various enzymes in body, including enzymes used for glucose metabolism and insulin homeostasis [82]. The consumption of wheat grain lowers the risk of type2- diabetes [83]. Wheat bran consists of lipid-soluble phytochemicals, alkylresorcinols (ARs), which plays a role in colon cancer prevention [84]. Wheat germ oil is rich in octacosanol, tocopherol, folate, β-carotene and essential fatty acids such as linoelic acid and linolenic acid [71]. A study showed that wheat germ oil can enhance the blood flow as well as immunity [70]. Rice (Oryza sativa) is one of the major cereals used as staple food [85]. Rice bran possesses most of the nutraceuticals such as dietary fibers (β-glucan, gum and pectin), antioxidants (tocopherols, tocotrienol and oryzanol) and α-lipoic acid [74]. Rice bran also contains Lutein and Zeaxanthin which reduce the chances of cataract [71]. Oat (Avena sativa) is rich in soluble fiber (β-glucan) and the consumption of this has a beneficiary role in glucose tolerance and blood lipid [86, 87]. Oat contains tocols (vitamin E) which reduces the risk of coronary heart disease (CHD) by reducing

Biological name

Triticum aestivum

Oryza sativa

Avena sativa

Linum usitatissimum

Pennisetum glaucum

Fagopyrum esculentum moench

Sorghum bicolor

Food

Wheat

Rice

Oat

Flax

Millet

Buckwheat

Sorghum

Table 7.2 Non-fermented functional foods.

3-deoxyanthocyanidins

Anticancerous activity

Antihaemorrhagic Hypotensive drug

Reduce the risk of degenerative diseases Anticancerous

Calcium, iron, potassium, magnesium, zinc, Phytic acid Niacin, B6 and folic acid, flavonides, flavones, phytosterols Thiamin-binding protein

Prevention of estrogen-dependent cancers

Reducing plasma LDL cholesterol Good for celiac diseases patients

Improve lipid profile in plasma Reduce chances of cataract Prevent lipid oxidation

Antioxidant activity Glucose metabolism and insulin homeostasis Anticancerous Enhance the immunity

Functional application

Omega-3 fatty acid, Enterodiol and Enterolactone,

Vitamin E, β-glucan

Dietary fibers (β-glucan, gum and pectin) Antioxidants (tocopherols, tocotrienols, and oryzanol), β-sitosterol (Lutein and Zeaxanthin)

Phenolic acid, Magnesium Alkylresorcinols, Octacosanol, Tocopherol Folate, Beta carotene

Products rich in

(Continued)

[71]

[71]

[71]

[74]

[72, 73]

[71]

[69, 70]

References

136 High Value Fermentation Products Volume 2

Biological name

Zea mays

Zingiber officinale

Allium sativum

Myristica fragans

Allium cepa

Corn

Ginger

Garlic

Nutmeg

Onion

Cont.

Food

Table 7.2

Sulphoxides Cepaenes

α- and β-pinenes Sabinen Eugenol, Methyl engenol, Neolignan, Safrol, Elemicin, Myristicin and Linalool

Allicin and ajoene

Gingerols, β-carotene, Shagaols, Caffeic acid, Salicylate, Curcumin Capsaicin

Thiamin, Folate, Dietary fiber, Vitamin-C, Pantothenic acid Cryptoxanthin, Phenolic compounds, Phosphorous andmanganese

Products rich in

Treat appetite loss Arteriosclerosis Digestive problems Asthmas and diabetes

Anticancerous activity Insecticidal activity

Anticancerous activity Increase immunity

Preventing of DNA damage in vitro Prevent ageing dependent penile vascular changes Impotency

Anticancerous activity Antioxidative effect

Functional application

(Continued)

[78]

[77]

[74]

[75, 76]

[71]

References

Functional Foods and Their Health Benefits 137

Biological name

Piper nigrum

Capsicum frutescens

Curcuma longa

Food

Black pepper

Chilli pepper

Turmeric

Table 7.2 Cont.

Curcumin

Phenolic compounds

Piperine, Piperamine and Piperyline

Products rich in

Anti-oxidant Anti-inflammatory immunomodulatory Anti-tumor properties

Inhibits iron absorption

Gastric juice secretion Anti-inflammatory

Functional application

[78]

[81]

[79, 80]

References

138 High Value Fermentation Products Volume 2

Functional Foods and Their Health Benefits 139 plasma LDL cholesterol [72]. Oat lacks gluten, so it can be used as food for patients with celiac disease [73]. Flaxseed (Linum usitatissimum) is known as nutraceutical because it is a good source of omega-3 fatty acid (α–linolenic acid) and phenolic compound lignin; therefore regular use of flaxseed provides specific health benefits [74]. Flaxseed contains enterodiol and enterolactone that are structurally similar to the naturally and synthetic estrogens; therefore it also shows weak estrogenic and anti-estrogenic activities, these may play a role in the prevention of estrogen-dependent cancers [74]. Flaxseed has also shown antitumor activity against colon cancer [88] and in the lungs of mice [89]. Regular consumption of flaxseed resulted in the reduction in the LDL cholesterol [90] as well as platelet aggregation [91].

7.3.2

Culinary Herbs and Spices

Many culinary spices and herbs (for example, clove, garlic, onion, ginger, pepper mustard and turmeric, etc.) have been used in food for centuries. These herbs and spices have been shown to possess numerous bioactive constituents, which have specific health benefits [92]. Ginger (Zingiber officinale) is known for its antimutagenic activity, induction of detoxification and preventing of DNA damage in vitro [75, 76]. Ginger constitutes a number of compounds that include gingerols, β-carotene, shagaols, caffeic acid, salicylate, curcumin and capsaicin [93]. Ginger can also treat peptic ulceration due to its inhibitory action on thromboxane synthetase [93]. Ginger root can prevent ageing dependent penile vascular changes and impotency [81]. Garlic (Allium sativum) contains numerous sulfur-containing compounds and some of this compounds showed chemopreventive activity [94]. Garlic has shown antitumor activity against colon cancer, gastrointestinal tract cancer, prostate cancer, mammary carcinoma, lung cancer, hepatocellular carcinoma and sarcoma and squamous cell carcinoma and esophagus [74]. Garlic contains allicin and ajoene that inhibit inducible nitric oxide syntheses and promote vasodilation [81]. The consumption of garlic and its components strengthen the immune system by increasing total white blood cell (WBC) count [95]. Nutmeg (Myristica fragans)oil exhibits anti-inflammatory, antibacterial, antifungal and insecticidal activities due to the presence of β- and α-pinenes and sabinen, eugenol, methyl engenol, neolignan, safrol, elemicin, myristicin and linalool [81]. Myristicin isolated from the nut (Figure 7.3) impacts exhibits an effective insecticidal activity [77]. Onion (Allium cepa) is used to treat appetite loss, arteriosclerosis, digestive problems colds, asthmas and diabetes [78]. The pungent smell and pharmacological activity of onions are due to the presence of sulphur-containing compounds like sulphoxides such as trans-5-(1-propenyl)-L-(+)-cysteine sulphoxide and cepaenes (α-sulphinyldisulphides) [78]. Black pepper (Piper nigrum) contains piperine, piperamine and piperyline which enhance the bioavailability of various therapeutically drugs [96]. Piperine has also been used for gastric juice secretion [79]. Other researchers have shown that pepper demonstrated impressive antioxidant [97, 98] and anti-inflammatory effects [97].

140 High Value Fermentation Products Volume 2

Figure 7.3 Nutmeg (Myristica fragans) (Source: https://media1.britannica.com/eb-media/77/170777004-E8858E53.jpg).

Chilli pepper (Capsicum frutescens) may interact with epithelial cells of the gastrointestinal tract to modulate their transport properties [99]. Chilli pepper contains phenolic compounds that bind to iron in the intestine and inhibit its absorption [81]. Turmeric (Curcuma longa) is the dried rhizome of plant Curcuma longa that is commonly used as a spice. It has been used for treating peptic ulcer and for its carminative effects [78]. It also shows properties including anti-oxidant, anti-inflammatory immunomodulatory and anti-tumor properties [100].

7.4

Conclusion

Functional foods have made their own position in the current food industry since their inception in 1984. With the increase in their popularity and development of newer products through cutting-edge research, they will create new market share. Research on the human microbiome would throw light on the influence of various microorganisms and their products from fermented foods on human health. There is great need of studies on the strains native to traditional fermented foods from which newer strategies for the improvement of human health could be achieved. Many whole foods also have gained interest as functional foods in recent times, and as a result their sales have considerably increased. This is mainly due to the advancement in food sciences, which

Functional Foods and Their Health Benefits 141 helps in highlighting the specific health promoting effect of otherwise uninteresting foods. Similarly, the advancement in the field of metagenomics, metabolomics and bioinformatics will help in deciphering key pathways involved in health-promoting factors of fermented functional foods and functional whole foods.

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8 Industrially Important Biomolecules From Cyanobacteria Y. P. Reddy, R. K. Yadav, K. N. Tripathi, A. Borah, P. Jaiswal and G. Abraham* Centre for Conservation and Utilization of Blue Green Algae, ICAR-Indian Agricultural Research Institute, New Delhi-110012, (India)

Abstract Cyanobacteria are photosynthetic organisms playing the role of primary producers inany ecosystem. Their cosmopolitan distribution coupled with faster growth rate and ability to fix atmospheric nitrogen makes the munique. Initially, these organisms have been exploited widely as biofertilizer for rice cultivation. These organisms are of great significance due to the presence of a variety of metabolites of pharmaceutical, biotechnological and industrial significance. Because of this potential, these organisms have been exploited as food, feed and neutraceuticals. Further, detailed understanding of these metabolites and the organism will help in augmenting their production and exploitation. In this review, attempts have been made to discuss the importance of these metabolites in view of the industrial prospects of biomolecules from cyanobacteria Keywords: Algae, bioactive compounds, biomolecules, food industry, neutraceuticals

8.1

Introduction

Cyanobacteria, commonly known as blue-green-algae, are a group of Gram-negative prokaryotes capable of performing oxygenic photosynthesis. They are considered to be one of the oldest life forms evolved on Earth, somewhere around 2.4 to 3.2 Ga [1–3] with Prochlorococcus (contains Chl b) as immediate ancestor [4]. Stanier and Van Niel [5] were the first to argue for their inclusion and classification under bacteria (photosynthetic bacteria) because of the prokaryotic nature of cells. Cyanobacteria are described as bacteria obtaining energy through photosynthesis and evolving oxygen as a by product [6]. The group consists of a heterogeneous assemblage of unicellular, colonial, filamentous to branched filamentous forms [7]. The cyanobacteria inhabit a wide spectrum of habitats ranging from aquatic to terrestrial environments as well as extreme habitats such as hot springs, hypersaline waters, deserts, and Polar Regions [8]. These organisms are unique and can easily survive on bare minimum requirement of *Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (147–164) © 2019 Scrivener Publishing LLC

147

148 High Value Fermentation Products Volume 2 light, carbon dioxide (CO2) and water [9, 10]. Because of this reason their cultivation is easy and it does not require any arable land. Cyanobacteria play a significant role in biogeochemical cycle of nitrogen, carbon and oxygen [11]. Some of them are able to fix atmospheric N2 either freely or in association with other organisms. Cyanobacteria degrade in the soil and release the fixed nitrogen in the form of ammonia, polypeptides, free amino acids, vitamins, and auxinlike substances [12]. Because of this property the cyanobacteria are used as biofertilizer for rice paddy cultivation. The nitrogen fixing cyanobacteria help in sustaining and improving the rice productivity of nitrogen-deficient paddy soils [13, 14]. In India, cyanobacteria have been effectively used in the cultivation of paddy [15, 16]. Dubey and Rai [17] established the agronomic potential of cyanobacteria in increasing yield as well as reducing the dependence on chemical nitrogen by 25%. Cyanobacteria have agronomic significance as biofertilizer due to the N2-fixation ability that helps them to grow successfully in habitats where little or no combined nitrogen is available. Recently, their ability to produce structurally novel and biologically active natural compounds has been recognized [18, 19]. Guiheneuf et al., [20] reported the importance of cyanobacteria as a valuable resource due to the presence of several valuable metabolites which can be exploited for biotechnological and industrial purposes. Carotenoids, phenolics, polysaccharides, fatty acids and phytohormones are some of the important compounds obtained from cyanobacteria. In the present chapter we have attempted to highlight the importance of some of the industrially important biomolecules from cyanobacteria.

8.2

Cultivation Methods for Cyanobacteria

Cultivation and harvesting of the biomass is important and in general the cyanobacteria can be cultivated and maintained in a culture room as batch culture. Selection of the appropriate strain that is best suited to the environmental and cultivation conditions is important in the production of high-quality algal biomass. Several factors influence the successful multiplication of the organism. The major factors are nutrients, pH alkalinity, light, cultural cell density and temperature. Contamination by other microorganisms also limits the successful multiplication of the cyanobacterial cultures. In the laboratory scale, the cyanobacteria is maintained as batch cultures in different types of media. Several media composition is available for the cultivation of cyanobacteria [21]. Various culture media commonly available for the cultivation of cyanobacteria is shown in Table  8.1. Based on the nitrogen fixation capacity of the cyanobacteria the medium may be supplemented with a nitrogen source such as nitrate, ammonia or urea. Cyanobacteria also take up inorganic and organic forms of carbon from the extracellular environment. Another essential macro-nutrient for growth is phosphorus. However, in phosphorus deficient the cyanobacteria may media utilize the storage form of phosphorous as polyphosphate reserves. Sulphur, calcium, magnesium and potassium are the other macronutirents required for the growth. The micronutrients required are molybdenum, iron, nickel, copper, zinc, cobalt, boron and manganese. However, economically important cyanobacteria used for commercial purposes can be mass multiplied in large

Industrially Important Biomolecules From Cyanobacteria 149 Table 8.1 Some common culture media used for the cultivation of cyanobacteria. Habitat

Media

Freshwater

Allen’s Blue-Green Algal Medium

Marine

References [22]

BG-11 Medium, Modified

[22–24]

Bold’s Basal Medium (BBM)

[25, 26]

Chu #10 Medium

[27]

Half-Strength Chu #10 Medium

[28]

COMBO Medium

[29]

D Medium

[30]

D11 Medium

[31]

Fraquil Medium

[32]

MA Medium

[33]

Spirulina Medium, Modified

[34]

ASN-III Medium

[35]

YBC-II Medium

[36]

F/2 Medium

[37, 37, 38]

K Medium

[39]

L1 Medium

[40]

Pro99 Medium

[41]

SN Medium

[42]

ANT Medium

[43]

raceways fitted with paddle wheel for agitation. They can also be maintained in small cemented tanks or plastic trays as per requirement and quality standards to be maintained. Bioreactors are used specially for the production of biodiesel, bioethanol and also to produce animal feed. On the basis of the design, the bioreactors have been classified in to open and closed bioreactors. Closed bioreactors are also known as photo-bioreactors. Different types of photobioreactors have also been developed on the basis of the availability of sunlight. However, the cultivation of algae in a photobioreactor narrows down the range of industrial application. Considerable amounts of wastewater generated from agro-industries have been used for the multiplication of cyanobacteria [44]. This may be considered as environment-friendly approach to reduce inorganic as well as organic pollutants. Cyanobacteria has been reported

150 High Value Fermentation Products Volume 2 Auxins

Vitamins Phytohormon es

Cytokinin

Phenolic compounds

Gibberellins

Flavonoids Plant Growth Promotion

Ethylene Abscisic acid

Fatty acid Amino acids

Lowering hydrolytic conductivity

Biodiesel Biogas

Polysacchari des

Bio - energy Heavy metals

Enhancing N&P availability

Textile dye Insecticide residue

Bio remediation

BGA

Improvemien to soil

Soil health

Oil spills Lowering EC

Lowering pH Antifungal Bio - control

Biodegradable plastics

Antiviral

Thickening and gelling agent

Antialgal Antibacterial

Lectins

Bio molecules

Medicine

Antioxidants

Antibiotic

Food

Antioxidan ts Minerals Natural Colorants

Anti HIV Anti inflamatory

Sun screen

Cosmetic Industry

Antiviral

Anti - aging Peptides

Anti - parasitic Anti cancerous

Protein MUFA

Vitamins PUFA

Figure 8.1 Multifaceted role of biocompounds found in cyanobacteria (BGA).

to have the capacity to degrade POPs, which are commonly used in industry and agriculture [45]. They observed that concentrations of ammonium, nitrate, nitrite and phosphate ions were considerably reduced in 10–15 days, following growth of Phormidium foveolarum in media with a maximum of 10% wastewater (7.9 mg/L of NH4+, 0.05 mg/L of NO3−, 0.12 mg/L of NO2−, and 0.03 mg/L of PO43−). Use of wastewater for algae cultivation offers an interesting option for clean bio-treatment coupled with the production of potentially valuable biomass, which can be used for production of value-added products. Harvesting the biomass is another challenge in the cultivation of cyanobacteria. The unicellular cyanobacteria being small in size pose problems during harvesting. But because of the larger size the filamentous forms can be easily harvested.

8.3

Multifaceted Role of Biocompounds Found in Cyanobacteria

An array of biocompounds having agricultural, pharmaceutical and industrial importance are produced by the cyanobacteria. In the present context, these biocompounds have been exploited for diverse purposes. The versatile applications of cyanobacterial metabolites are depicted in Figure 8.1.

Industrially Important Biomolecules From Cyanobacteria 151

8.3.1 Plant Growth Promoting Compounds Phytohormones are important in the growth and development of plants. Cyanobacteria have been reported to release extracellular plant growth promoting substances such as hormones. These compounds include auxins, gibberellins, cytokinins and abscisic acid [15, 46–48]. Studies conducted by Misra and Kaushik [49] and Karthikeyan et al., [50], showed the potential of cyanobacteria as plant growth regulators. Hussain et al., [51] showed the capability of endophytic Nostoc strains to produce phytohormones such as indole acetic acid and cytokinins in root cells of rice and wheat. The cyanobacteria accumulate and release a diverse group of phytohormones such as auxins, gibberellins, cytokinins and ethylene [52]. Recently, Gayathri et al., [53] reported plant growth promoting ability in several heterocystous and a unicellular cyanobacteria belonging to four different genera viz. Calothrix, Nostoc, Anabaena and Aphanothece isolated from diverse rice fields. For the first time they showed the production of free FAs volatiles by cyanobacteria along with known phytohormones e.g., IAA, IBA and cinnamic acid in the HPLC purified fraction of biomass wet extract (BWE). The cyanobacteria also secrete other compounds such as vitamins, amino acids and antibiotics with a potential role in plant growth promotion [54, 55]. Cyanobacterial inoculation in rice and wheat enhanced rice seed germination, root and shoot growth, root dry weight and chlorophyll [49, 56, 57]. Cyanobacterial inoculation has been linked with the accumulation of phenolic acids (gallic, gentisic, caffeic, chlorogenic and ferulic acids), flavonoids (rutin and quercetin) and phytohormones (indole acetic acid and indole butyric acid) in rice leaves [58]. It was observed that cyanobacterial inoculation resulted in systematic accumulation of chlorophyll, protein and total phenol besides induced levels of phenylpropanoids and phytohormones in rice leaves. In this context, it is important to screen cyanobacterial strains from wider range of agro-ecological habitats to assess their plant growth promoting potential. Thus, the exploitation of the plant growth promoting potential of cyanobacteria could be useful in reducing dependence on the synthetic and chemical plant growth promoting substances to a greater extent. This in turn would help in combating the pollution load due to chemicals.

8.3.2

Soil Health Improvement

Diazotrophic cyanobacteria play a key role in maintaining the nitrogen budget as well as fixing CO2 from the atmosphere. They play a major role in enhancing the plantavailable N in soil and yield improvement of rice plant as the conditions provided by the rice fields are favourable for growth and nitrogen fixation by these organisms [59]. Application of BGA in rice field not only results in addition of N but also increased the productivity of rice up to 10–15%. They provide 15–25 kg biologically fixed nitrogen, beside many other beneficial effects on soil quality [60]. Many cyanobacterial strains can utilize extracellular insoluble phosphate under both CN+ and CN- conditions A number of other cyanobacteria e.g., Anabaena, Nostoc, Tolypothrix, Aulosira and Anacystis have been reported to solubilize extracellular insoluble phosphates [61]. Cyanobacteria possess the ability to colonize bare areas of rocks and soil and play a critical role in the re-vegetation of such habitats as pioneers of inhospitable environments [8]. Singh [62] suggested that cyanobacteria could be used as a tool for reclamation of usar soils

152 High Value Fermentation Products Volume 2 because the organic matter and nitrogen added by the cyanobacteria in such soils helps binding of the soil particles, improving the soil permeability and aeration. Kaushik and Subhashini [63] observed that cyanobacteria could improve the physico-chemical quality of saline and alkali soils such as soil aggregation by lowering the pH, electrical conductivity, and hydraulic conductivity. In laboratory simulation experiments, Jaiswal et al., [64] showed that Nostoc calcicola, exhibited a tendency to lower the pH of ‘usar’ soil significantly and reported better growth and pigment content at 20% soil extract as compared to basal medium. Cyanobacteria are known producers of exopolysaccharides which help soil particles to bind together. Reclamation of the desert soils due to exopolysaccharides from cyanobacteria has been reported [65]. Mazor et al., [66] observed significant improvement of soil moisture due to exopolysaccharides produced by cyanobacteria. The cyanobacteria develop a superficial network of the trichomes/ filaments in the soil and therefore bind the soil particles, which results in enmeshing of the soil particles at depth [67].

8.3.3

Bioremediation

Rapid industrialization has resulted in the accumulation of toxic and hazardous chemicals in the environment. Among the photoautotrophs, cyanobacteria are relatively more tolerant to heavy metals [68]. Cyanobacteria are also known for their potential to degrade textile dyes [69–71]. Cyanobacteria have been used with limited success to decontaminate the toxic substances from the environment and they have shown tremendous potential for the treatment of environmental contaminates such as pesticides and heavy metals [72, 73]. The uptake of single or more metal ions (Cu, Pb, Zn, Ni, Cd and Cr) has been reported in several cyanobacterial strains such as Nostoc calcicola [74], Nostoc muscorum [75], Spirulina platensis [76] Oscillatoria anguistissima [77], and Microcystis sp [78]. Lincoln et al., [79] and Singh et al., [80] advocated the use of cyanobacteria as lowcost bioremediating agents for treatment of N-, P-rich dairy waste waters. Several cyanobacterial strains have been reported to accumulate and degrade organo-phosphorus and organo-chlorine insecticides [81]. Ability of certain cyanobacterial strains to form mats in the aquatic environment which helps in the bioremediation of oil spills has been reported [82, 83]. However, detailed studies need to be conducted using promising strains in a consortium mode to develop strategies for the reclamation of contaminated soils. This will lead to enhancing sustainability as well as agricultural productivity.

8.3.4

Compounds for Biocontrol

The secondary metabolites involve mostly polyphenolics which are not directly involved in the growth of cyanobacteria. The different phenolic compounds involve phenolic acids, flavonoids, tannins, and lignins [84]. Teuscher et al., [85] and Dahms et al., [86] reported a variety of biologically active compounds of antibacterial, antifungal, antialgal, and antiviral potential from the cyanobacteria. The presence of such compounds lead to the antagonistic effects of cyanobacteria against different plant diseases. For example, the phenolics have been reported to participate in providing defense against biotic and abiotic stresses [87, 88]. Therefore, the biocontrol potential of

Industrially Important Biomolecules From Cyanobacteria 153 some of these efficient strains of cyanobacteria can be exploited to enhance the agriculture yield. Availability of new techniques to characterize the cyanobacterial metabolites is a frontier area of research to obtain products of commercial value for maintaining sustainability in agriculture.

8.3.5 Bioenergy Applications Identification of alternative and environment-friendly renewable energy sources is essential to address the impending energy crisis and to combat global warming. Algal biofuel has been considered to be a feasible alternative to replace fossil-based fuels. Comparatively faster growth and multiplication of cyanobacteria is important in its exploitation as feed stock for the production of biofuel. Several species of microalgae have the capability of accumulating high amount of lipids which could lead to good oil yield [89]. Photosynthetic prokaryotes such as cyanobacteria and microalgae have been exploited recently for producing biodiesel [90]. Further, the cyanobacteria are able to produce molecular hydrogen which is important in considering them as an alternative to fossil fuel. Several cyanobacteria capable of hydrogen production has been identified [91–93]. Tiwari and Pandey [94], however, observed that the lower amount of hydrogen produced by the cyanobacteria is a serious bottleneck in exploiting this technology despite its potential to produce clean and green energy. The cyanobacterial biomass containing lignocellulosic compounds can be processed for syngas production [95]. Cyanobacterial biomass can also be used to produce biogas through anaerobic digestion or fermentation [96, 97]. The carbohydrates found in cyanobacteria are made up of cellulose and starch and are devoid of lignin and therefore can be easily converted to simple sugars for fermentation [98, 99]. Production of bioethanol is therefore considered as a viable option for producing green and clean fuel. However, the low productivity of the biomass coupled with low oil content is a serious constraint in the large exploitation of cyanobacteria as a potential candidate for biofuel production. Genetic engineering approaches appear to be promising to enhance the utility of cyanobacteria for biofuel production. In this context, a genetically engineered cyanobacterial system producing hydrocarbons and fatty acid derived alcohols through photosynthesis was successfully developed [100].

8.3.6 Medical Applications Several medicinally important biomolecules have been produced by cyanobacteria and mostly these biomolecules have been identified as having anti-inflammatory and anticancer properties [101]. The cyanobacterium Anabaena basta was found to produce an antibiotic bastadin [102]. Reshef et al., [103] showed antiviral activity by the cyanobacterium Oscillatoria raoi. Gunasekhara et al., [104] identified a species of the cyanobacterium, Lyngbya with immense anti cancerous property. They have identified two bioactive compounds such as dragonamide C and D. Similarly, the cyanobacterium Lyngbya majuscule shows tremendous antiparasitic potential against leishmania. Cryptophycin is an important biomolecule isolated from the cyanobacterium with anticancerous properties [105]. One of the cryptomycins, cryptomycin 52 has now entered advanced clinical trials against ovarian and lung cancers [106, 107]. Cyclic poly peptides having anti-HIV

154 High Value Fermentation Products Volume 2 activity was isolated from the cyanobacterium Lyngbya majuscule [108, 109]. Among the several cyanobacterial strains identified with potential medical applications Lyngbya majuscule offers tremendous potential against a number of diseases [110–112].

8.3.7 Applications in Food Sector Cyanobacteria are important in the food industry and several industries are already involved in the formulation of a number of products. They are reported to have a rich chemical composition (depends on microalgae species), and could be utilized as a food supplement or natural colorant [89, 113]. In this context, Spirulina platensis is important as it has a substantial amount of vitamins, minerals, fatty acids, proteins and antioxidants [108, 114, 115]. It is considered as one of the richest sources of vitamin B12 besides having substantial quantities of beta-carotene, riboflavin and thiamin [116]. Because of its richness in nutrients and digestability, Spirulina is an ideal source as a food supplement [117–119]. Sajilata et al., [120] observed that Spirulina platensis is rich in poly unsaturated fatty acids. Sharathchandra and Rajashekhar [121] examined total lipids and fatty acid composition in 13 species of freshwater cyanobacteria isolated from different aquatic habitats under laboratory conditions. They reported presence of palmitic acid (C16:0) in all the isolates followed by linoleic acid (C18:2). Some cynobacterial strains also showed the presence of long chain fatty acids (C20:1 and C24:0) in lower concentrations. In another study, a commercially important cyanobacteria Spirulina platensis has been shown to contain 15.8% lipid with 4.9% omega 3 fatty acid and 3.22% omega 9 fatty acid [122]. Maroneze et al., [123] reported production of ω−3 and ω−6, the fatty acids considered essential to the human metabolism from the cyanobacteria Aphanothece microscopia Nagelli. The fatty acids especially the poly unsaturated fatty acids (PUFA) produced by the cyanobacteria is nutritionally important. Because of this cyanobacterial strains have been used as ideal candidates in aquaculture [124]. Inclusion of Spirulina in the diet of people suffering from malnutrition improved their health [125]. Spirulina platensis is rich in pigments and therefore may be used as natural colourant [126].

8.3.8

Other Value-Added Biomolecules

Santillan [127] reported that in Spirulina the dominant polysaccharide is rhamnose and the carbohydrate production in cyanobacteria accounted up to 22% of its dry weight. Currently these polysaccharides could be exploited as thickening and gelling agents [128, 129]. Some cyanobacteria also produce important biomolecules of diagnostic importance known as lectins. Lectins are important in immunological studies and in the identification of diseases [130, 131]. Scytovirin, microvirin, agglutinin and cyanovirin-N are the most commonly present lectins identified from cyanobacteria [132–135]. Biologically active peptides such as aeruginosin and cyanopeptolin having a role in pharmaceutical industry have been identified from several cyanobacteria [136] Spirulina platensis is important from the perspective of cosmetic industry. Several secondary metabolites and photoprotective compounds synthesized by cyanobacteria find an important role in skin care products and anti-aging creams, etc [137–139]. Mycosporin like amino acids and Scytonemin are important compounds produced

Industrially Important Biomolecules From Cyanobacteria 155 by the cyanobacteria having importance as sunscreen compounds [140]. Suh et al., [141] observed that the mycosporine like amino acids improve the antioxidant status of the cells by quenching the superoxide radicals. Carotenoids such as β-carotene are important photoprotective compounds produced by cyanobacteria [142]. Similarly, phycocyanobilins produced by cyanobacteria are efficient quenchers of superoxide radicals [143, 144]. Several cyanobacteria have been exploited for the production of polyhydroxyalkanoates which is comparable to that of polypropylene [145]. The most commonly produced polyhydroxyalkanoate is poly 3-hydroxybutyrate. Campbell et al., [146], and Mallick et al., [147] observed the accumulation of polyhydroxyalkanoates in cyanobacteria. This is important in the exploitation of promising strains of cyanobacteria for the production on biodegradable plastic.

8.4

Industrial-Scale Production and Commercial Status

In order to achieve economical production of cyanobacteria it is important to shift the focus to large-scale production. Microalgal farming as well as the installation of photobioreactor systems is quite expensive. The cultivation of microalgae in outdoor systems poses several challanges due to limited control over the fluctuating environment and other complex interaction [148]. To combat this challenge, it is essential to optimize the microalgal culture systems with respect to reactor design [149, 150] and strain selection [151]. Successful culturing of the cyanobacteria under various conditions have been shown in earlier studies [44]. The short life cycle and efficiency to convert the captured solar energy into biomass makes cyanobacteria potential candidates for industrial applications. Further, these organisms are amenable to genetic engineering and therefore could be engineered to produce an array of fine chemicals. There is tremendous scope for the development of value-added products from the fine chemicals produced by the cyanobacteria. Further, recent advancements in molecular biology and biotechnology have provided impetus to the engineering efforts in cyanobacteria. Approaches based on genetic engineering have been attempted in cyanobacteria for achieving optimal growth and photosynthetic efficiency to obtain better quality of the desired products. Strain improvement of potentially useful cyanobacteria has been attempted to obtain high-quality products, and that is another important approach [152]. There are several reports on the application of genetic engineering in cyanobacteria [153–155]. The review by Ducat et al., [152] focuses on recent research highlights on the production of valuable compounds and natural products from cyanobacteria. Isolation and identification of compounds as well as economically feasible multiplication methods will lead to the generation of a number of products from cyanobacteria with high value.

8.5

Future Perspectives

Cyanobacteria are a rich source of biologically active compounds which could be exploited for a wide range of applications. These compounds find potential application in agriculture, the food industry, and the medicine, pharmaceutical and cosmetic industries. Most of these compounds are eco-friendly and therefore offer sustainability. Considerable developments have recently taken place regarding the identification of these compounds.

156 High Value Fermentation Products Volume 2 There has been a renewed interest to set up research and development infrastructure in the private sector. However, focused research using the advanced techniques and protocols must be conducted to enhance their utility in the agriculture and industry. Identification of promising strains is also an important pre-requisite in this direction. However, the full potential of these organisms is still untapped and cyanobacterial strains from inhospitable habitats need to be researched for their biotechnological exploitation. Active collaboration between basic sciences, chemistry, biotechnology, engineering and pharmacy needs to be encouraged. Strengthening the R&D activities will lead to the unraveling of important biomolecules from these pioneer organisms.

Acknowledgements The authors thank the Head, Division of Microbiology for facilities and encouragement. Financial assistance from ICAR-Indian Agricultural Research Institute, New Delhi is gratefully acknowledged.

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Industrially Important Biomolecules From Cyanobacteria 163 130. Rüdiger, H., Gabius, H.J., Plant lectins: occurrence, biochemistry, functions and applications. Glycoconj. J., 18(8), 589–613, 2001. 131. Kumar, S., Hahn, F.M., Baidoo, E., Kahlon, T.S., Wood, D.F., McMahan, C.M., et  al., Remodeling the isoprenoid pathway in tobacco by expressing the cytoplasmic mevalonate pathway in chloroplasts. Metab. Eng., 14(1), 19–28, 2012. 132. Bewley, C.A., Gustafson, K.R., Boyd, M.R., Covell, D.G., Bax, A., Clore, G.M., et  al., Solution structure of cyanovirin-N, a potent HIV-inactivating protein. Nat. Struct. Biol., 5(7), 571–578, 1998.10.1038/828 133. McFeeters, R.L., Xiong, C., O’Keefe, B.R., Bokesch, H.R., McMahon, J.B., Ratner, D.M, Bokesch, J.B., McMahon, D.M.R., et al., The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors. J. Mol. Biol., 369(2), 451–461, 2007.10.1016/j.jmb.2007.03.030 134. Ziemert, N., Ishida, K., Weiz, A., Hertweck, C., Dittmann, E., Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Appl. Environ. Microbiol., 76(11), 3568–3574, 2010. 135. Mandal, S., Rath, J., Extremophilic Cyanobacteria for Novel Drug Development. 7. Berlin, Springer, 2014. 136. Silva-Stenico, M.E., Silva, C.S., Lorenzi, A.S., Shishido, T.K., Etchegaray, A., Lira, S.P., et al., Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity. Microbiol. Res., 166(3), 161–175, 2011. 137. Shilpa, K., Varun, K., Lakshmi, B.S., An alternate method of natural drug production: Elciting secondary metabolite production using plant cell culture. J. of Plant Sciences, 5(3), 222–247, 2010. 138. Rastogi, R.P., Incharoensakdi, A., Characterization of UV-screening compounds, mycosporine-like amino acids, and scytonemin in the cyanobacterium Lyngbya sp. CU2555. FEMS Microbiol. Ecol., 87(1), 244–256, 2014. 139. Suh, S.-S., Hwang, J., Park, M., Seo, H., Kim, H.-S., Lee, J., et al., Anti-inflammation activities of Mycosporine-Like Amino Acids (MAAs) in response to UV radiation suggest potential anti-skin aging activity. Mar. Drugs, 12(10), 5174–5187, 2014.10.3390/ md12105174 140. Rastogi, R.P., Sonani, R.R., Madamwar, D., Cyanobacterial sunscreen scytonemin: Role in photoprotection and biomedical research. Appl. Biochem. Biotechnol., 176(6), 1551–1563, 2015.10.1007/s12010-015-1676-1 141. Suh, H.-J., Lee, H.-W., Jung, J., Mycosporine glycine protects biological systems against photodynamic damage by quenching singlet oxygen with a high efficiency. Photochem. Photobiol., 78(2), 109, 2003. 142. Aust, O., Stahl, W., Sies, H., Tronnier, H., Heinrich, U., Supplementation with tomatobased products increases lycopene, phytofluene, and phytoene levels in human serum and protects against UV-light-induced Erythema. International Journal for Vitamin and Nutrition Research, 75(1), 54–60, 2005.10.1024/0300-9831.75.1.54 143. Wagner, J.R., Motchnik, P.A., Stocker, R., Sies, H., Ames, B.N., The oxidation of blood plasma and low density lipoprotein components by chemically generated singlet oxygen. J. Biol. Chem., 268, 1993. 144. Kumar, J., Parihar, P., Singh, R., Singh, V.P., Prasad, S.M., UV-B induces biomass production and nonenzymatic antioxidant compounds in three cyanobacteria. J. Appl. Phycol., 28(1), 131–140, 2016.10.1007/s10811-015-0525-5 145. Loo, C.Y., Sudesh, K., Polyhydroxyalkanoates: bio-based microbial plastics and their properties. Malaysian Polymer Journal, 2, 31, 2007. 146. Campbell, J., Stevens, S.E., Balkwill, D.L., Accumulation of poly-beta-hydroxybutyrate in Spirulina platensis. J. Bacteriol., 149(1), 361, 1982.

164 High Value Fermentation Products Volume 2 147. Mallick, N., Gupta, S., Panda, B., Sen, R., Process optimization for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer production by Nostoc muscorum. Biochem. Eng. J., 37(2), 125–130, 2007. 148. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., et al., Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J., 54(4), 621–639, 2008.10.1111/j.1365-313X.2008.03492.x 149. Posten, C., Design principles of photo-bioreactors for cultivation of microalgae. Eng. Life Sci., 9(3), 165–177, 2009.10.1002/elsc.200900003 150. Scott, S.A., Davey, M.P., Dennis, J.S., Horst, I., Howe, C.J., Lea-Smith, D.J., et al., Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol., 21(3), 277–286, 2010. 151. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., Technical Report National Renewable Energy Laboratory–580, 1998. TP-. 152. Ducat, D.C., Way, J.C., Silver, P.A., Engineering cyanobacteria to generate high-value products. Trends Biotechnol., 29(2), 95–103, 2011. 153. Deng, M., Coleman, R., Applied Environmental Microbiology, 55(2), 523, 1999. 154. Jansson, C., Wullschleger, S.D., Kalluri, U.C., Tuskan, G.A., Phytosequestration: Carbon biosequestration by plants and the prospects of genetic engineering. Bioscience, 60(9), 685–696, 2010.10.1525/bio.2010.60.9.6 155. Niederholtmeyer, H., Wolfstädter, B.T., Savage, D.F., Silver, P.A., Way, J.C., Engineering cyanobacteria to synthesize and export hydrophilic products. Appl. Environ. Microbiol., 76(11), 3462–3466, 2010.

9 Augmenting Bioactivity of Plant-Based Foods Using Fermentation Sonam Chouhan, Kanika Sharma and Sanjay Guleria* Natural Product Laboratory, Division of Biochemistry, Faculty of basic Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Main Campus Chatha, Jammu, Jammu and Kashmir 180 009, (India)

Abstract Considerable attention has been paid in the last few years to bioactive compounds because of their health-promoting abilities for humans, such as the decrease in the occurrence of some degenerative diseases like diabetes and cancer, reduction in likelihood factors of cardiovascular diseases and also due to their anti-allergic, antioxidant, anti-inflammatory, anti-mutagenic, and anti-microbial effects. Fermentation is the most easy and safe traditional way of enriching useful bioactive compounds. Fermentation process is reported to upgrade biological properties of plants, herbs and vegetables. More specifically, fermentation is related to the decomposition and/or biotransformation of complex substrates into compatible components, thereby modulating the properties of the product or changing the quantity of certain bioactive compounds. Being originated from Japan, fermented plant extract (FPE) is a plant-based functional food that utilizes various plants as materials which are fermented with various microorganisms to create physical forms that are abundant in nutrients and active substances, thereby providing many health benefits. Keywords: Fermentation, plant extracts, antioxidant activity, antimicrobial activity

9.1

Introduction

Great attention has been paid in the last few years to bioactive compounds due to their health-promoting abilities for humans, such as the decrease in the incidence of some degenerative diseases like diabetes and cancer [1, 2], decrease in likelihood factors of cardiovascular diseases [3], anti-allergenic, antioxidant, anti-inflammatory, anti-mutagenic, and anti-microbial effects [4, 5], among others. Fermentation is an interesting choice for the enrichment of bioactive compounds, because of its ability to provide extracts with high quality and high activity while removing any harmful effects associated with the organic solvents. Fermented plant extract (FPE) is a type of plant functional food that is very popular in Japan, China, and some other Asian places. Various

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (165–184) © 2019 Scrivener Publishing LLC

165

166 High Value Fermentation Products Volume 2 plants including legumes, cereals, vegetables, fresh fruits and edible fungi (although not plant), medicinal and edible traditional medicine can be the materials for the synthesis of FPE. With plenty of nutrients and active substance, fermented plant extract provides numerous health benefits [6]. Fermentation has been used from ancient times for enhancing the shelf life, nutritional and organoleptic qualities of food [7]. Fermentation results in many biochemical alterations that result in an altered ratio of nutritive and anti-nutritive constituents and consequently, affects the properties of products, such as digestibility and bioactivity [8]. It results in an increased physiological and biochemical activities of biological substrates by modification of their original molecules [9]. These properties make it a useful technology for the production of new compounds as novel cosmeceutical and pharmaceutical agents. Due to varied species of microorganisms, fermentation possesses the potential to reduce or remove cytotoxicity of an herbal extract [10, 11]. Also, in elevating gastrointestinal health and skincare, the usefulness of many fermented natural products has been proven [12]. In the process of fermentation, production of bioactive compounds as secondary metabolites is started after the achievement of microbial growth [13]. Recently, the bioprocess of fermentation has been applied for producing as well as extracting bioactive compounds in the chemical, food, and pharmaceutical industries [14, 15]. This chapter provides an overview of the factors affecting the fermentation process and health-related benefits of fermented plant-based foods.

9.2

Effect of Fermentation on Bioactivity of Plant-Based Foods

One of the most useful techniques of bio-catalytical process is fermentation, which is regarded as an achievable means of producing new, active, and less harmful bioactive products as their generation would be otherwise inconvenient from either biological systems or chemical synthesis [16]. During the process of the breakdown of substrates metabolically, several compounds are released by microbes besides the usual products of fermentation (generally carbon dioxide and ethanol) particularly during the microbial growth in the lag phase. Also called secondary metabolites, these are the additional compounds spanning from various antibiotics to peptides, pigments, enzymes and growth factors [17]. Diverse biological activities are possessed by these compounds like anti-infective, anti-inflammatory and anti-cancer, and are therefore called bioactive compounds. The fermentation process is also reported to have beneficial impacts on the absorption and the bioavailability of herbal extracts through the facilitation of the synthesis or transformation of active constituents into their metabolites or by the generation of low molecular weight substances like aglycone from glycoside [18]. In addition to this, it has been indicated in several lines of conformation that the process of fermentation ameliorates the pharmacological properties and beneficial impacts of medicinal herbs that promote health and prevent disease. For instance, pharmaceutical fungal spp. mediated fermentation intensify the therapeutic efficiencies of some herbal medicines [19]. There is a positive relationship of the altered profile of secondary metabolites occurring during fermentation and variations in their mechanisms affecting biological activity with the therapeutic enhancement [20]. Particularly, it has been advocated that fermentation augment the number of bioactive constituents, such as

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 167 anti-oxidants, ameliorate the anti-inflammatory activity of numerous compounds and reduce the likelihood of hepatotoxicity instigated by ethanol [19, 21]. It has been found that Lactobacillus acidophilus mediated fermentation of Anoectochilus formosanus increases the total phenol content, thereby boosting its anti-oxidant activity [21]. It has been recommended that fermentation induced by bacteria is also capable of changing the structure of the flavonoids apart from the generation of the bioactive constituents of flavonoid. For example, fermentation caused by bacteria de-glycosylates, sulfates, or methylates flavonoids, which in turn modulates their absorption rate and metabolism in the liver [22]. More specifically, structural alterations in flavonoids mediated by bacterial fermentation result in acceleration of the absorption rate as well as the quantity absorbed which might ameliorate the bioavailability and bioactivity of the active constituents [23] and might have a contribution to the beneficial effect on bone [24]. As mentioned above, like bacteria, fungi also have a great possibility to manufacture bioactive compounds, such as anti-oxidants [20]. For example, after fermentation with filamentous fungi, the amount of total phenolics and anthocyanin plus anti-oxidative activity of black beans were increased [20]. During the fermentation process, numerous enzymes are known to be produced by fungi e.g., glycoside hydrolase, celluloseor xylan-degrading enzymes, and esterase that cause softening of the kernel structure, break down of cell walls of cereal and release of esterified and insoluble-bound nutrients [20]. Hydrolysis of glucosidic bonds of several phenolic compounds that exist chiefly in the form of conjugates with one or more sugar residues attached to hydroxyl groups is catalyzed by these enzymes [20]. For increasing the amount of free polyphenols and improving the nutraceutical activity of numerous substrates, hydrolysis of phenolic glucosides by enzymes is considered to be an efficient method [25]. Moreover, it is also demonstrated that isoflavone glucosides release lipophilic compounds such as genistin, due to the catalytic action of glucosidase during fungal fermentation causes enhancement of anti-oxidant activity in extracts [20]. Effect of fermentation on bioactivity of plant extracts is shown in Figure 9.1.

Effect of fermentation on bioactivity of plant based foods

Metabolic breakdown of substrates by microbes release several compounds like secondary metabolites, peptides, pigments, enzymes and growth factors

Improvement in the pharmacological properties that promote health and prevent disease Augmentation of the concentration of bioactive components like antioxidants

Improved bioavailability of compounds by structural changes, thereby, accelerating the absorption rate and bioavailability

Figure 9.1 Effect of fermentation on bioactivity of plant based functional foods.

168 High Value Fermentation Products Volume 2

9.3

Different Fermentation Procedures

The processes of fermentation can be grouped into two systems: submerged fermentation (SmF), based on cultivating microorganisms in a liquid medium containing nutrients, and solid state fermentation (SSF), that consists of the growth of microbes and product formation on solid particles in the absence (or near absence) of water; however, sufficient moisture is present in the substrate to permit the growth of microorganism and metabolism [26].

9.3.1

Solid State Fermentation (SSF)

In solid state fermentation, microorganisms are subjected to grow on solid materials under controlled states in the absence of free liquid [27]. Degradation of substrates occur very slowly but steadily, thus providing the chance of using the same substrate for longer periods of fermentation. Accordingly, the controlled delivery of products is facilitated by this technique. For fermentation with fungi and yeast, SSF is best suited because fungi and yeast require low moisture amount. As low water volume is involved in SSF, it has also a huge effect on the economy of the process mainly due to smaller size of fermenter, lowered downstream processing, lowered stirring and decrease costs of sterilization [13, 28]. Included variables of the process like pretreatment and particle-size of substrates, supplementation of growth medium, ingredients of the medium, sterilization of SSF medium, moisture content, density of the inoculum, water activity (aw), pH, temperature, agitation and aeration, have a considerable effect on the efficacy of solid state fermentation processes [29]. Consideration of these important factors should be done for the purpose of developing a victorious bioprocess under SSF states. Selection of a suitable microorganism strain and the solid support to be used are among some of the most important factors. In SSF, a variety of microorganisms, including fungi, yeasts and bacteria may be used. However, in the case of fermentation media having low moisture content, the most frequently used microorganisms are fungi and yeasts as they are capable of growing in environments with such characteristic, although, selection of the microorganism in solid state fermentation rely on the desired final product. Recently, in a study conducted on the bioprocessing of black beans for the preparation of koji by solid state fermentation using distinct food-grade filamentous fungi (specifically Aspergillus sp. and Rhizopus sp.), it was observed that the antioxidative properties of these beans was enhanced which might be associated with elevated levels of phenol and anthocyanin [30]. Moreover, it was also demonstrated that the enhanced phenolic content due to the β-glucosidase and α-amylase activities during solid state fermentation has an association in the improved antioxidant potential of fermented rice [31]. Nevertheless, enhancement in the antioxidant activity of the black bean koji differs with each microorganism that was used. For producing the compound of interest efficiently and economically, selecting the solid substrate in a right way is also of great importance. Mostly improvement in the yields of secondary metabolites can be achieved by suitably choosing substrate or mixture of substrates with appropriate nutrients [13].

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 169

9.3.2

Submerged Fermentation/ Liquid Fermentation (SmF/LF)

This involves growing microbes in an aqueous medium by using free flowing liquid substrates. Therefore, for microorganisms requiring high moisture like bacteria this fermentation process is prime suited. More specifically, in this process of fermentation cautious growth of selected microorganisms is required (bacteria and fungi) in closed vessels comprising of abundant nutrient broth (the fermentation broth). During this process, microbes liberate the bioactive compounds made by them into the fermentation broth. It is required to refill the nutrients repeatedly as microbes utilize the substrates quite rapidly. An additional benefit associated with submerged fermentation technique is that the purification of products is suitable. Extraction of secondary metabolites is the major significance of submerged fermentation. However, the rigorous preservation of sterility is a crucial step in this kind of fermentation than solid state fermentation [32].

9.4

Factors Affecting Fermentation Process

Among the environmental factors affecting food fermentation, pH is one of the most crucial environmental parameters. pH is closely related to change in the structure of phytochemicals as well as the growth of microbes during fermentation. Fermentation is also affected by alterations in temperature. During the fermentation process, maintenance of the optimal temperature results in enhanced microbial growth and activity of enzymes, and consequently, the benefits associated with fermentation are improved [33] (Table 9.1). Some of the microrganisms reported to be utilized in fermentation are Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus platarum, Lactobacillus rhamnosus, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Rhodotorula glutinis, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus oryzae and Cryptococcus flavus [33]. Process for production of fermented plant extracts is depicted in Figure 9.2. Microorganisms can benefit humans in many aspects through fermentation. Following are the main benefits associated with using microorganisms for the fermentation of plant extract [50–53]: 1. Extend shelf life: organic acid, ethanol, bacteriocins, etc., are among the metabolites that are produced for inhibiting the growth of harmful bacteria. 2. By impediment of pathogens or by decomposition of toxic compounds, food safety is improved. 3. Enhancement of the nutritional value by producing metabolites like proteins, essential fatty acids, vitamins and essential amino acids. 4. Enhancement in the organoleptic worth of food because of the synthesis of desirable acids and aromatic compounds plus enrichment of bioactive compounds.

Bacillus subtilis natto

Lactobacillus brevis

Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium catenulatum, Bifidobacterium breve, Bifidobacterium bifidum

Aspergillus oryzae M29

Lactobacillus fermentum, Lactobacillus MRS broth and MRS agar casei

Coprinus comatus

Lactobacillus casei, Lactobacillus MRS acidophilus, Bifidobacterium longum

Lactobacillus

Radix astragali

Artemisia princeps

Panaxnoto ginseng

Radix astragali

Ssanghwa- tang

Sophora flavescens

Anoectochilus formasanus Hayata

Oyalsum gisan

MRS broth

Liquid medium (20% potato extract liquid +2% dextran +1% KH2PO4 + 0.05% MgSO4

37 °C for 48 hr

(Continued)

[19]

[21]

[40]

27 °C for 3 days

37 °C for 60 hr

[39]

[38]

[37]

[36]

[35]

[34]

Reference

37 °C for 48 hr

30 °C for 3 days

37 °C for various time point

MRS broth

Potato Dextrose Agar (PDA)

30 °C for 3 days

37 °C for 48 hr

RT for 35 days

Temperature and time

Man Rogosa Sharpe(MRS) agar plates

-

Ginseng medium

Ganoderma lucidum

Ginseng

Media

Microorganism

Herbal formulation

Table 9.1 Fermentation conditions for certain herbal formulations.

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Microorganism

Lactobacillus acidophilus, Bifidobacterium longum, Leuconostoc mesenteroides

Phellinus linteus

Grifola frondosa

Bacillus licheniformis

Aspergillus niger

Streptococcus thermophilus

Lactobacillus amylophilus, Lactobacillus plantarum, Lactobacillus bulgaricus

Lactobacillus acidophilus

Lactobacillus plantarum

Codonopsis lanceolata

Red ginseng

Rhizoma gastrodiae

Rhizoma attractylodis Macrocephalae

Ginkgo biloba leaves

Cyclopia intermedia

Soshibo-tang

Jaeumganghwa- Tang

Rhizoma attractylodis Macrocephalae

Cont.

Herbal formulation

Table 9.1

Lactobacilli MRS broth

MRS medium

MRS broth

MRS medium

10 g of solid medium & 16 ml of nutritive salt

LB broth

Seed cultures

Potato Dextrose Broth (PDB)

Media

37 °C for 24 hr

37 °C for 24 hr

37 °C for 48 hr

37 °C for 1 day

30–40°C for 6 days

31 °C for 24 hr

25 °C for 8 days

25 °C for 5 days

30 °C for 48 hr

Temperature and time

[49]

[48]

[47]

[46]

[45]

[44]

[43]

[42]

[41]

Reference

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 171

172 High Value Fermentation Products Volume 2 Materials: fruits, vegetables, fungi etc

Pretreatment: Selection, cleaning, washing,blanching, peeling, shredding, enzymolysis

Bacteria

Fungi

Inoculation: Suitable probiotics selected, one kind or more, together in a row, isolated from nature or a commercial one

Fermentation:Naturally using a long time, sometimes with a starter culture

Time Temperature pH

Fermentation: Conditions optimization needed: Inoculum, temperature, time, formulation of the substrates etc.

Post-treatment: Sterilization, packing and storage.

Fermented Plant Extract

Figure 9.2 Schematic diagram of production process of fermented plant extracts [Partially adapted from Feng et al., [6]].

9.5

Bioactive Properties of Fermented Foods

Due to the presence of large quantities and varied kinds of bioactive substances, fermented plant extracts display several health-benefiting properties like antidiabetic, antioxidant, antimicrobial, etc. Various bioactive properties exhibited by fermented plant extracts are

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 173

Bioactive properties of fermented plant extracts

Antimicrobial effect

Antioxidative effect Anti-inflammatory effect

Antidiabetic effect

Other biological effects of fermented plant extracts: Cytotoxic activity Anti-hyperlipidemia potential Hepatoprotective effect Anti-hypertensive properties

Figure 9.3 Bioactive properties of fermented plant extracts.

shown in Figure 9.3. Molecules that hamper the oxidation of other molecules are called antioxidants and these antioxidant molecules are used widely in dietary supplements. Antioxidant activity refers to the total capacity of antioxidants to eliminate free radicals in the cell as well as in the food. Polyphenols are magnificent antioxidants because of a 30–40 dihydroxy group in their B ring and the galloyl ester present in the C ring of flavonoids, which are also important structures in the chelation of metal ion [54]. Likewise, Lee et al., reported that flavonoids possess strong antioxidative activity as their chemical structures have an o-diphenolic group, a 2–3 double bond conjugated with the 4-oxo function and hydroxyl groups in positions 3 and 5. Hydroxyl and peroxyl radicals are effectively scavenged by flavonoids, form complexes with metals, and inhibit lipid oxidation initiated by metal [55].

9.5.1

Antioxidant Effect

For the purpose of enhancing the supply of natural antioxidants, fermentation is a useful method as it enhances antioxidative activity by enhancing the liberation of flavonoids from plant-based foods. An important role is played by temperature in controlling the growth of microorganisms during fermentation and the antioxidative activity can be influenced by the growth of microorganisms. Enzymatic changes are induced by antioxidants, such as phenolic compounds during microbial fermentation [33]. Fermentation instigated structural breakdown in the cell walls of cereal may also result in the release and/or induction in the synthesis of various bioactive compounds [56]. For the manufacturing of bioactive compounds like antioxidants, fungi offer great possibilities [14]. It has also been demonstrated that after fermentation of black beans with filamentous fungi, the total phenolics and anthocyanin amounts were enhanced [57]. It has been reported in a study that after the process of fermentation, plant parts have elevated total phenols and the observed antioxidative activity may be due to the elevated amount of the total phenolic compounds [21]. Also, Monascus-fermented soybeans (MFS) possess antioxidative properties and might be promising antioxidants for

174 High Value Fermentation Products Volume 2 application in food products [58]. The antioxidant impacts of Damdusi (from Korea) extract was confirmed by Ahn et al., by examining it in both non-cellular systems and cellular systems. In non-cellular systems, scavenging activities on hydroxyl, DPPH, and hydrogen peroxide radicals, and high chelating ability and reducing power was shown. On the other hand, significant ROS (reactive oxygen species) scavenging ability, lipid peroxidation inhibition, and induced increase in levels of glutathione was shown in cellular systems [59]. Likewise, in the same year antioxidant activity of Radix astragali fermented by Aspergillus spp. was reported to be superior than unfermented Radix astragali against hydroxyl radicals, superoxide radicals, and peroxyl radicals [60]. Moreover, a study conducted in aged rats demonstrated that application of the fermented Panax ginseng extract (GINST) potentiated the anti-oxidant status caused during aging, thus resulting in the prevention of the oxidative stress as well as the free radicals associated incidence of age-related disorders [61]. The impact of liquid state fermentation (LSF) and solid state fermentation (SSF) of lentils for synthesis of water-soluble fractions with antioxidant properties was studied by Torino et al., and it was demonstrated that LSF performed either spontaneously (NF) or by Lactobacillus plantarum (LP) produced LSF water-soluble fractions having higher free amino groups, GABA amount and antioxidant activities than SSF [15]. Fermentation of ligulate florets of Chamomile anthodium by using native chamomile enzymes for hydrolyzing bound forms of apigenin to free aglycone resulted in antioxidant activity which was defined by electron spin resonance analysis for hydroxyl and superoxide radicals [62]. In 2017, Razak et al., reported the mix-cultured fermented rice bran extract possess antioxidant activities [63].

9.5.2

Antimicrobial Effect

During recent years, research and development of new antimicrobial agents remained a topic of growing interest for the purpose of combating microbial resistance. Therefore, screening of antimicrobial activity plus its evaluation methods is receiving considerable attention [64]. Accumulating proofs recommend that the antimicrobial activity of fermented plant-based foods is influenced by the microbial species present during the process of fermentation resulting in promising antimicrobial effect. Kantachote et al., investigated the antibacterial activity of household fermented plant beverages made in southern Thailand and reported that with the use of increasing amounts of beverages elevated inhibition of some pathogenic bacteria was resulted. Significant inhibition of Gram positive bacteria was observed whereas the beverages had varied inhibition on Gram negative bacteria [65]. Santos et al., reported that spontaneous aerobic fermentation of Theobroma cacao L. cacao husks produce an unpurified husk extract (CHE) with antimicrobial activity [66]. Likewise in the same year Bianchi et al., studied the impacts of four fermented beverages (with quinoa and soy extracts) towards human intestinal microbiota by utilizing a simulator. It was shown in the results that the synbiotic beverage displayed the best microbiological outcome in the ascending colon compartment, by growth stimulation of Lactobacillus spp., and Bifidobacterium spp., plus decreasing populations of Bacteroides spp., Clostridium spp., Enterococcus spp., and enterobacteria in this compartment [67]. Fermentation of ligulate florets of Chamomile anthodium by using native chamomile enzymes for hydrolyzing bound forms of apigenin to free aglycone resulted in antimicrobial activity for

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 175 eight microbial strains [62]. Recently, Dubey et al., reported that the fermented and green methanol extracts of honeybush Cyclopia intermedia were most effective against Staphylococcus aureus and Candida albicans respectively, whereas the green chloroform extract had the strongest activity for Streptococcus pyogenes [68].

9.5.3 Anti-Inflammatory Effect Trauma, microbial infection, or xenobiotic insults that elicit cell injury or death are among a number of factors that trigger a complex biological process called inflammation [69]. Consequences of inflammation are disrupted tissue homeostasis, release of a number of inflammatory mediators such as tumor necrosis factor- α (TNF-α) and interleukins (ILs) from leukocytes, monocytes and macrophages [70, 71], the family of NF-κβ/ Rel transcription factors in inflammation [72]. Inflammatory response are amplified by the activation of NF-kB by the regulation of transcription of different inflammatory mediators viz. IL-6, IL-1, TNF- α and cellular adhesion molecules (CAMs) [72]. A central role is played by IL-6 in acute inflammatory processes in many organs, including CNS, resulting in the initiation of many different brain pathologies like cerebral ischemia and excites toxic damage of brain [73]. Well documentation on the potential role of TNF- α and IL-1 for initiating and executing inflammation has been done [74]. It has been indicated in accumulating evidence that fermentation process can result in enhancing the anti-inflammatory activities of herbs. COX-2 which is a rate-limiting enzyme regulates the synthesis of various inflammatory mediators that are biologically active, including prostaglandin E2 (PGE2)and is also triggered in several carcinomas, suggesting the vital role of this enzyme in inflammation and tumor genesis [75]. COX-2 protein and production of PGE2 in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) [76]. Moreover, it has been shown in a study that obstruction of the translocation of NF-κβ p65 is caused by the fermented OYs due to decreased degradation of Iκβα as well as through phosphorylation of extracellular kinase that are regulated by signal [76] and p38 and c-Jun more strongly than the unfermented OY. Therefore, it has been inferred from these findings that the anti-inflammatory effect of OY is potentiated by fermentation through the obstruction of NF-κβ and MAPK pathway in the macrophage cells. Likewise, another study reported the obstruction of expression of COX-2 by BST204 to be NF-κβ-independent and shown to be mediated by the mTOR/p70 S6 kinase pathway as well as other pathways of unknown nature [76]. In a study performed by Oh et al., it was demonstrated that due to fermentation the obstructive impact of OY was increased significantly against the expression or production of many vital mediators that are pro-inflammatory like iNOS, COX-2, NO, TNF-α, PGE2 and IL-6 in RAW 264.7 cells [19]. Further, it has been demonstrated that co-treatment of Lipopolysaccharide (LPS) induced RAW 264.7 cells with fermented preparation of Rhizoma Atractylodis Macrocephalae (RAM) resulted in a considerable decreased activity of NF-κβ [44]. Moreover, inhibition in the activity of NF-κβα by fermented Artemesia princeps in LPS-stimulated peritoneal macrophages has also been reported [36]. Choi et al., reported that upon co-treatment of LPS-stimulated RAW 264.7 cells with fermented guava leaf extract could significant suppression in the transcriptional activity of NF-κβ in a concentration-dependent manner occurred [77].

176 High Value Fermentation Products Volume 2 Based on their experimental results, fermented guava leaf extract were recommended to cause obstruction in the activation of NF-κβ by the suppression of Iκβα deterioration instigated by Lipopolysaccharide [36].

9.5.4

Antidiabetic Effect

Diabetes Mellitus is a persistent progressive metabolic disorder that has life-threatening complications like cardiovascular diseases, neuropathy and retinopathy which may finally result in death. Characteristic of this disease is hyperglycemia with impaired lipid, carbohydrate and lipoprotein metabolism that result due to faults in insulin emanation (type 1 diabetes mellitus) or insulin action (type 2 diabetes mellitus) or both. In 2014, the estimated worldwide prevalence of diabetes was 9% among adults that were of the age 18 + years (Global status report on non-communicable diseases 2014, World Health Organization, Geneva, 2012). The 7th leading mortality source in 2030 will be diabetes as projected by the World Health Organization [78]. Pharmaceutical companies have achieved a remarkable advancement in the research and growth of antidiabetic agents. However, current antidiabetic drugs suffer from lack of efficiency despite these efforts and also have undesirable side effects, thereby, demanding new remedies [79]. It has been advocated in accumulating evidences that fermentation has beneficial impact on the antidiabetic effects of the herbal drugs. Kim et al., reported that fermented ginseng extract prevented β-cell damage in streptozotocin (STZ)-induced diabetic animal model, thereby, suggesting the preventive role of fermented ginseng extract in the progression of Type 1 diabetes [80]. It was reported in a study that fermented mung bean extracts considerably reduced the sugar levels in blood of glucose and alloxaninstigated hyperglycemic mice and this was due to improved plasma levels of insulin secretion in alloxan induced hyperglycemic mouse. The presence of elevated level of GABA and the free amino acid in the fermented mung bean extracts had a contribution in the improved antihyperglycemic impact of the fermented mung bean extracts [81]. Further, Lee et al., reported the improved antidiabetic effect of Morinda citrifolia (MC) through solid state fermentation by using KK-Ay diabetic mice as a model [82]. Decreased level of serum insulin and insulin resistance were the reasons associated for the hypoglycemic effect of fermented Morinda citrifolia (FMC). Likewise in the same year, Lim et al., reported that after administration of Bacillus subtilis MORI fermented soybean extracts BTD-1 (500 and 1000 mg/kg/day) in streptozotocin (STZ)-induced diabetic rats, the elevated level of plasma glucose was significantly reduced while the insulin level in plasma increased considerably thereby inhibiting hyperglycemia [83]. Ginseng is well documented for its antidiabetic effect. Various studies demonstrated the useful effect of fermented ginseng on the antidiabetic action. Jeon et al., reported decrease in the fasting blood glucose and HbA1c in type 2 DM mouse model by fermented ginseng extract (FGE) [84]. Further, it was indicated in a study that administration of fermented red ginseng (FRG) in old-aged ob/ob mice significantly decreased the blood glucose amount and increased the expression of insulin receptor, lipoprotein lipase, GLUT1, GLUT4, PPAR- γ, and phosphoenol pyruvate carboxykinase genes in key metabolic tissues, suggesting the task of fermented red ginseng (FRG) in the enhancement of insulin sensitivity [85].

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 177

9.5.5

Other Biological Effects of Fermented Plant Extracts

Fermentation of ligulate florets of chamomile anthodium by using native chamomile enzymes for hydrolyzing bound forms of apigenin to free aglycone was demonstrated to possess cytotoxic activity and this was evaluated using two human cell lines (human cervix carcinoma and human rhabdomyosarcoma) and murine fibroblasts [45]. In 2016, Christoph et al., reported cytotoxic impact of fermented wheat germ extract (FWGE) against nine human cancer cell lines with an IC50 value of 10 mg/ml [86]. In a study organized on rats by Ahren et al., it was found that L. plantarum DSM 15313 fermented blueberries powder had anti-hypertensive properties and may also decrease the possibility of cardiovascular diseases [87]. Similarly, consumption of fermented orange beverage produced remarkable safety against risk factors for cardiovascular diseases as compared to orange juice in healthy mice [88]. Oral administration of Monascus-FSE (fermented soybean extract) in hyperlipidemic rats remarkably decreased triglyceride amount, serum total cholesterol and low-density lipoprotein cholesterol levels. On the other hand, the amounts of high-density lipoprotein cholesterol were raised in hyperlipidemic rats, thereby indicating a hypolipidemic effect [89]. Also, fermented soybean extract (FSE) possess anti-hyperlipidemia potential [90]. Lee et al., [91] reported that fermented soybean extract when given for a short term at the concentration of 1 mg/mL exhibited a robust hindrance impact on the growth of MCF7 (null fibrosarcoma, human, KCLB 10121) and HT1080 (breast adenocarcinoma, human, KCLB 30022) with 58% and 64%, respectively. Likewise, fermented soy milk has a potential in reducing the threat of breast cancer [92]. The hepatoprotective impact of fermented ssanghwa-tang (SHT) upon fermentation with Lactobacillus fermentum on hepatotoxicity induced by CCl4 in rats has been demonstrated [93]. Similarly, Kim et al., reported hepatoprotective impacts of fermented Curcuma longa L. extract in rats with carbon tetrachloride-induced oxidative stress [94].

9.6

Conclusion and Future Perspectives

Considerable attention is being paid by people towards health and longevity due to improvement in living standards. Fermented plant extract (FPE) is a conventional and present-time plant functional food that is fermented by several microorganisms viz. yeasts, fungi and bacteria at certain standardized conditions of pH, temperature and moisture content for enhancing the production titers of bioactive compounds in the fermented plant extracts. During fermentation, either modification of existing bioactive compounds occur or new bioactive compounds are formed. Because of the existence of lots of bioactive substances in fermented plant extracts (FPEs), they have many benefits to human health, pharmaceutical industries, environment and other aspects. Nowadays, more and more production of fermented plant extracts (FPEs) is executed on an industrial scale and the production is becoming more standardized and safe. Moreover, there is great potential in the growth prospects of fermented plant extracts (FPEs). Further, tailoring in the alteration of fermentation process could be done for increasing the bioaccessibility of bioactive compounds. A yet unexplored potential is the manufacturing of bioactive compounds that could be mastered by making use of new fermentation process. Therefore, fermentation process could be expected to be utilized in the upcoming time for designing food having health promoting or health beneficial effects.

178 High Value Fermentation Products Volume 2

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Augmenting Bioactivity of Plant-Based Foods Using Fermentation 181 55. Đorđević, T.M., Šiler-Marinković, S.S., Dimitrijević-Branković, S.I., Effect of fermentation on antioxidant properties of some cereals and pseudo cereals. Food Chem., 119(3), 957–963, 2010. 56. Yu-Ling, L., Joan-Hwa, Y., Jeng-Leun, M., Antioxidant properties of water extracts from Monascus fermented soybeans. Food Chem., 106, 1128, 2008. 57. AHN, C.-B.U.M., JE, J.A.E.-Y., Ahn, B.C., Antioxidant activity of traditional korean fermented soybean (damdusi) extract on free radical-midiated oxidative system. J. Food Biochem., 35(4), 1242–1256, 2011. 58. Ramesh, T., Kim, S.W., Sung, J.H., Hwang, S.Y., Sohn, S.H., Yoo, S.K., et al., Effect of fermented Panax ginseng extract (GINST) on oxidative stress and antioxidant activities in major organs of aged rats. Exp. Gerontol., 47(1), 77–84, 2012. 59. Cvetanović, A., Švarc-Gajić, J., Zeković, Z., Savić, S., Vulić, J., Mašković, P, Aleksandra, G.S.J.C., Zoran, Z., Sasa, S., Jelena, V., Pavle, M., Gordana, C., et al., Comparative analysis of antioxidant, antimicrobiological and cytotoxic activities of native and fermented chamomile ligulate flower extracts. Planta, 242(3), 721, 2015. 60. Dang, R.A.L., Nur, R.A.Y., Anisah, J., Shaiful, S.A., Ainaa, K.A., Kamariah, L., Journal of the Saudi Society of Agricultural Sciences, 16, 127, 2017. 61. Chouhan, S., Sharma, K., Guleria, S., Antimicrobial activity of some essential oils—present status and future perspectives. Medicines, 4(3), 58, 2017. 62. Kantachote, D., Charernjiratrakul, W., Umsakul, K., Antibacterial activities of fermented plant beverages collected in southern thailand. J. of Biological Sciences, 8(8), 1280–1288, 2008. 63. Santos, R.X., Oliveira, D.A., Sodré, G.A., Gosmann, G., Brendel, M., Pungartnik, C., Antimicrobial activity of fermented Theobroma cacao pod husk extract. Genet. Mol. Res., 13(3), 7725–7735, 2014. 64. Bianchi, F., Rossi, E.A., Sakamotob, I.K., Adorno, M.A.T., Van de Wiele, T., Sivieria, K, Fernanda, A.E.R.B, Adornob, T.A.M., Wielec, D.V.T., Beneficial effects of fermented vegetal beverages on human gastrointestinal microbial ecosystem in a simulator. Food Res. Int., 64, 43, 2014. 65. Dubey, P., Meyer, S., Marnewick, J.L., Antimicrobial and antioxidant activities of different solvent extracts from fermented and green honeybush (Cyclopia intermedia) plant material. South African Journal of Botany, 110, 184–193, 2017. 66. O'Byrne, K.J., Dalgleish, A.G., Chronic immune activation and inflammation as the cause of malignancy. Br. J. Cancer, 85(4), 473–483, 2001. 67. Medzhitov, R., Origin and physiological roles of inflammation. Nature, 454(7203), 428–435, 2008. 68. Paterson, H.M., Murphy, T.J., Purcell, E.J., Shelley, O., Kriynovich, S.J., Lien, E., et al., Injury primes the innate immune system for enhanced Toll-like receptor reactivity. J. Immunol., 171(3), 1473–1483, 2003. 69. Korhonen, R., Lahti, A., Kankaanranta, H., Moilanen, E., Nitric oxide production and signaling in inflammation. Curr. Drug Targets. Inflamm. Allergy, 4(4), 471–479, 2005. 70. Hosomi, N., Ban, C.R., Naya, T., Takahashi, T., Guo, P., Song, X.Y., et al., Tumor necrosis factor-alpha neutralization reduced cerebral edema through inhibition of matrix metalloproteinase production after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab., 25(8), 959–967, 2005. 71. Keyel, P.A., How is inflammation initiated? Individual influences of IL-1, IL-18 and HMGB1. Cytokine, 69(1), 136–145, 2014. 72. Aoki, T., Narumiya, S., Prostaglandins and chronic inflammation. Trends Pharmacol. Sci., 33(6), 304–311, 2012.

182 High Value Fermentation Products Volume 2 73. Hla, T., Ristimäki, A., Appleby, S., Barriocanal, J.G., Cyclooxygenase gene expression in inflammation and angiogenesis. Ann. N. Y. Acad. Sci., 696(1), 197–204, 1993. 74. Seo, J.Y., Lee, J.H., Kim, N.W., Her, E., Chang, S.H., Ko, N.Y, Ko, Y.N., et  al., Effect of a fermented ginseng extract, BST204, on the expression of cyclooxygenase-2 in murine macrophages. Int. Immunopharmacol., 5(5), 929–936, 2005. 75. Choi, S.Y., Hwang, J.H., Park, S.Y., Jin, Y.J., Ko, H.C., Moon, S.W., et al., Fermented guava leaf extract inhibits LPS-induced COX-2 and iNOS expression in mouse macrophage cells by inhibition of transcription factor NF-κB. Phytother. Res., 22(8), 1030–1034, 2008. 76. Mathers, C.D., Loncar, D., Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med., 3(11), 442, 2006. 77. Chang, C.L.T., Lin, Y., Bartolome, A.P., Chen, Y.C., Chiu, S.C., Yang, W.C., Evidence-based Complementary and Alternative Medicine, 378657, 2013, 2013. 78. Kim, H.-J., Chae, I.-G., Lee, S.-G., Jeong, H.-J., Lee, E.-J., Lee, I.-S., Effects of fermented red ginseng extracts on hyperglycemia in streptozotocin-induced diabetic rats. J. Ginseng Res., 34(2), 104–112, 2010. 79. Yeap, S.K., Mohd Ali, N., Mohd Yusof, H., Alitheen, N.B., Beh, B.K., Ho, W.Y., et  al., Antihyperglycemic effects of fermented and nonfermented mung bean extracts on alloxaninduced-diabetic mice. J. Biomed. Biotechnol., 2012, 2012, 2012. 80. Lee, S.Y., Park, S.L., Hwang, J.T., Yi, S.H., Nam, Y.D., Lim, S.I., Evidence-based Complementary and Alternative Medicine, 163280, 2012d, 2012. 81. Lim, K.H., Han, J.H., Lee, J.Y., Park, Y.S., Cho, Y.S., Kang, K.D, Kyu, L.H., Ji-Hui, H., Jae, L.Y., Young, P.S., Yong, C.S., Kyung-Don, K., et al., Assessment of antidiabetogenic potential of fermented soybean extracts in streptozotocin-induced diabetic rat. Food Chem. Toxicol., 50(11), 3941, 2012. 82. Jeon, W.J., Oh, J.S., Park, M.S., Ji, G.E., Anti-hyperglycemic effect of fermented ginseng in type 2 diabetes mellitus mouse model. Phytother. Res., 27(2), 166–172, 2013. 83. Cheon, J.M., Kim, D.I., Kim, K.S., Insulin sensitivity improvement of fermented Korean Red Ginseng (Panax ginseng) mediated by insulin resistance hallmarks in old-aged ob/ob mice. J. Ginseng Res., 39(4), 331–337, 2015. 84. Otto, C., Hahlbrock, T., Eich, K., Karaaslan, F., Jürgens, C., Germer, C.T, Christoph, O., Theresa, H., Kilian, E., Ferdi, K., Constantin, J., Christoph-Thomas, G., et al., Antiproliferative and antimetabolic effects behind the anticancer property of fermented wheat germ extract. BMC Complement. Altern. Med., 16, 160, 2016. 85. Ahrén, I.L., Xu, J., Önning, G., Olsson, C., Ahrné, S., Molin, G., Antihypertensive activity of blueberries fermented by Lactobacillus plantarum DSM 15313 and effects on the gut microbiota in healthy rats. Clin. Nutr., 34(4), 719–726, 2015. 86. Escudero-López, B., Berná, G., Ortega, Á., Herrero-Martín, G., Cerrillo, I., Martín, F., et al., Consumption of orange fermented beverage reduces cardiovascular risk factors in healthy mice. Food Chem. Toxicol., 78, 78–85, 2015. 87. Pyo, Y.H., Seong, K.S., Hypolipidemic effects of Monascus-fermented soybean extracts in rats fed a high-fat and -cholesterol diet. J. Agric. Food Chem., 57(18), 8617–8622, 2009. 88. Nam, Y., Jung, H., Karuppasamy, S., Lee, J.Y., Kang, K.D., Hwang, K.Y., et  al., Antihyperlipidemic effect of soybean extract fermented by Bacillus subtilis MORI in mice. Lab. Anim. Res., 28(2), 123, 2012. 89. Lee, J.-S., Rho, S.-J., Kim, Y.-W., Lee, K.W., Lee, H.G., Evaluation of biological activities of the short-term fermented soybean extract. Food Sci. Biotechnol., 22(4), 973–978, 2013. 90. Takagi, A., Kano, M., Kaga, C., Possibility of breast cancer prevention: use of soy isoflavones and fermented soy beverage produced using probiotics. Int. J. Mol. Sci., 16(12), 10907– 10920, 2015.

Augmenting Bioactivity of Plant-Based Foods Using Fermentation 183 91. Kim, Y., You, Y., Yoon, H.G., Lee, Y.H., Kim, K., Lee, J., et al., Hepatoprotective effects of fermented Curcuma longa L. on carbon tetrachloride-induced oxidative stress in rats. Food Chem., 151, 148–153, 2014b. 92. Takagi, A., Kano, M., Kaga, C., Possibility of breast cancer prevention: use of soy isoflavones and fermented soy beverage produced using probiotics. Int. J. Mol. Sci., 16(12), 10907– 10920, 2015. 93. Eum, A.H., Lee, H.J., Yang, C.M., Shim, S.K., Lee, J.H., Ma, Y.J., African Journal of Traditional, Complementary, and Alternative Medicines, 8, 312, 2011. 94. Kim, Y., You, Y., Yoon, H.G., Lee, Y.H., Kim, K., Lee, J., et al., Hepatoprotective effects of fermented Curcuma longa L. on carbon tetrachloride-induced oxidative stress in rats. Food Chem., 151, 148–153, 2014.

10 Probiotic Intervention for Human Health and Disease Bilqeesa Bhat and Bijender Kumar Bajaj* School of Biotechnology, University of Jammu, Jammu-180 006, Jammu & Kashmir (India)

Abstract The gut microbiota influences the physiological, metabolic, genetic, and immunological attributes, and is recognized as a potential intervention target for the management of health and disease. Dysbiosis (disruption of gut microbiota) may have major health consequences leading to more disposition towards diseases/disorders, but it can be averted by intervention of probiotics. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The health benefits associated with probiotic consumption include prevention, treatment or management of diarrhoeal diseases, lactose intolerance, systemic infections, inflammatory bowel disease, allergies, immunomodulation, cancers, and cardiovascular diseases. Most commonly used probiotics include the species and strains of Lactobacillus, Bifidobacterium, and certain yeasts; however, bioprospecting of new/novel probiotics has been a continuous practice. Despite substantial health benefits, the underlying functional mechanism of action of probiotics is yet to be fully understood. The current chapter presents the recent trends on health intervention of probiotics. Keywords: Probiotics, health benefits, diarrhea, hypocholesterolemia, exopolysaccharides, antimicrobial potential

10.1

Introduction

The global hunger index (GHI) is a multidimensional statistical tool used to describe the hunger situation across the globe affecting people. To measure the progress and failures in the global fight against hunger and improve the availability and quality of food, various approaches have been adopted. In this context identification of bioactive food components and understanding their role as therapeutic agents in disease management and prevention has become a significant area of research. Probiotics is one of the novel approaches not only to tackle global food availability but to provide multiple health benefits associated with probiotic consumption [1].

*Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (185–210) © 2019 Scrivener Publishing LLC

185

186 High Value Fermentation Products Volume 2 Human gut is associated with a large and diverse population of microorganisms generally called microbiota. The term microbiota is a collective expression of microbial communities in a specific ecological niche and any alteration in it causes various diseases and disorders [2]. The microbiota is usually localized in skin, mouth, vagina, upper respiratory and gastrointestinal tract of host. Probiotics are now emerging as a promising key category of food supplement with advent of new methods of identification and characterization techniques. The increased consumers’ interest about the positive role underscores the need to determine new probiotic microorganisms. The search for probiotic candidates among lactic acid bacteria (LAB) isolated from different food sources may uncover new strains with promising health and probiotic properties [3]. Health benefits of probiotics include lactose metabolism, improved digestion, antimicrobial peptide secretion, and control of wide range of dermal infections, anticarcinogenic properties, immunostimulation, short chain fatty acid production, antiatherogenic and hypocholesterolemic attributes, vaginal or urinary tract infection prevention, neutraceutical and organoleptic properties, and also helps in averting the dysbiosis. Hence, the ability of a probiotic to protect the host from infection is a novel trait for promoting human health [2, 4–6]. Probiotics have come a long way in 100 years since Nobel laureate Elie Metchnikoff’s discovery in 1907, who suggested that “the dependence of the intestinal microbes on the food makes possible to adopt measures to modify the flora in our bodies and to replace the harmful microbes by useful microbes” [7]. Identification of bacteria associated with desirable and productive outcomes in animals may offer a direct approach to the identification of probiotics [8, 9]. The studies on human microbiome have revealed that even healthy individuals differ exceptionally in diversity and abundance of each habitat’s signature microbes widely, with strong niche specialization both within and among subjects [2, 10]. The probiotics for human health has been classified taxonomically in order to assign correct health benefits to characterize microbial strains in order to avoid uninformed marketing, both in terms of contents and possible effects [11]. The probiotic predominantly include lactic acid bacteria (LAB) viz. Lactobacillus, Lactococcus, Streptococcus, Enterococcus, and Bifidobacterium, some yeasts (Figure 10.1), and other bacterial strains [4]. The LAB are Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria, and produce lactic acid. These genera include various species, sub species and strains that have been characterized for various probiotic properties. Some of the species of genus Bifidobacterium includes Bifidobacterium ruminantium, B. longum subsp. longum, B. longum subsp. infantis, B. bifidum, and B. breve, [8, 9]. The major species studied Lactobacillus genus also include Lactobacillus plantarum, L. fermentum, L. casei, L. paracasei, L. acidophilus and L. sakei, and Lactococcus genus include Lactococcus lactis and L. garvieae as major species and others such as Leuconostoc mesenteroides strains [9]. Some of the newly studied and fully sequenced species include Lactobacillus. lactis subsp. lactis, L. lactis subsp. cremoris, L. garvieae, L. raffinolactis, L. plantarum, L. fujiensis, and L. piscium and some of them produce antimicrobial compounds, such as bacteriocins, nisin, and lactococcin [12]. In this direction various studies are being carried out by different research groups worldwide. Park et al., [13] have isolated 11 Lactobacillus strains from Kimchi,

Probiotic Intervention for Human Health and Disease 187

Streptococcus

Lactobacillus

Yeast

Probiotics

Enterococcus

Bifidobacterium

Leuconostoc

Figure 10.1 Commonly used probiotic microorganisms.

Korea's famous traditional fermented food, with L. brevis, L. plantarum and L. sakei as dominating populations in a range 3–4 × 108 cfu/mL and possess all the functional probiotic characteristics. Bifidobacterium species are one of the most predominant microorganisms to dominate colon of breast fed infants (Awasti et al., 2016). Probiotic bacteria have been assigned with generally recognized as safe (GRAS) status. The ability of probiotics to exert health benefiting effects on humans is a species- and strain-specific feature. Hence continuous research for isolating novel strains with probiotic potential is must [14]. The most efficient strains should survive the harsh gastrointestinal tract conditions. As safety status, the origin of the strains, their non-pathogenicity and the absence of antibiotic-resistant genes is assessed while evaluating the probiotics [2]. In a study, seven LAB namely Pediococcus acidilactici SDL 1402, P. acidilactici SDL 1405, P. acidilactici SDL 1406, Weissella cibaria SCCB 2306, Streptococcus thermophilus SCML 337, S. thermophilus SCML 300 and Enterococcus faecium SC 54 were isolated from Korean fermented soybean paste and all the seven isolates were sensitive to antibiotics in addition to strong gut colonization ability [15]. A recent study revealed the safety aspects of three Lactobacillus mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk. The presence bile salt hydrolase production (bsh) genes, intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified [3]. Studies have established an outstanding role for diet, gut microbiota and their metabolites like the short chain fatty acids (SCFAs) involved in the pathogenesis of several inflammatory diseases, such as asthma, arthritis, inflammatory bowel disorder (IBD), colon cancer and healing of wound. SCFAs can modulate the progression of inflammatory diseases and autoimmunity,

188 High Value Fermentation Products Volume 2 and reveal G protein coupled receptor (GPCRs) metabolite sensing and inhibition of histone deacetylases [2, 16]. In both Lactobacillus and Bifidobacterium strains, well documented probiotic bioactive and/or effector molecules have been found and the major molecules include surface-located molecules, S-layer proteins, exopolysaccharides, bacteriocins, vitamins, biosurfactants, various enzymes like bile salt hydrolases etc [17]. Health benefits of few probiotic isolates are presented in Table 10.1.

10.2

Various Sources of Probiotics

Progressively growing research interest in probiotic bacteria has contributed to their recognition as health-benefiting agents. The potential health benefits of probiotics include (Figure  10.2) antimicrobial agents like bacteriocin, alleviation of lactose intolerance, hypocholesterolemic effect, immune system modulation and prevention of antibiotic associated diarrhoea, inflammatory bowel syndrome (IBS) and allergies like atopic eczema and food allergy [8, 28]. Probiotic isolates Lactobacillus fermentum and six L. plantarum strains were isolated from Tunisian raw camel milk. All the seven strains have antibacterial activity against pathogens like Listeria monocytogenes, Staphylococcus aureus and Escherchia coli and with L. fermentum possessing high level of adhesion to human Caco-2 and HT29-MTX epithelial cells [5]. Lactic acid bacteria, such as Leuconostoc mesenteroides, L. citreum, Lactobacillus brevis, L. curvatus, L. sakei, L. plantarum and Pediococcus pentosaceus, were isolated from fermented sour dough. Furthermore, such probiotic strains were characterized for carbohydrate metabolism, antifungal activity, exopolysaccharide production, phytase and xylanase activities and adhesion ability to Caco-2 cells [29]. Also microbiological studies were performed on novel Faecalibacterium prausnitzii strains in order to understand the biodiversity and physiological diversity of such beneficial commensal species in human gut. F. prausnitzii possessed SCFA like butyric acid production potential which induces inter leukins (IL-10), anti-inflammatory molecule secretion. Such studies suggest that some F. prausnitzii strains could represent able candidates as next-generation probiotic [30]. Probiotics have become a therapeutic strategy with multiple immunogenic or antagonistic properties that improve human health. The Lactobacillus spp. isolated from cocoa bean was investigated for their ability to modulate the chemically induced inflammatory process called induced colitis. The Lactobacillus spp. prevents inflammation process by reducing the inflammatory infiltrate in rat models [20]. Probiotic isolates L. paracasei BRAP01 and L. acidophilus AD300 induce IFN-γ/IL-10 production in Taiwanese individuals and that L. reuteri BR101 was the most effective stimulator of anti inflammatory molecules like IL-10/IFN-γ that resulted in immune modulation [31]. Chronic kidney disease (CKD) is a major public health problem where diet based therapy has been an integral part of the medical management of patients. Such treatment patterns have shown acceptance of CKD dietary interventions among patient [32]. Probiotic bacteria have got diverse health benefits but few thrust areas have been identified where role of probiotics is now well established. It includes treatment of

Probiotic organims

Lactobacillus plantarum Lp3

Lactobacillus iners / L. crispatus

Lactobacillus fermentum

Lactobacillus fermentum

Lactobacillus brevis D6

Lactobacillus kefiranofaciens XL10

Enterococcus sp. and Lactobacillus sp.

Lactobacillus sanfranciscensis LBH1068

Lactobacillus rhamnosus GG or Saccharomyces boulardii

Kocuria SM1 and Rhodococcus SM2

Source

Tibetan yak milk

Vagina

Cocoa

Human oral samples

Artisanal smoked fresh cheese

Tibetan kefir grain

Instestines of Japanese quail

Mexican alcoholic fermented beverage

Different sources

Fish gut

Table 10.1 Health benefits of probiotics isolated from diverse sources.

Bacteriocinogenic potential against MDR human pathogens

Antibiotic associated diarrhea

Treatment of inflammatory bowel diseases

Antimicrobial activities

Novel functional food development

Immunomodulating properties

Antibacterial Activity

Colitis treatment

Bacterial vaginosis

Cholesterol lowering

Health implication

[27]

[26]

[25]

[24]

[23]

[22]

[21]

[20]

[19]

[18]

References

Probiotic Intervention for Human Health and Disease 189

190 High Value Fermentation Products Volume 2

Hypercholesterolemia Antimicrobial infections

Management of diabetes

Irritable bowl syndrome

Probiotic health benefits

Various types of cancers

Immunomodulation

Lactose intolerance Various types of diarrhea

Urinary tract infections

Figure 10.2 Various health benefits of probiotics.

various types of diarrhea, cardio vascular disease (CVD) management, diabetes and obesity, microbial infections etc.

10.3

Commercially Developed Probiotic Products

Several attempts have been made for development of probiotic products at commercial scale. In 1930s, Shirota (Japan) isolated strains from human intestine that exert health benefits to individuals and introduced his first product, Yakult, into the market containing Lactobacillus casei Shirot. Since, then probiotic products have been used globally, and gained significant popularity due to increasing awareness about health benefits of probiotic consumption [33]. Different probiotic products have emerged in the market with different formulations and brand names. A few important commercially available probiotics are presented in Table 10.2.

10.4

Global Market of Probiotics

The world market for probiotics was worth USD 27.9 billion in 2011 and is expected to reach USD 44.9 billion in 2018 [36]. Since the 1980s the world market has been dominated only by well-known probiotic strains like Lactobacillus rhamnosus GG, L. casei Shirota, and Bifidobacterium animalis lactis (BB12). The global market for functional foods and beverages has grown from $33 billion in 2000 to $176.7 billion in 2013, accounting for 5% of the overall food market. It has been estimated that probiotic foods comprise between 60 and 70% of the total functional food market [33]. Probiotic products with beneficial claims for people of all ages, gender, race, geographic location, and with different health status are globally

Probiotic culture

Lactobacillus casei Shirota

Lactobacilllus casei

Lactobacillus reuteri, L. acidophilus and L.casei

Lactobacillus acidophilus and Lactobacillus bifidus

Streptococcus thermophilus and Enterococcus faecium

Lactobacillus casei Shirota, Bifidus regularis

Bifidobacterium infantis 35624

Enterococcus LABSF 68

Escherichia coli Nissle 1917

Escherichia coli

L. caseirhamnosus Lcr 35

Lactobacillus johnsonii La1

Source

Human colon

Milk drink

Yogurt

Fermented milk

Yogurt

Human origin

Human origin

Intestinal tract and faeces of piglets and sows.

Swine

Urine

Vaginal flora

Feces of humans and animals

SVELTY® Gastro Protect

Ginophilus®

URO VAXOM®

Mutaflor

Bioflorin

Align

Yakult®

Gaio

Ofilus

Symbalance

Actimel orange

Probiotic dairy foods

Food product

Table 10.2 Commercial probiotic products, sources and developer companies [33–35].

Nestlé

Probionov

Apsen

Ardeypharm

Cerbios–Pharma

Procter & Gamble

Yakult Honsha Co. Ltd.

MD Foods

Yoplait

Tonilai

Danone

Yakult Honsha Co.

Company

(Continued)

Probiotic Intervention for Human Health and Disease 191

Probiotic culture

Lactobacillus johnsonii La1 and Acidophilus bacteria

L.L. casei (Defensis)

Bifidobacterium breve Bb-03,B. lactis Bi-07, B. lactis Bl-04, B. longum Bl-05, Lactobacillus acidophilus La-14,L. bulgaricus Lb-64,L. brevis Lbr-35,L. casei Lc-11, Lactococcus lactisLl-23, L. plantarumLp-115, L. paracaseiLpc-37, L . rhamnosus Lr-32, L . salivarius Ls-33, Streptococcus thermophilus St-21

L.L. acidophilus NCFM™

L. acidophilus, L. casei, B. bifidus

L. acidophilus LA-5 and Bifidobacterium

Feces of humans

Kumis

Multiple sources

Conventional foods

Multiple sources

Cottage cheese

Cont.

Source

Table 10.2

Biorich®

Yógourmet Products

Chr.Hansen A/S

Lyo-San,Inc.

Danisco A/S

Danisco A/S

Flora FIT®

HOWARU®Premium Probiotics

Danone

Nestlé

Company

Actimel®

LC1 Yogurt®

Food product

(Continued)

192 High Value Fermentation Products Volume 2

Probiotic culture

L. acidophilus, L. casei, Bifidobacterium lactis

L.L. helveticus R0052, B. longumR0175

L.L. acidophilus, L. casei, B. lactis

Lactobacillus helveticus MTCC 5463, L. rhamnosus MTCC 5462

Multiple sources

Multiple sources

Multiple sources

Human gut

Cont.

Source

Table 10.2

Supplements and dairy products

Heini`s Yogurt Cultured Cheese

XoBiotic™ squares

Chocolate Probiotic Bars Chocolate Crisp®

Food product

Anand Agriculture University, Gujarat

Bunker HillCheese Company

MXI Corp.™

ATTUNE

Company

Probiotic Intervention for Human Health and Disease 193

194 High Value Fermentation Products Volume 2 marketed but such products are not accessible to people of developing countries [25]. A detailed account of some of the most prominent probiotic applications is given in the sections below.

10.5

Probiotic Production From Fermentation Process

Fermentation is one of the traditional food processing technologies. In developing countries where access to refrigeration is not available easily, fermentation is a widely used practice to increase the shelf life, safety, and stability of foods. Fermentation of foods enhances palatability, sensory quality, nutritional value of food products, may increase the availability of proteins and vitamins, and reduces the undesired and toxic compounds. Fermented food products have been the major carriers of probiotics. The major lactic acid bacteria associated with fermented plant products includes Lactobacillus plantarum, L. fermentum, L.acidophilus, Lactococcus lactis and Leuconostoc mesenteroides strains [37, 38]. Among LAB isolated from meat products, Lactobacillus sakei is most studied species due to its role in the fermentation of sausage and its prevalence during cold storage of raw meat products and L. sakei possesses multifaceted health benefits. The L. sakei improves microbial quality and safety of meat based food products at the end of storage [9]. In fermented foods, LAB display numerous antimicrobial activities mainly due to production of organic acids, bacteriocins and antifungal peptides. Bacteriocin application in sourdough, fermented sausage, and cheese has been studied during laboratory fermentations as well as on pilot-scale level [37, 38]. Various probiotics have been examined for their ability to ferment the dietary fibre carbohydrates such as β-glucan, xylan, xylo-oligosaccharides (XOS) and arabinoxylan by lactobacilli, bifidobacteria, enterococci or Escherichia coli [39]. Various fermented foods, and dairy products, are used as probiotic carriers and such products have positive health effects on the host. The probiotics must survive in the product and become active when entering the host gastrointestinal tract [38]. Lactic acid bacteria and bifibobacteria strains were isolated during the processing of Iberian dry fermented sausages, human and pig faeces. All isolates were identified as lactobacilli originated from human faeces (Lactobacillus casei and L. fermentum) and pig faeces (L. reuteri, L. animalis, L. murinus, and L. vaginalis). Pediococcus acidilactici strains were isolated from Iberian dry fermented sausages and pig faeces, whereas the large number of Enterococcus strains were identified as Enterococcus faecium, with this species being isolated from Iberian dry fermented sausages, and human and pig faeces [40].

10.6

Health Implications of Gut Microbiota Dynamics

Probiotics have been used for both prevention and treatment of multiple diseases. Torres-Maravilla et al., [25] has studied the potential of a novel probiotic Lactobacillus sanfranciscensis LBH1068 strain to treat inflammatory bowel disease (IBD). An effort to understand the safety and the risk-benefit balance of probiotics, a study was carried out to understand the probiotic intervention in irritable bowel syndrome (IBS) and IBD. The result revealed that adverse events vary widely among these two diseases [41]. Balance in gut microbiota has well defined role to play in various chronic

Probiotic Intervention for Human Health and Disease 195 manifestations of gut structure. The changes in composition of gut microbiota are critical to the pathogenesis of diverticular (pouches along digestive tract) complications such as diverticulitis and peridiverticular abscesses wherein increased number of proteobacteria and actinobacteria have been seen in patients as compared with healthy individuals. The study found that available data shows insufficient evidence of probiotic benefits in patients with diverticular disorders [42]. Vemuri et al., [43] studied the role of probiotics in IBD, a major public health concerns worldwide. The study revealed that the composition of beneficial microbes in the gut is comparatively higher but in many cases like malnutrition, use of antibiotics and any underlying diseases, favors colonization and invasion of pathogens leading to various disorders. Hence, consumption of probiotic bacteria has shown many beneficial effects in various gastrointestinal and inflammatory disorders. Another clinical study indicates that there is no substantial evidence that supports use of prebiotics or probiotics in Crohn’s disease. However, a few convincing evidences for the use of probiotics in pouchitis, both primary prevention and maintenance of remission have been demonstrated [44]. Awasti et al., [10] isolated three bifidobacterial strains (NBIF-2, NBIF-5 and NBIF-7) of human origin that demonstrated appreciable probiotic characteristics like antioxidant, antimutagenic and cholesterol assimilation properties. Alteration in gut microbiota composition (Dysbiosis) may play a significant role in pathogenesis of various inflammatory and autoimmune disorders. A meta-analysis of published data on probiotics has revealed efficacy in treatment of ulcerative colitis and in pouchitis, while little discouraging results in Crohn’s disease upon probiotic intervention. Some data supports the supplementation of probiotic in prevention of rotavirus diarrhea [12]. Harata et al., [45] reported for the first time a correlation between the gut microbiota and blood lipid level in Japanese cedar pollinosis (JCP), a challenging public health issue. Supplementation of fermented milk containing probiotics may be a prospective target and such supplementation has shown some beneficial effects on blood lipid levels in JCP patients. The IBD usually comprises Crohn’s disease, ulcerative colitis, and pauchitis and its manifestation usually occurs due to presence of many commensal enteric and pathogenic bacteria. Probiotic intervention has been reported to play an efficient role in management of IBDs and related pathologies [46]. Dysbiosis may drive gastrointestinal and extra gastrointestinal disease by interaction with the epithelial barrier and mucosal immune system or by production of various metabolites. Currently there is no curative therapy for chronic inflammatory conditions such as IBD, and the available therapies are usually used to suppress inflammation rather than curing it. Therefore, the modulation of gut microbiota with interventions like fecal microbiota transplant or phage therapy may be of great help [47].

10.6.1

Probiotics and Cardio Vascular Diseases, Blood Pressure, Obesity and Diabetes

Convincingly diet is considered one of the most influential lifestyle factors contributing to the rise in cardio vascular diseases (CVD) and diabetes in both developed and developing countries. Various clinical studies have shown that consumption of probiotics in adequate amount may improve blood pressure (BP); however, the mechanism involved

196 High Value Fermentation Products Volume 2 has multiple approaches and need further in-depth elucidation. Arterial hypertension (AH) is one of the most prevalent risk factors for CVD and ischemia, a major cause of death worldwide. Research has provided some insights about lowering of blood pressure with probiotic intervention and modulation of gut microbiota by interacting with ingested polyphenols [48]. A lot of research supports an impact of probiotic consumption on overall lipid profile and cholesterol levels in particular. Madjd et al., [49] conducted a clinical trial comparing the effect of daily probiotic consumption with low-fat conventional yogurt on weight loss in healthy obese women following an energy-restricted diet. In this clinical trial, a total of 89 participants were randomly assigned to either of intervention groups. But after 12 weeks of observation, no significant differences were found for fasting plasma glucose, high-density lipoprotein cholesterol, or triglycerides within both groups. Another study found that oral spore based probiotic supplementation reduced symptoms indicative of “leaky gut syndrome” and supplementation was associated with a 42% reduction in serum endotoxin at 5 hr post-prandial compared to a 36% increase in placebo at the same time point. It was also associated with a 24% reduction in serum triglycerides as compared to control [50]. Obesity is another alarming public health issue worldwide, affecting both developed and emerging countries. Obesity is characterized by an abnormal excess of white adipose tissue, a major risk factor for manifestation of diabetes, CVD and various types of cancers [51]. Intestinal microbiota has been proposed to have an impact on the energy balance, hence potential determinant of obesity [52]. Role of probiotics on obesity, insulin resistance syndrome (IRS), type two diabetes (T2D) and non-alcoholic fatty liver disease in human randomized clinical trials have revealed that probiotic consumption had beneficial effects on IRS, and plasma lipid levels. Various probiotic supplements and symbiotics have been reported to have positive impact on carbohydrate metabolism, fasting blood glucose, insulin sensitivity, and for reducing the metabolic stress in T2D diabetic subjects [53]. Gut microbiota has been recently established to have a role in the development of disorders, like obesity, and T2D. Therefore, any modulation of gut microbiota may be an effective therapeutic approach in management and control of CVD and other metabolic disorders [54].

10.6.2 Cholesterol Reducing and Total Lipid Profile Influencing Potential of Probiotics Elevated cholesterol levels are leading cause of global mortality, and hypercholesterolaemia is a major risk factor for various other cardiovascular disorders. Different drugs like statins are widely used to contain elevated circulating cholesterol levels and the associated adverse side effects. However, the high mortality rates associated with CVD suggest these measures are insufficient to manage and control the disorder. Probiotic intervention has evolved as a novel approach in this condition [55]. Tsai et al., [56] have studied various mechanisms involved in cholesterol lowering include bile salt hydrolase enzyme action (BSH), cholesterol precipitation, deconjugation of bile salts by BSH, etc. (Figure 10.3).

Probiotic Intervention for Human Health and Disease 197

Bile salt deconjugation Short chain fatty acids

Conversion of cholesterol to coprostanol

Cholesterol precipitation

Cholesterol assimilation

Cholesterol lowering properties

Cholesterol lowering from micelles

Cholesterol adherence to cell wall

Figure 10.3 Various mechanisms used by probiotics for lowering cholesterol.

Probiotic bacteria Lactobacillus plantarum Lp3, isolated from traditional fermented Tibetan yak milk, was evaluated for its cholesterol reducing potential in in vitro assays. Administration of L. plantarum Lp3 to rats fed on high cholesterol diet displayed a notable reduction in serum and liver cholesterol and triglycerides levels, and reduced the lipid deposition in the cytoplasm of rat’s liver tissue signifying its potential in treatment of hyperlipidemia [57]. The number of lactobacillus strains were qualitatively and quantitatively evaluated during in vivo experimentation on male Wistar rats fed at a daily dose of probiotics (1 × 109 CFU/mL) for 21 days, and it was recorded that a number of lactobacilli strains could deconjugate bile salts and most of them modulated the blood lipid profile resulting from a high-fat diet [58]. Aboseidah et al., [59] isolated probiotic bacteria Enterococcus faecium Y1 from infants stool and found that E. faecium Y1 showed reduction of 71.6% in cholesterol level. The results were confirmed by gas chromatography analysis of the fermentation extract, and it showed that the end product of cholesterol was 5á-cholestane-3à, 25-diol (C27H48O2). Zhang et al., [60] isolated ten probiotic strains from healthy infant fecal samples and found that an isolate Enterococcus faecium WEFA23 showed the cholesterol reducing ability at dose of 1.89 ± 0.07 mg/1010 CFU, glycodeoxycholic acid hydrolase activity (1.86 ± 0.01 U/mg), and strong adhesion capacity to Caco-2 cells (17.90 ± 0.19%). Results also indicated that administration of E. faecium WEFA23 to rats with high-fat diet-induced metabolic syndrome (MS) improved almost all key markers of MS, including obesity, hyperlipidemia, hyperglycemia, and insulin resistance. Lactococcus lactis KX881768, Lactobacillus plantarum KX881772, Lactococcus lactis KX881782 and Lactobacillus plantarum KX881779 isolated from camel milk exhibited remarkable cholesterol removal abilities with few showing very promising fermentation profiles [61]. The consumption of Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of dietinduced weight gain in wild-type C57BL/6J mice fed on high-fat diet. Such data supports

198 High Value Fermentation Products Volume 2 assessment of such strains in human trials [55]. Kim et al., [62] found that Lactobacillus kefiri DH5 isolated from kefir has potential to inhibit the development of obesity and nonalcoholic fatty liver disease (NALFD) in mice fed on 60% high-fat diet (HFD) apart from having hypocholesterolemic activity of about 51.6% in in vivo experiment. An overall conclusion of such study revealed that L. kefiri DH5 exerts anti-obesity effects by reduction of cholesterol in the lumen and upregulation of PPAR-α gene (transcription factor and a major regulator of lipid metabolism in the liver) in adipose tissues, FABP4, and CPT1 expression in the epididymal adipose tissues. Current research has also provided us insights about raised cholesterol levels as a major risk factor for CVD. One more study in this direction was carried out in which daily body restore (DBR), a proprietary blend of 9 probiotic for its effects on cholesterol metabolism. Consumption of DBR supplementation in hypercholesterolemic mice for 4 and 8 weeks showed significant decreases in blood concentrations of low-density lipoprotein (LDL, 47%) and increases in high-density lipoprotein (HDL, 32%) but no changes in triglyceride concentrations in 4 weeks and low-density lipids (LDL) concentrations were dramatically reduced by 78% and HDL was increased by 52% relative to control mice after 8 weeks [63]. Zhang et al., [60] studied the effects of Bifidobacterium bifidum, Lactobacillus casei, and L. plantarum on HFD induced hyperlipidemia and liver injury in diet modified C57BL/6N mice for a period of 6 weeks. A significantly reduction in HFD induced body weight gain, hyperlipidemia, liver fat accumulation and reactive oxygen species (ROS) production was observed. Furthermore, the role of probiotic-fermented soymilk may decrease the production of TNF-α, and oxidative stress and induce adipose leptin and triacylglycerol hydrolase (TGH) production. Various lactobacilli strains have been used to decrease serum lipid levels. Eight new potential probiotic strains including L. acidophilus, L. casei, L. paracasei, and L. rhamnosus were used to decrease the serum lipid levels of male hamsters fed on a high-fat (12%) and high-cholesterol (0.2%) diet. When treated with various doses of probiotics (200, 400, and 800 mg/kg), the different hamsters groups presented different levels of recovery of serum LDL-cholesterol, triglycerides, and total cholesterol levels in a dose dependent manner. Such study provides an alternative to cholesterol reduction drugs like rosuvastatin, where results have confirmed the effectiveness of the probiotics as suitable choice due to lack of drug-like side effects [64]. Modulation of gut microbiota via maternal probiotic intervention during pregnancy and breastfeeding may potentially change future disease course in both mother and infant [65]. Some evidence suggests the possibility that probiotics may influence blood pressure but the mechanism in such cases largely remains unexplored. More research is needed to determine species- and strain-specific effects of probiotics in healthy participants with different degrees of stress sensitivity [66].

10.6.3 Probiotic Intervention in Neurological Disorders Dysbiosis, a state of altered gut microbiota composition in humans have also have been implicated in various neuropsychiatric conditions such as depression, autism and Parkinson’s disease but as a causal secondary effect [67]. There are scientific evidences in abundance that advocates probiotic consumption as a successful strategy for management and treatment of different types of diarrheal diseases, immune modulation, colon cancer and other inflammatory disorder prevention [15]. The effectiveness of

Probiotic Intervention for Human Health and Disease 199 probiotics consumption in treatment or prevention of neurological diseases is evolving as new domain of research [68]. Number of preclinical studies have suggested that gut microbiota is currently viewed as a key regulator of an even bidirectional discourse between the gut and the brain which is referred to as gut-brain axis. Health benefits exerted by probiotics may also play an important role in neuro-developmental and neuro-degenerative disorders [67]. Dolan et al., [69] explored the role of probiotics in balancing mental well-being and neurological issues like anxiety, depression, hyperactivity, autism, cerebral and multiple sclerosis. Mangiola et al., [70] studied the importance of gut microbiota impairment in the etiopathogenesis of autism, dementia and mood disorder. The evidence of microbiota alteration in inflammatory disorders like schizophrenia, which is a major depressive and bipolar disorder, have been well established. The advancement of the therapeutic tools for the modulation of gut microbiota is warranted for a better management of autism spectrum and mood disorders.

10.6.4 Health Benefits of Probiotic Exopolysaccharides Exopolysaccharides (EPS) of microbial origin are generally composed of monosaccharide residues and their derivatives. EPS produced by food grade LAB are mainly used for improving texture, rheology and mouth feel of milk-based functional foods [69]. Uroic et al., [22] isolated Lactobacillus brevis D6 carrying a 45 kDa S-layer protein that was found to be involved in its increased survival rates, an important prerequisite for exertion of probiotic attributes in host gut. The S-layer was also found to play a role in adhesion potential to extracellular matrix proteins and Caco-2 intestinal epithelial cell line in in vitro assay. The Lactobacillus kefiranofaciens XL10, isolated from Tibetan kefir grain, produced extracellular polysaccharide (EPS), that helped bacterium to adhere to the mucous tissue and colonized the ileum of the mice based on fluorescence imaging, flow cytometry, and qPCR indicating its large probiotic properties [23]. The Lactobacillus fermentum CFR 2195 based EPS were found to contribute to specific rheology, texture of novel food products and finds applications even in other therapeutics. Structural and composition analysis of EPS was carried and typical polysaccharide nature and flakes like structure have been established through various analytical techniques [71]. Anticancer and antidiabetic studies of Lactobacillus plantarum RJF4 derived EPS have been studied. EPS has displayed total antioxidant capacity (32%), DPPH radical scavenging ability (23.63%) and reduction potential (50%) and cholesterol lowering potential (42.24%). The α-amylase inhibition (40%) assay of EPS revealed its anti-diabetic property. Cytotoxicity studies revealed its cancer cell specific anti-proliferative effects and toxicity to MiaPaCa2-pancreatic cancer cell line in dose dependent manner. It was confirmed that EPS remained nontoxic to normal fibroblast cells [69].

10.6.5

Antimicrobial Potential of Probiotics

Probiotic bacteria confer many advantages to humans and animals by providing natural protection against a wide range of bacterial pathogens such as Salmonella typhymurium, Eschericia coli, Enterococcus fecalis, Klebsiella pneumoniae, micrococcus ssp., Staphylococcus aureus, Salmonella typhimurium, etc [2]. Probiotics are nonpathogenic

200 High Value Fermentation Products Volume 2 microorganisms with documented evidence of its potential application in prevention and treatment of various infections. Probiotics exert antimicrobial activity against various pathogens involved in manifestation of diseases like diarrhea, atopic eczema, dental carries, colorectal cancers, and many more gastrointestinal disorders. Scientific evidence indicates a surge in the demand for probiotics in clinical applications [6]. Many studies have found that indiscriminate antibiotic use in human and animal food has resulted in the emergence of antibiotic-resistant bacterial strains. Probiotics are seen as a novel alternative to reduce disease burden due to emerging pathogens [72]. A study found that probiotic Lactobacillus plantarum possessed the ability to inhibit food-borne pathogens, a mould (Mucor spp.) found in fermented sausages [73]. Ghatani and Tamang [74], isolated probiotic bacterial strains like L. plantarum YD5S and YD9S, L. pentosus YD8S, L. paraplantarum YD11S, Enterococcus lactis YHC20 and E. faecium YY1 from indigenous fermented yak milk products with cholesterol lowering potential. Probiotic biosurfactant are studied as a new approach to check various infections in clinical and food setups. The Lactobacillus helveticus was isolated and studied for biosurfactant production and subsequently evaluated for its antimicrobial and antiadhesive potential. Lactobacillus helveticus was used to counteract effectively against the initial deposition of biofilm and to reduce the overall biofilm forming potential by a range of pathogenic microorganisms [75]. Pediococcus strains have been studied for production of pediocin, an effective antilisterial bacteriocin [76]. Microbial modification owing to antibiotics and probiotic consumption, and fecal transplant has been effective in the treatment of conditions such as recurrent Clostridium difficile infection, pouchitis, and IBS [77]. Probiotic isolates Enterococcus faecium, E. durans and Lactobacillus salivarius isolated from quail’s (Coturnix coturnix Japonica) intestine were studied for antimicrobial activities against indicator bacterium, i.e. Staphylococcus aureus, Escherichia coli and Salmonella typhimurium. All the strains were able to produce antimicrobial activity against all the three indicator pathogenic strains [78]. A study carried by Kawai et al., [79] on two strains of Lactobacillus plantarum 122 (oral cavity) and L. fermentum ALAL020 (soy milk) showed strong growth inhibition effects and the major antibacterial substance produced by L. plantarum 122 was thought to be sodium lactate. Probiotics Kocuria SM1 and Rhodococcus SM2 recovered from intestines of rainbow trout (Oncorhynchus mykiss, Walbaum) were characterized. Secondary metabolites of both probiotics were inhibitory to Acinetobacter baumannii, Vibrio anguillarum and V. ordalii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Significantly, Rhodococcus SM2 displays a high degree of bacteriocinogenic potential against multidrug resistant human pathogens [27]. The Lactobacillus fermentum showed excellent antimicrobial activity against various pathogens including Pseudomonas aeruginosa and Klebsiella pneumoniae. The antibacterial molecules suggest that it is a compound with low molecular weight and with highly hydrophilic component [21]. Various modes involved in elimination of pathogens include inhibition of pathogen replication by producing antimicrobial substances like bacteriocins, competition for nutrients and space, antitoxin production, biosurfactant production, inhibition of virulence, antiadhesive (antibiofilm) and antiinvasive effects, and coaggregation ability [6, 27]. To evade antibiotic resistance, a better understanding of probiotic and different

Probiotic Intervention for Human Health and Disease 201 pathogen interactions is a must to understand for effective use of probiotics to control different infections [75].

10.6.6

Probiotics for Bacterial Vaginosis

A major part of the vaginal microbiota is composed of Lactobacillus species. Women are most often diagnosed with bacterial vaginosis (BV) and analysis of the bacterial communities revealed that BV negative women are dominated by either Lactobacillus iners or L. crispatus [19]. Various probiotic formulations containing Lactobacilli strains have reduced BV symptoms and have improved the tolerant vaginal microflora profile. Several combinations of metronidazole, clindamycin or tinidazole with lactobacilli probiotic preparations have displayed promising effects in treatment of BV and help in maintenance of healthy vagina [80]. High recurrence (40% within 3 months and 50% within 6 months) of BV even after metronidazole or vaginal clindamycin treatment has invoked the need for alternate therapeutics for BV treatment. It has been proposed that addition of probiotics containing lactobacilli species may increase efficacy of such treatments by reducing vaginal pH by production of lactic acid, stimulate the immune system and produce hydrogen peroxide [81]. Administration of probiotic suppositories SYNBIO®gins (Lactobacillus rhamnosus IMC 501® and L. paracasei IMC 502®) in women on daily basis for 7 days contributed to a significant increase in the lactobacilli level in patients and it was found that it could be useful in restoring and maintaining a normal vaginal microbiota [82]. In a study, suitability of oral probiotics for antenatal and vaginal health care was evaluated in a randomized, placebocontrolled, triple-blind, and parallel group trial. Oral administration of Lactobacillus rhamnosus GR-1and L reuteri RC-14 (109 cfu) was given to pregnant women for 8 weeks. The primary outcome indicated that proportion of normal vaginal microbiota decreased from 82.6% to 77.8% in the treatment group compared to placebo where 79.1–74.3% decrease in vaginal microbiota was observed [83].

10.6.7 Probiotics for Treatment of Diarrhea A number of studies have been carried out so far to document the probiotic potential for therapeutic and preventive strategy in patients with diarrheal diseases. However, considering that probiotics have strain-specific effects, major focus has been shifted to individual probiotic strains. Bacillus coagulans MTCC 5856, a probiotic strain with claimed efficacy in diarrheal treatment, was also evaluated for its safety and efficacy in diarrhea predominant irritable bowel syndrome (IBS) patients [36] in a double blind placebo controlled multi-centered clinical trial. Thirty-six patients were divided into control and test groups, who received B. coagulans MTCC 5856 tablet (2 × 109 cfu/day) for 90 days in place of placebo. A significant decrease in the clinical symptoms like bloating, vomiting, diarrhea, abdominal pain and stool frequency in a patient group receiving B. coagulans MTCC 5856 was observed, with an overall decrease in severity of disease [84]. For treatment of digestive discomfort, patients are prescribed with H2 blockers and proton pump inhibitors (PPIs) and antacids which upon long-term consumption cause serious health complications. Clinicians are exploring the possibility of probiotic consumption as a novel approach to alleviate symptoms of digestive tract discomfort

202 High Value Fermentation Products Volume 2 [85]. Different probiotic strains have been effective in antibiotic associated diarrhea or inflammatory bowel disease for instance. Rotavirus cause infectious diarrhea among infants in most of the cases while as Escherichia coli is particularly responsible for acute diarrhea among travelers. The bacterial pathogens mostly include Salmonella, Campylobacter, and Shigella. Studies support potential of probiotics in eliminating such infections by contending for available nutrients and binding sites, reducing gut pH, producing short chain fatty acids, bacteriocins, biosurfactants etc. which may be specific or non-specific in their action [86]. Probiotic strains like Lactobacillus rhamnosus GG, Saccharomyces boulardii and Lactobacillus reuteri DSM 17938 are recommended by European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) in addition to rehydration as therapy for treatment of acute gastroenteritis in children [87]. A major GI disorder identified is antibiotic-associated diarrhea (AAD) in both children and adults. Although not too many clinical studies have been done, L. rhamnosus GG [88] and S. boulardii [89] probiotic consumption has been recommended in certain cases [26]. In C. difficile–associated diarrhea (CDAD) case administration of S. boulardii and fecal microbiota transplantation appears as a practical treatment option for excluding C. difficile from the human gut. Rodriguez et al., [90] studied the effect of a probiotic Saccharomyces boulardii in prevention and treatment of antibiotic induced diarrhea in both children and adults and it was found that freeze drying did not affect nutrients and bioactive compounds and such probiotics could be incorporated in different foods.

10.6.8 Probiotics for Urinary Tract Infections Urinary tract infections (UTIs), one of most common infections worldwide, face high recurrence rates and increasing antimicrobial resistance. Probiotic bacteria, especially of the genus Lactobacillus, are considered a promising preventive and/or treatment therapy against UTIs. It has been observed that people with neuropathic bladder, such as from spinal cord injury, are at significant risk of UTI. Probiotics have been recommended to reduce colonization of uropathogens and to manage the dual problems of infection and antibiotic resistance. Results have shown uncertainty in prevention of urine infections in people with bladder dysfunction upon probiotic consumption. Hence, further studies are needed in this direction [91]. Use of probiotics as a potential prevention strategy to reduce risk of infection in a postoperative state in abdominal surgery and a reduction in UTIs from abdominal surgeries was of low quality owing to imprecision such as less patient number considered for study. Thus larger multicentered trials are needed to establish the certainty in results [92]. In postmenopausal women with recurrent UTI, the role of intestinal microbiota in the development of onset of UTIs was studied. It was found that the intestinal microbiota was not predictive for new-onset UTIs [93]. Probiotics as a non-antibiotic prevention strategy to prevent UTI in post-menopausal women in several in vivo and in vitro studies have been investigated. The effectiveness of probiotic therapy with combinations of Lactobacillus reuteri RC-14 plus L. rhamnosus GR-1 and/or L. rhamnosus GG plus Bifidobacterium BB-12 in preventing UTI in people with spinal cord injury as compared to placebo was

Probiotic Intervention for Human Health and Disease 203 evaluated and it was found that probiotics might be seen as effective in preventing UTI [94]. The adherence of Lactobacillus strains and clinical uropathogenic strains to T24 epithelial bladder cells was studied and it was found that L. salivarius UCM572 and L. acidophilus 01 significantly inhibited the adherence of the five uropathogens to T24 cells. The in vitro results suggested that inhibition of the adherence of uropathogens to epithelial bladder cells by different lactobacillus strains may be one of the novel means to prevent UTIs [95]. In another study, cell free supernatant of Lactobacillus plantarum LPS10 was examined for its inhibitory effect against clinical urogenital bacterial strains and Candida albicans isolated from UTI infections. CFS obtained from this probiotic showed a promising antibacterial and anti candidal results. It was found that L. plantarum LPS10 possessed the capability to be used as a probiotic against pathogens involved in urogenital infections [96].

10.7

Conclusions

Probiotic application in treatment and managements of various diseases and disorders is an ever-expanding discipline. Probiotics act through diverse mechanisms affecting the composition or function of the commensal microbiota. Certain probiotic interventions have shown promising results in such disorders as atopic dermatitis, necrotising enterocolitis, pouchitis, cancers, gut inflammation, diabetes and CVD, and other related disorders. Further studies are required to characterize the microbiota pattern in diseases so that the microbiota can be remodelled to a more robust and resilient form so that subjects attain a disease-free state by probiotic administration. More intensive research is needed to ensure the progress and success of probiotics in this area, and evaluation of different probiotic strains, their dose and the product formulations for realizing their effective health benefits.

Acknowledgement Dr. Bijender Kumar Bajaj (BKB) gratefully acknowledges financial support in the form of Research Projects from Department of Science and Technology (DST), Council of Scientific and Industrial Research (CSIR), University Grants Commission (UGC), Department of Biotechnology (DBT), and Indian Council of Medical Research (ICMR); BKB gratefully acknowledges various Agencies for Research Fellowships: IAS, Durham University, UK (COFUND-International Senior Research Fellowship Durham University, UK), Commonwealth Scholarship Commission, UK (Commonwealth Fellowship at IBERS, Aberystwyth University, UK), ERUSMUS-MUNDUS (Invited Professor Scholarship, University of Naples, Italy), VLIR-UOS (University of Antwerp, Belgium) and ASM-IUSSTF (Indo-US Professorship at OARDC, Ohio State University, USA). Ms Bilqeesa Bhat gratefully acknowledges the Council of Scientific and Industrial Research (CSIR) for CSIR-Senior Research Fellowship for Doctoral Research. Authors thank the Director, School of Biotechnology, University of Jammu, Jammu, for necessary laboratory facilities.

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11 Saccharomyces - Eukaryotic Probiotic for Human Applications Alok Malaviya1,*, Shruti Malviya2, Anil Agarwal3, Madhvi Mishra1 and Valina Dalmida1 1

Applied and Industrial Biotechnology Laboratory, Department of Life Sciences, CHRIST (Deemed to be University), Hosur Road, Bengaluru-560029 (India) 2 National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bellary Road, Bengaluru-560065(India) 3 Department of Chemistry, CHRIST (Deemed to be University), Hosur Road, Bengaluru-560029 (India)

Abstract Probiotics are viable microorganisms which are meant to confer health benefits to host after ingestion. Any probiotic strain has a special characteristic to survive in the extremely acidic and hostile conditions of stomach and intestine. Among all the commercially available probiotic strains, prokaryotes constitute the bulk of it, with quite a few belonging to eukaryotic yeasts. Eukaryotic probiotics are very limited and currently there are only two yeast strains (Saccharomyces boulardii and Kluyveromyces sp.), which are approved for human consumption and are available commercially in market. S. boulardii has been reported to have tremendous therapeutic potential. The main mechanism of action for S. boulardii includes strong antagonistic effect against a number of enteric pathogens, trophic effects on the intestinal mucosa, neutralisation of bacterial toxins as well as modification of host cell signaling pathways involved in inflammatory and non-inflammatory intestinal disease. Pertaining to these advantages, S. boulardii have been reported to be exceptionally effective against diarrheal diseases and intestinal inflammatory conditions including inflammatory bowel disease (IBD). Increasing scientific reports confirming the therapeutic potential of eukaryotic probiotics and their advantages over prokaryotic probiotic strains have dramatically increased the worldwide interest in these probiotics. Keywords: Diarrheal diseases, eukaryotic probiotic, inflammatory bowel disease, Saccharomyces boulardii

11.1

Introduction

The term “probiotic” was originated from the Greek words “pro” and “bios” which means promoter of life, helping in a natural way to improve the overall health status of *Corresponding author: [email protected] Saurabh Saran, Vikash Babu and Asha Chaubey (eds.) High Value Fermentation Products Volume 2, (211–230) © 2019 Scrivener Publishing LLC

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212 High Value Fermentation Products Volume 2 host [1]. The World Health Organization (WHO) defines probiotic as “viable microorganisms that can provide benefits to human (and animals) health when administered in adequate amounts”. Any non-pathogenic microorganism, irrespective of its origin, capable of surviving in the digestive tract of host and exerting such host beneficial effects could be a probiotic candidate [2]. In recent years, the definition of a probiotic has changed primarily because of the recognition that probiotic bacteria can influence the physiological outcomes distant from the gut lumen. Moreover, the activation of local mucosal protective mechanisms and the modulation of adaptive immune effector functions can influence protection levels and the degree of inflammation at all mucosal sites. These observations shifted the concept of probiotics from a narrow range of dairy isolates to the concept of ‘immunobiotics’ [3]. Nowadays probiotics are quite common in our daily diet due to increasing scientific evidence of their beneficial effects and commercial interests [2, 4]. Most of the commonly used probiotics are prokaryotic in nature. However, there are quite a few eukaryotic probiotics conferring health and nutritional benefit to human and animals. Among the eukaryotic probiotics, Saccharomyces sp. and Kluyveromyces sp. (yeast) are the only commercially available eukaryotic species whose efficacy and usefulness has been confirmed by solid scientific evidence. The pH of the stomach ranges from 0.8 to 1.5 and it rises towards the distal part of the GI tract, which makes it an extremely hostile environment for most of the microbes. The presence of yeast in such condition can be explained by their resistance to intestinal fluids (e.g., bile and pancreatic juice) and the short transit time in the duodenum. Yeast can grow well in the pH range of 7–8, optimal growth being observed between pH 4.5 and 6.5. Most of them can grow at pH 3.0, and some species can even tolerate a pH as low as 1.5. Therefore, yeasts are considered among good probiotic candidates as probiotics entering the GI tract must be resistant to local stresses. The human intestinal system has been shown in Figure 11.1. Saccharomyces cerevisiae var boulardii (henceforth mentioned as S. boulardii) is one of the yeast probiotic, approved for human consumption. Besides S. boulardii, other S. cerevisiae strains (Saccharomyces cerevisiae Sc47) have been approved as

Liver

Stomach (empty: pH 0.8-1.5; up to 1012 bacteria/ml)

Gall bladder (bile acid)

Small intestine (pH 4-7; up to 107 bacteria/ml)

Large intestine (pH 5.5-7; up to 1012 bacteria/ml)

Figure 11.1 Human intestinal system (Adapted from a graphic of David Carlson: http://www.carlsonart.com/lifescience/index.html).

Saccharomyces - Eukaryotic Probiotic for Human Applications 213 animal feed additives and veterinary probiotics [5–7]. S. boulardii was first discovered when a French scientist, Henri Boulard, observed that drinking of a tea made of the tropical fruits mangosteen and lychee helped the people to become refractory to the symptoms of Cholera. Analysis of the skin of those fruits resulted in the isolation of S. boulardii, which later proved to have probiotic properties [8]. The genetic material of S. boulardii is significantly different than that of S. cerevisiae. Hennequin et al., [9] identified a unique and specific microsatellite allele characterizing S. boulardii that distinguishes it from other strains of S. cerevisiae at metabolic and physiological level, particularly with respect to growth yield and resistance to temperature and acidic stresses. This might be attributed to its survival fitness in the harsh environmental conditions found in gastrointestinal (GI) tract having low pH, bile salts and pancreatic proteases. These properties confer S. boulardii with excellent probiotic potentials. In addition to S. boulardii, Kluyveromyces marxianus fragilis B0399 is the only yeast strain available in the market, which is approved as a probiotic strain for application in animal feed as well as human consumption [10]. Several in vitro and in vivo experimental evidences demonstrated that K. marxianus B0399 is able to modulate the immune response, reducing the proinflammatory cytokine levels, thus being useful for mitigation of the effect of several diseases, such as irritable bowel syndrome [11–13].

11.2

Advantages of Eukaryotic Probiotics Over Prokaryotic Probiotics

Bacteria are the predominant colonizers (~99%) in gut microbiome and hence most of the currently used probiotics belong to prokaryotes. This is evident by a list of approved probiotic strains by Food Safety and Standards Authority of India presented in Table 11.1. A comparison of the cellular properties of prokaryotic (Bacteria) and eukaryotic (Yeast) probiotics has been presented in Table 11.2. Eukaryotic probiotics are more advantageous as compared to prokaryotic probiotics, pertaining to their natural resistance to many antibiotics and inability to horizontal gene transfer. Presently, development of antimicrobial resistance by the pathogenic bacteria has become an important public health concern. Antimicrobial resistance occurs both vertically (inherent or natural resistance of bacterial species or genus) and horizontally because of the transfer of genes between bacteria. The mammalian GI tract provides favorable conditions for the transfer of genetic material between bacterial species and under such favorable conditions, the prokaryotic probiotics and intestinal microbiota can easily acquire as well as transfer the antibiotic resistance genes to unwanted bacterial pathogens [15]. In case of eukaryotic probiotics, no such transfer of genetic material takes place. Moreover, probiotic yeast (S. boulardii) could be used even during antibiotic treatments, which is not possible with bacterial probiotics [3].

214 High Value Fermentation Products Volume 2 Table 11.1 List of approved probiotic strains by Food Safety and Standards Authority of India [14]. Sl. no.

Approved probiotic strains

Prokaryotes 1

Lactobacillus acidophilus

2

Lactobacillus plantarum

3

Lactobacillus reuteri

4

Lactobacillus rhamnosus

5

Lactobacillus salivarius

6

Lactobacillus casei

7

Lactobacillus brevis

8

Lactobacillus johnsonii

9

Lactobacillus delbrueckii sub-sp. bulgaricus

10

Lactobacillus fermentum

11

Lactobacillus caucasicus

12

Lactobacillus helveticus

13

Lactobacillus lactis

14

Lactobacillus amylovorus

15

Lactobacillus gallinarum

16

Lactobacillus paracasei

17

Lactobacillus gasseri

18

Lactobacillus delbrueckii

19

Bifidobacterium bifidum

20

Bifidobacterium lactis

11

Bifidobacterium breve

22

Bifidobacterium longum

23

Bifidobacterium animalis

24

Bifidobacterium infantis

25

Streptococus thermophiles

26

Bacillus coagulans (Continued)

Saccharomyces - Eukaryotic Probiotic for Human Applications 215 Table 11.1 Cont. Sl. no.

Approved probiotic strains

Eukaryotes 1

Saccharomyces boulardii

2

Saccharomyces cerevisiae

11.3

Probiotic Properties of Approved Yeast Strains

For a microbe to be a potential probiotic candidate, it needs to exhibit certain physiological and metabolic characteristics which include: (i) resistance to gastric acidity, (ii) bile acid resistance, (iii) antimicrobial activity against potentially pathogenic bacteria, (iv) ability to reduce pathogen adhesion to surfaces, and (v) bile salt hydrolase activity [16]. In addition to these properties, which are deemed essential for an organism to be considered as a probiotic strain, yeast probiotic strains (Saccharomyces sp.) exhibits several other useful properties. These include a broad range of nutritional requirements, ability to tolerate temperature, pH, oxygen and osmotic stress, ability to secrete various useful enzymes, antibiotic resistance as well as antimicrobial activities against wide range of pathogens, which makes them a suitable probiotic candidate [17]. Another probiotic yeast, Kluyveromyces B0399 is different from commonly used Lactobacillus, Bifidobacterium & Saccharomyces sp. It is capable of performing bioregulatory action and rebalancing of the intestinal flora. It efficiently stimulates development of the good gut flora (e.g., endogenous Bifidobacteria), even at low dosage (>100 times less then S. boulardii & Bifidobacteria). It efficiently colonizes intestine adhering to enterocytes of the intestinal epithelium. It has strong antimicotic action (particularly against “Candida albicans”) and efficiently compete against pathogens through: a) direct adherence to epithelium, b) direct competition for nutrients, c) promotion of epithelial cell growth and reinforcement of the major gut barrier against infections, d) by decreasing pH it turns the local environment unsuitable for the growth of certain pathogens (e.g., E.coli). It modulates immune response through: a) fine modulation of the level of anti- and proinflammatory cytokines, possibly attenuating the proinflammatory effect in inflammatory disorders such as Irritable Bowel Syndrome (IBS) and Celiac Disease, among others; b) as a rich source of prebiotics, it gives rise to immunostimulating beta-glucans and oligosaccharides (GOS, MOS, FOS). In the following section, we will discuss some of the essential probiotic properties of yeast based probiotics.

11.3.1

Survival in Gastrointestinal Tract

To be considered as a potential probiotic, microorganisms need to have the ability to survive in the harsh conditions of gastrointenstinal (GI) tract. Literature reports suggest that eukaryotic probiotics can tolerate the GI conditions, both in vitro (simulated conditions) and in vivo conditions. It has been observed that eukaryotic probiotic strains are not only able to survive the harsh GI condition, but they were able to exert positive

Properties

Size

Cell wall composition

pH optima

Temperature (Range)

Tolerance to acidic environment in stomach

Tolerance to bile salts

Antibiotic resistance

Ability to horizontally transfer genetic material

Presence in gut microbiome

Ability to colonize in gut

Synergistic effects on other microbes

Ability to produce antagonistic compounds

Ability to neutralize enterotoxins

Sl. no.

1

2

3

4

5

6

7

8

9

10

11

12

13

Yes

Low

Yes

Low

Sporadic ( 99%)

Yes

No

Yes

Yes

10–80°C

6.5–7.5

Peptidoglycan, lipoteichoic acid (LTA), lipopolysaccharide (LPS)

Small

Bacterial probiotics

216 High Value Fermentation Products Volume 2

Saccharomyces - Eukaryotic Probiotic for Human Applications 217 effects [2]. Yeast strains belonging to Saccharomyces, Debaryomyces, and Kluyveromyces species have been found to be extremely tolerant to bile salts (up to 0.3%) and low pH (upto 2.5) under simulated in vitro conditions [18–20]. Similar observations have been reported during in vivo studies using S. boulardii, where these strains have been found to survive and flourish in the gut conditions, exhibited by their high population in host gut [2].

11.3.2 Stress Tolerance Capability Another desirable property for a microbe to be considered as an ideal probiotic candidate is their ability to tolerate stress conditions. During their industrial processing, the probiotic strain has to go through highly stressed conditions (e.g., freezing, drying, heating (thermal stress), osmotic stress, etc.) irrespective of their origin. Finally, after going through all these processing steps, the probiotic strains should be in viable conditions. Yeast strains have the capability to quickly adapt themselves in response to changing environmental conditions. Yeast strains can tolerate heat shocks by accumulation of trehalose, followed by expression of heat shock proteins (HSP 104), antioxidant enzymes and involvement of plasma membrane ATPase [21].Yeast strains are also tolerant to broad range of osmotic stresses. Osmotic stresses could lead to the cell shrinkage culminating towards cell death, and yeast strains possess various mechanisms to avoid water depletion during osmotic stress. Accumulated trehalose as well as gene products of GPD1, HAL1 and HAL3 helps yeast cells to overcome various osmotic stresses [11]. All these properties help yeast strains to cope up with the processing stresses (osmo stress and heat stress). During storage, probiotic strains go through stress caused by low storage temperature. It is equally important that probiotic strains don’t lose their viability while stored at low temperatures. Yeast cells have inherent capability to resist freezing temperatures and it was found that S. boulardii cells stored at −20 °C showed specific growth rate and biomass production similar to those normal cells [2].

11.3.3 Ability to Adhere with Gastrointestinal Tract Adherence to gut mucosal surface is considered to be yet another and crucial property of any organism with probiotic potential. It is believed that this property ensures their presence in gut environment for longer period of time, helping them to exert their beneficial effects. Yeast probiotic strains of Saccharomyces, Debaryomyces, Kluyveromyces, Candida species have shown excellent adhesive properties during in vitro studies [19, 20]. However, their adherence capability differs based on sources, species and strains.

11.4

Pharmacodynamics of S. Boulardii

It has been found that S. boulardii survives the gastric environment in lyophilized form, and can be detected alive throughout the entire digestive system. S. boulardii is also

218 High Value Fermentation Products Volume 2 resistant to proteolysis and to various antibiotics [22]. When S. boulardii is taken simultaneously with amoxicillin, its population in gastrointestinal tract was found to be doubled. However, nystatin completely eliminates S. boulardii if taken together. A gap of 4–6 hr between intake of antifungal agent (Nystatin) and S. boulardii was found to have no effect on survival of probiotic yeast [23]. Various effects of yeast probiotics on human host have been presented below:

11.4.1 Effect on Enteric Pathogens S. boulardii directly interacts with gut microbiota and exerts strong antagonistic effect against a number of enteric pathogens like C. albicans, E. coli, Shigella, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, C. difficile, Vibrio cholerae, and Entamoeba histolytica. It has also been reported to alter the Helicobacter pylori structure [22]. S. boulardii can exert its beneficial effect against various enteric pathogens by two main mechanisms: (i) production of factors that neutralized bacterial toxins, and (ii) modulation of the host cell signaling pathway implicated in pro inflammatory response during bacterial infection.

11.4.2

Neutralization of Bacterial Toxins

S. boulardii helps with neutralisation of bacterial toxins. Initially the antitoxin action of S. boulardii was demonstrated in cases of C. difficile infection. The antitoxin action is mediated by production of two enzymatic proteins of 120 and 54 kDa respectively. 120 kDa protein help in proteolysis of C. difficile toxin A and its receptor, while the 54 kDa protease inhibits the binding of toxin A and B to their membrane receptors, thereby checking the enterotoxic and cytotoxic effects caused by C. difficile [24–26]. Additionally, 120 kDa protein of S. boulardii has also been reported to check the metabolic changes in intestinal mucosa, caused by Vibrio cholera. Yet another antitoxin factor produced by S. boulardii has been described in cases of cholera toxin (CT), where 120 kDa protein was found to inhibit the CT-stimulated chloride secretion by reducing the formation of cyclic AMP. Dephosphorylation of endotoxins such as LPS from E. coli O55B5 has also been reported by the probiotic strain of S. boulardii [22].

11.4.3 Modification of Host Cell Signalling Enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) shares a common pathogenic mechanism, where pathogenic E. coli adhere to mucosal surface and subsequently leads to changes in the integrity of tight-junction permeability and activation of signaling pathways (mitogen activated protein kinase (MAPK) and the transcription factor NF-κB) that stimulated IL-8 synthesis. It has been reported that S. boulardii inhibits the phosphorylation of the myosin light chain (MLC) which are associated with a cytoskeletal protein involved in intercellular tight-junctions control. S. boulardii has also been reported to modify host cell signalling and prevent apoptosis and synthesis of tumor necrosis factor alpha (TNFα) during EPEC– or EHEC infection [27, 28].

Saccharomyces - Eukaryotic Probiotic for Human Applications 219

11.4.4

Trophic Effect on Intestinal Mucosa

S. boulardii has been reported to exert trophic effects when ingested. It stimulates the expression of enzymes involved in nutrient digestion such as sucrase and leucine aminopeptidase [29]. D-glucose absorption could also be improved by S. boulardii. It induces and stimulates the production of glycoproteins (hydrolases, transporters, secretory IgA, receptor for polymeric immunoglobulin) and polyamines (spermidine, spermine, and putrescine) in brush borders of microvilli [22]. Buts et al., [29] have indicated the production of polyamines as S. boulardii’s mediator of trophic effect.

11.4.5 Anti-Inflammatory Effect S. boulardii also possess anti-inflmmatory and immunological effects [30]. Antiinflammatory property is mediated by decrease in the secretion of proinflammatory cytokine IL-8 by inhibiting MAP kinase and NF-κB signal transduction pathways [31]. Additionally, it has been found that S. boulardii (i) inhibits dendritic cell-induced activation of naive T-Cells and, (ii) migration of lymphocytes [22]. S. boulardii has also been found to protect from histological damage, suppress NF-κBactivation, and inhibit proinflammatory cytokine gene expression. A small (