Biotechnology of Bioactive Compounds: Sources and Applications [1 ed.] 1118733495, 978-1-118-73349-3, 0471926914, 9780471926917

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Biotechnology of Bioactive Compounds

Biotechnology of Bioactive Compounds Sources and applications EditorS

Dr. Vijai Kumar Gupta Lead researcher, Molecular Glycobiotechnology Group, department of Biochemistry, School of Natural Sciences, National University of ireland Galway, Galway, ireland

Dr. Maria G. Tuohy Lecturer and Head, Molecular Glycobiotechnology Group, department of Biochemistry, School of Natural Sciences, National University of ireland Galway, Galway, ireland

Co-EditorS

Dr. Mohtashim Lohani Assosciate Professor, department of Biotechnology, integral University Lucknow (UP), india

Dr. Anthonia O’Donovan Lead researcher, Molecular Glycobiotechnology Group, department of Biochemistry, School of Natural Sciences, National University of ireland Galway, Galway, ireland

this edition first published 2015 © 2015 by John Wiley & Sons, Ltd Registered Office John Wiley & Sons, Ltd, the Atrium, Southern Gate, Chichester, West Sussex, Po19 8SQ, UK Editorial Offices 9600 Garsington road, oxford, oX4 2dQ, UK the Atrium, Southern Gate, Chichester, West Sussex, Po19 8SQ, UK 111 river Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell. the right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, designs and Patents Act 1988. 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 the UK Copyright, designs and Patents Act 1988, without the prior permission of the publisher. designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. the publisher is not associated with any product or vendor mentioned in this book. Limit of Liability/disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. it is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. if professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Biotechnology of bioactive compounds : sources and applications / [edited by] dr. Vijai K. Gupta, dr. Maria tuohy, dr. Anthonia o’donovan, dr. Mohtashim Lohani. pages cm includes index. iSBN 978-1-118-73349-3 (cloth) 1. Pharmaceutical biotechnology. 2. Food–Biotechnology. 3. Bioactive compounds–Biotechnology. i. Gupta, Vijai Kumar, editor. ii. tuohy, Maria G., editor. iii. o’donovan, Anthonia, editor. iV. Lohani, Mohtashim, editor. tP248.65.F66B639 2015 664–dc23 2014037008 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Cover image: iStock © ilbusca Set in 9.5/13pt Meridien by SPi Publisher Services, Pondicherry, india

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2015

Contents

List of contributors, ix Foreword, xvii Preface, xix

Section I: Bioactive compounds from diverse plant, microbial, and marine sources 1 Bioactive compounds from vegetable and fruit by-products, 3 B. De Ancos, C. Colina-Coca, D. González-Peña, and C. Sánchez-Moreno 2 Bioactive compounds in fresh-cut fruits: occurrence and impact

of processing and cold storage, 37 María Elida Pirovani, Andrea Marcela Piagentini, and Franco Van de Velde 3 Pressurized hot water extraction of polyphenols from plant material, 63

José Rodrigo Vergara-Salinas, José Cuevas-Valenzuela, and José R. Pérez-Correa 4 Bioactive compounds in cereals: technological and nutritional

properties, 103 Bartłomiej Makowski, Justyna Rosicka-Kaczmarek, and Ewa Nebesny 5 Antimicrobials from medicinal plants: research initiatives, challenges,

and the future prospects, 123 Anita Pandey and Vasudha Agnihotri 6 Coccoloba uvifera as a source of components with antioxidant activity, 151

Maira Segura Campos, Jorge Ruiz Ruiz, Luis Chel Guerrero, and David Betancur Ancona 7 Bioactive compounds and medical significance of some endangered

medicinal plants from the Western Ghats region of india, 163 Manoharan Melvin Joe, Abitha Benson, Muniappan Ayyanar, and Tongmin Sa 8 Fungal bioactive compounds: An overview, 195

Gerardo Díaz-Godínez 9 Arbuscular mycorrhizal fungi: Association and production of bioactive

compounds in plants, 225 Marcela C. Pagano and Partha P. Dhar

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Contents

10 Extremophiles as source of novel bioactive compounds with industrial

potential, 245 Mohamed Neifar, Sameh Maktouf, Raoudha Ellouze Ghorbel, Atef Jaouani, and Ameur Cherif 11 New trends in microbial production of natural complex bioactive

isoprenoids, 269 Rama Raju Baadhe, Ravichandra Potumarthi, Naveen Kumar Mekala, and Vijai K. Gupta 12 Production of C-phycocyanin and its potential applications, 283

Mohammed Kuddus, Poonam Singh, George Thomas, and Athar Ali

Section II: Chemistry, biotechnology, and industrial relevance 13 Glycosides: From biosynthesis to biological activity toward therapeutic

application, 303 Maria Henriques L. Ribeiro 14 trehalose mimics as bioactive compounds, 345

Davide Bini, Antonella Sgambato, Luca Gabrielli, Laura Russo, and Laura Cipolla 15 Virtual screening and prediction of the molecular mechanism

of bioactive compounds in silico, 371 Bashir A. Akhoon, Krishna P. Singh, Madhumita Karmakar, Suchi Smita, Rakesh Pandey, and Shailendra K. Gupta 16 Steroids in natural matrices: Chemical features and bioactive

properties, 395 João C.M. Barreira and Isabel C.F.R. Ferreira 17 Bioactive compounds obtained through biotechnology, 433

Gustavo Molina, Franciele M. Pelissari, Marina G. Pessoa, and Gláucia M. Pastore 18 Metabolic engineering of bioactive compounds in berries, 463

Ivayla Dincheva, Ilian Badjakov, and Violeta Kondakova 19 Food-derived multifunctional bioactive proteins and peptides:

Sources and production, 483 Dominic Agyei, Ravichandra Potumarthi, and Michael K. Danquah 20 Food-derived multifunctional bioactive proteins and peptides:

Applications and recent advances, 507 Dominic Agyei, Ravichandra Potumarthi, and Michael K. Danquah

Contents

Section III: Biochemistry and nutraceutical or health-related applications 21 An overview of the molecular and cellular interactions of some bioactive

compounds, 527 Amro Abd Al Fattah Amara 22 Bioactive compounds as growth factors and 3d matrix materials

in stem cell research, 555 Naveen Kumar Mekala, Rama Raju Baadhe, and Ravichandra Potumarthi 23 Phytosterols: Biological effects and mechanisms of hypocholesterolemic

action, 565 Rafaela da Silva Marineli, Cibele Priscila Busch Furlan, Anne y Castro Marques, Juliano Bicas, Gláucia Maria Pastore, and Mário Roberto Maróstica, Jr. 24 overview of the role of food bioactive compounds as complementary

therapy for celiac disease, 583 Antonio Cilla, Laia Alemany, Juan Antonio Giménez, and José Moisés Laparra 25 Bioactive lipid components from ruminant milk and meat:

the new face of human health, 599 Malgorzata Szumacher-Strabel, Mohamed El-Sherbiny, Adam Cieslak, Joanna Szczechowiak, and Hanna Winiarska 26 the milk fat globule membrane: A potential source of health-promoting

glycans, 631 Sarah A. Ross, Jonathan A. Lane, Michelle Kilcoyne, Lokesh Joshi, and Rita M. Hickey 27 Seaweed and milk derived bioactive peptides and small molecules

in functional foods and cosmeceuticals, 669 Maria Hayes, Melani García-García, Ciarán Fitzgerald, and Tomas Lafarga index, 693

vii

List of contributors

Vasudha Agnihotri, PhD Knowledge Products and Capacity Building, G. B. Pant institute of Himalayan Environment and development, Almora, Uttarakhand, india Dominic Agyei, PhD department of Chemical Engineering, Monash University, Clayton, Victoria, Australia Bashir A. Akhoon department of Bioinformatics, CSir – indian institute of toxicology research, Lucknow, india Athar Ali, PhD department of Biotechnology, Jamia Hamdard, New delhi, india Amara A. Amro, PhD department of Protein research, Genetic Engineering and Biotechnology research institute, City for Scientific research and technology Applications, Egypt David Betancur Ancona, PhD Facultad de ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán, México Muniyappan Ayyanar, PhD department of Botany and Microbiology, AVVM Sri Pushpam College, Poondi – 613503, thanjavur, tamil Nadu, india. Rama Raju Baadhe, PhD department of Biotechnology, National institute of technology, Warangal, india Ilian Badjakov, PhD AgroBioinstitute, Sofia, Bulgaria João C. M. Barreira, PhD Centro de investigação de Montanha (CiMo), ESA, instituto Politécnico de Bragança, Bragança, Portugal Abitha Benson, PhD department of Plant Biotechnology, School of Biosciences and technology, Vit University, Vellore, india

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List of contributors

Juliano Bicas, PhD department of Chemistry, Biotechnology and Bioprocess Engineering, Federal University of São João del-rei, Minas Gerais State, Brazil; department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil Davide Bini, MSc department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, italy Maira Segura Campos, PhD Facultad de ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán, México Adam Cieslak, PhD Poznan University of Life Sciences, department of Animal Nutrition and Feed Management, Poland Antonio Cilla, PhD Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia, Valencia, Spain Laia Alemany, PhD Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia, Valencia, Spain Laura Cipolla, PhD department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, italy C. Colina-Coca, BSc Quality and Safety, institute of Food Science, technology and Nutrition (iCtAN), Spanish National research Council (CSiC), Madrid, Spain José Cuevas-Valenzuela, PhD department of Chemical and Bioprocesses Engineering, ASiS, Anillo de Ciencia y tecnología ACt1105, Pontificia Universidad Católica de Chile, Santiago, Chile Michael K. Danquah, PhD department of Chemical Engineering, Monash University, Clayton, Victoria, Australia; department of Chemical Engineering, Curtin University of technology, Sarawak, Malaysia B. De Ancos, PhD department of Characterization, Quality and Safety, institute of Food Science, technology and Nutrition (iCtAN), Spanish National research Council, Madrid, Spain

List of contributors

xi

Partha P. Dhar, PhD Plant Production department, College of Food and Agricultural Sciences, King Saud University, Saudi Arabia Gerardo Díaz-Godínez, PhD Laboratory of Biotechnology, research Center for Biological Sciences, Universidad Autónoma de tlaxcala, tlaxcala, Mexico Ivayla Dincheva, MSc AgroBioinstitute, Sofia, Bulgaria Mohamed El-Sherbiny, MSc Poznan University of Life Sciences, department of Animal Nutrition and Feed Management, Poland; National research Centre, department of dairy Sciences, Cairo, Egypt Isabel C. F. R. Ferreira, PhD Centro de investigação de Montanha (CiMo), ESA, instituto Politécnico de Bragança, Bragança, Portugal Ciarán Fitzgerald, PhD Food BioSciences department, teagasc Food research Centre, dublin, ireland Cibele Priscila Busch Furlan, MSc department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil Luca Gabrielli, PhD department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, italy Melani García-García, MSc Food BioSciences department, teagasc Food research Centre, dublin, ireland Raoudha Ellouze Ghorbel, PhD Unité Enzymes et Bioconversion, Ecole Nationale d’ingénieurs de Sfax, Université de Sfax, Sfax, tunisia Juan Antonio Giménez, PhD department of Chemistry and Biodynamics of Food, institute of Animal reproduction and Food research of the Polish Academy of Sciences, olsztyn, Poland D. González-Peña, BSc department of Characterization, Quality and Safety, institute of Food Science, technology and Nutrition (iCtAN), Spanish National research Council (CSiC), Madrid, Spain

xii

List of contributors

Luis Chel Guerrero, PhD Facultad de ingeniería Química, Universidad Autónoma de Yucatán, Mérida, Yucatán, México Shailendra K. Gupta, PhD department of Bioinformatics, CSir – indian institute of toxicology research, Lucknow, india; department of Systems Biology and Bioinformatics, University of rostock, Germany Vijai K. Gupta, PhD Molecular Glycobiotechnology Group, department of Biochemistry, School of Natural Sciences, National University of ireland Galway, Galway, ireland Maria Hayes, PhD Food BioSciences department, teagasc Food research Centre, dublin, ireland Rita M. Hickey, PhD teagasc Food research Centre, Cork, ireland Atef Jaouani, PhD Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de tunis, Campus Universitaire, tunis, tunisia Manoharan Melvin Joe, PhD department of Microbiology, School of Life Science VELS University, Pallavaram, Chennai- 600117, tamilnadu, india Lokesh Joshi, PhD Glycoscience Group, National Centre for Biomedical Engineering Science, National University of ireland, Galway, ireland Madhumita Karmakar department of Bioinformatics, CSir – indian institute of toxicology research, Lucknow, india Michelle Kilcoyne, PhD Glycoscience Group, National Centre for Biomedical Engineering Science, National University of ireland, Galway, ireland Violeta Kondakova, PhD AgroBioinstitute, Sofia, Bulgaria Mohammed Kuddus, PhD department of Biochemistry, College of Medicine, University of Hail, Hail, Saudi Arabia

List of contributors

xiii

Tomas Lafarga, PhD Food BioSciences department, teagasc Food research Centre, dublin, ireland Jonathan A. Lane, PhD teagasc Food research Centre, Cork, ireland José Moisés Laparra, PhD Microbial Ecology and Nutrition research Group, institute of Agrochemistry and Food technology, National research Council (iAtA-CSiC), Valencia, Spain Bartłomiej Makowski, MSc institute of Chemical technology of Food, technical University of Łódź, Poland Sameh Maktouf, PhD Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Sfax, Tunisia Rafaela da Silva Marineli, MSc Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil Mário Roberto Maróstica, Jr., PhD Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil Anne y Castro Marques, PhD Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil Naveen Kumar Mekala, PhD Department of Biotechnology, National Institute of Technology Warangal, India Gustavo Molina, PhD Laboratory of Bioflavors, Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, Brazil; Institute of Science and Technology, Food Engineering, University of Jequitinhonha and Mucuri, Diamantina, Brazil Ewa Nebesny, PhD Institute of Chemical Technology of Food, Technical University of Łódź, Poland Mohamed Neifar, PhD Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Campus Universitaire, Tunis, Tunisia

xiv

List of contributors

Marcela C. Pagano, PhD Departamento de Física, Federal University of Minas Gerais, Belo Horizonte, Brazil Anita Pandey, PhD Biotechnological Applications, G. B. Pant Institute of Himalayan Environment and Development, Almora, Uttarakhand, India Rakesh Pandey Central Institute of Medicinal and Aromatic Plants, Lucknow, India Gláucia M. Pastore, PhD Laboratory of Bioflavors, Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, Brazil Franciele M. Pelissari, PhD Institute of Science and Technology, Food Engineering, University of Jequitinhonha and Mucuri, Diamantina, Brazil José R. Pérez-Correa, PhD Department of Chemical and Bioprocesses Engineering, ASIS, Anillo de Ciencia y Tecnología ACT1105, Pontificia Universidad Católica de Chile, Santiago, Chile Marina G. Pessoa, PhD Laboratory of Bioflavors, Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, Brazil Andrea Marcela Piagentini Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química, Universidad Nacional del Litoral, Santa Fe, Argentina María Elida Pirovani, PhD Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química, Universidad Nacional del Litoral, Santa Fe, Argentina Ravichandra Potumarthi, PhD Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia Maria Henriques L. Ribeiro, PhD Research Institute for Medicines, Faculdade de Farmácia, Universidade Lisboa, Lisbon, Portugal Justyna Rosicka-Kaczmarek, PhD Institute of Chemical Technology of Food, Technical University of Łódź, Poland

List of contributors

xv

Sarah A. Ross, BSc Teagasc Food Research Centre, Cork, Ireland; Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland Jorge Ruiz Ruiz, BSc Departamento de Ingeniería Química-Bioquímica, Instituto Tecnológico de Mérida, Mérida, Yucatán, México Laura Russo, PhD Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy Tongmin Sa, PhD Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea C. Sánchez-Moreno, PhD Quality and Safety, Institute of Food Science, Technology and Nutrition (ICTAN), Spanish National Research Council, Madrid, Spain Antonella Sgambato, MSc Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy Krishna P. Singh Department of Bioinformatics, CSIR – Indian Institute of Toxicology Research, Lucknow, India Poonam Singh, PhD Department of Molecular and Cellular Engineering, SHIATS, Allahabad, India Suchi Smita, PhD Society for Biological Research and Rural Development, Lucknow, India; Department of Systems Biology and Bioinformatics, University of Rostock, Germany Joanna Szczechowiak, MSc Poznan University of Life Sciences, Department of Animal Nutrition and Feed Management, Poland Malgorzata-Szumacher-Strabel, PhD Poznan University of Life Sciences, Department of Animal Nutrition and Feed Management, Poland

xvi

List of contributors

George Thomas, PhD Department of Molecular and Cellular Engineering, SHIATS, Allahabad, India Franco Van de Velde, PhD Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química, Universidad Nacional del Litoral, Santa Fe, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Santa Fe, Argentina José Rodrigo Vergara-Salinas, PhD Department of Chemical and Bioprocesses Engineering, ASIS, Anillo de Ciencia y Tecnología ACT1105, Pontificia Universidad Católica de Chile, Santiago, Chile Hanna Winiarska, PhD Poznan University of Medical Sciences, Department of Pharmacology, Poland Ameur Cherif, PhD LR11-ES31 Biotechnology and Bio-Geo Resources Valorization, Higher Institute for Biotechnology, Biotechpole Sidi Thabet, University of Manouba, 2020 Ariana, Tunisia

Foreword

Industrial biotechnology encompasses the application of biotechnology-based tools for traditional industrial processes and the manufacturing of bio-based products from renewable feedstocks. Microorganisms, enzymes, and plantderived compounds form the basis of a suite of technologies and processes that a diverse group of companies, researchers, and scientists are seeking to develop for commercial use. The World Economic Forum estimated that by 2020 the market for biofuels, bio-based bulk chemicals and plastics, and bioprocessing enzymes will approach $95 billion. The possibility of utilization of microorganisms or vegetables for obtaining molecules’ potential in many industrial sectors can place any nation in a prominent position on the international scene. This theme currently has received great attention in science, technology, and innovation policies as fundamental to the future development of a country. One of the more concrete possibilities of use of microbial and vegetable biodiversity in sustainable development of a country is in the agricultural, food, and pharmaceutical sectors, which could put the country in a relevant world position in the production of bioactive compounds in a sustainable way. In this scenario, the use of biomolecules is highlighted as a sustainable practice to be encouraged. The wide biodiversity of the planet yet unexplored can be an infinite source of bioactive compounds with industrial applications. Some examples of applications are related to the use of microorganisms and microalgae for production of biomolecules, mainly for the pharmaceutical and food industries. Plants, fruits, and residues of fruit-processing industries can be a source of many industrial compounds. This book describes the current stage of knowledge of production of bioactive compounds from microbial, algal, and vegetable sources. In addition, the molecular approach for screening bioactive compounds is also discussed as well examples of applications of these compounds on human health. Marcio Anthonio Mazutii Department of Chemical Engineering, Federal University of Santa Maria, Santa Maria, Brazil

xvii

Preface

Since time immemorial, natural products have not only been used as dyes and colors all over the world, but they have also made a tremendous contribution through pharmacognosy to a myriad of drugs currently used in medicine. Today bioactive compounds are central in high-value product development in the chemical industry. Bioactive compounds have been identified from diverse sources and their therapeutic benefits, nutritional value and protective effects in human and animal healthcare have underpinned their application as pharmaceuticals and functional food ingredients. The end of the 19th century witnessed a decline in the level of interest in natural products, mainly due to the escalating expectations of scientists and industries regarding the identification of new compounds and their potential synthesis. However, shortly afterward, concern for the environment through the excessive use of synthetic chemicals, along with the growing need to search for new and better drugs, provided a fresh impetus for the analysis of bioactive compounds and their sources, with the focus again being on their pharmacological application, either directly or as synthetic derivatives of parent natural compounds. This book considers recent developments in the field of pharmacognosy, especially the interdisciplinary efforts to identify new sources of bioactive compounds and biological applications. The orderly study of biologically active products and the exploration of potential biological activities of these secondary metabolites, including their clinical applications, standardization, quality control, mode of action, and potential biomolecular interactions, has emerged as one of the most exciting developments in modern natural medicine. This book provides specific examples of current research and development in natural products biochemistry. Information on diverse sources of bioactive compounds, ranging from microorganisms and algae to plants and dietary foods (Chapters 1–12), as well as selected bioactivities and biotechnological and biomedical potential reviewed in Chapters 13–27. The bioactive compounds profiled include compounds such as C-phycocyanins, glycosides, phytosterols, and natural steroids. An overview of the usage of bioactive compounds as antioxidants, anti-inflammatories, and antiallergics and in  stem cell research is presented (Chapters 21–27), with an overview provided in Chapter 21 on overall medicinal applications of plant-derived compounds.

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Preface

This book has been designed to be an informative text for undergraduate and graduate students of biomedical chemistry who are keen to explore the potential of bioactive natural products and also provides useful information for scientists working in various research fields where natural products have a primary role. Vijai Kumar Gupta and Maria G. Tuohy

SECTION I

Bioactive compounds from diverse plant, microbial, and marine sources

CHAPTER 1

Bioactive compounds from vegetable and fruit by-products B. De Ancos, C. Colina-Coca, D. González-Peña, and C. Sánchez-Moreno Department of Characterization, Quality and Safety, Institute of Food Science, Technology and Nutrition (ICTAN), Spanish National Research Council (CSIC), Madrid, Spain

1.1 Introduction Consumers’ contemporary eating habits are changing in consonance with 21stcentury lifestyles and as a result of better understanding of the effects of food on health and quality of life. Nowadays, there is increasing demand on the part of consumers for fresh or processed fruits and vegetables, mainly as juices, canned, frozen, or minimally processed products (fresh-cut, easy-to-eat, or easy-toprepare), among other products, that are microbiologically safe, and at the same time offering biological properties beyond nutritional factors. Numerous studies have demonstrated that phytochemicals in fruits and vegetables are the major bioactive compounds with human health benefits. Another key feature that consumers are demanding is food without synthetic additives because the synthetic molecules are suspected to cause or promote negative health effects. In line with the present tendency to consume healthy, safe foods free of synthetic additives, consumers demand natural ingredients and additives capable of not only maintaining the initial quality of food, but at the same time providing healthy properties (reducing the risk of disease) that go further than nutritional requirements. It has been evident that the consumption of food rich in phytochemicals, as well as food enriched in them, ensures the desirable antioxidant status and helps in prevention of development degenerative diseases. Moreover, the processing of fruits and vegetables produces high amounts of by-products such as peels, seeds, stones, residual pulp, discarded whole pieces, etc., rich in phytochemical compounds (phenolic compounds, carotenoids, dietary fiber, vitamin  C, minerals, etc.) that can be used as a low-cost source to obtain functional ingredients.

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Bioactive compounds from vegetable and fruit by-products

1.2 Beneficial health effects obtained by consuming vegetable and fruit products rich in phytochemicals In recent years, an increasing consumption of vegetables and fruits in the diet has been observed, due to the fact that the consumer has a greater knowledge of the beneficial properties obtained. Numerous epidemiological studies have shown a direct relationship between a diet rich in fruits and vegetables and lower incidence of degenerative diseases such as certain types of cancer, cardiovascular diseases, macular degeneration, aging, and others (Liu et  al. 2000; Michels et al. 2000; Kris-Etherton et al. 2002; trichopoulou et al. 2003; Willcox et al. 2003; dauchet et al. 2006; ordovás et al. 2007; Liu 2013). this effect has been attributed to the presence of certain compounds in the food owing to determined biological activities related to health benefits known as bioactive or phytochemical compounds (Liu 2013). the biological activity of these compounds (dietary fiber, carotenoids, phenols, vitamins A, C, and E, glucosinolates, organosulphur compounds, sesquiterpenic lactones, etc.) has been studied by means of in vitro, in vivo, and human intervention studies. in general, phytochemicals could be defined as chemical substances that can be found in vegetable products, giving them physiological properties beyond the nutritional considerations. the beneficial mechanisms resulting from the consumption of fruits and vegetables are as yet not well known. they seem to be related to synergistic or additive interactions between the phytochemicals that could affect different pathways such as modulation of steroid hormone concentration and detoxifying enzymes; reduction of plaque aggregation and blood pressure; changes in cholesterol and hormone metabolism, antioxidant, antiviral, and antibacterial activity; stimulation of the immune response; reduction of inflammatory processes; antimutagenic and anticarcinogenic properties; and prevention and delay of cardiovascular diseases (Liu 2013; Yu and Ahmedna 2013). it is a fact that fruits and vegetables can be processed for economical and logistical reasons in order to improve their commercial shelf-life and digestibility, in accordance with the consumer habits of each country or to facilitate the consumption by special groups (children, pregnant women, older adults, patients with certain pathologies, etc.). in addition to traditional thermal processing—such as frozen, canned, or pasteurized vegetable products, etc.—there is growing interest in the development of new processing systems that minimally modify or improve the nutritional and health properties related to the consumption of fruits and vegetables. Among these new food processing technologies, researchers, industrialists, and distributors have been focused on the development of minimal processing technologies for producing vegetable products with minimally modified sensorial and nutritional characteristics such as “fresh-cut vegetables” and “ready-to-eat processed vegetables” (González-Aguilar et al. 2005; oms-oliu et al. 2010; Artes and

Bioactive compounds from vegetable and fruit by-products

Canned 8%

Frozen 6%

5

Others 2%

Fresh-cut 4%

Fresh 80%

Figure 1.1 distribution of vegetable products consumed in Europe.

Allende  2005). Figure  1.1 shows the distribution of vegetable products more frequently consumed in Europe. At present, Europeans preferably consumed fresh vegetable products (80%), and only 20% of vegetable production is consumed processed, mainly canned (8%), frozen (6%), or fresh-cut products (4%).

1.3 By-products from vegetable and fruit processing to obtain phytochemicals there are important food mass losses throughout the supply chain that lead to edible food for human consumption. Food losses take place at the production, postharvest, and processing stages in the food supply chain. Food losses occurring at the end of the food chain (retail and final consumption) are called “food waste,” which relates to retailers’ and consumers’ behavior (Parfitt et al. 2010). Fruit and vegetable processing generates large quantities of solid and liquid waste such us peels, seeds, stones, fruit pomace from the juice industry, and blanching liquid from the frozen vegetable industry, among others. Figure 1.2 shows the percentage of the initial weight of fruits and vegetables that are discarded at each phase of plant food chain in different regions. it is noteworthy that in most countries, the percentage of the initial weight of vegetables and fruits that are lost or discarded is over 40%, becoming more than 50% in less industrialized countries (sub-Saharan Africa, North Africa, Central and East Asia, etc.). the losses in agricultural production between 15 and 20% dominate for industrialized regions (Europe, North America, etc.), mostly due to postharvest fruit and vegetable grading caused by quality standards set by retailers. Waste at the end of the food supply chain is also substantial, with 15–30% of purchases mass discarded by consumers (Gustavsson et al. 2011). in developing regions, losses in agricultural production dominate total losses throughout the food supply chain. Losses during postharvest and distribution stages are also

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Bioactive compounds from vegetable and fruit by-products

Fruit and vegetable losses in food chain 60% 50% 40% 30% 20% 10% 0%

Europe

North America

Consumption

Asia Africa SubIndustrialized Saharan Distribution

North Africa South & & Southeast East Central Asia Asia

Processing

Postharvest

Latin America

Agriculture

Figure 1.2 Percentage of fruit and vegetable production discarded at different phases of the plant food chain in different regions.

severe, which can be explained by deterioration of perishable crops in the warm and humid climate of many developing countries, as well as by seasonality that leads to unsaleable gluts (FAO 2011). Every year in Europe there are more than 190 million tons of food losses and wastes from fruit and vegetable processing, which are responsible for 20–30% of the negative environmental impact that industrialized countries account for. Discarded fruit and vegetable peels, seeds, stones, or whole pieces are considered waste if they are not used as sources of phytochemicals or other valuable products. If this waste is used for obtaining phytochemicals, then it is considered as fruit and vegetable processing by-products. Figure  1.3 shows the flow diagram of most usual vegetable processing. this diagram shows that the first stages (1–7) are similar for the majority of processed vegetables (canned, frozen, minimally processed, etc.), and also they are the processing steps that generate the largest amount of solid by-products. thus, in step 4, “selection and classification,” whole product damaged either by mechanical injury or fungal attack is removed, and also the pieces that do not have the characteristics of size and maturity required by commercial-quality parameters are discarded. Also, in step 6, “elimination of not edible fraction,” roots, leaves, pods of green peas and beans, dry outer layers of onion or garlic, outer leaves of lettuce, artichoke, or corn cobs, among others, are removed. the weight of discarded product is quite variable depending on the type of vegetable. For example, in the case of the artichoke and celery, the waste generated can be approximately 50–60% of the initial fresh weight of the vegetable, reaching 75% in the processing of peas (Larrosa et al. 2002). Finally, in step 7, “peeling and cutting,” peels, seeds, and stones are discarded, generating by-products with a high phytochemical concentration (Ayala-Zavala et al. 2011).

Bioactive compounds from vegetable and fruit by-products

1. Harvesting, precooling, and cold transport

By-products

7

2. Raw material reception

3. Cold storage

4. Selection and classification

By-products

5. Washing whole vegetable

6. Elimination not edible fraction

By-products

7. Peeling and cutting

By-products

a. Fresh-cut

8a. Washing and disinfection

9a. Draining and centrifuging

10a. Weighing and modified atmosphere packaging (MAP) 11a. Storage, distribution, and retail at refrigeration temperature

b. Canned 8b. Blanching, cooling, and draining 9b. Weighing, and packaging (or aseptic packaging)

c. Frozen

8c. Blanching, cooling, and draining 9c. Freezing

10b. Sterilization (or pasteurization)

10c. Weighing and packaging

11b. Storage, distribution, and retail at room temperature (or refrigeration)

11c. Storage, distribution, and retail at frozen temperature

Figure 1.3 Vegetable processing flow diagram.

Fruit juices are the main processed fruit product commercialized in the whole world. Practically 40% of citrus fruit world production is processed as juices in different forms, mainly obtained from concentrate or by direct extraction, and stored at room temperature or under refrigeration (Figure  1.4). Processing of citrus fruit produced great amounts of by-products such as pulp, seeds, or fruit pomace. Figure 1.4 shows the flow diagram for obtaining juice from other types of fruits such as apples, stone fruit, grapes, or wild fruits that also produce large amounts of such by-products as peels, seeds, and stones. From an economic point of view, the food losses produced during fruit and vegetable processing is an important problem because the cost of their management influences the final price of the product, which significantly increases due to that activity. Furthermore, disposal of this material is a serious problem as there are significant legal requirements related to its manipulation. Environmental legislation in the European Union is demanding for the treatment of waste,

8

Bioactive compounds from vegetable and fruit by-products

1. Harvesting, precooling,

2. Raw material reception

and cold transport By-products

3. Cold storage

4. Selection and classification

Citric fruits

5. Washing whole fruits

By-products

By-products

Other fruits 6. Elimination not edible fraction

6. Juice extraction By-products

7. Crushing (fruit puree) 7. Centrifuging and filtering (separation of floating pulp)

a. Juice from concentrate 8a. Concentration/evaporation and cooling (65° brix) 9a. Frozen storage of concentrated juice (–20°C)

b Juice not from concentrate 8b. Pasteurization and cooling

9b. Cold storage

8. Thermal treatment (enzymatic inactivation) 9. Pressing

10a. Centrifugation

10b. Enzymatic treatment, centrifugation

Cloudy juice 10a. Reconstitution, thermal

10b. Pasterurization and

11. Pasterurization and

treatment, aseptic packaging

aseptic packaging

aseptic packaging

11b. Storage, distribution, and

12. Storage, distribution, and

retail at room temperature

retail at room temperature

(or refrigeration)

(or refrigeration)

11a. Storage, distribution, and retail at room temperature

Clarified juice

Figure 1.4 Fruit processing flow diagram.

according to Directive 2006/12/EC that was subsequently improved in Directive 2008/98/EC. This Directive establishes the obligation of member states to implement measures to reduce industrial waste by recycling, recovery, and reuse as an energy source or other procedures to extract valuable raw materials. The total or partial recovery of these wastes or by-products produced during fruit and vegetable processing or in other steps of the food supply chain involves significant advantages in economic, social, and environmental considerations. Generally, these products have been reused as animal feed, compost for farmland, or in biomass used in the production of fuels such as bioethanol (Lenucci et al. 2013). At present, producers and the food industry are looking for innovative ways of using these wastes as by-products for further exploitation on the production of additives or supplements with high nutritional and functional value. one of these innovative research lines has revealed agro-industrial by-products as a major source of phytochemicals that can be used as natural additives and functional ingredients in the formulation of new foods. therefore, recovery and recycling of these by-products could be economically attractive to industries.

Bioactive compounds from vegetable and fruit by-products

9

There are several reviews that summarize the published studies related to obtaining phytochemicals from agro-industrial by-products and their application in the design of new functional foods. thus, Schieber et al. (2001) conducted one of the first reviews on the utility of fruit and vegetable processing by-products as inexpensive raw material for the production of phytochemicals with high nutritional value and potential as antioxidants (vitamin C, phenolic compounds, carotenoids, tocopherols, and minerals) (Moure et al. 2001). Since then numerous studies have been published on obtaining phytochemicals from fruit and vegetable processing by-products (djilas et  al. 2009; Aguedo et  al. 2012; Kalogeropoulos et  al. 2012; o’Shea et  al. 2012; Wijngaard et  al. 2012; Yu and Ahmedna 2013). At present, increased consumption of tropical fruits and derivatives (juices, purees, canned, fresh-cut, etc.), due to their high nutritional value and beneficial health properties, has made the processing of these fruits generate a large amount of by-products rich in phytochemicals, which can be used as natural additives with different activities (antioxidants, antibrowning, antimicrobials, colorants, texturizers, etc.) (Ayala-Zavala et al. 2011; Correia et al. 2012). one of the major bioactive compounds obtained from agro-industrial byproducts are polyphenols with antioxidant and anti-inflammatory properties (Larrosa et al. 2002; Balasundram et al. 2006; djilas et al. 2009; Correia et al. 2012; Yu and Ahmedna 2013). Polyphenols are characterized by having at least one aromatic ring with one or more than one hydroxyl group attached. Polyphenols are generally classified into classes and subclasses based on their chemical structures (Crozier et  al. 2009). Four major classes of polyphenols found in fruit and vegetable by-products are phenolic acids, flavonoids, lignans, and stilbenes. Phenolic acids are divided into hydroxybenzoic and hydroxycinnamic acids. the hydroxycinnamic acids are more common than hydroxybenzoic acids, and they mainly include gallic acid, p-coumaric acid, caffeic and chlorogenic acids, and also ferulic and sinapic acids. these acids are rarely found in free form and are usually extracted as glycosylated derivatives or esters of quinic acid, shikimic acid and tartaric acid. Flavonoids are the most numerous of the phenolic compounds in plant products and are divided in several subclasses: flavonols, flavones, flavan-3-ols, flavanones, anthocyanidins and isoflavones, and other minor components of the diet such as coumarins or chalcones. the main dietary flavonols, kaempferol, quercetin, isorhamnetin, and myricetin, are most commonly found as O-glycosides. Also flavones such as apigenin and luteolin occur as 7-O-glycosides. Flavan-3-ols range from the simple monomers (+)-catechin and its isomer (−)-epicatechin, which can be hydroxylated to form gallocatechins and also undergo esterification with gallic acid, to complex structures including oligomeric and polymeric proanthocyanidins, which are also known as condensed tannins. the most common anthocyanidins are pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin, which are  invariably found as sugar conjugates known as anthocyanins. Flavanones are present in especially high concentrations in citrus fruits. the most common

Bioactive compounds from vegetable and fruit by-products

10

O

COOH HO

OH

O HO

OH HO

OH Gallic acid

OH

O

O

O

OH

O

OH

O

HO

OH

O

O OH

HO

OH

OH OH

OH

Chlorogenic acid

Narirutin OH OH

OH O

HO HO

O

O

OH

HO

O

OH OH

OH

O

Luteolin-7-O-glucoside

OH

OH

O

HO OH OH

O O HO

OH

OH Resveratrol

Quercetin-3-O-glucoside OH HO

OCH3 OH + O O

HO

OH OH

OH

OH

HO

HO OH

O

O

O

OCH3 OH OH

HO

O

OH OH

OH

n 4' OH

HO

8 8a O 2 6

4a

3 4

1' 2'

OH

OH

OH

Malvidin-3-O-glucoside

Proanthocyanidins

Figure 1.5 Major polyphenols found in vegetable and fruit processing by-products.

flavanone glycoside is hesperetin-7-O-rutinoside (hesperidin) along with narigenin-7-O-rutinoside (narirutin). Major stilbenoids found in foods of plant origin are resveratrol and its glucosides. Figure 1.5 shows the main polyphenols that can be extracted from fruit and vegetable processing by-products. Carotenoids consist of a conjugated backbone composed of four isoprene units forming a C40 carbon skeleton. there are two general classes of carotenoids: carotenes and xanthophylls. Carotenes consist only of carbon and hydrogen atoms; beta-carotene and lycopene, mainly present in carrots and tomatoes, are the most common carotenes. Xanthophylls have one or more oxygen atoms; lutein is one of the most common xanthophylls. Lycopene is the main carotenoid that can be obtained from fruit and vegetable processing by-products (Figure 1.6). these agro-industrial by-products also have a high content in dietary fiber with numerous beneficial physiological functions (Larrauri 1999; Garcia-Herrera et  al. 2010; Aguedo et  al. 2012). Soluble and insoluble dietary fiber can be extracted from fruit and vegetable by-products, the proportion between them being an important factor influencing the physiological function of this bioactive

Bioactive compounds from vegetable and fruit by-products

11

Lycopene

Figure 1.6 Major carotenoids found in vegetable and fruit processing by-products.

Beta-carotene

compound. Thus, insoluble dietary fiber mainly acts on the intestinal tract to produce mechanical peristalsis while soluble dietary fiber can also influence available carbohydrate and lipid metabolism. o’Shea et al. (2012) summarized a large part of the studies published about the use of by-products from fruit and vegetable processing to obtain dietary fiber and other phytochemicals that may be used as functional ingredients. in the following sections are described the main by-products obtained from the processing of vegetables and fruits used as raw material for the production of phytochemicals that can be used as functional ingredients.

1.4 Vegetable by-products 1.4.1 Lettuce (Lettuca sativa L.) Lettuce (Lactuca sativa L.) is an annual plant of the sunflower family Asteraceae. it is most often grown as a leaf vegetable, but sometimes for its stem and seeds. World production of lettuce in 2011 was 24.5 million tons and China was the first producer with 13.43 million tons. it is also notable that the lettuce production in North America and Europe was 4.13 and 2.55 million tons, respectively. Lettuce is most often used for salads, although it is also seen in other types of foods, such as in soups, sandwiches, and wraps; it can also be grilled. At present, in the context of the increasing demand from consumers for fresh-cut vegetables, lettuce salads account for 61% of all fresh-cut products commercialized in the world. Lettuce by-products are rich in phenolic compounds. Numerous epidemiological studies have shown that a diet high in plant foods rich in phenolic compounds decreases the risk of cardiovascular disease and cancer. the beneficial health effects of the consumption of foods rich in polyphenols are associated with their antioxidant and anti-inflammatory properties. Polyphenols’ properties result in the reduction of serum levels of cholesterol, triglycerides, fatty acids, and low-density lipoprotein (LdL) and the increase of high-density lipoprotein

12

Bioactive compounds from vegetable and fruit by-products

(HDL). Furthermore, they have a high capacity to inhibit cell proliferation, favoring the reduction of cell growth rate and apoptosis. The importance of phenolic compounds in the diet is enormous. The daily consumption of phenolic compounds in the Mediterranean countries is approximately 1100 mg/day, which is 10 times higher than the intake of vitamin C and 100 times greater than β-carotene and vitamin E, although this depends on cultural habits and family traditions (Crozier et al. 2009; Mitjavila and Moreno 2012). the phenolic composition of lettuce by-products varies with the lettuce variety as well as with the climatic conditions and agricultural practices used (frequency of irrigation, type of fertilizer, etc.). At present, there is a growing consumer demand for salads of a single variety of lettuce (iceberg, Batavia, trocadero, Lollo roso, oak leaf, or romaine), or mixtures of leaves of different varieties. there is also growing consumer demand for baby leaf salads, mainly lettuce or other leafy vegetables such as spinach, chard, watercress, and rocket salad. this increase in the consumption of lettuce salads has caused the amount of lettuce by-products to rise significantly. the by-products of lettuce processing are mainly outer leaves and stems. it is worth noting that the outer leaves of lettuce have a higher content of phenolic compounds than the inner leaves (Hohl et al. 2001). table  1.1 shows the phenolic composition of by-products obtained from lettuce processing, shown according to the main phenolic families, variety of lettuce, and solvent used in the extraction procedure (Llorach et al. 2004). Generally, the major phenolic fraction of lettuce is made up of caffeic acid derivatives (90%), mainly esterified with quinic acid (chlorogenic acid and isochlorogenic), tartaric acid (chicoric acid), and malic acid (Llorach et al. 2008). Table 1.1 Phenolic compounds from lettuce and escarole by-products. Variety (extractant) Romana (water) (methanol) Iceberg (water) (methanol) Baby (water) (methanol) Escarole (water) (methanol)

Total Phenols (µg/g fw)

Total Flavonols (µg/g fw)

Total Flavones (µg/g fw)

496.00 221.00

84.22 85.15

46.33 30.42

211.05 108.10

21.84 24.38

7.14 9.20

1088.00 1215.20

157.70 320.23

5.80 21.70

420.50 415.43

346.32 407.00

nd nd

fw, fresh weight; nd, not detected. Source: Llorach et al. (2004).

Bioactive compounds from vegetable and fruit by-products

13

Flavonoid compounds in lettuce leaves represent a minor fraction (5%) of the total phenolic compounds and are mainly composed by flavonols and flavones. The most common flavonols found in lettuce are derivatives of quercetin and kaempferol conjugated with glucose and rhamnose. Flavone compounds, principally luteolin derivatives, have also been identified. Additionally, anthocyanin compounds have been identified in the pigmented leaves of lettuce, mainly derivatives of cyanidin. Frequently, the anthocyanins in red lettuce are cyanidin-3-O-(6-malonylglucoside) and cyanidin-3-O-glucoside (Llorach et al. 2008). thus, the majority of flavonols in lettuce are quercetin derivatives such as quercetin-3-O-glucoside, quercetin-rutinoside, and quercetin-glucuronide. these compounds are present at low levels in green lettuce but are more abundant in red varieties. Kaempferol derivatives were found only in escarole as kaempferol-3-Oglucuronide and kaempferol-3-O-(6-O-malonylglucoside). Luteolin is a flavone rarely present in green lettuces but is more abundant in pigmented lettuces such as the Lollo roso variety, where it was found as luteolin-7-glucuronide, luteolin7-rutinoside, and luteolin-7-glucoside. therefore, all phenolic compounds that have been described in lettuce are also present in the by-products resulting from its processing. Although the concentration of phenolic compounds in lettuce is relatively low, the high consumption of this product in the majority of the countries makes it one of the major sources of phenolic compounds in the human diet.

1.4.2 Tomato (Solanum lycopersicum L.) tomato is the fruit of the plant Solanum lycopersicum L. that belongs to the Solanaceae family. tomato is consumed in many ways, either raw or as an ingredient in many dishes, sauces, purees, salads, and juices. tomato production is the fourth agricultural product in volume in the world after rice, wheat, and soybeans, with a production of 160 million tons (FAo 2011). tomatoes and processed tomato products, either sliced or in form of sauces, juices, or purees, have a high content of micronutrients (vitamin C and E, folate, and minerals), dietary fiber, and phytochemicals: phenolic compounds and mainly carotenoids such as lycopene and β-carotene (Sánchez-Moreno et al. 2008). the consumption of tomatoes and tomato products has been associated with a reduced risk of certain cancers such as prostate cancer (Giovannucci 2002). tomato product consumption also shows a high protective effect against cardiovascular diseases due to important antioxidant and antiplatelet activities as well as reduction of blood lipid levels (George et  al. 2004; Fuentes et  al. 2013). Health protection associated with the consumption of processed tomato products has been highlighted by numerous in vivo studies. these studies have shown the reduction of certain markers of lipid oxidation and inflammation such as the oxidation of LdL cholesterol and F2-isoprostanes (Burton-Freeman et  al. 2012). the protective effect of the consumption of processed tomato products has been primarily associated with the presence of lycopene

14

Bioactive compounds from vegetable and fruit by-products

(Giovannucci 2002), highly concentrated in processed tomato products and in their by-products, mainly in the peel (Chang et  al. 2006). Besides the peel, tomato processing leads to other different types of by-products such as discarded whole tomatoes, seed, and pulp. the phytochemical concentration of tomato by-products depends on different factors such as tomato variety, ripeness stage, and processing conditions. Generally, the production of juices, sauces, or tomato paste produces a solid residue consisting of 56% peels and 44% seeds (Schieber et al. 2001). the industrial yield of tomato-derived products can vary between 95 and 98% of the initial fresh weight; thus, if we consider an approximate average yield of 96%, the solid residue or by-product produced could be 4% of the raw tomato weight. it was found that tomato processing by-products—mainly peels, seeds, and pulp—have a phytochemical qualitative composition similar to the fresh tomato fruit. Generally, the peel is the by-product with the highest concentration of lycopene and phenolic compounds, while seeds also have phenolic compounds and a high content of unsaturated fatty acids, primarily linoleic acid (Schieber et al. 2001). the byproducts obtained in tomato paste processing (seed plus peel) has a similar amount of total phenolic compounds and antioxidant activity, measured according to dPPH and FrAP methodologies, than the raw whole tomato. Hydroxycinnamic acids such as caffeic and chlorogenic acids predominate in raw tomato, and tomato by-products are rich in flavonoids, mainly naringenin (87%) (Kalogeropoulos et al. 2012) (table 1.2). However, when the concentration of lycopene in the different parts of the tomato fruit was calculated in fresh

Table 1.2 Phytochemical compounds extracted from whole raw tomato and its corresponding by-product formed by peels and seeds. Compound Carotenoids (µg/g dw) Lycopene β-Carotene α-Tocopherol (µg/g dw) Sterols (µg/g ps) β-Sitosterol Stigmasterol Campesterol Polyphenols (µg/g dw) Hydroxycinnamic acids Phenolic acids Flavonoids Naringenin (% Flavonoids) dw, dry weight; ns, no significant. Source: Kalogeropoulos et al. (2012).

Whole Raw Tomato

By-product

Significance

1013.2 ± 89 86.1 ± 4.4 85.8 ± 5.9

413.7 ± 80 149.8 ± 86 155.7 ± 10

** ** **

91.5 ± 2.2 67.3 ± 2.5 10.8 ± 0.8

378.8 ± 53 151.7 ± 19 65.6 ± 5.8

** ** **

105.5 ± 2.4 120.3 ± 2.3 51.8 ± 2.3 8.3 ± 0.7

120.8 ± 8.3 128.1 ± 7.6 378.7 ± 62 63.5 ± 4.6

ns ns ** **

Bioactive compounds from vegetable and fruit by-products

15

Table 1.3 Phytochemical compounds extracted from tomato by-products. By-product

Phytochemical

Concentration

Source

Whole

Lycopene

29 µg/g fw

Peel

Lycopene

486 µg/g fw

Peel (enzymatic extraction with pectinase) Whole Peel plus seed

Lycopene

1590 µg/g fw

Lycopene Lycopene

Lycopene

1013 µg/g dw (38.8 µg/g fw) 414 µg/g dw (78.12 µg/g fw) 310 µg/g dw

Choudhari & Ananthanarayan (2007) Choudhari & Ananthanarayan (2007) Choudhari & Ananthanarayan (2007) Kalogeropoulos et al. (2012)

Baysal et al. (2000)

Lycopene

465 µg/g dw

Baysal et al. (2000)

Lycopene Lycopene Phenols Phenols Phenols Phenols Phenols Fiber

864 µg/g dw 19.8 µg/g fw 92–270 µg/g fw 104–400 µg/g fw 127 µg/g fw 291 µg/g fw 220 µg/g fw 50% dw

Knoblich et al. (2005) Kaur et al. (2008) George et al. (2004) George et al. (2004) Toor & Savage (2005) Toor & Savage (2005) Toor & Savage (2005) Valle et al. (2006)

Peel plus seed (solvent extraction) Peel plus seed (SFE + 5% ethanol) Peel plus seed Peel Pulp Peel Pulp Peel Seed Peel plus seed

Kalogeropoulos et al. (2012)

fw, fresh weight; dw, dry weight; SFE, supercritical fluid extraction.

weight, the tomato peel has a concentration of lycopene (486 μg/g fresh weight [fw]) significantly greater than the whole tomato (29 ug/g fw) (Choudhari and Ananthanarayan 2007) (Table 1.3). the use of enzymes capable of hydrolyzing the cell walls such as cellulases and pectinases can increase by 107% and 206%, respectively, the extraction of lycopene from tomato peels (Choudhari and Ananthanarayan 2007) (table  1.3). in addition, extraction systems using supercritical extraction with carbon dioxide in the presence of ethanol may increase up to 50% the quantity of lycopene extracted from tomato peels (of 309–465 μg/g dry weight) (Baysal et al. 2000). in addition to lycopene, tomato-derived products are also rich in phenolic compounds, having shown that tomato peel and seeds have a higher concentration of phenolic compounds than pulp (table 1.3) (Kalogeropoulos et al. 2012). the phytochemical composition of the by-products obtained from vegetable processing makes them valuable sources of nutritional ingredients for obtaining a great variety of functional foods. table 1.4 shows some examples of functional foods obtained from tomato by-products.

16

Bioactive compounds from vegetable and fruit by-products

Table 1.4 Foods functionalized with ingredients obtained from vegetable processing by-products. Vegetable

By-product

Phytochemical

Food

Functionalization

Source

Tomato

Peel (powder)

Lycopene

Burger

Tomato pulp

Lycopene Fiber Fenoles Lycopene β-carotene Phenols

Ketchup

Increase lycopene level Natural thickener

García et al. (2009) Farahnaky et al. (2008)

Phenols

Tomato juice

Phenols

Beverages

Waste

Phenols

Honey candy

Calvo et al. (2008) Larrosa et al. (2002) Larrosa et al. (2002) Stoll et al. (2003) Durrani et al. (2011)

Outer leaves or blanching liqueur Peel

Phenols

Tomato juice

Increase carotenoid level Increase phenol level Increase phenol level Increase phenol level Increase shelf-life to 6 months at 30°C Increase phenol level

Fiber

Wheat bread

Increase fiber level

Kaack et al. (2006)

Peel (powder) Onion Carrot

Artichoke

Potato

Peel plus outer coats Crownes plus tips Waste

Sausage Tomato juice

Larrosa et al. (2002)

1.4.3 Artichoke (Cynara scolymus L.) Artichoke (Cynara scolymus L.) is a flowering plant cultivated as food that belongs to the Asteraceae family, native to the Mediterranean area. World production of artichokes in 2011 was 1.5 million tons, and the Mediterranean countries were the major producers: Italy (474.550 tons), Egypt (202.458 tons), and Spain (182.120 tons) (FAO 2011). Artichoke processing discards approximately 50–60% of the initial fresh weight as a by-product mainly formed by outer leaves and part of the stem. These by-products are very rich in phenolic compounds. Furthermore, some processing technologies require previous blanching to inactivate spoilage enzymes (polyphenoloxidase, peroxidase, etc.). This pre-treatment results in a high amount of solid and liquid (water blanching) wastes with a high concentration of valuable phenolic compounds and dietary fiber (Table  1.5) (Femenia et al. 1998; Larrosa et al. 2002). Consumption of artichoke or derived products has been shown to produce health benefits, especially hepatoprotective, anticancer, and hypocholesterolemic effects (Llorach et al. 2002). Artichoke consumption has also significant antioxidant properties due to its high content in caffeic acid derivatives such as

Bioactive compounds from vegetable and fruit by-products

17

Table 1.5 Phytochemicals extracted from artichoke by-products. Vegetable

By-product

Phytochemical

Concentration*

Artichoke

Blanching leaves Blanching liqueur Blanching leaves Blanching liqueur

Phenols Phenols Phenols Phenols

4320 µg/g fw 6380 µg/mL 4400 µg/g fw 6600 µg/mL

*Chlorogenic acid equivalents; fw, fresh weight. Source: Larrosa et al. (2002).

chlorogenic acid (5-O-caffeoylquinic). This vegetal also presents a high content in flavonoids, especially glucosides and rutinosides, derivatives of apigenin and luteolin, and derivatives of cyanidin-caffeoylglucoside. Therefore, solid and liquid by-products from artichoke processing are an important source of phenolic compounds with antioxidant properties and a protective effect on health. However, due to the heat treatment applied to the artichoke, some phenolic compounds are converted into their isomers. Thus, the cynarin (1,3-O-dicaffeoylquinic acid) and neochlorogenic acid (3-O-caffeoylquinic acid) found in artichoke-derived products come from the isomerization of 1,5-O-dicaffeoylquinic acid and chlorogenic acid, respectively. Despite such phenolic isomerization, artichoke by-products have a high antioxidant capacity that makes them very useful as functional ingredients (Llorach et al. 2002). table 1.4 shows examples of functional foods obtained by using artichoke by-products.

1.4.4 Carrot (Daucus carota L.) Carrot (Daucus carota L.) is a root vegetable that belongs to the Apiaceae family. this plant is known for its characteristic orange color, although there are also some purple, red, yellow, and white varieties. World production of carrots in 2011 was 35.66 million tons and more than half were grown in China (FAo 2011). Carrot is a root vegetable that is mostly consumed in the Mediterranean diet (fresh, frozen, canned, dehydrated, etc.). Nowadays carrots are extensively commercialized as a fresh-cut product or minimally processed vegetable as mini carrots or strips and sticks that have been peeled, washed, sliced, or diced. Carrots are used in a variety of ways—in salads and soups, for example. Also, it is one of the principal components of purees destined for baby food and healthy beverages combined with other vegetables. Carrot processing produces different types of by-products: whole pieces eliminated due to defects, crowns and root tips, and peelings. thus, by-products from carrot processing may have a phytochemical composition qualitatively similar to those of the whole carrot that usually depends on the variety, cultivation conditions (irrigation, fertilizer, etc.), and processing conditions. Carrot gets its characteristic bright orange color from β-carotene, and lesser amounts of α-carotene

18

Bioactive compounds from vegetable and fruit by-products

Table 1.6 Phytochemicals extracted from different carrot varieties. Color variety

Total phenols (mg GAE/g fw)*

Chlorogenic acid (µg/g dw)

Purple-orange Purple-yellow Red Dark orange Orange Yellow White

38.7 ± 5.4a 15.0 ± 1.1b 2.27 ± 0.1c 1.66 ± 0.1c 2.34 ± 0.05c 1.97 ± 0.6c 2.35 ± 0.32c

18790 ± 38a 771.0 ± 22e 7661 ± 4.9b 334.2 ± 12d 1347 ± 2.3c 610.1 ± 40c 631 ± 0.56c 1334.7 ± 71a 1150 ± 0.30c 816.3 ± 17b 306 ± 1.59c 52.0 ± 17f 978 ± 2.78c 17.6 ± 11f

Total carotenoids (µg/g dw)

Lycopene (µg/g dw)

β-carotene (µg/g dw)

α-carotene (µg/g dw)

128 ± 17d 239 ± 8c 187 ± 18c 940 ± 54a 579 ± 79b 30 ± 8e 2.8 ± 4e

18.9 ± 2c 3.68 ± 2.03b 83.7 ± 4c 2.02 ± 0.28b 1.74 ± 0.7c 419.4 ± 49a 382 ± 18a 7.76 ± 0.89b 228 ± 141b 5.09 ± 1.52b 1.86 ± 1.2c 0.32 ± 0.10b 0.5 ± 0.33c 0.35 ± 0.0

*GAE, gallic acid equivalents; fw, fresh weight; dw, dry weight. Different letters in the same column indicate significant differences (p < 0.05). Source: Sun et al. (2009).

and γ-carotene. α- and β-carotenes are partly converted into vitamin A in humans. Carrot processing by-products present different types of phytochemicals, mainly carotenoids, but also phenolic compounds. The concentration of these phytochemicals varies depending on the variety and the process employed, as shown in Table  1.6. All carrot varieties have a very high concentration of chlorogenic and caffeic acids derivatives, including 3-O- and 5-O-caffeoylquinic acid, 3-O-p-cumorylquinic and 5-O-feruoylquinic acid, and 3,5-dicaffeoylquinic acid. derived compounds from p-hydroxybenzoic and ferulic acids have also been identified. it is noteworthy that purple carrot varieties have a higher total phenolic concentration than the other varieties (table  1.6), with 5-O-caffeoylquinic acid (540 μg/g dw) concentration 10 times higher than other varieties. Moreover, purple carrot varieties showed anthocyanins in their phenolic composition and also higher antioxidant capacity (dPPH and ABtS) than other varieties (Sun et al. 2009). Furthermore, orange carrot varieties have higher concentrations of total carotenes (lutein plus α-carotene plus lycopene plus β-carotene) than other varieties. Generally, β- and α-carotene are the major carotenoids in orange carrots, ranging between 13–40% and 44–79% of the total, respectively, while lycopene is the principal carotenoid (419 μg/g dw) in red varieties (table 1.6) (Sun et al. 2009). the solid residue obtained from carrot processing also has high dietary fiber content. thus, 63.6% of the dry weight of the solid residue is composed of total fiber, soluble fiber being 50% of the total (Chau et al. 2004). this solid residue also contains high concentrations of phytochemicals mainly phenolic and carotenoid compounds that can be used to obtain

Bioactive compounds from vegetable and fruit by-products

19

functional ingredients with antioxidant properties. It is also significant that solid carrot by-products have the advantage of not transferring undesirable flavors to the food to which it is added (o’Shea et al. 2012). table 1.4 shows some examples of functional food obtained by adding carrot by-products.

1.4.5 Onion (Allium cepa L.) onion (Allium cepa L.) is the bulb of the onion plant and the vegetable most widely cultivated of the genus Allium that belongs to the Liliaceae family. World onion production in 2011 was 86 million tons. onion is cultivated and used around the world and it is an important vegetable of the Mediterranean diet. onions can be found as fresh, frozen, canned, caramelized, dehydrated, pickled, and chopped, and also as fresh-cut products. onion is often chopped and consumed fresh in salads, or cooked and fried as part of many dishes. onion varieties of many different colors are known: yellow, brown, red, purple, and white. onion by-products are mainly made up of the outer peel, the next two layers, the top and bottom of the bulb, the roots, and whole pieces discarded because of mechanical damage, microbial contamination, or deformation (Benítez et  al. 2012). this waste cannot be used neither for animal feed due to its strong aroma nor as fertilizer because it decomposes quickly due to the growth of plant pathogens (Sclerotium cepivorum). its destruction by incineration presents serious inconveniences due to air pollution and high economical cost because of its large water content. therefore, researchers and producers are investigating new and more advantageous systems for reusing these by-products as a source of functional ingredients. thus, these residues can be used to obtain functional ingredients as the onion has in its chemical composition numerous biologically active phytochemicals. the beneficial health effects resulting from onion intake have been linked to their antioxidant, anti-inflammatory, and antimicrobial properties, among others (Griffiths et  al. 2002; González-Peña et  al. 2013). in fact, onion consumption has been associated with a significant reduction in the risk of cardiovascular disease and certain types of cancer (Hertog et al. 1993; roldánMarín et al. 2009a, 2010). the beneficial health effects of onion have been linked to the high concentration of bioactive compounds such as flavonoids, sulphur compounds like sulphoxides of S-alk(en)yl-L-cysteine (ACSos), fructooligosaccharides such as inulin, and dietary fiber (Griffiths et al. 2002; Benitez et al. 2012; González-Peña et al. 2013; Colina-Coca et al. 2013, 2014). onion is one of the major sources of dietary flavonoids in Europe, containing mainly two subclasses of this polyphenolic group: anthocyanins, responsible for the purple-red of some varieties, and flavanols such as quercetin and its derivatives, which are responsible for the yellowing of the pulp and the brown skin of other varieties. it is noteworthy that flavonols, mainly quercetin derivatives, are in a much higher concentration (280–400 mg/kg) in the onion when compared with other vegetables (100 mg/kg in broccoli, 50 mg/kg in apple, or 30 mg/kg in

20

Bioactive compounds from vegetable and fruit by-products

tea). It is also noted that the concentration of flavonols in the outer skin of onions is higher than in the other parts of the onion, especially in browncolored ones (Benítez et  al. 2011). the most abundant flavonols in this vegetable are derivatives of quercetin, mainly quercetin-4’-O-glycoside and quercetin-3,4’-O-diglucoside, in addition to small quantities of isorhamnetin4’-glycoside and other quercetin glycosides (González-Peña et  al. 2013). Moreover, some onion varieties have other quercetin, kaempferol, and isorhamnetin glycosides, and also anthocyanins. thus, red onions, besides having a composition rich in flavonols as yellow onions, have a high concentration of anthocyanins (250 mg/kg), consisting mainly of cyanidin-3-O-(6’-malonylglucoside). therefore, by-products formed by the brown external peel, the first flesh layer, and the top cut and the bottom of the bulb may be used as sources of functional ingredients because they are very rich in dietary fiber (insoluble fraction) and flavonols (quercetin derivatives), demonstrating an important antioxidant, anti-inflammatory, and protective properties against cardiovascular disease. Numerous studies have shown that the brown outer peel has the highest concentration of quercetin such as aglycone and calcium, and the top and bottom parts of the bulb have the highest concentration of minerals. therefore, outer onion layers rich in these compounds can be used as raw material for obtaining flavonols and dietary fiber, while the inner layers are better sources of fructans and sulphoxides of S-alk(en)yl-L-cysteine. As an example of the high concentration of phytochemicals presented in onion by-products, table  1.7 shows the composition of onion cv. recas and its byproducts (Benítez et al. 2011). Numerous published studies have investigated the potential of such by-products as raw materials for the production of bioactive ingredients (roldán-Marín et al. 2009a, 2009b, 2010; Benítez et al. 2011;

Table 1.7 Phytochemical from whole onion cv. recas and its by-products.

Whole onion Brown outer peel Outer flesh layers Inner flesh layers Upper and lower cut

Total phenols (mg GAE/g dw)1

Total flavonoids (mg/g dw)2

Total ACSOs (µmol/g dw)

Total dietary fiber (mg/g dw)

AA (FRAP) (µmol Fe2+/g dw)3

17.3 ± 1.3 52.7 ± 0.9 19.7 ± 1.6 9.4 ± 0.6 30.5 ± 2.0

10.3 ± 0.3 43.1 ± 41.8 19.5 ± 0.7 7.0 ± 0.1 25.9 ± 0.7

23.8 4.6 29.9 54.2 22.2

291 750 312 222 667

83.5 ± 1.8 227.8 ± 3.2 105.1 ± 0.6 28.7 ± 1.7 156.1 ± 1.6

1 GAE, gallic acid equivalents; 2 Quercetin equivalents; dw, dry weight; ACSOs, S-alk(en)yl-L-cysteine sulphoxides; 3 AA, antioxidant activity. Source: Benítez et al. (2011).

Bioactive compounds from vegetable and fruit by-products

21

González-Peña et al. 2013; Colina-Coca et al. 2013, 2014). table 1.4 shows an example of functional food obtained by adding onion by-products.

1.4.6 Potato (Solanum tuberosum L.) Potato is a starchy, tuberous crop than belongs to the Solanaceae family. the word potato may refer to the plant itself, in addition to the edible tuber. the world production of potatoes in 2011 was about 373 million tons. China is the largest producer of potatoes in the world, and almost one-third of the world’s production is harvested in China and india (88.4 and 42.3 million tons) (FAo 2011). Potatoes can be prepared in many ways: whole or peeled, cut up, with seasoning or without. Generally, the potato has to be peeled and cut before being processed for consumption with the aim of swelling the starch granules (boiled, steamed, baked, grilled, fried, dehydrated, etc.). Most common potato dishes consist of boiled potatoes (served hot or cold in salads), mashed potatoes, or fried potatoes as chips. Peeled and sliced potato generates many by-products each year, which are made up mainly of peels. these by-products have a high content of dietary fiber, carbohydrates, starch, and phenolic compounds whose concentrations vary depending on the potato variety (table  1.8). Whole potato fiber content with peel (2 g) is equivalent to that of many whole-grain breads, pastas, and cereals. in general, potato contains vitamins and minerals, as well as different phytochemicals, such as carotenoids and natural phenols. Phenolic compounds in potatoes are mostly in soluble form (free phenols, soluble esters, and glycosides) Table 1.8 Phenolic compounds from whole potatoes and their corresponding by-products. Potato variety Product Van Gogh Whole-boiled-peeled Fresh peel Boiled peel Rosamunda Whole-boiled-peeled Fresh peel Boiled peel Nicola Whole-boiled-peeled Fresh peel Boiled peel

Chlorogenic acid (µg/g fw)*

Total phenols (µg/g fw)

41 ± 2 260 ± 25 230 ± 0.3

100 340 440

8.6 ± 1.5 150 ± 11 130 ± 2.3

19 250 230

91 ± 2.8 230 ± 15 270 ± 11

170 350 450

*Caffeic acid quivalents; fw, fresh weight. Source: Mattila and Hellström (2007).

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Bioactive compounds from vegetable and fruit by-products

and to a lesser degree in the insoluble form due to the phenols attached to the cell wall. Ninety percent of potato phenolic compounds in soluble form in the pulp and skin are hydroxycinnamic acid derivatives, fundamentally chlorogenic acid derivatives. Other phenolics found in potatoes are 4-O-caffeoylquinic acid (crypto-chlorogenic acid), 5-O-caffeoylquinic (neo-chlorogenic) acid, and 3,4-dicaffeoylquinic and 3,5-dicaffeoylquinic acids. The purple-colored varieties have a higher content of anthocyanins and flavonoids than the white-fleshed varieties. It is noteworthy that the peel and the adjacent flesh have phenolic compound concentration and antioxidant activity up to 50% higher than the rest of the pulp (Albishi et al. 2013). the main soluble phenolic compound in potato peel is chlorogenic acid and its derivatives (Mattila and Hellström 2007). Phenolic compound concentration in boiled potato peel varies from 230 to 450 μg/g fw. this concentration was significantly higher than that found in boiled whole potato (100–170 μg/g fw) (table 1.8). thus, by-products of the processing of potatoes, constituted mainly by peels, are excellent raw materials for obtaining functional ingredients (Mattila and Hellström 2007) (table  1.8). there are numerous published studies that found strong correlations between the intake of foods rich in phenolic compounds such as chlorogenic acid and antioxidant and anti-inflammatory properties as well as beneficial health effects such as antitumor properties and glycemic index reduction. therefore, a natural ingredient derived from potato peels would be suitable for consumers with diabetes (o’Shea et  al. 2012). table  1.4 shows an example of low-glycemic bread obtained by adding a powder by-product from potato peels.

1.4.7 Beet (Beta vulgaris L.) Consumption of leaves (Beta vulgaris L. var. cycla) and beetroot (Beta vulgaris L. var. rubra) is common in the Mediterranean diet and are increasingly used as an ingredient in salads or cooked dishes worldwide. the main producers are the russian Federation (47.64 million tons), France (38.10 million tons), and the United States (26.21 million tons). By-products of sugar beet processing are mainly composed of leaves, crowns, and outer layers of the root and pulp. traditionally, beet by-products have been reused in animal feed for the production of fertilizers, natural bio-absorbent for the pretreatment of wastewater, or for obtaining alcohols and biofuels. However, these by-products are an important source of biologically active compounds such as fiber, betaine, betalains, polyphenols, minerals, etc., which have a significant value as functional ingredients (Pyo et  al. 2004; Stintzing and Carle 2007; Canadanovic-Brunet et al. 2011; Ninfali and Angelino 2013). Beet roots have nitrogen-soluble pigments known as betalains, which consist primarily of two types of compounds: red pigments called betacyanins, betanin being the majority, and the yellow pigments or betaxantinas such as vulgaxanthin. it is estimated that the average concentration of betalains in red beet root is 1.2 g/kg fw. in fact, there is a commercial natural food coloring called “red beet”

Bioactive compounds from vegetable and fruit by-products

23

Table 1.9 Phytochemicals extracted from different beet varieties. Variety

Plant area

Total phenols (mg/g dw)

Leaves Roots Seeds

11.12 ± 0.56 0.72 ± 0.04 1.88 ± 0.76

7.92 ± 0.39 0.88 ± 0.05 1.55 ± 0.08

192.8 ± 9.6 8.54 ± 0.43 49.10 ± 2.76

Leaves Roots

12.76 ± 0.76 1.77 ± 0.08

11.64 ± 0.81 1.44 ± 0.15

200.3 ± 11.2 18.21 ± 0.86

Total flavonoids (mg/g dw)

ORAC (μmolTE/g dw)

Beta vulgaris var. cicla

Beta vulgaris var. rubra

dw, dry weight. Source: Ninfali and Angelino (2013).

(E162), consisting mainly of betanin obtained from beet root. The industrial yield of this coloring is 0.5 g of betanins per kilogram of beet root (Stintzing and Carle 2007). Beets and their by-products are also a good source of dietary fiber, vitamin C, and minerals such as potassium, manganese, zinc, copper, iron, and folic acid. Beet by-products have a high concentration of nitrates. The concentration of phenolic compounds is also important in beets, with a greater concentration of total phenols, total flavonoids, and antioxidant capacity values (measured by ORAC methodology) in leaves than in roots (Table 1.9) (Ninfali and Angelino 2013). the major phenolic compounds described in the leaves of red beet (var. cycla) are syringic acid (44 mg/100 g), followed by caffeic acid (15 mg/100 g ) and coumaric acid (11 mg/100 g). Also ferulic acid, vanillic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, and the flavonoid kaempferol are found in red beet leaves (Pyo et al. 2004). the leaves and seeds of beet (var. cycla) have a high concentration of a flavonoid derived from apigenin called vitexin, whose anticancer properties are widely recognized (Ninfali and Angelino 2013). Beet products (juices, dried powder) and its by-products have been used in traditional medicine for thousands of years. the beneficial effects of the consumption of derivatives of red beet root are largely related to the presence of the betalains, which have antioxidant (free radical scavenger), anti-inflammatory, antitumor, and hypoglycemic properties. it is important to highlight their protective effect against cardiovascular disease by reducing blood pressure, platelet aggregation and lipid levels, and blood cholesterol. it also has protective properties of liver cells (Kanner et al. 2001; Ninfali and Angelino 2013). Beetroot has an N-methylated amino acid called trimethylglycine (N, N, N-trimethylglycine) or betaine. its concentration in sugar beet root is relatively high, 1.0–1.5% on a dry solid basis. the dry residue from the processing of beet normally contains a range between 3 and 8% of betaine. its main physiological functions are to protect cells under stress (osmoprotectant effect), while serving as a source of methyl groups necessary for the formation of many biochemical pathways.

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Bioactive compounds from vegetable and fruit by-products

Historically, we have used betaine supplementation to control excess blood homocysteine (caused by a hereditary condition known as homocystinuria) and reduce heart disease, stroke, cancer, and Alzheimer disease risk (Sacan and Yanardag 2010). Therefore, beet processing by-products may also be considered in the design of functional foods as raw materials suitable for obtaining biologically active natural ingredients.

1.5 Fruit by-products 1.5.1 Fruits Citrus is one of the most important fruit crops worldwide, with a production in 2011 of 131.20 million tons, China being the first producer with 29.99 million tons followed by Brazil (22.01 million tons) and the United States (10.70 million tons). There is also remarkable production in Mediterranean countries such as Spain (5.77 million tons), Italy (3.84 million tons), and Egypt (3.73 million tons) (FAO 2011). The main use of citrus in food industries includes fresh juice (mainly orange juices) and citrus-based beverages. Practically 40% of citrus fruit production goes to the production of juices. During the production of citrus juices, approximately 50–60% of fresh fruit weight account as by-product. These large amounts of processed citrus fruits result in very large amounts of by-products. The main citrus processing by-products are whole fruit discarded in the selection process, peel, pulp, and seeds. The citrus peel is the main by-product, which represents 50% of fresh fruit weight. Citrus peel is traditionally dried and used in animal feed. Nowadays, citrus peel is an important source of valuable products such as volatile flavoring compounds, used in the cosmetic and perfume industries, phenolic and carotenoid compounds, and phytochemicals such as limonoids, oxygenated terpenoids (djilas et  al. 2009; Lagha-Benamrouche and Madani 2013). Also, citrus peel is an important source of fiber since it is very rich in pectins (rodríguez et al. 2006). the pulp, separately in different steps of citrus juice production, represents 5% of fresh fruit weight and is a valuable by-product for the recuperation of phenolic and carotenoid compounds. Citrus seeds represented only 0–1.5% of fresh fruit weight, depending on the variety, and can be used for the extraction and recuperation of terpenoids. in general, citrus peels have higher concentrations of dietary fiber and phenolic compounds than their corresponding peeled fruit. thus, Figure 1.7 shows that the contents of total soluble and insoluble dietary fiber in peels of several citrus fruits were significantly higher than in the peeled fruit. regarding phenolic compounds, table  1.10 shows that the total phenolic content in the peels of oranges, lemons, and grapefruit was approximately 15% higher than those found in the peeled fruits (Gorinstein et al. 2001). Like the total phenol content,

Bioactive compounds from vegetable and fruit by-products

18

Soluble fiber

Total fiber

25

Insoluble fiber

16 14 Mg/100g dw

12 10 8 6 4 2 0 Orange peeled

Orange peel

Lemon peeled

Lemon peel

Grapefruit peeled

Grapefruit peel

Figure 1.7 total soluble and insoluble fiber content in peel and pulp of citrus fruits.

Table 1.10 total phenolic content, phenolic acids, and antioxidant activity in citrus peel and their peeled fruit. Variety

Total phenols*

Ferulic acid*

Sinapic acid*

p-Cumaric acid*

Caffeic acid*

TRAP** (nmol/ml)

Orange peeled Orange peel Lemon peeled Lemon peel Grapefruit peeled Grapefruit peel

154 ± 10.2 179 ± 10.5 164 ± 10.3 190 ± 10.6 135 ± 10.1 155 ± 10.3

34.1 ± 3.1 39.2 ± 4.0 38.8 ± 4.1 44.9 ± 4.2 27.1 ± 3.0 32.3 ± 3.1

30.71 ± 3.1 34.90 ± 3.1 36.40 ± 3.1 42.10 ± 4.1 27.30 ± 2.9 31.90 ± 3.0

24.10 ± 2.2 27.90 ± 2.5 31.30 ± 3.1 34.90 ± 3.4 10.80 ± 1.1 13.10 ± 1.3

8.10 ± 0.8 9.50 ± 0.8 12.10 ± 0.8 14.20 ± 1.3 5.0 ± 0.5 5.6 ± 0.5

2111 ± 199 3183 ± 311 4480 ± 398 6720 ± 601 1111 ± 102 1667 ± 161

*mg/100 g fresh weight. **TRAP, total radical-trapping antioxidant potential. Source: Goristein et al. (2001).

antioxidant capacity (TRAP) was significantly higher in peels than in the peeled fruits. The same results were obtained for the hydroxycinnamic acids (ferulic, sinapic, p-coumaric, and caffeic acids) found in the citrus fruits (Table 1.11). Citrus peel also contains higher amount of flavonoids than the edible portion. Neoericitrin, naringin, and neohesperidin are the main flavanones in the peels of sour orange (C. aurentium), lemon (C. limon), and bergamote (C. bergamia Fantastico). Hesperidin is the main flavonoid in Valencia, Navel temple, and Ambersweet orange peels, and naringin is the most abundant flavonoid in grapefruit. Also, diosmetin derivatives are the flavone compounds found in navel orange and lemon peels (Wang et al. 2008). Comparing the results found in eight different citrus fruits produced in taiwan, Wang et  al. (2007, 2008) found that both total flavonoid and total carotenoid contents were higher in the

26

Bioactive compounds from vegetable and fruit by-products

Table 1.11 total flavonoid and carotenoid content (mg/g dw) in peel and edible portion of different citrus fruits. Citrus fruit Scientific name

Part of citrus fruit

C. reticulata Blanco

Peel Edible portion Peel Edible portion Peel Edible portion Peel Edible portion

C. Sinensis (L.) Osbeck C.reticulate x C. sinensis C. Limon (L.) Bur

Total flavonoids

Total carotenoids

49.2 ± 1.33 11.2 ± 0.32 35.5 ± 1.04 15.7 ± 0.43 39.8 ± 1.02 11.1 ± 0.34 32.7 ± 1.06 21.6 ± 0.57

2.04 ± 0.036 0.198 ± 0.0008 0.445 ± 0.008 0.080 ± 0.002 1.59 ± 0.011 0.336 ± 0.005 0.110 ± 0.001 0.061 ± 0.001

dw, dry weight. Source: Wang et al. (2007, 2008).

peels than in the edible part of the fruits (Table  1.11). thus citrus peel is an important source of flavonoids that have a wide range of biological effects, such as antioxidative, anticancer, antiviral, and anti-inflammatory activities (Harborne and Williams 2000). Citrus fruits have another health benefit: phytochemicals called limonoids, very high-oxygenated terpenoids. Limonoids appear in large amounts in citrus juices and citrus pulp as water-soluble glucoside derivatives, and in citrus seeds as water-insoluble limonoid aglicons. these are the compounds responsible of the bitterness in unripe fruits and are converted to nonbitter glucoside derivatives during fruit ripening. the main limonoids found in citrus fruit by-products are limonin, nomilin, and nomilinic. Limonoids have antiviral, antifungal, antibacterial, antineoplastic, and antimalarial activities. Some of them, such as azadirachtin, are also insecticides (djilas et al. 2009).

1.5.2 Apples (Malus domestica) Apple fruit (Malus domestica) belongs to the Rosaceae family and is harvested in the majority of countries in the world, with total production in 2011 of 75.48 million tons, China being the first producer with 50% of world apple production, with 35.98 million tons, followed by Europe with 15.19 million tons and the United States with 4.27 million tons. Apple pomace is the main by-product obtained by crushing and pressing during the clear juice recovery, and represents 25–35% of the fresh fruit weight. Apple pomace, consisting of peel, seeds, core, stems, and exhaustive soft tissue, has been widely studied for its antioxidant properties and beneficial effects on human health that have been demonstrated by numerous in  vitro assays such as strong inhibitory activity of tumor-cell proliferation and human LdL cholesterol oxidation (djilas et  al. 2009; Grigoras et  al. 2013; thilakarathna et  al. 2013). Several studies have

Bioactive compounds from vegetable and fruit by-products

27

identified the presence of important bioactive compounds such as polyphenols, minerals, dietary fiber, and also terpenoids. Apple pomace has been shown to be a good source of polyphenols, which are predominantly localized in the peels and are less extracted into the juice. Major phenols identified included derivatives of benzoic acids (gallic acid), hydroxycinnamic acids (chlorogenic acids), flavanols (catechin), flavonols (rutin and quercetin), and dihydrochalcones (phloridzin and phloretin-2′-xyloglucoside). Among the triterpenes, ursolic acid and oleanolic acid were the most abundant. These compounds also have many beneficial health properties such as anti-inflammatory, antimicrobial, antimycotic, antioxidant, antiviral, liver protective, immunomodulatory, hemolytic, or cytostatic effects (Muffler et al. 2011). the phenolic profile and antioxidant capacity of apple pomace is mainly related to the apple cultivars employed in fruit juice processing, the growing conditions of the apple tree, and the season (diñeiro García et al. 2009; Grigoras et al. 2013).

1.5.3 Grapes (Vitis vinifera) Grapes are a fruit that belongs to genus Vitis of the Vitaceae family. Grapes are an important world crop, with a production of 58.5 million tons in 2011, China being the first producer (9.17 million tons) followed by the United States (6.75 million tons) and Mediterranean countries such as italy (7.11 million tons), France (6.58 million tons), and Spain (5.80 million tons). Commercially cultivated grapes can usually be classified as either table or wine grapes, based on their intended method of consumption: eaten raw (table grapes) or used to make wine (Vitis vinifera). therefore, grapes can be eaten raw or used to produce wine, juice, jam, jelly, grapeseed oil, raisins, and vinegar. Approximately 71% of world grape production is used to produce wine, 27% as fresh fruit, and 2% as dried fruit. Grape pomace is a by-product of the wine industry and represents about the 20–25% of the weight of grapes crushed for wine production. the composition of grape pomace varies considerably depending on the grape variety and technology used to produce wine. Grape pomace is constituted by peels (skins), seeds, and stems, and is very rich in extractable phenolic compounds (10–11% of dry weight), mainly anthocyanins, catechins, procyanidins, flavonol glycosides, phenolic acids, and stilbenes (Yu and Ahmedna 2013). the seeds constitute a considerable portion of the grape pomace, amounting to 38–52% on a dry matter basis. Grape seeds are rich in phenolic antioxidants such as phenolic acids, flavonol glycosides, flavan-3-ols (catechin, epicatechin, and epicatechin-3-O-gallate), and stilbenes as resveratrol, while grape skins contain abundant anthocyanins. Grape seeds also contain 13–19% of oil rich in unsaturated fatty acids (mainly linoleic acid), about 11% of protein, and 60–70% of nondigestible carbohydrates, and other antioxidants such as tocopherols and beta-carotene (djilas et al. 2009). Flavan-3-ols are detected in the grape pomace derived from seeds and are catechin, epicatechin, epicatechin-3-O-gallate, gallocatechin, and their polymers.

28

Bioactive compounds from vegetable and fruit by-products

Flavan-3-ols easily condenses into oligomeric procyanidins and polymeric compounds (condensed tannins). The dimeric procyanidins found in grape pomace are procyanidin B1, procyanidin B2, and procyanidin B4, and the trimeric procyanidins C1 and C2. Polyphenol composition of grape pomace depends on the grape variety, the growing area, climate, maturity, and wine vinification method. Thus, red grape varieties are very rich in anthocyanins, mainly located in the skin (69–151 mg/ kg fw); meanwhile, flavan-3-ols are the main phenolic compounds in white varieties (52–81 mg/kg fw) (Cantos et al. 2002). there are numerous published studies about the polyphenolic composition of grape pomace from different grape varieties in different wine-producing regions in the world (e.g., Yu and Ahmedna 2013). thus, pomace from grape varieties widely produced in Brazil (Cabernet Sauvignon, Merlot, Bordeaux, and isabel) showed that total phenol content ranged from 46.23 mg/g in Merlot to 74.75 mg/g in Cabernet Sauvignon, and total anthocyanins ranged from 7.02 mg/g in Cabernet Sauvignon and 11.22 mg/g in Bordeaux, being catechin the major nonanthocyanin compound identified (150 mg/kg). the anthocyanin content of grape pomace also varies with the variety and wine vinification method employed. Anthocyanins identified in grape pomace include 3-O-monoglucosides and acetylglucosides of delphinidin, cyanidin, petunidin, peonidin, and malvidin. Malvidin-3-O-glucoside was found to be the predominant anthocyanin. resveratrol is another important polyphenol found in grape pomace, which comes mainly from the skin. resveratrol content varies with the grape variety and maturity. Muscadine grapes contain more resveratrol than other types of grapes. the average resveratrol content in white grape skin is 8.64 mg/100 g of dry mass and in white grape seeds is 1.42 mg/100 g of dry mass. Some studies have shown that resveratrol content in grapes could be increased by postharvest technologies such as cold storage or UV irradiation. it is important to note that although certain amounts of resveratrol transfer into wine, the majority remains in the grape pomace (Yu and Ahmedna 2013). Finally, grape pomace contains a great content of nonextractable polyphenols, mainly consisting in high-molecular-weight proanthocyanins and polyphenols complexed with protein and cell wall polysaccharides. the nonextractable polyphenols was quantified in red grape pomace in 67 mg/g of dry mass and in white grape pomace in 1.68 mg/g of dry mass (Pérez-Jimenez et al. 2009). Health benefits of consumption of grape pomace extracts are known for producers, researchers, the food industry, and the nutraceutical industry for many years. Numerous in vitro and in vivo studies have demonstrated that grape pomace phenolic compounds have many health benefits such as antimutagenic and anticarcinogenic activity, in addition to antioxidant and anti-inflammatory activities, prevention and delay of cardiovascular diseases, increase in lifespan, and delayed onset of age-related markers. in addition to antioxidant and beneficial health properties, there are numerous studies that show antibacterial activity of grape

Bioactive compounds from vegetable and fruit by-products

29

pomace extracts. Also, resveratrol, which is rich in grape skin, has antifungal and antibacterial activities. There are numerous reviews that summarize the most recent studies about the phytochemical composition of grape pomace and their biological properties and human health benefits (Yu and Ahmedna 2013).

1.5.4 Tropical fruits Tropical fruit production, trade, and consumption have increased significantly due to their attractive sensory properties and a growing recognition of their nutritional and health-promoting properties (Ayala-Zavala et al. 2011; Correia et  al. 2012). Examples of tropical crops include mango, papaya, pineapple, passion fruit, acerola, cashew apple, guava, longan, jackfruit, avocado, tamarind, sapodilla, and others. there is now a special interest from researchers, producers, health authorities, and the food, nutraceutical, and pharmaceutical industries in the study of pulps and by-products of tropical fruit to isolate specific phytochemicals for application in nutraceutical supplements, dietary additives, and new food and pharmaceutical products. these studies also contribute to the recovery of agro-industrial process waste, with major industrial, economic, and environmental impact (Ayala-Zavala et al. 2011). Seeds, peels, and residual pulp are generated as solid by-products of the fruit processing industry. For instance, mango, papaya, and pineapple by-products represent from 35 to 60% of the fruit weight. the most bioactive compounds found in tropical fruit by-products are vitamins C and E, dietary fiber, and phenolic compounds. in general, vitamin C is uniformly distributed in fruits, carotenoids occur mainly in the external pericarp and peel, and phenolic compounds are located preferentially in peel and seeds. table  1.12 shows the phenolic content in different parts of the fruit; it is noteworthy that the peel has higher phenolic concentration than the pulp. Also, table 1.12 shows the total

Table 1.12 total phenolic content (g/kg dw) of by-products from different mango cultivars. Mango cultivar

By-product

Tommy Atkins

Peel Seed Peel Seed Peel Seed Peel Seed

Kent Van Dyke Fafá

Source: Barreto et al. (2008).

Total phenolic (g/kg dw) 25.13 200.05 91.21 191.25 59.09 70.10 52.28 149.33

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phenolic and carotenoid content of several tropical fruit by-products compared with their corresponding pulps. Therefore, tropical fruit by-products that are composed mostly of peels and seeds are a good source of phenolic compounds with biological activities beneficial for human health (Ayala-Zavala et al. 2011). taking into account the potential anti-inflammatory and antioxidant properties of tropical fruit by-products, numerous studies on the composition of bioactive compounds of these by-products have been reported and summarized in recent reviews (Ayala-Zavala et al. 2011; Correia et al. 2012; Silva et al. 2014).

1.6 Pretreatment and extraction systems An important step in the production of phytochemicals from plant processing byproducts is the stabilization and preparation of these for extraction (o’Shea et al. 2012). the difficulties found for the stabilization and preparation of by-products are: •  Heterogeneous starting material (cultivars, processing conditions, etc.), which makes it difficult to control yields and final prices. •  Biological instability of the by-products due to the high microbial load of these wastes, which can accelerate the degradation of phytochemicals and other nutritional compounds such as proteins, in addition to making the product unsafe. •  High water content (70–90%) of the plant by-products that makes transport to the recycling plant more difficult due to its high weight. Also pretreatments for the extraction of phytochemicals from by-products with high water content are more complicated. due to the high water content of some by-products (71% in tomato, 90% in artichoke, and 82% in beet), the drying process (60 °C) and subsequent pressing involves high cost (Peschel et  al. 2006). Furthermore, pressing presents an additional problem since the water obtained has a high content of organic substances and has to be recycled. •  Oxidation of by-products with a high fat content (avocado) can lead to the development of unpleasant odors from the oxidation of fatty acids. •  Enzymatic activity of plant residues. the enzymes remain active and may accelerate the degradation process, which leads to the loss of valuable phytochemicals or bioactive compounds. the thermal treatment or blanching (85–100 °C) prior to the extraction of phytochemicals from by-products may be useful to inactivate the enzymes that cause various degradative processes such as enzymatic browning. As an alternative to traditional blanching, ohmic heating can be employed, which is a homogeneous and faster electrical heating that reduces the loss of heat-sensitive phytochemicals such as vitamin C (icier 2010). •  Pretreatments. For extracting phytochemicals from vegetable by-products, various pretreatments are required: wet grinding of the by-product (for reducing the particle size of wet residue) and drying (oven, lyophilization) and grinding the dried extract to achieve the required particle size (o’Shea et al. 2012.). the

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31

stabilization of the by-products before being subjected to extraction is a critical phase that requires the study of the most suitable conditions to prevent degradation of phytochemicals or bioactive compounds. For example, in the case of onions, mild heat treatment (pasteurization) and the freezing/freeze-drying of their by-products have been the best pretreatments to preserve the stability of their phytochemicals (roldán et al. 2008; Benítez et al. 2011). •  Extraction. the traditional method of solid–liquid extraction using a Soxhlet with organic solvents is a process that requires time and large amounts of solvents (Ayala-Zavala et al. 2011). Enzymatic treatments with enzymes such as pectinase and cellulose, capable of degrading the cell wall constituents and to facilitate the extraction of phytochemicals such as lycopene from tomato skins, have been also employed in the industry (Choudhari and Anantharayan 2007). An interesting alternative to conventional solvent extraction is supercritical fluid extraction, primarily with supercritical carbon dioxide (SC-Co2), and also the extraction with solvent under pressure generally known as extraction with subcritical water (Wijngaard et al. 2012). these technologies have the disadvantage of being expensive but may be of interest in terms of economic value and functional characteristics of phytochemical compounds extracted. this is the case of lycopene, a valuable functional ingredient of high economic value, that lost its biological activity as a consequence of degradation and isomerization caused by heat treatment (Lennuci et al. 2010). Also being studied is the use of new extraction technologies such as low-intensity electrical pulses, ultrasonic, and microwave (Wijngaard et al. 2012). Finally, it should be kept in mind that before using functional ingredients obtained from plant processing by-products, additional studies are needed, such as toxicological studies, to ensure that the ingredient is free of pesticides and other undesired or toxic substances. Bioactivity studies are also required to enable us to determine the bioaccessibility and bioavailability of phytochemicals extracted from these by-products. regulation (EC) 1924/2006 on nutrition claims on foods requires that any declaration (within the permitted) has to be based on scientific evidence. therefore, the correct characterization of plant by-products and their corresponding extracts is critical for potential commercialization. therefore, the industrial production of phytochemicals derived from byproduct processing plants and their use as functional ingredients in foods requires the coordination of interdisciplinary studies from food technologists, food chemists, nutritionists and toxicologists. in conclusion, agro-industrial by-products are a good source for obtaining phytochemicals with high antioxidant activity and other beneficial health properties. in addition, the exploitation of these abundant and low-cost renewable resources could be anticipated for the pharmaceutical, nutraceutical, and food industries with the opportunity of developing new nutraceutical and/or pharmaceutical products. Also, this form of recuperation of by-products is an interesting way to reduce industrial waste, cost, and environmental impact

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generated by the habitual destruction of by-products, with the added value of obtaining phytochemicals with beneficial health properties. From the point of view of the consumer, the general use of natural additives to replace synthetics is a healthy advantage. In general, additives from vegetable processing by-products are perceived by consumers as a more natural ingredient; besides, its consumption can provide beneficial health effects.

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CHAPER 2

Bioactive compounds in fresh-cut fruits: Occurrence and impact of processing and cold storage María Elida Pirovani1, Andrea Marcela Piagentini1, and Franco Van de Velde1,2 1

Instituto de Tecnología de Alimentos, Facultad de Ingeniería Química Universidad Nacional del Litoral, Santa Fe, Argentina 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Santa Fe, Argentina

2.1 Introduction Epidemiological studies have noted a consistent association between the consumption of diets rich in fruits and vegetables and a lower risk for chronic diseases, including cancer, heart disease, and stroke, and better diabetes control and risk of obesity (Hannum 2004). Protection against degenerative diseases by fruits and vegetables has been attributed to the fact that these plant foods may provide an optimal mix of phytochemicals, such as natural antioxidants like phenolic compounds, carotenoids, vitamins C and E, fiber, and other bioactive compounds (de Ancos et al. 2000; Leong and Shui 2007). Phenolic compounds comprise a wide variety of molecules with one phenol ring, such as phenolic acids and phenolic alcohols, but also molecules that have a polyphenol structure (i.e., several phenolic groups). The content of phenolic compounds in fruits and vegetables depends on various factors, such as genotypic differences, preharvest climatic conditions, and postharvest handling procedures (Aaby et al. 2007). There are hundreds of different phenolic compounds in plant foods, but about two-thirds of those most commonly consumed are flavonoids and about one-third are phenolic acids (Scalbert and Williamson 2000). Flavonoids are classified into anthocyanins, flavones, isoflavones, flavanones, flavonols, and flavanols. Otherwise, phenolic acids can be further classified in hydroxybenzoic and hydroxycinnamic acid derivatives (Ignat et al. 2011). As an example of flavonoids, anthocyanins are important soluble pigments that are responsible for the shiny orange, pink, red, violet, and blue colors in the flowers and fruits of some plants (Castañeda-Ovando et al. 2009). Flavonoids in particular are potent antioxidants, and phenolic acids may be fairly good antioxidants,

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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depending on their structures (Hannum 2004). Both, flavonoids and phenolic acids may work synergistically with other antioxidants such as ascorbate and tocopherol and seem to have a sparing effect on these vitamins (Croft 1998). Several other properties such as antibacterial, antiviral, anti-inflammatory, antiallergic, antimutagenic, and anticarcinogenic activities are associated with phenolic compounds as well (Friedman 1997; Velioglu 1998; Reyes and CisnerosZevallos 2003). Carotenoids are found in fruits and vegetables as carotenes (unsaturated hydrocarbons) and xanthophylls (oxygenated derivatives). Generally, the main carotenoids in vegetables are lutein, β-carotene, violaxanthin, and neoxanthin, whereas in fruits xanthophylls are usually found in a greater proportion. Carotenoids are liposoluble, stable, and able to color food products from yellow to red (Hill and Johnson 2012). Ascorbic acid is a potent antioxidant that has the capacity to eliminate several different reactive oxygen species, keeps the membrane-bound antioxidant α-tocopherol in the reduced state, and has a role in stress resistance (Davey et al. 2000; Hernández et al. 2006). In general, fruits are considered the best dietary vitamin C food sources (ascorbic acid plus dehydroascorbic acid content), with black currants being especially rich at 200 mg 100 g–1, strawberries at 40–60 mg 100 g–1, and citrus fruits at 30–50 mg 100 g–1. However, not all fruits contain such levels; apples, pears, and plums represent only a very modest content of vitamin C (3–5 mg 100 g–1). Although the health-promoting effects of vegetables are generally accepted, the average intake of fruits and vegetables is still below the recommendations of the World Health Organization (400 g per person per day) (World Health Organization Europe 2003; Vandekinderen et al. 2008). However, in the last years, there has been an increasing demand for fresh-cut, minimally processed, or ready-to-eat fruits and vegetables as a consequence of their convenience and freshness. These products are defined by the International Fresh-Cut Produce Association (2002) as “any fruit or vegetable or any combination thereof that has been physically altered from its original form, but remains in a fresh state.” Minimal processing of vegetables and fruits involves the use of a combination of procedures, such as selection, prewashing, peeling, cutting, washing–disinfection, and packaging (Figure 2.1). these operations may cause an increase in product respiration, biochemical changes, and microbial spoilage and, therefore, detrimental effects on quality (Allende et al. 2006; Plaza et al. 2011). Bioactive compounds as phenolics and vitamins can be affected by minimal processing, and the healthy potential of fresh-cut fruits and vegetables could be different to the nonprocessed products. the objective of this chapter is to deal with the scientific, technical, and practical aspects related to the processing of some fresh-cut fruits (apples, oranges, peaches, and strawberries), focusing on its consequences over the changes of the bioactive compounds’ content and their antioxidant capacity.

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Receiving Refrigerated (0–5°C or 6–12°C) storage Inspection/sorting/grading/triming Prewashing/disinfection Coring/peeling Size reduction operations Washing/disinfection Preservation treatments Dewatering Weighting/packaging

Figure 2.1 Fresh-cut fruits diagram:

major unit operations at industry and commercial chain.

Refrigerated storage, transportation, and distribution Retailing and food service

2.2 Factors affecting quality and phytochemical content of fresh-cut fruits The basic requirements for preparation of fresh-cut fruits are high-quality raw material; strict hygiene and good manufacturing practices; low temperatures during processing; careful cleaning and/or washing before and after peeling; use of mild processing aids in wash water for disinfection or prevention of browning and texture loss; minimization of damage during peeling, cutting, slicing, and shredding operations; gentle draining to remove excess moisture; correct packaging materials and methods; and correct temperature during distribution and handling (Garret 2002). Fresh-cut fruits are generally more perishable than whole fruits because they have been subjected to physiological stress caused by wounding (OdriozolaSerrano et al. 2010). The process of peeling, coring, chopping, slicing, dicing, or shredding injures cells, releasing their content at the sites of wounding. Respiration increases when fruits are wounded, causing accelerated consumption of sugars, lipids, and organic acids, and an increase in ethylene production, which induces ripening and causes senescence. Moreover, subcellular compartmentalization is disrupted at the cut surfaces and the mixing of substrates and enzymes, that are normally separated, can initiate reactions that normally do not occur (González-Aguilar et al. 2005).

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Each process operation must be designed appropriately to obtain a fresh-cut fruit that maintains its quality and provides the maximum of their bioactive potential. In many fruits, the peel should be removed because it is not edible. However, in some cases, it is desirable to design the product with skin or peel since it provides valuable bioactive compounds. Piagentini et al. (2009) found that apple peel has higher antioxidant capacity than fruit flesh. Washing–disinfection is an essential operation to eliminate foreign matter, microorganisms on the surface of the fruit, and cellular fluids produced by cutting (Pirovani et al. 2004). Compounds derived from chlorine are the most widely used disinfectants in the fresh-cut vegetable industry but their reaction with organic matter may result in the formation of carcinogenic halogenated disinfection by-products, such as trihalomethanes and haloacetic acids. For this reason, in recent years, the use of alternative disinfectants as chlorine dioxide, ozone, organic acids, peracetic acid, and hydrogen peroxide are gaining interest (Ölmez and Kretzschmar 2009). Some problems may take place during the washing–disinfection operation. First, the loss of pigments, vitamins, and other compounds by oxidation, because the disinfectant generally used is strong. Moreover, the operation may also produce a deleterious effect on the color of vegetables by inducing browning or bleaching of the vegetable tissue (Vandekinderen et al. 2008). On the other hand, the cutting of the tissue and the immersion in the disinfectant solution could enhance the lixiviation process of solutes. Therefore, washing–disinfection may affect appearance and color and reduce nutrients and bioactive compounds of fresh-cut fruits. The combination of different preservation methods such as modified atmosphere packaging, low temperature storage, or the addition of preservatives may be an excellent way to preserve the original quality attributes of these products (Alzamora et al. 1998). Atmospheres low in O2 (1–5%) and high in CO2 (5–10%) have been shown to extend the shelf-life of fresh-cut fruit by reducing respiration, ethylene biosynthesis, and the proliferation of aerobic microorganisms. However, if atmospheric modification during storage results in excessively low O2 and high CO2 concentrations in package headspace, this situation can stimulate the growth of anaerobic psychotrophic microorganisms and the production of undesirable metabolites (Soliva-Fortuny et al. 2002). Metabolic rates of fruits and vegetables are directly related to storage temperatures within a given range. The higher the rate of respiration, the faster the produce deteriorates. Lower temperatures slow respiration rates, as well as ripening and senescence processes, which prolongs the storage life of fruits and vegetables (González-Aguilar et al. 2005). Several chemical treatments can be used on fresh-cut fruit elaboration mainly for controlling browning development, loss of firmness, and decay. The antioxidant preservatives used may also reduce bioactive compound loss, maintaining the health-related attributes of the whole fruits. As fresh-cut fruits are associated by

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consumers to healthy food, the use of synthetic preservatives is not recommended; therefore, the use of natural antioxidants to extend fresh-cut fruit shelf-life is being considered. Cisneros-Zevallos (2003) proposed that controlled abiotic stress treatments— such as wounding, temperature, altered gas composition, and heat shock—may be used as tools by the fresh-cut fruit industry to create healthier products by enhancing their nutraceutical content. These abiotic stresses affect phytochemical accumulation or loss by inducing an increase or reduction in key enzyme activities of secondary metabolic pathways, for instance, in the phenylpropanoid pathway enzyme, phenylalanine ammonialyase (PAL). As many of these types of abiotic stresses are associated with the operations to obtain fresh-cut fruits, a complete knowledge of the impact of minimal processing applied to a specific plant commodity will allow obtaining of healthier fresh-cut fruits.

2.3 Raw material The content of bioactive compounds and the antioxidant capacity of fresh-cut fruit products, as well as their physicochemical characteristics, depend on the quality of the whole fruit, which may change depending, among other things, on genetic and preharvest factors and the physiological state (Piagentini et al. 2009; Wolfe et al. 2003; de Ancos et al. 2009). Numerous authors found significant differences in the content of bioactive compounds of fresh-cut products, depending on the variety of the fruit used as raw material. Among the preharvest factors, temperature and light intensity have a great influence on the content of bioactive compounds. Moreover, the optimum stage of maturity is another factor determining the quality in terms of nutritional composition of minimally processed vegetables (Chen et al. 2012; de Ancos et al. 2009; Martin et al. 2011; Piagentini et al. 2009; Williner et al. 2003). The elaboration of minimally processed fruits should start with the selection of the most suitable variety, evaluating characteristics such as flesh texture, browning development on the cut surface, and antioxidant capacity, among other physicochemical attributes (de Ancos et al. 2009; Martín et al. 2012; Piagentini et al. 2012; Seipel et al. 2009). Special attention should also be paid to the polyphenol content and profile since they contribute to color, browning development, bitterness, and astringency (Khanizadeh et al. 2008). Reig et al. (2013) evaluated the antioxidant capacity, quality, and anthocyanin and nutrient contents of 106 peach cultivars from different breeding programs. High variability was found among cultivars within each quality trait, for example, a five-fold range (2.17–12.07 g of malic acid L–1), six-fold range (144.20–711.73 μg of Trolox g–1 of FW), and 11-fold range (0.70–11.43 mg of cyanidin-3-glucoside kg–1 of FW) were observed in titratable acidity, relative antioxidant capacity, and anthocyanin content, respectively. The breeding program within each fruit type

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had significant effects on the quality. Nevertheless, within each breeding program, there is high variability among cultivars. Therefore, these results could help to make decisions for the selection of new peach cultivars with better-quality performance, good sensory characteristics, and high health benefits for consumers. Cantin et al. (2009) evaluated the antioxidant capacity and contents of total phenolics, anthocyanins, flavonoids, and vitamin C in 218 genotypes from 15 peach and nectarine breeding progenies. It was found that the phytochemical profile varied depending on peach/nectarine and yellow/white flesh-color qualitative traits. On the other hand, antioxidant capacity was linearly correlated to total phenolic content, but no correlation was found for vitamin C versus any other phytochemical trait. These results suggest the importance of genetic background on the antioxidant profile of peaches and nectarines and stress its relevance for selecting new peach and nectarine genotypes rich in bioactive compounds to benefit consumers’ health. Martín et al. (2011) analyzed different cultivars of peaches to be used as raw materials for minimal processing. They reported the total phenolic content and antioxidant capacity of four cultivars of peaches (Prunus persica L.), three with yellow flesh (Early Grande, Flordaking, and Hermosillo) and the other with white flesh (Tropic Snow). It was found that the concentration of phenolic compounds was higher for yellow-fleshed cultivars Early Grande and Hermosillo (0.97 and 1.12 mg GAE g–1), being 50% lower for Tropic Snow and Flordaking. The values found for the Tropic Snow peach agree with those determined by Gil et al. (2002) for white-fleshed cultivars Champagene and Snow Giant. Moreover, the values determined for Early Grande and Hermosillo were higher than those determined by the same authors for other five cultivars of yellow-fleshed peaches. The antioxidant capacity was higher for the three yellow-fleshed cultivars Early Grande, Flordaking, and Hermosillo (0.32–0.35 mg ascorbic acid equivalent g–1), being 50% lower for Tropic Snow. Despite the lower concentration of phenolic compounds, Flordaking presented the same antioxidant capacity than the other yellowfleshed peaches, possibly due to its carotenoid content (Martín et al. 2011). These results do not agree with those of Gil et al. who that found that the antioxidant capacity of white-fleshed peaches was higher than yellow-fleshed cultivars. Martín et al. (2011) found that Tropic Snow and Hermosillo cultivars, which exhibited the highest acidity and lower polyphenoloxidase (PPO) activity, were those that developed less browning on the cut flesh, despite the significant difference found between their phenol contents. Taking into consideration other determinations, such as higher yield, phenolic content, antioxidant capacity, and good firmness and less browning of the Hermosillo cultivar, it was concluded that it presented a nutritional advantage over the other three cultivars studied for being minimally processed (Martín et al. 2011). Apples are an excellent source of several phenolic compounds and also have high total antioxidant capacity. The nature and distribution of these phenolics differs between the flesh and the peel of the apple. Among others, apple flesh

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contains catechins, procyanidins, phloridzin, phloretin glycosides, caffeic acid, and chlorogenic acid; the peel possesses all of these compounds and has additional flavonoids not found in the flesh, such as quercetin glycosides (Wolfe et al. 2003). Several authors reported that the content of polyphenolic compound varied among the apple cultivars (McGhie et al. 2005; Seipel et al. 2009; Tsao et al. 2003). Piagentini et al. (2009) determined total phenolic content and antioxidant capacity of five apple cultivars for minimal processing. The highest phenolic content in apple flesh was found in the Red Delicious cultivar, followed in decreasing order by Eva, Caricia, Granny Smith, and Princesa. The total phenolic content in apple peel was about two to five times higher than that found for the apple flesh for all cultivars. The peel of Caricia and Red Delicious cultivars had about 1.80 times more concentration than the peel of the lowest cultivar, Eva. The antioxidant capacity of Red Delicious flesh was about 1.75 times greater than the flesh antioxidant capacity of the other four cultivars. It was also found that the apple peel with the highest antioxidant capacity corresponded to Red Delicious, followed by Caricia, Granny Smith, Princesa, and Eva. The antioxidant capacity of apple peel was about two to four times higher than the flesh for all apple cultivars. Other authors also reported that apple peel contained higher phenolics compound concentration and antioxidant activity compared to apple flesh (Droguodi et al. 2008; Wolfe et al. 2003; Tsao et al. 2003; Chinnici et al. 2004, Khanizadeh et al. 2008). A positive correlation was found between total phenolics content and antioxidant activity in flesh and peel tissues, suggesting that phenols have a significant contribution to the total antioxidant capacity of apples. Moreover, some researchers reported that the antioxidant activity of apples depended on their phenolics composition in a qualitative and quantitative way (McGhie et al. 2005 Drogoudi et al. 2008; Piagentini et al. 2009). Browning susceptibility of apple cultivars would be mainly related to the amount and type of phenols and PPO activity together with ascorbic acid content and acidity that may also influence browning (Amiot et al. 1992). Piagentini et al. (2012) reported that Red Delicious apples had higher phenolic content, antioxidant capacity, and browning development than Granny Smith and Princesa cultivars. On the other side, the latter two cultivars showed the highest values of firmness and juiciness. Therefore, according to this work, Princesa and Granny Smith cultivars would be more suitable for minimal processing. The results reported indicated that fresh-cut apple polyphenol consumption could be increased through appropriate cultivar choice and/or avoiding apple peel removal (Wolfe et al. 2003; Chinnici et al. 2004; Hagen et al. 2007; Piagentini et al. 2009). Strawberry is an important source of bioactive compounds due to its high level of vitamin C and phenolics. The main phenolic compounds are anthocyanins, which are responsible for the fruit color, with reported concentrations of up to 0.65 mg g–1 FW (Lopes Da Silva et al. 2007). Van de Velde et al. (2013a) evaluated the health-related compounds of two strawberry cultivars (Camarosa and Selva). It was found that vitamin C content

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ranged from 0.412 to 0.476 mg g–1 FW in the Camarosa cultivar and from 0.287 to 0.510 mg g–1 FW in the Selva cultivar. It was reported that Camarosa strawberries presented higher total phenolic and anthocyanin content, and consequently better antioxidant capacity. Moreover, there were differences in the phenolic compound profiles for both cultivars. The Camarosa cultivar presented higher content of anthocyanidins, and Selva showed higher total ellagic acid content. The anthocyanins and the ellagitannins are the groups with the highest contributions to total antioxidant capacity in strawberries, while it was estimated that vitamin C contributes 15–30% to this capacity (Lopes Da Silva et al. 2007; Pincemail et al. 2012). The content of total ellagic acid in Camarosa was in agreement with the data reported by Williner et al. (2003) for the same strawberry variety (mean value: 0.616 mg g–1 FW). Moreover, total ellagic acid content in both strawberry cultivars was slightly lower than those reported by da Silva Pinto et al. (2008), ranging from 0.17 to 0.47 mg g–1 FW. The higher antioxidant capacity determined in Camarosa strawberries was in accordance with the higher anthocyanin content observed for these berries. However, Selva strawberries presented higher ellagic acid content, which leads to a good level of antioxidant capacity in this cultivar (Van de Velde et al. 2013a). Large differences in ellagic acid content in strawberries have been found among cultivar ripening stages and also among tissues. Green fruit pulp has been found to contain about twice as much ellagic acid as red fruit pulp (Wang et al. 1994; Maas et al. 1991). Williner et al. (2003) determined the ellagic acid content in strawberries and other common fruits, evaluating the effects of ripening stage and the tissue and seasonal variability in selected strawberry cultivars. Strawberries showed significantly higher levels of ellagic acid than many other fruits (apple, banana, kiwi fruits, orange, pear, pineapple, plum, and tangerine), ranging from 0.0163 to 0.178 mg g−1 FW. The ellagic acid content of strawberries was the highest in green fruit, intermediate in midripe fruit, and lowest in fullripe fruit. Cultivars Chandler and Camarosa contained the highest amounts of ellagic acid in strawberries with edible value (0.0684 and 0.0616 mg g−1 FW, respectively), while Oso Grande and Milsei exhibited the lowest (0.0285 and 0.0286 mg g−1 FW, respectively). Moreover, the content of this phytochemical was higher when considering pulp with achenes compared with pulp without achenes (Williner et al. 2003). Oranges have appropriate morphological and physiological characteristics for the preparation of fresh-cut products. They are also a good source of nutritional and bioactive antioxidant compounds, including vitamin C and phenolic compounds (Del Caro et al. 2004; Plaza et al. 2011). The flavanone glycosides, such as hesperidin, naringin, and narirutin, are the most important water-soluble phenols. Other compounds, such as limonoids (triterpene derivatives); some flavones, such as sinensetin and nobiletin; and phenylpropanoids, such as hidroxycinnamates, have high antioxidant potential and health-promoting capacity (Kaur and Kapoor 2001), but the most significant antioxidant is vitamin C.

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Del Caro et al. (2004) reported that the contribution of the total flavonoids to antioxidant activity was much lower than the contribution of vitamin C. However, Plaza et al. (2011) suggested that in addition to vitamin C, it is necessary to take into account the possible synergistic effect of the other phytochemicals. Studies on minimally processed orange fruit have focused on blond cultivars (Del Caro et al. 2004; Plaza et al. 2011; Van de Velde et al. 2013b). However, Rapisarda et al. (2006) evaluated the suitability of three clones of a pigmented (blood) cultivar (Tarocco) to be minimally processed. Tarocco is an orange cultivar with a brilliant red flesh, a distinctive and agreeable fragrance, as well as large size and balanced levels of sugars and acids. In addition, this fruit contains high levels of antioxidant compounds including vitamin C, flavanones, hydroxycinnamic acids, and anthocyanins. It was reported that Tarocco is the orange cultivar that is richest in vitamin C, containing between 60 and 90 mg 100 mL–1. These high levels of antioxidant compounds and distinctive sensory characteristics convert the Tarocco orange in a very suitable cultivar to be minimally processed (Rapisarda et al. 2006).

2.4 Effect of minimal processing: Major operations 2.4.1 Cutting Fresh-cut products are cut in different types of shapes, and the cutting shapes could influence the degree of damage caused in the product (Rivera-Lopez et al. 2005). Van de Velde et al. (2013b) found that, on the day of processing, Valencia Late sweet oranges cut in wedges and slices did not show significant differences in ascorbic acid content, antioxidant capacity, and total phenol content compared with whole oranges. Van de Velde (2012) studied the impact of processing Camarosa strawberries with different degrees of cutting (i.e., whole without hull, half, and quartered). It was found that ascorbic acid, vitamin C, phenolic, and anthocyanin contents were higher in whole without hull fruits, decreasing with the degree of cutting. For instance, quartered strawberries lost 13.3, 16.0, 12.6, and 18.9% more of ascorbic acid, vitamin C, phenolics, and anthocyanins, respectively, than whole strawberries. The greater the injury practiced in the strawberries, the more the oxidation and/or lixiviation of bioactive compounds. However, there were no significant differences on the antioxidant capacity of whole without hull, half, and quartered strawberries. Moreover, Castro et al. (2002) showed that the effect of removing the stem from strawberries (cultivar Camarosa) did not induce changes in the bioactive compounds. However, the effect of cutting strawberries in pieces (from 3 to 18 mm) and the exposure of cut surfaces to air induced losses of ascorbic acid as high as 50% after only 5 minutes, although after 30 minutes the losses were not significantly higher. On the other hand, authors reported that anthocyanin and phenolic content showed a slight decrease in the cut strawberries after 30 minutes.

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2.4.2 Washing–disinfection Chlorine derivates are effective for microbiological decontamination but their reaction with organic matter may result in the formation of carcinogenic halogenated disinfection by-products, such as trihalomethanes and haloacetic acids (Ölmez and Kretzschmar 2009). Based on these concerns, researchers are trying to replace the chlorine in washing–disinfection operation and to incorporate alternative disinfectants to maintain or improve visual quality, shelf-life, and security of fresh-cut products. In this sense, peracetic acid (PAA), commercially available as a quaternary equilibrium of acetic acid, hydrogen peroxide, PAA, and water, has been a suitable alternative taking into account microbiological decontamination (Vandekinderen et al. 2009). Peracetic acid is a sanitizing agent that does not react with proteins to cause toxic or carcinogenic compounds and its decomposition products are only oxygen and acetic acid (Silveira et al. 2008). In this sense, Van de Velde et al. (2013c) proposed to model the changes in total anthocyanin, ascorbic acid, and vitamin C content and color of fresh-cut strawberries from two cultivars (Selva and Camarosa) as a consequence of washing– disinfection by immersion in solutions of peracetic acid at different concentrations (0–100 mg L–1), contact times (10–120 seconds), and temperatures (4–40°C). The reduction of anthocyanin and ascorbic acid content were principally affected by peracetic acid concentration and processing time, both cultivars behaving in the same way. Doing a prediction at 80 mg L–1 PAA, maximum concentration suggested by the United States Code of Federal Regulations (CFR; 2007) for vegetables washing, 120 seconds and 22°C, the total anthocyanin reduction was approximately 30% and the ascorbic acid reduction was approximately 37%. These predicted values demonstrated the possible losses of bioactive compounds after the washing–disinfection operation for both strawberry cultivars. With respect to vitamin C, each strawberry cultivar was affected differently by washing–disinfection variables. Camarosa cultivar lost approximately 10% vitamin C at any condition in the experimental domain assayed. However, in the case of the Selva cultivar, the vitamin C reduction was affected by the levels used in the operation variables. Working at 80 mg L–1 PAA and 120 s, it resulted in a predicted reduction of approximately 30%. Van de Velde et al. (2013d) optimized the fresh-cut strawberries washing–disinfection operation based on bioactive compound retention (ascorbic acid and total anthocyanins) and microbial load reduction. It was concluded that when the objective is to maximize the total microbial reduction with losses in ascorbic acid and total anthocyanins of no more than 10%, the predicted operative variable levels should be set at 100 mg L–1 PAA, 24°C, and 50 seconds. At these conditions, the washed fresh-cut strawberries showed 1.8 log UFC g–1 microbial count reduction and 19.2 and 22.5% anthocyanin and ascorbic acid reduction, respectively. On the other hand, when the objective is to maximize the retention of ascorbic acid and total anthocyanins with moderate microbial reduction, the operative variable levels should be set at 20 mg L–1 PAA, 18°C, and 52 seconds. Washed fresh-cut strawberries at these conditions showed 0.8 log UFC g–1 microbial count reduction, 12.8% reduction

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of total anthocyanins, and 6.7% reduction of ascorbic acid. Based on the latter results and the economic convenience of lesser peracetic acid consumption, the latter conditions were recommended.

2.4.3 Chemical treatment Color and texture are some of the main sensory attributes considered when fresh-cut fruit freshness is evaluated. During the peeling and slicing of fruits, a damage signal happens rapidly, resulting in a change in their sensory properties. One of the color changes that occur in fresh-cut fruit is the result of enzymatic browning, being one of the main factors that limit its shelf-life (Rico et al. 2007; Piagentini et al. 2012; Toivonen and Brummell 2008). Enzymatic browning, which causes changes in the natural fruit color, is due to the enzymatic oxidation of the phenolic compounds by the action of the enzyme polyphenol oxidase (PPO) in the presence of oxygen (Gil et al. 2005). Color preservation is, after safety, an important attribute to be preserved because frequently a product is selected for its appearance, particularly its color. Furthermore, preservation treatments may not only prevent or delay deteriorative reactions, but also maintain or increase bioactive compound content. Control of enzymatic browning has always been a challenge for the food industry. Among the most effective methods for the inhibition or delaying of browning development are treatment with antioxidants, pH adjustment, cooling, heat treatment, and exclusion of oxygen. One of the common ways of solving this problem is the reduction of the quinones formed by PPO or the inhibition or inactivation of PPO. Treatments with antioxidant compounds such as ascorbic acid and their derivatives (alone or in combination with citric acid) have been used in numerous studies on fruits and vegetables in concentrations ranging from 0.5 to 4.0%, and its effect to prevent browning has been shown (Toivonen and Brummell 2008; Piagentini et al. 2012; Rodríguez-Arzuaga et al. 2013). Thus, it has been reported that inhibition of 90–100% of the activity of the enzyme PPO occurs in apple cubes using a solution of 1% ascorbic acid and 0.2% citric acid. Furthermore, the ascorbic acid, acting as a reducing agent, prevents the reduction of phenolic content (Soliva-Fortuny and Martín-Belloso 2003). Cocci et al. (2006) reported that the ascorbic acid and total phenolic content and antioxidant activity of Golden Delicious fresh-cut apples dipped with an aqueous solution of 1% ascorbic acid and 1% citric acid were higher than nontreated samples over the 8 days of refrigerated storage. Similar results were found for Granny Smith and Princesa fresh-cut apples treated with the same antioxidant solution (Depetris et al. 2009). Rodriguez Arzuaga et al. (2013) reported that Granny Smith fresh-cut apples treated with an aqueous solution of 1% citric acid and 1% ascorbic acid and 0.5% calcium chloride had higher values in texture and overall appearance than nontreated apples, and had values of total phenolic content and antioxidant activity similar to the fruit used as raw material after 7 days of refrigerated storage. Acidifying substances as phosphoric, citric, or malic acid can inhibit PPO, reducing the pH and/or chelating the copper present in the fruit (Jiang et al. 2004).

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Reducing agents such as ascorbic acid, cysteine, or sulfites are also used to reduce enzymatic browning, decreasing the concentration of oxygen present. However, these sulfites are questioned because of their adverse health effects. Therefore, it has been established the need to evaluate the inhibitory effect of natural and nontoxic compounds on the activity of enzymes involved in enzymatic browning and potential applicability in the food industry to replace synthetic antioxidants. The inhibitory effect of honey, onion extract, and green tea, among others, on PPO activity was evaluated by several authors (Jeon and Zhao 2005; Kim et al. 2005; Lee et al. 2007; Soysal 2009). The inhibitory activity of some of these natural compounds on the PPO is due to their content in phenolic compounds. On one side, phenolic compounds are oxidized enzymatically, contributing to the browning of fresh-cut fruits. However, on the other hand, these compounds are important secondary metabolites that can act as neutralizing free radicals and a source of natural antioxidants. Nowadays, food industry uses food grade antioxidants, particularly phenols, to prevent spoilage and maintain its nutritional value. Currently, there is a market trend to incorporate natural additives as a response to consumer demands as a simple and effective way to improve the nutritional and sensory quality and extend the shelf-life of foods (De la Rosa et al. 2011). An example of this would be the use of the mate, a widely consumed, traditional beverage in Argentina, Brazil, Paraguay, and Uruguay, which consists of an infusion of dried and ground leaves of yerba mate (Ilex paraguariensis St. Hill., Aquifoliaceae) (Rodriguez Arzuaga and Piagentini 2013; Heck and Mejia 2007). The high content of polyphenols and antioxidant capacity of this infusion make it a potentially useful treatment for inhibiting the development of enzymatic browning in fresh-cut fruits. Martin et al. (2010) applied infusions of 1 and 2% of yerba mate, alone or combined with ascorbic and/or citric acid, to enzymatic extracts of Princesa apple PPO and concluded that yerba mate infusions have a significant inhibitory effect on the enzyme activity, and the combination of it with citric and ascorbic acid increased the enzyme inhibition, showing a synergistic effect. A preliminary study reported that the application to Granny Smith fresh-cut apples of an infusion of 2% yerba mate combined with 1% citric and ascorbic acids reduced browning development, without any detrimental effect on the sensory attributes, increasing their health-related properties. The results showed an increase of about 74% in the phenolic content and 62% in the antioxidant activity with respect to the raw material (Rodriguez Arzuaga and Piagentini 2013).

2.5 Effect of atmosphere composition and temperature during storage Once the product has been prepared (peeled, cut, sanitized, etc.), it must be packaged in bags or trays (Figure 2.1). this technology is commonly known as modified atmosphere packaging (MAP). it can be done with or without injection

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of a protective atmosphere above the product, which are known as active and passive MAP, respectively. This means that the packaged product is exposed to different conditions of temperature, time, and CO2 and O2 gas concentration during storage, transport, distribution, and marketing. The modified atmosphere during storage is the result of the simultaneous action of the respiration of the vegetable product and gas exchange with the atmosphere outside the bag or container. Therefore, a dynamic equilibrium is established through the film until it reaches a stationary atmosphere. The concentrations of oxygen and carbon dioxide that are reached at a given temperature will depend on the weight and size of the product, its respiratory activity, the microbial load, the permeability of the film, and the headspace volume of the package (Sandhya 2010). The selection of the appropriate atmosphere for the cut product depends on the fruit, cultivar, maturity, style of cutting, and other specific product characteristics and storage environmental conditions, mainly temperature. The benefits of modified atmosphere packaging (reduction in respiration rate, inhibition of ethylene production and action, delaying of ripening, and maintenance of sensory quality) for fruits and vegetables have been well described recently by Sandhya (2010) and Zhuang et al. (2014). However, there is little information on the effect of MAP on the nutritional and health potential of fruits. The operative conditions (temperature and gas concentrations) used during MAP could modify bioactive compounds such as vitamin C, carotenoids, and phenolics including anthocyanins. Major effects of traditional passive and active MAP (low oxygen and high carbon dioxide concentration) and special active modified atmosphere on the bioactive compounds of strawberries, oranges, and apples during storage are summarized in Table 2.1. With respect to oranges, Van de Velde et al. (2013b) studied the changes on fresh-cut products in passive MAP for 10 days at 3°C. oranges were processed as wedges or slices. the wedges and slices did not show changes on total phenol content during storage. However, they showed changes in ascorbic acid content and antioxidant capacity. Both ascorbic acid content and antioxidant capacity decreased 30% in this period. the antioxidant capacity presented a high correlation with the ascorbic acid content, which would indicate the great contribution of ascorbic acid to the total antioxidant capacity in citrus fruits. the total phenols were constant during refrigerated storage at 3°C and showed no good correlation with antioxidant capacity, indicating their minor contribution to the healthy potential of citrus. Similarly, Plaza et al. (2011) studied the effect of minimal processing and storage in MAP (equilibrium-modified atmosphere: 19% o2 to 1.6–2.6% Co2) on the health-related attributes of orange fruit. oranges were prepared as whole fruits, hand-peeled fruits, and manually separated segments, and stored at 4°C for 12 days. the total carotenoid content showed a significant increase (88% of the initial value) for the whole oranges after 12 days, whereas no significant changes were observed for segmented or peeled oranges. With regard to vitamin C, there was a significant decrease throughout the 12 days of cold storage for all samples (19–24% for ascorbic acid and 15–23% for total

Orange: wedges

Strawberries and other fresh-cut fruits Strawberries: whole

Special MAP

Air

Active and passive MAP

Special MAP (individual effect and combined with other technologies) Special MAP

Strawberries: quartered

Strawberries: quartered

Orange: Whole, peeled, and segments Orange: wedges and slices

Passive MAP

Passive MAP

Product

Technology of storage

2.5kPa O2 + 7kPa CO2 10kPa O2 + 5kPa CO2 21kPa O2 60 kPa O2 80 kPa O2 21 days at 4°C

MAPs: 80% O2 and 10% CO2 both compensated with N2. 12 days 2°C Initial composition 80 kPa O2 and temperature (5-20°C).

MAPs: 75% O2 and 60% O2 compensated with N2. 8 days at 2°C 6 days at 5°C

10 days at 3°C

12 days at 4°C

Treatment conditions

Losses of anthocyanins and antioxidant capacity were estimated by a Weibull kinetic model, and losses of vitamin C by first-order kinetic model. The temperature dependency of rate constants for each antioxidant property was modeled through the non-Arrhenius approach. At >21kPa O2: Quercetin content enhanced, phenolic acids (p-coumaric, hydroxibenzoic and ellagic acid) and vitamin C greater losses. Anthocyanins increased at hot-water > cold-water. The G. frondosa hot-water extract showed high scavenging ability on superoxide anions. Total phenols, flavonoids, ascorbic acid, and α-tocopherol are the major antioxidant components found in the various Grifola frondosa extracts. Based on EC50 values ( hot-water > cold-water extracts. Scavenging abilities of water extracts from three samples on hydroxyl radicals were 53.4–80.1% at 20 mg/mL. Chelating abilities of cold- and hot-water extracts on ferrous ions were higher than those of ethanolic extracts. Contents of total phenols were in a descending order: fruit bodies (8.62–12.38 mg/g) > mycelia (5.84–7.85 mg/g) > filtrate (4.80– 5.57 mg/g). Overall, three extracts from fruit bodies were more effective in antioxidant properties assayed than those from mycelia and filtrate. Ethanolic extracts were more effective in antioxidant properties assayed, except for scavenging abilities on hydroxyl radicals (Lee et al. 2007). The antioxidant activity of fruiting bodies of four edible mushrooms (Pleurotus sajor-caju, Volvariella volvaceae, Agaricus bisporus, and Pleurotus ostreatus) was investigated. The antioxidant activity, peroxidase, number of ascorbate oxidase units, and catalase activity were significantly more with Agaricus bisporus than the other edible mushrooms used in the study. All the activities measured are to find out for the presence of reactive oxygen species, which play an important role in cell death and signal transduction (Surekha et al. 2011). Pleurotus ostreatus M2191 and PBS281009 cultivated using the batch system produced an average of between 0.1–2 Exopolysaccharides (EPS) and 0.07– 1.5 g/L/day intracellular polysaccharides (IPS). The antioxidant effect is relatively similar to that of the ascorbic acid, particularly for PBS281009. The

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antioxidant activity of the polysaccharides included the scavenging activity against DPPH and ABTS radicals, the reducing power, as well the chelating ability. A good correlation between the DPPH and ABTS scavenging activity was determined (Vamanu 2012). Analyses from different stages of Pleurotus djamor (Fr.) Boedijn. revealed that it contains 1.2–2.7 mg/g of total phenols on a fresh-weight basis. Quantitative estimation of total phenols and antioxidants revealed that the species showed highest amount of phenols and antioxidants in juvenile bud stage (1-day stage) and gradually decreased, but at post-mature (4 days old) condition, amount of total phenol again increased (Saha et al. 2012). The bioluminescent mushroom Omphalotus nidiformis reported the occurrence of active compounds such as phenol, flavonoid, alkaloid, terpenoid, and saponins. The total phenolic content of the methanol extract was 1.901 ± 0.011 mg gallic acid equivalent/g of extract. The total flavonoid content was estimated as 0.29 mg quercetin equivalent/g of dried methanol extract. The dot-blot assay and TLC-DPPH screening method indicated the presence of antioxidant compounds. Radical scavenging activity of the mushroom (IC50 450 μg/mL) was also observed (Shirmila and Radhamany 2012). Total phenolic compounds and antioxidant activity of five popular Vietnamese edible mushrooms (Pleurotus ostreatus, Volvariella volvacea, Lentinula edodes, Auricularia polytricha, and Ganoderma lucidum) were studied. All mushrooms, except Auricularia polytricha, contained large amounts of total free phenolic compounds, which exhibited significant antioxidant capacity of the extracts. In addition, the total bound phenolic content of Ganoderma lucidum was also high, resulting in high antioxidant capacity of the extract (Hung and Nhi 2012). The acetone, methanol, and hot-water extracts of Lentinus lepideus were prepared and assayed for their antioxidant activity. The hot-water extract showed the strongest β-carotene-linoleic acid inhibition compared to the other extracts. At 8 mg/mL, the methanolic extract showed a high reducing power. The acetone and methanol extracts were more effective in scavenging DPPH radicals than the hot-water extract. The strongest chelating effect was obtained from the methanolic extract. Gallic acid, chlorogenic acid, vanillin, naringin, naringenin, formononetin, and biochanin-A were detected in the acetonitrile and hydrochloric acid (5:1) solvent extract (Yoon et al. 2011). Some mushrooms were collected from different sites/forest areas of south Kashmir and their antioxidant activity was evaluated. Sarcoscypha coccinia showed the highest antioxidant activity, followed by Cantharella cibarius, Bovista plumbea, Coprinus comatus, and C. atramentarius, respectively. All the extracts of mushrooms showed positive correlation with the standard oxidant, catechol (Wani et al. 2010). Antioxidant activity of Cantharellus friessi, Cantharellus subcibarius, and Cantharellus cinerius collected from the northwestern Himalayan region of India was compared with Pleurotus florida. The total phenol contents showed major

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antioxidants components ranging from 9.55 to 16.8 mg/g in different mushrooms. The antioxidant activity of Cantharellus friessi was significantly higher than other mushrooms (Kumari et al. 2011). The methanolic extracts of 24 dried wild edible mushroom (Agaricus bisporus, Chlororhyllum rhacodes, Macrolepiota procera var. procera, Amanita rubescens var. rubescens, Pleurotous dryinus, Armillaria ostoyae, Pleurotous ostreatus, Polyporus squamosus, Boletus edulis, Boletus pseudosulphureus, Leccinum scabrum, Suillus luteus, Lepista nuda, Lepista personata, Hydnum repandum, Lactarius deliciosus, Lactarius piperatus, Lactarius salmonicolor, Lactarius volemus, Russula delica, Russula integra var. integra, Russula nigricans, Russula vinosa, and Boletus erythropus var. erythropus) were analyzed for antioxidant activity in different assays, namely, ferric antioxidant reducing power (FRAP), scavenging activity on DPPH radicals, and total phenolic content. The extracts from Leccinum scabrum showed the most potent radical scavenging activity, showing 97.96%. The EC50 of Pleurotous dryinus and Lactarius piperatus methanolic extracts were 24.71 and 24.12 mg/mL, respectively. Total phenolics in the methanolic extracts were the highest in Boletus edulis. On the other hand, dry matter and ascorbic acid were determined in 24 dried wild edible mushrooms. The determined amounts of ascorbic acid and total phenolic compounds found in the mushroom extracts were of very low concentrations (Keleş et al. 2011). The antioxidant activity of mycelia from 21 wild mushrooms—Agaricus bresadolanus, Auricularia auriculajudae, Chroogomphus rutilus, Fomes fomentarius, Ganoderma lucidum, Gloeophyllum trabeum, Gymnopus dryophilus, Infundibulicybe geotropa, Inocybe flocculosa var. crocifolia, Inocybe catalaunica, Lentinula edodes, Lentinus sajor-caju, Lycoperdon excipuliforme, Macrolepiota excoriata, Morchella esculenta var. rigida, Morchella intermedia, Omphalotus olearius, Pleurotus djamor, Postia stiptica, Rhizopogon roseolus, and Stropharia inuncta—were investigated. Antioxidant properties of ethanol, chloroform, and water extracts of these 21 mycelia were studied by two methods: the scavenging activity against DPPH and ABTS radicals. Among the 21 mushroom extracts, Omphalotus olearius displayed the most potent antioxidant activity (Kalyoncu et al. 2010). Three species of medicinal mushrooms are commercially available in Taiwan: Ganoderma lucidum (Ling-chih), Ganoderma tsugae (Sung-shan-ling-chih), and Coriolus versicolor (Yun-chih). Methanolic extracts were prepared from these medicinal mushrooms and their antioxidant properties studied. At 0.6 mg/mL, Ganoderma lucidum, Ganoderma lucidum antler, and Ganoderma tsugae showed excellent antioxidant activity (2.30–6.41% of lipid peroxidation), whereas Coriolus versicolor showed only 58.56%. At 4 mg/mL, reducing powers were in the order Ganoderma tsugae (2.38) ≈ Ganoderma lucidum antler (2.28) > Ganoderma lucidum (1.62) > Coriolus versicolor (0.79). At 0.64 mg/mL, scavenging effects on the DPPH radical were 67.6–74.4% for Ganoderma and 24.6% for Coriolus versicolor. The scavenging effect of methanolic extracts from Ganoderma lucidum and Ganoderma lucidum antler on hydroxyl radical was the highest (51.2 and 52.6%) at 16 mg/mL,

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respectively. At 2.4 mg/mL, chelating effects on ferrous ion were in the order Ganoderma lucidum antler (67.7%) > Ganoderma lucidum (55.5%) > Ganoderma tsugae (44.8%) > Coriolus versicolor (13.2%). Total phenols were the major naturally occurring antioxidant components found in methanolic extracts from medicinal mushrooms. Overall, Ganoderma lucidum and Ganoderma tsugae were higher in antioxidant activity, reducing power, scavenging and chelating abilities, and total phenol content (Mau et al. 2002). Tolypocladium sp. Ts-1 was isolated from the fruiting body of a wild Cordyceps sinensis, one of the best known traditional Chinese medicine and health foods. The antioxidant activities of hot-water extracts from cultured mycelia of Tolypocladium sp. were assessed. The extracts showed superoxide dismutase activity of 35.6 U/mg protein and are effective in scavenging superoxide radical in a concentration-dependent fashion with IC50 value of 1.3 mg/mL. DPPH radical scavenging activities of the extracts reached more than 75.2% at the concentrations of 3–6 mg/mL. The extracts showed moderate reducing power and ferrous ion chelating activity (Zheng et al. 2008).

8.2.2 Antihypercholesterolemic and antihyperlipidemic compounds The main plasmatic lipids including cholesterol and triglycerides circulate associated to proteins and are transported as macromolecular complexes called lipoproteins. People with hyperlipidemia have high levels of blood lipids, putting them at risk for heart disease or stroke. Hypercholesterolemia is the presence of elevated levels of cholesterol in the blood. Many years of high cholesterol lead to accelerated atherosclerosis, which may be expressed in a number of cardiovascular diseases: coronary artery disease (angina, heart attack), stroke and ischemic stroke, and peripheral vascular disease. There are some recommendations on dietary and life habits together with the use of drugs to decrease the cholesterol and lipids in the blood. However, studies of the production of bioactive compounds in fungi with antihyperlipidemic and antihypercholesterolemic effects has increased in recent years. Lovastatin is a hypocholesterolemic agent and has been demonstrated as a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-Co A) reductase in cholesterol biosynthesis. Lovastatin decreases the plasmatic concentration of low-density lipoprotein (LDL) by regulating cholesterol synthesis and consequently synthesis of LDL receptors. Currently there are five statins in clinical use. Lovastatin and pravastatin (mevastatin derived) are natural statins of fungal origin, while symvastatin is a semisynthetic lovastatin derivative. Atorvastatin and fluvastatin are fully synthetic statins, derived from mevalonate and pyridine, respectively. In addition to the principal natural statins, several related compounds, monacolins and dihydromonacolins, isolated fungal intermediate metabolites, have also been characterized. All natural statins possess a common polyketide portion, a hydroxy-hexahydro naphthalene ring system, to which different side chains are linked. The

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biosynthetic pathway involved in statin production, starting from acetate units linked to each other in head-to-tail fashion to form polyketide chains, has been elucidated by both early biogenetic investigations and recent advances in gene studies. Natural statins can be obtained from different genera and species of filamentous fungi. Lovastatin is mainly produced by Aspergillus terreus strains, and mevastatin by Penicillium citrinum. Pravastatin can be obtained by the biotransformation of mevastatin by Streptomyces carbophilus and simvastatin by a semi-synthetic process involving the chemical modification of the lovastatin side chain (Manzoni and Rollini 2002). Statins including lovastatin reduce vascular smooth muscle cell proliferation and migration, prevent LDL oxidation and foam cell formation, reduce inflammatory response associated to atherosclerosis, normalize coagulation and fibronolysis, and improve endothelial function. All these properties seem to be mediated by intermediary isoprenoid compounds from the HMG-Co A reductase metabolic pathway and do not depend on cholesterol concentration in the medium (Álvarez et al. 1999). Lovastatin was evaluated in mushrooms. Among fruiting bodies, Pleurotus ostreatus (Japan) and Agaricus bisporus contained the highest amount of lovastatin (606.5 and 565.4 mg/kg, respectively). Among mycelia, Cordyceps sinensis and Antrodia salmonea contained the highest (1365 and 1032 mg/kg, respectively) (Chen et al. 2012). The effect of carbon and nitrogen sources on lovastatin production by Aspergillus terreus was studied. Glutamate and histidine gave the highest lovastatin production level. When glucose and glutamate were present together, lovastatin synthesis was initiated when glucose consumption leveled off. When Aspergillus terreus grew on lactose, lovastatin production was initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A three-fold higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract (Hajjaj et al. 2001). The production of cholesterol synthesis—inhibiting molecules, by five different strains of Aspergillus oryzae grown on a complex liquid medium, was studied. After growing these strains in crude organic-phase extracts and specific fractions, compounds were found that inhibit the cholesterol synthesis in human hepatic T9A4 cells in vitro at enzyme sites downstream of dihydrolanosterol. This was evidenced by using different radioactively labeled precursors, namely acetate, mevalonate, 24,25-dihydro- [24,25-3H2]-lanosterol, or [3-3H]-lathosterol (Hajjaj et al. 2005). Lovastatin production by Aspergillus terreus in both shaking-flask and 5-L fermentor cultivations in presence of oxygen carriers (n-dodecane, n-tetradecane, or n-hexadecane) was studied. In the shaking-flask cultivation, an addition of 2.5% (w/v) n-dodecane to the medium gave about a 1.4-fold increase (0.51 g/L)

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in lovastatin production. This improvement was related to morphological changes in the fungal cells, and resulted mainly from the formation of small, uniform, compact pellets of Aspergillus terreus. In contrast, in the 5-L fermentor cultivation, lovastatin production was only one-ninth of that achieved without the n-dodecane addition independent of the pellet density. This adverse effect of adding the oxygen carrier to the medium was attributed to an uncontrolled, high dissolved oxygen level (higher than 60%), which resulted in unfavorable morphological changes and the formation of star-like pellets. However, when this dosage of n-dodecane was added 24 hours after the start of the 5-L fermentor cultivation, lovastatin production was further enhanced due to its foam suppressing activity in the fungal fermentation (Lai et al. 2002). Lovastatin production by Aspergillus terreus JPM 3 grown in both solid-state and submerged fermentation was evaluated. Solid-state fermentation was better than submerged fermentation. The lovastatin yield by Aspergillus terreus isolate JPM 3 was higher in the case of wheat bran as a substrate (982.3 μg/g). Wheat bran was found to be the best solid substrate for increased lovastatin production, followed by sorghum grains, rice bran, and paddy straw (Jaivel and Marimuthu 2010). The effect of agitation and aeration regimens in stirred bioreactors on the fungal pellet morphology, broth rheology, and lovastatin production of Aspergillus terreus ATCC 20542 was studied. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology, and the lovastatin titers. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin (Casas-López et al. 2005). The effect of some physical and chemical factors on lovastatin production was investigated. Shaking conditions showed negative effect on lovastatin production to a great extent. The highest yield has been observed in 8 days’ incubation. Fermentation at 30 °C was optimal for lovastatin production. Lovastatin productivity was optimal at alkaline pH 8.5. The highest level for lovastatin production has been found in cultures grown on oat meal. However, the use of glucose as a carbon source resulted in a repression of lovastatin productivity. Oat meal (20 g/L) was the optimal concentration for lovastatin production. The use of urea as a nitrogen source in the production medium led to an increase in lovastatin production. Also, methionine as an amino acid resulted in an increase in lovastatin production. Nutritional improvement increases the productivity level 3.45 times compared to the original fermentation medium (54.5 μg/mL) (Osman et al. 2011).

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The antihypercholesterolemic and antioxidative effects of ethanolic extract from Pleurotus ostreatus were evaluated. Oral administration of the mushroom extract (500 mg/kg b.wt.) and chrysin (200 mg/kg b.wt.) to hypercholesterolemic rats for 7 days resulted in a significant decrease in mean blood/serum levels of glucose, lipid profile parameters, and hepatic marker enzymes and a concomitant increase in enzymatic and nonenzymatic antioxidant parameters. The hypercholesterolemia-ameliorating effect was more pronounced in chrysintreated rats than in extract-treated rats, being almost as effective as that of the standard lipid-lowering drug, lovastatin (10 mg/kg b.wt.) (Anandhi et al. 2012). Phytosterols and β-glucans have been shown to inhibit the absorption of exogenous cholesterol. Edible mushrooms are good sources of phytosterol-like structures such as ergosterol, fungisterol, and other derivatives since they are constitutive compounds of the hyphae membranes. On the other hand, edible mushrooms contained polysaccharides and, depending on the species, they showed high levels of β-glucans. Some mushroom species have certain molecules different from lovastatin (since statins were not detected) capable of affecting endogenous cholesterol synthesis by inhibiting the HMG-Co A reductase (Gil-Ramirez 2011). Lentinus lepideus has been widely used for nutritional and medicinal purposes. The effects of dietary Lentinus lepideus on plasma and feces biochemical and on the liver histological status were investigated in hypercholesterolemic rats. A diet containing 5% Lentinus lepideus fruiting bodies reduced plasma total cholesterol, triglyceride, low-density lipoprotein, total lipid, phospholipids, and the ratio of low-density to high-density lipoprotein. Body weight was reduced. The diet did not adversely affect plasma biochemical and enzyme profiles. Lentinus lepideus reduced significantly plasma β- and pre-β-lipoprotein, while α-lipoprotein content was increased (Yoon et al. 2011). Comparative effects of oyster mushrooms on plasma and fecal lipid profiles and on liver and kidney function were evaluated. Feeding of hypercholesterolemic rats a 5% powder of Pleurotus ostreatus, Pleurotus sajor-caju, and Pleurotus florida reduced the plasma total cholesterol level by 37%, 21%, and 16%, respectively, and reduced the triglyceride level by 45%, 24%, and 14%, respectively. LDL/HDL ratio decreased by 64%, 45%, and 41% for Pleurotus sajor-caju, Pleurotus ostreatus, and Pleurotus florida fed rats, respectively. Mushroom feeding also reduced body weight in hypercholesterolemic rats. However, it had no adverse effect on plasma bilirubin, creatinin, and urea nitrogen level. Mushroom feeding also increased the total lipid and cholesterol excretion in the feces (Alam et al. 2009). The feeding of 5% powder of the fruiting bodies of Pleurotus eryngii to hypercholesterolemic rats reduced their plasma total cholesterol, triglyceride, low-density lipoprotein, total lipid, phospholipids, and LDL/ HDL ratio by 24.05%, 46.33%, 62.50%, 24.63%, 19.22%, and 57.14%, respectively (Alam et al. 2011).

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8.2.3 Antitumor compounds Mushrooms have proven to be a very important source of bioactive compounds and it is also of great importance those showing antitumor and immunostimulating activity, since tumor diseases are a major global health problem. Antitumor compounds are both of low and high molecular weight. Recently, some of these compounds identified by their activity were confirmed. Basidiomycetes mushrooms contain biologically active polysaccharides in fruit bodies, cultured mycelium, and culture broth. These polysaccharides with antitumor action are of the group β-glucans; these have β-(1 → 3) linkages in the main chain of the glucan and additional β-(1 → 6) branch points. High-molecularweight glucans appear to be more effective than those of low molecular weight. Their activity is especially beneficial in clinics when used in conjunction with chemotherapy. Mushroom polysaccharides prevent oncogenesis, show direct antitumor activity against various allogeneic and syngeneic tumors, and prevent tumor metastasis. Polysaccharides from mushrooms do not attack cancer cells directly, but produce their antitumor effects by activating different immune responses in the host. The antitumor action of polysaccharides requires an intact T-cell component; their activity is mediated through a thymus-dependent immune mechanism (Wasser, 2002). The Polyporaceae family has known effects against cancers of the stomach, esophagus, and lungs. Antitumor polysaccharides are considered to be biological response modifiers or immunopotentiators because of their action mechanism. In Japan three different polysaccharide antitumor agents have been isolated from the fruiting body, mycelium, and cultured medium from three mushroom species. As the result of screening for growth inhibition of cultured cancer cells, some terpenoids, steroids, and other compounds have been found to have antitumor activity. These compounds, low-molecular-weight components, exhibit cytotoxicity and are being considered as chemotherapy agents (Mizuno et al. 1995). TML-1 and TML-2 were two lectins isolated from the mushroom Tricholoma mongolicum. They did not differ appreciably in their pH stability and cationic requirement for hemagglutinating activity. They both stimulated the production of nitrite ions and activated the macrophages in mice. The two lectins were able to inhibit the growth of implanted sarcoma 180 cells by 68.84% and 92.39%, respectively. The growth of tumor cells in the mouse peritoneal cavity was also inhibited by the two lectins with TML-2 expressing a greater potency (Wang et al. 1996). Ganoderma species have long been used traditionally as an immunomodulating and antitumor agent, with recent studies validating its use in chemotherapeutics. Crude dichloromethane, ethanol, water, and polysaccharide extracts of Ganoderma lucidum were evaluated for their ability to suppress the expression of HPV 16 E6 (the E6 region of the HPV 16 genome encodes for an oncoprotein responsible for the pathogenesis of the disease). All crude extracts of Ganoderma lucidum presented HPV 16 E6 suppression, with the dichloromethane extract being the most active

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compared to the other crude extracts. The crude dichloromethane extract of Ganoderma lucidum possesses flavonoids, terpenoids, phenolics, and alkaloids (Lai et al. 2010). Antiproliferative and immunomodulatory properties of a protein fraction designated as Cibacron blue affinity eluted protein (CBAEP) isolated from fruit bodies of Termitomyces clypeatus, Pleurotus florida, Calocybe indica, Astraeus hygrometricus, and Volvariella volvacea were evaluated. This protein fraction (10–100 μg/ mL) mediated antiproliferative activity on several tumor cell lines through the induction of apoptosis. Also, the isolated protein fraction from all five mushrooms had a stimulatory effect on splenocytes, thymocytes, and bone marrow cells. Furthermore, it enhanced mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide (NO). The highest immunostimulatory activity was determined in the CBAEP from Termitomyces clypeatus and the highest antiproliferative activity from Calocybe indica (Maiti et al. 2008). The water-soluble polysaccharide (POP), with a molecular mass of 2.4 × 104 Da, was obtained from the fruiting body of Pleurotus ostreatus. Structure features of the purified polysaccharide were investigated by a combination of chemical and instrumental analysis. Preliminary tests in vitro showed POP is capable of enhancing concanavalin A (ConA)- or lipopolysaccharide (LPS)-induced lymphocyte proliferation, which suggested that POP could be a potential immunostimulating agent for use in functional foods or medicine against both pathogens and cancer (Sun and Liu 2009). Genus Paecilomyces sp. isolated from the inner barks of three kinds of pharmaceutical plants, Taxus mairei, Cephalataxus fortune, and Torreya grandis, has a high positive rate of antitumor and antifungal activity. Antitumor activity was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and antifungal activity was determined by observing fungal growth inhibition (Huang et al. 2001). Twenty-eight fungal endophytes were isolated from agarwood (Aquilaria sinensis) by strict sterile sample preparation and were classified into 14 genera and four taxonomic classes (Sordariomycetes, Dothideomycetes, Saccharomycetes, and Zygomycetes) based on molecular identification. Of the 28 isolates, 13 (46.4%) showed antimicrobial activity against at least one of the test strains by the agar well diffusion method, and 23 isolates (82.1%) displayed antitumor activity against at least one of five cancer cell lines by MTT assay (Cui et al. 2011). Alkaline-soluble antitumor polysaccharide was prepared from the cell wall of the mushroom Flammulina velutipes. The backbone(s) of the polysaccharide is mainly composed of β-(1 → 3)-D-linked glucose and its molecular weight was estimated to be about 200 kD. The polysaccharide was found to be nontoxic by brine shrimp assay. When injected into mice intraperitoneally, the polysaccharide triggered proliferation of splenic lymphocytes and also vascular dilation and hemorrhage (VDH) response. The polysaccharide exhibited potent antitumor activity against sarcoma SC-180 in vivo but not in vitro (Leung et al. 1997).

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It has been reported that the degree of beta-linked branching at position 6 in glucans was remarkably related to the antitumor activity. Structure and antitumor activity of Smith-type degradation products (OL-2-I, OL-2-II, and OL-2-III) of an alkali-soluble glucan, OL-2, isolated from a crude fungal drug “Leiwan” (Omphalia lapidescens) were investigated. Methylation analysis suggested that OL-2-I was a (1 → 3)-β-D-glucan with approximately one branch at every three main chain glucosyl units at each C-6 position; OL-2-II was a (1 → 3)-β-D-glucan with approximately one branch at 24 main chain glucosyl units at each C-6 position (number of all main chain glucosyl units is on average). OL-2-I, OL-2-II, and OL-2-III, which were Smith-type degradation products of OL-2, showed potent antitumor activity against the solid form of sarcoma 180 in ICR mice. These results indicated that the degree of beta-linked branching at position 6 was remarkably related to the antitumor activity (Saito et al. 1992). Fomes fomentarius, grown in submerged fermentation produced under optimal culture condition, the maximum exopolysaccharide concentration reached 3.64 g/L, which is about four times higher than that obtained in the basal medium. Furthermore, the EPS from Fomes fomentarius has a direct antiproliferative effect in vitro on SGC-7901 human gastric cancer cells in a dose- and timedependent manner. Moreover, it was about three times that exopolysaccharide at noncytoxicity concentration of 0.25 mg/mL could sensitize doxorubicin (Dox)-induced growth inhibition of SGC-7901 cells after 24 hours of treatment (Chen et al. 2008). Fomes fomentarius produced 2.86 g/L of intracellular polysaccharide in a 15 L stirred tank bioreactor, which were about twice that of the basal medium. Furthermore, the ethanol extract of mycelia and intracellular polysaccharide had a direct antiproliferative effect on human gastric cancer cell lines SGC-7901 and MKN-45 in a dose-dependent manner. In contrast, human normal gastric cell line GES-1 was less susceptible to extract of mycelia and intracellular polysaccharide (Chen et al. 2011). Water-soluble polysaccharides were extracted by alcohol precipitation from the fermented broth of edible Pleurotus citrinopileatus. These extracts, referred to as SPPC, had a molecular mass of more than 105 Da and were largely made up of glucose and mannose. SPPC was fed to mice that had artificial pulmonary metastatic tumors. Changes in the percentage of the numbers of tumor cells and immune cells were determined by flow cytometry. Daily feeding of SPPC at a dosage of 50 mg/kg to tumor-bearing mice for 12 days resulted in a significant increase in the number of T cells, CD4+ cells, CD8+ cells, and macrophages, compared with mice that were not fed any SPPC. The proliferation rate of the pulmonary sarcoma lesions slowed down (Wang et al. 2005). A glucan extracted with hot water from the basidiomycete Pleurotus pulmonarius had potent anti-inflammatory and analgesic (antinociceptive) activities, possibly by the inhibition of pro-inflammatory cytokines (Smiderle et al. 2008).

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8.2.4 Antibacterial compounds Aureobasidium pullulans, Citeromyces matritensis, Cryptococcus laurentii, Rhodotorula glutinis, and Sporobolomyces roseus produced antibacterial compounds inhibitory to both Pseudomonas fluorescens and Staphylococcus aureus in an overlay bioassay. In  contrast, isolates of Candida albicans, Filobasidium uniguttulatum, Saccharomyces cerevisiae, Torulaspora delbruckii, Tremella foliacea, Trichosporon beigelii, and Trichosporon dulcitum obtained from soil or from culture collections did not produce inhibitory compounds when screened by the same procedure. Two distinct antibiotics were produced by an isolate of Aureobasidium pullulans in liquid culture during both the logarithmic and the stationary phases of growth (McCormack et al. 1994). Ganoderma applanatum collected from Tamarix aphylla trees in southern Iraq was used to isolate, purify, and identify two bioactive chemical compounds (G1 and G2). The chemical formula of G1 is C20H34O4, which belongs to the Tanin group and G2 is C21H28O2 and belongs to the Terpenoides group. The antimicrobial bioactivities of the purified compounds against bacterial strains Escherichia coli and Staphylococcus aureus were tested using a disc diffusion agar method. Purified G1 and G2 compounds exhibited good bioactivities against the tested bacteria (Muhsin et al. 2011). Two edible Nigerian macro-fungi Lycoperdon pusilum and Lycoperdon giganteum were assayed in vitro for their antimicrobial activities using water, methanol, and ethanol as extractive solvents. The best inhibitory zone was recorded in ethanol extract of Lycoperdon giganteum against Proteus vulgaris. The best antifungal activity was recorded in Lycoperdon giganteum extract against Microsporum boulardii. The minimum inhibitory concentration for the ethanolic extract was between 0.75 and 4.0 mg/mL for bacteria, and between 9.00 and 13.75 mg/mL for fungi (Jonathan and Fasidi 2003). Seven coumarins were isolated from CH2Cl2 and MeOH extracts from aerial parts of Mexican tarragon (Tagetes lucida Cv. Asteraceae: Campanulatae). The antibacterial activity of these compounds was determined on Bacillus subtilis, Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Salmonella typhi, Salmonella sp., Shigella boydii, Shigella sp., Enterobacter aerogenes, Enterobacter agglomerans, Sarcina lutea, Staphylococcus epidermidis, Staphylococcus aureus, Yersinia enterolitica, Vibrio cholerae (three El Tor strains—CDC-V12, clinic case, and INDRE-206—were obtained from contaminated water), and V. cholerae (NO-O1). The most active compounds against Gram-positive and -negative bacteria were the dihydroxylated coumarins. In addition, they showed interesting activity against Vibrio cholerae. Coumarins were the most effective compounds against Gram-negative bacteria. The extract MeOH/ CH2Cl2 (1:4) at 0.4 μg/disk inhibited the growth of E. coli and P. mirabilis (40%), K. pneumoniae (31.1%), Salmonella sp. (35.5%), and Shigella sp. (0%) at 72 hours of culture (Céspedes et al. 2006). The antimicrobial activity of 10% ethyl acetate extract of fruiting bodies of Pleurotus sajor-caju, Volvariella volvaceae, Agaricus bisporus and Pleurotus ostreatus were investigated. Agaricus bisporus and Pleurotus ostreatus showed significant

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inhibitory activity against Staphylococcus aureus, which is a normal inhabitant in humans (Surekha et al. 2011). The antibacterial and antifungal activity of methanol and aqueous extract of fruit bodies from Phellinus on five selected bacterial pathogens—Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, and Streptococcus mutans—and five fungal strains—Penicillium sp., Aspergillus fumigatous, Aspergillus niger, Aspergillus flavus, and Mucor indicus—were evaluated. The fruit body of Phellinus showed potential antimicrobial activities against the selected strains and maximum inhibition zone 42 mm was recorded from 200 mg of aqueous extract of Phellinus fruit body against Pseudomonas aeruginosa and minimum (5 mm) by the above pathogen at 50 mg of methanol extract. The methanolic extract showed the maximum antifungal activity 35 mm inhibition zone was recorded from 200 mg of extract against Aspergillus flavus and minimum 3 mm by 50 mg of extract against Penicillium sp. (Balakumar et al. 2011). Fungal endophytes (Cordyceps memorabilis, Phomopsis longicolla, Dothideomycete sp., and one unindentified) isolated from Trichilia elegans were assayed against five pathogenic bacteria. The extraction by ethyl acetate from Cordyceps memorabilis inhibited growing of Enterococus hirae, Micrococcus luteus, and Escherichia coli. Fungal Phomopsis sp. inhibited Micrococcus luteus, Enterococus hirae and Salmonella typhi, Dothideomycetes sp., and G8-25 inhibited Micrococcus luteus and Enterococus hirae (Rhoden et al. 2012).

8.2.5 Antiviral compounds Medicinal mushroom antivirals are a diverse group of chemical structures ranging from relatively low-molecular-weight compounds to high-molecular-weight carbohydrates and proteins. There is a paucity of information on the manner in which these antivirals interfere with viral replication. Mushroom-derived triterpenes and ubiquitin-associated peptides are examples of novel structures with potentially unique mechanisms of antiviral activity (Pirano 2006). A new highly oxygenated triterpene named ganoderic acid alpha has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with 12 known compounds. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of  7.8 μg/mL for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3  beta-5 alpha-dihydroxy-6 beta-methoxyergosta7,22-diene, ganoderic acid alpha, ganoderic acid H, and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17–0.23 mM (El-Mekkawy et al. 1998). A protein of 10 425 Da was purified from the edible mushroom Rozites caperata and shown to inhibit herpes simplex virus types 1 and 2 replication with an IC50 value of ≤5 μM. The protein designated RC-183 also significantly reduced the severity of HSV-1-induced ocular disease in a murine model of keratitis, indicating in vivo efficacy. HSV mutants lacking ribonucleotide reductase and thymidine kinase were

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also inhibited, suggesting the mechanism does not involve these viral enzymes. Antiviral activity was also seen against varicella zoster virus, influenza A virus, and respiratory syncytial virus, but not against adenovirus type VI, coxsackie viruses A9 and B5, or human immunodeficiency virus (Piraino and Brandt 1999). A peptide isolated from the enzymic hydrolysate of oyster Crassostrea gigas has high inhibitory activity on herpetic virus (Zeng et al. 2008). The aqueous extract of Agaricus blazei Murill ss. Heinem was assessed to its antiviral action against herpes simplex type 1 (HSV-1) and bovine herpes type 1 (BoHV-1) in HEp-2 cell culture. Viral replication inhibition was evaluated by plaque assay and immunofluorescence test. The extract demonstrated virucide action for both viruses, being more effective against HSV-1, inhibiting its infectivity in 78.4 and 73.9% at the concentrations of 50 and 100 μg/mL, respectively; moreover, reduction in 47% the number of fluorescent cells was observed for both concentrations. The extract also showed discrete therapeutic activity (Bruggemann et al. 2006). Two water-soluble substances, GLhw and GLlw, and eight methanol-soluble substances, GLMe-1–8, were prepared from carpophores of Ganoderma lucidum. These substances were examined for their antiviral activities against five strains of pathogenic viruses such as HSV-1 and HSV-2, influenza A virus, and vesicular stomatitis virus (VSV) Indiana and New Jersey strains in vitro. Antiviral activities were evaluated by the cytopathic effect inhibition assay and plaque reduction assay. Five substances—GLhw, GLMe-1, -2, -4, and -7—significantly inhibited the cytopathic effects of HSV and VSV. In the plaque reduction assay, GLhw inhibited plaque formation of HSV-2 with 50% effective concentrations of 590 and 580 μg/ml in Vero and HEp-2 cells, and its selectivity indices (SI) were 13.32 and 16.26. GLMe-4 did not exhibit cytotoxicity up to 1000 μg/mL, whereas it exhibited potent antiviral activity on the VSV New Jersey strain with an SI of more than 5.43 (Eo et al. 1999).

8.2.6 Antihyperglycemic effect Diabetes is a global epidemic, characterized by failures in the production or action of insulin, producing chronic hyperglycemia and altering the metabolism of carbohydrates, fats, and proteins. Diabetes represents a big health problem and health care in many cases is insufficient. A lot of research is being done to find alternatives to current treatments. Some studies have shown that fungi have hypoglucemientes capabilities, which represent a potential alternative to help combat this epidemic. The effect of Maitake (Grifola frondosa) on insulin concentration in streptozotocin-induced diabetic rats was evaluated. It was postulated that the bioactive substances present in Maitake can ameliorate the symptoms of diabetes (Horio and Ohtsuru 2001). The effect of water-soluble polysaccharides extracted from submerged fermented medium of Pleurotus citrinopileatus on hyperglycemia and damaged

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pancreatic cells in rats with streptozotocin (STZ)-induced diabetes was evaluated. The fasting blood glucose levels of diabetic rats fed with water-soluble polysaccharides were 44% lower than the negative controls (Hu et al. 2006). Using male Wistar rats injected with saline (normal rats), streptozotocin (STZ-NT rats), or streptozotocin plus nicotinamide (STZ + NT rats), we investigated the hypoglycemic activity of orally ingested fruiting bodies (FB), submerged culture biomass (CM), or the acidic polysaccharide glucuronoxylomannan (GXM) of Tremella mesenterica, an edible jelly mushroom. The results demonstrated that FB ingestion significantly attenuated the elevated blood glucose levels in an oral glucose tolerance test (OGTT) in STZ-NT rats. However, in STZ + NT rats, FB, CM, and GXM ingestion significantly attenuated the increases in food and water intake, 2-hour postprandial blood glucose concentrations, and blood glucose levels in OGTT. Moreover, FB and GXM ingestion significantly decreased serum concentration of fructosamine in STZ + NT rats (Lo et al. 2006). The antidiabetic effect of the crude exopolysaccharides (EPS) produced from submerged mycelial culture of Phellinus baumii in STZ-induced diabetic rats was investigated. The produced EPS consisted of two different heteropolysaccharides and two proteoglycans. The food intake of the diabetic control rats (STZ) was increased by 28.1%, whereas body weight gain was reduced by 44.1% as compared to the nondiabetic animals (NC). The plasma glucose level in the EPS-fed rats (EPS) was substantially reduced by 52.3% as compared to the diabetic rats (STZ), which is the highest hypoglycemic effect among mushroom-derived materials documented in literature (Hwang et al. 2005). Glucuronoxylomannan (AC) from the fruiting bodies of Tremella fuciformis exhibited a significant dose-dependent hypoglycemic activity in normal mice and also showed a significant activity in STZ-induced diabetic mice, by intraperitoneal administration (Kiho et al. 1994). The administration of Agaricus campestris mushroom in the diet (62.5 g/kg) and drinking water (2.5 g/L) countered the hyperglycemia of streptozotocin-diabetic mice. An aqueous extract of mushroom (1 mg/mL) stimulated 2-deoxyglucose transport (2 · 0-fold), glucose oxidation (1 · 5-fold), and incorporation of glucose into glycogen (1 · 8-fold) in mouse abdominal muscle. It was demonstrated that there was antihyperglycemic, insulin-releasing, and insulin-like activity in Agaricus. campestris (Gray and Flatt 1998). Dehydrotrametenolic acid, found in several polypores including Wolfiporia cocos, Laricifomes officinalis, and Laetiporus sulphureus Murrill, acts as an insulin sensitizer in glucose tolerance tests and reduces hyperglycemia in mice with noninsulin-dependent diabetes (Sato et al. 2002).

8.2.7 Antiallergic compounds Extracts of many fungi have shown antiallergic activity, which are very important, because this could be an alternative for the treatment of allergic diseases. The inhibitory effects of edible higher Basidiomycetes mushroom extracts from Hypsizygus marmoreus (Peck) Bigel., Flammulina velutipes (Curt.: Fr.) P. Karst.,

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Pholiota nameko (T. Ito) S. Ito et Imai, and Pleurotus eryngii (DC.: Fr.) Quel. on oxazolone-induced type IV allergy in male ICR mice were investigated. Oral administration of the ethanol extracts of these mushroom species for 3 days before the challenge showed significant antiallergic effects at a dose of 250 mg/kg bw/day. However, oral administration of the extracts for 3 days before the sensitization had no antiallergic effect at the same dose. A single percutaneous administration of the mushroom extracts together with the challenge also resulted in significant antiallergic effects at a dose of 0.1 mg/ear. The, results suggest that ethanol extracts of these mushroom species suppress the hypersensitive immune response such as the inflammation in delayed allergy (Sano et al. 2002). The MeOH extract of Ganoderma lucidum has an inhibitory action on histamine release from rat mast cells. From the physiologically active fraction of the extract, along with the known triterpenes ganoderic acids A and B, two new triterpenes were isolated and named ganoderic acids C and D. The structures of ganoderic acids C and D were determined to be 3β, 7β, 15α-trihydroxy-11,23-dioxo-5αlanost-8-en-26-oic acid, and 7β-hydroxy-3,11,15,23-tetraoxo-5α-lanost-8-en26-oic acid, respectively. Ganoderic acids C and D were shown to inhibit histamine release from rat mast cells. Quantitative analysis of these triterpenes was performed for the purpose of crude drug quality control (Kohda et al. 1985).

8.2.8 Angiotensin I converting enzyme inhibitor A cell-free extract of Saccharomyces cerevisiae containing the angiotensin I converting enzyme (ACE) inhibitory peptide was treated in a successive simulated gastric-intestinal bioreactor (step 1: amylase digestion, step 2: gastric fluid digestion, step 3: intestinal fluid digestion) to illustrate the absorption pattern of antihypertensive ACE inhibitory peptide, and the ACE inhibitory activities of each step were determined. Total ACE inhibitory activities of step 1, step 2, and step 3 were 55.96%, 80.09%, and 76.77%, respectively. The peptide sequence of each step was analyzed by MS/MS spectrophotometry. Eleven kinds of representative peptide sequences were conserved in each step, and representative new peptides including RLPTESVPEPK were identified in step 3 (Jang et al. 2011). D-Mannitol, one of the main phytochemicals of the edible Tamogi-take mushroom (Pleurotus cornucopiae), was found to inhibit an ACE. The antihypertensive effect of D-Mannitol and hot-water extract of Tamogi-take mushroom was demostratd in spontaneously hypertensive rats by oral administration (Hagiwara et al. 2005).

8.3 Conclusion Fungi have a variety of biologically active compounds beneficial to health, so it can be recommended as food or by administration of the bioactive compounds in therapeutic processes; however, even more research must be done to elucidate each of the bioactive compounds as well as their biological activities. These studies

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may involve finding new species and growth conditions of the fungus, as well as strategies for obtaining bioactive compounds without denaturation, and their therapeutic application.

Acknowledgments We wish to thank the Mexican Council of Science and Technology for supporting research in the area of production of fungal bioactive compounds (Project No. 156406).

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CHAPTER 9

Arbuscular mycorrhizal fungi: Association and production of bioactive compounds in plants Marcela C. Pagano1 and Partha P. Dhar2 1 2

Departamento de Física, ICEx, Federal University of Minas Gerais, Belo Horizonte, Brazil Plant Production Department, College of Food and Agricultural Sciences, King Saud University, Saudi Arabia

9.1 Introduction Interest in the potential of microorganisms for biotechnological processes focusing on the production of bioactive compounds (Molina et  al. 2012) and for understanding the bioactive zones of allelochemicals of plants (Barto et al. 2011) has discovered that they have essential functions in ecosystems with higher biodiversity. This has become more serious after researchers found no substitute for primary forests with regard to tropical biodiversity (Gibson et al. 2011) and, thus, most of the bioactive compounds present in pristine sites could be lost with increasing deforestation rates. Presence of symbiotic endophytes, particularly AM fungi, play important roles in the succession, stabilization, and productivity of forest plant species (van der Heijden et al. 1998; Kiers et al. 2000; Castelli and Casper 2003). In this sense the diversity of endophytes present in ecosystems with greatest biodiversity, such as the tropical and temperate rain forests, have increased in importance (Strobel, 2003). It is known that plants interact with other organisms (biotic factors) and tolerate infection or damage by herbivory and also symbioses and parasitism (Schulze et al. 2002). Currently, interest in biotic factors such as allelochemicals (secondary metabolites involved in plant–plant and plant–microorganism interactions) has also increased. Plants represent economically efficient sources to search for new bioactive compounds with various applications (e.g., pathogenic microorganism inhibition in fish and prawn tanks) (Sumthong and Verpoorte 2007). However, there is evidence that the bioactive compounds of medicinal plants are products of the plant itself or of the endophytes living inside the plant (Miller et al. 2012). In this sense, bioactive compounds that have beneficial health effects have also been

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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seen as extra-nutritional constituents that occur in small quantities in vegetal food (Kris-Etherton et al. 2002). The endophytes may provide protection and survival conditions to their host plant by producing several substances that may also have potential for use in industry, agriculture, and medicine (Strobel 2003). From the point of view of natural product detection, endophytes associated with medicinal plants have the potential to produce anticancer, antibacterial, and antifungal compounds (Miller et al. 2012). That is why new directions in microbial ecology have a need for integration of microbial physiological ecology, population biology, and process ecology since microorganisms have a great diversity of evolutionary adaptations (Schimel et al. 2007) and it has become difficult to identify and culture them. It is interesting to mention that biotechnology offers new strategies that could be used to develop transgenic plants with improved production of biodynamic compounds. In this point of view, more research is needed on plant genetic variability concerning bioactive content as affected by plant development, environmental and agronomic conditions, and symbioses. Moreover, germplasm collected from distant or isolated sites, tolerant mutants, and wild species can be exploited for improved tolerant genotypes in other areas. Thus, the study of important medicinal plants and their associated microbial symbionts are also addressed here. This chapter examines the current information on the potential of microorganisms for biotechnological processes focusing on the production of bioactive compounds. Specifically, arbuscular mycorrhizal fungi (AMF) and root fungal endophyte associations that may have a synergistic or antagonistic influence are  discussed. We begin by explaining plant services showing the bioactive compounds produced. We then review recent reports on their aboveground interactions focusing on AMF and dark septate endophyte (DSE) fungi. The last endophyte type was included due to its presence in most plants and also in plants colonized by mycorrhizas. Finally, we highlight the rising number of reports on AMF found in medicinal plants and their importance both for humans and natural systems.

9.2 Plant fungal endophytes Of paramount significance are the effects of global change on soils: increased soil temperatures, increased nutrient availability, increased ground instability in mountainous regions, and increased erosion from floods. Plant responses to stresses are complex (and include stress avoidance or tolerance); however, root symbionts alleviate the plant and soil stresses (see Pagano and Cabello 2013). As nutrient and water limitations increase, plants can allocate more photosynthate to the hyphae of the most common plant symbionts, mycorrhizas, to

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increase soil resource uptake. This can be seen particularly in high-latitude and high-altitude ecosystems (see Simard and Austin 2010). Two major types of fungal symbionts associate with plants: (1) endophytic fungi, which are located within plant tissues and may be associated with roots, stems, and/or leaves; and (2) mycorrhizas, which reside only in roots but spread out into the rhizosphere. It is known that endophytes can confer habitat-adapted fitness benefits such as symbiosis, which permits plants to persist in high-stress habitats (Rodriguez et al. 2009). Moreover, plant endophyte associations are represented in the fossil record in the same geological time frame as mycorrhizal symbioses (Singh et al. 2011). Some authors have reported that fungal endophytes consist of three basic ecological groups: the mycorrhizal fungi, the balansiaceous or grass endophytes, and the nonbalansiaceous taxa (see Schulz and Boyle 2005). By and large, the bioactive compounds of medicinal plants are products of the plant itself or of the associated endophytes. From the perspective of natural product discovery, the potential for endophytes extracted from medicinal plants to produce anticancer, antibacterial, and antifungal compounds (Miller et al. 2012) has been confirmed. In this sense, search for unique antimicrobial metabolites from endophytes is urgently needed since levels of drug resistance by plant and human pathogens are increasing. Substances produced by endophytic fungi may also be used in agroindustries for the biological control of pests and diseases. Endophytes provide a broad variety of bioactive secondary metabolites with unique structure, including alkaloids, benzopyranones, flavonoids, phenolic acids, quinones, steroids, terpenoids, tetralones, and xanthones, to name just a few (Tan and Zou 2001). Such bioactive metabolites are found to have wide-ranging application as agrochemicals, antibiotics, immunosuppressants, antiparasitics, antioxidants, and anticancer agents (Strobel 2003). Thus, the number of published papers (SCOPUS 2014) on plant endophytic fungi and bioactive compounds is ever increasing.

9.3 Arbuscular mycorrhizal fungi and biocompounds Plants provide several services such as provisioning of plant products, erosion control, invasion resistance, pathogen and pest regulation, and soil fertility regulation (Quijas et al. 2010). Moreover, plants provide bioactive compounds with various applications including medicines. Choice of the plant material to search for bioactive compounds may be based on previous research, local knowledge, chemotaxonomy, or new insights. The new activity of the compound is usually demonstrated by bioassays such as antifungal, antibacterial, antiviral, or antinematode activity. In order to detect active compounds, different chemical techniques (thin layer chromatography and nuclear magnetic resonance spectrometry) are commonly employed (Sumthong and Verpoorte 2007).

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As plants are sessile organisms exposed to natural climatic or edaphic stresses and to environmental changes from human activities, they interact with aboveground (Zheng and Dicke 2008) as well as below-ground (Kuyper and Goede 2005) microorganisms. Copious reports on endophytes have been published; however, mycorrhizas, which were more studied, are not typically viewed in the same mode as endophytes (Hyde and Soytong 2008). Additionally, the numbers of worldwide AM plants (3,617 species in 263 families) (Wang and Qiu 2006), totaling more than 3,941 (over 324 AM plant species compiled by Pagano 2012), and estimated at 200,000 (Kuyper and Goede 2005). The number of AM plants is much higher than that of symbionts, for example, 98 species of rhizobia according to (www.rhizobia.co.nz) and ~230 AMF species (www.mycobank.org), both distributed in 13 genera. It is worth noting that there is evidence that all plants in native ecosystems associate with fungi and other microbes (bacteria, yeast) on their leaf and root surfaces, rhizosphere and internal tissues in a symbiotic way that benefit their fitness (Ganley et al. 2004; van der Heijden et al. 2006). However, much of these interactions are still unknown. It is known that plants participate in mutualistic and parasitic endosymbiosis with varied microorganisms, ranging from Gram-negative (Rhizobium, Nostoc) and Gram-positive bacteria (Frankia), to oomycetes (Phytophthora), Chytridiomycetes, Glomeromycetes (AMF), and true fungi (Erysiphe, ascomycete; Puccinia, basidiomycete) (see Parniske 2000). Among them Rhizobium, actinorrhizal, and mycorrhizal symbionts have been well studied and considered the main symbionts associated with plant roots. Additionally, both legumes and actinorrhizal plants can also associate with AMF (see Singh et al. 2011). AMF and rhizobia are the most interesting plant symbionts. The first is the most widespread symbiosis among plants and can offer several benefits (Smith and Read 2008; Pagano 2012). On the other hand, the second involves almost exclusively legumes (Fabaceae) known to form nitrogen (N)–fixing root nodules, which results in N gains for the plant (Sprent 2008); most of them generally associate with AMF. The DSE fungal association (see below) is found less frequently in AMF plants; however, little attention has been paid to DSE until 1998. In this review we focused on mycorrhizas and DSE, as they are two promising plant–microorganism interactions with great benefits for future biotechnological research. In the AMF symbiosis of plant roots the fungi are categorized as biotrophic because they rely on living plant tissue to support their growth (see Parniske 2000). Moreover, under stress conditions, plant symbionts such as AMF are able to modify plant physiology to deal with the changed environmental factors (Miransari et  al. 2008). In this sense, there has been increasing interest on developing the potential biotechnological applications of fungal endophytes for improving plant stress tolerance and sustainable production of food crops

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Sampled roots

Fixation in FAA or alcohol solution

Clearing (KOH 1M)

(a)

Bleaching (0.5 % NH4OH + 0.5 % H2O2 v/v in water) (Kormanik and McGraw, 1982) Acidification (HCl 5%)

Staining (Phillips and hayman, 1970)

Microscopic observation

(b)

Mycorrhizal / DSE evaluation (McGonigle et al. 1990; Trouvelot et al. 1986)

Quantification of fungal structures

Determination of plant mycorrhizal / DSE status (c)

Figure 9.1 Protocol for studying the presence of root endophytes in plants. Roots of plants are (a) stained for AMF/DSE colonization. Determination of fungal structures including asseptate hyphae and vesicles (b) and melanized microesclerotia (c) are required (Photo Credit: M. Pagano). FAA, 70% formalin-acetic acid-alcohol.

(Singh et  al. 2011). Several reports have also demonstrated the benefit for plant health when associated with AMF (Azcón and Barea 2010; Barea et al. 2005a,b). The mycotrophic status of plants is an important tool for various purposes: seedling production, plant cultivation, ecological restoration, endangered species protection, and biomining, to name a few. A protocol for studying presence of root endophytes in plants is presented in Figure 9.1. Additionally, this information can be used to differentiate plant functional types with regard to nutrient

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uptake strategy (Cornelissen et al. 2003) and plant mycorrhizal types (Sathiyadash et al. 2010), or to screen for plant stress tolerance (Pagano and Cabello 2013). This is also relevant to detect the presence of biocompounds in plants (plant secondary metabolisms are changed during the formation of AMF symbiosis). In this sense, Ceccarelli et al. (2010) showed the effects of AMF symbiosis on polyphenolic production by artichoke plants inoculated with AMF (Glomus mosseae, Glomus intraradices, and a combination of them), through microcosm and also field experiments. They found AMF inoculation as a useful biotechnological tool to enhance plant biosynthesis of secondary metabolites with health-promoting activities. Additionally, the authors pointed out the need for more research on plant genetic variability concerning polyphenolic contents as affected by plant development, environmental and agronomic conditions, and AMF symbioses. The agronomic potential of the use of biocompounds related to AMF is now beginning to be studied in more detail. This can be very useful in countries with greater adoption of no-tillage practices, where research dealing with AMF is appreciated for determining appropriate land management and as a background for successful inoculation (Sieverding 1991; Miranda 2008; Verbruggen et  al. 2013). In this sense, important cropping regions such as the Argentine Pampas, which account for more than 90% of grain production (mainly soybean and wheat), are the object of multiple studies (Pagano and Covacevich 2011); among them long-term studies by Schalamuk et al. (2006, 2011, 2013), for example. Those studies have showed the behavior of AMF communities and the benefits resulting from indigenous or inoculated AMF in wheat under tillage and nontillage during different phenological crop stages (see also Schalamuk and Cabello 2010). The authors stressed that soils that are not mechanically disturbed (similarly to nontillage soils), presenting higher diversity of plants of varying age, can host different AMF as most of plant species have been previously mentioned as mycorrhizal (Wang and Qiu 2006). In this sense, understanding the effect of the interactions of AMF and biocompounds realized by crop plants and weeds is relevant for managing mycorrhizal populations. However, in certain conditions, the AMF that predominate in nontillage sites may not necessarily be beneficial to the crops (Schalamuk et al. 2011). In those experiments, wheat (Triticum aestivum L.), a crop plant associated with AMF, was cultivated since 1995; however, in the fallow, the plots were invaded by weeds, mostly Cynodon dactylon (L.) Pers., which also associate with AMF (see Wang and Qiu 2006). Schalamuk et al. (2013) compares the cultivated site with their boundaries (not cultivated for more than 20 years), which presented the following plant species: Ammi visnaga (L.) Lam., Avena fatua L., Briza minor L., Cynara cardunculus L., C. dactylon, Deyeuxia viridiflavescens (Poir.) Kunth, Ipomea purpurea (L.) Roth, Lolium perenne L., Plantago lanceolata L., Vicia sp., and Xanthium cavanillesii Schouw ex Didr. Seven of them have been found associated to AMF; however, four plant species have no report of their mycorrhizal status (Wang and Qiu 2006). This indicates that most of plants in noncultivated sites can have AMF association and can act as a propagule

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source for AMF dispersion, which could also affect the need for crop inoculation. The significance of crop edges in preserving weed biodiversity was also highlighted by Romero et al. (2008). In this sense, it was reported that herbaceous weeds can stimulate differences in dominant AMF species, delivering a wider spectrum of AMF for colonizing crop roots (Radić et al. 2012). Additionally, the allelochemical realized by the roots of weeds may have negative effects on native plant species (e.g., phytotoxins), which seems to be influenced by soil characteristics (Thorpe et al. 2009). It was assumed that AMF are able to protect seedlings against allelopathy (Pellissier and Souto 1999). According to these results, by manipulating allelopathy and AMF we can suppress unwanted weeds in field crops, avoiding herbicide applications. It is known that AMF can improve plant growth and production under different conditions, including various soil stresses (heavy metals, compaction, drought, and salinity). The promotion of plant growth under stress is due to their benefits, which include establishment of extensive hyphal networks and secretion of biochemicals like glomalin, which enhance water and nutrient uptake, ameliorating soil structure (Miransari 2010). These benefits are the reason why the abundance and diversity of mycorrhizas in natural as well as in manipulated ecosystems are now more exhaustively researched (see below) in spite of the fact that the species accumulation curves in general do not attain 100%. For example, the study of Barea et  al. (2011) compiled the diversity of mycorrhizas found in semiarid Mediterranean ecosystems in southeast Spain. It showed the benefit of mycorrhizal fungi present in plant establishments, especially in systems with nutrient deficiency, drought, soil disturbance, and other environmental stresses involved in soil degradation. Interestingly, MonroyAta and García-Sánchez (2009) also showed better water relations, plant growth, and survival in semiarid plants of Mexico associated with AMF. They tested species of Fabaceae, Cactaceae, and Agavaceae in the greenhouse (some of them at field conditions), showing the magnitude of AMF inoculation, in spite of the strong herbivory present. DSE were also observed but not registered (MonroyAta, personal communication, June 3, 2013). In Argentinean arid and semiarid regions a total of 225 AMF plant species occurred in different vegetal types such as Jarillal and Puna (see Pagano et al. 2012), some of them also associated with DSE. In the dry Puna ecosystem (2000–4400 m.a.s.l.), 10 AMF species were found, Glomus being the predominant genus. In Brazil, for example, reports from highland fields as well as deciduous forests (see Pagano and Araújo 2011; Pagano 2012) pointed out a total of ~28 AMF plant species and at least 36 AMF species that occurs in those ecosystems (Pagano et al. 2013). Additionally, Carvalho et al. (2012) reported 49 AMF species in the highland fields of Brazil, of which 23 AMF species are in common with reports of Pagano et al. (2013). These studies highlighted the appreciation of fungal endophytes showing that the potential use of microorganisms was underestimated in

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the past. In the future, accumulated evidence will be a useful tool for determining where AMF interactions may be especially intense and profitable. Recent evidence has been reported for below-ground common mycorrhizal networks that interconnect multiple plant species and also transfer allelochemicals among plants. Those networks increase the bioactive zones of allelochemicals in natural environments, with significant consequences for interspecies chemical interactions and facilitation in plants (Barto et al. 2011). It is recognized that water and nutrients move between plants through the common mycorrhizal networks (Querejeta 2003; Allen 2007), and it is likely that signals inducing plant defenses are also transported (Song et  al. 2010). This will result in an increasing interest in developing the potential biotechnological applications of fungal endophytes for invasive plant suppression or allelophatic tree cultivation. In this sense it is known that the production of allelochemicals is regulated by diverse factors (temperature, light intensity, water and nutrient availability, soil texture, and associated microorganisms). Allelopathy can affect plant germination rates, seedling growth, mycorrhizal function, insect and bacterial growth, nitrification, or litterfall decomposition (Blanco 2007). Thus, multiple factors influence allelochemical production and toxicity, including nutrient availability, soil moisture and texture, solar radiation, and temperature (Blanco 2007). Furthermore, factors related to stress can increase the biological activity of allelochemicals (Inderjit et  al. 2005). The allelopathic compatibility of mixed plants may be crucial to determine the success of agroforestry systems since most of the agroforestry species have negative allelopathic effects on food and fodder crops (Rizvi et  al. 1999). In this regard, both AMF and ectomycorrhiza (EM) were detected in plants such as Eucalyptus camaldulensis (monoculture and mixed plantations) (Pagano and Scotti 2008) cultivated in Brazil, showing their importance for both native and exotic tree fitness in arid environments. Lastly, there are still few reports that indicate that AM colonization could enhance bioactive compounds (approximately 10 scientific papers were published in SCOPUS database since 2007). Nevertheless, Yuan et al. (2007) compiled information on accumulation of secondary metabolites in AMF plants and concluded that AMF may be exploited as a bioinoculant to increase the essential oil concentration of some medicinal plants. This is of special interest since the use of biocompounds in the pharmaceutical industry is widespread and an increasing number of plants are officially included by pharmacopeias.

9.4 Ectomycorrhizas and biocompounds Due to the scarce number of effective antibiotics against diverse bacterial species, and few new antimicrobial agents in development, interest for natural biocompounds (bioprospection) and for developing the potential biotechnological applications of fungal endophytes is increasing. Endophytes provide a variety of

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bioactive secondary metabolites with unique structure, including alkaloids, benzopyranones, flavonoids, phenolic acids, quinones, steroids, terpenoids, tetralones, and xanthones (Tan and Zou 2001). Such bioactive compounds have wide-ranging applications as agrochemicals, antibiotics, immunosuppressants, antiparasitics, antioxidants, and anticancer agents (Strobel 2003). In this sense, new findings related to the production of allelochemicals, their persistence in the rhizosphere, and their influence on sensitive plant species and on other organisms will result in enhanced utilization of natural products as pesticides or as pharmaceuticals and nutraceuticals (Inderjit et  al. 2005). It is worth noting, moreover, that as soil factors affect allelochemical production, search for novel compounds can be based on material collection from distant sites (altitude and latitude), unexplored areas, and ecosystems with severe conditions. Plants growing under water stress can increase allelochemical production (water soil potential controls the rate of microorganism activity, allelochemical movement, and leaching). Additionally, soil texture (e.g., arid zones with coarse texture) and herbicides, heavy metals, or other allelochemicals also affect allelochemical toxicity (see Blanco 2007). Mycorrhizal macrofungi can also present bioactive compounds. Reis et  al. (2011) identified bioactive compounds (studied for their bioactive and chemical properties by in vitro assays) in five edible mycorrhizal species (Amanita caesarea, Cortinarius anomalus, Cortinarius violaceus, Lactarius volemus, and Suillus luteus) from northeast Portugal. Also in that country, Carvalho et al. (2013) studied the mycorrhizal symbionts of chestnuts (the most important export product of the Portuguese fruit sector and also for timber and wild edible mushroom commercialization). They pointed out that Paxillus involutus was the best EM fungus to  associate with Castanea sativa; however, considering bioactive compound production, Pisolithus arhizus was more efficient since it allowed an increase in the contents of sugars and tocopherols. Many bioactive compounds, also containing antifungal agents, were isolated from Xylaria (Ascomycetes) residing in different plant hosts. Compounds with antifungal activity against Candida albicans (Pongcharoen et al. 2008; Boonphong et al. 2001) and against herpes simplex virus type 1 (Pittayakhajonwut et al. 2005) were reported. As it was shown above, some economically important species can associate with AMF and EM. This is the case of some Eucalyptus species (see Carrenho et al. 2008; Pagano et al. 2010). In general, AM colonization has been reported only in young Eucalyptus species that usually form EM (Chilvers et  al. 1987); however, 5-year-old plantation of E. camaldulensis presented an inverse relationship of AM/EM colonization (Pagano et al. 2008). During the rainy season the AMF colonization decreased, while the EM colonization increased, but in the dry season EM was reduced. EM improves water balance of host plants, reduces impacts on trees from root pathogens, and mobilizes essential plant nutrients directly from soil (Smith and  Read, 2008). Moreover, eucalypts associate with numerous species of EM

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(Scleroderma, Pisolithus, Hymenogaster, Hydnangium, and Laccaria), Pisolithus tinctorius (Pers) Cocker and Couch being the most important fungus to increase plant growth in forestation (Garbaye et al. 1988) and to improve seedling growth at low phosphorus fertilization (Mason et al. 2000). Moreover, some rhizomorph-forming EM fungi establish an extensive mycelia network connected by different hyphal strands (rhizomorphs), which transport water and nutrients over long distances (Read 1984; Pagano 2011). We discuss AM further later in the chapter.

9.5 Plants, arbuscular mycorrhizal fungi, and DSE Endophytes from medicinal plants have become the focus of research for bioactive compounds. Hyde and Soytong (2008) stressed that due to their high diversity and ease to apply statistics and to study, the study of plant endophytes has expanded. Endophytes provide nutrients and establish a buffer from external environmental stresses and microbial competition (see Schulz and Boyle 2005). In the review of Hyde and Soytong (2008), 12 definitions of endophytes were compiled; however, they discuss the differences among mycorrhizas, leaf endophytes, and their exceptions, as well as mention the early mutualistic symbioses on fossil trees. In the same review, they pointed out the history and advances on endophytic studies. Mycorrhizas have synchronized plant-fungus development and nutrient transfer at specialized interfaces (Brundrett 2004). In  the other plant–endophyte relationships three main features are lacking: a cellular interface with specialized structures (arbuscules), synchronized development between the plant and the fungal associates, and significant benefits for both partners (Brundrett 2006). Other root endophytic associations comprise a diverse group of fungi, the best known being the dark septate endophytes (DSE), so called for the reason that colonizing hyphae are both septate and melanized (Jumpponen and Trappe 1998; Mandyam and Jumpponen 2005; Sathiyadash et al. 2010). DSE are root symbionts found in herbaceous and tree species (Mandyam and Jumpponen 2005; Urcelay et al. 2011; Urcelay 2012; Pagano et al. 2012), ferns (Fernández et al. 2012), and some aquatic plants (Marins et al. 2009). DSE are found usually in agronomic plants (e.g., Barrow and Aaltonen 2001; Barrow 2003; Chaudhry et al. 2005; Likar et al. 2008; Newsham et al. 2009; Newsham 2011; Stevens et  al. 2010) and comprise pigmented and phialidic hyphomycetes as Phialocephala fortii, Chloridium pauciflorum, Cadophora (=Phialophora) finlandia, and Leptodontidium orchidicola, which belong to Helotiales (Ascomycota) (Upson et al. 2009) or Pleosporales (Ascomycota) (Jumpponen and Trappe,1998). Being potentially beneficial to injurious for the host, DSE are considered biotrophic facultative (Jumpponen and Trappe 1998). While some root DSE colonizes many  herbaceous plants and trees are mutualistic, others become pathogenic (Jumpponen 2001; Sieber 2002).

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DSE range from mutualism to parasitism (Jumpponen 2001; Mandyam and Jumpponen 2005) and whether they would be considered mycorrhizal or not still remains controversial (Smith and Read 2008). Notably, research on AMF has allowed increasing studies on DSE fungi, as the same methodology permit their observation in roots (see Figure  9.1). Moreover, because of this facility, most researchers on AMF have begun to register DSE in the same analyzed root samples (see below). Analyzing the root colonization of pteridophytes in Puerto Blest (Valdivian temperate forest of Argentina), Fernández et al. (2012) found that ~50% of the species presented AMF; and surprisingly all the species contained DSE in their roots. Those authors suggested the adaptation of DSE to associate with both AMF and nonmycorrhizal plants in that region. Additionally, reports from other regions of Argentina (“Chaco”) have also shown most plant species associated with AMF and DSE (Urcelay et al. 2012). Moreover, a shrubby ericaceous species (Gaultheria poeppiggi DC) was found associated with ERM (ericoid mycorrhizal) fungi, DSE, and AMF in the Córdoba mountains in central Argentina at 1,500–2,500 m elevation (Urcelay 2002). Some plant species occurring above 3,500 m elevation (e.g., in the Peruvian Andes, Venezuelan páramo, Bolivian Andean highlands, Puna) are colonized by AMF, which might be nutritionally important in adverse environments (Barrow and Osuna 2002; Schmidt et al. 2008). Pioneer studies by Lugo in Argentina indicate that 20 native grass species were colonized by AMF, three grasses presented exclusively AM (Cynodon dactylon var. biflorus, Microchloa indica, and Muhlenbergia rigida) and 17 grasses also associated with DSE (see Pagano et al. 2012). AMF were always found in the roots of Puna grasses, in general at low root colonization levels; however, DSE mean root colonization was lower than AMF. Presence of AMF and DSE will help to understand plant communities and how these will respond to environmental change and, consequently, their influence on biocompound production. Additionally, South American nearthreatened species (e.g., Polylepis australis) presented AMF–DSE association (Menoyo et al. 2007) or AMF (e.g., Cordia oncocalix and Schinopsis brasiliensis) (Pagano and Araujo 2011). Furthermore, with regard to DSE, a high diversity was isolated from Oryza glumaepatula (a wild rice species) growing in oligotrophic sites in Amazonia, Brazil, investigating two different vegetation covers (cerrado and forest) (Pereira et al. 2011). They also showed that inoculated selected DSE, which benefit plant health, in wild and commercial rice have increased biotechnological potential for crop plants. Lastly, there are still few reports that indicate that DSE colonization could enhance bioactive compounds (of approximately 87 scientific papers on DSE, only one was published in SCOPUS database since 2010 in relation to bioactive compounds).

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Aiming to increase the studies on the dynamics of AM/DSE colonization and nutrient uptake, Sathiyadash et  al. (2010) showed that dual association with DSE fungi and AMF occurred in half of 50 south Indian grasses. Those authors suggested that the lack of correlation between AM and DSE colonization levels can indicate the lack of influence between them. Interestingly, some nonmycorrhizal plants frequently associate with DSE, such as two plant species studied by Sathiyadash et al., and some species such as five herbs studied by Pagano et al. (2013) did not present any colonization signal during the wet season, which needs more studies. Other authors (Chaudhry et al. 2006) observed an inverse relationship between DSE and AMF colonization levels, pointing out that further studies on the dynamics of AM/DSE colonization and nutrient uptake would yield more information on the ecological relevance of these fungi.

9.6 Arbuscular mycorrhizal fungi and medicinal plants Some 80% of the world’s population still depends on plants for primary healthcare; even today in Western medicine, and despite progress in synthetic chemistry, some 25% of medicaments are originated from plants (Farnsworth and Soejarto 1991). The detection of new plant bioactive compounds with the potential for chemical modification will also increase their phytomedical significance. The indicated compounds are screened using new research equipment and new technology including molecular biology (Cordell 2000; Cordell and Colvard 2005; Yaniv and Bachrach 2005; Pieters and Vlietinck 2005). Interestingly, ecological function of secondary products may present medicinal effects for humans (plant defense products toward microbial pathogens could demonstrate useful as antimicrobial medicines in humans) (Briskin 2000). In Brazil, for example, the antimicrobial reaction of secondary metabolite produced by endophytic fungus Diaporthe helianthi isolated from Luehea divaricata Mart. (Tiliaceae) was confirmed on the human pathogenic bacteria Enterococcus hirae, Escherichia coli, Micrococcus luteus, Salmonella typhi, Staphylococcus aureus, phytopathogenic Xanthomonas asc. phaseoli, and phytopathogenic fungi, showing their biotechnological potential (Specian et al. 2012). Therefore, ethnobotanical research has become increasingly appreciated in the development of healthcare and conservation programs in different parts of the world (Barboza et al. 2009). Such increased interest in plants as a source of novel pharmacophores notices their chemical diversity and versatility, not matched by synthetic chemistry libraries. However, the contribution of endophytic compounds in medicinal plants is relatively unknown (Miller et al. 2012). With regard to AMF symbioses and medicinal plants (approximately 106 scientific papers were published in SCOPUS database since 2000), the little evidence and the focus on Glomus species, such as Glomus intraradices (e.g., Sawilska

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and Jendrzejczak 2013) and Funneliformis mosseae, which do not represent the whole AMF biodiversity, speak in favor of the need of urgent studies on AMF species and selected medicinal plants. In this way, recent interest in the effect of different AMF species on medicinal plants showed 15 plant species associated with AMF (Zubek et al. 2012). Among those reports, only a recent review (Zeng et al. 2013) stresses the benefit of AMF for cultivated medicinal plants, improving their quality and active compound composition. However, authors stressed that the cultivation system of important medicinal plants need more studies (Padmavathi and Ranjini 2011). Additionally, studies on the AM/DSE colonization of 107 medicinal and aromatic plants in the Western Ghats region of southern India revealed that 79 were AM and 38 harbored a DSE association (Muthukumar et al. 2006).

9.7 Conclusion In the introduction to this chapter, we briefly described plant stress factors and the benefits that mycorrhizal fungi provide to their plant hosts as well as for human health and food crops. We showed the increasing interest in developing the potential biotechnological applications of fungal endophytes for multiple purposes improving plant stress tolerance and sustainable production of food crops. Throughout the chapter, we have showed that stress affects soil physical and chemical properties, influencing the population, diversity, and activities of soil microbes, including symbiotic fungal populations. To know the mycotrophic status of plant species is essential, and research on AMF has increased the studies on DSE fungi, as the same methodology permits their observation in roots. Additionally, effects of anthropogenic alterations (tillage) were discussed due to the great implication in the manipulation of AMF species able to colonize plants in agricultural soils. This chapter argues that AMF, which can have greater effect on plant growth, can also increase the amount of biodynamic compounds; however, to develop technologies, protocols, and controlled laboratory experiments is crucial. Finally, further research is needed to study plant biocompounds and their interactions with mycorrhizas.

Acknowledgments M. Pagano is grateful to the Council for the Development of Higher Education at  Graduate Level, Brazil (CAPES) for the postdoctoral scholarships granted. Dr. P. P. Dhar acknowledges the support of the Deanship of Scientific Research, King Saud University, Saudi Arabia.

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CHAPTER 10

Extremophiles as source of novel bioactive compounds with industrial potential Mohamed Neifar1, Sameh Maktouf2, Raoudha Ellouze Ghorbel2, Atef Jaouani1, and Ameur Cherif3 1

Laboratoire Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis,Campus Universitaire, Tunis, Tunisia Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Sfax, Tunisia 3 LR11-ES31 Biotechnology and Bio-Geo Resources Valorization, Higher Institute for Biotechnology, Biotechpole Sidi Thabet, University of Manouba, 2020 Ariana, Tunisia 2

10.1 Introduction Extremophiles are microbes that can live and reproduce at extremes of pH (>pH 8.5, 45°C, 500 atmospheres), salinity (>1.0 M NaCl), and in high levels of radiations, recalcitrant substances, or heavy metals (Podar and Reysenbach 2006). They have adapted to thrive in ecological niches such as deep-sea hydrothermal vents, hot springs, salterns, and hypersaline lakes. Most of the extremophiles that have been identified to date belong to the domain of the Archaea. However, many extremophiles from the eubacterial and eukaryotic kingdoms have also been identified and characterized (Rothschild and Manicinelli 2001; El Hidri et al. 2013; Guesmi et al. 2013; Jaouani et al. 2014). Extremophiles are important not only because of the fundamentals of their biochemical and structural biodiversities but, also, because of their enormous potential as sources of enzymes and other biological materials with applications in biotechnology and industry (Table  10.1). The growing number of genomes available from extremophiles will greatly aid the discovery and identification of novel enzymes that have not been detected by functional screening procedures (Van den Burg 2003). However, the identification of potentially useful enzymes is only a first step. The production of extremophilic biomass is very important to provide sufficient material for biomolecule isolation and characterization, eventually revealing particular features of industrial interest (Schiraldi and De Rosa 2002). Biomass production can be improved either by optimization of the medium composition or by varying the specific fermentation procedures. The availability of good cloning and expression systems can also be used for Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Table 10.1 Extremophiles and some applications of their biocatalysts and biomolecules in biotechnology and industry (Demirjian et al. 2001; Chiara and Mario 2002; Schiraldi and De Rosa 2002; Van den Burg 2003). Extremophiles

habitat

Enzymes and biomolecules

Representative applications

Thermophiles

Psychrophiles

Hyperthermophile (T > 85°C) Thermophile (65–85°C) Moderate thermophiles (45–65°C) Low temperature (T 8; NaCl up to 33%) have been mainly found in extremely alkaline saline environments, such as the Rift Valley lakes of East Africa and the western soda lakes of the United States (Wiegel and Kevbrin 2004). The genome of a haloalkaliphilic archaeon from highly alkaline soda lakes, Natronomonas pharaonis, has revealed several adaptations to this environment (Falb et al. 2005).

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These include an overall modification of the proteome to increase the fraction of acidic amino acids and reduce protein hydrophobicity, a coating of the cell membrane with glycoproteins and secreted enzymes attached by lipid anchors, and an efficient transport system for heavy metals and nitrogen compounds, which are scarce in hypersaline environments (Gomes and Steiner 2004). Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica (Karan et  al. 2013). Its genome sequence was recently completed and analyzed by comparative genomics, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures (Anderson et al. 2011; Karan et al. 2013). The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations (4–4.5 M NaCl and KCl) and both low (50°C) temperatures. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry (Karan et al. 2013). Novel thermoactive and salt-tolerant α-amylases have been identified in thermophilic halophiles (Mijts and Patel 2002; Tan et al. 2008; Jabbour et al. 2013). Since this group of enzymes has a very wide spectrum of industrial applications— the sugar, animal nutrition, baking, brewing, and distilling industries; production of cakes and starch syrups; preparation of digestive aids; and pharmaceutical industries (Kiran and Chandra 2008)—there is an increase in the demand for extremophilic α-amylases that have activity and stability characteristics suitable for the harsh conditions required by the industrial processes (Jabbour et al. 2013). Geodermatophilus obscurus is a polyextremophilic actinobacterium frequently isolated from stressful environments such as rock varnish in deserts and mountains (Ivanova et  al. 2010). It has demonstrated remarkable resistances to a range of damage caused by ionizing radiation, desiccation, ultraviolet radiation, oxidizing agents, and electrophilic mutagens (Gtari et  al. 2012). A highly thermostable esterase from G. obscurus G20 has been recently purified and characterized (Jaouani et al. 2012). It showed great potential as a novel biocatalyst for many food applications due to its extremely intrinsic stability at high temperatures and its resistance to organic solvents and metal ions. The halophilic alkalithermophiles are a novel group of polyextremophiles that grow optimally under the combined conditions of extreme salinity (>2 M NaCl), alkaline pH (pH > 8.5), and elevated temperature (>50°C). Halophilic alkalithermophiles are found within both the Bacteria and Archaea (Bowers et al. 2009; Mesbah and Wiegel 2009, 2012). Life under these conditions undoubtedly involves the development of unique physiological characteristics, phenotypic properties, and adaptive mechanisms that enable control of membrane permeability, control of intracellular osmotic balance, and stability of the cell wall, intracellular proteins, and other cellular constituents (Mesbah and Wiegel 2012). These microorganisms, called polyextremophiles, produce enzymes that are functional under extreme conditions. Consequently, the unique properties of

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these extremophilic biocatalysts have resulted in several novel applications of these enzymes in food, pharmaceutical, and other industrial processes (Kumar et al. 2011; Prakash et al. 2012).

10.11 Conclusion The extremophilic nature of thermophiles, psychrophiles, alkaliphiles, acidophiles, halophiles, piezophiles, radiophiles, and metalophiles has stimulated intense efforts to understand the physiological adaptations for living in extreme environments and to probe the potential biotechnological and industrial applications of their stable cellular components. This is particularly true of their enzymes (called extremozymes), which are able to remain catalytically active under extremes of temperature, salinity, pH, pressure, radiations, and metals. Many interesting enzymes and special metabolites have been isolated, purified, and characterized from extremophilic microbes and some of them have potential industrial uses. However, several technical difficulties have prevented the largescale industrial application of biomolecules from extremophilic sources, the most important being the availability of these compounds. Novel developments in the cultivation and production of extremophiles but also developments related to the cloning and expression of their genes in heterologous hosts, will increase the number of enzyme-driven transformations in food, pharmaceutical, and other industrial applications. With continued development of appropriate molecular tools as well as better insight into structure–function principles, a new battery of biomolecules will become available to meet the growing biotechnological interest in these extremophilic products.

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Sharma, A., Y. Kawarabayasi and T. Satyanarayana. 2012. acidophilic archaea and bacteria: acid stables biocatalysts and their potential applications. Extremophiles 16:1–19. Sheng, X.F., J.J. Xia, C.Y. Jiang, L.Y. He and M. Qian. 2008. Characterization of heavy metalresistant endophytic bacteria from rape Brassica napus roots and their potential in promoting the growth and lead accumulation of rape. Environ. Pollut. 156:1164–1170. Shi, X.W., M.L. Sun, B. Zhou and X.Y. Wang. 2009. Identification, characterization, and overexpression of a phytase with potential industrial interest. Can. J. Microbiol. 55:599–604. Simonato, F., S. Campanaro, F.M. Lauro, A. Vezzi, M. D’Angelo, N. Vitulo, G. Valle and D. H. Bartlett. 2006. Piezophilic adaptation: a genomic point of view. J. Biotechnol. 126:11–25. Sinha, R. and S.K. Khare. 2012. Isolation of a halophilic Virgibacillus sp. EMB13: Characterization of its protease for detergent application. Ind. J. Biotechnol. 11:416–426. Slonczewski, J.L., M. Fujisawa, M. Dopson and T.A. Krulwich. 2009. Cytoplasmic pH measurement and homeostasis in bacteria and archaea. Adv. Microb. Physiol. 55:1–317. Sterner, R. and W. Liebl. 2001. Thermophilic adaptation of proteins. Crit. Rev. Biochem. Mol. Biol. 36:39–106. Tan, T., B. Mijts, K. Swaminathan, B. Patel and C. Divne. 2008. Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme–substrate complex and an N domain with a role in binding raw starch. J. Mol. Biol. 378:850–868. Turner, P., G. Mamo and E.N. Karlsson. 2007. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Fact. 6:1–9. Valls, M., and V. De Lorenzo. 2002. Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol. Rev. 26:327–338. Van den Burg, B. 2003. Extremophiles as a source for novel enzymes. Curr. Opin. Microbiol. 6:213–218. Verma, M.L. and S.S. Kanwar. 2010. Purification and characterization of a low molecular mass alkaliphilic lipase of Bacillus cereus MTCC 8372. Acta Microbiol. Immunol. Hung. 57:191–207. Vezzi, A., S.M. Campanaro, F. D’Angelo, N. Simonato, F.M. Vitulo, A. Lauro, G. Cestaro, B. Malacrida, N. Simionati, C. Cannata, D.H. Romualdi and G. Valle. 2005. Life at depth: Photobacterium profundum genome sequence and expression analysis. Science 307: 1459–1461. Wang, W., M. Sun, W. Liu and B. Zhang. 2008. Purification and characterization of a psychrophilic catalase from Antarctic Bacillus. Can. J. Microbiol. 54:823–828. Wani, P.A., M.S. Khan and A. Zaidi. 2007. Chromium reduction, plant growth-promoting potentials, and metal solubilization by Bacillus sp. isolated from alluvial soil. Curr. Microbiol. 54:237–243. Wei, G., F. Lianmei, Z. Wenfei, F. Yunyun, Y. Jianfu and T. Ming. 2009. Isolation and characterization of the heavy metal resistant bacteria CCNWRS33-2 isolated from root nodule of Lespedeza cuneata in gold mine tailings in China. J. Hazard. Mater. 162:50–56. Wiegel, J.V. and V. Kevbrin. 2004. Alkalithermophiles. Biochem. Soc. Trans. 32:193–198. Wierzbicka-Wos, A. Cieslinski, H. Wanarska, M. Kozlowska-Tylingo, K. Hildebrandt, P. and Kur, J. 2011. A novel cold-active beta-D-galactosidase from the Paracoccus sp. 32d-gene cloning, purification and characterization. Microb. Cell. Fact. 10:1475–2859. XiangSheng, Y., C. XingLin, X. XianZhong and Z. RunYing. 2011. Cold-adaptive alkaline protease from the psychrophilic Planomicrobium sp. 547: Enzyme characterization and gene cloning. Adv. Polar. Sci. 22:49–54. Yang, J. and H. Dang. 2011. Cloning and characterization of a novel cold-active endoglucanase establishing a new subfamily of glycosyl hydrolase family 5 from a psychrophilic deep-sea bacterium. FEMS Microbiol. Lett. 325:71–76.

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CHAPTER 11

New trends in microbial production of natural complex bioactive isoprenoids Rama Raju Baadhe1, Ravichandra Potumarthi2, Naveen Kumar Mekala1, and Vijai K. Gupta3 1

Department of Biotechnology, National Institute of Technology Warangal, India Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia 3 Molecular Glycobiotechnology Group, Department of Biochemistry, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland 2

11.1 Introduction Microbial fermentation processes have been widely used for the production of foods and beverages since ancient times (Alba-Lois and Segal-Kischinevzky 2010). Technological developments in genetic engineering endorsed microbial synthesis of heterologous compounds such as hormones, growth factor, therapeutic proteins, vaccines, and genetically modified organisms/crops by insertion of corresponding gene of interest. Observations from the natural world and diversity of natural compounds inspired researchers to engineer beyond a single heterologous gene product. In specific, these new applications focused on the manipulation of sets or combinations of proteins, or enzymes that acted in concurrence in a cell, within metabolic pathways to convert energy and precursor chemicals into desired natural and non-natural products (Smolke 2009). Traditional chemical synthesis for the production of value-added chemicals and major pharmaceuticals is a well-established field. Nevertheless, some complex molecules are very difficult to be synthesized through chemical methods. Enzymes established significant expertise at the synthesis of very complex molecules through environmentally friendly methods (Smolke 2009). Approaches that involve biological catalysts and avoid organic solvents, heavy metal catalyzers, and strong acids and bases are currently employed upon synthetic chemistry-based routes. In addition, extraction and purification of valuable natural products from native plant sources is difficult as these compounds accumulate in very small amounts, and need many steps and solvents for extractions and purification; for example, it would take approximately six 100-year-old Pacific yew trees to provide enough

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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taxol (paclitaxel) to treat one cancer patient (Horwitz 2004; Jeandet et al. 2013). At present, Escherichia coli and Saccharomyces cerevisiae are employed for the microbial synthesis of all possible natural products of interest, though new platform microorganisms are being developed (Marienhagen and Bott 2013). This chapter summarizes the engineering of microbes for the biosynthesis of isoprenoid products, with a particular emphasis on bioactive compounds.

11.2 Isoprenoids Isoprenoids (terpenoids) are the most structurally diverse class of natural compounds commonly produced in plants (Croteau et al. 2000). Basic isoprene (C5) unit (carbon number) categorize terpenoids as mono terpenoids (C10), sesqui terpenoids (C15), di terpenoids (C20), sesterterpenoids (C25), tri terpenoids (C30), tetra terpenoids (C40), and polyterpenoids (Cn) (Ruzicka 1959). More than 55,000 terpenes have been isolated and characterized, consistently doubling in their numbers each decade (Breitmaier 2006; McGarvey and Croteau 1995). Isoprenoids have diverse functional roles in plants such as growth, defense, and development (McGarvey and Croteau 1995). Based on these characteristic features, terpenoids have prominence in the pharmaceutical, fragrance, and biofuel industries, shown in Table  11.1 (Breitmaier 2006; Peralta-Yahya et  al. 2012). Terpenoids, being secondary metabolites produced in very small quantities and scaled up with existing plant and microorganism strains (Chang and Keasling 2006), are not Table 11.1 Biological activities and commercial applications of classical isoprenoids (modified from Maury et al. 2005). Class

Biological activity

Commercial applications

Examples

Monoterpenoids

Signal molecules, defense agents

Limonene, menthol, camphor

Sesquiterpenoids

Antibiotic, antitumor, antiviral, immunosuppressive, and hormonal activities Hormonal activities, antitumor properties Cytostatic activities Membrane components Antioxidants, photosynthetic components, pigments, and nutritional elements

Flavors, fragrances, cleaning products, anticancer agents, antimicrobial agents Flavors, fragrances, potential pharmaceuticals, biofuels

Diterpenoids Sesterterpenoids Triterpenoids Tetraterpenoids

Anticancer agents None as yet Biological markers Food additives (colorants, antioxidants), anticancer agents

Juvenile hormone, cubebol, artemisinin, bisabolene α-santalene, valencene Gibberellins, phytol, taxol Haslenes Sterols, hopanoids β-carotene, lycopene,

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cost-effective. With commercial and medicinal uses of plant terpenoids on the rise, there is a need to increase the yield of terpenoid biosynthesis.

11.2.1 Biosynthesis of isoprenoids All isoprenoids originate from the five-carbon basic unit, isopentenyl-pyrophosphate (IPP), and its isomer dimethylallyl-pyrophosphate (DMPP), both resulting from either the mevalonate (MEV) pathway, frequently found in the eukaryotic cytoplasm, or the 2Cmethyl- D-erythritol-4-phosphate (MEP) pathway, which is observed in the eubacteria such as Escherichia coli and Streptomyces species (Kuzuyama and Seto 2003; Rohmer et al. 1993; Baadhe et al. 2012) as well as in plant plastids (Figure 11.1). The MEP pathway does not exist in animals or fungi, but both pathways are active in higher-level plants such as Artemisia annua, Arabidopsis thaliana, and Helianthus annuus. Almost all organisms have the ability to produce isoprenoid precursors and therefore can offer IPP, DMAPP, and other subsequent isoprenoid backbone precursors, as shown in Figure  11.1 (Hunter 2007). Due to the established Sugar metabolism

MEV pathway

MEP pathway

IPP

DAMP

GPP

Isoprene

Monoterpenes Sesquiterpenes

FPP Triterpenes Diterpenes GGPP

Sesterpenes Tetraterpenes

Figure 11.1 Graphic representation of isoprenoid biosynthesis from isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP) to important precursors of various isoprenoids. FPP, farnesyl-pyrophosphate; GGPP, geranyl geranyl pyrophosphate; GPP, geranyl-pyrophosphate; MEP, 2Cmethyl-D-erythritol-4-phosphate; MEV, mevalonate pathway.

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technology developments for analysis and manipulations of the omic’s data of E. coli and S. cerevisiae, they have been engineered for production of plant isoprenoids (Tyo et al. 2007). In addition, both E. coli and S. cerevisiae demonstrated as prominent hosts for the creation of isoprenoids and other (plant) natural products (Marienhagen and Bott 2013).

11.3 Metabolic engineering of the mevalonate (MEV) pathway In yeast, the mevalonate pathway (Figure  11.2) is primarily involved in the synthesis of an end product called ergosterol, an essential element involved in yeast membrane permeability and fluidity (Joseph et al. 2006). The pathway consists of two distinct parts: An early part of the mevalonate pathway supplies intermediate farnesyl diphosphate (FPP) for the synthesis of a number of other essential cellular constituents such as hemes, quinones, dolichols, or isoprenylated proteins and the other part of the pathway involved for the synthesis of ergosterol. In the mevalonate pathway, three molecules of acetyl-coenzymeA (CoA) couple to yield 3- hydroxy-3-mehtylglutarylCoA (HMG-CoA), which is subsequently reduced by the enzyme HMG-CoA reductase to yield mevalonicacid (MVA). In the next two steps, mevalonate kinase and mevalonate5phosphatekinase catalyze MVA to form mevalonate5-diphosphate, which is subsequently decarboxylated to yield isopentenyl pyrophosphate (IPP) Furthermore, IPP and DAMP gives geranyl phosphate, which, finally converted to FPP, leads to the production of geranyl geranyl pyrophosphate (GGPP) (Baadhe et al. 2012).

11.3.1 Metabolic engineering interventions for improved production of isoprenoids through the mev pathway 11.3.1.1 3-hydroxy-3-methyl-glutaryl-CoA reductase (hMGR) The rate-limiting step of the pathway catalyzed by HMG-CoA reductase and regulation takes place at the levels of transcription, translation, post-translational modification, and degradation. This generally occurs in two forms: HMG1 and HMG2. Among the two enzymes, HMG1 is considered quite stable whereas HMG2 undergoes mevalonate products-induced degradation. Truncated and soluble tHmg1p catalytic domain overexpression causes accumulation of a large amount of squalene (Hampton and Garza 2009). Overexpression of tHMG1 was applied to improve the amorphadiene and artemisinic acid production in yeast (Ro et al. 2006; Baadhe et al. 2013b). Construction of a point mutation by substituting lysine 6 by an arginine (K6R) in HMG2 signified its resistance toward ubiquitination (Kampranis and Makris 2012).

Acetate ACS1/ACS2 Acetyl-CoA

Gene Upregulation ALD6, SeACS, HMG1, HMG2, IDI1, ERG20 BTS1, LPP1, DPP1

ERG10/AtoB Aceto acetyl-CoA

Protein Engineering HMGR, GGPPS, TPSs Protein Fusion HMGS, AtoB, ERG20, PTS, BTS1, DPP1, CDS, TPs, ADS, GPS, eAS

Sterols

Gene Downregulation ERG9 LPP1, DPP1

HMGS

Squalene

hMG-CoA ERG9 HMG1, HMG2

DAMP ERG20 IDI1

Mevalonate ERG8, ERG12, ERG19

DPP1

GGPS

ERG20 GPP

FPP

GGPP BTS1

LPP1

Geranyl geraniol

IPP

Figure 11.2 Outline of terpene biosynthesis in yeast indicating the genes involved and the metabolic engineering contributions. AtoB, acetoacetyl-CoA synthase/thiolase; CDS, copalyl diphosphate synthase; CPP, copalyl diphosphate; DTS, diterpene synthase; eAS, epi-aristolochene synthase; PTS, patchoulol synthase; SeACS(L641P), Salmonella enterica acetyl-CoA synthase mutant L641P.

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11.3.1.2 Squalene synthase (ERG9) Squalene synthase (ERG9) catalyzes the first committed step for synthesis of sterols by converting two FPP into squalene. Since the majority of FPP is involved in the synthesis of sterols, limited substrate is available for isoprenoid synthesis, hence suppression of ERG9 one way to increase FPP availability. Nevertheless, complete deletion ERG9 affects the growth of the cell since sterols are involved in the maintenance of cell membrane integrity. Replacement of native promoter (ERG9) with repressible promoter (MET3) with varying concentrations of methionine repressed the transcription of ERG9 (Gardner and Hampton 1999). This approach in combination with overproduction of tHMG1 improved the production of major sesquiterpenoids such as amorphadiene, cubebol, valencene, and patchoulol in various studies (Ro et al. 2006; Asadollahi et al. 2008; Baadhe et al. 2013a, Baadhe et al. 2013b). However, in these studies, accumulation of farnesol as a by-product was observed, thus indicating destitute regulation of the promoter. Consequently, to provide tighter control of ERG9 expression, glucoseregulated promoters PHTX1 and PHTX2 were tested and PHTX1 was shown to be efficient in downregulating ERG9 expression under glucose-limiting conditions. In addition, regulating the transcript stability and heterozygous deletion of the gene in a diploid strain increased the monoterpenes and sesquiterpenes (Ignea et al. 2011). 11.3.1.3 Farnesyl diphosphate synthase (ERG20) The ERG20 enzyme catalyzes the formation of GPP and later FPP by the condensation of IPP and DMAP. The overexpression of ERG20 did not improve amorphadiene production (Ro et al. 2006). But under control of a strong promoter (PGAL1), it improved the production of some terpenoids (Ignea et  al. 2011). Mutations in the lysine 197 of ERG20 (K197E) increased the formation of GPP and, consequently, geraniol productivity by the expression of geraniol synthase; thus, it can be used for improved production of monoterpenes (Blanchard and Karst 1993; Fischer et al. 2011). 11.3.1.4 Acetyl-CoA metabolism Apart from contribution in energy generation through the TCA cycle, acetylCoA is also involved in the initiation of MEV pathway (Figure 11.3). The pyruvate dehydrogenase bypass converts pyruvate into acetyl-CoA by the action of pyruvate decarboxylase, cytosolic acetaldehyde dehydrogenase (ALD6), and acetyl-CoA synthetase (ACS). Thus, Overproduction of heterologous, acetaldehyde dehydrogenase (ALD6) from Salmonella enterica (L641P), together with tHMG1 expression, attained considerable enhancements in amorphadiene production (Shiba et  al. 2007). Later the push–pull–block approach (Chen et  al. 2013) improved the acetyl-CoA supply toward the MEV pathway (Figure 11.3). This strategy involved a push of carbon from ethanol via acetaldehyde to cytosolic acetyl-CoA and a block of further conversion of acetyl- CoA by competing

New trends in microbial production of natural complex bioactive isoprenoids

Sugars

275

Ethanol ADH2

Pyruvate

Acetaldehyde ALD6

Pyruvate

Acetate Acs SE L641P Acetyl-CoA

TCA cycle

ERG10

Aceto Acetyl-CoA

MLS1 Cellular lipids

GYC

CIT2 MEV Pathway

Figure 11.3 Illustration of the push–pull–block strategy for improving the production of

acetyl-CoA metabolism by acetyl-CoA in the cytosol.

pathways. The push part of the strategy involved overexpression of the endogenous ADH2 gene, encoding alcohol dehydrogenase, by using the glucoseregulatable HXT7 promoter, as well as constitutive overexpression of ALD6, encoding NADP-dependent aldehyde dehydrogenase, and a codon-optimized ACS variant (L641P) from S. enterica (acsSE L641P), encoding acetyl-CoA synthetase. ACSSE L641P contains a point mutation that prevents the enzyme from being inhibited by acetylation (Starai et al. 2005). Overexpression of ADH2 ensures that ethanol produced byAdh1p during growth on glucose can be converted back to acetaldehyde and from here further to acetyl-CoA. To ensure pulling of acetyl-CoA toward the products of interest, ERG10 is overexpressed, which encodes acetyl-CoA C-acetyl transferase that catalyzes the conversion of acetyl-CoA to aceto-acetyl-CoA (AcAcCoA). Finally, the block part involves the reduction precursor acetyl-CoA loss by avoiding the involvement and consumption of acetyl-CoA in the glyoxylate cycle (GYC), inhibiting the key enzymes peroxisomal citrate synthase, encoded by CIT2 and cytosolic malate synthase, encoded by MLS1.

11.3.1.5 Uptake control (UPC2) Upc2p and Ecm22p are two highly homologous zinc cluster proteins that regulate a number of ERG genes in the yeast ergosterol biosynthetic pathway and of DAN/TIR gene products. They relocate from intracellular membranes to perinuclear foci on sterol depletion; they positively regulate transcription by binding to

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sterol response elements in the promoters of the target genes. The upc2-1 mutant contains a single amino acid change (G888D) within the activation domain of the protein. Overexpression of upc2-1 in combination with tHMG1 and PMET3-ERG9 had a prominent effect on the synthesis of amorphadiene (Ro et al. 2006; PeraltaYahya et al. 2011; Westfall et al. 2012)

11.3.1.6 Geranylgeranyl diphosphate synthase (BTS1) The enzyme catalyzes the synthesis of GGPP by using FPP and IPP as substrates for ubiquinone biosynthesis and geranylgeranylation of proteins for membrane attachment. GGPP is a precursor for diterpene and carotenoid biosynthesis (Figure 11.1). Overexpression of BTS1 has been combined with lycopene cyclase/phytoene synthase (CrtYB) and phytoene desaturase (CrtI) from xanthophyllomyces dendrorhous to generate β-carotene and with cytochrome-P450 hydroxylase (CrtS) to produce astaxanthin (Verwaal et al. 2007; Ukibe et al. 2009). 11.3.1.7 Lipid phosphate phosphatase (LPP1) and diacylglycerol pyrophosphatase (DPP1) Downregulation of ERG9 in mammalian species was shown to lead to conversion of FPP to farnesol which was also understood in yeast strains (Kuranda et al. 2010). It has been hypothesized that dephosphorylation of FPP and GPP may be a mechanism to alleviate the possibly toxic effects of substrate accumulation. LPP1 and DPP1 are two enzymes initially recognized as phosphatidic acid hydrolases and later as dephosphorylate isoprenoid phosphates (Faulkner et al. 1999). A modest increase in the production of the sesquiterpene α-santalene and a 24% drop in farnesol accumulation was observed with the deletion of DPP1 (Scalcinati et al. 2012). However, other studies targeting at high sesquiterpene production did not perceive noteworthy improvements (Takahashi et al. 2007; Albertsen et al. 2011). Still, when LPP1 and DPP1 were overexpressed fused to BTS1, they employed a strong positive effect on geranylgeraniol production, with DPP1 exerting the strongest effect, yielding 2.9-fold higher levels of geranylgeraniol (GGOH) than simple co-expression of the genes (Tokuhiro et  al. 2009). 11.3.1.8 Protein engineering interventions for improved production of isoprenoids Protein engineering strategies includes the increase of product yield to interfere with the cyclization chemistry of the terpene synthases, which improves enzyme specificity or alters the products out of a specific enzyme. Extensive studies on E. coli and metabolic enzymes sequences noted that Gly and Pro were significantly less frequently mutated than other amino acid enzymes. To know their effects on the catalytic activity, an engineered version of tHMGR (tHMGRG9), with nine mutated residues, revealed a 2.5- to 3-fold increase in the production of mevalonate compared to the wild-type tHMGR. Similarly, HUM-G6

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(an engineered version of humulene synthase bearing six such substitutions) improved sesquiterpene production by 80-fold. Further integration of the tHMGR-G9 and HUM-G6 mutants into the same host resulted in a three- to four-fold improvement in growth and nearly a 1000-fold overall improvement in sesquiterpene production.

11.3.1.9 Metabolic channelling by expression of chimeric enzymes Heterologous expression of pathways/enzymes in microbial hosts is complex due to the presence of numerous native host enzymes and tight regulation of the host machinery. Heterologous product formation is not only affected by the host environment but also by the loss of intermediate metabolites through diffusion, degradation, or by competitive pathways. In order to avoid such intermediate loss and make heterologous expression more efficient, enzymes catalyzing successive reactions are often fused in close proximity to each other by using linkers. Linkers are the sequence coding for amino acids, which separates the two proteins in space with a small distance, allowing them to fold properly without restraints from each other. Consequently, the substrate was channeled between active sites of two or more sequential enzymes of a pathway, without allowing free diffusion of the intermediates. Subsequently it reduces the transit time required for the intermediates to spread the enzyme that catalyzes the next step in the reaction (Baadhe et al. 2013b). Fusions of ERG20 to PTS (FPPS-PTS) and amorphadiene synthase (ADS) (FPPS-ADS) were tested for the production of patchoulol and unveiled higher yields than the individual enzymes. Sometimes orientation enzymes during the fusion also affects their catalysis. C-terminal fusion of EYFP to a sesquiterpene synthase (STS) caused a significant reduction in enzymatic activity. A similar effect was observed with ADS-FPPS and PTS-FPPS (Albertsen et al. 2011; Baadhe et al. 2013b). In another approach, fusion of STS or FPPS to targeting proteins of organelles improved the isoprenoids. The cytochrome C oxidase subunit 4 isoform 1, mitochondrial (COX4), a mitochondrial targeting sequence, diverts pathway to the organelles where FPP pools are naturally present for the synthesis of ubiquinone, heme A, etc. The fusion FPPS or STS to COX4 and combined with cytosolic tHMG1 overexpression had a significant improvement in sesquiterpene production (Farhi et al. 2011).

11.4 Metabolic engineering of the MEP pathway for isoprenoids production Numerous studies have reported the engineering of the MEP pathway to increase the supply of isoprenoid precursors in E. coli. Balancing the pool of glyceraldehyde-3-phosphate and pyruvate, or overexpression of 1-deoxy-D-xylulose

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5-phosphate synthase (DXS; encoded by the gene dxs) and IPP isomerase (encoded by idi), drives the increased carotenoid buildup in the cell. Nevertheless, enhancements in isoprenoid production were noted, as well as some limitations raised by the native host by controlling the MEP pathway in E. coli. To circumvent this pathway, expressing the S. cerevisiae mevalonate-dependent pathway in E. coli-abundant isoprenoid precursors was observed, but that sometime inhibited the growth. The synchronized expression of a synthetic ADS resulted in high-level production of amorphadiene and circumvented growth inhibition (Martin et al. 2003) The E. coli MEP pathway was reconstructed in S. cerevisiae by cloning seven major genes of the pathway for isoprenoid production and the pathway was shown able to endure growth of yeast, under inhibition of it native MEV pathway. The resulting strain harboring valencene synthase produced up to 1 mg/L of valencene, but it is much less compared to other reported studies; however, optimization of expression of the MEP pathway in yeast may improve isoprenoid production (Maury et al. 2008). Nonfunctionality of the MEP pathway in S. cerevisiae is revealed by the lack of enzyme activity of IspG and/or IspH, which catalyze the last two reactions of the MEP pathway.

11.5 Modeling and simulation approaches Advancements in analytical technologies for analysis of omic’s data and modeling tools enabled the construction of metabolic models of the host organisms. Minimization of the metabolic adjustments algorithm (MOMA) recognized GDH1 as a conceivable target, which could swing the metabolic flux toward the ergosterol pathway. The gene encodes a glutamate dehydrogenase involved in ammonium metabolism in yeast and requires NADPH for its function. The conversion of HMG-CoA to mevalonate is a NADPH required step; thus, deletion of GDH1 was assumed to be beneficial for carbon flux through the mevalonate pathway by increasing the pool of available NADPH for HMGR. In yeast there are two other glutamate dehydrogenase enzymes encoded by GDH2 and GDH3. GDH3 appears to have arisen from genome duplication of GDH1, while GDH2, unlike the other two, is an NADH-dependent enzyme. Deletion of GDH1 in cells expressing cubebol synthase led to approximately 85% increase in the final titer (Asadollahi et al. 2009). Redox cofactors NAD (H) and NADP (H) are essential for the transfer of reducing equivalents in many enzymatic reactions and availability of these intracellular pools are very important for metabolic engineering. These cofactors species activities and specificities are manipulated by using genetic, metabolic, and protein engineering techniques. Identifying a potential target among the hundreds of reactions is tedious. A novel cofactor modification

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analysis (CMA) within the constraints-based flux analysis framework was a good approach to identify the target species. Analysis of isoprenoid synthesis pathway revealed that the IPP yield can be improved either by decreasing NADPH consumption or by increasing NADPH regeneration through modification of substrate specificity either from NADPH to NADH or from NAD to NADP, respectively. Between the enzyme targets identified by CMA, the alteration of cofactor specificity of glyceraldehyde-3-phosphate dehydrogenase from NAD to NADP was found to cause the best IPP yield improvement in both E. coli and S. cerevisiae (Chung et al. 2013).

11.6 Conclusions Contemporary developments indicate great ability for the production of isoprenoid compounds in either E. coli or S. cerevisiae host platforms. Many attempts have been made for increasing flux towards isoprenoid precursors by overexpressing, upregulating, and downregulating the endogenous genes. Still much effort continuing for improving the intermediate fluxes. Some successful attempt made, but there is still a thrust driving toward improving the acetyl- CoA precursor flux. The application and combination of the latest tools and strategies in systems biology, synthetic biology, protein engineering, process engineering, high-throughput technologies for cultivation, screening and analytics, and metabolic engineering of microorganisms for isoprenoid significantly enlarge the number as well as commercialization of isoprenoids.

References Alba-Lois, L. and C. Segal-Kischinevzky. 2010. Beer & wine makers. Nat. Edu. 3:17. Albertsen, L., Y. Chen, L. S. Bach, S. Rattleff, J. Maury, S. Brix, J. Nilsen and U. H. Mortensen. 2011. Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes. Appl. Environ. Microbiol. 77:1033–1040. Asadollahi, M. A., J. Maury, K. Møller, K. F. Nielsen, M. Schalk, A. Clark and J. Nielsen. 2008. Production of plant sesquiterpenes in Saccharomyces cerevisiae: effect of ERG9 repression on sesquiterpene biosynthesis. Biotechnol. Bioeng. 99:666–67. Asadollahi, M. A., J. Maury, K. R. Patil, M. Schalk, A. Clark and J. Nielsen. 2009. Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering. Metab. Eng. 11:328–334. Baadhe, R. R., N. K. Mekala, S. R. Palagiri and S. R. Parcha. 2012. Development of petri net-based dynamic model for improved production of farnesyl pyrophosphate by integrating mevalonate and methylerythritol phosphate pathways in yeast. Appl. Biochem. Biotechnol. 167:1172–1182. Baadhe, R. R., N. K. Mekala, S. R. Parcha and Y. P. Devi. 2013a. Optimization of amorphadiene production in engineered yeast by response surface methodology. 3 Biotech, 1–8. 10.1007/ s13205-013-0156-y.

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Baadhe, R. R., N. K. Mekala, S. R. Parcha and Y. Prameela Devi. 2013b. Combination of erg9 repression and enzyme fusion technology for improved production of amorphadiene in saccharomyces cerevisiae. J. Anal. Method. Chem. 2013. http://dx.doi.org/10.1155/2013/140469. Blanchard, L. and F. Karst.1993. Characterization of a lysine-to-glutamic acid mutation in a conservative sequence of farnesyl diphosphate synthase from Saccharomyces cerevisiae. Gene. 125:185–189. Breitmaier, E. 2006. Terpenes: flavors, fragrances, pharmaca, pheromones. John Wiley & Sons. pp. 223. Chang, M. C. and J. D. Keasling. 2006. Production of isoprenoid pharmaceuticals by engineered microbes. Nat. chem. biol. 2:674–681. Chen, Y., L. Daviet, M. Schalk, V. Siewers and J. Nielsen. 2013. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab. Eng. 15:48–54. Chung, B. K. S., M. Lakshmanan, M. Klement, B, Mohanty and D. Y. Lee. 2013. Genome-scale in silico modelling and analysis for designing synthetic terpenoid-producing microbial cell factories. Chem. Eng. Sci. 103:100–108. Croteau, R., T. M. Kutchan and N. G. Lewis. 2000. Natural products (secondary metabolites). Biochem. Mol. Biol. Plant. 24:1250–1319. Farhi, M., E. Marhevka, T. Masci, E. Marcos, Y. Eyal, M. Ovadis, H. Abeliovich and A. Vainstein. 2011. Harnessing yeast subcellular compartments for the production of plant terpenoids. Metab. Eng. 13:474–481. Faulkner, A., X. Chen, J. Rush, B. Horazdovsky, C. J. Waechter, G. M. Carman and P. C. Sternweis. 1999. The LPP1 and DPP1 gene products account for most of the isoprenoid phosphate phosphatase activities in Saccharomyces cerevisiae. J. Biol. Chem. 274:14831–14837. Fischer, M. J., S. Meyer, P. Claudel, M. Ragdoll and F. Karst. 2011. Identification of a lysine residue important for the catalytic activity of yeast farnesyl diphosphate synthase. Protein. J. 30:334–339. Gardner, R. G and R. Y. Hampton, 1999. A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes. J. Biol. Chem. 274:31671–31678. Hampton, R. Y. and R. M. Garza. 2009. Protein quality control as a strategy for cellular regulation: lessons from ubiquitin-mediated regulation of the sterol pathway. Chem. Rev. 109: 1561–1574. Horwitz, S. B. 1994. How to make taxol from scratch. Nature. 367:593. Hunter, N. 2007. The non-mevalonate pathway of isoprenoid precursor biosynthesis. J. Biol. Chem. 282:21573–21577. Ignea, C., I. Cvetkovic, S. Loupassaki, P. Kefalas, C. B. Johnson, S. C. Kampranis and A. M. Makris. 2011. Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microb. Cell. Fact. 10:1–18. Jeandet, P., Y. Vasserot, T. Chastang and E. Courot. 2013. Engineering microbial cells for the biosynthesis of natural compounds of pharmaceutical significance. Biomed. Res. Int. 2013. doi:10.1155/2013/780145. Kampranis, S. C. and A. M. Makris. 2012. Developing a yeast cell factory for the production of terpenoids. Comput. Struct. Biotechnol. J.3: e201210006, http://dx.doi.org/10.5936/ csbj.201210006. Kuranda, K., J. François and G. Palamarczyk. 2010. The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae. FEMS. Yeast. Res. 10: 14–27. Kuzuyama, T. and Seto, H. 2003. Diversity of the biosynthesis of the isoprene units. Nat. Prod. Rep. 20:171–183. Marienhagen, J. and Bott, M. 2013. Metabolic engineering of microorganisms for the synthesis of plant natural products. J. Biotechnol. 163:166–178.

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Marienhagen, J. and M. Bott, 2013. Metabolic engineering of microorganisms for the synthesis of plant natural products. J. Biotechnol. 163:166–178. Martin, V. J., D. J. Pitera, S. T. Withers, J. D. Newman and J. D. Keasling. 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21:796–802. Maury, J., M. A. Asadollahi, K. Møller, M. Schalk, A. Clark, L. R. Formenti and J. Nielsen. 2008. Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae. FEBS. Lett. 582:4032–4038. Maury, J., M. A. Asadollahi, K. Møller, A. Clark and J. Nielsen. 2005. Microbial isoprenoid production: an example of green chemistry through metabolic engineering. In Biotechnology for the Future ed. by J. Nielsen pp. 19–51. Springer-Verlag, Berlin Heidelberg. McGarvey, D. J. and R. Croteau. 1995. Terpenoid metabolism. Plant Cell. 7:1015. Peralta-Yahya, P. P., F. Zhang, S. B. Del Cardayre and J. D. Keasling. 2012. Microbial engineering for the production of advanced biofuels. Nature. 488:320–328. Peralta-Yahya, P. P., M. Ouellet, R. Chan, A. Mukhopadhyay, J. D. Keasling and T. S. Lee. 2011. Identification and microbial production of a terpene-based advanced biofuel. Nat. Commun. 2:483. Ro, D. K., E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu,., K. A. Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Y. Chang, S.T. Withers, Y. Shiba, R. Sarpong and Keasling, J. D. 2006. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature. 440:940–943. Rohmer, M., M. Knani, P. Simonin, B. Sutter and H. Sahm. 1993. Isoprenoid biosynthesis in bacteria: A novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 295:517–524. Ruzicka, L. 1953. The isoprene rule and the biogenesis of terpene compounds. Experientia. 9:357–367. Scalcinati, G., C. Knuf, S. Partow, Y. Chen, J. Maury, M. Schalk, L. Daviet, J. Nilsen and V. Siewers. (2012). Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode. Metab. Eng. 14: 91–103. Shiba, Y., E. M. Paradise, J Kirby, D. K. Ro and J. D. Keasling. 2007. Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids. Metab. Eng. 9:160–168. Smolke, C. 2009. The metabolic pathway engineering handbook: tools and applications (Vol. 2). CRC Press, Taylor & Francis. pp. 584. Starai, V. J., J. G. Gardner and J. C. Escalante-Semerena. 2005. Residue Leu-641 of acetyl-CoA synthetase is critical for the acetylation of residue Lys-609 by the protein acetyltransferase enzyme of Salmonella enterica. J. Biol. Chem. 280:26200–26205. Takahashi, S., Y. Yeo, B. T. Greenhagen, T. McMullin, L. Song, J. Maurina-Brunker R. Rosson, J. P. Noel and J. Chappell. 2007. Metabolic engineering of sesquiterpene metabolism in yeast. Biotechnol. Bioeng. 97:170–181. Tokuhiro, K., M. Muramatsu, C. Ohto, T. Kawaguchi, S. Obata, N. Muramoto, M. Hirai, H. Takahashi, A. Kondo, E. Sakuradani and S. Shimizu. 2009. Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. Appl. Environ. Microbiol. 75:5536–5543. Ukibe, K., K. Hashida, N. Yoshida and H. Takagi. 2009. Metabolic engineering of Saccharomyces cerevisiae for astaxanthin production and oxidative stress tolerance. Appl. Environ. Microbiol. 75:7205–7211. Verwaal, R., J. Wang, J. P Meijnen, H. Visser, G. Sandmann, J. A. Van den Berg and A. J. Van Ooyen. 2007. High-level production of beta-carotene in Saccharomyces cerevisiae by successive

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CHAPTER 12

Production of C-phycocyanin and its potential applications Mohammed Kuddus1, Poonam Singh2, George Thomas2, and Athar Ali3 1

Department of Biochemistry, College of Medicine, University of Hail, Hail, Saudi Arabia Department of Molecular and Cellular Engineering, SHIATS, Allahabad, India 3 Department of Biotechnology, Jamia Hamdard, New Delhi, India 2

12.1 Introduction Since the advent of technology, science has explored many concealed natural resources. The chemicals used in the past decade were expensive and caused biohazards. Therefore, research in finding safe and less expensive natural bioactive compounds has been started. In the early period, the immunodiffusion assays followed by radio-immunoassays and enzyme-linked immunoassays provide information of significant magnitude. In these assays, different synthetic fluorochromes were used and today it explores a separate field of interest, called fluorescent immunoassay (Kasten 1993). As the synthetic fluorochromes have biohazards and are carcinogenic in nature, such as ethidium bromide, a new approach to research was needed to find out novel and safe chemical compounds. Phycobiliproteins are natural proteins commonly present in cyanobacteria and red algae possessing a spectrum of applications and extensively used for fluorescent application in clinical and immunological analysis along with their therapeutic value (Sekar and Chandramohan 2007). Phycobiliproteins are brilliantly colored, highly fluorescent, water-soluble protein components of the photosynthetic light-harvesting antenna complexes found in blue-green algae, red algae, and cryptomonads. Based on their colors, these proteins are classified into two groups: phycocyanin (blue) and phycoerythrin (red). The phycocyanins include C-phycocyanin (C-PC), R-phycocyanin (R-PC), and Allophycocyanin (APC). Phycobiliproteins are assembled into an organized cellular structure known as phycobilisomes that are attached in regular arrays to the external surface of the thylakoid membrane and act as major light-harvesting pigments in cyanobacteria and red algae. The term “phycobilisome” was first coined by Gantt and Conti (1966) on the basis of their size and shape, as visualized by an electron microscope. The electron micrographs showed a series of large granules aligned regularly on the thylakoid membranes of different cyanobacteria and red alga, Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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which were about twice the size and of similar shape of ribosomes, and attracted the attention of early biologists due to their brilliant colors. The phycobilisomes allow the pigments to be arranged geometrically in a manner that helps to optimize the capture of light and transfer of energy. The phycobiliproteins were introduced as a novel class of fluorescent dye in 1982 (Oi et  al. 1982). These naturally occurring fluorescent macromolecules had immediate widespread use in diagnostic assays and in diverse research applications (Kronick 1986; Glazer and Stryer 1990). The phycobiliproteins serve as valuable fluorescent tags with numerous applications in flow cytometry, fluorescence-activated cell sorting, histochemistry, and, to a limited degree, in immunoassay. These applications exploit the unique physical and spectroscopic properties of phycobiliproteins (Glazer 1994). In addition, because of the high molecular absorptivity of these proteins at visible wavelengths, they are convenient markers in such applications as gel electrophoresis, isoelectric focusing, and gel exclusion chromatography (Sarada et al. 1999; Rito-Palomares et al. 2001). C-phycocyanin belongs to a family of phycobiliproteins that are well suited as a fluorescent reagent for immunological analysis because they have a broad excitation spectrum and fluorescence with a high quantum yield. Phycobiliproteins are easily isolated as a pigment protein complex, which are soluble in water and very fluorescent (Glazer 1982). It is a stable protein and contains multiple chromophore prosthetic groups, which are responsible for the fluorescent properties of this protein. They are attached to the surrounding protein structure via thioether linkage involving cystein residues. Chromophore prosthetic groups are constructed from linear or open tetra-pyrol rings and are structurally related to the bile pigment biliverdin. The four main chromophore types present in algal and cyanobacterial species are phycocyanobilin (PCB), phycoerythrobilin (PEB), phycourobilin (PUB), and cryptoviolin, whereas C-PC has only phycocyanobilin.

12.2 Production of C-phycocyanin The cyanobacteria are a potential source of C-PC pigments along with rodophytes (Table 12.1). Their cultivation without organic substrates can be an economical advantage over the other microorganisms and an optimized production of relevant compounds under controlled conditions is conceivable (Kreitlow et al. 1999). Pure phycocyanins from crude algal extracts are usually obtained by a combination of different techniques such as salt precipitation and chromatography. The various aspects of C-PC production have been reported by Eriksen (2008). According to the above report, production of C-PC includes four different options: photoautotrophic, mixotrophic, heterotrophic, and recombinant production (Table 12.2).

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Table 12.1 C-phycocyanin-producing organisms (Kuddus et al. 2013). C-PC-producing organisms

Reference

Anabaena marina Anabaena sp. Aphanizomenon flosaquae Arthronema africanum Coccochloris elabens Cyanidium caldarium

Ramos et al. 2010 Moreno et al. 1995; Shukia et al. 2008 Benedetti et al. 2006 Minkova et al. 2007 Kao et al. 1973 Kao et al. 1975; Oranda et al. 1975; Troxler et al. 1981; Stec et al. 1999 Graverholt 2004 Contreras-Martel et al. 2007 Patel et al. 2005 Kao and Berns 1977 Wang et al. 2012 Shukia et al. 2010 Soni et al. 2006 Satyanarayana et al. 2005; Soni et al. 2008 Minkova et al. 2003 Herrera et al. 1989; Rito-Palomares et al. 2001; Abd El-Baky and El-Baroty 2012; Muthulakshmi et al. 2012 Boussiba and Richmond 1979; Zhang and Chen 1999; Doke 2005; Chen et al. 2006; Patil et al. 2006; Niu et al. 2007; Zhu et al. 2007; Duangsee et al. 2009; Mohite and Wakte 2011; Walter et al. 2011 Singh et al. 2010; Cherdkiatikul and Suwanwong 2014 Rogner et al. 1990 Nield et al. 2003 MacColl et al. 1974 Abalde et al. 1998; Adir et al. 2001 Gupta and Sainis 2010

Galdieria sulphuraria Gracilaria chilensis Lyngbya sp. Mastigocladus laminosus Microcystis Nostoc, Phormidium Oscillatoria quadripunctulata Phormidium fragile Spirulina fusiformis Spirulina maxima Spirulina platensis/ Arthrospira platensis

Spirulina sp. Synchacystis sp. Synechochoccus elongates Synechochoccus lividus Synechochoccus vulcanus Synechococcus sp.

Table 12.2 Different methods of C-PC production (Kuddus et al. 2013). Method

Characteristics

Photoautotrophic production

An outdoor method of C-PC production by using open ponds. Mostly A. platensis, used for dry weight production. Production is carried out in an enclosed reactor. Specific growth rate of mixotrophic cultures correspond to sum of photoautotrophic- and heterotrophic-specific growth rates. Higher growth rate of A. platensis is found in mixotrophic indoor cultures compared to photoautotrophic outdoor cultures. Production is not limited by incident light intensity. The unicellular rhodophyte Galdieria sulphuraria is a candidate for heterotrophic production of C-PC. Recombinant protein production is an option for heterotrophic synthesis of C-PC. Recombinant C-PC has been produced in photoautotrophic Anabaena.

Mixotrophic production

Heterotrophic production

Recombinant production

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12.3 Photoautotrophic production This method is predominantly used at tropical and subtropical locations. This is an outdoor method of C-PC production by photoautotrophic cultures of cyanobacterium grown in open ponds (Pulz 2001; Carlozzi 2003; Jimenez et al. 2003). Spirulina platensis has been commonly chosen as a host for C-PC production due to its availability. It is among a few photoautotrophic microorganisms that can be grown in open ponds without being outcompeted by contaminating organisms, although contaminants do appear in open S. platensis cultures (Richmond and Grobbelaar 1986; Richmond et al. 1990). Worldwide production of S. platensis has been increasing since 1980 (Borowitzka 1999; Pulz and Gross 2004) and the majority of its dry weight (>3,000 metric tons) produced worldwide annually, are used for health products and animal feed additives (Pulz and Gross 2004; Spolaore et al. 2006).

12.4 Mixotrophic production In this method cultivation of blue-green alga including S. platensis is to be carried out in an enclosed reactor. The mixotrophic cultivation results in faster growth and increased maximal biomass concentrations compared to photoautotrophic cultures (Marquez et  al. 1993; Vonshak et  al. 2000; Chojnacka and Noworyta 2004). The comparative data of Eriksen (2008) on the productivities of C-PC contents also showed a higher growth rate in the mixotrophic indoor cultures than in the photoautotrophic outdoor cultures of S. platensis. The specific growth rate of mixotrophic cultures grown on glucose correspond to the sum of the photoautotrophic- and heterotrophic-specific growth rates (Marquez et al. 1993).

12.5 Heterotrophic production The use of organic compounds for growth of microorganisms is defined as heterotrophy and the organism’s dependence on organic compounds synthesized by other organisms is known as heterotrophs. The basic culture medium composition for heterotrophic cultures is similar to the autotrophic culture except for organic carbon. Nowadays heterotrophic cultures are gaining increasing interest for producing a wide variety of microbial metabolites at all scales. However, heterotrophic cultures have some major limitations also, such as a limited number of microbial species that can grow heterotrophically, increase in the costs of adding an organic substrate, inhibition of growth by excess organic substrate, and inability to produce light-induced metabolites (Perez-Garcia et  al. 2011). The heterotrophic microbial processes are easier to scale up with regard to reactor size, mixing, gas transfer, and productivity since high surface-to-volume

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ratios are not mandatory. The unicellular rhodophyte, Galdieria sulphuraria, is a candidate for heterotrophic production of C-PC. Its natural habitat is hot, acidic springs, so the optimal growth conditions are found at temperatures above 40°C and it is able to utilize a variety of carbon sources (Gross and Schnarrenberger 1995). The properties of C-PC from heterotrophic G. sulphuraria resemble those of C-PC from other cyanobacterial sources. Schimidt et al. (2005) and Graverholt and Eriksen (2007) investigated the growth and C-PC production by G. sulphuraria 074G strains. The Arthospira strains can also grow heterotrophically on glucose and fructose in darkness.

12.6 Recombinant production Production of recombinant protein is an alternative method for synthesis of heterotrophic C-PC. The requirement of recombinant proteins has increased as it is more applicable in some areas. An essential condition for sufficient recombinant C-PC production is the efficient expression of the C-PC-producing gene. Production of phycobiliprotein such as C-PC is more challenging than production of other recombinant proteins. Recombinant C-PC, in which cpcA and cpcB genes were fused to His6 tags for affinity chromatographic purification, has been produced in photoautotrophic Anabaena species, which naturally synthesize and insert phycocyanobilin into C-PC (Cai et al. 2001). These fusion proteins were expressed as stable C-PC complexes. Coding sequences for different biospecific recognition domains were also fused to stabilized C-PC fusion constructs, and the expressed multidomain fusion proteins were used as fluorescent probes (Eriksen 2008). Gene engineering has resulted in the production of recombinant C-PC with novel functions. In heteromorphic hosts, recombinant holo C-PC α-subunit has been expressed in Escherichia coli. The His6 tags have allowed purification by affinity chromatography, and domains with affinities for specific biological structures have been incorporated (Tooley et al. 2001; Guan et al. 2007).

12.6.1 Isolation of C-phycocyanin C-phycocyanin is a major phycobiliprotein found in all cyanobacteria. It is usually isolated from Spirulina; however, some other cyanobacterial genera have also been reported. Isolation and purification of C-PC is a multistep process that includes fractional salt precipitation and various chromatographic methods (Gantar et al. 2012). Currently, a wealth of literature is available by various authors on the methods of C-PC isolation, which is summarized in a related review (Sekar and Chandramohan 2007). According to the available data, the process of C-PC isolation involves various steps such as cell disruption or breakage, crude isolation, purification, drying, and characterization of the end products. According to the nature of organisms, a variety of physical and chemical methods are used for cell disruption. The common physical method includes sonication, cavitation, osmotic

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shock, and repeated freezing and thawing. In chemical methods, usage of acids, alkali, detergents, enzymes, and their combination thereof are reported. In general, combinations of a variety of physical and chemical methods are exploited for cell breakage. After cell breakage, clarification by centrifugation was performed and the product is primarily isolated from the supernatant. It includes fractionation using ammonium sulphate or other salts, dialysis, and polyethylene glycol precipitation. Further purification is usually achieved by column chromatographic methods using adsorbents, molecular sieves, anion exchangers, or their combinations. According to the nature of organisms, a combination of physical and chemical methods with primary isolation and purification of the product is adapted. For drying the pigment, only freeze drying was found to be suitable. It is up to the technologist to choose any of these methods and their combination to achieve maximum recovery of the product. The characterization of C-PC is required for the molecular understanding of various structures of phycobilisomes and this provides a basis for the modeled reconstruction of the pigment complex. In order to exploit this natural-colored substance, the C-PC is to be extracted from the phycobilisome and purified. Extraction of the phycobiliprotein from cyanobacteria and microalgae are very difficult because of the extremely resistant cell wall and the small size of the microbes (Stewart and Farmer 1984; Wyman 1992). However, various methods can be employed for extraction of phycobiliproteins, but no standard technique to quantitatively extract the C-PC pigment from microalgae exists (Wilshire et al. 2000). A method that works well in one organism may not be the method of choice for another organism (Ranjitha and Kaushik 2005). The above various methods referring to cell disruption have certain limitations, so some methods are combined in order to break the cell completely. C-phycocyanin has been extracted by resuspending the biomass in 0.1M phosphate buffer, pH 7.0 (Doke 2005; Oliveira et al. 2008), or 0.5M (NH4)2SO4 (Niu et al. 2007). Doke (2005) found that most C-PC could be extracted from biomass dried at low temperatures. At 25°C, 80 mg C-PC per gram dried biomass could be extracted compared to just 16.5 mg per gram from biomass dried at 50°C. Oliveira and coworkers (2008) found that high drying temperatures in two different dryers decreased the amount of extractable C-PC from A. platensis. From wet biomass, C-PC has been extracted by subjecting the biomass to cycles of freezing at –25 to –15°C or in liquid nitrogen, and thawing at 4 to 30°C (Abalde et  al. 1998; Zhang and Chen 1999; Minkova et  al. 2003; Doke 2005; Soni et al. 2006). When compared to alternative methods, freeze–thaw cycles have been the most efficient way to extract C-PC from wet cyanobacterial biomass (Abalde et  al. 1998; Doke 2005). C-PC has also been extracted after mechanical cell disruption (Boussiba and Richmond 1979; Schimidt et al. 2005), lysozyme treatment (Boussiba and Richmond 1979), sonication (Abalde et  al. 1998; Furuki et al. 2003), and high-pressure exposure (Patil et al. 2006; Patil and Raghavarao 2007). It has also been described that live Klebsiella pneumonia effectively lyses A. platensis and extract C-PC in 24 hours (Zhu et al. 2007).

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12.6.2 Purification and characterization of C-phycocyanin The purified C-PC is required for determination of their structure and function along with industrial and pharmaceutical applications. Also, the required purity of C-PC varies according to the anticipated use of the product such as the role it plays in commercial applications, particularly if used as fluorescence tags. In general, the purity of C-PC is evaluated by using the absorbance ratio of A620/A280. C-PC preparations with A620/A280 greater than 0.7 was considered as food grade, while A620/A280 of 3.9 was reactive grade and A620/A280 more than 4.0 as analytical grade. The purity of C-PC is directly related to the cost of processing (Muthulakshmi 2012). Usually C-PC has been purified by using a series of chromatographic columns but in recent years some new purification techniques/methods such as the rivanol method and the two-phase extraction method have been used. Rivanol method was used to purify C-PC from Spirulina fusiformis; the purity of C-PC (A620/A280) was 4.3 (Minkova et  al. 2003). Aqueous two-phase extraction has some advantages over one phase such as short time, high recovery rate, and concentrated product. Crude extract of C-PC from Spirulina platensis is subjected to aqueous two-phase extraction, and purity was increased from 1.18 to 5.22 (Patil et  al. 2006). Herrera et  al. (1989) combined ultra-filtration, charcoal adsorption, and spray drying to obtain C-PC with A620/A280 of 0.74 and a yield of 34%, while additional chromatographic steps were included to purify C-PC to A620/A280 of 3.91 and a yield of 9%. Ammonium sulphate precipitation combined with a variety of chromatographic principles has been employed to obtain C-PC of food, reactive, and analytical grades (Boussiba and Richmond 1979; Abalde et al. 1998; Zhang and Chen 1999; Minkova et al. 2003; Benedetti et al. 2006; Soni et al. 2006; Minkova et al. 2007; Niu et al. 2007). Also two-phase aqueous extraction was developed into an efficient method for C-PC purification (RitoPalomares et  al. 2001), which resulted in highly pure C-PC preparations and high yields (Patil et al. 2006; Patil and Raghavarao 2007). Single-step hydrophobic interaction chromatography is recently reported to result in extremely pure C-PC with an A624/A280 value of 4.52 (Soni et al. 2008). A number of methods that have been used to purify C-PC from cell extracts are described by Eriksen (2008). After purification the protein should be characterized to know their properties before using it in any industrial or biotechnological process. The literature showed that C-PC is composed of αβ heterodimers, each heterodimer containing three linear tetrapyrrole chromophores, referred to as phycocyanobilins. Three αβ units oligomerize as disc-shaped trimers (αβ)3, which in turn form hexamers (αβ)6 that stack one above the other, forming the rod-like structure of phycobilosome (Glazer 1989). The C-PC structure has been determined by X-ray crystallography at a resolution of 1.45 Å (Nield et al. 2003). Twelve crystal structures of phycocyanins were determined with the ultimate goal to obtain a physical picture of energy absorption and transfer. Adir (2005) analyzed the molecular understanding of various structures of phycobilisomes and suggested that the structural information obtained from the components

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provides the basis for the modeled reconstruction of this pigment complex. In a recent study, phycocyanin was purified from Anabaena variabilis CCC421 through ammonium sulfate precipitation, dialysis and anion exchange chromatography, with a purity of 2.75. Purified phycocyanin was found to contain two subunits of 17 and 18 kDa, which were identified as α and β subunits by SDS-PAGE and MALDI-TOF (Chakdar et al. 2014).

12.6.3 Applications of C-phycocyanin Due to increasing awareness of the environmental hazard of synthetic colors, biological sources of natural colors have been in great demand, especially in the food and cosmetics industries. Cyanobacteria possess a broad range of colored components including carotenoids, chlorophyll, and phycocyanin (Gantt 1975). C-phycocyanin is commonly used as a natural dye in food and cosmetics and replaced synthetic dyes. Also, phycocyanin has significant antioxidant, antiinflammatory, hepatoprotective, and radical scavenging properties as they are nontoxic and noncarcinogenic (Romay and Gonzalez 2000). Phycobiliproteins including C-PC is mainly used as fluorescent markers of cells and macromolecules in biomedical research and in highly sensitive fluorescence techniques (Glazer 1994). C-PC has also been used in a variety of immunological assays and as fluorescent labels for cell sorting because of its high molar absorptivity and other phycobiliproteins at visible wavelength are convenient markers in such applications as gel electrophoresis, isoelectric focusing, and gel exclusion chromatography. This has further enhanced the scope of the diagnostic industry. C-PC can be used for the detection of multiple myeloma cells and as a potential therapeutic agent in oxidative stress-induced disease (Bhat and Madyastha 2000). The pharmaceutical industry demands highly pure C-PC with an absorption ratio (A620/A280) of 4 and the food industry demands a ratio of 2 (Bhaskar et al. 2005). Some of the specific applications of C-PC are described in detail next.

12.7 C-phycocyanin as a natural dye Most of the blue-green algae contain C-PC as a major phycobiliprotein. The intense blue color in blue-green algae is due to the presence of phycocyanin, which emits red fluorescence on excitation. This pigment has a single visible absorbance maximum between 615 and 620nm. It has maximum fluorescence emission at around 640nm. The molecular weight of pigment is between 70 and 110 kDa and is composed of two subunits of α and β chain, which occur in equal numbers. However, the exact number of αβ pairs may vary among different species. In addition to absorbing light directly, this intensely blue pigment accepts quanta from phycoerythrin by fluorescent energy transfer in organisms in which phycoerythrin is present. Also, the C-PC pigment is widely used as a natural dye

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for various purposes due to its deep and intense blue color. They are well suited as a fluorescent reagent without any toxic effect for immunological analysis since they have a broad excitation spectrum and fluorescence with a high quantum yield (Hardy 1986). They can be used as a valuable fluorescent probes for analysis of cells and molecules (Kulkarni et al. 1996).

12.8 Application of C-phycocyanin as an additive in food and cosmetics The phycocyanin extracted from microalgae Spirulina has been used as a naturally occurring colorant for food-additive purposes (Kato 1994; Hirata et  al. 2000) but is limited by its lower stability to heat and light (Jespersen et al. 2005). Recent studies showed that phycocyanin is used as a colorant in jelly gum and coated soft candy, despite its lower stability toward heat and light (Jespersen et al. 2005). Some studies have addressed the functionality of C-PC in foods as color stabilizers (Jespersen et al. 2005; Mishra et al. 2008) and rheological properties (Batista et al. 2006). C-PC from A. platensis is also marketed as a food and cosmetics colorant in Japan (Prasanna et al. 2007). Several literatures also mentioned that limited customer preference in consuming blue foods might have minimized the industry’s interest in coloring food with C-PC (Glazer and Stryer 1984). More attention has been paid on the use of C-PC as a nutraceutical, particularly in health foods in which dried A. platensis is the functional component. Along with the nutritional value of C-PC, whole cyanobacteria are recommended to stimulate the immune system and possess anti-inflammatory, antioxidant, anticancer, antiviral, and cholesterol-lowering effects (Jensen et al. 2001). The largest intake of C-PC by humans is in nonpurified form through A. platensis health food products (Spolaore et al. 2006). In conclusion, phycocyanin is used as a natural colorant in many food items, including ice cream, dairy products, chewing gum, soft drinks, and jellies, as well as in cosmetics such as lipsticks, eye shadows, and eyeliners (Pandey et al. 2013).

12.9 Diagnostic applications of C-phycocyanin In comparison to other fluorophores, phycocyanin has a high molar extinction coefficient and fluorescence quantum yield along with large stoke shifts. Phycobiliproteins such as phycocyanin conjugated to immunoglobins and avidin that developed into fluorescent probes and have obtained broad application in histochemistry, flow cytometry, fluorescence-activated cell sorting, fluorescence microscopy, and fluorescence immunoassays (Glazer and Stryer 1984; Sun et al. 2003; Sekhar and Chandramohan 2007). The use of C-PC in fluorescent probes is dependent on chemical cross-linking of peptides to form stable

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trimers (Sun et al. 2006). Absorbance and fluorescence emission spectra of the chemically stabilized C-PC trimers are very similar to native C-PC trimers, except that their coefficients are actually increased. These chemically stabilized C-PC trimers can be used as fluorescent probes with spectral properties different from other phycobiliproteins. Also complete phycobilisomes from A.  platensis composed of C-PC have been chemically stabilized, combined with streptavidin, and used as fluorescent probes in cytometry (Telford et al. 2001). Also genetically stabilized C-PC fusion proteins fused to biospecific recognition domains have been used directly as biospecific fluorescent probes (Cai et  al. 2001, Eriksen 2008). The other applications of phycocyanins as fluorescent probes include the utilization of in vivo fluorescence from phycocyanin for online monitoring of growth in cyanobacterial cultures (Sode et  al. 1991), detection of toxic cyanobacteria in drinking water (Izydorczyk et al. 2005) and remote sensing of cyanobacteria in natural water bodies (Simis et  al. 2005). Badakere et  al. (1982) and Kulkarni et  al. (1996) reported the possibility of staining RBCs, WBCs, platelets, lymphocytes, nucleated cells, and genomic DNA with C-PC. Singh et al. (2010) also concluded that partially purified C-PC could be used as a substitute of ethidium bromide and may be applied for immunological analysis and DNA staining.

12.10 Nutraceutical and pharmaceutical applications A nutraceutical is defined as a product isolated or purified from foods that provides medical or health benefits and as generally sold in medicinal forms. Pharmaceutical products are an essential component of medicine that is used in the medical diagnosis, cure, treatment, or prevention of disease. Purified C-PC has both nutraceutical and pharmaceutical potentials. Along with C-PC, Spirulina platensis—a filamentous cyanobacterium—is extensively used in human nutrition and health as a food and dietary supplement due to its variety of nutritional components (Cohen 1997). Phycocyanin from different cyanobacteria has also been reported to exhibit a variety of pharmacological activities, such as anticancerous, antioxidant, anti-inflammatory, neuroprotective, hepatoprotective, hypocholesterolemic, and radical scavenging properties (Pandey et  al. 2013). A  variety of impaired physiological conditions are reported to be relieved by C-PC administration (Liu et al. 2000; Cherng et al. 2007; Sathyasaikumar et al. 2007). It has also been observed that C-PC can inhibit cell proliferation (Liu et al. 2000), induce apoptosis in cancerogenic cell lines (Dasgupta et  al. 2001), and affect gene regulation in mammalian cell lines (Cherng et al. 2007). The antioxidant and radical scavenging activities of C-PC from different cyanobacteria are well documented (Bhat and Madyastha 2000; Romay and Gonzalez 2000; Dasgupta et al. 2001; Upasani and Balaraman 2003; Bermejo et al. 2008; Soni et  al. 2008). Enhanced radical scavenging activities have been reported in

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selenium-enriched C-PC obtained from A. platensis grown in Se-enriched medium (Chen et al. 2006; Huang et al. 2007). However, intact C-PC may not be the dominant functional antioxidants in vivo. These observations have launched a further interest in C-PC as a nutraceutical or pharmaceutical with other possible health effects.

12.11 Anticancerous activity The C-PC from cyanobacterium Spirulina paletnsis has been reported to inhibit the growth of cancer cells and induce apoptosis in cancerous cell lines (Liu et al. 2000; Subhashini et al. 2004; Roy et al. 2007). It was also observed that the inhibitory effects of C-PC on the growth of human leukemia K562 cells are in a dose- and time-dependent manner. Also, the purified PC of S. platensis and its hydralysates have apparent inhibition effects on the proliferation of HeLa cells in vitro (Xueqing et  al. 2008). Li et  al. (2009) also suggested that C-PC decreased the number of HeLa cells. It was indicated that the β subunit of C-PC has potential anticancer fragment  in vitro (Shenfeng 2005). Also, a recombinant apo-C-PC β-subunit has been observed to inhibit cell proliferation and cause apoptosis in carcinoma cells (Wang et al. 2007). One of the recent findings indicates that C-PC could be potentially useful for treatment of LPS-related acute lung injury by inhibiting inflammatory responses and apoptosis in lung tissues (Leung et al. 2013).

12.12 Future prospects In addition to their nutritional values, biomass from cyanobacteria are also considered as a source of protein and C-PC. The cultivation of cyanobacteria under controlled optimum conditions and at low cost of its downstream processing would be an economical advantage to execute its current demand. Along with different factors affecting production and composition of C-PC, light quality and its intensity have high significant value. The commercial potential of C-PC has some major obstacles such as widespread utilization and increasing product yield. These problems may be solved through linkages between laboratory researchers and industrial technologists. The various approaches directed toward low-cost production and harvesting technologies along with evaluation of new environmental conditions for cyanobacterial production may be useful. This chapter summarizes some recent developments in production and applications of C-PC. However, efforts have to be made in order to achieve economical overproduction of C-PC by rDNA technology and increase its nutritional and pharmacological value by protein engineering and by other techniques.

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SECTION II

Chemistry, biotechnology, and industrial relevance

CHAPTER 13

Glycosides: From biosynthesis to biological activity toward therapeutic application Maria Henriques L. Ribeiro Research Institute for Medicines, Faculdade de Farmácia, Universidade Lisboa, Lisbon, Portugal

13.1 Introduction Cancerous, infectious, and neurodegenerative diseases are a health problem all over the world. More than 70% of all cancer deaths occur in low- and middleincome countries, where resources available for prevention, diagnosis, and treatment are limited or nonexistent. On the other hand, the 20th century brought deep demographic changes in industrialized countries due to birth and mortality rate imbalances, leading to an increase in the older population, which, as expected, will continue in 21st century. Around 2050–2060 it is expected that up to 25–30% of the population will be age 65 and older (Bustacchini et al. 2009). Therefore, the increase of age-related disease incidence is foreseen, shifting drug research into new fields. Among the diverse group of age-related diseases are atherosclerosis, hypernatremia, metabolic syndrome, cancer, prostate enlargement, osteoporosis, osteoarthritis, insulin resistance, age-related macular degeneration, dementia, cognitive dysfunction, and Alzheimer’s and Parkinson’s diseases. A relevant economic resource for the pharmaceutical, cosmetic, and food industries are natural products, a highly available source of bioactive compounds, useful to improve the well-being and the quality of human life. In line with this assumption, the academic and industrial interest for the health benefits of glycosides has increased. In fact, glycosides have important pharmacological applications, with emphasis as cardiac drugs, anti-inflammatories, neuroprotectors, analgesics, antirheumatics, laxatives, and some claim to reduce capillary fragility and, more recently, as anticancer agents (e.g., amygdalin commercialized in the United States as Laetrile). It should be mentioned that flavonoids have important properties such as antioxidant, anti-inflammatory, antidemential, and anticarcinogenic, among others. One of the main factors influencing the bioavailability of these

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

303

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compounds is the chemical structure of the compound. Most of the glycosides in foods exist as polymers or in glycosylated forms. The sugar group is known as the glycone and the nonsugar group is the aglycone. In native forms the glycosides are not absorbed and have to be hydrolyzed by intestinal enzymes or by colonic microflora before absorption. Anthocyanins represent an exception, since the intact glycosides can be absorbed and are detected in the circulation. The explanation for this may lie in the instability of the aglycone form or in specific mechanisms of absorption or metabolism for anthocyanins, as suggested by many studies. The specific chemical structure of glycosides, as well the type of the sugar in the glycoside, determines their rate and extent of intestinal absorption. The enzymatic hydrolysis of glycosides is held by glycosidases, which catalyze the hydrolysis of terminal nonreducing residues with release of glucose. One of the problems associated with some glycosides in therapeutics is their scarce availability in nature, especially some (de)glycosylated forms. Nowadays, biocatalysis with the biosynthesis of new or “old” glycosides is an important approach to solve this problem.

13.2 Glycosides Glycosides are molecules with a sugar bound to another functional group via a glycosidic bond. Usually they are nonreducing compounds, which hydrolysis by chemical reagents or enzymes yield one or more reducing sugars, beyond the products of hydrolysis. Sugars exist in α and β isomeric forms (Table  13.1). Examples of α linkage are in sucrose, glycogen, and starch. Theoretically, both α and β glycosides are possible, but in reality the natural glycosides are of β type.

13.3 Classification Glycosides can be classified (Table 13.1) according to the type of glycosidic linkage, chemical group of the aglycone involved in the acetal union, nature of the simple sugar, number of monosaccharides in the sugar moiety, and the chemical nature of the aglycone. Additional classifications have been developed based on (1) physiological or pharmacological activity (“therapeutic classification”) (e.g., laxative glycosides, cardiotonic glycosides); (2) plant families; and (3) correlation to the parent natural glycoside, as primary glycosides (e.g., amygdalin, purpurea glycoside A) and secondary glycosides (e.g., prunasin, digitoxin). The sugars in glycosides include (1) monosaccharides (e.g., glucose in salicin, rhamnose in ouabain), (2) disaccharides (e.g., gentiobiose in amygdalin), (3) trisaccharides (e.g., strophanthotriose), (4) tetrasaccharides (e.g., purpurea glycosides), (5) rare sugars (e.g., deoxy sugars), and (6) sugar linked in one position to the aglycone, rarely in two positions as sennosides.

O

OH

O

O

N

N

OH

OH

OH

OH

OH

N

N

NH2

OH

– –

–

– –

–

–

Glucuronides (glyc: ac glucuronic)

–

–

Arabinosides (glyc: arabinose)

Mannosides (glyc: mannose)

Glucosides (glyc: glucose)** Galacosides (glyc: galacose)

Nature of the simple sugar

C-glycoside (Cgroup)

*aglycone involved in the acetal union. **glyc, glycone.

– –

HO

–

HO

HO

HO HO

N-glycoside (NHgroup)

S-glycoside (SHgroup)

β-glycosides (β-sugar)

O

O-glycoside (OHgroup)

α- glycoside (α-sugar)

OH

Chemical group of the aglycone*

Type of glycosidic linkage

Table 13.1 Classification of glycosides.

– –

–

–

–

–

Triosides (three monosaccharide); e.g., strophanthotriose

Monoside (one monosaccharide); e.g., salicin Biosides (two monosaccharide); e.g., gentobioside

Number of monosaccharides in the sugar moiety

Flavonoidal G (aglycones: 2-phenyl chromone structure) Sulphur containing thioglycosides (aglycones contain sulphur) Alkaloidal glycosides (aglycone: alkaloidal in nature); e.g., glucoalkaloids of solanum species

Chromone glycosides (aglycones: derivatives of benzo-δ-pyrone)

Coumarin G (aglycones: derivative of benzo α-pyrone)

Steroidal G (aglycones: steroidal in nature, derived from cyclopentanoperhydrophenanthrene)

Anthracene or anthraquinone G (aglycones: anthracene der.)

Cyanogenic G (aglycones: nitriles or derivatives of hydrocyanic acid)

Alcoholic and phenolic glycosides (aglycones: alcohols or phenols) Aldehydic G (aglycones: aldehydes)

Chemical nature of the aglycone

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Glycosides: From biosynthesis to biological activity

Moreover, depending on the type of sugar moiety, glycosides can be categorized into glucosides, galactosides, fructosides, or glucuronides. The conversion of sugars to glucuronide by glucuronidation, known as phase II metabolism, is an essential method for the excretion of toxic chemicals from the human body.

13.4 Hydrolysis Glycosides can be hydrolyzed by acids, alkalis, or enzymes.

13.4.1 Acid Hydrolysis Acetal linkage between the aglycone and glycone is more unstable than that between two individual sugars within the molecule. The glycosides are hydrolysable by acids (e.g., HCl, H2SO4, among others), except C-glycosides. The glycosides containing 2-deoxy sugars are unstable toward acid hydrolysis even at room temperature.

13.4.2 Alkali hydrolysis Glycosides can be hydrolyzed by mild or strong alkali, namely NH4OH and NaOH, in different concentrations.

13.4.3 Enzymatic hydrolysis In recent years the use of enzymes to modify structure and improve physicochemical and biological properties of various natural products has been of great scientific and industrial interest, due to the large availability, high efficiency, low cost, wide substrate spectrum, ease of operation, and environmental friendliness (Benkovic and Hammes-Schiffer 2003). In this field, cellulases, pectinases, and β-glucosidase have been extensively used to enhance the extraction of intracellular contents of cells or glycosidases and lipases on the modification of compound chemical structures into their analogues with improved biologic activities. The production of these enzymes has been extensively studied. An example is β-glucosidase, an important component of the cellulase complex that not only hydrolyzes cellobiose and short-chain oligosaccharides to glucose, but also removes the inhibitory effect of cellobiose on β-1,4-endoglucanase and exoglucanase, increasing the overall rate of cellulose biodegradation. Recently, β-glucosidase purified to homogeneity from a culture supernatant of a fungus Penicillium simplicissimum and its kinetic properties were studied, using the glycoside salicin as substrate (Bai et al. 2013). Enzymatic hydrolysis is specific for each glycoside. The same enzyme can hydrolyze different glycosides; however, α and β stereoisomers of the same glycoside are usually not hydrolyzed by the same enzyme (e.g., α-glycosides hydrolyzed by maltase, invertase, and α-glycosidase). Glycosides can be hydrolyzed by β-glycosidase, α-L-rhamnosidase, naringinase, hesperidinase, and snailase, among other hydrolases (Table 13.2).

Glycosides: From biosynthesis to biological activity

307

Table 13.2 Examples of enzymes used for glycoside deglycosylation. Substrate

Product

Enzyme

Reference

Daidzin Genistin Hesperidin Naringin Naringin Naringin

Daidzein Genistein Hespertin Naringenin Naringenin Prunin

Kusaikin et al. 2011 Kusaikin et al. 2011 Furtado et al. 2012 Zhang et al. 2012 Ribeiro 2011 Vila-Real et al. 2011

Rutin Rutin

Quercetin Quercetin

β-D-glycosidase β-D-glycosidase Hesperidinase β-glycosidase Naringinase β-D-glucosidase (naringinase) Snailase β-glycosidase

Wang et al. 2012 Zhang et al. 2012

β-glycosidase (EC 3.2.1.21), an exocellulase—also called gentiobiase, cellobiase, emulsin, elaterase, aryl-β-glucosidase, β-D-glucosidase, β-glucoside glucohydrolase, arbutinase, amygdalinase, p-nitrophenyl β-glucosidase, primeverosidase, amygdalase, linamarase, salicilinase, and β-1,6-glucosidase—is an enzyme that acts upon β1- > 4 bonds linking two glucose or glucose-substituted molecules (i.e., the disaccharide cellobiose). β-glycosidase has specificity for a variety of β-D-glycoside substrates, catalyzing the hydrolysis of terminal nonreducing residues with release of glucose. Naringinase, an α-rhamnopyranosidase, expresses activity on α-L-rhamnosidase (E.C. 3.2.1.40) and β-D-glucosidase (E.C. 3.2.1.21). This hydrolytic enzymatic complex has wide occurrence in nature and has been reported in plants, yeasts, fungi, and bacteria (Chen et al. 2014; Mukund et al. 2014). It is commercially attractive due to its potential usefulness in the pharmaceutical and food industries (Ribeiro 2011; Puri 2012). It is particularly useful in the biotransformation of steroids, antibiotics, and mainly of glycoside hydrolysis. The deglycosylation of glycopeptide antibiotics, flavonoids, or glycolipids has been achieved successfully (Puri and Banerjee 2000; Vila-Real et al. 2007, 2010; Marques et al. 2007; Ribeiro et al. 2008, 2010; Amaro et al. 2009). Moreover, it has been used in citrus juice debittering and in the wine industry. Many natural glycosides—which include naringin, rutin, quercitrin, hesperidin, diosgene, and ter-phenyl glycosides—containing terminal α-L-rhamnose and β-D-glucose can act as substrates of naringinase (Vila-Real et al. 2010; Ribeiro 2011; Puri 2012). An example of naringin hydrolysis by naringinase is in Figure 13.1. The hydrolysis of naringin into prunin by α-L-rhamnosidase activity of naringinase liberates one molecule of rhamnose whereas the hydrolysis of prunin by β-D-glucosidase into naringenin liberates one molecule of glucose. The enzymatic hydrolysis of glycosides by β-D-glucosidase involves the formation of a covalent glycosyl-enzyme intermediate (Xu et al. 2013). Structurally, rhamnosidases are classified into family 78 of glycoside hydrolases and are characterized by the presence of Asp567 and Glu841 in their active site (Puri 2012). The overall structure consists of five domains, four of which

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Glycosides: From biosynthesis to biological activity

Naringin

Prunin

α-L-rhamnosidase

+ β-D-glucosidase

+

Rhamnose

Glucose

Naringenin

Figure 13.1 Hydrolysis of naringin to prunin and naringenin by α-L-rhamnosidase and β-D-glucosidase activities of naringinase.

are β-sandwich structures, designated as domains N, D1, D2, and C, and an (α/α) 6-barrel structure, designated as domain A (Puri, 2012). Acidic residues generally function as general acid and base catalysts in glycoside hydrolases. During the refinement of the three-dimensional structure, two strong electron densities regions were identified—one for the calcium located near Val423 in the loop domain D2 and the other for the calcium located near Lys953 in domain C, indicating that activity of the rhamnosidase may be dependent on divalent cations (Puri 2012). Hydrolysis of the glycosidic bond occurs exclusively by one of two mechanisms, either with retention or inversion of the anomeric configuration. In both mechanisms, a pair of carboxylic acids is utilized in the active site. Glu572 in combination with Asp567 or Glu841 are key residues in the catalytic mechanism, thus indicating putative general acid–base catalysis obeyed by rhamnosidase (Puri, 2012).

Scale bioprocess design The bio-based and green processing approach can be advantageously used for the design of new production processes of glycosides or for the improvement of those currently under use. The biological systems are able to operate under mild environmental conditions to perform complex chemical conversions with high specificity and efficiency. A bioprocess optimization involves three interconnected design steps: (1) biocatalyst design, (2) medium design, and (3) process design. Initial optimization studies are usually carried out in small scale under conditions as close as possible to the large-scale approach (Figure 13.2).

Glycosides: From biosynthesis to biological activity

Microreactor/ shaken flask

Mechanical stirred reactors type

Batch Discontinuous operation mode

309

Plug flow reactor type

Packed bed reactor (PBR) or CSTR Continuous stirred tank reactor Fluidized bed reactor (PBR)

Continuous operation mode

Figure 13.2 Different types of bioreactors.

Newer technological developments in the field of biocatalyst design, including immobilization, are likely to provide a more rational and cost-effective way for the use of biocatalysts in the pharmaceutical and food industries, in medicine, and in the development of bioprocess devices. The continuous or repeated use of biocatalysts over an extended time gap is typically sought for both economic and technical issues. Immobilization of biocatalysts has many advantages in large-scale processing, namely biocatalyst reuse, ease of separation of biocatalysts from reaction media, continuous mode of operation, prevention of contamination of the process product, higher enzyme concentrations, and higher superficial area to reaction, among others. A set of guidelines can be considered in the quest for the design of an effective system based on immobilized biocatalysts. The selection of the suitable combination of technique and support for immobilization is largely conditioned by the nature of the enzyme and of the substrate(s)/product(s) and by the application. Therefore, the carrier must be such as to provide facile, secure immobilization with good interaction with substrates. Simultaneously, the carrier must also display a shape, mechanical stability, size and nature of the pore, and morphology compatible with the intended application (Cao and Schmid 2005). The developments of so-called hydrogels and thermoreactive water-soluble polymers have attracted attention in this field of biotechnology. Such gels with a water content of about 96% provide a microenvironment for the immobilized enzyme close to that of the soluble enzyme with minimal diffusion restrictions. These polymers are generally glassy in the dehydrated state but swell to become an elastic gel upon water penetration. Swelling characteristics of the hydrogels depend on the presence of salts and the degree to which the acetate groups are replaced by hydroxyl groups (Varshosaz and Koopaie 2002). There are a variety of methods used to immobilize biocatalysts, the most common being adsorption, covalent binding, entrapment, and cross-linking. The oldest and simplest method for enzyme and cell immobilization is adsorption.

310

Glycosides: From biosynthesis to biological activity

Immobilization by adsorption includes reversible surface interactions between enzymes or cells and the support material. Some advantages of adsorption techniques include (1) little or no damage to enzymes/cells, (2) simple and fast immobilization, (3) no chemical changes of support or enzyme/cells, and (4) reversible process to allow regeneration with fresh enzymes/cells. Major disadvantages of adsorption to a support include nonspecific binding and overloading on the support and, above all, leakage of enzymes/cells from the support. A suitable support matrix will allow surface interaction between the support matrix and the enzyme (hydrogen bonding, electrostatic forces, and hydrophobic effects), without conformational changes that negatively affect the active site or overall activity. The covalent binding of enzymes to solid support is one of the most effective ways to prevent enzyme leaching. This is achieved by the reaction of enzyme amino acid residues (–NH2, –CO2, –SH) with organic functional groups bound to a support matrix. Moreover, immobilization of an enzyme on a carrier decreases the degree of movement of the enzyme, which often leads to loss of the native activity. The physical entrapment or encapsulation of an enzyme in a porous matrix with restricted apertures may prevent enzyme leakage. It is difficult to achieve a ship-in-a-bottle type structure with a crystalline matrix. However, amorphous materials such as sol-gels can encapsulate enzymes and minimize leaching (Tran and Balkus 2012). In order to overcome the mass transfer limitation currently associated with enzyme immobilization, many efforts have been made to immobilize enzymes onto surfaces of nano-scaled materials such as nanoparticles, nanotubes, mesoporous materials, and nanofibrous membranes (Wang et al. 2009). These nanoparticles and nanofibers can maximize surface area and improve the activity of the biocatalyst relative to the classical carrier structure of a relatively bulky nature. Enzymes can even be fabricated into nanoparticles themselves by forming cross-linked enzyme aggregates (CLEAs). This later configuration has particular advantages, such as highly concentrated enzyme activity in the catalyst and high stability and low production costs, since no carrier is required (Ribeiro and Rabaça 2011). Nevertheless, the application of enzymes immobilized on nanoparticles or as aggregates presents some specific challenges, namely related to recovery and environmental hazards, which can be overcome using magnetic particles. In contrast, nanofibrous supports are generally in the form of easily handled free-standing mats (Wang et al. 2009). For the optimum design of a product-scale reactor system, the data on a small scale (model) must be translated to the large scale or vice versa. The characterization of the engineering environment in miniature bioreactors has proven less complex than similar endeavors for shaken vessels, at least down to the milliliter scale (Nunes et al. 2013). Microtiterplates with a number of wells comprised within 4 and 96 and different shapes commonly used in the development of bioprocesses are available

Glycosides: From biosynthesis to biological activity

311

(Figure 13.2). The shape of the well, along with the static surface area-to-volume ratio and shaking intensity, has been shown to affect biocatalyst activity in bioconversion systems requiring oxygen. In Figure 13.2 are the major types of reactors based on operation and agitation mode (Nunes et al. 2013). Batch processes operate in closed systems. The substrate is added at the beginning of the process and products removed only at the end. Batch reactors are the most commonly used type of reactor, namely when soluble enzymes are utilized as catalysts. In most cases no recovery of the enzyme is attempted. On the other hand, when immobilized enzymes are used in a batch operation, the recovery of the biocatalyst is carried out at the end. During this recovery procedure some loss of immobilized enzyme can occur as well as inactivation of the enzyme due to repeated recovery cycles. High operational stability is required. Furthermore, the use of a simple batch reactor does not take advantage of the potential of continuous operation, a major feature of immobilized enzyme systems. Continuous reactors can be grouped into two basic types: continuous-flow stirred tank reactor (CSTR) and tubular reactors (plug-flow reactor [PFR]) and fluidized-bed reactor [FBR]) (Figure 13.2). These reactors are different especially in mixing performance. Consequently, conditions within the CSTR are the same as the outlet stream, while in the PFR the conditions vary with length from inlet to outlet. The CSTR operates under uniform conditions of low substrate and high product concentrations. Several factors can give rise to deviations from a plugflow pattern in a packed-bed reactor. Temperature and velocity gradients normal to the flow direction and substrate diffusion in the axial direction are the most frequently occurring problems, and even small deviations from the idealized flow pattern can alter the kinetics of the reaction. The production of glycosides has been developed in different types of bioreactors (Nunes et al. 2012, 2013).

13.5 Chemical properties and identification Common general physical and chemical properties for glycosides are quite difficult due to their structural diversity. Most glycosides are water soluble and soluble in alcohols and some are either less soluble or insoluble in nonpolar organic solvents, with the exception of chloroform and ethylacetate. In fact, more sugar units in a glycoside lead to more solubility in polar solvents. Glycosides are water mixed with different proportions of methanol or ethanol (most suitable extracting solvent), while nonpolar organic solvents are generally used for the de-fating process. The extraction of glycosides is mainly carried out with the solvent systems previously mentioned and the isolation of glycosides from plants usually involve drying at 100 °C; boiling water or alcohol; boiling with acetone; cold acid pH treatment; extraction at very low temperatures; de-fating or purification of the plant

312

Glycosides: From biosynthesis to biological activity

material (e.g., in the case of seeds); extraction of the glycosides constituents by alcohol, water, or dilute alcohols; or for dry material sometimes ether saturated with water. The process is followed by concentration of the alcoholic extract, addition of water (sometimes hot water), extraction of nonglycosidic impurities by organic solvents and precipitation of water-soluble impurities by lead acetate, and finally purification of the aqueous extract with glycoside(s), namely by crystallization. The qualitative tests used on glycoside identification depend on the chemical nature of the aglycone and on the sugar part (Table 13.3). Glycosides do not reduce Fehling’s solution. The reducing sugars of glycosides (C-glycosides are exceptions) can be estimated before and after hydrolysis (acidic or enzymatic) (Table 13.3).

Table 13.3 Tests used on the identification of the aglycone and sugars of glycosides. Glycosides

Tests Liebermann’s Borntrager’s Nh4Oh, AlCl3, Na bikrate Silver FeCl3 paper sulphate + AgNO3 test test

Steroidal + structure Anthraquinone –

–

–

–

–

–

–

–

–

+ (reddish coloration with alkaly) –

Cyanogenetic

–

–

Sulphur

–

–

Aglycone Flavonoid (Genin)

+ – (characteristic color) – yellow - > red color – –

–

– Black precipitate

Tests Special tests based on the chemical structure of the glycoside Sugar Keller Killiani’s part Examples: 1. Scillarin A [acid hydrolysis] → → → Scillaridine A + Scillabiose 2. Scillabiose [Scillabiase] → → → Rhamnose + glucose 3. Amygdalin [amygdalase] → Prunasin + glucose

Determination of the glycosidic linkages

Color reactions based on the sugar moiety

Examples: Examples: 1. Use of α and β 1. DNS method glycosidases 2. Acid hydrolysis of glycosides, immediate optical activity measurement of the resulting solution

Glycosides: From biosynthesis to biological activity

313

Glycosides hydrolysis can be followed by the 2,4-dinitrosalicylic acid (DNS) method acid (Miller 1959). This method allows for the quantification of reducing sugars formation (e.g., rhamnose and glucose). The DNS macroassay was modified into a microassay using a newly designed 96 microwell plate by Nunes et al. (2010), allowing higher repeatability, speed, large sample analysis number, and sample volume reduction. Glycosides, and the respective aglycone and glycone, can be quantified by HPLC-UV (high-performance liquid chromatography–ultraviolet), HPLC-RI (refractive index), or HPLC-PDA (photo-diode array) on C18 reverse-phase column and detection of absorbance at 280 nm (Ribeiro and Ribeiro 2008; Ni et al. 2013). Identification of glycosides, namely flavonoid glycosides from rutinosides or polyphenol glycosides, can be carried out by HPLC-LC-MS analysis (Sun et al. 2009; Vila-Real et al. 2011). Recently, a UPLC-PDA (ultra-performance liquid chromatography–photo-diode array) detector was developed for the simultaneous determination of six anthraquinone glycosides (Wang et al. 2013).

13.6 Biologic activity and therapeutic applications Glycosides have important biologic activities (Table 13.4). The main therapeutic applications are as cardiac drugs (cardiotonic glycosides; e.g., digitalis glycosides, strophanthus, squill), anti-inflammatories (e.g., naringin, naringenin), antipyretics (e.g., salicin), antirheumatics, analgesics (e.g., methylsalicylate [hydrolytic product of gaultherin]), and antivirals (e.g., glycyrrhizin, prunin), demulcent, expectorant, and antispasmodic action, laxatives (e.g., anthraquinone glycosides of senna, aloes, rhubarb, cascara, frangula), and counter-irritants (e.g., thioglycosides and their hydrolytic products). Some glycosides are claimed to reduce capillary fragility (e.g., flavonoidal glycosides, rutin, hesperidin) and more recently as anticancer agents (e.g., amygdalin) (Acton 2013). The development of promising novel cytotoxic solasodine rhamnosyl glycoside drugs offers not only gains in specificity and efficacy but also in safety, tolerability, nonresistance, and convenience in the treatment of patients with cancer (Cham 2013). Some of the biologic activities of glycosides are related to their antioxidant properties, which can directly quench free radicals, inhibit enzymes of oxygenreduction pathways, and sequester transient metal cations. Studies concerning the structure-activity of glycosides are important in the research process for new anti-inflammatory agents. The glycosidic residue of certain compounds influences not only pharmacokinetics but also pharmacodynamics (Kren and Martinova 2001). The glycosidic residue is responsible for the reduction in hydrophobicity of the compound, reducing the solubility within the cell membrane. In fact, several studies demonstrated the interference of glycosylation in anti-inflammatory activity (Ribeiro et al. 2008; Amaro et al. 2009; Hostetler et al. 2012). These authors showed that pure flavone aglycones and aglycone-rich

Hesperetin

Geraniin

Genistein

Obesity Hepatocellular carcinoma

Allergy

Obesity

Mouse adipocytes HepG2

Hs68 SH-SY5Y PC12 Rat basophil leukemia RBL-2H3

Huh7 liver hepatoma Ishikawa endometrial adenocarcinoma 3 T3-L1 mouse embryo fibroblasts PC12

HepG2

Genipin

Hepatocellular carcinoma PON1 Endometrial cancer

PC12

Vero

HepG2

Human colon adenocarcinoma Caco-2 PC12

Cell type

Formononetin

Fisetin

Diosmetin

Hepatocellular carcinoma Dengue virus type-2 infection

Cancer

8-hydroxydaidzein

Calycosin

Disease

Glycosides

Table 13.4 Glycosides preclinical studies in vitro.

Inhibit degranulation by suppression of pathway signals and inhibited phosphorylation of Akt, which leads to the suppression of cytokines Inhibit TNF-α-stimulated FFA secretion Anti-invasive agent that suppresses MMP-9 enzymatic activity via NF-κB and AP-1 signaling pathway

Protection against D-galactose-induced oxidative stress mediated by decreased intracellular ROS and binding activity of NF-κB (antioxidant activity) Cytoprotective and antioxidant effects Cytoprotective and antioxidant effects

Potential inducer of the anti-atherogenic enzyme paraoxonase-1 Inhibit the proliferative effects of estrongen (adjuvant for endometrial cancer treatment and prevention) Inhibition of leptin synthesis

Neuroprotection mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals Induce apoptosis through ROS/JNK signaling

Enhances citotoxicity of epirubicin, an antineoplastic agent (adjuvant for cancer treatment and prevention) Neuroprotection mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals Cytostatic effects via CYP-catalyzed metabolism, activation of JNK and ERK, and P53/P21 up-regulation Antidengue virus replication activity

Effect

Yoshida et al. 2010 Lee et al. 2010

Ling et al. 2012 Ling et al. 2012 Ling et al. 2012 Murata et al. 2012

Hsieh et al. 2011

Phrakonkham et al. 2008

Schrader et al. 2012 Sampey et al. 2011

Khanal et al. 2012

Yu et al. 2009

Androutsopoulos et al. 2013 Zandi et al. 2011

Yu et al. 2009

Lo 2013

Reference

Cancer

Naringenin chalcone

Naringenin derivatives

Obesity Neuroinflammatory injury Prevention of leukemia

Allergy

Hepatocellular carcinoma

Mucous hypersecretion Cancer activity Obesity

Naringenin

N101-2*

Luteolin

Hesperidin derivatives

–

Hesperidin

Hepatocellular carcinoma Diabetes Osteoporosis

Disease

Glycosides

HCT116 human colon cancer

Human cervical (HeLa) cancer 3 T3-L1 adipocytes

C6 glioma HBE16 human airway epithelial

Human leukemia THP-1

Mouse adipocytes Glial

SiHa CaSki Rat basophil leukemia RBL-2H3

PC12

HepG2 Raw264.7 Bone marrow mononuclear HepG2

Human peripheral blood lymphocytes HepG2

Cell type

Anticancer activity Improves adipocyte metabolic functions and exerts insulinsensitizing effects Anticancer and antioxidative effect

Induces apoptosis through downregulation of Akt and caspase-2 activation Antiproliferative effects Attenuates mucous hypersecretion

Inhibit degranulation by suppression of pathway signals and inhibited phosphorylation of Akt, which leads to the suppression of cytokines Inhibit TNF-α-stimulated FFA secretion Inhibits inflammatory signaling in glial cells

Cytostatic effects via CYP-catalyzed metabolism, activation of JNK and ERK, and P53/P21 upregulation Neuroprotective effects through scavenging free radical–induced oxidative stress Apoptotic effects

Inhibited acetaldehyde-induced matrix metalloproteinase-9 gene expression α-glucosidase inhibition, antihyperglycemic activity Decreased the differentiation into osteoclasts

Effective radioprotector against ϒ-irradiation induced in vitro

Effect

(Continued)

Yoon et al. 2013

Krihnakumar et al. 2011 Horiba et al. 2010

Sabarinathan et al. 2011 Yang et al. 2011

Park et al. 2008

Yoshida et al. 2010 Vafeiadou et al. 2009

Murata et al. 2012

Kim et al. 2012

Androutsopoulos et al. 2013 Pavlica et al. 2010

Zhang et al. 2012 Kim et al. 2011

Yeh et al. 2009

Kalpana et al. 2009

Reference

Gastric cancer (Daunorubicin resistant) Alzheimer´s disease

Antiamyloidogenic and antioxidant activities

APPswe

APPswe

Human Gastric carcinoma (EPG85-257P) EPG85-257RDB

PC12

Antiamyloidogenic and antioxidant activities

Cardioprotective effects through the induction of activation of AMPK and eNOS Neuroprotective effects through scavenging free radical–induced oxidative stress Antiproliferative impact, mainly through induction of apoptosis

Modulates Nrf2 and glutathione-related defenses

HepG2

Human aortic endothelial

Hypoglycemic agents through regulation of glucose metabolism Attenuates EGF-induced MUC5AC secretion Hypoglycemic agents through regulation of glucose metabolism Enhancement of immunomodulatory effects of interferon-β Potential protective effect on t-BHP-caused injury in hepatocytes through the induction of metallothioneins

Protective effects against doxorubicin-induced apoptosis

Effect

HepG2 A549 HepG2 PBMC HepG2

H9C2

Cell type

*N101-2, diethyl 5,7,4´-trihydroxy flavanone N-phenyl hydrazon.

Rutin

Quercetin

Neohesperidin

Gastric cancer

Cardiomyopathyassociated with doxorubicin Diabetes Diabetes Diabetes Multiple sclerosis Environmental oxidant-induced liver damage Hepatocellular carcinoma Alzheimer´s disease

Naringenin-7-Oglucoside

Naringin

Disease

Glycosides

Table 13.4 (Continued)

Jiménez-Aliaga et al. 2011

2012

Borska et al.

Pavlica et al. 2010

Granado-Serrano et al. 2012 Jiménez-Aliaga et al. 2011 Shen et al. 2012

Zhang et al. 2012 Nie et al. 2012 Zhang et al. 2012 Sternberg et al. 2008 Weng et al. 2011

Han et al. 2008

Reference

Glycosides: From biosynthesis to biological activity

317

extracts effectively reduced TNF-α production and inhibited the transcriptional activity of NF-κB, while glycoside-rich extracts showed no significant effects. In addition, it was demonstrated that the deglycosylation of flavones increased cellular uptake and cytoplasmic localization (Hostetler et al. 2012). The higher efficacy of aglycones rather than glycosides to reduce TNF-α and NF-kB activity is consistent with other studies showing that neither diosmetin 7-rutinoside nor apiin-reduced nitric oxide (NO) and TNF-α in response to LPS, whereas apigenin effectively decreased these inflammatory mediators (Shanmugan et al. 2008). Oxidative stress through oxidative damage and redox imbalance is known to play an important role in the pathogenesis of neurodegenerative diseases (Chung et al. 2009), namely Alzheimer’s disease. The molecular inflammation mechanism may be a cause or part of disease progression, contributing to the aging process as well as to the chronicity of age-related diseases (Chung et al. 2006). The association between free radical reactions and age-related diseases is the basis of antioxidant therapy against such diseases (Harman 2003; Khansari et al. 2009). Afterward, besides endogenous antioxidants that belong to the antioxidant defense system in the body, there are also exogenous antioxidants provided by nutrition. Some of these antioxidants belong to the glycosides family, some of them common in higher plants. Several in vitro studies (preclinical trials) have demonstrated multiple beneficial effects of glycosides in prevention or in therapeutics of different pathologies (Table  13.4). These effects have attracted increasing interest because they can prevent certain chronic diseases such as cancer, cardiovascular, and neurodegenerative pathologies.

13.7 Case studies: From production to therapeutic application 13.7.1 Alcoholic and phenolic glycosides Alcoholic and phenolic glycosides are part of the free primary alcoholic group; aglycones are phenolic in nature. Salicin and arbutin are examples of this type of glycoside.

Biosynthesis of salicin Salicin is obtained from different species of Salix (Salix fragilis) and can be hydrolyzed by acid, giving glucose and the phenolic ether, saliretin, a condensation product of two molecules of saligenin (Figure 13.3). Salicin can also be hydrolyzed by enzymatic hydrolysis with β-glycosidase into saligenin (salicyl alcohol) and glucose (Figure 13.3). The oxidation of saligenin gives salicylic acid. Salicin has been used in the treatment of fever and rheumatism. Additionally—as it is better tolerated in

318

Glycosides: From biosynthesis to biological activity

OH HO

OH OH

O HO

O

Salicin

Acid

Enzyme

HO

HO

HO

O

+

HO

O

OH

O

HO HO

HO Saligenin

HO

OH

OH

HO

OH

OH

Glucose

Saliretin

Glucose

Figure 13.3 Enzymatic and acid hydrolysis of salicin.

(a)

OH

OCH3

(b)

OH OH O

HO

O O

HO OH

O

HO HO

OH H

Figure 13.4 Arbutin (a) and methylarbutin (b).

the stomach than sodium salicylate, aspirin, and other antipyretics and antiinflammatory agents—salicin is now used as an analgesic-antipyretic in case of periodic fever.

Biosynthesis of Arbutin Arbutin is a phenolic glycoside (hydroquinone β-D-glucopyranoside) found in bearberry leaves Arectostaphyllos uva ursi. Acidic or enzymatic (β-glycosidase) hydrolysis of arbutin yields glucose and hydroquinone (Figure 13.4). This hydrolysis product, hydroquinone, is the responsible for the main therapeutic applications of arbutin as a diuretic and as a bactericide. Additionally, methylarbutin (methyl ether of arbutin) (Figure 13.4), found in Uva ursi leaf, contributes to the diuretic and urinary antiseptic action.

Glycosides: From biosynthesis to biological activity

319

13.7.2 Aldehyde Glycosides One of the most important aldehyde glycosides is vanillin, mainly obtained from the glycosylated form glucovanillin. It is a glycoside constituent of green vanilla pods.

Biosynthesis of Vanillin Vanillin (aglycone) is mainly obtained by the enzymatic action of β-glycosidase on the glycoside (Figure 13.5) from the pods of vanilla plants. Moreover, it can be prepared from the glycoside coniferin, lignin, or from the phenolic volatile oil constituents eugenol (Figure 13.5). The main application of vanillin is as a flavoring agent.

13.7.3 Cyanogenic Glycosides Cyanogenic glycosides yield hydrocyanic acid as one of their hydrolytic products. These glycosides are obtained from plants that are toxic. The majority of cyanogenic glycosides are derived from benzaldehyde cyanohydrin (carbonyl compound - aglycone part) (Figure 13.6).

Biosynthesis of Amygdalin Amygdalin is the most widely distributed cyanophore glycoside, occurring in several Prunus species, mainly in bitter almonds (Prunus amygdalus var. amara, family Rosaceae). Amygdalin is a gentiobioside of D-mandelonitrile. Gentiobioside is a reducing disaccharide consisting of two molecules of β-glucose linked by β-1,6 linkage. Different enzymes, such as amygdalase, prunase, or β-glycosidase, can hydrolyze amygdalin. Amygdalin is composed of two molecules of glucose, one of benzaldehyde, which induces an analgesic action, and one of hydrocyanic acid, an antineoplastic compound. Amygdalin can be used in medicine, mainly for prevention and treatment of migraine, hypertension, and chronic inflammation. Since the 1950s, a purportedly nontoxic intravenous form of amygdalin, an anticancer drug, was patented as Laetrile.

13.7.4 Anthraquinone Glycosides There are different types of anthraquinone glycosides depending on the aglycon, namely O-glycoside (aglycone moiety: 1,8 dihydroxyanthraquinone), O-glycoside (aglycone moiety: partially reduced 1,8 dihydroxyanthraquinone, e.g., oxanthronetype), C-glycoside (aglycone structure: anthrone), and O-glycoside (aglycone moiety: di-anthrone; e.g., sennosides, with a C-C bridge between the anthranol units) (Figure 13.7). The anthraquinone glycosides can be extracted and hydrolyzed by boiling with acids. The aglycones can be extracted from the acidic solution with ether or benzene. The aqueous layer acquires a deep red color, after shaking the ether or benzene layer with aqueous alkali or ammonia solution, leading to the

O

HO

O

Hydrolysis

O

O

O

O

O

O OH

OH

HO

HO

Oxidation

O OH

OH

O

Glucovanillin

OH

HO HO

Vanillin

O

KOH Oxidation

HO O

HO

O

HO

O

OH

O

O

OH

OH

Coniferin

Coniferyl alcohol

Figure 13.5 Hydrolysis of vanillin from glucovanillin, coniferin, and eugenol.

Vanillin

Eugenol

Isoeugenol

Vanillin

OH

O

HO Sugars

HCN

N

OH O

HO

Mandilonitrile glycosides

O Benzaldehyde

N

OH

O

Mandilonitrile O

HO

N O

N

OH

O

O HCN

OH

O

Sugars

HO

O OH

HO OH

HO HO

Acetone

Acetone cyanohydrin

Figure 13.6 Mandilonitrile glycoside synthesis.

OH

OH

Linamarin

HO Amygdalin

Prunasin

322

Glycosides: From biosynthesis to biological activity

O

O

OH

4H

O Anthraquinone

Anthrone

Anthranol

2H

O O

Oxanthrone

Figure 13.7 Anthraquinone glycosides.

formation of anthraquinone salts. Borntrager’s reaction can distinguish anthraquinones from anthrones and anthranols, which do not give the test unless they are converted to anthraquinone by oxidation with mild oxidants such as hydrogen peroxide or ferric chloride. The biologic activity of anthraquinone glycosides is dependent on glycosylation, hydroxylation, oxidation level, and the nature of substances at C-3. An example is the purgative action of anthracene-bearing drugs, owed to their anthracene glycoside content rather than the content of free anthracene aglycones. In this case glycosylation is the limiting step for activity because the sugar moiety transports the aglycone to the site of action in the large intestine. Hydroxylation of C-1, C-8 is essential for activity. The increase in hydroxylation leads to higher solubility. Pharmacological activity is also dependent on the degree of oxidation at positions C-9 and C-10. Higher oxidation level at these positions caused lower activity. In fact, anthrones and anthranols are more active than their corresponding oxanthrones, which consecutively are more active than their corresponding anthraquinones. Complete reduction of C-10 and C-9 leads to complete loss of activity. Additionally, the nature of compounds at the C-3 position influences the activity. CH2OH derivative (e.g., in aloe emodin) is more active than those with CH3 substitution. Anthraquinone glycosides containing a dimer are more active than with a monomer.

13.7.5 Cardiac glycosides The aglycones of all cardiac glycosides are steroidal in nature (Figure 13.8). The glycone portion at position C-3 of cardiac glycosides may contain four monosaccharide molecules linked in series; a single aglycone may have linked a monoside, a bioside, a trioside, or a tetroside. The sugars in cardiac glycosides are deoxy

Glycosides: From biosynthesis to biological activity

CH3

R

323

Lactone ring

H

H

OH

O

Sugar

H

Figure 13.8 Cardiac glycosides.

sugars, such as digitoxose, cymarose, thevetose, D-glucose, and L-rhamnose. The glycone part has a great influence on the solubility and on the rate of absorption and distribution of the glycosides to the site of action. As a matter of fact, a small change in the molecules such as the location of the group OH modifies the cardiac activity or even eliminates it completely. The saturation and/or cleavage of the lactone ring wipe out cardiac activity. Moreover, closely related cardiac glycosides differ greatly in the rate of absorption, duration of action, and cumulative effect. Cardiac active glycosides are characterized by the presence of the following structural features: (1) β-OH group at position C-3, always involved in a glycosidic linkage to a mono, di, tri, or tetra saccharide; (2) β-OH group at position C-14; (3) unsaturated 5- or 6-membered lactone ring at position C-17, in the β configuration; (4) the A/B ring junction is usually (cis), while the B/C ring junction is always (trans) and the C/D ring junction is in all cases (cis); and (5) additional OH groups may be present at C-5, C-11, and C-16. The cardiac glycosides include cardenolides and bufadienolides. The cardiac glycosides act as cardiotonic agents. Their main characteristics include highly specific action on cardiac muscle, with tone increase, excitability, and contractility and, as a consequence, the weakened heart function will be more efficient. Therefore, purified extracts or synthetic analogues have been used for the treatment of congestive heart failure and cardiac arrhythmia. The main therapeutic applications of cardiac glycosides, besides cardiotonics, are rheumatic heart disease, atherosclerosis, and diuretics through the distension of the capillary of the kidneys.

Biosynthesis of cardenolides Cardenolides are cardiac glycosides, α-β unsaturated 5-membered lactose ring in position C-17, represented by the digitalis and strophanthus group. The digitalis glycosides contain angular methyl group at C-10, whereas strophanthus glycoside is characterized by the presence of an aldehyde (CHO) or a primary alcohol (CH2OH) group at C-10. The digitalis glycosides have the origin on D. purpurea, D. lanata, D. lutea, and D. thapsi. They include the aglycones digitoxigenin (lanatoside A), gitoxigenin

324

Glycosides: From biosynthesis to biological activity

(a)

O

O (b)

O

O R1

R

HO H

H Sugar

O

H

H

OH

R2 OH

HO H

H

Figure 13.9 (a) Cardenolides (Strophanthus glycosides: R=CHO or CH2OH), (b) Digitalis glycosides (Digitoxigenin: R1 and R2=H; Gitoxigenin: R1=H and R2=OH; Digoxigenin R1=OH and R2=H).

(lanatosin B), and digoxigenin (lanatoside B). The glycoside lanatoside A besides the aglycone digitoxigenin, has the glycone acetyl-digitoxin, digitoxin, and glucose; the lanatoside B has gitoxigenin and the glycones acetyl-gitoxin, gitoxin, and glucose; the lanatoside C [(3β,5β,12β)- 3-{[β-D- glucopyranosyl(1 → 4)- 3-O-acetyl- 2,6-dideoxy- β-D-ribo- hexopyranosyl- (1 → 4)- 2,6-dideoxy- β-D- ribo- hexopyranosyl- (1 → 4)- 2,6-dideoxy- β-D- ribo- hexopyranosyl] oxy}- 12,14-dihydroxycard- 20(22)- enolide] has digoxigenin and the glycones, 3-acetyldigitoxose, two digitoxoses, and glucose (Figure 13.9). Cardenolides can be hydrolyzed by acid or enzymes into aglycones and sugar residues. Enzymatic hydrolysis of lanatosides A, B, and C can be carried out by βglucosidases with the release of glucose and the secondary glycosides acetyldigitoxin, acetylgitoxin, and acetyldigoxin (Pekic and Lepojevic 1991), respectively. Digitoxin, gitoxin, and digoxin are obtained by the action of alkali on their acetyl-derivatives. The strophanthus glycoside can be isolated from the strophanthus species. These glycosides include K-strophanthoside (trioside: Strophanthidin 3-diglucosylcymarose), K-strophanthin B (bioside), and cymarin (a monoside). From the seeds of Strophanthus gratus another glycoside named ouabain or (G-strophanthin) can be isolated, which yield by hydrolysis rhamnose and the aglycone ouabagenin (Figure 13.10). A scalable route to the polyhydroxylated steroid ouabagenin with an unusual take on the age-old practice of steroid semisynthesis was developed by Hans et al. (2013). During the design of the synthesis, the incorporation of both redox and stereochemical resulted in efficient access to more than 500 milligrams of the key precursor toward ouabagenin and to the discovery of innovative methods for carbon-hydrogen (C-H) and carbon-carbon activation and carbon-oxygen bond homolysis (Hans et al. 2013). Due to the medicinal

Glycosides: From biosynthesis to biological activity

325

O O

HO HO

OH HO

HO

OH OH O

O

OH

Figure 13.10 Ouabain (G-strophanthin).

O O

Sugar

O

OH

Figure 13.11 Bufadienolides (Squill glycosides: R1=OH, R2=H; Bufotoxin: R1 and R2=ester group).

relevance of the cardenolides in the treatment of congestive heart failure, a variety of ouabagenin analogs could potentially be generated from the key intermediate as a means of addressing the narrow therapeutic index of these molecules (Hans et al. 2013).

Biosynthesis of bufadienolides Bufadienolides are cardiac agents with a doubly unsaturated 6-membered lactone ring in position C-17 (Figure 13.11). This group includes the squill glycosides. Its aglycones differ from those of the cardenolides in the number of the doubly unsaturated lactone ring in position C-17 and at least in one double bond in the steroid nucleus. They include glucoscillarin, scillarin A, and proscillaridin A. Bufadienolides were originally isolated from traditional Chinese plants, widely used in traditional remedies for the treatment of several ailments, such as

326

Glycosides: From biosynthesis to biological activity

infections, rheumatism, inflammation, cancer, and disorders associated with the central nervous system (Kamboj et al. 2013). Also, they increase the contractile force of the heart by inhibiting the enzyme Na+/K + –ATPase. However, one problem of these compounds was its toxicity to livestock. Structural changes in functionality could significantly alter its cytotoxic effects (Kamboj et al. 2013). Recently, a series of bufadienolides isolated from toad venom showed antitumor activities, with reduced cytotoxicity (Liu et al. 2013). In vitro studies showed that bufalin, a cardiotonic steroid, and cinobufagin had the potential to induce differentiation and apoptosis of tumor cells, mainly leukemia, prostate cancer, gastric cancer, and liver cancer (Yin et al. 2012). The inhibition of α1β1 suggested being the first step in the cancer growth inhibition effects of cardiac glycosides. This feature was also true for some cancer types that overexpress the NaK α1β1 but not the α3β1 complex, such as gliomas, melanomas, and renal cell carcinomas. The expected pattern of behavior of the glycoside and aglycone was correlated, as in the case of hellebrigenin and hellebrin (Banuls et al. 2013). Hellebrigenin, with its free C3 position, may be derivatized into novel analogs to increase the selectivity for the α1 subunit, making optimized hellebrigenin analogs novel weapons to combat gliomas, melanomas, and renal cell carcinomas that overexpress the α1 subunit. Banuls et al. (2013) suggested that there is a direct impact of the dramatic reduction in the oxygen consumption rate in cardenolide- and bufadienolide-treated cells on mitochondrial oxidative phosphorylation.

13.7.6 Tannins Tannins are phenolic constituents widely distributed in plants. They can be defined as high-molecular-weight phenolic plant constituents that can be detected by Glodbeater’s skin tanning test and quantified. Gallic acid, catechin, flavan-3,4-diol, and chlorogenic acid are simple molecular weight compounds that usually coexist with true tannins (Figure 13.12).

Biosynthesis of tannins Tannins are hydrolyzed by acids or enzymes and give phenolic acids, called phenolic acid glycosides (gallic or ellagic) and glucose. Tannins of gallic acid are gallitannins and those of ellagic acid are ellagitannins. Dry distillation of hydrolyzable tannins gives pyrogallol, the class named pyrogallol tannins. The condensed tannins are derived from catechin and flavan, 3,4-diol, giving catechol. Gallic acid (3,4,5 trihydroxy benzoic acid) is an important intermediate compound in the synthesis of the antibacterial drug trimethroprim. It is also a substrate for the chemical or enzymatic synthesis of propyl gallate, an important antioxidant. Gallic acid can be produced by hydrolysis of tannic acid with acid, alkali, or by microbial enzyme tannase. A new producer, Aspergillus tamarii, was able to produce extracellular tannases in submerged cultures.

Glycosides: From biosynthesis to biological activity

HO

OH O

O

OH

O

HO O

OH

HO HO

OH OH

OH

HO

327

Glucogallin

Gallic acid

OH OH OH HO

O

O O OH

HO

OH O Flavan-3,4-diol

OH Chlorogenic acid

Figure 13.12 Chemical structure of tannins.

13.7.7 Sulphur glycosides Several plants of the family Cruciferae yield glycosides containing sulphur (thioglycosides), which hydrolysis yields volatile agycons of thiocyanate structure (e.g., mustard oils). Sinigrin and sinalbin are two glycosides occurring, respectively, in black and white mustard seeds (Figure 13.13). Black and white mustard seeds, due to their content in thioglycosides, are used as rubefacients and counter-irritants. The hydrolysis of sinigrin gives a glucose, allyl isothiocyanate (volatile oil of mustard) and potassium acid sulphate, whereas the hydrolysis of sinalbin gives a phenolic isothiocyanate (acrinyl isothiocyanate), glucose, and sinapine.

13.7.8 Flavonoid glycosides Classification Flavonoids comprise one of the most common groups of plant glycosides. The biosynthesis of flavonoids starts with the removal of the amino group of phenylalanine, leading to phenylpyruvate. The thiamine pyrophosphate, from the pyruvate dehydrogenase complex, makes the oxidative decarboxylation of two molecules of phenylpyruvic acid, with the production of two active aldehyde molecules, which together with a C1-fragment (-CHO) in an oxidative step leads to the nucleus of phenyl-γ-pyrone (chromen-4-one) (Havsteen 2002). Multiple hydroxylation in different positions leads to the production of individual

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Glycosides: From biosynthesis to biological activity

OH

OH

HO O O S

O

O S

N

HO

OH

O

HO

S

N

S O

HO

OH

O–

O

O

OH O

OH Sinigrin

N+

O O

HO O Sinalbin

Figure 13.13 Sinigrin and sinalbin.

flavonoids through oxidation/reduction steps and derivatization of the hydroxyl groups with methyl groups (Gauthier et al. 1998), carbohydrates (Matern et al. 1981), and isoprenoids. Theoretically, considering a possible combination of all these sources of variability, thousands of different flavonoid compounds can occur (Havsteen et al. 2002). Flavonoids can be divided into various classes on the basis of their molecular structure. The six main groups of flavonoids, the best-known members of each group, and the natural source in which they are present are listed in Table 13.5. The molecular structure of each group of flavonoids is given in Figure 13.14. Flavones are derived from the skeleton structure of 2-phenylchromen-4-one, which is derived from the basic nucleus, chromen-4-one. The group of flavones includes the flavonols rutin, isoquercetin, and quercetin as well as the flavanones naringin, prunin, and naringenin (Figure 13.15). Flavanones and flavonols have the same basic nucleus of flavones. Flavanones are derived from the 2,3-dihydro-2-phenylchromen-4-one nucleus and flavonols are derived from the 3-hydroxy-2-phenylchromen-4-one nucleus. Naringin and rutin, among flavones, are two rutinosides derived from the disaccharide rutinose (rhamnose and glucose) and the aglycones naringenin and quercetin, respectively. Prunin and isoquercetin are glucosides from naringenin and quercetin, respectively. The scarcity of these compounds makes them very expensive to isolate. Additionally, flavonoid family compounds are very difficult to separate due to

Glycosides: From biosynthesis to biological activity

329

Table 13.5 Main groups of flavonoids, individual compounds, and natural sources. Class

Compound

Natural Sources

Anthocyanins

Cyanidin Petunidin Delphinidin Malvidin Apigenin Luteolin Kaempferol Myricetin Quercetin Fisetin Hesperitin Naringenin Catechin EGCG* Daidzein Genistein Glycitein

Berries Strawberries, red wine, tea Black grape Strawberry, red wine, plum, rhubarb, red cabbage, cherry Apple skins, celery Capsicum pepper Broccoli, leek Broccoli, blueberry, apple, tomato, red wine, fruit peels Lettuce, olives, onions Citrus fruit Citrus peel Lemon juice Red wine, beans Green tea, black tea, red wine, cherry, blackberry, apple, grape Soybeans, boiled Miso Tofu, tempeh, soy milk

Flavones Flavonols

Flavanones

Catechins Isoflavones

*Epigallocatechin gallate.

O

O

O

O

O OH

Flavanone

Flavanonol

O Flavone

O + O

O

OH Flavanol

O Isoflavone

Anthocyanidin

Figure 13.14 The molecular structure of each group of flavonoids.

their structural similarity and facility in decomposition (Havsteen et al. 2002). As consequence, their market price is very high. Moreover, the practical applications are often strongly limited due to low solubility and stability in hydrophilic media. To improve the hydrophobic nature of flavonoids, a solution is their hydrolysis, leading to the cleavage of some sugar groups, directly implicated in the biologic properties of these compounds.

OH

OH HO

OH HO

OH

OH HO

HO

O

O

OH

O

HO

HO

O

O

O

O O

HO

O

O

O

OH

OH

OH

O

OH

Naringenin

Prunin

Naringin

OH OH

HO

OH

HO

O

OH OH

O

O OH

O

OH

O

HO OH

HO

OH O O

OH

O

OH

HO

O

O O

OH OH

O OH OH OH

Rutin

HO

OH

Isoquercetin

Quercetin

Figure 13.15 Molecular structures of the flavanones naringin, prunin, and naringenin and the flavonols rutin, isoquercetin, and

quercetin.

Glycosides: From biosynthesis to biological activity

331

Production of flavonoids The absorption and utilization of flavonoids in the human body strongly depend on the number and position of sugar moieties at the aglycon (Hollman et al. 1999; Scalbert and Williamson 2000). The majority of natural flavonoids are flavonoid glycosides poorly absorbed by the human body, solely after hydrolysis by bacterial enzymes in the intestine, whereupon their aglycones are absorbed. Therefore, flavonoid extracts need to be converted from a majority composition in glycoside to an aglycone composition for better absorption and higher bioactivity in the human body for dietary and medicinal uses. The use of selected enzymes can lead to the glycosylation of the molecule of flavonoids (Kim et al. 1999; Ko et al. 2013) or contrary to the hydrolysis of the glycoside (Vila-Real et al. 2011). One approach is the extraction and transformation of plant flavonoids in a single cost-effective and environmentally friendly process. Flavonoid acidic hydrolysis has some drawbacks, namely low yield, difficulties in purification of product, and the use of corrosive and toxic acids. Chemical methods are concerns over adverse environmental impact, safety, and waste. In addition, when a chemical method is used, many protection/ deprotection steps are required to obtain selective functionalization because of the numerous reactive hydroxyl groups in flavonoid structures. An exception to these drawbacks is the Fischer synthesis; however, it is only applicable to a limited range of aglycons. Unlike acid hydrolysis, enzymes exhibit excellent regio and stereoselectivity toward the synthesis of heat-sensitive and isomerically pure aglycons (Wang et al. 2012). The use of enzymatic hydrolysis offers some advantages, such as an increase in the selectivity and hydrolysis reaction rate, easy separation of product from substrate, and improvement of product stability. Several trials have been carried out to obtain flavone aglycones with different enzymes (Table  13.2). Additionally, other types of enzymes have been tested for flavonoid biocatalysis, such as lipase, transferase, isomerase, esterase, or protease. Biocatalysis Lipases were the most frequently used enzymes in the biocatalysis of flavonoids. Candida Antarctica lipase B (CALB) has been the enzyme mostly used. An example was the acetylation of two flavonoid glycosides, rutin and isoquercitrin, through molecular modeling using CALB catalysis (Wang et al. 2010). The results showed that the aglycon part of both rutin and isoquercitrin was localized at the entrance of the binding pocket, stabilized by hydrogen bond and hydrophobic interactions (Wang et al. 2010). Only the primary 6′-OH of the isoquercitrin glucose and the secondary 4′-OH of the rutin rhamnose were expected to be acetylated, while the acetylation occurred only on 3′-OH, 5′OH, and 7-OH hydroxyls when Pseudomonas cepacea lipase (PSL-C) was used (Wang et al. 2010).

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Glycosides: From biosynthesis to biological activity

Transferases are the enzymes that transfer a chemical group from one compound (donor) to another (acceptor). NovQ is a prenyltransferase, a useful biocatalyst for the synthesis of prenylated flavonoids, capable of catalyzing the transfer of a dimethyl allyl group to the B-ring of flavonoids (Wang et al. 2010). The incubation with naringenin and genistein yielded two products with a dimethylallyl group at C-3’ or O-4’ in the B-ring; naringenin was most highly converted to its prenylated derivatives with 98.3% yield under suitable reaction conditions (Ozaki et al. 2009). Enzymatic glucosylation with glycosyltransferases has also been used to enhance the water solubility of aglycones and flavonoids (Xiao et al. 2009). The flavonol quercetin is usually found glycosylated on one or more of its five hydroxyl groups, in order to increase its solubility and stability (Haddad et al. 2006). Hesperidinase has been reported to catalyze the formation hesperetin-7-glucoside and hesperitin, which modulated the bioavailability of hesperidin and changed the absorption site from the colon to the small intestine (Nielsen et al. 2006; Furtado et al. 2012). The cyclization of chalcone to form flavanone, which plays a central role in flavonoid biosynthetic pathways, can be carried out by chalcone isomerase, catalyzing the transformation of chalcone and 6’-deoxychalcone into (2S)naringerin and (2S)-5-deoxyflavanone (Wang et al. 2010). The structure and mutational analysis of chalcone isomerase suggested a mechanism in which shape complementarity of the binding cleft locks the substrate into a constrained conformation (Jez et al. 2000; Tian and Dixon 2006; Ruiz-Pernia et al. 2007). Laccase has been used for the production of a new flavonoid. Myceliophthora laccase was used on the production of a flavonoid polymer with rutin as a substrate in a mixture of buffer and methanol (Kurisawa et al. 2003). This polymer showed enhanced physiological properties compared with native rutin and under selected conditions showed improved superoxide scavenging activity and inhibition effects on human low-density lipoprotein oxidation initiated by 2,2’-azobis (2-amidinopropane), dihydrochloride. The oxidation of catechin in the presence of gelatin has been catalyzed by a laccase to synthesize the gelatin– catechin conjugate (Chung et al. 2003). The conjugates had a good scavenging activity against superoxide anion radicals and showed an amplified inhibition effect on human low-density lipoprotein oxidation. The conjugation of green-tea catechin with amine-substituted octahedral silsesquioxane was catalyzed by horseradish peroxidase (Ihara et al. 2005). The conjugate showed improved activity against superoxide anion compared to intact catechin and strongly inhibited xanthine oxidase activity, allowing amplification of the beneficial physiological properties of flavonoids. The pectinolytic and cellulolytic cellulases were characterized for mainchain and side-chain polysaccharide hydrolyzing activities and also against pure samples of various flavonoids previously identified in bergamot peel to determine various glycosidase activities (Mandalari et al. 2006). Commercial cellulose

Glycosides: From biosynthesis to biological activity

333

preparations from Trichoderma viride was also found to show transglucosylation activity toward (+)-catechin and (–)-epigallocatechin gallate using dextrin as a glucosyl donor (Noguchi et al. 2008). Trichoderma viride cellulase (EC 3.2.1.4), a commercially available cell-wall-degrading enzyme, to assist the extraction of flavonoids from plants, leading to a remarkable increase of flavonoid aglycone contents in the extract, was used in the mild ethanol − water extractant, while the activity of the enzyme in cell wall degradation was employed simultaneously to increase the extraction yield. Flavonoid glycosides, rutin, and flavonoid disaccharide monoglycosides were acylated by the catalytic action of the protease subtilisin in anhydrous pyridine. The acylation occurred with high yield, with rutin giving a single monoester on its glucose moiety (Danieli et al. 1995). In the flavonoid acylation, the proper enzyme often plays a multiple roles and significantly influences the regioselectivity of the reaction. Hydrolases are the enzymes that catalyze hydrolytic reactions, namely the hydrolysis of a glycosidic bond (Metzler 2001; Buckow 2007).The enzymatic deglycosylation of both naringin and rutin comes out as a suitable process in order to produce the respective glucosides and aglycones, due to the specificity and mild conditions of enzymatic reactions, avoiding additional purification steps and preserving unstable products. Naringinase from Penicillium decumbens was used for flavonoid glycosides deglycosylation into aglycones.

Microbial biotransformation Microbial transformation is an effective tool for the structural modification of bioactive natural and synthetic compounds including flavonoids. Microbial factories have advantages such as rapid growth, ease of growth, genetic manipulations, increased product selectivity, high-level production on natural product biotransformation, and reduced usage of toxic chemicals while conserving energy usage (Chebil et al. 2007). Metabolic engineering of microorganisms provided an alternative method of supplying valuable natural products that occur at low concentrations. Biotransformations of flavonoid by many microorganisms including species of Aspergillus and Bacillus have been studied. Apergillus niger strain was used for the regioselective O-demethylation of the flavones tangeretin (1) and 3-hydroxytangeretin (6) into their 4’-Odemethylated metabolites. Aspergillus oryzae was used for the biotransformation of genistein into shoyuflavone B (Wang et al. 2010). Molds belonging to Aspergillus niger species are effective biocatalysts for transformations of flavonoids in diversity of the transformation products and high yield. Bacillus was chosen to be used in transformations of flavonoids because of its safety, rapid growth, and ease of scale-up for mass cultures. Bacillus subtilis natto NTU-18 in black soy milk could effectively hydrolyze the glycosides from isoflavone and fermented black soy milk has the potential to be applied to selective estrogen receptor modulator products (Kuo et al. 2006). However, the hydrolysis

334

Glycosides: From biosynthesis to biological activity

of those glycosides to aglycones does not enhance the bioavailability of isoflavones in humans (Richelle et al. 2002). The solubility can also be enhanced for puerarin when it was transglycosylated by using Bacillus stearothermophilus maltogenic amylase, 14 and 168 times higher than that of puerarin (Kulikov et al. 2009). Escherichia coli have also been utilized as a drug factory for the production of a diverse array of important pharmaceuticals. In most cases, productivity is greatly limited by the low availability of intracellular precursors. However, engineered E. coli strains, which express the plant flavonoid biosynthetic pathways, allow high-yield flavonoid and its derivatives production through traditional metabolic engineering techniques (Leonard et al. 2008). Recombinant E. coli cells, containing four genes for a phenylalanine ammonialyase—cinnamate/ coumarate, CoA ligase, chalcone synthase, and chalcone isomerase—in addition to the acetyl-CoA carboxylase, have been established for the efficient production of (2S)-naringenin from tyrosine and (2S)-pinocembrin from phenylalanine. The engineered E. coli cells using the flavanone 3β-hydroxylase and flavonol synthase genes from the plant Citrus species was applied to produce kaempferol from tyrosine and galangin from phenylalanine (Miyahisa et al. 2006).

Biologic activity of flavonoids The biological activities of flavonoids include a broad spectrum, from a neuroprotection effect, to anticancer and antibacterial activities, to prevention of endothelial disfunctions, to inhibition of bone reabsorption, among others. A  vast number of pharmacological activities are shared by flavonoids. Free radical scavenging and antioxidant and anti-inflammatory activities are the most common, but plenty other activities are described, including antitumor, atherosclerosis-preventive activity through the protection of human low-density lipoprotein (LDL) against oxidation, hypocholesterolemic activity through the inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG-CoA reductase), antithrombotic activity by preventing platelet aggregation, antiallergic activity, inhibition or killing of many bacterial strains, inhibition of viral enzymes, destruction of some pathogenic protozoans, and even stimulation of some hormones and neurotransmitters (Havsteen 2002). The treatment of neurodegenerative diseases (e.g., Alzheimer’s disease) based on flavones focuses on targeting the inflammation process, behind the progression of the disease. The anti-inflammatory therapeutic approach is based on the action to prevent oxidative stress and consequent inflammation, acting as antioxidants through free radical scavenging. Flavones are a very large subgroup within the flavonoids to whom much attention has been dropped, in order to find compounds of potential pharmacological use. As part of the flavonoids group, flavones share with them common biosynthetic routes, appearance in certain plant organs, as well as certain pharmacological activities based on similar mechanisms of action (Havsteen 2002).

Glycosides: From biosynthesis to biological activity

335

Among flavonoids, the glucosides (prunin and isoquercetin) and the aglycones (naringenin and quercetin) are scarcer than the rutinosides (naringin and rutin). Naringin is reported to show in vitro free radical scavenging activity and in vivo antioxidant (Rajadurai et al. 2007) and anti-inflammatory activity (Amaro et al. 2009). It also shows atherosclerosis preventive activity, by hypocholesterolemic and antihyperlipidemic activities (Jung et al. 2006; Lee et al. 2008; Kim et al. 2006; Morikawa et al. 2008). In cancer therapy, naringin can potentially be used as a antineoplastic agent (Ali et al. 2009) as well as a chemotherapy adjuvant. Diabetes is also a potential field of application, due to its hypoglycemic activity, by increasing hepatic glycolysis and glycogen concentration and/or by lowering hepatic gluconeogenesis (Jung et al. 2006). Other potential target diseases are osteoporosis, cerebral ischemia, septic shock, and even anxiety. Prunin shows hypocholesterolemic activity, antithrombotic activity (Itoh et al. 2009), and activity as a chemotherapy adjuvant (Han et al. 2008). Naringenin shows a higher antioxidant activity and hydroxyl and superoxide radical scavenger efficiency than naringin. It also has anti-inflammatory activity (Amaro et al. 2009; Ribeiro et al. 2008; Vafeiadou et al. 2009) and immunomodulatory activity acting against asthma. The hypocholesterolemic activity shown in vivo (Jeon et al. 2007) is helpful against atherosclerosis disease, while hypoglycemic activity may be useful for diabetes. In cancer naringenin can be used in adjuvant chemotherapy, or as a potential antineoplastic agent in oral carcinogenesis, in endogenous hormone 17β-estradiol-dependent cancers, and malignant melanoma. Osteoporosis and hepatitis C infection are other potential targeting diseases. Rutin exhibits free radical scavenging (Sun et al. 2009) and antioxidant activities as well as antineoplastic activity, where it may lead to the inhibition of breast cancer tumor proliferation through the inhibition of vascular endothelial growth factor (VEGF). Isoquercetin shows free radical scavenging activity, antioxidant activity higher than rutin (Sun et al. 2007), and in vivo anti-inflammatory activity. Isoquercetin might be highly useful in the treatment of asthma (Fernandez et al. 2005), diabetes, and obesity through the inhibition of the glucose transport across intestinal cells, via glucose transporter-2 (GLUT2), while rutin does not. Quercetin shows free radical scavenging activity; however, in high concentrations it can show pro-oxidant activity. As isoquercetin, quercetin exhibits potential activity against obesity through the inhibition of glucose and fructose transport by GLUT2 (Kwon et al. 2007). A phase II clinical trial for the therapeutic use of quercetin is currently running. Quercetin is also active against cancer, as a tyrosine kinase inhibitor, asthma, and hypertension (Emura et al. 2007).

13.7.9 Saponins Saponins are widely distributed in plants. They are classified according to the aglycon part in steroidal type and triterpinoidal type C30. Saponins have the glycosidic linkage at position 3 (Figure 13.16).

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Glycosides: From biosynthesis to biological activity

CH3 OH O

CO OH

O

O

O Testosterone

Progesterone

O

CH2OH

O

O

HO

CO OH

O Diosgenin

Cortisone

Figure 13.16 Molecular structure of different saponins.

Saponins are excellent emulsifying agents and were formerly used as detergents to replace soap. They have hemolytic properties, being toxic when injected into the bloodstream; however, they are not harmful when taken orally. The steroidal saponins are structurally related to synthetic compound adrenocorticoids and therefore are suitable precursors in the hemisynthesis of the hormones (e.g., diosgenin [sapogenins]) isolated from the rhizome of Dioscoria species. Saponins increase the rate of absorption of many active pharmacological substances (e.g., cardiac glycosides). Besides being a valuable flavoring and sweetening agent, many saponins are used as expectorants (e.g., Ipeca, Senaga, and liquorice), as their contents stimulate bronchial secretion, activate the ciliary epithelium of the bronchi, and have antispasmodic action. All these activities are attributed to the saponin glycyrrhizin.

Production of ginsenosides Panax ginseng is a well-known medicinal plant that has been used as a tonic and home medicine in Oriental countries since ancient times. Ginsenosides, a group of tetracyclic triterpene glycosides also known as saponins, are the principal bioactive constituents of P. ginseng. The pharmacological activities of ginsenosides

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337

have been demonstrated in relation to its antiplatelet, hypocholesterolemic, antitumor, and immunomodulatory functions, and improving central nervous system functions. This triggers an increasing demand of ginsenosides for clinical trials and applications in the future. Plant cell culture has been considered a promising alternative for the mass production of secondary metabolites such as ginsenosides (Huanga et al. 2013). Crude preparations of beta-galactosidase from Aspergillus oryzae and lactase from Penicillium sp. were found to produce two minor saponins, ginsenoside Rg(2) [6-O-(alpha-L-rhamnopyranosyl-(1– > 2)-beta-D-glucopyranosyl)-20(S)protopanaxatriol] and ginsenoside Rh(1) (6-O-beta-D-glucopyranosyl-20(S)protopanaxatriol), respectively, in high yields. Moreover, a naringinase preparation from Penicillium decumbens readily gave a metabolite, ginsenoside F(1) (20-O-betaD-glucopyranosyl-20(S)-protopanaxatriol), as the main product, with a small amount of 20(S)-protopanaxatriol from a protopanaxatriol-type saponin mixture (Ko et al. 2003). Various structure-similar plant secondary metabolites such as ginseng saponins (ginsenosides) possess different or even totally opposite biological activities. Intentional manipulation of the ginsenoside heterogeneity in cellular biosynthesis is of great interest and significance (Yue et al. 2008). In vitro production of saikosaponins using cultured cells or roots of B. falcatum L. has been studied for saikosaponin production (Katagiri and Sugimoto 2012).

13.8 Conclusion Biocatalysis and biotransformation of glycosides to modify their physicochemical and biological properties are of a great scientific and industrial interest. Enzymatic transformation of glycosides has been achieved using different enzymes, mainly glycosidases. Merging the potential of microbial genetics and biocatalysis with biological and chemical diversity would offer a brighter future for natural product (glycosides) drug discovery.

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CHAPTER 14

Trehalose mimics as bioactive compounds Davide Bini, Antonella Sgambato, Luca Gabrielli, Laura Russo, and Laura Cipolla Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy

14.1 Introduction Trehalose (Figure 14.1) is a nonreducing disaccharide formed by an α,α-1,1-glycosidic bond between two glucose units, the only anomer out of the possible three (α,β1,1-, β,β-1,1-, and α,α-1,1-), found in living organisms. Trealose is widespread throughout the biological world (Elbein 1974; Thevelein 1996; Strom and Kaasen 1993), and can be found in plant, insect, and microbial cells often linked to physiological stresses such as heat and osmotic shock, sporulation, and dehydration. This disaccharide presents several distinctive physical properties, including high hydrophilicity and chemical stability, nonhygroscopic glass formation and the lack of internal hydrogen bond. The widespread distribution of α,α-trehalose among different species suggests a relevant role of this molecule in the biology of such living organisms. Trehalose behaves as an energy source in some organisms, whereas it plays structural or transport roles in others (Takayama and Armstrong 1976). It can be also found in signaling or regulation pathways, or as a protection for membranes and proteins during cellular stresses, such as cold, dessiccation, heat, and anoxia (Crowe et al. 1987). Therefore, depending on the specific biological system taken into consideration, a variety of functions have been proposed for trehalose, ranging from an external carbon source, stored as compatible solute by photosynthetic bacteria, or a structural component of the cord factor in mycobacteria (Crowe et al. 1988; Strom and Kaasen 1993). In yeast and filamentous fungi, large amounts of trehalose are accumulated both as a reserve source and as protection against stress conditions. Several species of insects contain trehalose in the “fat body” and in the hemolymph, as an energy source during flight.

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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OH HO

OH OH

HO

HO HO

O OH

O

HO

O OH

HO

HO α,α-trehalose

OH 7 OH 5 6 4 N

HO 3 2

HO NH 1' HO

5'

NH

6' OH OH

2'

1 S

3' OH 4'

Validoxylamine A

OH HO

O

OH OH

1-Thiatrehazolin

Figure 14.1 Chemical structure of the naturally occurring trehalose anomer and some relevant

mimetics.

14.2 Trehalose processing enzymes and trehalose mimetics as bioactive compounds 14.2.1 Trehalases Trehalose hydrolysis is catalyzed by the glycolytic enzyme trehalase (EC3.2.1.28), first observed in Aspergillus niger (Bourquelot 1983) and then by Fischer in 1895 from S. cerevisiae (Elbein 1974), and afterward reported in several other organisms within the plant and animal kingdoms. Trehalases belong to the GH37 family of the Carbohydrate-Active Enzyme (CAZy) classification (Coutinho and Henrissat 1999). From a mechanistic point of view, it is an inverting glycosidase (Defaye et al. 1983), hydrolyzing the glycosidic bond with inversion of configuration at the anomeric center. Trehalose hydrolysis is a relevant, and somehow essential, process of several organisms; it is essential for insect flight (Thompson 2003) and growth resumption of resting cell or spore germination in fungi. The first three-dimensional structure of a trehalase (Tre37A from E.coli), in complex with known inhibitors validoxylamine A and 1-thiatrehazolin (Figure  14.1), was solved only in 2007 (pdb entries 2JF4 and 2JG0) (Gibson et al. 2007). The structure of Tre37A consists of an (α/α)6 barrel, with the subsites +1 for the leaving-group and −1 as the “catalytic” site.

14.2.2 Trehalase inhibitors Trehalose analogues have potential as bioinsecticides, fungicides, or antibiotics due to their ability to inhibit trehalase, a key enzyme in trehalose metabolism of fungi, insects, and some bacteria. A variety of mimetics of trehalose have been synthesized over the years and inhibition studies revealed that several compounds are able to specifically block trehalase activity in a micromolar or submicromolar range.

Validoxylamines The validamycin complex is produced by Streptomyces hygroscopicus subsp. limoneus and includes validamycins A, B, C, D, E, F, and G and validoxylamines A, B, and G (Figure 14.2) (Iwasa et al. 1971; Horii et al. 1972; Kameda et al. 1986). Validamycins A, C, D, E, and F contain validoxylamine A as a common moiety,

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D-gluco-dihydro-validoxylamine A OH HO HO

Valienamine

O

OR5

HO 7 4 3

NH OH HO

OH

1 5

OH HO HO HO

2

R6O HO HO

R1

NH ' 6' 5

1'

7'

HO HO

2'

NH OH HO

R3

OH

4'

OR2 OR4

Valiedamine hydroxyvalidamine or Valiolamine

OH OH

L-ido-dihydro-validoxylamine A Validoxylamine A Validoxylamine B Validoxylamine G

R1=H R1=H R1=OH

R2=H R2=H R2=H

R3=H R3=OH R3=H

R4=H R4=H R4=H

R5=H R5=H R5=H

R6=H R6=H R6=H

Validamycin A Validamycin B Validamycin C Validamycin D Validamycin E

R1=H R1=H R1=H R1=H R1=H

R2=H R2=H R2=H R2=α-D-Glc R2=H

R3=H R3=OH R3=H R3=H R3=H

R4=β-D-Glc R4=β-D-Glc R4=β-D-Glc R4=H R4=α-D-Glc R4=β-D-Glc

R5=H R5=H R5=α-D-Glc R5=H R5=H

R6=H R6=H R6=H R6=H R6=H

Validamycin F Validamycin G

R1=H R1=OH

R2=H R2=H

R3=H R3=H

R4=β-D-Glc R4=β-D-Glc

R5=H R5=H

R6=H R6=H

Figure 14.2 Validoxylamines structures.

but differ from each other in the number, the site, and/or the type of glycosidic attachment to validoxylamine A (Figure 14.2). As shown in Table 14.1, the inhibitory effect of validoxylamines was found to be a potent competitive inhibitors against various trehalases (Asano et al. 1990); validoxylamines and validamycins, except for validamycin C, also showed a potent inhibitory activity against insect trehalase (Spodoptera litura and termites) (Jin and Zheng 2009). Validoxylamine A was found to be the most potent inhibitor against the insect trehalase (Ki value: 4.7 × 10–10 M for S. litura and 3.2 × 10–6 M for termites; Table  14.2), showing also insecticidal activity in S. litura (Asano et al. 1990), while Validamycin A is the most active compound against sheath blight of rice plants caused by the phytopathogenic fungus Rhizoctonia solani (Table  14.1) (Asano et al. 1987).

2.4 · 10–6 – –

2.5 · 10 –4

4.2 · 10–7 – –

4.9 · 10–9 2.0 · 10–8 – – – – 3.0 · 10–6 3.1 · 10–7

2.4 · 10–9

1.0 · 10–3

Rat (intestine)

Porcine (kidney)

1.9 · 10–4

1.4 · 10–8 3.4 · 10–8 – – – – 5.4 · 10–6 3.0 · 10–8

Porcine (intestine)

8.2 · 10–9 – –

5.3 · 10–4

1.1 · 10–10 4.0 · 10–7 – – – – 1.1 · 10–6 1.9 · 10–8

Rabbit (kidney)

M.s., Mycobacterium smegmatis; S.l., Spodoptera litura; R.s., Rhizoctonia solani.

Validoxylamine A Validoxylamine B Validoxylamine C Validoxylamine D Validoxylamine E Validoxylamine F Validoxylamine G D-gluco-D-hydrovalidoxylamine A L-ido-D-idrovalidoxylamine A Validamycin A Validamycin B Validamycin G

Compound

Table 14.1 Inhibitory activity (IC50) of validoxylamines against several trehalases.

7.4 · 10–6 – –

8.4 · 10–3

3.0 · 10–9 1.9 · 10–6 – – – – 3.2 · 10–6 7.4 · 10–7

Baker’s yest

IC50 (M)

4.2 · 10–4 – –

1.1 · 10–8 2.6 · 10–6 – – – – 1.0 · 10–7 1.9· 10–3 1.8 · 10–6

M.s.

4.8 · 10–8 6.6 · 10–6 – – – – 5.9 · 10–6 5.3· 10–8 8.4· 10–4 3.7 · 10–7 1.8 · 10–6 7.9 · 10–7

S.l.

1.4 · 10–7 1.6 · 10–3 7.4 · 10–6

–

7.2 · 10–5 3.5 · 10–5 – 1.5 · 10–3 – – 5.2 · 10–6 –

R.s.

6.1 · 10–6 4.0 · 10–6 –

–

–

4.3 · 10–5 5.9 · 10–6 – – – –

Termites

Trehalose mimics as bioactive compounds

349

Table 14.2 Ki values of some validoxylamines with different trehalases. Compd.

Porcine Rat Rabbit intestine intestine kidney

Baker’s yeast

M.s.

S.l.

Termite

R.s.

Ki (M) Validamycin A Validamycin B Validoxylamine A Validoxylamine B

7.8 × 10–10 3.1 × 10–7 1.2 × 10–10 2.7 × 10–10 4.9 × 10–9

4.7 × 10–8 4.02 × 10–4 1.9 × 10–7 2.69 × 10–4 4.7 × 10–10 3.2 × 10–6 1.9 × 10–9 1.9 × 10–7 1.03 × 10–5

M.s., Mycobacterium smegmatis; S.l., Spodoptera litura; R.s., Rhizoctonia solani.

Trehazolin and its analogues as trehalase inhibitors Trehazolin (1, Figure  14.3) is a pseudodisaccharide isolated from the culture broth of Micromonospora strain SANK 62390 (Ando et al. 1991). It was tested as an inhibitor of purified pig kidney trehalase (Kyosseva et al. 1995), silkworm trehalase (Uchida et al. 1995a), porcine trehalase (Ando et al. 1991), and Rhizoctonia solani trehalase (Table  14.3) (Ando et al. 1995a). Trehazolin 1 was found to be a very powerful and competitive inhibitor (IC50 of about 1.9 × 10–8 M) selective for the kidney trehalase. The interest in the structure and inhibitory-activity connection of this unique inhibitor has prompted Uchida et al. (1995a, 1995b) to synthesize different structural analogues (2–12) of trehazolin 1, which contain chemically modified cyclitols or sugar portions. The inhibitory activity (IC50) against silkworm trehalase of the analogues 2–12 is reported in Table 14.4, together with the reference compounds trehazolin (1), the analogues 6 and 8, and the diastereoisomers thereof. Removal of each hydroxyl function of the 6,7,8-trihydroxy-2-oxa-4-azabicyclo-[3.3.0]octane moiety caused a decrease in the inhibitory potency, as well as deoxygenation of the sugar part. All the three 6-, 7- and 8-hydroxyl groups, including 6-hydroxymethyl, are therefore considered to be topologically essential in mimicking those of the α-D-glucopyranose residue of the natural substrate. On the other hand, the cyclopentane part containing the cyclic isourea acts as a mimic of the postulated charge-distributed flattened half-chair transition state of the glycone during hydrolysis. Only the 5a’-carba-analogue 12 (Uchida et al. 1995b) conserves the nanomolar inhibitory activity of the parent compound against trehalases. The effects of structural modifications at the oxazoline ring have been less studied: the only synthesized analogue of this type reported is compound 9 (Uchida et al. 1995a), which contains an imidazoline ring, but also two other concomitant structural modifications: a 5a-carbaglucose, to confer stability toward hydrolytic cleavage without compromising activity, and a 4-de(hydroxymethyl)cyclitol moiety, a modification that has been shown (Uchida et al. 1994a) to lower the inhibitory activity by 100 times with respect to the parent

Trehalose mimics as bioactive compounds

350

6' OH 4' HO HO

4'

O 2'

5'

5'

HO HO

1'

3'

N

4'

O 2'

N 3

3 N

2O

4

1

6

Y 7

8

N

2O

OH

5

H

4

4N

6' 4' HO HO 3'

6'

OH 5'

4'

X 2'

HO HO 1'

6' 4' HO HO

1' OH

3'

H

3 N

2Y 1

OH 5'

N

N

5 8

5'

1' OH N

OH 7

6 OH

5

HO

N

2O 8

3 OH

OH

OH

OH 6 7 OH

OH

10

8 X=Y=O, R=H 9 X=CH2, Y=NH, R=H 12 X=CH2, Y=O, R=CH2OH 13 X=O, Y=S, R=CH2OH

4 5

1

2 4

OH

H

3

1 6

OH

O 2'

H N9

7O

R

1

X

F

8

4

O2

6 X=H, Y=OH 7 X=OH, Y=H

2' 3'

OH

3

Y

OH 5

1 X=Y=Z=OH 2 X=H, Y=Z=OH 3 X=Z=OH, Y=H 4 X=Y=OH, Z=H

7 8

OH

OH

N

5

6

6

X Z

OH

H

OH 7

8

OH

H

5

1

1'

3'

OH

H

O 2'

5'

HO HO

1'

3'

OH

6' O H

OH

6'

11

Figure 14.3 Structure of trehazolin and its analogues. Table 14.3 Inhibitory activity (IC50) of trehazolin against pig kidney,

silkworm, porcine, and R. solani trehalase. Compound

Trehazolin

IC50 (M) Pig kidney

Silkworm

Rhizoctonia solani

Porcine

1.9 × 10–8

4.9 × 10–8

6.6 × 10–8

3.7 × 10–9

compound 1. Since compound 9 is 1000 times less potent than trehazolin against silkworm trehalase, it can be concluded that the oxazoline to imidazoline modification is deleterious to inhibitory activity, by roughly an order of magnitude. With this in mind, Chiara et al. (2005) decided to synthetize the corresponding

Trehalose mimics as bioactive compounds

351

Table 14.4 Inhibitory activity (IC50) of trehazolin analoguesa

against silkworm trehalase. Compound

Inhibitory activity IC50(M)

Trehazolin (1) Diastereoisomer of 1a 6-Deoxy 2 7-Deoxy 3 8-Deoxy 4 6-Deoxy-6-epi 5 6,7-Diepi 6 Diastereoisomer of 6a 6-Epi 7 6-Dehydroxymethyl 8 Diastereoisomer of 8a Cyclic guanidine 9 Validoxylamine-type 10 6’-Deoxy-6’-fluoro 11 5a’-carbaglucose 12

4.9 · 10–8 1.2 · 10–6 2.1 · 10–5 6.3 · 10–5 >2.7 · 10–4 >2.7 · 10–4 9.2 · 10–7 2.6 · 10–5 3.0 · 10–7 7.8 · 10–6 1.6 · 10–7 5.9 · 10–5 >5.0 · 10–4 2.3 · 10–7 4.9 · 10–8

The diastereoisomers contain the corresponding enantiomeric cyclopentanepolyol moieties.

a

thiazoline analogue 13 and to assay its inhibitory activity against commercially available porcine kidney trehalase. Compound 13 resulted to be a nanomolar inhibitor of this enzyme, although with an IC50 value (IC50 83 nM, Ki = 30.4 nM) approximately five times higher than that of parent 1 (IC50 19 nM, Ki = 2.1 nM) (Ando et al. 1995b). Although trehazolin and its analogues have specific and strong inhibitory activity against trehalase in vitro, they do not show any insecticidal activity toward insects in vivo; due to their hydrophilicity they cannot penetrate insect skin and reach the trehalase target. In order to increase the hydrophobicity and penetrating ability, Qian et al. (2001) designed and synthesized a new group of compounds (14–23, Figure 14.4) on the basis of the mechanism of action of trehazolin (Uchida et al. 1994b, 1995b; Rhinehart 1990; Danzin and Ehrhard 1987) and its structural model (having a N = C − NH unit, heterocyclic moiety, and polyhydroxyl groups). To increase the hydrophobicity they introduced a benzene ring and fluorine atoms, which also have the possibility to form hydrogen bonds with the active site of trehalase. The structures of the synthesized compounds and their IC50 values are given in Figure  14.4. The experiments in vitro showed that compounds 14–23 have modest trehalase inhibitory activity (pIC50 around 2.21–4.45), with compound 20 having the highest bioactivity. As a general trend, the inhibition activities of oxazolines are higher than those of thiazolines. Compounds 14–23 were screened for their insecticidal activity toward the adult of wild fruitfly Drosophila

Trehalose mimics as bioactive compounds

352

CH2OH

N

CH2OH

N R

X

Compounds

pIC50

14 X=O, R=2-F 15 X=S, R=2-F 16 X=O, R=4-F 17 X=S, R=4-F 18 X=O, R=2,4-F2 19 X=S, R=2,4-F2 20 X=O, R=3-Cl,4-F 21 X=S, R=3-Cl, 4-F 22 X=O, R=2,3,4-F3 23 X=S, R=2,3,4-F3

4.21 3.08 3.58 3.24 3.05 2.21 4.45

3.62 4.07 3.15

Figure 14.4 Structure formulas and inhibitory activities of compounds 14–23 against porcine

trehalase.

HO

OH

H 7

6 5

7' 4N

1

2

HO OH

8 CH2OH

25

H N

OH

HO OH

H N

CH2OH 28

HO

OH OH

N

3

24

HO

H

H

HO

N

H

OH N

OH

CH2OH 30

24 25 26 27 28 29 30 31 32 33

55 160 310 310 450 12 0.34

27

HO OH

IC50(µM)

CH2OH

26 R=H 31 R=β-D-glucopyranosyl HO

Compound OH

CH2OH

CH2OH

OH

OH

H

OR

N

CH2OH 29

OH

RO

H N

OH OH CH2OH

32 R=H 33 R=α-D-glucopyranosyl

Figure 14.5 Structures of polyhydroxylated pyrrolizidines 24–33 and their biological activity against porcine kidney trehalase.

melanogaster. They did not show any insecticidal activity, but showed interesting flight-inhibition activity (compound 21 was the most potent). Moreover, it was found that compounds 14–23 have obvious larvicidal activity toward the wild fruitfly Drosophila melanogaster and compound 21 was the most potent.

Polyhydroxylated pyrrolizidine alkaloids as trehalase inhibitors Polyhydroxylated pyrrolizidine alkaloids 24–33 (Figure 14.5) were isolated from the pods of the legume Alexa leiopetala (Nash et al. 1988a). Although the wide class of pyrrolizidine alkaloids bears a carbon substituent at C-1 (Wrobel 1985; Robins 1995), alexine is the first example of a pyrrolizidine alkaloid with a carbon substituent at C-3. At about the same time, australine 25 was isolated from seeds of the Australian legume Castanospermum australe and found to be 7a-epi-alexine from X-ray crystallographic analysis (Molyneux et al. 1988). The isolation of

Trehalose mimics as bioactive compounds

353

1-epi-australine 26 (Harris et al. 1989; Nash et al. 1990) and 3-epi-australine 27 (Nash et al. 1988b) from the same plant was later reported, and 7-epi-australine 28 (Denmark and Herbert 1998, 2000) was synthesized by Denmark et al. Accurate search for polyhydroxylated pyrrolizidines in the seeds of Castanospermum australe led Kato et al. (2003) to the discovery of three new alkaloids: 2,3-diepi-australine 29, 2,3,7-triepi-australine 30, and 1-epi-australine-2-O-βD-glucopyranoside 31. In Figure  14.5 are reported the inhibitory activities (IC50), against porcine kidney trehalase, of alkaloids 24–31 and highly oxygenated pyrrolizidines, casuarine 32 (Nash et al. 1994), and its 6-O-α-Dglucopyranoside 33 (Figure 14.5). These two compounds were also synthesized (Bell et al. 1997; Denmark and Hurd 2000; Cardona et al. 2009); the first total synthesis of 6-O-α-D-glucopyranosyl casuarine was achieved by Cardona et al. (2009) and allowed to perform more reliable biological tests on chemically pure compound. The most noteworthy inhibition activity was shown for compound 33 (IC50 = 0.34 μM) that inhibited the enzyme in a competitive manner, with a Ki value of 0.018 μM. It is now well accepted that the trehalase catalytic site contains two subsites (Gibson et al. 2007; Cardona et al. 2009; Asano et al. 1996; Silva et al. 2004), a catalytic site and a recognition site. The extremely high affinity of a pseudodisaccharide inhibitor derives from the synergistic interactions of an alkaloid unit and a sugar (or cyclitol) unit with the two subsites (Asano 2003). Compounds 32 and 33 proved their strong and selective inhibitory properties also toward E. coli trehalase Tre37A (Cardona et al. 2009). Compound 33 (Ki = 12 nM) resulted to be around a thousand-fold more potent than 32 (Ki = 17 μM), indicating that the glucose moiety contributes considerably to binding. Two new casuarine-6-O-α-D-glucoside analogues 34 and 35 (Figure 14.6) were synthetized by Cardona et al. (2010) in order to generate highly potent trehalase inhibitors with potential insecticidal activity. Compounds 33–35 were tested against C. riparius trehalase, Tre37A, and commercial porcine

Porcine kidney, Ki

E. coli Tre 37A, Ki

C. riparius, Ki

33

11 nMa

12 nM

34 35

138 nM >10 μM

86 nM 2.8 μM

0.66 nM 22 nM 157 nM

OH HO HO

O

R

HO O

Ki = 18 nM for 33 isolated from natural source (purity about 60%). a

7 6

OH

H 7' 5 4N

1 2

OH

3

33 R=OH 34 R=H 35 R=CH2OH

OH

Figure 14.6 Casuarine-6-O-α-D-glucoside (33) and its analogues (34, 35) and their biological

activity.

354

Trehalose mimics as bioactive compounds

kidney trehalases (Figure 14.6). The potency of 33, 34, and 35 toward the three trehalases showed a similar trend, with the most potent inhibition afforded with compound 33. The functional group at the C7 position has a critical effect on the inhibitory activity of these analogues; the activity is decreasing in the order OH > H > CH2OH. It is interesting to observe, however, that compounds 33 and 35 are at least one order of magnitude more potent toward the C. riparius trehalase than the porcine trehalase. Compound 33 is the most potent insect trehalase inhibitor described to date with a Ki value against C. riparius lower than that displayed by validoxylamine A toward the porcine kidney trehalase (Ki = 2 nM) (Asano et al. 1990; Kyosseva et al. 1995) and an order of magnitude lower than that calculated for trehazolin with other insect species (Ki = 10–8 M toward locust flight muscle trehalase) (Wegener et al. 2003; Kyosseva et al. 1995). However, in terms of selectivity, compound 35 is probably the most interesting one, showing more than 60-fold selectivity between C. riparius and porcine kidney trehalase. To further investigate the potentiality of the pyrrolizidine ring of casuarine, a number of derivatives were assayed as inhibitors of trehalase from C. riparius (Forcella et al. 2010). The structures included in this study are illustrated in Figure  14.7, and their effect is reported in Table  14.5. None of the compounds tested resulted to be more effective than casuarine itself: thus, the hydroxyl group in position 7 of casuarine seems again to play a fundamental role in enhancing inhibitory activity. In particular, substitution of the hydroxyl group with a hydroxy methyl group dramatically increased the Ki value (273-fold in C. riparius), whereas removing the hydroxyl group (7-deoxycasuarine) had a relatively modest effect with a 4.9-fold increase of the Ki value. Reduction of the size of the heterocyclic ring from pyrrolizidine to pyrrolidine (as in 6-deoxy-DMDP and DAB-1) caused a sensible decrease in the inhibitory activity with similar values against C. riparius larvae and porcine kidney trehalase. HO HO

H

HO

OH

N

H

HO

OH

OH

N

H HO

OH

OH

N

OH

OH Casuarine (32)

OH 7-Deoxycasuarine

7-Homocasuarine HO

HO

OH

OH

OH

OH

OH

N H

N H

DAB-1

6-Deoxy-DMDP

Figure 14.7 Structure of casuarine and analogues used as inhibitors of trehalase.

Trehalose mimics as bioactive compounds

355

Table 14.5 Inhibition by casuarine derivatives of membrane-bound trehalase from C. riparius larvae and trehalase from porcine kidney. Compound

7-Deoxycasuarine 7-Homocasuarine 6-Deoxy-DMDP DAB-1 Casuarine (32)

Porcine kidney

C. riparius IC50(μM)

Ki

IC50(μM)

Ki

1.2 ± 0.04 65.5 ± 1.5 52.4 ± 2.2 19.0 ± 0.9 0.25 ± 0.03

0.59 ± 0.02 33.0 ± 0.8 25.6 ± 1.1 9.3 ± 0.4 0.12 ± 0.01

21.5 ± 2.8 214.0 ± 28.8 110.8 ± 16.4 10.6 ± 2.4 12

11.7 ± 1.4 107.0 ± 14.4 55.4 ± 8.2 5.3 ± 1.2 –

Piperidine and pyrrolidine-based trehalase inhibitors It is well known that polyhydroxy pyrrolidine and piperidine (Asano 2003; Compain and Martin 2007), are powerful glycosidase inhibitors due to the presence of endocyclic nitrogen, which is able to mimic the transition state of the enzymatic reaction. Pyrrolidine alkaloids such as DAB-1 (Figure 14.7) and deoxynojirimycin (Figure 14.8) have been recently found in the latex exuded from Mulberry leaves (Morus spp.) and their toxic effect toward Eri silkworm (Samia ricini) larvae has been evidenced. These results suggest that these sugarmimic alkaloids exert toxicity by inhibiting trehalase (Hirayama et al. 2007; Konno et al. 2006). Hence, several pyrrolidine- and piperidine-based compounds (such as 36–57, Figure 14.8) have been designed and tested as trehalase inhibitors (Asano 2003; Bini et al. 2011, 2012). A series of inhibitors (36–43) were synthesized, introducing a substituent of the iminoglucitol ring of deoxynojirimycin (Forcella et al. 2010), and assayed on Chironomus riparius trehalase activity. Their effect is reported in Table 14.6. Compound 37 resulted the most active molecule in a competitive manner (Ki = 25 μM). The second most active inhibitor was compound 41, but its effect seemed to be noncompetitive as well as that of inhibitors 42 and 43. The remaining four compounds of the series did not show trehalase inhibition. For comparison, the same assay was carried out using porcine kidney trehalase; only compound 37 showed slight inhibitory activity. MDL 25,637, that efficiently inhibits trehalase (Table 14.7) and, to a lesser extent, isomaltase > sucrase > glucoamylase > maltase, was used as reference compound (Rhinehart et al. 1987). Compounds 44–48 (Figure 14.8) were tested for their inhibitory activity against porcine trehalase. Preliminary screening assays at a fixed concentration (1 mM) of potential inhibitors were carried out, and dose–response curves were established for the most active compounds to determine the Ki values. The nojirimycin dimer, compound 44, was the most active derivative of the series, showing 100% inhibition at a concentration of 1 mM, and IC50 = 88 μM and Ki = 44 μM (Table 14.7). Another set of compounds (49–57) has been synthesized and evaluated as potential

HO

HO

HO

HO

NH

HO

HO

O N

36 HO

O

HO

NH2

HO

N

HO NH

HO

NH (CH2)4

HO

HO

N

NHBn

HO

HO

NH2

HO OH

HO

N H

46 α-arabino-α-arabino 47 β-ribo-β-ribo 48 α-arabino-β-ribo

HO

OH

54

HO OH

CONH2

HO

OH

55

N

HO

OH

OH

53

52

51 N

CONH2

HO

H N

NH

HO

HO

C8H17

HO

HO

N

HO

OH

H N

OH

HO

56

C8H17

57

Figure 14.8 Structure of deoxynojirimycin, MDL 25,673, and other iminosugar-based trehalase inhibitors (36–57). Table 14.6 Inhibition by deoxynojirimycin derivatives of membrane-bound trehalase from C. riparius larvae and trehalase from porcine kidney. Data represent relative activities and are expressed as a percentage of a control without inhibitors. Inhibitor (100 μM)

36 37 38 39 40 41 42 43

Trehalose concentration C.riparius

Porcine kidney

0.5 mM

2.5 mM

50 68 59 46 101 91 100 95

OH

OH

HO

HO

HO

50 H N

OH

H N

OH

OH

49

43

HO

O N

CH3

OH OH

44 X=NH 45 X=O

O

O

HO OH

OH HO

O

N

42

OH

MDL 25,673

O

HO

O CH3

X

OH

HO

HO

HO

OH

O

O

HO

39

38

HO

OH HO

OH

CH3 OH

O

N

O

HO

OH

41

OH

O

HO

O

HO

40 OH

HO

NH2 OH

OH

37

N

O CH3

OH

O

HO

NH

N

HO

OH

O

HO N

HO

OH

Deoxynojirimycin

O

HO

N H

N

CH3 HO

HO OH

HO

HO

O

O

HO

O

103 97 106 75 94 102 98 103

Trehalose mimics as bioactive compounds

357

Table 14.7 Inhibitory effect of deoxynojirimicin, MDL 25637, and compounds 44 and 49–57 on various trehalases. Compd. C. riparius

Porcine kidney

Porcine int.

Rat int.

Rabbit Baker’s M.s. S.l. kidney yeast

IC50 (μM) Ki (μM) IC50 (μM) Ki (μM) IC50 (μM)

IC50 (μM)

DNJ

2.83

1.39

5.96

2.98

26

74

40

27

420

87

MDL 25637

–

–

0.14

–

–

0.25

–

–

–

–

44 49 50 51 52 53 54 55 56 57

– 349 31 9.7 350 290 NI* NI* 277 NI*

– – – – – – – – – –

88 NI* 154 109 NI* NI* NI* NI* 537 NI*

44 – – – – – – – – –

– – – – – – – – – –

– – – – – – – – – –

– – – – – – – – – –

– – – – – – – – – –

– – – – – – – – – –

– – – – – – – – – –

Int., intestine; M.s., Mycobacterium smegmatis; S.l., Spodoptera litura. *No inhibition.

inhibitors of both porcine and insect trehalase. Compounds 50, 51, and 56 proved to be active against both enzymes with selectivity towards the insect glycosidase, while compounds 49, 52, and 53 behaved as inhibitors only of insect trehalase (Table 14.7). The most active and specific inhibitor was compound 51, characterized by a nojirimycin ring with a propyl group at C-1. Compared to lead 1-deoxynojirimycin, the presence of the propyl group in 51 causes a slight decrease of activity, but nevertheless imparting a ten-fold selectivity toward insect trehalase. In general, the collected data clearly indicate that the catalytic sites of trehalases from porcine kidney and insects have different recognition requirements, which can be exploited for the future design of specific inhibitors.

14.3 Trehalose-processing mycolyltransesterase enzymes Trehalose-based glycolipids are particularly abundant in the pathogenic bacterium M. tuberculosis: trehalose is found in the mycobacterial cell envelope as the glycolipids trehalose dimycolate (TDM, or cord factor) and trehalose monomycolate (TMM; Figure 14.9) (Hoffmann et al. 2008), together with other

358

Trehalose mimics as bioactive compounds OH

OH

O

O OH

O

HO

+

OH

O

HO

O

O

TMM

OH

HO

OH

HO

OH

HO

OH

HO TMM

RO

RO Ag85A, Ag85B, Ag85C OR

OH

O

O OH

O

HO

+

O TDM

OH

HO

OH

RO

HO

Trehalose

OH

O

HO

O HO

OH

HO

OH

HO

OH

O

R= 8

10-20 25-30

Figure 14.9 Ag85 catalyzed transesterification reaction.

glycolipids such as pentaacyl trehalose, triacyl trehalose, and sulfolipid-1 (SL-1). Tuberculosis is an infection that has plagued mankind for millennia and still remains a major worldwide health problem, causing almost 2 million deaths annually, mainly located in the developing world (World Health Organization 2008, www.who.int/tb/en/). TDM and TMM are based on a common trehalose core, acylated mainly with mycolic acids (Chatterjee 1997) that are long chain (C60–C90) cyclopropanated lipids; essential for M. tuberculosis, they are relevant for bacterial outer membrane structure, virulence, and persistence within the host (Barry and Mdluli 1996). Due to the relevance of SL-1 and TDM trehalose-based lipids to mycobacterial viability and virulence, several inhibitors of the enzymes involved in their biosynthesis have been designed over the years.

14.3.1 Mycolyltransesterase enzymes Trehalose is anchored into the mycobacterial cell wall as mono- or di-mycolates by the action of the extracellular proteins mycolyltransesterases Ag85A, Ag85B, and Ag85C (Figure 14.9). Mycolyltransesterases Ag85A, Ag85B, and Ag85C (i.e., antigen 85A, 85B, and 85C) are extracellular proteins showing immunogenicity, whose enzymatic activity was first described in the 1980s in a cell-free extract from Mycobacterium

Trehalose mimics as bioactive compounds

359

smegmatis catalyzing the synthesis of TDM (Kilburn et al. 1982). Despite wide studies on their immunologic and enzymatic activities, the Ag85 family remains scarcely understood. All Ag85 isoforms are able to perform the reversible transesterification between two units of TMM, affording TDM and free trehalose (Figure 14.9). The Ag85 enzymes have high structural and sequence homology (Anderson et al. 2001; Ronning et al. 2000, 2004), featuring a hydrophobic fibronectin-binding domain and an α,β-hydrolase fold. Active sites are highly conserved, possessing a classical catalytic triad made up of a histidine, aspartic acid, or glutamic acid and serine, a hydrophobic tunnel to accommodate the lipids and two trehalose binding sites. The presence of the catalytic triad suggests a reaction mechanism similar to that of serine hydrolases, in which the formation of a covalent acyl-enzyme (serine-mycolic acid) intermediate is attacked by the 6-hydroxyl of trehalose (Ronning et al. 2000), as reported in Figure 14.10A. Structural analysis of Ag85B complexed to trehalose highlighted that positions 4′ and 2 of the disaccharide are directed outward toward solvent, suggesting that these positions might tolerate substitutions. Indeed, the synthesis and assays of a carbohydrate library showed that Ag85 enzymes have exceptionally broad substrate specificity (Mbackus et al. 2011). This allowed exogenously added

HO

O (A)

HO TMM HO HO

O OH

OH

O

HO

S125

OH R

O

HO

(B)

O

O

Mycolic acid COOH

O Tetrahedral transition state HO HO

49

HO

O OH

O

OH

O

HN S

OH

HO

O -O

O

O

S125

Mycolic acid covalently attached to Ag85C

OH O

O

HO

O OH

O

OH OH

HO

O HO HO

OH TMM R

O

H2N

O

OH

HO

O R

R

O HO

OH HO

O HO

S125 +

S125

R HO

HO

O

HN

HO HO

OH HO

O

O

OH OH

50

OH

O O

HO O OH

O

OH

O TDM

OH R

O

HO HO HO

HO 51

OH

O

O F

OH OH

O

Figure 14.10 (A) Catalytic mechanism for Ag85 proteins; (B) Structure of FITC-trehalose and

other sample structures used in the reported study.

360

Trehalose mimics as bioactive compounds

synthetic probes to be specifically incorporated into M. tuberculosis growing in vitro and within macrophages. In general, the assays revealed a strong selectivity for trehalose-like disaccharides over monosaccharides, but a surprising substrate tolerance for all tested trehalose analogues. Alterations on every position of the sugar scaffold were tolerated, including C-1 methyl groups at the crowded anomeric linkages (i.e., FITC-trehalose 49, Figure 14.10B), positive charges, such as in the 2-amino-trehalose 50, and even stereochemically “incorrect” 2,2′-di-fluoroα,β-manno-trehalose 51. The fluorescein-containing trehalose probe 49 was incorporated by growing microorganisms, thereby originating fluorescent bacteria. Furthermore, addition of FITC-trehalose to M. tuberculosis allowed selective and sensitive detection within infected mammalian macrophages. These studies suggest that analogues of trehalose may prove useful as probes.

14.3.2 Mycolyltransesterase inhibitors Potential inhibitors of Ag85C enzymes have been synthesized and their activity against the protein and in cell culture was studied. A series of phosphonates and sulfonates was suggested as potential transition-state (Figure 14.10A) analogues of Ag85C. The inhibitors were designed with a phosphonate or sulfonate moiety mimicking the ester functionality, alkyl chains of different lengths mimicking the mycolic acid side chain, and substituted benzyl alcohols, N-(ϖ-hydroxyalky)phthalimide, 2-phenylethanol, or 4-(phthalimido)butanol as trehalose mimetics (Figure  14.11) (Gobec et al. 2004, 2007; Kovac et al. 2006). Also, simple alkylphosphonic acids were synthetized and assayed in vitro for their inhibition of recombinant M. tuberculosis antigen 85C mycolyltransferase activity (Table 14.8). Compounds containing phthalimido or 3-phenoxybenzyl moiety and lipophilic C4–C7 alkyl chain, linked together by an ethyl phosphonate or phosphonic acid moiety, resulted in the most active inhibitors (IC50 values in the low μM range for compounds 62, 70, and 75). Surprisingly, also alkylphosphonic acids 59 and 60 were very active, probably acting as nonspecific “promiscuous” inhibitors; in this series medium-size alkyl chains (C6 or C7) are preferred over shorter (C4) or longer (C9) ones. For all these analogs, substitution of the alkyl chain with a phenylethyl or phthalimido-butyl side chain caused inactivation or sensible decrease the inhibitory activity. Starting from a common 6,6’-dideoxytrehalose core, two sets of trehalose analogues with different modifications at the deoxy positions were also synthesized (Figure 14.12) and evaluated for their antimycobacterial activity against M. tuberculosis H37Ra, a panel of clinical isolates of M. avium, or  M.  smegmatis (Rose et al. 2002; Wang et al. 2004). 6,6’-Diamino-6,6’dideoxytrehalose 89 and its diazido precursor 88 were both inactive, but relevant activity was found among the sulfonamides, N-alkylamines, and the amidines.

O n

O

OEt

P

OH

n

P

n

O

OEt O

O

OEt O

68, n=1, x=1 69, n=1, x=2 70, n=4, x=1 71, n=6, x=1 72, n=6, x=2 73, n=11, x=1 O

n

P

O

O

P

O 83 O

82

P

N

O P

N

N

O P

N O

OEt O

84

O

O

85

O

O

O O OEt

O

OEt OH

O P

N O

86

Figure 14.11 A series of potential Ag85C inhibitors.

Table 14.8 Inhibitory activity of compounds 58–87 against Ag85C. Compound

Inhibitory activity IC50(μM)

Compound

Inhibitory activity IC50(μM)

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72

430.72 3.56 1.06 471.31 2.01 42.55 21.03 14.83 862.29 101.6 10.0 50.74 1.31 87.09 25.67

73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

40.99 4.39 1.47 NI* NI* NI* 4.3 NI* NI* NI* 348 NI* NI* 225 110

*No inhibition.

OEt O

O

O

OEt O

O

P

xN

80, x=1 81, x=2 O

O

O

OEt O

O

76, n=1 77, n=5

O

P

N

S n O O

xN

O O

O

O

OH

74, n=3, x=2 75, n=4, x=2

S n O O 78, n=1 79, n=5

OMe OMe

O

O

OMe

O

67, n=3

O xN

OEt

P

n

62, n=1 63, n=3 64, n=4 65, n=6 66, n=11

58, n=1 59, n=3 60, n=4 61, n=6 O

P

87

OEt OH

362

Trehalose mimics as bioactive compounds

R2

R1 O

O O

HO HO

88 R1 = R2 = N3 89 R1 = R2 = NH2 90 R1 = R2 = CH3(CH2)3SO2NH91 R1 = R2 = CH3(CH2)7SO2NH92 R1 = R2 = CH3(CH2)15SO2NH93 R1 = R2 = C6H5SO2NH94 R1 = R2 = [CH3(CH2)11]NH95 R1 = R2 = CH3(CH2)7NH-

OH

HO

OH OH

96 R1 = R2 = CH3(CH2)11NH97 R1 = R2 = CH3(CH2)17NH98 R1 = R2 = [CH3(CH2)3CH(Et)CH2]2N99 R1 = R2 = CH3(CH2)5NH100 R1 = R2 = CH3(CH2)9NH101 R1 = R2 = [CH3(CH2)3CH(Et)CH2NH102 R1 = CH3(CH2)3CH(Et)CHNH;R2 = OH 103 R1 = NH2;R2 = CH3(CH2)10C(NH)NH-

104 R1 = R2 = CH3(CH2)10C(NH)NH105 R1 = R2 = H2NC(NH)NH106 R1 = R2 = C8H17 107 R1 = HR2 = C8H17 108 R1 = R2 = NH(CO)NHC9H19 109 R1 = R2 = NH(CO)C9H19 110 R1 = R2 = CH2(CO)NHR 111 R1 = R2 = CH2(CO)NHNH(CO)C7H15

Figure 14.12 Structure of trehalose dimycolate analogues tested against several Mycobacterium

strains.

Some structure–activity relationships could be drawn from this library. The C8-alkylsulfonamido compound 91 was the most active compound, while the C16, C4, and phenyl analogues resulted as inactive; C8, C10, and C12 N-alkylamines (95, 96, and 100, respectively) showed good activity against the mycobacterial panel. The C6 mimetic 99 and the branched-C8 (2-ethylhexyl) 101 evidenced the broader activities against different mycobacteria strains. 6,6’-Bis(tertiaryamino) derivatives 94 and 98 were less active than their bis(secondary-amino) counterparts 96 and 101. Double substitution at both 6 and 6’ positions proved to be significant for activity, since monosubstitute derivative 102 resulted as inactive, while its 6,6’-disubstituted counterpart 101 was slightly active; the same trend was observed with the C12 bis(amidine) 104 and its monosubstituted counterpart 103. None of the synthesized dimycolate analogues presented strong activity; nevertheless, 6,6’-difunctionalized compounds were more active than monosubstituted derivatives.

14.4 Trehalose-processing sulfotransferase Since 2002, the biosynthesis of SL-1 has been deeply investigated (Kumar et al. 2007), the sulfation of trehalose being the first committed step mediated by a specific sulfotransferase (Figure 14.13) (Mougous et al. 2004). After sulfation, several proteins are involved in the acylation process, even if the biosynthetic order of these acylation reactions is not fully elucidated (Kumar et al. 2007). Sulfotransferases are best studied in eukaryotes, while little information is known about this class of enzymes in bacteria (Mougous et al. 2002a). Sulfotransferases (STs) catalyze the transferring of a sulfuryl group from a general donor, 3′-phosphoadenosine5′-phosphosulfate (PAPS; Figure 14.13), to different acceptors such as carbohydrates, proteins, and other low-molecular-weight molecules.

Trehalose mimics as bioactive compounds

OH

OH O

HO HO

O

HO HO

-O SO 3

StfO

HO O

T2S O OH

OH

Trehalose HO

O

OH

PAPS

HO

PAP

O

NH2

NH2 PAPS O

O

N O

N

O

S

P

O

OH

O -2O

N

PAP

N N

OH OH

OH

-O

363

-O

O

N

O

P

N N

O

OH 3PO

OH

-2O PO 3

OH

Figure 14.13 Mechanism of trehalose sulfation.

In particular, sulfotransferase O, SftO, was recognized as the enzymes catalyzing 2-O-sulfation of trehalose (Figure 14.13) (Mougous et al. 2004), the first committed step in the biosynthesis of SL-1. The enzyme has a rigorous substrate specificity, showing a marked decreasing activity upon minor structural modifications to trehalose. Gal(1,1-α,α)Glu, a synthetic epimer of trehalose, is sulfated 68-fold less efficiently than trehalose. Replacement of one glucose moiety of trehalose with a phenyl group, retaining the α-linkage (α-phenyl glucoside), reduced enzyme activity by 3,000-fold compared to the natural substrate, while no activity was reported with the β-linked isomer (β-phenyl glucoside), thus evidencing a strong preference for the α-glycoside. Free glucose and unnatural stereoisomers of trehalose, α,β (neo-trehalose) and β,β (iso-trehalose) were not sulfated by StfO. Many details of SL-1 biosynthesis still remain unclear (Kumar et al. 2007).

14.5 Acyl2SGL mimetics: Novel potential antitubercular vaccines Although the production of sulfated metabolites is common in bacteria (Mougous et al. 2002b), Mycobacterium tuberculosis has a particularly high production of SL-1 (Figure 14.14). It was found that high levels of this sulfoglycolipid are correlated to strain virulence (Goren et al. 1974). Due to its positioning on the outer envelope, SL-1 has been thought to be directly involved in host–pathogen interactions (Daffe and Draper 1998). Also, diacylated sulfoglycolipid (acyl2SGL; Figure  14.14) was recently identified (Gilleron et al. 2004) and resulted in characteristics of M. tuberculosis. It is characterized by a 2’sulfate-trehalose core, esterified in position 2 with a palmitic or stearic acid, and in position 3 with a hydroxyphthioceranoic moiety.

364

Trehalose mimics as bioactive compounds

OH

O

HO

OH

O

7

14

O

HO3SO O

O O

O

SL-1 (sulfolipid I)

14

O

O

7

14 OH OH

O O O

OH O O

5

OH

15

OH n = 2-9

O O

O O

O HO

SO-3Na+

acyl2SGL

OH OH

Figure 14.14 Chemical structure of sulfatide I (SL-1) and acyl2SGL.

Novel potential antitubercular vaccines were developed from acyl2SGL structure: a diacylated trehalose sulfate library (Figure 14.15) was prepared and all compounds were tested for their ability to activate the acyl2SGL-specific T cells (Guiard et al. 2008). Biological data showed that T-lymphocyte activation was highly dependent on the length, position, and structure of the fatty-acid residues linked to the trehalose core. In general, all active compounds possess a saturated or monounsaturated polymethylated fatty acid (S configuration) at the 3-position of the trehalose core. The length and the specific positions of the fatty acyl chains were crucial to immunogenicity. Shortening of the 2-O-acyl chain of the SGL from a palmitoyl (compound 122) to an octanoyl moiety (compound 124), decreased the activity. When the 3-O-acyl group was shortened from a trimethyltetracosanoyl chain to a trimethyloctanoyl chain (compounds 121 and 127, respectively), an even more pronounced effect on the activity was observed. Moreover, the introduction of unsaturation at the γ,δ-position of the 3-O-fatty acid (compound 128) and the use of a methylated fatty acid with R stereochemistry (compound 126) afforded inactive products. The protective effect of these synthetic sulfoglycolipids against tuberculosis infection deserves further investigation, and, in summary, the bacteriocidal nature of synthetic compounds against Mycobacterium may be relevant for the development of new therapies.

Trehalose mimics as bioactive compounds

HO

OH O

O

HO

O HO

O -O S 3

Compound acyl2SGL

365

OH O O

O O

R2

R1 R2

R1 palmitoyl /stearoyl

n = 2-9 n OH

112

palmitoyl

palmitoyl

113

palmitoyl

HO

114

palmitoyl

triacontanoyl

115

palmitoyl

116

palmitoyl

117

palmitoyl

118

palmitoyl

119

palmitoyl

120

palmitoyl

121

palmitoyl

122

palmitoyl

123

palmitoyl

124

octanoyl

125

tetracosanoyl

126

palmitoyl

127

palmitoyl

128

palmitoyl

O

O R/S O O O O O O O O O O O O O O

Figure 14.15 Structure of acyl2SGL analogues as potential antitubercular vaccines.

14.6 Conclusions Trehalose is a relevant biomolecule in several species, such as mycobacteria, fungi, and insects. The design of inhibitors of trehalose-processing enzymes can offer new perspectives as biocides and antitubercular therapies.

366

Trehalose mimics as bioactive compounds

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Jin, L.-Q. and Y.-G. Zheng. 2009. Inhibitory effects of validamycin compounds on the termites trehalase. Pestic. Biochem. Phys. 95:28. Kameda, Y., N. Asano, T. Yamaguchi, K. Matsui, S. Horii and H. Fukase. 1986. Validamycin G and validoxylamine G, new members of the validamycins. J. Antibiot. 39:1491. Kato, A., E. Kano, I. Adachi, R. J. Molyneux, A.A. Watson, R.J. Nash, G.W.J. Fleet, M.R. Wormald, H. Kizu, K. Ikeda and N. Asano. 2003. Australine and related alkaloids: easy structural confirmation by 13C NMR spectral data and biological activities. Tetrahedron: Asymm. 14:325. Kilburn, J.O., K.K. Takayama and E.E.L. Armstrong. 1982. Synthesis of trehalose dimycolate (cord factor) by a cell-free system of Mycobacterium smegmatis. Biochem. Biophys. Res. Commun. 108:132. Konno, K., H. Ono, M. Nakamura, K. Tateishi, C. Hirayama, Y. Tamura, M. Hattori, A. Koyama and K. Kohno. 2006. Mulberry latex rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars. P. Natl. Acad. Sci USA 103:1337-1341. Kovac, A., R.A. Wilson, G.S. Besra, M. Filipic, D. Kikel and S. Gobec. 2006. New lipophilic phthalimido- and 3-phenoxybenzyl sulfonates: inhibition of antigen 85C mycolyltransferase activity and cytotoxicity. J. Enzym. Inhib. Med. Ch. 21:391. Kumar, P., M.W. Schelle, M. Jain, F.L. Lin, C.J. Petzold, M.D. Leavell, J.A. Leary, J.S. Cox and C.R. Bertozzi. 2007. PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor sulfolipid-1. Proc. Natl. Acad. Sci. U.S.A. 104:11221. Kyosseva, S. V., Z. N. Kyossev and A. D. Elbein. 1995. Inhibitors of pig kidney trehalase. Arch. Biochem. Biophys. 316:821. Mbackus, K., H.L. Boshoff, C. Sbarry, O. Boutureira, M.K. Patel, F. D’Hooge, S.S. Lee, L. Evia, K. Tahlan, C. Ebarry and B.G. Davis. 2011. Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis. Nature Chem. Biol. 7:228. Molyneux, R. J., M. Benson, R. Y. Wong, J. E. Tropea and A. D. Elbein. 1988. Australine, a Novel Pyrrolizidine Alkaloid Glucosidase Inhibitor from Castanospermum australe. J. Nat. Prod. 51:1198. Mougous, J.D., C.J. Petzold, R.H. Senaratne, D.H. Lee, D.L. Akey, F.L. Lin, S.E. Munchel, M.R. Pratt, L.W. Riley, J.A. Leary, J.M. Berger and C.R. Bertozzi. 2004. Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis. Nature Struct. Mol. Biol. 11:721. Mougous, J.D., M. D. Leavell, R. H. Senaratne, C. D. Leigh, S. J. Williams, L. W. Riley, J. A. Leary and C. R. Bertozzi. 2002b. Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach. Proc. Natl. Acad. Sci. USA. 99:17037. Mougous, J.D., R.E. Green, S.J. Williams, S.E. Brenner and C.R. Bertozzi. 2002a. Sulfotransferases and sulfatases in mycobacteria. Chem. Biol. 9:767. Nash, R. J., L. E. Fellows, A. C. Plant, G. W. J. Fleet, A. E. Derome, P. D. Baird, M. P. Hegarty and A. M. Scofield. 1988a. Isolation from Castanospermum australe and x-ray crystal structure of 3,8-diepialexine, (1R,2R,3S,7S,8R)-3-hydroxymethyl-1,2,7-trihydroxypyrrolizidine [(2S,3R,4R,5S,6R)-2-hydroxymethyl-1-azabicyclo[3.3.0]octan-3,4,6-triol]. Tetrahedron 44: 5959. Nash, R. J., L. E. Fellows, J. V. Dring, G. W. J. Fleet, A. E. Derome, T. A. Hamor, A. M. Scofield and D. J. Watkin. 1988a. Isolation from Alexa leioptela and X-ray crystal structure of alexine, (1R, 2R, 3R, 7S, 8S)-3-hydroxymethyl-1,2,7-trihydroxypyrrolizine [(2R, 3R, 4R, 5S, 6S)-2hydroxymethyl-1-azabicyclo [3.3.0] octan-3,4,6-triol], a unique pyrrolizidine alkaloid. Tetrahedron Lett. 29:2487. Nash, R. J., L. E. Fellows, J. V. Dring, G. W. J. Fleet, A. Girdhar, N. G. Ramsden, J. M. Peach, M. P. Hegarty and A. M. Scofield. 1990. Two alexines [3-hydroxymethyl-1,2,7-trihydroxypyrrolizidines] from Castanospermum australe. Phytochem. 29:111.

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Nash, R. J., P. I. Thomas, R. D. Waigh, G. W. J. Fleet, M. R. Wormald, P. M. Lilley, Q. De and D. J. Watkin. 1994. Casuarine: A very highly oxygenated pyrrolizidine alkaloid. Tetrahedron Lett. 35:7849. Qian, X., Z. Liu, Z. Li and G. Song. 2001. Synthesis and quantitative structure-activity relationships of fluorine-containing 4,4-dihydroxylmethyl-2-aryliminooxazo(thiazo)lidines as trehalase inhibitors. J. Agric. Food Chem. 49:5279. Rhinehart, B. L. 1990. Castanospermine-glucosides as selective disaccharidase inhibitors. Biochem. Pharmacol. 39:1537. Rhinehart, B. L., K. M. Robinson, P. S. Liu, A. J. Payne, M. E. Wheatley and S. A. Wanger. 1987. Inhibition of intestinal disaccharidases and suppression of blood glucose by a new alphaglucohydrolase inhibitor--MDL 25,637. J. Pharmacol. Exp. Ther. 241:915–920. Robins, D. J. 1995. The Alkaloids: Chemistry and Pharmacology, Academic Press, New York, pp. 1–61. Ronning, D.R., T. Klabunde, G.S. Besra, V.D. Vissa, J.T. Belisle and J.C. Sacchettini. 2000. Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines. Nature Struct. Biol. 7:141. Ronning, D.R., V. Vissa, G.S. Besra, J.T. Belisle and J.C. Sacchettini. 2004. Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity. J. Biol. Chem. 279:36771. Rose, J.D., J.A. Maddry, R.N. Comber, W.J. Suling, L.N. Wilson and R.C. Reynolds. 2002. Synthesis and biological evaluation of trehalose analogs as potential inhibitors of mycobacterial cell wall biosynthesis. Carbohydr. Res. 337:105. Silva, M.C.P., W.R. Terra and C. Ferreira. 2004. The role of carboxyl, guanidine and imidazole groups in catalysis by a midgut trehalase purified from an insect larvae. Insect Biochem. Mol. Biol. 34:1089. Strom, A.R. and I. Kaasen. 1993. Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression. Mol. Microbiol. 8:205. Takayama, K. and E.L. Armstrong. 1976. Isolation, characterization, and function of 6-mycolyl6’-acetyltrehalose in the H37Ra strain of Myocobacterium tuberculosis. Biochemistry. 15:441. Thevelein, J. M. 1996. Regulation Of Trehalose Metabolism And Its Relevance To Cell Growth And Function, In The Mycota, ed.by Brambl R, Marzluf GA. Springer, Berlin Heidelberg New York, pp 395–414. Thompson, S. N. 2003. Trehalose - the insect “blood” sugar. Adv. Insect. Physiol. 31:206. Uchida, C., H. Kitahashi, S. Watanabe and S. Ogawa. 1995b. Synthesis of trehazolin analogues containing modified sugar moieties. J. Chem. Soc. Perkin Trans. 1. 1707. Uchida, C., H. Kitahashi, T. Yamagishi, Y. Iwaisaki and S. Ogawa. 1994a. Synthesis of trehazolin analogues containing modified aminocyclitol moieties. J. Chem. Soc. Perkin Trans. 1. 2775. Uchida, C., T. Yamagishi and S. Ogawa. 1994b. Total synthesis of the trehalase inhibitors trehalostatin and trehazolin, and of their diastereoisomers. Final structural confirmation of the inhibitor. J. Chem. Soc. Perkin Trans. 1. 589. Uchida, C., T. Yamagishi, H. Kitahashi, Y. Iwaisaki and S. Ogawa. 1995a. Further chemical modification of trehalase inhibitor trehazolin: structure and inhibitory-activity relationship of the inhibitor. Bioorg. Med. Chem. 3:1605. Wang, J., B. Elchert, Y. Hui, J.Y. Takemoto, M. Bensaci, J. Wennergren, H. Chang, R. Raia and C.-W. T. Chang. 2004. Synthesis of trehalose-based compounds and their inhibitory activities against Mycobacterium smegmatis. Bioorg. Med. Chem. 12:6397. Wegener, G., V. Tschiedel, P. Schlöder and O. Ando. 2003. The toxic and lethal effects of the trehalase inhibitor trehazolin in locusts are caused by hypoglycaemia. J. Exp. Biol. 206:1233. World Health Organization. 2008. Global Tuberculosis Control Report. Wrobel, J. T.1985. The Alkaloids: Chemistry and Pharmacology. Academic Press, New York, pp. 327–385.

CHAPTER 15

Virtual screening and prediction of the molecular mechanism of bioactive compounds in silico Bashir A. Akhoon1, Krishna P. Singh1, Madhumita Karmakar1, Suchi Smita2,4, Rakesh Pandey3, and Shailendra K. Gupta1,4 1

Department of Bioinformatics, CSIR – Indian Institute of Toxicology Research, Lucknow, India Society for Biological Research and Rural Development, Lucknow, India 3 CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow, India 4 Department of Systems Biology and Bioinformatics, University of Rostock, Rostock, Germany 2

15.1 Introduction The term virtual screening (VS) was coined in the late 1990s to describe the use of computational algorithms for the identification of novel bioactive molecules. Virtual screening is defined as the search for bioactive molecules within a database of interest that matches a given query structure. The query structure may be a pharmacophore, ligand, or receptor structure. A variety of bioactive compounds have been used to understand the functional role of biomolecules (Harding et al. 1989; Liu et al. 1991; Flanagan et al. 1991; Nishi et al. 1994; Kudo et al. 1999; Yoshida et al. 1990; Whitesell et al. 1994; Stebbins et al. 1997; Prodromou et al. 1997). To develop new chemical probes of biological systems, identification of the target proteins of bioactive compounds is the preliminary requisite. Among various methods to elucidate biomolecule function, VS is notable where small molecular compounds are used as probes to elucidate the function of the molecule (protein or nucleic acid) and many virtual screening tools have proved their applicability (McInnes 2007). VS can be an alternative approach or complement to high-throughput screening (HTS) to narrow down the range of molecules to be tested in vitro and in vivo, which in turn can greatly reduce the economical investment in chemical synthesis and/or preliminary testing. Even it allows the in silico screening of compounds, which are physically not available and can be synthesized on demand. VS can be broadly divided into two categories, namely ligand-based and structure-based.

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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15.2 Ligand-based virtual screening (LBVS) LBVS methods are based on the fact that ligands similar to an active ligand are more likely to be active than random ligands and identifies the new ligands just by evaluating the similarity between candidate ligands and known active compounds. This type of screening uses the information present in known active ligands rather than the structure of a target protein for lead identification and such methods are only chosen when no three-dimensional structure of the target protein is available. LBVS methods can be divided into three major classes: Similarity searching including two-dimensional fingerprints. This method is based on the principle that structurally related molecules should have similar properties. Hence, databases having structurally similar structures with unknown biological activity are likely to contain some compounds with activity of interest. Pharmacophore methods. A set of structural features in a ligand or a threedimensional arrangement of molecular features/fragments that are directly related to the ligand’s recognition at a receptor site and its biological activity is known as a pharmacophore. Such methods identify the pharmacophore pattern common to a set of known active compounds and subsequently make usage of this pattern in a three-dimensional (3D) substructure search. Machine learning methods. These methods depend on the training set data that contain known active and inactive molecules. Model goodness depends on the size of data used to train the model, the value you would like to predict, the input parameters, etc. (Eckert and Bajorath 2007).

15.3 Structure-based virtual screening (SBVS) SBVS involves the identification of a potential ligand-binding site on the target molecule and in this approach knowledge about the 3D structures of the target proteins is essential to perform in silico high-throughput screening. The determination of the structure of a target protein is carried out by NMR, X-ray crystallography, or homology modelling. Homology modelling refers to constructing an atomic-resolution model of the “target” protein from its amino acid sequence by in silico searching for relatively one or more experimentally resolved 3D structures as templates. Since 3D structure of a target protein acts as an initializing staircase in SBVS, the modelling of such targets should be properly handled. Due to an ever increasing number of structures resolved and stored in the protein data bank (PDB) and other databases, SBVS studies have remarkably increased. However, to achieve the success of the SBVS process, users should be able to make proper selection of the docking procedure. In general, all docking programs make prediction of ligand orientation and conformation into the active site,

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373

Citation index

AutoDock DOCK GOLD Glide FlexX FRED LigandFit CDOCKER Surflex-Dock ParaDockS Molegro Virtual Docker Fleksy Hex

Figure 15.1 Citation index of various docking software (data collected until February 2013). For color detail, please see color plate section.

followed by a measure of its fitness into the chosen binding site. Usually, the docking algorithms are divided into three main categories: Systematic methods. These methods explore all degrees of freedom in a receptor molecule to place ligands into the desired active site and involve incremental construction, conformational search, Hammerhead algorithm, etc. Random or stochastic methods. Such methods include Monte Carlo and genetic algorithms (GAs). While the former method generates several ligand configurations into the protein binding site and then scores the configurations in a multistep procedure, the latter one takes advantage of the principles of biological competition and population dynamics to place the ligand in an active site. Simulation methods (molecular dynamics). These methods are based on Newton’s equation of motion for an atomic system where atomic forces and masses are used to determine atomic positions over a series of very small time steps and forces between the particles and potential energy are defined by molecular mechanic force fields. Some of the popular docking programs using the above searching methods are FlexX (incremental construction approaches), GOLD and AutoDock (genetic algorithms), Glide (systematic incremental search techniques), DOCK (shapebased algorithms), and LigandFit (Monte Carlo simulations). Figure 15.1 shows the citation index of various routinely used docking tools.

15.3.1 Computational tools for virtual screening With the continuous improvement in computationally intensive algorithms and advances in computer hardware, various docking programs have been developed for high-throughput virtual screening (HTVS) and inverse virtual screening (IVS)

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of molecular interactions. HTVS involves docking many ligands against one or a few receptors, while IVS docks many receptors against one or a few ligands. VS programs are a useful adjunct to the time-consuming and expensive wet bench experiments. Some open-source or low-cost VS computer programs are even used at institutional and nonprofit budgets to discover new therapeutic candidates. Table 15.1a–c summarizes some of the most popular VS software, Web resources, and commonly screened chemical databases, respectively. The criterion to select Table 15.1a Commonly used docking programs for virtual screening. Software

Search strategy

Docking

Source/ Free Availability for academia

AutoDock

Lamarckian genetic algorithm (LGA) Geometric hashing algorithm Genetic algorithm (GA)

Rigid/flexible

Yes

http://autodock.scripps.edu

Rigid

Yes

http://dock.compbio.ucsf.edu

Rigid/flexible

No

Monte Carlo simulated annealing algorithm Fragment-based algorithm Chemgauss4 scoring function Monte Carlo conformational search Genetic algorithms (GAs) Monte Carlo and Genetic algorithms (GAs) Genetic algorithm

Flexible

No

Flexible

No

Rigid/flexible

Yes

Rigid/flexible

No

http://www.ccdc.cam.ac.uk/ products/life_sciences/gold/ http://www.schrodinger.com/ productpage/14/5/ http://www.biosolveit.de/flexx/ index.html?ct=1 http://www.eyesopen.com/ oedocking http://accelrys.com

Rigid/flexible Rigid/flexible

No No

http://www.chemcomp.com http://accelrys.com

Rigid/flexible

No

Surflex

Using the Hammerhead scoring function

Flexible

No

ICM

Monte Carlo minimization procedure Optimization algorithm MolDock algorithm

Flexible

No

Rigid Rigid

Yes No

Flexible

No

http://www.vlifesciences.com/ products/VLifeMDS/VLifeDock.php http://www.tripos.com/index. php?family=modules,Simple Page,,,&page=Surflex_Dock http://www.molsoft.com/ docking.html http://www.paradocks.org http://www.molegro.com/ mvd-product.php http://www.cmbi.ru.nl/software/ fleksy/

Rigid

Yes

http://hex.loria.fr

Rigid

Yes

Flexible

Yes

http://www.bioinformatics. ku.edu/files/vakser/gramm/ http://www.nmr.chem.uu.nl/ haddock/

DOCK GOLD Glide FlexX FRED LigandFit MOE CDocker VLifeDock

ParaDockS Molegro Virtual Docker Fleksy Comprises an ensemblebased soft-docking using FlexX-Ensemble Hex Parametric docking and superposition algorithms GRAMM Fast Fourier Transform (FFT) algorithm HADDOCK CPORT algorithm

Virtual screening and prediction of the molecular mechanism

375

Table 15.1b Important Web resources for virtual screening. Resource

Availability

SwissDock DockingServer 1-Click Docking

http://www.swissdock.ch http://www.dockingserver.com/web https://mcule.com/apps/1-click-docking/?utm_source=ccl&utm_medium=maillist& utm_campaign=1-click-docking http://blaster.docking.org https://mcule.com/subscribe/packages/ http://docking.utmb.edu http://www.scfbio-iitd.res.in/dock/pardock.jsp http://flexpepdock.furmanlab.cs.huji.ac.il/ http://bioinfo3d.cs.tau.ac.il/PatchDock/ http://medock.csbb.ntu.edu.tw http://zhanglab.ccmb.med.umich.edu/BSP-SLIM/ http://www.biodrugscreen.org http://genome.jouy.inra.fr/GPCRautomdl/cgi-bin/welcome.pl http://abcis.cbs.cnrs.fr/LIGBASE_SERV_WEB/PHP/kindock.php http://iscreen.cmu.edu.tw http://idtarget.rcas.sinica.edu.tw http://dock.bioinfo.pl

Blaster Mcule docking Docking At UTMB Pardock FlexPepDock PatchDock MEDock BSP-SLIM BioDrugScreen GPCRautomodel kinDOCK iScreen idTarget MetaDock

Table 15.1c Databases commonly used for screening by the virtual screening programs. Database

Type

No. of compounds

Website

PubChem ChEMBL NCI Set ChemSpider TCM ZINC ChemBridge Specs Asinex Enamine Maybridge WOMBAT ChemNavigator ACD

Public Public Public Public Public Public Commercial Commercial Commercial Commercial Commercial Commercial Commercial Commercial

>8,000,000 structures 1,376,469 structures >140,000 structures >28,000,000 structures 37,170 compounds >21,000,000 compounds >900,000 compounds >1,500,000 compounds >600,000 compounds >1,640,000 compounds >56,000 molecules 331,872 compounds >91,500,000 compounds >7,000,000 compounds

MDDR

Commercial

>150,000 compounds

http://pubchem.ncbi.nlm.nih.gov https://www.ebi.ac.uk/chembldb/index.php http://dtp.nci.nih.gov/index.html http://www.chemspider.com http://tcm.cmu.edu.tw http://zinc.docking.org http://www.chembridge.com http://www.specs.net http://www.asinex.com http://www.enamine.net http://www.maybridge.com http://www.sunsetmolecular.com http://www.chemnavigator.com http://accelrys.com/products/databases/ sourcing/available-chemicals-directory.html http://accelrys.com/products/databases/ bioactivity/mddr.html

a particular VS program depends on the desired scientific objective, available computer hardware access, and financial resources. Some successful virtual screening case studies are highlighted in the Table 15.2.

rDock GOLD

DOCK

Chk1 DPP-IV

ICM

280,000 compounds

CATS, TOPAS

EGFR kinase

∼ 700,000 drugs 500,000 compounds

FlexX-Pharm Ligand Scout

Chk1 kinase Acetyl cholinesterase (AChE) Cannoboid Receptor (CB-1) Hsp90 thymidine monophosphate kinase SARS coronavirus

World Drug Index Database (WDI) rDock FlexX-Pharm

1,381 compounds

Maybridge database

AMPA receptor

Aromatase (CYP19)

∼ 200,000 compounds > 110,000 compounds

DOCK

CK2 Kinase

∼ 700,000 drugs 10,000 fragment-sized compounds ∼315,000 compounds

20,000 compounds

14,000 compounds

∼ 400,000 compounds

634 compounds

CONAN

Thrombin

No. of compounds screened

Softwares

Targets

Table 15.2 Some successful stories of virtual screening.

Identification of novel lead series of EGFR kinase inhibitor

Discovery of novel noncovalent inhibitors of SARS coronavirus Constitutes a successful target for the treatment of breast cancer Identification of 10 diverse inhibitors of Chk1 Identification of novel S1-binding fragments

Shape-feature-based computational method is used to rapidly filter compound libraries Compounds docked into the ATP binding site of a human CK2α homology model For synthesis and identification of potential ligands for lead optimization processes Identification of four novel classes of inhibitors Showed significant, dose-dependent and long-lasting inhibitory activities Assembly of molecular fragments and topological pharmacophore similarity measure New class of inhibitor with low to submicromolar potency To find new antitubercular leads

Comments

Cavasotto et al. 2006

Foloppe et al. 2006 Rummey et al. 2006

Schuster et al. 2006.

Liu et al. 2005

Barril et al. 2005 Gopalakrishnan et al. 2005

Rogers-Evans et al. 2004

Lyne et al. 2004 Rollinger et al. 2004

Barreca et al. 2003

Vangrevelinghe et al. 2003

Srinivasan et al. 2002

References

644 compounds 5,000,000 compounds 17 compounds 24,245 compounds

FlexX

ZINC database

AutoDock

COPICAT

NEDD8-activating enzyme (NAE)

Histone deacetylases (HDAC) arylamine N-acetyltransferase NAD+-DNA ligase PIK3CG, PARP1, and ACACA

ZINC database

∼44,000 compounds

GPCR CCR5

90,000 compounds

65 compounds

AutoDock, FlexX, and GOLD and multiple scoring functions GOLD and Surflex

CYP2D6

No. of compounds screened

Softwares

Targets

Identification of novel nonpeptide ligands for the GPCR CCR5 Focused virtual libraries in hit finding and lead optimization of HDAC inhibitors Alternative lead discovery approaches such as fragment-based screening Can be used as antibacterial drug candidate for the prevention of a wide range of bacterial diseases Protocol useful for combining in silico screening and experimental evidences for identifying target proteins of small molecules. Explained the structure-based drug discovery strategy for NAE inhibitors in silico.

Procedure for metabolic site prediction of CYP2D6 substrates

Comments

Zhong et al. 2012

Kobayashi et al. 2012

Akhoon et al. 2011

Schneider 2010

Price et al. 2007

Kellenberger et al. 2007

de Graaf et al. 2006

References

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Virtual screening and prediction of the molecular mechanism

15.3.2 Integrating virtual screening and Quantitative Structure Activity Relationship (QSAR) method QSAR method is based on machine learning and statistical approaches to develop quantitative relationships between chemical descriptors and biological activities of compounds to be tested experimentally. The predictive QSAR models can be helpful in screening large virtual libraries to find out putatively active compounds and prioritize them for experimental testing. These strategies shift the traditional focus of QSAR modelling from obtaining statistically significant training set models in the direction of exploiting validated models to prioritize chemicals for subsequent biological evaluation. QSAR modelling is very beneficial to fulfill the ultimate needs of experimental medicinal chemists to discover novel bioactive compounds. There are various reports in the literature on QSAR studies but only a few authors have used QSAR models for VS. One such example is the study carried out by Mahmoudi and coworkers (2006) where they attempted to use the QSAR model generated by using 395 compounds tested against Plasmodium falciparam (training set). The model was used to screen 2,000 compounds from the Merk Index, and 22 compounds were selected and evaluated in vitro. Finally, they have identified six new compounds with antimalarial activities at nanomolar concentration. The above example clearly shows the integration of QSAR in VS.

15.3.3 General steps involved in QSAR studies Step 1. Dataset of Molecules: A library of chemical compounds of interest margin with known biological activity like half maximum inhibitory concentration (IC50) or reproducible potency (EC50) can be used for model development and validation. Such activity values can be retrieved from various databases such as PubChem’s BioAssay. Step 2. Descriptor Calculation: In a common aspect, descriptors are numerical representations of specific molecular features. Numerous software packages are available to calculate a wide variety of descriptors for various chemical structures. Examples include Dragon, JOELib, and ADAPT. The descriptors can be mainly classified into four categories: geometrical, topological, electronic, and hybrid. Geometric descriptors can be described by shape and 3D coordinates. Before these descriptors can be calculated, the geometry of the structure must be optimized. Some geometric descriptors are moment of inertia, molecular surface area and volumes, and shadow descriptors. Topological descriptors include the topology of molecules. These descriptors describe features such as path lengths and connectivity. Examples include connectivity indices, distance edge vectors, and eccentricity indices. Electronic descriptors include various features related to the electronic environment. Major descriptors of this group include HOMO and LUMO energies, electronegativity, and various atom-centered partial charge descriptors.

Virtual screening and prediction of the molecular mechanism

379

Hybrid descriptors are usually combinations of both electronic or topological descriptors and geometric descriptors. Examples include the charged partial surface area (CPSA), hydrophobic surface area (HPSA), and hydrogen bonding descriptors. Step 3. QSAR Set Generation: The generation of QSAR sets is an important step in the modelling process. For the chosen dataset of molecules, three mutually exclusive sets are usually generated. The first set, known to be a training set, is mainly used for model-building. The learning algorithm used to build the model uses this set to characterize the dataset based on features present in the training set. The second set is the cross-validation set and is used in the case of linear regression models. The second set is mainly used to allow the monitoring of the error rate during training. If the model is linear, then the training set and cross-validation set are combined together. At last, the prediction set is a subset of the dataset, which is not used for modelbuilding. All sets are collectively known to be QSAR sets. When the features that are present in the training set are not adequately presented in the prediction set, then the model predictive capability will be poor. In a broader sense, the QSAR set should be produced in such a way that the various features present in the dataset should be proportionally represented in each individual QSAR set. The most common way to design the cross-validation dataset is leave-one-out (LOO). Step 4. Model Development: After developing a good QSAR set we have to reduce the original descriptor data to a more convenient size and then choose a number of optimal descriptor subsets so that we can then proceed to build a set of models and choose the best one. Usually the QSAR, which relates variations in biological activity to variations in the values of computed (or measured) properties for a series of molecules, takes the form of a linear equation: Biological Activity = Const + (c1×P1) + (c2×P2) + (c3×P3) +…….. + (cn×Pn) where the parameters P1 through Pn are computed for each molecule in the series and the coefficients c1 through cn are calculated by fitting variations in the parameters and the biological activity. The excellence of any QSAR will be totally dependent on the quality of the data, which is used to derive the model. The quantification of QSAR data is totally dependent upon the correlation coefficient (r). Correlation coefficient measures how strongly the observed data tracks the fitted regression line. If any kind of error has taken place in the model or in the data, it will lead to a bad fit. This indicator of fit to the regression line is calculated as:

r2 = Regression Variance/Original Variance Where the Regression Variance is calculated as “Original Variance” minus “Variance” around the regression line. The Original Variance is the sum-of-thesquares distances of the original data from the mean.

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The value for r2 falls between 0 and 1. When r2 becomes 0, it means there is no relationship between activity and the parameter(s) selected for the study and r2 equal to 1 means there is a perfect correlation among them. The genetic function approximation (GFA) algorithm (Rogers and Hopfinger, 1994) offers a new way for the problem of building a QSAR and a Quantitative Structure Property Relationship (QSPR) models. In a comparative study, they implemented GFA algorithm that allows the construction of models competitive with, or superior to. Those produced by standard techniques and allowed users to build models with higher-order polynomials, splines and gaussians in addition to linear polynomials models. Step 5. Prediction of Activity Using QSAR Model Developed: After completion of model-building, the final step is to investigate the predictive ability of the build model (i.e., to examine the model on a subset of the dataset that has not been used during the model development process [the prediction set or test set]). The statistical results that come from the prediction set can give us some indication of the model’s predictive ability. The general statistics used for linear models are r2.

15.3.4 Pitfalls in virtual screening Although VS has been established as a powerful alternative to HTS, VS is not yet a fully mature technology. Undoubtedly, many virtual screening tools have proved their applicability; there are certain issues that cut down the accuracy of the VS programs. For example, missing or limited structural information, poor scoring functions, imprecise understanding of the properties of drug-like molecules, inability to map 3D properties onto 2D structures, incorrect assessment of existing SAR data, and poor docking strategies create major hurdles in the VS process. The pitfalls of VS can be divided into four categories: (1) those concerning erroneous assumptions and expectations; (2) those concerning data design and content; (3) those relating to the choice of software; and (4) those concerning conformational sampling as well as ligand and target flexibility. The detailed descriptions of all these problems are elaborated in the article published by Scior and coworkers (2012). The careful consideration in these areas would certainly advance the application of VS programs. The user is always in doubt whether there are some methods that perform better than others and if yes, in what particular situations they are well suited. Furthermore, of the multitude of settings, parameters, and datasets in the software that the practitioner can choose from, VS becomes much more erroneous if the user is lacking the background knowledge of the VS resource. To avoid the pitfalls by making the user aware of published and unpublished problems, we would recommend that the user read the shortcomings, failures, and technical traps of VS methods highlighted by Scior and coworkers (2012).

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15.4 Reverse docking approach for predicting biological activity of compounds Several docking programs (Table 15.1) have been used to find ligands that recognize the 3D structure of a given target obtained by X-ray crystallography, NMR spectroscopy, or even by homology modelling. Virtual screening methods focus on searching chemical space for chemicals that can specifically bind to a protein target. But in the real world there may exist unknown off-target proteins that can bind to the ligand molecules unexpectedly, raising severe side effects. At the same time, it would be useful to find unknown therapeutic uses in ligand repositioning. Identification and validation of ligand targets from among thousands of candidate macromolecules is still a challenging task (Totrov and Abagyan 2008; Rao et al. 2009). Thus, there is a need for an efficient computational method to find putative binding proteins for a given compound from either genomic or protein databases, and subsequently to use various experimental procedures to validate such computational results. The computational approach that involves docking search against a protein database instead of a drug molecular database is known as reverse docking. In many cases, targets not annotated as ligand ability or not stored in a target database previously may exhibit high binding fitness and hence are highly likely to be real binding targets. Therefore, reverse docking approaches are receiving increasing interest to find unknown targets of natural products and existing drugs (Sotriffer et al. 2008; Cai et al. 2006; Scior et al. 2012). Some popular reverse docking tools are TarFisDock and PharmMapper. We can design a series of programming scripts that will invoke docking software core code such as AutoDock to combine the docking procedure with a target protein database like a protein drug target database (PDTD), which contains thousands of protein entries with known 3D structures and will dock a small molecule (ligand) to all proteins in the database one by one automatically. The lowest docking energy score and other criteria such as inhibition constant (Ki) can be used to assess screening results.

15.4.1 Case study of reverse docking approach The reverse docking methodology can be applied in various ways. Here we present two case studies where the reverse docking approach was used to identify the mechanistic insight of biological activities of artemisinic acid and D-pinitol.

15.5 Predicting molecular targets responsible for antiaging properties of artemisinic acid Step 1. Chemical Structure of Artemisinic Acid: The 3D structure of artemisinic acid (PubChem ID: CID 10922465), as shown in Figure 15.2, was retrieved using the PubChem database (http://pubchem.ncbi.nlm.nih.gov) available at the National Center for Biotechnology Information (NCBI).

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H32

H33 C12

H27 H30 H29

C11 H26

H20 C2

C9

H22

C3

C7

C4

C8 H25

H36 C13 H35

C15

H34

C10

H28

C6

01 9

H24

C14

C16

H37 C17

02 H38

Figure 15.2 Three-dimensional structure of artemisinic acid.

Step 2. Molecular Targets of Artemisinic Acid: The chemical structure of artemisinic acid was submitted to PharmMapper (http://59.78.96.61/ pharmmapper/submit_file.php), an open web server used for finding potential molecular targets for given small molecules like drugs, natural products, or compounds with unidentified binding sites using the pharmacophore mapping approach. PharmMapper uses a genetic algorithm to optimize the pharmacophore mapped poses. The server needs just a chemical structure either in Mol2 or SDF format as its input. Users can provide the email address along with the job description and can upload the desired chemical structure. Here, we submitted the artemisinic acid structure retrieved from PubChem as our input. For easy understanding of the server usage, the home page of the server has been shown in Figure 15.3. After structure uploading, users will be prompted to the next page where various parameters can be adjusted as per the requirement of the user. The various available parameters in the server are shown in Figure 15.4.

15.5.1 Basic options available in PharmMapper Generate Conformers. PharmMapper adopts a semi-rigid pharmacophore mapping protocol resulting in multiple conformations of the query molecule. The user can generate a conformer ensemble either online or offline. Cyndi is used as default option to generate conformers if the parameter is set to “TRUE.” Maximum conformations to be generated in Cyndi are set as 300 by default.

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Figure 15.3 Homepage of PharmMapper showing the required input data fields for the server.

Figure 15.4 The choice of parameter options available for target fishing using PharmMapper.

Select Targets Set. Currently there are 7,302 pharmacophore models in PharmTargetDB and 2,241 of them are annotated as the potential protein targets in humans. Due to the limited size of human protein pdb subsets, more pharmacophore models from other origins (like other mammals, prokaryotes, and eukaryotes) with sequential homology to corresponding human proteins are also enclosed. All the 7,473 models are included in the target identification by default, but the users can also switch to “Human Targets Only” if they want to focus only on current available human protein targets. Number of Reserved Matched Targets. Here the user can fix the maximum number of top ranked potential target candidates to be reserved in the “Get Result” page (the default is 300 targets).

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15.6 Advanced options Users can also optimize the input parameters in the advanced options dialog by fixing various cut-offs like fit value cutoff, vector angle cutoff, weight of hydrophobic score (hydrogen bond donor, hydrogen bond acceptor, positive charged center, negative charged center, aromatic ring), and minimum number of hydrophobic center (hydrogen bond donor, hydrogen bond acceptor, positive charged center, negative charged center, aromatic ring). Here in our case the maximum number of reserved matched targets was kept as a default and only the human protein dataset was selected as a targets for searching potential human targets for artemisinic acid. After uploading the mol2/sdf file, press the “Submit” button to set the desired parameters of PharmMapper. After clicking on the “Submit” button, users will be automatically directed to the result page, as shown in Figure 15.5. PharmMapper output page (Figure 15.6) displays the potential human target candidates ranked in descending order on the basis of PharmMapper’s fit score for artemisinic acid. The calculated fit score evaluates the pairwise alignment between the ligand pose and pharmacophore model. By clicking on the ‘+’ symbol of the first column of each row provides information about the pharmacophore model and visualization of ligand-pharmacohore superposition presented in the drop-down subwindows.

Figure 15.5 Snapshot of Result page showing various potential human targets (ranked by Fit Score), along with their PDB IDs.

Virtual screening and prediction of the molecular mechanism

GLY125

385

SER38

PHE126 VAL124 Artemisinic Acid

GLY123

ALA40

LYS100 ASP42 ASNS H-Bonds Donor

MET86

Acceptor

Figure 15.6 Artemisinic acid docked in the active pocket of the heat shock protein 90. The active

site residues are labelled and the surface is colored as per H-bond donor and acceptor residues. For color detail, please see color plate section.

Step 3. Sorting of Antiaging Targets in the Human Aging Genomic Resources (HAGR) Database: The PDB IDs of the top 100 protein targets of artemisinic acid were converted to their respective SwissProt IDs using UniProt ID Mapper/converter and such targets were searched in HAGR (http://genomics. senescence.info/) for their antiaging activity. HAGR is a freeware Web-based research database for obtaining aging-related gene information using several bioinformatics approaches such as functional genomics, network analyses, systems biology, and evolutionary analyses. The database features important resources such as (1) GenAge: a manually curated database consisting of human genes; (2) AnAge: a major curated database for studying aging and longevity in animals; (3) GenDR: a database containing genetic manipulations induced by dietary restriction for lifespan extension and constant gene expression changes. The SwissProt IDs of human targets generated from the PharmMapper server were manually matched with all the human aging genes datasets available at the GenAge HAGR database for antiaging target fishing. We noticed that among 100 potential targets of artemisinic acid, only 11 human genes were involved in the aging process and such targets are listed in Table 15.3. In the HAGR model system, homologues aging genes in lower organisms can also be identified. Here we tried to identify the human homologues in C. elegans for all the 11 genes and observed eight aging genes in C. elegans that were homologues to human. Figure 15.6 shows the docking orientation of artemisinic acid in C. elegans heat shock protein 90 (PDB ID: 4GQT) involved in aging process.

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Virtual screening and prediction of the molecular mechanism

Table 15.3 Antiaging targets of artemisinic acid. Protein name

hAGRID

hGNC symbol

PDB ID

Fit Score

C. elegans homologs

Tyrosine-protein phosphatase non-receptor type 1 Heat shock protein HSP 90-alpha Insulin receptor Mitogen-activated protein kinase 14 Glutathione S-transferase P 3-phosphoinositidedependent protein kinase 1 Cyclin-A2 Glycogen synthase kinase-3 beta Proto-oncogene tyrosine-protein kinase ABL1 Tyrosine-protein kinase JAK2 Androgen receptor

33

PTPN1

1JF7

4.099

No

74

HSP90AA1

1UY8

3.911

daf-21

42 168

INSR MAPK14

2AUH 1KV1

3.791 3.772

157

GSTP1

18GS

3.753

87

PDPK1

1OKZ

3.709

daf-2 MAPK14: pmk-1 (P38 Map Kinase family) GSTP1: gst-23 (Glutathione S-Transferase) PDPK1: pdk-1 (PDK-class protein kinase)

177 97

CCNA2 GSK3B

1PKD 1Q3D

3.605 3.577

78

ABL1

2HZI

3.52

215

JAK2

2B7A

3.508

No

110

AR

3B67

3.433

No

CCNA2: ZK507.6 (ZK507.6) GSK3B: gsk-3 (Glycogen Synthase Kinase family) ABL1: abl-1 (related to oncogene ABL)

15.7 Predicting molecular targets of D-pinitol responsible for antioxidant activity in a C. elegans model using computational approaches D-pinitol, an alicyclic polyalcohol is a naturally occurring compound and is found in various plants, trees, and foods such as soy. It is a methyl-inositol extract that promotes the transport of glucose and glycogen synthesis. The immense medicinal potential of D-pinitol is mainly due to its antioxidative (Govindarajan et al. 2003), antidiabetic (Govindarajan et al. 2007), anti-inflammatory, antifertility (Rathi et al. 2004), antiulcer (Dharmani et al. 2005), and analgesic (Lai et al. 2009) properties, besides possessing insulin-like effects, via driving creatine and other nutrients into muscle cells. The molecular insight of antioxidative property shown by D-pinitol is still not investigated in detail. Here, we present several bioinformatics resources to predict the molecular targets of D-pinitol, which reveal the molecular mechanism of its antioxidant activity previously assessed by several researchers in the C. elegans model.

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387

15.7.1 Identification of molecular targets for D-pinitol The reverse docking approach was used to predict the molecular targets of D-pinitol using the Potential Drug Target Database (PDTD) (Gao et al. 2008). PDTD is a web-accessible database that currently contains 1,207 proteins covering 841 known and potential drug targets with structures in the Protein Data Bank (Berman et al. 2000). PDTD provides quick screening of potential binding proteins for any drug-like molecule using the TarFisDock server (Li et al. 2006), which works on a reverse ligand-protein docking approach (Gupta et al. 2011; Chen et al. 2001; Paul et al. 2004; Muller et al. 2006). The TarFisDock server tries to dock the ligand into an already known active site of protein using a PatchDock algorithm (Schneidman-Duhovny et al. 2003) where the surfaces of receptor and ligand are first divided into patches and then further use a geometric hashing algorithm to indentify maximum surface shape complementary while minimizing the number of steric clashes. The server generates the target list in the increasing order of interaction energy (Eint), which is collectively expressed with the van der Waals and electrostatic interaction: lig rec

Eint i 1 j 1

Aij

Bij

a ij

b ij

r

r

332.0

qi q j Drij

where i and j are the atoms in ligand and receptor, rij, is the distance between the receptor and ligand atom, Aij, Bij and a, b are the parameters and exponent of van der Waals repulsion and attraction, q is the charges on the atoms, D is dielectric function, and conversion factor 332 converts the electrostatic energies into kcal/mol.

15.7.2 Gene ontology (GO) analysis of the targeted proteins In order to predict the molecular processes, biological functions, and metabolic pathways associated with the proteins targeted by D-pinitol, we performed gene ontology analysis using the PANTHER server (Protein ANalysis THrough Evolutionary Relationship; www.pantherdb.org). For this, PDB IDs of targeted proteins were converted into UniProt IDs using a ID Mapper/Converter tool available on UniProt server (www.uniprot.org/mapping/) and uploaded to a Panther Version 8.0 database for the assignment of GO terms. All the proteins that are involved in the oxygen and reactive oxygen species metabolic processes were filtered and analyzed for the mechanism of antioxidative activities shown by D-pinitol.

15.7.3 Identification of protein homologues in C. elegans To validate the antioxidant property of D-pinitol in vivo, we used the C. elegans model system. For all the probable proteins responsible for antioxidant property of D-pinitol, we searched the homologs in C. elegans using the BLAST/BLAT search tool available on WormBase (www.wormbase.org/tools/blast_blat).

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Virtual screening and prediction of the molecular mechanism

Immune system process (3)

Cell adhesion (1)

Cell cycle (19)

Cell communication (73)

Metabolic process (89) Cellular process (74)

Generation of precursor metabolites and energy (2)

Developmental process (43)

Response to stimulus (3)

System process (16) Reproduction (4)

Transport (13)

Cellular component organization (1)

Apoptosis (7)

Figure 15.7 Pie chart showing various biological processes that might get affected by D-pinitol treatment. Number below each biological process represents total number of potential enzymes involved in respective biological processes and targeted by D-pinitol in C. elegans. For color detail, please see color plate section.

Analysis of the top 10% of proteins (106 proteins) returned by the TarFisDock server was carried out to find the potential molecular targets for D-pinitol. To identify the targets for antioxidant activity, screening of only those proteins were performed, which are involved in oxidative stress-related processes. This was achieved by mapping the GO terms using the PANTHER server. Figure  15.7 highlights various biological processes governed by potential enzymes targeted by D-pinitol. In total, six important enzymes were identified as potential targets for D-pinitol and were involved in oxidative stress-related processes (Table 15.4). For all six enzymes mentioned in Table 15.4, amino acid sequences were downloaded from UniProt and a BLAST/BLAT search was performed using a sequence alignment tool available on WormBase. Table  15.5 highlights all the potential homologues from C. elegans for all the enzymes targeted by D-pinitol in Table 15.4. The interaction of D-pinitol with GSTP is shown in Figure 15.8. A total of five hydrogen interactions can be seen in the figure, with SER65, ASP98, and GLN51 amino acid residues of GSTP in the binding cavity.

Virtual screening and prediction of the molecular mechanism

389

Table 15.4 Enzymes involved in oxidative stress-related process and targeted by D-pinitol. S. No.

Enzyme targeted

UniProt ID

PDB ID

Class

1 2 3 4 5 6

Glutathione S-transferase P Nitric oxide synthase Glutathione S-transferase A3 Glutathione S-transferase theta-2 Mitogen-activated protein kinase 10 Signal transducer and activator of transcription 1-alpha/beta

GSTP1_HUMAN NOS2_HUMAN GSTA3_HUMAN GSTT2_HUMAN MK10_HUMAN STAT1_HUMAN

18GS 4NOS 1TDI 3LJR 1JNK 1YVL

Transferase Oxidoreductase Transferase Transferase Transferase Signalling protein

Table 15.5 Blast result from C. elegans protein database with the enzymes targeted by D-pinitol. Enzyme targeted by D-pinitol

homologous C. elegans enzyme (WormBase id)

E-value

Glutathione S-transferase P Nitric oxide synthase

GST-1 (WP:CE00302) EMB-8 (WP:CE02018) GST-37 (WP:CE21504) GST-31 (WP:CE22421) JNK-1, isoform a (WP:CE27574) STA-1, isoform C (WP:CE22342)

Glutathione S-transferase A3 Glutathione S-transferase theta-2 Mitogen-activated protein kinase 10 Signal transducer and activator of transcription 1-alpha/beta

% Identities

% Similarity

1e-42 2e-52

39 26

58 43

2e-18

29

50

1e-3

24

42

1e-157

69

83

2e-35

26

45

Previous studies have already reported the upregulation of the glutathione S-transferase gene in C. elegans during oxidative stress conditions (Tawe et al. 1998; Leiers et al. 2003). Another protein nitric oxide synthase has similarity with EMB-8 protein (i.e., NADPH-cytochrome P450 reductase associated with increased production of ROS). Similarly, D-pinitol also targets the JNK-1 isoform a in C. elegans that promotes DAF-16 translocation into the nucleus in the stress condition, which in turn activates several genes to increase stress resistance (Gami and Wolkow 2006). Oxidative stress also activates STAT1, which is one of the important signalling molecules that transmit signals from the cell surface in response to several cytokines/growth factors (Osuka et al. 2010). Thus computational data clearly indicate that D-pinitol will show the antioxidative activities in the C. elegans model system as most of the stress-related enzymes targeted by D-pinitol have homologues in C. elegans. Identification of novel compounds with antioxidative property in multiple species are highly desirable, both as a tool for further research along with potential

390

Virtual screening and prediction of the molecular mechanism

Gln51

Tyr7 Leu52

Tyr49 Pro53

Tyr63 Arg13

lle10

Gln64 Lys102 Ser65 Asp98 lle65 Cys101 Asp94

Asn66 Arg100

Glu97

Figure 15.8 Interaction of D-pinitol with GSTP, generally upregulated in the stress conditions. Amino acid residues in the binding cavity are labelled. Surface is generated around D-pinitol and colored on the basis of hydrogen bond donor/acceptor. Donors are colored in green and receptor acceptors in cyan. Hydrogen bonds are shown with dotted green lines. For color detail, please see color plate section.

therapeutic avenues for ROS related diseases. The present case study provides use of various computational protocols to understand the mechanism and targets responsible for the antioxidant property of D-pinitol. These protocols may be further generalized to screen other therapeutic compounds with antioxidant properties.

15.8 Future directions Here we address some key problems that need to be tackled for successful prediction of bioactive compounds by VS. Although we need improvement to optimize the accuracy of rapid quantitative predictions of one-to one interactions between a ligand and a single target, future virtual screening should account for many-to-many relationships. The static view of both targets and ligands should be replaced by dynamic descriptions of molecules and molecular dynamics simulations can help us to sample conformational ensembles of targets and ligands. While it might be a potential approach to identify chemotypes for which

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391

receptor–ligand complex formation is dominated by enthalpy changes rather than entropy changes, for accuracy enhancement, the predictions should come from both enthalpic and entropic contributions. For prioritization of the various suggested ligand poses, much progress has been made in the improvement of scoring functions but such functions are still grossly inaccurate (Totrov and Abagyan 2008; Rao et al. 2009; Sotriffer et al. 2008). Therefore, there is an emerging need for the development of new scoring functions to evaluate the fitness between the docked compound and the target. After treatment of the aforementioned problems, the accuracy of future predictions of novel bioactive compounds by VS would be significantly improved that could provide a major boost to the realization of the full potential of virtual screening. However, although virtual screening through docking is one of the important computational tools for the preliminary identification of lead molecules, docking methods concern the application of scoring functions that largely fail to estimate ligand binding energies in reasonable agreement with experiments. Docking techniques still lack reliable simulation of the flexibility of both ligands and receptor and inclusion of water molecules that play an important role in the complex formation are ignored during the docking process. Hence, MM/PBSA or MM/GBSA, a popular method for calculating binding affinities of biomolecular complexes, combined with the molecular dynamics simulations, can be used to rescore the docked complex to remove false-positives obtained from the docking methods and to calculate the binding free energy of the ligands with good accuracy. This approach will act as a post-docking filter in further enriching our virtual screening results. Furthermore, free energies will be decomposed into insightful interaction and desolvation components.

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CHAPTER 16

Steroids in natural matrices: Chemical features and bioactive properties João C.M. Barreira and Isabel C.F.R. Ferreira Centro de Investigação de Montanha (CIMO), ESA, Instituto Politécnico de Bragança, Bragança, Portugal

16.1 Introduction Steroids share the same fixed three-dimensional framework (Figure 16.1). The early history of steroids derives almost exclusively from two well-known compounds: cholesterol (I) and cholic acid (II) (Figure 16.2). These molecules are available in large quantities from natural sources, explaining why they were the first steroids to be obtained in pure crystalline form (Lednicer 2010). Many of their chemical properties are determined by the steric properties of the nucleus, which incorporates over half a dozen chiral centers, not counting the side chain. Cholesterol, for example, can in theory consist of no fewer than 512 stereo isomers. However, this compound occurs as a single chiral species, as do virtually all other steroid-based products (Lednicer 2010). Structural features are also relevant in determining the biological activity (estrogens, for instance, are characterized as having an aromatic A ring) and the reaction sequence used during the preparation of potential drugs based on steroids (Lednicer 2010). Sterols (from the Greek word steros, meaning “solid”) are special forms of steroids, with a hydroxyl group at C3 and a cholestane (Figure  16.3) backbone (Fragkaki et al. 2009) that can be found in animals, plants, fungi, and microorganisms (Bernal et al. 2011). In this chapter, sterols are referred by their common (trivial) names. To be acquainted with alternative names, systematic names, molecular masses, and CAS (Chemical Abstracts Service) registry numbers, the excellent review conducted by Moreau et al. (2002) may be helpful. Sterols derive from hydroxylated polycyclic isopentenoids having a 1,2-cyclopentanophenanthrene structure (Abidi 2001) and comprise the largest proportion of the unsaponifiable fraction of lipids (Lagarda et al. 2006). These compounds have a wide range of biological activities and physical properties.

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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18 12

17

11 13

19 1

C

9

8

2 A 3

10

D 14

16

15

B 7

5 4

Figure 16.1 Basic steroid structure.

6

O

OH OH

H H H H

H

H OH

HO HO

H (I)

(II)

Figure 16.2 Chemical structures of cholesterol (I) and cholic acid (II).

H H

H (III)

Figure 16.3 Chemical structure of cholestane (III).

Plant sterols (i.e., phytosterols) (Figure  16.4), in particular, are important for the health and nutrition industries (Abidi 2001). Cholesterol (Figure  16.1) is the most well-known animal sterol, but other common steroids found in animals include testosterone (X), epitestosterone (XI), progesterone (XII), and estradiol (XIII) (Figure 16.5). Three distinct classes of sterols—estrogens, progestins, and androgens (differing from each other in both biological activity and structure)—are among the chemical substances commonly known as sex hormones, which direct the reproductive function in mammals (Lednicer 2010).

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H

H

H H HO

H

HO

H

H

H

HO

(IV)

(V)

(VI)

H H H

H

H H

H

HO

H

H

HO

H

HO

(VII)

(VIII)

(IX)

Figure 16.4 Examples of structures of sterols ubiquitous in plants: (IV) 4,4-dimethylsterol (cycloartenol); (V) 4-methylsterol (obtusifoliol); (VI) 4-desmethylsterol (campesterol); (VII) stigmasterol; (VIII) β-sitosterol; (IX) ∆5-avenasterol. O OH H

OH

OH

H H

O

H

H

H

(X)

H

H

(XI)

H

H

H

HO

O

O

H

(XII)

(XIII)

Figure 16.5 Examples of structures of sterols commonly found in animals: (X) testosterone; (XI) epitestosterone; (XII) progesterone; (XIII) estradiol.

On the other hand, steroids are commonly associated with androgenicanabolic compounds (mainly analogues of testosterone) due to the frequent exposés of the usage of these drugs by athletes seeking to enhance their performance. The androgens in question, however, comprise only a single, relatively small class of biologically active steroids. What may be called athletic androgens is in reality overshadowed by a large diversity of compounds sharing the same tetracyclic nucleus (Lednicer 2010). In fact, a large number of therapeutic drugs also possess steroidal skeleton in their respective chemical structure (Figure 16.6). The administration of exogenous steroids produces positive effects including muscle growth, appetite stimulation, increased red blood cell production, and bone density (Bhasin et al. 1996; Frisoli et al. 2005). In medical therapy, steroids have been used in treating several illnesses, including topical diseases, inflammation, anemia, neoplasia, rebuilding of muscles after a debilitating disease, and osteoporosis in postmenopausal women (Basaria et al. 2001).

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O HO

O OH

O

O

HO

O

O

HO H F

H H

F

H

O

O (XIV)

(XV)

Figure 16.6 Chemical structures of dexamethasone (XIV) and betamethasone (XV).

However, natural steroidal hormones such as testosterone (X), progesterone (XII), and their synthetic analogs have been found to cause neurobiological, developmental, reproductive, and immunological effects, as well as genotoxicity and carcinogenicity. Furthermore, the continuous exposure to corticosteroids may lead to long-term consequences (ocular medical disorder such as cataracts, ocular hypertension, and open-angle glaucoma) on the health of susceptible individuals (Yadav et al. 2012).

16.2 Structure and classification The basic skeleton of a steroid possesses four fused rings, out of which three are cyclohexane and one is a cyclopentane. In most steroids, methyl groups are present at C10 and C13 and an alkyl chain (or substituted alkyl chain) may also be present at C17. In many steroids, functional groups like OH, CHO, CO, or COOH may also be attached to the ring or present in the alkyl chain (Yadav et al. 2012). Steroid nomenclature is puzzling because it lacks complete international standardization. Herein, the nomenclature (Figure  16.7) recommended by IUPAC-IUB (Moss 1989) was adopted. The four rings are commonly denoted by the capital letters A, B, C, and D reading from left to right. The ring letter designation is used only in discussion sections for publications, since they have no role in formal nomenclature (Lednicer 2010). Systematic names for steroids are based on a set of hypothetical hydrocarbons (Figure 16.8). Omitting the methyl group 19 at C10, for example, affords gonanes (XVI), more commonly known as 19-nor steroids. Estranes (XVII) comprise an important part of oral contraceptives. Androstanes (XVIII), the compounds that support male reproductive function, include a methyl group at C10. The other so-called sex hormones, the pregnanes (XIX), retain the intact 19-carbon atom nucleus and in addition support a two-carbon side chain at position 17. Glucocorticoids, like cortisol (XX), best known for their anti-inflammatory activity, are also named as derivatives of pregnane. The nucleus depicts the most

Steroids in natural matrices: Chemical features and bioactive properties

399

242 21 241

22 18 12 11 19 1 9

2

HO

A 4

3

10

C 8

17

13 14

B

D

24 25

20 23

26

16 27

15

7

5 6

29

28

Figure 16.7 Nomenclature for phytosterols according to IUPAC-IUB (Moss, 1989).

H H

H H

H

H

(XVI)

H

H

H

H

H

H

(XVIII)

(XVII)

O HO

H

H

H H

OH

HO

H

H

H

O (XIX)

(XX)

Figure 16.8 Chemical structures of some representative steroids: (XVI) gonane; (XVII) estrane; (XVIII) androstane; (XIX) pregnane; (XX) cortisol.

generalized structure that serves as the base for molecules with larger side chains at C17, such as cholesterol and ergosterol and their derivatives (Lednicer 2010). In nature, sterols can be found as free sterols or as four types of conjugates in which the 3-β-hydroxyl group is esterified to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty acyl hexose (Lagarda et al. 2006). These particular steroids contain a total of 27–30 carbon atoms (the number of carbon atoms in the biosynthetic precursor squalene oxide) in which a side chain with at least seven carbon atoms is attached at C17 (Abidi, 2001). Sterols can be described and organized in three groups based on the number of methyl groups at C4: two (4-dimethyl), one (4-monomethyl), or none (4-desmethyl). 4-Dimethylsterols and 4α-monomethylsterols are metabolic intermediates in the biosynthetic pathway leading to 4-desmethyl phytosterols, but they are usually present at low levels in most plant tissues (Moreau et al. 2002).

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16.3 Bioavailability of steroids In plant as in animal cells, the plasma membrane is greatly enriched in sterols relative to other cell membranes. Through interaction with phospholipids in a 1:2 stoichiometry, sterols condense the bilayer, reduce bulk fluidity and permeability, and broaden or eliminate phospholipid phase transitions. Sterol–phospholipid interactions influence membrane functions such as simple diffusion, carriermediated diffusion, and active transport, and also modulate the activities of membrane-bound enzymes or receptors (Moreau et al. 2002). While cholesterol is present in animals in relatively high abundance, plants, with very few exceptions, produce negligible amounts of this compound. Reported phytosterol data for some plant foods and vegetable oils have shown that nuts and oils contain higher levels (≥1%) of sterols than fruits and vegetables ( HPLC > SFE. The sensitivity order may vary depending on the sterol structures and detectors coupled to the

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chromatographic instruments. GC-FID (or MS, when peak identity confirmation is needed) can be considered the method of choice for the determination of phytosterols in foods and diets (Lagarda et al. 2006). In fact, GC-FID is the most commonly used method to quantitate sterols in various sample matrices due to the large linear mass range of response of the system. All quantitation requires statistical method validation in the context of reproducible retention times, precision, recovery studies with spike samples, and absolute response factors. Losses of sterol analytes during isolation and separation must be corrected for the final results of analyte quantification by using internal standardization with standards not present in samples or radioisotopes. Evaluation of the repeatability of the best extraction efficiency can further reduce analytical errors in estimating sterols in assay samples (Abidi 2001). After extraction, additional sterols are sometimes liberated by subsequent acid or alkaline hydrolysis, suggesting that there may be pools of “bound” or “evasive” sterols in sample tissues. Additional research is necessary to provide a better understanding of the optimal extraction methods that are required to ensure complete sterol extraction. Hydrolysis methods should be optimized, and enzymatic methods may prove to be superior to acidic and/or alkaline hydrolyses (Moreau et al. 2002). As compared to GC, quantitative analysis of sterols by HPLC is somewhat limited. Using dose–response calibration curves, HPLC-PDA is useful for quantitation of specific sterols (Abidi 2001). It should also be noted that each matrix is unique, and conditions have to be selected to optimize the accuracy/yield of all forms of sterols in different sample types. On the other hand, faster procedures must be developed to facilitate the control of functional foods with added phytosterols (Lagarda et al. 2006). The identification of sterols might be enabled by developing methodologies such as APCI-MS, electrospray ionization, MALDI-TOF, or 13C NMR. The hyphenations of LC-MS and other separation and analytical techniques have been explored in efforts to improve the speed and efficiency of discovery of novel drugs from natural sources (Wu et al. 2013). In terms of biological activity, additional research is mandatory in order to understand the underlying mechanisms that support inflammatory response regulation, anti-hypercholesterolemic, antitumoral, and antioxidant activities. The development of nonsteroidal selective androgen receptor modulators (SARMs) may offer better dissociation of biological effects when compared to anabolic steroids. Furthermore, it may allow the therapeutic targeting of specific tissues and organs (Kicman 2008). Age-associated changes in systemic sex steroid levels have profound effects on human health. It is now clear that the abrupt cessation of ovarian estrogen biosynthesis at menopause impacts greatly upon systemic markers of inflammation, inflammation associated with acute skin wounds, and the incidence and progression of specific inflammatory conditions. Notably, estrogens are

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antiatherosclerotic in females, while androgens fulfill a similar role in males. At the cellular level, estrogens and androgens influence such processes as chemotaxis and activation, as well as the secretion of inflammatory cytokines and adhesive interactions between inflammatory cells and the endothelium (Gilliver 2010).

Acknowledgments The authors are grateful to CIMO (Strategic Project No. PEst-OE/AGR/ UI0690/2011). João C.M. Barreira also thanks Fundação para a Ciência e a Tecnologia, POPH-QREN, and FSE for his grant (No. SFRH/BPD/72802/2010.

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CHAPTER 17

Bioactive compounds obtained through biotechnology Gustavo Molina1,2, Franciele M. Pelissari2, Marina G. Pessoa1, and Gláucia M. Pastore1 1

Laboratory of Bioflavors, Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, Brazil 2 Institute of Science and Technology Food Engineering, UFVJM, Diamantina, Brazil

17.1 Introduction Modern trends have drawn attention to the role that diet plays in human health. Indeed, several epidemiological studies have indicated that high intake of plant products helps to reduce the risk of a number of chronic diseases, such as atherosclerosis and cancer (Gosslau and Chen 2004; Gundgaard et al. 2003). Economic reasons and time constraints have led the drug industry to rely heavily on synthetic products. However, many of the currently available medications cause problems; for example, human pathogenic bacteria, fungi, and viruses have developed drug resistance. Therefore, finding new therapeutic agents to effectively treat diseases in humans, plants, and animals is desirable (Strobel and Daisy 2003; Strobel 2003; Zhang et al. 2005). In this sense, some microorganisms that produce bioactive natural compounds have emerged as a potentially useful alternative to resolve the issues related to drug safety and human health (Strobel et al. 2004). Methods to obtain bioactive substances include extraction from a natural source, chemical synthesis, and microbial production via fermentation. The former method poses disadvantages: it depends on seasonal, climatic, and political features and may culminate in ecological problems. Hence, developing innovative strategies to obtain bioactive compounds is essential (Bicas et al. 2009). Because bioactive compounds find potential application in the food, chemical, and pharmaceutical industries, the interest in designing bioprocesses to produce or extract these substances from natural sources has increased (Martins et al. 2011). A relatively recent review by Newman and Cragg (2007) listed all the agents approved from 1981 to 2006. A significant number of natural drugs that microbes and/or endophytes synthesize appeared on the list. Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Biotechnological techniques that use different microorganisms are promising ways to establish inexhaustible, cost-effective, and renewable sources of highvalue bioactive compounds. In this context, this chapter focuses on the role that microorganisms play in bioactive compound synthesis, paying particular attention to antioxidant, anticancer, and antimicrobial agents. This chapter shows why it is important to include microbes during drug screening; it also presents the biotechnological process as an innovative approach to obtain novel compounds. Additionally, this chapter describes bioactive compounds on the basis of their different actions, including examples that illustrate their potential use in humans. Figures 17.1 to 17.3 contain the structures of some bioactive compounds that microorganisms can generate.

17.2 Antioxidant compounds An antioxidant compound is any substance that can delay, prevent, or remove oxidative damage to a target molecule even when the antioxidant is present at lower concentration than the oxidizable substrate (Halliwell 2007). Antioxidants act in various ways: they can (1) prevent free lipid radical formation and thus inhibit free radical oxidation reactions (preventive oxidants); (2) interrupt autoxidation chain reaction propagation (chain-breaking antioxidants); (3) quench singlet oxygen; (4) operate synergistically with other antioxidants; and (5) convert hydroperoxides into stable compounds (Carocho and Ferreira 2013). Compounds bearing antioxidant activity are extremely important—they effectively prevent the harm that reactive oxygen species (ROSs) and oxygenderived free radicals elicit, avoiding countless pathological effects such as DNA damage, carcinogenesis, and cellular degeneration (Huang et al. 2007; Seifried et al. 2007). In addition, they may help to prevent and treat ROS-linked diseases such as cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/ reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer and Parkinson diseases), rheumatoid arthritis, and aging (Valko et  al. 2007). Many antioxidant compounds possess anti-inflammatory, antiatherosclerotic, antitumor, antimutagenic, anticarcinogenic, antibacterial, and antiviral activities in higher or lower levels (Owen et  al. 2000; Cozma 2004; Halliwell 1994). Interestingly, many of the beneficial effects that diet has on human health result from compounds displaying antioxidant activity. For example, vegetables contain important antioxidants, mainly vitamins C and E, carotenoids, and phenolic compounds, in their composition (Krinsky 2001; Shi et al. 2001). The food industry has employed antioxidants as food additives to prevent lipid peroxidation. Although synthetic antioxidants have found wide application in food processing, researchers have reassessed them to detect possible toxic and carcinogenic components that originate during their degradation (Maeura et al. 1984; Ito et  al. 1985). These health concerns have led to the hypothesis that

Bioactive compounds obtained through biotechnology

HO

OH

O

OMe

435

OH

OH MeO OH O OH

O

O

O

HO OH Isopestacin

HO

Graphislactone A

OH

O

O

Candidusin B O

O O

O

O N

O

O

O

O O

O

N H Carbazomycin

N

Ascorbic acid

Benthocyanin

β-Carotene

Figure 17.1 Structure of some antioxidant compounds obtained through biotechnology.

O

O O

O O

O O

O

O

O

H O

O O

H OO H O

O O

O

O

O N

O O

N

N O

O

O

H

O

Podophyllotoxin

Taxol

9-Methoxycamptothecin

N N

O N

N

O

O

O O

Tubericidin

H

O

P

O

O

O

H O O

H

O Ergosterol

Mevastatin

Figure 17.2 Structure of some anticancer agents obtained through biotechnology.

natural antioxidants can potentially replace their synthetic counterparts. The only issue is that natural compounds usually cost more and have inferior effect. Thus, finding safer and more effective and inexpensive natural antioxidants have become highly desirable. In this scenario, microbial sources appear as a potential source to obtain natural antioxidants (Ishikawa 1992).

O O O

O

α-Phellandrene oxide

β-Maaliene

Phenyllactic acid

O

O

N O S N N

O N

O

N O

N

N

O

S N

N N O

N

O

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Figure 17.3 Structure of some antimicrobial compounds obtained through biotechnology.

Erythromycin C

O

Bioactive compounds obtained through biotechnology

437

In the following sections we present antioxidant compounds obtained through biotechnology and discuss the processes and microorganisms involved in their biosynthesis.

17.2.1 Fungal metabolites Fungal endophytes represent a promising source of antioxidants (Pimentel et al. 2011). Some examples include Pestacin and Isopestacin (Figure 17.1) generation by Pestalotiopsis microspore, an endophytic fungus isolated from the plant Terminalia morobensis (Papua New Guinea) (Strobel et al. 2002; Harper et al. 2003). Pestacin is believed to have 11 times greater antioxidant activity than Trolox, a vitamin E derivative; this activity primarily derives from cleavage of an unusually reactive C–H bond and, to a lesser extent, from O–H abstraction (Harper et al. 2003). As for isopestacin, it is structurally similar to flavonoids and exerts its antioxidant action by scavenging both superoxide and hydroxyl radicals that are free in the medium. Besides acting as antioxidants, pestacin and isopestacin exhibit antimycotic and antifungal activities, respectively (Strobel et al. 2002). Liu and coworkers (2007) evaluated the antioxidant activity of an endophytic Xylaria sp. isolated from the medicinal plant Ginkgo biloba. The methanol extract exhibited strong antioxidant capacity; “phenolic” and “flavonoid” compounds existed among the 41 identified compounds. Huang and coworkers (2007) investigated the antioxidant action of endophytic fungal cultures of medicinal Chinese plants and its correlation with the total phenolic content. These authors suggested that the phenolic content accounted for the antioxidant activity of the endophytes. The phenolic metabolite called Graphislactone A (Figure 17.1) isolated from the endophytic fungus Cephalosporium sp. IFB-E001 residing in Trachelospermum jasminoides displayed stronger in vitro free radical scavenging and antioxidant activities than the standards butylated hydroxytoluene (BHT) and ascorbic acid, co-assayed in the study (Song et al. 2005). One study assessed antioxidant compounds production from Aspergillus candidus 3153 d in a 5-L fermentor (Yen and Chang 2003). The authors observed higher and similar inhibition of ethyl acetate broth extract peroxidation (IP% =/93%) as compared with α-tocopherol and BHA at 200 mg.mL–1, respectively. The researchers isolated and identified the antioxidant components as 3,3ƒ-dihydroxyterphenyllin, (3,3ƒ-DHT), 3-hydroxyterphenyllin (3HT), and candidusin B (CANB) (Figure 17.1). These antioxidants were not cyto/genotoxic toward human intestine 407 (Int 407) cells or mutagenic toward Salmonella typhimurium TA98 and TA100 (Yen et al. 2001). In previous studies, the same authors optimized fungal extract production (Yen and Lee 1996). They used various model systems to assess activity and found that fungi could be a potential antioxidant in food products (Yen and Lee, 1997). Some fungal strains can generate exopolysaccharides with useful biological activities (Wang and Luo 2007; Xu et  al. 2009; Kimura et  al. 2006; Du et  al. 2009) including antioxidant action (Sun et al. 2009; Guo et al. 2010, 2013). One

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example is the Aspergillus versicolor LCJ-5-4 exopolysaccharide (EPS) (Chen et al. 2012). Certain fungal strains synthesize intracellular polysaccharides (IPS), for example, a submerged culture of Pholiota adiposa SX-02 (Deng et al. 2011). Chen et al. (2011) characterized a homogeneous EPS extracted from a mangrove endophytic fungus Aspergillus sp. Y16; this EPS, designated As1-1, consisted mainly of mannose and small amounts of galactose (molecular weight about 15 kDa). These same authors used scavenging assays involving 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals to evaluate the in vitro antioxidant activity of As1-1; they demonstrated that this compound could be a potential antioxidant and food supplement. Some microorganisms can also ferment low-cost substrates. Through solidstate fermentation (SSF), these microorganisms can enhance the properties of a given substrate and, consequently, its biological activity (Nigam 2009). Table 17.1 presents some examples of microorganisms capable of fermenting low-cost substrates to enhance their properties or to obtain novel compounds with biological potential. Some examples include the use of pomegranate husks together with Aspergillus niger GH1 (Aguilar et  al. 2008) as well as the by-product cranberry pomace as substrates to obtain ellagic acid and other phenolic compounds (Vattem and Shetty 2003; Zheng and Shetty 2000). Fermentation can also increase the phenolic compounds content in certain food products, thereby improving their antioxidant activity (Martins et  al. 2011). Yao et  al. (2010) proved that black soybeans fermented with Table 17.1 Enhancement of the properties and biological activity of low-cost substrates through fermentation processes. Microorganism

Substract

Product

Technique*

Reference

Lentinus edodes

Cranberry pomace Black soybeans Beans

Ellagic acid

DPPH; β-carotene oxidation DPPH; FRAP DPPH; Fe2+ chelating activity DPPH; ABTS

Vattem and Shetty, 2003 Yao et al. 2010 Lee et al. (2008)

Aspergillus sp. Aspergillus awamori Rhizopus oligosporus Aspergillus oryzae Trichoderma harzianum Phanerochaete chrysosporium Rhizopus oryzae

Peas seeds Wheat grains Soybean Green coconut husk Powder of Teri pod cover

Phenolic content Antioxidant potential Phenolic Content Phenolic content

DPPH; ABTS

Starzynska-Janiszewska et al. 2008 Bhanja et al. 2009

Phenolic acids and flavonoids Vanillin

Several

Singh et al. 2010

-

Barbosa et al. 2008

Gallic acid

-

Kar et al. 1999

*Antioxidant activities using the 1,1-diphenyl-2-picrylhydrazine (DPPH), ABTS*(+) [2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] and the ferric-reducing antioxidant power (FRAP) assays.

Bioactive compounds obtained through biotechnology

439

microorganisms, such as Aspergillus sp., at 30°C for 3 days yielded significant antioxidant levels as compared with nonfermented black soybeans [Glycine max (L.) Merrill]. These authors assessed the antioxidant content, including total phenolics and total flavonoids, and the antioxidant activities using the 1,1-diphenyl-2-picrylhydrazine (DPPH) and the ferric-reducing antioxidant power (FRAP) assays. The results revealed that fermented black soybean preparations exhibited antioxidant activities, attributed to the total phenolic and flavonoid content (Yao et al. 2010). Lee et al. (2008) employed Aspergillus sp. and Rhizopus sp. to boost the antioxidant properties of beans used to produce koji. The antioxidant activity of the fermented beans might have resulted from increased phenol and anthocyanin contents. Wheat grains also benefited from fermentation with Aspergillus oryzae and Aspergillus awamori—these microorganisms increased the grains phenolic content and antioxidant properties (Bhanja et al. 2009). Similarly, fermentation with Cordyceps sinensis improved the chemical composition and bioactivity of stale rice (Zhang et al. 2008). Apart from generating new molecules in conventional media, these fermentation processes could potentially convert inexpensive agroindustrial residues into a variety of high-value compounds, including bioactive phenolic compounds with antioxidant potential (Martins et al. 2011).

17.2.2 Bacterial metabolites Literature papers have reported that some bacteria produce biotechnological compounds with antioxidant potential; one example is Carbazomycin, presented in Figure 17.1 (Kato 1994). Also, benthocyanins A (Figure 17.1), B, and C and benthoenin isolated from Streptomyces prunicolor and Carquinostatin A obtained from Streptomyces exfoliates display antioxidant activity comparable to that of vitamin E. These compounds suppress glutamate toxicity and exert brain-protectin activity in the hippocampal neuron system (Shin-ya 1994). Actinobacteria isolated from different sources constitute an infinite pool of novel chemicals and secondary metabolites. Among various genera, Streptomyces, Saccharopolyspora, Amycolatopsis, and Micromonospora are interesting biocatalysts to promote the synthesis of metabolites with biological activities (Karthik et al. 2013) such as antioxidant, antifungal, and anti-inflammatory actions (Karthik et al. 2011). Marine members of the family Flavobacteriaceae furnish carotenoids like β-carotene, astaxanthin, and zeaxanthin, which are commercially important natural products (Bhosale and Bernstein 2004; Ueda 2007; Asker et al. 2007). Prabhu et  al. (2013) used two novel bacteria isolated from sandy beaches on the southwestern coast of India to examine how carbon sources affected zeaxanthin production. The authors identified the sources as belonging to the genus Muricauda and showed that microbial zeaxanthin scavenged nitric oxide in vitro, inhibited lipid peroxidation, and displayed higher 2,2-diphenyl-1-picryl hydrazyl scavenging activity than commercial zeaxanthin (Prabhu et al. 2013).

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Over the past years, several strategies involving different bacterial strains have simplified L-ascorbic acid synthesis (Hancock and Viola 2002). This vitamin has antioxidant character and is crucial to several physiological functions (Bremus et al. 2006), and its structure is presented in Figure 17.1. Several strains, such as those from the genera Gluconobacter, Acetobacter, Pseudomonas, and Erwinia, can synthesize this vitamin through different pathways and metabolic intermediates (Boudrant 1990; Bremus et al. 2006). Microbiological biotransformations that employ reasonable raw materials have received particular attention; recent advances in genetic engineering have driven microorganism industrial production (Bremus et al. 2006). Like fungi, bacteria can afford polysaccharides with antioxidant potential, such as those reported by Raza et al. (2012), who used the strain Pseudomonas fluorescens WR-1. Fang et al. (2013) described an innovative method to improve antioxidant EPS production from Bacillus licheniformis; the authors achieved 185.01 mg L–1 EPS after treating the bacteria with 12.5% n-hexane or 5% xylene for 3 hours. The crude EPS exhibited strong scavenging activities toward superoxide and hydroxyl radicals in vitro (Fang et al. 2010).

17.3 Anticancer compounds Cancer is a disease characterized by uncontrolled cell growth, invasion, and metastasis. Cancer starts when different cell types lose control of their activities due to environmental factors (chemicals, ionizing radiation, and infection). Damage and mutations in the DNA of these cells also trigger cancer by affecting the cell cycle, replication, and differentiation (Patel et al. 2010; Padilla and Furlan 2009). Conventional anticancer treatment includes chemotherapy, radiation therapy, surgery, and immunotherapy, besides chemically synthesized antitumor drugs or drugs derived from natural sources (Chang et al. 2011). However, many anticancer therapies have nonspecific toxicity, elicit severe side effects, and may be ineffective against some cancer types (Pimentel et al. 2011). For this reason, scientists have searched for alternative techniques to treat cancer. In fact, drug discovery and the development of new active compounds currently constitute one of the priorities of biotechnology (Patyar et al. 2010; Padilla and Furlan 2009). Anticancer compounds can occur in a variety of natural sources such as plants (Mans et al. 2000; Cragg and Newman 2005; Zhang et al. 2005), animals, marine invertebrates (Schwartsmann et al. 2001), and microorganisms (Patel et al. 2010). Hence, using microorganism-derived antitumor drugs has become a promising, more effective, and less toxic alternative in cancer therapy (Chang et al. 2011). This section describes some recent and the most relevant data on anticancer compounds production by microorganisms. Table 17.2 summarizes some examples of anticancer compounds obtained via biotechnology, their application, and the concentration obtained.

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Table 17.2 Examples of anticancer compounds obtained via biotechnology, their application, and the concentration obtained. Microorganism

Product

Concentration

Application

Reference

Cladosporium cladosporiodes Metarhizium anisopliae Pestalotiopsis terminaliae

Paclitaxel

800 µg.L–1

Mitotic inhibitor

Paclitaxel

846.1 µg.L–1

Mitotic inhibitor

Zhang et al. 2009 Liu et al. 2009

Paclitaxel

211 µg.L–1

Mitotic inhibitor

Phialocephala fortinii

Podophyllotoxin

189 µg.L–1

Unidentified ungal strain Fusarium solani

10-hydroxycamptothecin

677 µg/L

Purgative, antirheumatic, and antitumor Antineoplastic agent

9-Methoxycamptothecin

0.45 µg/g

Agaricus blazei

Ergosterol

n.d.

Bacillus thuringiensis

Parasporin

n.d.

Micromonospora

Diazepinomicin

n.d.

Salinispora tropica

Salinosporamide A

n.d.

Inhibits the DNA enzyme topoisomerase Inhibits tumor growth Cytocidal activity against human cancer cells Cytotoxicity against glioma and breast and prostate cancer cells Induces apoptosis in myeloma cells

Gangadevi and Muthumary, 2009 Eyberger et al. 2006 Min and Wang, 2009 Shweta et al. 2010 Takaku et al. 2001 Ohba et al. 2009 Charan et al. 2004

Feling et al. 2003

n.d., not described.

17.3.1 Fungal metabolites Several fungal strains produce natural compounds with anticancer activity, mainly because the orders Ascomycetes and Basidiomycetes rely on a large biodiversity (Pimentel et al. 2011). These microorganisms constitute interesting biocatalysts to synthesize novel microbe-derived metabolites. This section brings detailed examples and covers the most important fungal genus regarding anticancer compound production. The diterpenoid Taxol (Figure 17.2) constitutes the most important anticancer compound synthesized by an endophyte fungus and approved by the Food and Drug Administration. Taxol was first isolated from the plant Taxus brevifolia. The fungus Taxomyces andreanae was the first of other taxol-producing organisms to be reported; this fungus provided an alternative strategy to obtain a cheaper and more promptly available product. Later, different genera of fungal

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endophytes were described to produce taxol (Bhimba et  al. 2012; Pimentel et al. 2011; Gangadevi and Muthumary 2007). This compound exerted its anticancer activity via a unique mechanism of action: it reduced or interrupted cell growth and proliferation by preventing tubulin depolymerization during cell division (Gangadevi and Muthumary 2007). In fact, this class of microorganisms, known as endophytes, can synthesize important biological compounds and a variety of bioactive secondary metabolites with unique structure, as previous reviews have described (Pimentel et al. 2011; Strobel and Daisy 2003). It is believed that tropical endophytes provide more active natural products than the temperate ones; in addition, tropical endophytes can synthesize a larger number of secondary metabolites than the fungi obtained from other substrates. The evolution of these organisms has enabled them to produce the same bioactive compound as their host plant (Strobel et al. 2004). Since the year when an endophytic fungus was first reported to produce bioactive compounds, researchers have been devoted to finding new anticancer-producing microorganisms. Through microbial fermentation and genetic engineering, some paclitaxel-producing fungi isolated from Taxus improved paclitaxel production. Paclitaxel concentrations reached 800 and 846.1 μg.L–1 using Cladosporium cladosporiodes and Metarhizium anisopliae, respectively, both of which were isolated from plants belonging to the genus Taxus (Zhao et al. 2010). Podophyllotoxin (PDT) is a precursor for the chemical synthesis of anticancer substances such as etoposide, teniposide, and etophose phosphate and its structure is presented in Figure 17.2. This compound occurs naturally in Sinopodophyllum plants, but two endophytic Phialocephala fortinii strains can produce PDT with yields ranging from 0.5 to 189 μg.L–1 in liquid culture suspension. The fungus Trametes hirsute also yields PDT and its glycoside in Sabouraud broth culture (Zhao et al. 2010). The endophyte Entrophospora infrequens in culture suspended in either shake flasks or bioreactor affords camptothecin and its analogue 10-hydroxycamptothecin. The fungus Fusarium solani synthesizes 9-methoxycamptothecin (Figure  17.2) and 10-hydroxycamptothecin in Sabouraud dextrose broth. These results confirm that endophytes can potentially produce anticancer and other bioactive substances (Zhao et al. 2010). The endophytic fungi isolated from Thai medicinal plants constitute another source of biologically active substances. From the 360 morphologically distinct isolates, 60 isolates act against the human oral epidermoid carcinoma (KB) cell line, whereas 48 display activity against a breast cancer cell line; however, the taxonomy of the active metabolite in the isolates has not been identified (Wiyakrutta et  al. 2004). Another endophytic fungus, Pestalotiopsis terminaliae, isolated from the medicinal plant Terminalia arjuana, exhibits anticancer action. This strain yields taxol at 211 μg.L–1. The taxol extracted from the fungal culture extract has strong cytotoxicity against human cancer cells (Gangadevi and Muthumary 2009).

Bioactive compounds obtained through biotechnology

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Another study isolated six new fungal strains from mangrove plants. These strains had the potential to generate yet unknown active metabolites and included a new anticancer-producing fungus identified as Hypocrea lixii. Cytotoxicity tests revealed that the extracts from the isolated fungi had positive effects on laryngeal carcinoma cell lines and human breast adenocarcinoma cells. However, researchers must accomplish further studies to identify the mechanism of action of these compounds and to obtain novel anticancer drugs (Bhimba et al. 2012). Takaku et al. (2001) isolated ergosterol (Figure 17.2) from the lipid fraction of Agaricus blazei Murill, a fungus that Brazilians use to prevent cancer, diabetes, hyperlipidemia, arteriosclerosis, and chronic hepatitis. The authors showed that ergosterol intraperitoneal and oral administration inhibited tumor growth without causing the usual side effects of cancer chemotherapy drugs. The results about the mechanism of action of ergosterol suggested that this substance suppressed tumor growth by directly inhibiting the angiogenesis induced by solid tumors. Indeed, this was the first report of ergosterol as an antiangiogenic substance (Takaku et al. 2001). To identify fungal strains with the ability to provide new metabolites, including anticancer compounds, it is necessary to conduct premolecular screening to look for the key genes involved in natural products biosynthesis. Genes that encode a polyketide synthase (PKS) and a nonribosomal peptide synthetase (PS) are great targets in these studies—polyketides, nonribosomal peptides, and polyketides/peptide hybrids are some of the most common biologically active natural products that microorganisms generate. Ireland et al. (2003) amplified, cloned, sequenced, and analyzed fragments of the genomic DNA of 161 fungi. Over 70% of the cloned PS-like gene fragments bore little similarity to fungal or bacterial PS genes with known or unknown functions. The authors proposed that these genes were the ones with the greatest potential to synthesize novel chemicals, and that fungi containing uncharacterized PS and/or PKS genes had the genetic potential to synthesize new compounds if cultured under conditions that supported metabolite gene expression. The presence of these genes suggested that the chemical screening programs alone did not unveil the great metabolic diversity of fungal metabolism (Ireland et al. 2003). Microorganisms also afford other kinds of drugs, such as mevastatin (Figure 17.2), and may inhibit tumor angiogenesis. However, the FDA has not yet approved their use. Despite this difficulty, microorganisms remain as the most promising sources of natural antitumor drugs. Efforts have been made to find novel microorganisms that produce anticancer drugs as well as new bioactive compounds that can help to treat cancer (Chang et al. 2011).

17.3.2 Bacterial metabolites Because there exist countless microorganisms with a varied potential to produce metabolites, several authors have screened biocatalysts that could generate new anticancer agents. Phonnok et al. (2010) tested 394 extracts, mainly from

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bacteria, to evaluate their antiproliferative activity against four cancer cell lines: cervical cancer (HeLa), liver cancer (HepG2), breast cancer (MCF-7), and monocytic leukemia (U937). The authors used African green monkey kidney (Vero), the normal cell line. Four extracts presented positive results: Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus sp., and the yeast Candida tropicalis. The microbial crude extracts induced apoptosis: DNA fragmentation and caspase-3 activation occurred in various cancer cells, accounting for the antiproliferative activity determined by the MTT assay. However, the structure and mechanism of action of the bioactive compounds still deserve further investigation (Phonnok et al. 2010). Some bacterial products such as endotoxins display anticancer property. Indeed, these toxins can destroy tumors or help to produce vaccines, as attested by cancer therapy tests (Patyar et  al. 2010). Vaccines produced with the Pseudomonas aeruginosa lipopolysaccharide (LPS) significantly prolonged remission and survival in patients with myelogenous leukemia, as compared with untreated patients. The protein azurin elicited cancer regression in mice harboring human melanoma-azurin entered the cytosol of a human melanoma cell line and reached the nucleus, where it formed a complex with the tumor suppressor protein p53, stabilizing the disease. Protein p53 arrested tumor growth and induced apoptosis, leading to melanoma regression (Yamada et  al. 2002; Chakrabarty 2003). Cyanobacteria are particularly attractive as natural sources of bioactive molecules. They can produce such compounds in culture medium, to afford complex molecules that are difficult to achieve by chemical synthesis. Some cyanobacteria can yield tubericidin (Figure 17.2) and toyocamycin, besides macrolides such as scytophycin B isolated from Scytonema pseudohofmanni, which is cytotoxic to the human nasopharyngeal carcinoma cell line (Borowitzka 1995). The cyanobacterial metabolites like 9-deazaadenosine and dolastatin 13 are also cytotoxic to tumor cells (Namikoshi and Rinehart 1996). Thirty-five bacterial strains were isolated from six marine algae and their organic extracts were used to evaluate their anticancer activities. The strains Cc51 isolated from Centroceras clavulatum, Sm36 isolated from Sargassum muticum, and Eb46 isolated from Endarachne binghamiae exhibited anticancer activity when tested against HCT-116 colon cancer cells (Villarreal-Gómez et al. 2010). In another study, Jörn Piel (2004) isolated the genes of a symbiotic marine bacterium and found that they belonged to a prokaryotic genome, which conducted the biosynthesis of almost the entire portion of a pederin-type anticancer agent. Identification of gene architecture had important consequences for the production of known and novel pederin-type anticancer agents and also for marine biotechnology in general (Piel et  al. 2004). Wagner-Döbler et  al. (2002) also screened and identified novel marine microorganisms. The authors evaluated the new metabolites for their bioactivity and antitumor action (Wagner-Döbler et al. 2002).

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Recently, a new protein produced by Bacillus thuringiensis has been described to display anticancer properties. Parasporin (PS) has a strong, preferential cytocidal activity against human cancer cells of various origins, but only when digested with proteases (Ohba et al. 2009). Serratia marcescens and other Gram-negative bacteria produce prodigiosin (PG), a natural red pigment with selective cytotoxicity against cancer cells, p53-independent proapoptotic effect, and antimetastatic activity. Using PGs to develop antitumor drugs is advantageous compared with other natural drugs: it requires shorter preparation time; provides a different pathway to treat cancer; and exhibits immunosuppressive, antifungal, antiproliferative, and proapoptotic properties (Chang et al. 2011). Actinomycetes belong to the phylum Actinobacteria. They produce different types of secondary metabolites with biological activities and can potentially function as therapeutic agents. Therefore, scientists have been devoted to isolating novel actinomycetes from both marine and terrestrial sources (Lam 2006). Marine actinomycetes have attracted great attention—they have unique metabolic and physiological ability to survive in extreme habitats. They can produce antitumor compounds, probably because they are closely related to marine eukaryotic organisms, including mammals (Olano et al. 2009). The actinomycete Micromonospora produces diazepinomicin, which has in vitro and in vivo cytotoxicity against glioma and breast and prostate cancer cells in mouse models. The preclinical development of this substance as an anticancer agent has been completed. Salinosporamide A, isolated from Salinispora tropica, is an active proteasome inhibitor that induces apoptosis in myeloma cells through a mechanism that is distinct from that followed by the commercial proteasome inhibitor anticancer drug Bortezomib. This represents the first clinical cancer treatment candidate produced by the saline fermentation of an obligate marine actinomycete (Lam 2006). Saha et  al. (2006) isolated four marine actinobacteria and obtained a substance that was active against human leukemia cells. The authors were able to grow the biocatalyst, belonging to the genus Streptomyces sp., in a bioreactor; they assayed the supernatant and found that, at 0.05 μg.mL–1, it inhibited the growth of human leukemia cell line by 54% as compared with cytosine arabinoside at 200 μg.mL–1 (Saha et al. 2006). Bacteria from the genus Streptomyces constitute a source of a variety of bioactive compounds with anticancer properties, such as lavanducyanin (Imai et al. 1989), renastacarcin (Sasaki and Otake 1975), BE-18591; Kojiri et al. 1993), NA22598A (Kuwahara et al. 1997), and four types of trioxacarcins. Maskey and Ijaz (2004) described the structures of some of these compounds. Mukhtar et al. (2012) optimized some parameters to produce antitumor and antibiotic substances from Streptomyces capoamus in batch fermentation at 30°C, 7.5 of pH, and 72 hours of incubation time. The latter authors also used 2% maltose as carbon source, 2% corn steep liquor as nitrogen source, and 48-hour-old inoculum at a concentration of 8% (v/v) to obtain antitumor compounds from Streptomyces capoamus (Mukhtar and Ijaz 2012).

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On the basis of this approach, it is possible to expect that bacterial diversity will become a source of important products in the future of cancer treatment.

17.3.3 Important alternatives to support the search for new anticancer metabolites The metabolic engineering of microorganisms is an important strategy to produce drugs and drug precursors. This type of engineering consists in purposely modifying cellular metabolism using recombinant DNA and other molecular biology techniques to establish new metabolic pathways to obtain the desired product. By enforcing or removing the existing metabolic pathways, it should be possible to enhance product formation. In metabolic engineering, one can use a Saccharomyces cerevisiae strain to heterologously express eight taxoid biosynthetic genes and produce taxadien-5α-acetoxy-10β-ol, a precursor of taxol. Other anticancer and antitumor compounds could also originate from such a process; for example, the heterologous expression of rebeccamycin biosynthetic genes in Streptomyces albus could lead to indolocarbazole compounds; a strain of Escherichia coli expressing an echinumycin gene cluster could afford echinomycin; and the expression of plant sesquiterpenes biosynthetic genes and the downregulation of  ERG9 in Saccharomyces cerevisiae together with methionine addition to the medium could prompt the yeast to produce valencene, cubebol, and pathooulol (Lee et al. 2009). Chemicals used in cancer treatment usually bear complex structures and are difficult to synthesize. Employing natural compounds that occur in plants or animals may negatively impact the environment by depleting resources and polluting the extraction site, not to mention that plants have a long life cycle. Using microorganism-produced substances is therefore attractive and offers many advantages: microbial fermentation can furnish the target product in a controlled and consistent manner using relatively inexpensive substrates; microorganisms grow at a faster rate than higher organisms; and metabolic engineering is easier to perform in microorganisms than in mammalian and plant cells, which should accelerate anticancer compound production (Lee et al. 2009). Bioprocesses are expensive, so scientists have attempted to reduce costs related to raw materials and/or final products purification. In general, the price of the carbon sources used in the culture medium impacts the final cost. In this sense, a cheap alternative is to use industrial residues, such as molasses, as starting materials to make the industrial-scale process viable. By means of submerged fermentation, Chimilovski et al. (2011) used Grifola frondosa together with sugar cane and soy molasses as carbon sources to obtain EPS, including β-glucans; the latter possess immunostimulatory and antitumor activities. Sugar cane molasses medium gave the best results: 22.93 ± 3.37 g L–1 dry biomass and 5.14 ± 0.26 g L–1 EPS. Hence, using alternative carbon sources is an interesting, less costly option to increase anticancer compounds production (Chimilovski et al. 2011).

Bioactive compounds obtained through biotechnology

447

Solid-state fermentation increases active compound production, is less expensive, and demands lower energy input, simple fermentation medium, and lower water consumption. Mishra (2006) described that a new isolated strain of Aspergillus niger produced L-Asparaginase from agricultural waste by solid-state fermentation. This enzyme derived mainly from submerged fermentation and was useful to treat acute lymphoblastic leukemia. The authors reported economically attractive L-asparaginase production from untreated biomass residues that constituted promptly available raw material in agriculture-based countries. Also, the fact that L-asparaginase had maximum activity at near physiological temperature made it a valuable asset in cancer treatment (Mishra 2006). Despite the great potential of this area, several drawbacks exist: controlled pH, temperature, agitation, and illumination are necessary; production costs may still be unattractive; and the purification and isolation processes are complex. Therefore, further studies must be conducted to improve the production of this enzyme, reduce costs, and better understand enzyme activity, so as to develop novel effective cancer therapeutics (Chang et al. 2011).

17.4 Antimicrobial compounds Antimicrobial agents can inhibit or kill microorganisms (e.g., bacteria and fungi) by breaking their cell wall, interrupting their metabolism, or binding to their DNA and preventing their replication. Consequently, these agents stop microbe multiplication in the body. It is possible to group antimicrobial medicines according to the microorganisms that they primarily act against; for example, antibacterial (commonly known as antibiotics) and antifungal agents act against bacteria and fungi, respectively. Medications can also be classified according to their function: antimicrobials that kill microbes are called microbicidal; those that merely inhibit microbe growth are designated microbiostatic (Bryskier 2005). Most microbiologists classify antimicrobial agents into two groups: antibiotics— natural substances that certain groups of microorganisms produce—and synthetic chemotherapeutic agents. A hybrid substance consists of a semisynthetic antibiotic—a chemist modifies a molecular version that the microbe produces to  achieve the desired properties. Furthermore, chemical synthesis can yield some antimicrobial compounds originally discovered as microorganism products (Bryskier 2005). Using substances with antimicrobial properties has been common practice for at least 2,000 years. Ancient Egyptians and ancient Greeks employed specific molds and plant extracts to treat infection. More recently, microbiologists such as Louis Pasteur and Jules Francois Joubert reported that laboratory animals injected with the causative agent of anthrax (Bacillus anthracis) and common saprophytic bacteria did not develop the disease. In this case, the saprophyte used oxygen and starved the anthrax organism (Pasteur and Joubert 1877).

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Bioactive compounds obtained through biotechnology

In  the following years, this inhibitory action was found to stem from many mixed cultures, a phenomenon known as microbial antagonism (Foster and Woodruff 2010). In the majority of cases, antagonisms lead a microorganism to produce metabolites that are toxic to or inhibit other microorganisms. In 1928, Alexander Fleming discovered a fungus, known as Penicillium rubens, that produced a natural antimicrobial agent named penicillin. In 1942, this substance successfully eliminated a Streptococcus infection. Later, penicillin acted effectively against many other infectious diseases such as gonorrhea, strep throat, and pneumonia, which were potentially fatal to patients until then (Nobelprize.org 2014). Rapid isolation of streptomycin, chloramphenicol, and tetracycline soon followed. By the 1950s, these and several other antibiotics were being used in the clinical setting. Today, numerous antimicrobial agents exist to treat a wide range of infections.

17.4.1 Antibacterials Antibacterials or antibiotics aid in treating bacterial infections and are among the most prescribed drugs worldwide. Unfortunately, clinicians are increasingly concerned about their effectiveness because resistant bacteria have emerged (Maestro and Sanz 2007). The presence of antibiotic-resistant bacteria in food can be a threat to public health: antibiotic resistance determinants can transfer to other bacteria of clinical significance via genetic transference mechanisms such as conjugation and transformation (Sunde and Nordstrom 2006; Van et al. 2007; Walsh et al. 2007). Over the last decades, antibiotics have been used to treat bacterial infections in humans and animals. However, many studies have demonstrated that antibioticresistant bacteria occur in various environments due to uncontrolled discharge of urban and animal wastewater, which may contaminate food of animal or plant origin (Kumar et al. 2005; Silva et al. 2006; Watkinson et al. 2007). Researchers have pursued novel strategies to develop antibiotics, for example, combinatory chemistry tools. Nevertheless, at present, the pharmaceutical industry produces only a few new antibiotic agents (Coates and Hu 2007). Strategies to obtain new drugs include organic synthesis (Abbanat et al. 2003), drug pharmacokinetics modification using nanotechnology (Jeong et al. 2007), and search for molecules with unexploited mechanisms of action (Lockwood and Mayo 2003). In the past, natural products constituted the main source of antibiotics or antibiotic prototypes (Butler and Buss 2006). First, microorganisms that produce β-lactam antibiotics were discovered (Demain and Elander 1999), which was followed by production of polyketides, aminoglycosides, macrolides, glycopeptides, quinolones, β-lactams, and the modern oxazolidinones and daptomycin (Singh and Barret 2006). Although synthetic molecules display effective antimicrobial properties, natural products remain a promising alternative (Newman and Cragg 2007). Literature papers have described novel methods and technologies to discover new drugs from microbial sources (Luzhetskyy

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et al. 2007). The screening of novel strains unveils microorganisms that have not yet been assayed for their antibacterial activity (Donadio et  al. 2007); these microorganisms could produce innovative molecules or useful templates to develop new antibiotics (Sofia and Boldi 2006). It is also advisable to search for original sources of microorganisms—the environment can affect microbial metabolism, so researchers have isolated antibiotics-producing microorganisms from the most diverse habitats such as endophytes of terrestrial plants and sea organisms, among others (Gunatilaka 2006; Gandhimathi et al. 2008). Fleming et  al. (2010) isolated 31 coliform strains from salad, cheese, and meat products sold in commercial establishments in the city of Rio de Janeiro; the authors tested the strains for antibiotic resistance and antimicrobial substance production. Thirteen strains (41.9%) were resistant to at least one tested antibiotic; one of these strains was actually resistant to nine different antibiotics. Two strains (6.4%), identified as Klebsiella ozaenae and Raoultella terrigena, inhibited the indicator strains Escherichia coli LMIFRJ and Salmonella enterica I; the antimicrobial substances that the former strains produced were sensitive to proteolytic enzymes, suggesting that they were bacteriocins. Although they had similar action spectrum, bacteriocins were different. Both strains inhibited E. coli, Klebsiella, Enterobacter, and Salmonella strains, including antibiotic-resistant ones. Therefore, these bacteriocins, named klebicin K and raoultellin L, could potentially act against some foodborne pathogens. Arasu et  al. (2013) isolated Streptomyces strains from the marine region of Andra Pradesh, India. Among the 210 screened Streptomyces strains, 64.3% exhibited activity against Gram-positive bacteria, 48.5% were active against Gram-negative bacteria, 38.8% displayed action against both Gram-positive and Gram-negative bacteria, and 80.9% possessed significant antifungal activity. However, primary screening revealed that Streptomyces sp. AP-123 exhibited significant antimicrobial activity against all the tested bacteria as compared with other Streptomyces strains. Hence, the authors used hexane and ethyl acetate to  extract antimicrobial substances from spent medium in which Streptomyces sp. AP-123 grew at 30°C for 5 days. Elution of the crude extract using varying concentrations of solvents and chromatographic purification afforded a compound that the authors identified as a polyketide-related antibiotic. The compound displayed significant antibacterial activity against Gram-positive and Gram-negative bacteria as well as potent cytotoxic activity against the cell lines, namely, Vero (Green monkey kidney) and HEP2 (laryngeal carcinoma cells) in vitro. Barros et al. (2013) tested the ability of three psychrotrophic Gram-negative bacilli isolated from Chilean Patagonian cold freshwater rivers to produce bioactive metabolites. The strains biochemical properties and 16S rRNA gene analysis aided strain identification. The authors obtained the metabolite fractions that possessed antibacterial activity by solvent extraction and partially characterized them by gas chromatography–mass spectrometry (GC-MS). Molecular analysis helped to identify the three Patagonian strains as Pseudomonas sp. RG-6

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(Pseudomonas brenneri 99.6% identity), Pseudomonas sp. RG-8 (Pseudomonas trivialis 99.6% identity), and Yersinia sp. RP-3 (Yersinia aldovae 99.5% identity). These extracts inhibited both Gram-positive and Gram-negative bacteria but not Listeria monocytogenes. The chemical structure of the active molecule remains to be elucidated, although GC-MS analysis of the filtrates suggested that compounds like sesquiterpenes derivatives from β-maaliene (Figure 17.3) or δ-selinene could account for the antibacterial activity. Microbial biotransformation converts terpene derivatives into compounds that constitute an important source of natural pharmaceutical, fragrance, and aroma substances. Işcan et al. (2012) studied how 16 different strains of microorganisms (bacteria, fungi, and yeasts) biotransformed the monoterpene α-phellandrene. The authors initially screened transformation metabolites by thin-layer chromatography (TLC) and GC-MS and further characterized them by nuclear magnetic resonance spectroscopy (NMR). Among the six characterized metabolites, the investigation by Iscan et  al. was the first to report 6-hydroxypiperitone, α-phellandrene epoxide (Figure  17.3), cis-p-menth-2en-1-ol, and carvotanacetone, which originated from (–)-(R)-α-phellandrene. The authors subjected the substrate and the metabolite 5-p-menthene-1,2-diol to in vitro antibacterial and anticandidal tests. The metabolite showed moderate-to-good inhibitory activities against various bacteria and especially against Candida species as compared with its substrate (–)-(R)-a-phellandrene and standard antimicrobial agents. Isnansetyo and Kamei (2009) wrote a review about the bioactive substances (antimicrobial, antiviral, and cytotoxic agents) that marine isolates of Pseudomonas produce; while Peirú et al. (2005) developed an Escherichia coli strain that can generate the potent antibiotic erythromycin C (Figure  17.3) by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. Antibiotics help to prevent the spread of pathogenic bacteria; however, many antibiotics are broad-spectrum drugs that kill bacterial species indiscriminately (Riley and Wertz 2002). Bacteriocins have a relatively narrow killing activity spectrum; some constitute pathogen-specific designer drugs. Given the diversity of naturally produced bacteriocins, identifying effective bacteriocins against specific human pathogens may be a relatively simple task (Riley and Wertz 2002). In addition, the use of bacteriocins may reduce the need for chemical additives in food and minimize the intensity of food processing techniques, contributing to production of healthier foods (Osmanagaoglu et  al. 2011). In recent years, researchers have focused on lactic acid bacteria (LAB) from different sources; these bacteria synthesize bacteriocins considered to be safe food biopreservatives that gastrointestinal proteases can degrade (Facklam and Elliott 1995). A variety of industrial applications relevant to both human and animal health have employed these probiotic compounds; no side effects have been reported. Hence, it is relevant to identify new strains with useful characteristics.

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To date, the bacteriocin nisin (presented in Figure 17.3), produced by Lactococcus lactis, has been the most thoroughly studied bacteriocin and has been applied as a food additive (Delves-Broughton et  al. 1996). Substantial work has been conducted to verify nisin effectiveness against various spoilage and pathogenic microorganisms such as L. monocytogenes and to evaluate its potential application in different food products (Freitas et al. 2008; Schillinger et al. 2001; Staszewski and Jagus 2008). Other bacteriocins such as pediocin are also potentially applicable in foods, although they have not received approval as antimicrobial food additives (Naghmouchi et al. 2007). Abbasiliasi et al. (2012) isolated LAB from a range of traditional fermented products to synthesize bacteriocin-like inhibitory substances. They screened a total of 222 LAB strains from fermented milk products in the form of fresh curds, dried curds, ghara (a traditional flavor enhancer prepared from whey), and fermented cocoa bean. Biochemical methods and 16S rDNA gene sequencing revealed 11 LAB isolates that produced antimicrobial substances, namely Lactococcus lactis, Lactobacillus plantarum, and Pediococcus acidilactici strains. The Kp10 (P. acidilactici) cell-free supernatant inhibited Listeria monocytogenes the most strongly. Further analysis identified the antimicrobial substance produced by Kp10 as proteinaceous in nature and active over a wide pH range. Kp10 (P. acidilactici) was catalase-negative, able to produce β-galactosidase, resistant to bile salts (0.3%) and acidic conditions (pH 3), and susceptible to most antibiotics. Therefore, Kp10 could be potentially used to produce probiotic and functional foods. Yang et al. (2012) isolated and identified bacteriocinogenic LAB from various cheeses and yogurts and evaluated their antimicrobial effects on selected spoilage and pathogenic microorganisms in vitro as well as on a food commodity. The agar diffusion bioassay helped to screen LAB that synthesized bacteriocin or bacteriocin-like substances (BLS) using Lactobacillus sakei and Listeria innocua as indicator organisms. Of the 138 assayed LAB isolates, 28 inhibited the indicator bacteria. 16S rRNA gene sequencing identified these as strains of Enterococcus faecium, Streptococcus thermophilus, Lactobacillus casei, and Lactobacillus sakei subsp. sakei. The authors assessed the antimicrobial activity of eight isolates against L. innocua, Escherichia coli, Bacillus cereus, Pseudomonas fluorescens, Erwinia carotovora, and Leuconostoc mesenteroides subsp. mesenteroides using the agar diffusion bioassay at 5 and 20°C. They also investigated how selected LAB strains affected L. innocua inoculated onto fresh-cut onions. Organic acids and/or H2O2 produced by LAB and not the BLS had strong antimicrobial effects on all the tested microorganisms, except for E. coli., Ent. faecium, Strep. Thermophiles, and Lact. Casei; they also effectively inhibited the growth of natural microflora and L. innocua inoculated onto fresh-cut onions. According to the authors, the bacteriocinogenic LAB present in cheeses and yogurts may be potentially applicable as biopreservatives in foods.

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17.4.2 Antifungals Antifungals kill or prevent further fungi growth. Clinicians use antifungal agents to treat infections such as athlete’s foot, ringworm, and thrush; these agents function by exploiting differences between mammalian and fungal cells. Antifungals kill the fungal organism without harming the host. Unlike bacteria, both fungi and humans are eukaryotes, so fungal and human cells share similarities at the molecular level. Thus, an antifungal drug may attack other cells apart from the fungus in the infected organism. Consequently, some antifungals may have life-threatening side effects if not used properly (Bryskier 2005). Several antifungal compounds have been isolated from bacterial cultures. Magnusson and Schnürer (2001) were the first to report that a Lactobacillus coryniformis subsp. coryniformis strain produced a broad-spectrum proteinaceous antifungal compound. Gerez et al. (2009), Magnusson et al. (2003), Ström et al. (2002), and Lavermicocca et  al. (2003) described a potential antifungal compound known as phenyllactic acid (Figure 17.3). Rouse et al. (2008) separated a possible cyclic dipeptide from Pc. pentosaceous. Yang and Chang (2010) were the first to show the antifungal activities of cyclo(Leu–Leu) produced by LAB, a class of 2,5-diketopiperazines. Recently, the biological properties of 2,5-diketopiperazines have attracted attention (McCleland et  al. 2004). Dal Bello et  al. (2007) identified lactic acid, phenyllactic acid, and two cyclic dipeptides [cyclo (L-Leu-L-Pro) and cyclo (L-Phe-L-Pro)] as the major components accounting for the antifungal activity of L. plantarum FST 1.7. Because these authors also observed antifungal activities in other fractions, they believed that more antifungal compounds existed in addition to the ones that they detected. Li et al. (2011) screened Lactobacillus casei AST18 as an antifungal lactic acid bacterium. More recently (Li et al. 2012), these authors detected the antifungal properties of the cell-free culture filtrate (CCF) from L. casei AST18; they obtained the antifungal compounds of CCF by ultrafiltration and semi-preparative highperformance liquid chromatography (HPLC) and determined them by GC-MS. CCF was sensitive to pH and heat treatment but unaffected by treatment with trypsin and pepsin. Ultrafiltration and semi-preparative HPLC yielded two CCF fractions with antifungal activities: fractions 1 and 4. The main antifungal agent in fraction 1 was lactic acid. In fraction 4, GC-MS detected three small potential antifungal substances: cyclo-(Leu-Pro), 2,6-diphenyl-piperidine, and 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a;1’,2’-d]pyrazine. The antifungal activity of L. casei AST18 resulted from a synergistic effect among lactic acid and cyclopeptides. Digaitiene et al. (2012) isolated five lactic acid bacteria (LAB) strains from rye  sourdoughs to investigate antimicrobial substance production. According to these authors, the supernatants of the analyzed LAB inhibited growth of up to 15 out of 25 indicator bacteria strains as well as up to 25 out of 56 LAB strains isolated from rye sourdoughs. Moreover, these five LAB were active against ropes-producing Bacillus subtilis and the main fungi causing bread mold

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spoilage—Aspergillus, Fusarium, Mucor, and Penicillium. Lactobacillus sakei KTU05-6 displayed the best antibacterial properties and was resistant to heat treatment even at 100°C for 60 minutes. Antibacterial substances produced by LAB may be a good co-starter culture to ensure sourdough stability and to avoid bacterial and fungal spoilage of the end product. The Bacillus amyloliquefaciens strain NJN-6 produces antagonistic substances that act against Fusarium oxysporum (Yuan et  al. 2012). Ultrasound-assisted extraction (UAE) of dried leavening with n-butanol afforded the active antifungal substance. HPLC analysis with electrospray ionization mass spectrometry (ESI-MS) showed that the antimicrobial substances consisted of three homologues of the iturin A family with molecular weights of 1043, 1057, and 1071 Da as well as two homologues of the fengycin family with molecular weights of 1477 and 1491 Da. Compared with traditional extraction techniques, UAE is a  simple, cheap, and environmentally friendly method to isolate and identify lipopeptides and other active compounds. Many studies have shown that Bacillus controls multiple soil-borne diseases, including Rhizoctonia solani in potato, rice blast and sheath blight in rice, and Fusarium wilt in cotton (Brewer and Larkin 2005; Leelasuphakul et  al. 2006; Safiyazov et al. 1995). Wiyada (2012) authored a review about the Bacillus beneficial substances related to plants, humans, and animals. Competition, antagonism, systemic resistance induction, and plant growth promotion are mechanisms through which organisms accomplish biological control. Identification of the antifungal substances involved in producing antagonistic effects may provide a better understanding of the underlying antifungal mechanisms. Despite the increased resistance to antimicrobial agents among important pathogenic microorganisms worldwide, the number of new antimicrobial agents launched into the market has declined steadily over the past decades. Continuing research exists to find new effective antimicrobials, much of which takes place in academic centers and small biotechnology companies rather than large pharmaceutical enterprises. While classic screening methods and chemical modification of known antimicrobial agents continue to produce potential leads for new antimicrobial agents, researchers are looking into a range of other approaches, which include searching for potentiators of the activity of known antimicrobial agents and developing hybrid agents, novel membrane-active drugs, and inhibitors of bacterial virulence and pathogenesis. Scientists are also exploring new bacterial targets such as bacteriophages and their lytic enzymes (Moellering 2011). Given the amount of investigation that is currently underway, it is clear that, although the antibiotic pipeline is not as promising as it was half a century ago, it is expected to soon allow for the production of novel metabolites with biological activities. For more detailed information on antimicrobial, antioxidant, and anticancer agents from microbial sources, please consult the references authored by Newman and Cragg (2007) and Firáková et al. (2007).

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17.5 Conclusion Microorganisms have proven to be rich sources of novel natural compounds with a wide spectrum of biological activities and high level of structural diversity. However, the food and pharmaceutical industries still only modestly apply them to obtain compounds of interest, although useful microorganisms that can accomplish a large scope of reactions are widely available worldwide. Hence, biotechnological techniques using different microorganisms appear to  be promising alternatives to establish an inexhaustible, cost-effective, and renewable resource of high-value bioactive products. This chapter focused on the role of microorganism bioactive compounds production, the importance of including microbes when screening for novel drugs, and the biotechnology process as a new strategy to obtain such compounds.

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CHAPTER 18

Metabolic engineering of bioactive compounds in berries Ivayla Dincheva, Ilian Badjakov, and Violeta Kondakova AgroBioInstitute, Sofia, Bulgaria

18.1 Introduction Metabolic engineering involves the targeted and purposeful alteration of metabolic pathways found in an organism to achieve better understanding and use of cellular pathways for chemical transformation, energy transduction, and supramolecular assembly (Lessard 1996). In essence, metabolic engineering leads to the establishment of new metabolic pathways and suppression or removal of existing pathways to enhance the formation of a desired product by recombinant DNA technology (Stephanopoulos 1999). The interest in metabolic engineering is stimulated by potential commercial applications for production of useful secondary metabolites that determine important aspects of human food quality (taste, color, and smell) and are widely used in the manufacture of medicines, dyes, fungicides, insecticides, flavors, and fragrances. The extension of metabolic engineering to produce desired compounds presents fundamental information applied to plant development and physiology. Genetic modification by transformation allows stable alterations of the biochemical processes involved in important traits such as yield and nutritional value. To improve the phenolic content of fruits, a novel field of interest is based on results obtained using elicitors, agrochemicals that were designed to improve resistance to plant pathogens. Although elicitors do not kill pathogens, they trigger plant defense mechanisms, one of which is to increase the levels of phenolic compounds (Ruiz-Garcia et al. 2013).

18.2 Engineering of berry secondary metabolites Genetic engineering of a secondary metabolic pathway aims to increase/decrease the quantity of a certain compound(s) or to transfer a pathway or part of a pathway to other plant species (Verpoorte and Memelink 2002). Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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In this connection, two general approaches have been followed. First, methods have been employed to change the expression of one or a few genes, thereby overcoming specific rate-limiting steps in the pathway, to shut down competitive pathways, and to decrease catabolism of the product of interest. An enzymatic step in the pathway can be knocked out, for example, by reducing the level of the corresponding mRNA via antisense, co-suppression, or RNA interference technologies, or by overexpressing an antibody against the enzyme. Second, attempts have been made to change the expression of regulatory genes that control multiple biosynthesis genes (Verpoorte and Memelink 2002). Regulatory genes control the expression of structural genes, leading to the production of proteins called transcriptional factors. They play an important role in regulating secondary metabolism pathways more than structural genes that control only a single step (Broun 2004).

18.2.1 The basic flavonoid upstream pathway of anthocyanin biosynthesis Among the secondary metabolite groups in plants, flavonoids are the most common group of plant polyphenolic secondary metabolites. Currently there is broad interest in the effects of dietary polyphenols on human health. In addition to the potent antioxidant activity of many of these compounds in vitro, an inverse correlation between the intake of certain polyphenols and the risk of cardiovascular diseases, cancer, and other age-related diseases has been observed in epidemiological studies (Harborne 2000). Enhancing flavonoid biosynthesis in chosen crops may provide new materials that have the potential to be used in food designed for specific benefits to human health. The best-studied route at the genetic level is the flavonoid biosynthesis pathway leading to the formation of anthocyanins. Most of the structural and several regulatory genes involved in this pathway have now been cloned. Anthocyanins, synthesized via the flavonoid pathway, are a class of crucial phenolic compounds that are fundamentally responsible for the color of flowers, berries, and vegetables. As the most important natural colorants in berries and their products, anthocyanins are also widely studied for their numerous beneficial effects on human health. In recent years, the biosynthetic pathway of anthocyanins in grapes has been thoroughly investigated (Mazza 1995). They are synthesized via the flavonoid pathway, which shares the same upstream pathway with proanthocyanidins, until the formation of anthocyanidins by the catalysis of anthocyanidin synthase (ANS), also known as leucoanthocyanidin dioxygenase (LDOX) (Gollop et al. 2001; Springob et al. 2003; Fujita et al. 2005). In the past, ANS used to be considered the first key enzyme that could lead the flavonoid flux into the anthocyanin branch (He et al. 2010). Recent work has revealed that ANS also plays a significant role in the biosynthesis of proanthocyanidins (Dixon et al. 2005; Xie and Dixon 2005; Tian et al. 2008). Anthocyanidins are not only direct substrates for the anthocyanin synthesis, but also can be catalyzed by

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anthocyanidins reductase (ANR) to produce (2R,3R)-cis-flavan-3-ols, which is an important group of substrates for the proanthocyanidins synthesis (Gargouri et al. 2009). Consequently, the whole pathway can be divided into two parts: the basic flavonoid upstream pathway and the specific anthocyanin downstream branch. The basic flavonoid upstream pathway has been investigated extensively at both the biochemical and genetic levels. Previous studies revealed that the enzymes acting earlier in the flavonoid pathway are usually encoded by larger gene families, whereas the enzymes acting late in the pathway are commonly encoded by one single active gene (Sparvoli et al. 1994). This may result from the fact that the earlier-acting genes usually possess more diverse metabolic functions, which requires correspondingly diverse control of gene expressions by using gene duplication. In contrast, the later-acting genes normally point to simpler and more specific metabolic functions and the further duplication of these genes is usually unnecessary. Generally, by the action of chalcone synthase (CHS), three malonyl-CoA molecules and one p-coumaroyl-CoA molecule can condense to produce a naringenin chalcone (Durbin et al. 2000; Tian et al. 2006a). In grapes (V. vinifera), there are at least three genes encoding CHS—CHS1 (AB015872), CHS2 (AB066275), and CHS3 (AB066274)—which are transcribed under different controls (GotoYamamoto et al. 2002; Ageorges et al. 2006). Their product, the naringenin chalcone, is not only used in the synthesis of anthocyanins or proanthocyanidins, but also in the formation of other phenolic compounds. Thus, the three different CHSs may act in three different pathways to produce different secondary metabolites (Tian et al. 2006a). Furthermore, although the expressions of these Chs genes at both transcript and protein levels are relatively low, their high biosynthetic efficiency may also guarantee their high production. After the action of chalcone isomerase (also known as chalcone-flavanone isomerase [CHI]), the naringenin chalcone can be modified to its isomer naringenin flavanone stereospecifically and quickly, which initially consists of the basic three rings of the  general C6-C3-C6 flavonoid skeleton (Jez and Noel 2002). It is worth noting  that the product of CHI is almost absolutely the biologically active (2S)-flavanone, which is crucial to the subsequent reactions. The ‘B’ ring of  the naringenin flavanone can be further hydroxylated by flavonoid 3’-hydroxylase (F3’H) or flavonoid 3’5’-hydroxylase (F3’5’H) to produce eriodictyol or pentahydroxyflavanone, respectively (Bogs et al. 2006). All of the three (2S)-flavanones can be modified by the catalysis of flavanone 3β-hydroxylase (F3H, also known as FHT) to produce the corresponding dihydroflavonols. Furthermore, the direct enzymatically oxidized product of naringenin flavanone, dihydrokaempferol, is also the potential substrate for F3’H and F3’5’H to produce the corresponding dihydroflavonols, dihydroquercetin, and dihydromyricetin, respectively.

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In grapes, there are at least two putative F3Hs (CAA53579 and P41090), the expressions of which are quite similar to that of the CHS (CAA53583) or ANR (CAD91911) (Waters et al. 2005). Recently, four genes encoding F3’H— F3’h1 (AB213602), F3’h2 (AB213603), F3’h3 (AB213604), and F3’h4 (AB213605)—and one gene encoding F3’5’H—F3’5’h (AB213606)—were identified and characterized in grapes. The expression of the four F3’h genes are different from each other in various grape organs, and the expression of F3’5’h is higher than that of F3’h at the transcriptional level in the grape skin of some V. vinifera cultivars, such as Cabernet Sauvignon and Dornfelder, which explains their different anthocyanin composition. After these modifications, dihydroflavonol 4-reductase (DFR) can reduce these dihydroflavonols to their corresponding leucoanthocyanidins. These reactions also have extremely high stereospecificity; the absolute configuration of their products was determined to be (2S,3S,4S). Anthocyanins were synthesized with the increased DFR activity by inducing Pi deficiency and feeding of elevated dihydroquercetin in a novel cell suspension culture initiated from nonpigmented grape cells. Leucoanthocyanidins, besides being used to synthesize the corresponding anthocyanidins, can also be reduced to their corresponding (2R,3S)-trans-flavan-3-ols by the action of leucoanthocyanidin reductase (LAR), which is the direct substrate for proanthocyanidin polymerization (Bogs et  al. 2005; Pfeiffer et  al. 2006). Furthermore, even the leucoanthocyanidins themselves are also considered as a class of potential substrates for proanthocyanidin biosynthesis. Although DFR has been widely studied in numerous plants, until recently little has been known, especially in grapes, about its structural and biochemical properties. Recently, the crystal structure of the V. vinifera DFR was determined, and the crucial region of substrate binding and recognition was also confirmed (Petit et al. 2007). The promoter of the gene encoding the grape DFR (CAA72420.1) was cloned and analyzed. A specific sequence of the DFR promoter might be involved in the expression of the gene in grape berries and white light, calcium, and sucrose can induce the DFR gene expression, suggesting a UV receptor signal transduction pathway might be involved in the induction of the DFR gene (Gollop et al. 2002). As mentioned above, ANS also plays a pivotal role in the biosynthesis of both anthocyanins and proanthocyanidins. The colorless leucoanthocyanidins can be oxidized to their corresponding colored anthocyanidins by the catalysis of ANS, with the help of ferrous iron. As a member of the 2-oxoglutarate-dependent oxygenase family, just like F3’H and flavonol synthase (FLS), ANS usually possesses multiple functions except the basic role mentioned above, for it usually catalyzes the production of dihydroflavonols or flavonols. The promoter of the grape ANS gene (CAA53580) was cloned and analyzed. Similar to that of DFR, the ANS promoter also has several putative DNA binding motifs and can be induced by light, calcium, and sucrose.

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18.2.2 Specific pathway for the anthocyanin modification Consequent to the activity of ANS, colored anthocyanidins are formed, but their immediate modification, mainly by glycosylation, methylation, and acylation, is very necessary for their stabilization as vacuolar anthocyanins. Mutants of red grapes lacking the expression of the genes for the initial glycosylation of anthocyanidins usually accumulate no anthocyanins in their berries, though the intact anthocyanidin biosynthetic pathway exists. In the last decade, by cloning the related genes in various plants, our appreciation of the specific anthocyanin downstream branch has been advanced greatly. Glycosylation is an important modification for increasing the hydrophilicity and stability of anthocyanins because the anthocyanidins are inherently unstable under physiological conditions. In plants, UDP-glucose anthocyanidin flavonoid glucosyltransferase (UFGT), especially the anthocyanidin/anthocyanin glycosyltransferases, catalyze the O-glycosylation of anthocyanidins or anthocyanins, which recognize either anthocyanidins or anthocyanins as sugar acceptors and UDP-sugars as the sugar donors. Several other sugar donors are also be used occasionally by grape UFGT, such as UDP-galactose, UDP-rhamnose, UDP-xylose, UDP-glucuronic acid, and UDP-arabinose. In V. vinifera grapes, anthocyanidins can only be O-glycosylated at the C3 position with the addition of glucoses by the activity of UFGT. Thus, V. vinifera UFGT used to be called 3-Oglucosyltransferase (3-GT) for short. Normally, the UFGT expression is only detected in berry skin after the onset of ripening specifically, whereas most of the upstream genes may express constitutively in different organs and tissues at diverse levels. The UFGT enzyme that has been isolated from V. vinifera cell suspension cultures shows highest activity with cyanidin as acceptor, but it can also use delphinidin greatly as well as pelargonidin, peonidin, petunidin, and malvidin at lower levels at its optimal pH 8.0 (Do et al. 1995). Furthermore, there are no differences in the UFGT gene coding and promoter sequences between white grapes and their red skin sports, but the UFGT gene only expresses in the red sports. This result revealed the significant role of UFGT in anthocyanin biosynthesis and suggested that its expression is under extremely strict regulation of some genetic transcriptional factors (Kobayashi et al. 2001). Its expression has high organ and time specificity, as well as high substrate specificity (Offen et al. 2006). In grapes, the biosynthetic pathway for pelargonidin-3-O-glucoside is intact, and the relatively wide substrate specificity of the structure genes—for example, CHI, F3H, DFR, ANS, and UFGT—cannot get rid of the possibility of its existence. However, our recent research supported the evidence of the presence of pelargonidin-3-O-glucoside in grapes at trace levels. The comparatively higher activity of F3’H and F3’5’H in grapes may explain this question. In this pathway, most of the naringenin and dihydrokaempferol (the precursors of pelargonidin) were used to form the flavonoids with more than one hydroxyl group, and the following products are used in the formation of corresponding

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anthocyanins or flavonols. As a result, there is little flux pointing to the production of pelargonidin anthocyanins or kaempferol glycosides. Thus, the major flux is pumped to the “side path,” not the “central way” that leads to the synthesis of pelargonidin-3-Oglucoside. This assumption can be reasonably explained by the analysis of anthocyanin or flavonol profiles in many V. vinifera varieties. In most non–V. vinifera grapes or their hybrids, O-glycosylation can also occur at both of the C3 and C5 positions to form anthocyanidin-3,5-O-diglucoside, while only trace amounts of 3,5-O-diglucoside can be found in some European cultivars (Goldy et al. 1989; Lamikanra 1989; Kim et al. 2010; Zhao et al. 2010). A recent research paper reported that the cloning of the nonfunctional allele of the functional 5-O-glucosyltransferase (5-GT) from the heterozygous hybrid cultivar. Comparison and analysis of the sequences revealed that two mutations of this gene inactivated its 5GT function in V. vinifera grapes and resulted in the absence of anthocyanidin-3,5-Odiglucosides. In the plant kingdom, it was reported that almost 90% of the anthocyanins were modified by methylation of the six common anthocyanidins. Of these three methylated anthocyanidins, peonidin, petunidin, and malvidin comprise 20% of the total reported anthocyanidins. The participation of S-adenosyl-L-methionine (SAM), O-methyltransferase (OMT, also known as anthocyanin O-methyltransferase [AOMT]) can mediate the methylation of the hydroxyl groups at the C3’ positions or both at the C3’ or C5’ positions on the B rings of the anthocyanins (Ibrahim et al. 1998). In grapes, significant OMT activity on anthocyanins requires the presence of divalent cations such as Mg2+, and belongs to the Class I OMTs. The hydroxyl group at the C4’ position on the B ring of the anthocyanins is seldom methylated. In a much earlier report, a highly specific OMT was partially purified and characterized from a cell suspension of V. vinifera L. cv. Gamay Fréaux. This was found to almost absolutely catalyze the transfer of the methyl group of SAM to the hydroxyl group at the C3’ position of cyanidin-3-O-glucoside, but to have a  low affinity for cyanidin and did not methylate either cyanidin-3-O-pcoumaroylglucoside or delphinidin (Bailly et al. 1997). Interestingly, a recently found novel divalent cation-dependent OMT from the grapevine seems to have lower substrate specificity. It can mediate the methylation of both anthocyanins and flavonols and prefers 3’,5’ methylation when 3’,4’,5’ hydroxylated substrates are available. Further studies indicated that it is located in cytoplasm and had the highest expression at the onset of ripening, indicating it plays a crucial role in biosynthesis of anthocyanins in grape berries (Hugueney et al. 2009). Acylation is one of the most common modifications of plant phenolics, including anthocyanins, resulting in greatly increased structural diversity of anthocyanins from the addition of aromatic and/or aliphatic constituents linked to the C6” positions of the glucosyl groups. Without the protection of acylation, anthocyanins can be easily and quickly decolorized in neutral or weakly acidic aqueous solutions. Besides its beneficial effect on color stabilization, acylation

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can also promote color intensity for the anthocyanins, for the stacking of the polyphenolic moieties of the acylated anthocyanins can further stabilize the color of anthocyanins and may play an important role in the blue color shift (Yonekura-Sakakibara et al. 2009). The acylation of anthocyanins in plants are catalyzed by the action of anthocyanin acyltransferases (ACT, also known as AAT), which have really high substrate specificity, for both the anthocyanin acceptors and the acyl group donors. In plants, there are mainly two types of ACTs that are classified based on the acyl group donors: the BAHD family using acyl-CoA and the serine carboxypeptidaselike (SCPL) group using acyl-activated sugars (D’auria 2006). Although in some important V. vinifera cultivars the acylated anthocyanins can account for more than 60% of the total anthocyanin content, until now there has been no report about the exact genes required for ACTs in grapes. As shown above, anthocyanins are synthesized by an extremely complex network of all the structural enzymes in the pathway. Color variation of the grape berries accord with the particular pattern of genotype-specific expression of the whole set of genes involved in anthocyanin biosynthesis in a direct transcript– metabolite–phenotype relationship. For an efficient production of anthocyanins in different steps, it is speculated that all of the key enzymes involved in the anthocyanin biosynthesis are associated with each other to form a multi-enzyme complex.

18.2.3 Transcriptional regulation of anthocyanin biosynthesis As the secondary metabolites of the flavonoid pathway, anthocyanins are synthesized under the complex regulation of multiple regulatory genes at the transcriptional level (Winkel-Shirley 2001; Koes et al. 2005). Generally, the basic flavonoid upstream pathway of anthocyanin biosynthesis (down to the synthesis of anthocyanidins) is under the control of several different  families of regulatory genes in plants, such as MYB transcriptional factors, MYC transcriptional factors (encoding basic helix–loop–helix proteins, bHLH), and WD40-like proteins, which also play crucial roles in the regulation of other flavonoid products, such as flavonols and proanthocyanidins (Xie and Dixon 2005; Tian et al. 2008). Flavonoid pathway genes are known to be coordinately induced and transcription factors that directly regulate the expression of the structural genes of the pathway have been identified in several species (Jaakola 2013). Regulation of the pathway occurs by the interaction of DNA-binding R2R3 MYB transcription factors and MYC-like basic helix–loop–helix (bHLH) and WD40repeat proteins (Allan et al. 2008; Matus et al. 2008). The expression pattern and the DNA-binding specificity of MYB proteins and, to some extent, bHLH proteins also determine the subset of genes that is activated, whereas WD40 proteins seem to have a more general role in the regulatory complex (Hichri et al. 2010). Plant MYB proteins are involved in the regulation of the pathways of diverse secondary metabolites, signal transduction, developmental changes, and disease

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resistance (Czemmel et  al. 2009). MYB genes contain structurally conserved, 100–160-bp DNA-binding domains comprising single or multiple repeats. The R2R3 MYB genes associated with the flavonoid pathway feature two repeats and represent the most abundant class of MYB genes in plants. Most of the MYBs involved in the control of flavonoid biosynthesis are positive regulators that enhance the expression of the structural flavonoid pathway genes. Repressors have also been characterized, such as FaMYB1 in strawberries (Fragaria x ananassa Duch.) and VvMYB4 in the berries of grapevine (Aharoni et al. 2001). In fruits, particularly in the grapevine, the regulation of flavonoid biosynthesis has been studied intensively and 14 flavonoid biosynthesis-related R2R3 MYB family members have been described (Fournier-Level et al. 2010). It is known that different R2R3 MYB family members can control separately the biosynthesis of the end products of different flavonoid pathway branches leading to anthocyanins, flavonols, and proanthocyanins (Akagi et al. 2009). The biosynthesis of anthocyanins in grapevine berries is regulated by VvMYBA1 and VvMYBA2, which are homologues of Arabidopsis AtMYB75, AtMYB90, AtMYB113, and AtMYB144 (Azuma et  al. 2008). The homologues of these transcription factor genes have also been shown to be involved in the regulation of anthocyanin biosynthesis in members of the rosaceous family (Kui et al. 2010). Polymorphisms of the VvMYBA family have been reported to be responsible for variations in anthocyanin content in different grapevine varieties (Walker et  al. 2007). The MYB proteins are believed to be key components in the allocation of specific gene expression patterns, whereas the bHLH proteins, other members of the regulatory complex, may have overlapping regulatory targets. In fruits, bHLH proteins involved in flavonoid biosynthesis have been characterized in grapevine fruits and strawberries (Matus et  al. 2010; Xie et  al. 2012). In grapevine fruits, the bHLH transcription factor VvMYC1 was shown to interact with different MYB proteins (Vv MYB5a, VvMYB5b, VvMYBA1/A2, VvMYBPA1) to induce promoters of flavonoid pathway genes involved in biosynthesis of anthocyanins and proanthocyanidins.

18.2.4 Metabolic engineering for improved tolerance to pathogens and pests Enormous progress has been made in the understanding of plant responses to biotic stresses, mainly of plant-pathogen interactions, and several strategies to develop pathogen and pest resistant plants by metabolic engineering are being tackled (Campbell et al. 2002; Grover and Gowthaman 2003; Tenllado et al. 2004). The resistance process, mediated by the accumulation of endogenous salicylic acid (SA), a metabolite downstream the biosynthetic pathway initiated by phenylalanine ammonialyase (PAL), is called systemic acquired resistance (SAR) and is based on the induction of secondary metabolic pathways and the increased synthesis of products, phenolic compounds among them, by this metabolism as a response to pathogen attack (Iriti et al. 2005).

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Such strategies include the utilization of chitinases, which degrade chitin in fungal cell walls. Several chitinase-enriched transgenic plants have shown enhanced resistance against B. cinerea, including cucumber (Tabei et al. 1998; Kishimoto et al. 2004) and tobacco (Terakawa et al. 1997). Strawberry phenolic metabolism was modified by introducing the stilbene synthase gene (NS-Vitis3) from frost grape (Vitis riparia Michx). The effects of the transgene were studied at the DNA, RNA, and protein levels, and by comprehensive metabolite screening. All these approaches together allowed the interpretation of the unexpected consequences of the genetic modification, eventually leading to the characterization of a metabolite class not previously known to be present in strawberries. Elicitation is a process used to mimic the natural reactions of plants toward various environmental stresses and has been successfully applied to plants and plant cell cultures to induce secondary metabolite production (Dixon 2001).

18.3 Enhanced secondary metabolite production by elicitation Elicitors are compounds stimulating any type of plant defense. This broader definition of elicitors includes both substances of pathogen origin (exogenous elicitors and compounds released from plants by the action of the pathogen (endogenous elicitors). Elicitors could also be used as enhancers of plant-secondary-metabolite synthesis and could play an important role in biosynthetic pathways to enhanced production of commercially important compounds. The increased production, through elicitation, of the secondary metabolites from plant cell cultures has opened up a new area of research, which could have important economical benefits for the bio industry. Elicitors could also be used as enhancers of plant-secondary-metabolite synthesis and could play an important role in biosynthetic pathways to enhanced production of commercially important compounds. To improve the phenolic content of fruits, a novel field of interest is based on results obtained using elicitors, agrochemicals that were primarily designed to improve resistance to plant pathogens. In the absence of any attack, these defense mechanisms may be induced by physical or chemical elicitation. Physical elicitors include, for example, high and low temperatures and ultraviolet and gamma radiation. The stilbene content of Monastrell grapes irradiated with UV-C light was found to be higher than in control grapes and the final wine made from UV-C-irradiated grapes was about 2- and 1.5-fold enriched in resveratrol and piceatannol, respectively, compared with the control wine (Cantos et al. 2003). Postharvest treatments involving UV irradiation increased the level of trans-resveratrol in apples (Crifò et  al. 2011) and of volatile and nonvolatile phenols in blueberries (Eichholz et  al. 2011).

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Cold stress in blood oranges induced transcriptomic modifications directed toward increased flavonoid biosynthesis (Crifò et al. 2011). Similarly, postharvest carbon dioxide treatments induced proanthocyanidin synthesis in grapes (Becatti et al. 2010). Chemical elicitors, such as chitosan, benzothiadiazole (BTH), harpin, and 1-methylciclopropane, among others, are agrochemicals that can mimic the action of the signaling molecules salicylic acid (SA) and jasmonates (JA) and their derivates, or simulate the attack of a pathogen. These molecules may interact with receptors in the plant, activating defense responses and triggering, in some cases, a hypersensitive reaction. The incidence of disease caused by Penicillium expansum in BTH-treated peaches was lower than in nontreated ones. Besides the higher resistance, the treated fruits presented higher levels of phenolic compounds, lignin, and chlorogenic acid (Liu et al. 2005). The oxidative burst in tomatoes can be elicited by hyphal wall components isolated from Phytophthora spp. as an internal emergency signal to induce the metabolic cascade involved in active defense (Doke et al. 1996). For this reason, elicitors were primarily designed to improve plant resistance against pathogens. These compounds do not kill pathogens but trigger plant defense mechanisms, among them the production of increased levels of phenolic compounds. The effect of the application of different elicitors to plants also proved a useful technique for improving their phenolic content.

18.3.1 Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) This compound is an analog of salicylic acid with a molecular weight of 136.17. It was synthesized during a project directed at synthesizing sulfonylurea herbicides, where the formation of 2-benzylthio-3-furanylbenzoic acid methyl ester was expected rather than BTH. The ability of BTH in triggering SAR was soon discovered (Kunz et al. 1997). BTH induces the activation of the enzyme PAL, as observed after postharvest treatment in mangoes (Zhu et al. 2008) and peaches (Liu et al. 2005). In both studies, an increase in total phenolic compounds was also observed. Other enzymes from plant metabolism were activated by this elicitor, including glucose6-phosphate dehydrogenase, shikimate dehydrogenase, tyrosine ammonia lyase, PAL, cinnamate-4-hydroxylase (C4H), 4-coumarate/coenzyme A ligase (4-CL), and dihydroflavonol 4-reductase (DFR) (Cao et al. 2010). Postharvest treatments in bananas and mangoes also resulted in the activation of polyphenol oxidase (PPO) and peroxidases (POD) and an increased total phenolic content (Zhu and Ma 2007; Lin et al. 2011). However, the effect on flavonoid metabolism might be species-dependent since PAL was inhibited, whereas POD and PPO were activated by postharvest BTH treatment in loquat (Zhu et al. 2007).

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Besides the above-mentioned studies on enzyme activities, the overall effect of BTH on polyphenolic compounds has been investigated in a variety of fruits. For instance, preharvest treatment with BTH of strawberries in greenhouses has proved to be useful for preventing powdery mildew and increasing the content of quercetin and kaempferol (Anttonen et al. 2003), it also enhanced the accumulation of ellagic acid, ellagitannins, p-coumaric acid, gallic acid, and kaempferol hexose in leaves, and kaempferol malonylglucoside in fruits (Hukkanen et al. 2007), while increasing the amount of quercetin and kaempferol in berries (Karjalainen et  al. 2002). Field treatments of grapevine with BTH improve resistance to Botrytis cinerea and enhance resveratrol and anthocyanin biosynthesis (Iriti et al. 2004). Similar studies also found an increase in total polyphenols in berry skin, particularly the proanthocyanidin fraction (Iriti et  al. 2005; Fumagalli et  al. 2006), and in the anthocyanin, flavonol, and proanthocyanidin content of grapes and the color of the corresponding wines (Ruiz-Garcia et al. 2012). Postharvest treatment with BTH resulted in an enhancement of the phenolic and anthocyanin contents of strawberries (Cao et al. 2011).

18.3.2 Methyl jasmonate (mej) MeJ is a plant volatile derived from JA with a molecular weight of 224.3. It has similar activity to JA in plants and so is able to activate the enzymes responsible for the biosynthesis of polyphenols, such as the PAL enzyme. The activation of PAL following postharvest application of the elicitor has been confirmed in many studies in fruits such as lychees (Yang et  al. 2011), peaches (Jin et  al. 2009), apples, plums, table grapes, and strawberries (Heredia and CisnerosZevallos 2009) with a subsequent increase of total phenols. The activation of CHS, STS, UDP glucose flavonoid-O-transferase (UPGT), proteinase inhibitors, and chitinase gene expression has also been reported in preharvest treatments of grapevine with MeJ. Such activations triggered the accumulation of both stilbenes and anthocyanins in cells (Belhadj et al. 2008). In a different fruit, red raspberries, the enhancement in the levels of myricetin, quercetin, and kaempferol has also been reported after postharvest treatment with MeJ (Moreno et al. 2010a). Several other studies on different fruits describe how MeJ affects polyphenol compounds. For example, postharvest treatment with MeJ resulted in higher amounts of total phenols and anthocyanins in strawberries (Moreno et al. 2010b) and bayberries, in which an increase of other flavonoids was also found (Wang et  al. 2010). Finally, preharvest treatment with MeJ has been shown to enhance the levels of flavonoids in blueberries (Percival and Mackenzie, 2007), blackberries (Wang et al. 2008), apples (Shafiq et al. 2011), and grapes (Ruiz-Garcia et  al. 2012) and resveratrol levels in strawberries (Wang et al. 2007) and grapevine cultivars (Vezzulli et al. 2007; Esna-Ashari and Pour 2011).

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18.3.3 Harpin Harpin is a heat-stable, glycine-rich protein of bacterial origin. This protein was first described as being produced by the plant pathogen Erwinia amylovora, the bacterium that causes fire blight in pear, apple, and other rosaceous species (Wei et al. 1992). Harpin is able to provoke a hypersensitive response in nonhost plants. This response is characterized by a rapid localized cell death at the site of the invasion (He et al. 1993), for which reason it is able to act as a chemical elicitor. Moreover, this protein activates ROS burst, SA, and JA/ethylene signal transduction pathways that confer SAR to different plants (Dong et  al. 1999, 2004; Li et al. 2012b). Harpin has also been described as an elicitor able to activate enzymes such as PAL from the polyphenol biosynthesis pathway. Examples of this have been found in several fruits, for instance, in postharvest-treated peaches and jujube, with a subsequent increase in total phenols (Danner et al. 2008; Li et al. 2012a).

18.3.4 Chitosan Chitosan is a polysaccharide resulting from the deacetylation of chitin, the linear polymer of (1-4)-β-linked N-acetyl-D-glucosamine. It is obtained from the outer shell of crustaceans such as crabs and shrimps. This polysaccharide has a positive charge that confers specific physiological and biological properties that are found useful in different industries such as the cosmetics, food, biotechnology, pharmacology, medicine, and agriculture industries (Bautista-Banos et al. 2006). Even though it has antimicrobial properties, there is strong evidence that it can act as an elicitor by inducing the production of callose and phenolics in susceptible plants (Gozzo 2003). The extent of the antimicrobial action of chitosan is influenced by factors such as its molecular weight (MW) and degree of acetylation (DA). However, it is difficult to find a clear correlation between these two characteristics and the antimicrobial activity. In general, as the DA increases, the antimicrobial activity is enhanced, since chitosan with a high DA dissolves in water completely, leading to an increased chance of interaction between chitosan and the negatively charged cell walls of microorganisms. Similarly, as the MW increases, chitosan activity against pathogens increases, but, above a certain value, the effect is reversed (Badawy and Rabea 2011; Zhang et al. 2011). Applications of chitosan in the field proved to be effective in controlling postharvest diseases in strawberries (Reddy et al. 2000; Romanazzi et al. 2000) and in jujubes, where it activated defense-related enzymes to reduce postharvest decay (He et al. 1993). Postharvest applications of a coating composed of chitosan and Origanum vulgare L. essential oil at subinhibitory concentrations were able to control Rhizopus stolonifer and Aspergillus niger in grapes (Dos Santos et al. 2012). Many pre- and postharvest treatments with chitosan have demonstrated that this compound can activate the enzyme PAL and increase total polyphenols in table grapes, controlling storage gray mold (Romanazzi et al. 2002) and activating PPO (Meng et al. 2010). It may also enhance the activity of defense-related enzymes in bananas (Meng et  al. 2012) and increase the amount of total

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polyphenols in strawberries (Mazaro et  al. 2008). In addition, chitosan has proved to be effective at controlling powdery mildew and at increasing the total polyphenol content of grapes. Moreover, wines made from chitosan-treated grapes showed a higher total polyphenol content and antiradical power than those made from fungicide-treated and untreated grapes (Iriti et al. 2011).

18.3.5 Other elicitors Many other chemical substances have been studied as possible elicitors in different fruits. For example, oxalic acid and calcium chloride enhanced defenserelated enzyme activities—such as β-1, 3-glucanase, PAL, POD, and PPO—and reduced disease incidence caused by Alternaria alternata in pears (Tian et  al. 2006b); Burdock flucto-oligosaccharide and oligandrin inhibited postharvest disease caused by B. cinerea in tomatoes and activated PAL, enhancing the biosynthesis of phenolic compounds (Wang et  al. 2009, 2011); phosphite and acibenzolar-S-methyl induced the synthesis of trans-resveratrol in apples (Sautter et  al. 2008) and, finally, potassium silicate increased the amounts of catechin and epicatechin in avocados (Tesfay et al. 2011).

18.4 Conclusion Metabolomics and other omics technologies are increasingly being adopted to support metabolite-engineering projects, and they should become a standard feature in the future. The understanding of metabolic networks by identifying (often unexpected) correlations and links between different metabolites will thereby facilitate the process of hypothesis generation. In order to approach the goal of metabolic engineering that is based on comprehensive metabolic control analysis, however, innovations in technology and methods are required.

References Ageorges, A., L. Fernandez, S. Vialet, D. Merdinoglu, N. Terrier, and C. Romieu. 2006. Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries. Plant Sci. 170:372–83. Aharoni, A., C. H. R. De Vos, M. Wein, Z. K. Sun, R. Greco, A. Kroon, J. N. M. Mol, and A. P. O’connell. 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant Journal. 28:319–32. Akagi, T., A. Ikegami, T. Tsujimoto, S. Kobayashi, A. Sato, A. Kono, and K. Yonemori. 2009. DkMyb4 Is a Myb Transcription Factor Involved in Proanthocyanidin Biosynthesis in Persimmon Fruit. Plant Physiology. 151:2028–45. Allan, A. C., R. P. Hellens, and W. A. Laing. 2008. MYB transcription factors that colour our fruit. Trends in Plant Science. 13:99–102. Anttonen, M., A. Hukkanen, K. Tiilikkala, and R. Karjalainen. 2003. Benzothiadiazole induces defence responses in berry crops, In: Berry Crop Breeding, Production and Utilization for a New Century, ed. by P. Hicklenton, and J. Maas, pp. 177–82.

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CHAPTER 19

Food-derived multifunctional bioactive proteins and peptides: Sources and production Dominic Agyei1, Ravichandra Potumarthi1, and Michael K. Danquah1,2 1 2

Bioengineering Laboratory, Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia Department of Chemical Engineering, Curtin University of Technology, Sarawak, Malaysia

19.1 Bioactive peptides: Overview Certain food formulations, aside from offering nutrition, are capable of performing biological functions in living body systems to influence health positively. The biological activities exhibited by such formulations are attributable to the presence of certain biomolecules in forms and quantities that ultimately trigger beneficial health effects when administered into the body. Over the past few decades, research has uncovered several bioactive components from food-derived macro-biomolecules such as carbohydrate polymers, lipids, and proteins, among which protein-derived biomolecules are the most diverse and most widely studied (Danquah and Agyei 2012). Some food protein molecules and their hydrolysates have been shown to trigger certain “hormone-like” responses in vivo and/or in vitro (Korhonen 2009). These protein hydrolysates are also called bioactive peptides and can be defined as specific protein fragments, which, once ingested and absorbed, have the ability to meet two conditions: (1) it must impart a physiologically measurable biological effect that impact body functions or conditions and (2) it must ultimately influence health positively (Kitts and Weiler 2003; Korhonen and Pihlanto 2006). The second caveat is very important since it excludes potential damaging effects such as toxicity, allergenicity, antigenicity, and mutagenicity—all of which are forms of “bioactivities” (Möller et al. 2008). Bioactive peptides usually contain 2–20 amino acid residues (Kim and Wijesekara 2010) and can be obtained from several food proteins. Bioactive peptides can be released from proteins found in almost all organisms on the taxonomic level (Table 19.1). The number of bioactive peptides obtainable from food proteins is therefore limitless and offers alternatives for individuals who are averse to products from one taxonomic group. For example, alternative Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Table 19.1 Sources of bioactive peptides. Source of bioactive peptide Animal products

Milk and fermented milk products (yogurt, Tarag, sour milk, etc.) Eggs

Cheese Beef Pork Seafood Fish

Plant sources

Chicken Rice and rice products (sake) Corn Soy and soy products Brassica carinata Wheat Broccoli

Algae

Fungi (mycoproteins)

Sweet potato Garlic (Allium sativum) Pulse crops (pea, chickpea, and lentil) Capsicum annuum, Nigella sativa, Sesamum indicum, and Cuminum cyminum Red algae Macroalgae Microalgae; Chlorella vulgaris 87/1, Spirulina platensis Mushroom Fusarium venenatum Brewer’s yeast Tricholoma giganteum

Reference(s) (Hayes et al. 2007; Peng 2009; Atanasova and Ivanova 2010) (Mine and Kovacs-Nolan 2006; Chalamaiah et al. 2010; You et al. 2010) (Silva et al. 2006; Sienkiewiczszłapka et al. 2009) (Jang and Lee 2005) (Li et al. 2007a; Jang et al. 2008) (Battison et al. 2008; Kim and Wijesekara 2010) (Klompong et al. 2007; Hermannsdottir et al. 2009; Yang et al. 2009) (Cave 2004) (Saito et al. 1994; Takahashi et al. 1994; Li et al. 2007b) (Miyoshi et al. 1995; Kim et al. 2009) (Yamauchi and Suetsuna 1993; Gibbs 2004; Kong et al. 2008) (Pedroche et al. 2007) (Motoi and Kodama 2003; Horiguchi et al. 2005) (Lee et al. 2006a; Hartmann and Meisel 2007) (Ishiguro et al. 2012) (Kunio 1998) (Roy et al. 2010) (Orlovskaya et al. 2010)

(Aneiros and Garateix 2004) (Lordan et al. 2011) (Morris et al. 2009; Sheih et al. 2009) (Lu et al. 2010) (Sun et al. 2004) (Brownsell 2001) (Kanauchi et al. 2005) (Hyoung Lee et al. 2004)

Food-derived multifunctional bioactive proteins and peptides

Gastrointestinal system

Immune system

Nervous system

Mineral chelation Anorexigenic Antimicrobial Anticariogenicity Antigastric

Antimicrobial Immunomodulatory Cytomodulatory

Opioid antagonistic and agonistic acivities Antiamnesic Anxiolytic

485

BIOACTIVITIES

Calcium binding properties

Antihypertensive Antioxidative Antithrombotic Hypocholesterolemic Hypolipidemic

Musculoskeletal system

Cardiovascular system

Figure 19.1 Bioactivities of milk protein–derived peptides and the influences body systems.

sources such as plants and mycoproteins exist for vegetarians who may be opposed to the isolation of bioactive peptides from animal proteins. Bioactive peptides are often involved in various biological functions depending on their amino acid composition and sequence. They have numerous applications in the health, pharmaceutical, and food industries due to the many physiological responses they trigger, including antihypertensive, opioid agonists or antagonists, immunomodulatory, antithrombotic, antioxidant, anticancer, anxiolytic, antiamnesic, anorexigenic, and antimicrobial activities that influence various major body systems such as digestive, cardiovascular, nervous, musculo-skeletal, and immune (Gibbs 2004; Korhonen and Pihlanto 2006; Hartmann and Meisel 2007; Möller et al. 2008; Korhonen 2009; Yang et al. 2009) (see Figure 19.1). The discovery of bioactive peptides has therefore been the subject of growing commercial and research interest in the past few decades (Korhonen and Pihlanto 2006).

19.2 Multifunctional food peptides Proteins, due to their three-dimensional shapes are among the key biomolecules that exhibit a strong structure–function relationship. It is therefore no wonder that protein hydrolysates also carry this trait. The type, amount, and sequence of amino acids of a bioactive peptide are what determine the functional property of

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the peptide. Structural characteristics such as the type (i.e. presence of cysteine groups that form disulphide bridges) and the nature (charge, basicity, and hydrophobicity) of key amino acids in the primary sequence influence the threedimensional (3D) conformation of a peptide, thus affecting its function (Hancock and Sahl, 2006; Korhonen and Pihlanto 2006). The binding characteristics of bioactive peptides, on the other hand, have been attributed largely to the secondary structure rather than the tertiary structure (Kaur et al. 2007). That key amino acids play significant roles in determining bioactive peptide functionalities is demonstrated by a number of studies. Most antimicrobial peptides have been known to be short, hydrophobic, and cationic in nature (Hancock and Sahl 2006). Also, the amino acid proline, usually present at the carboxyl terminal end, plays a significant role in the well-studied ACE inhibitory lactotripeptides (valine-proline-proline and isoleucine-proline-proline). Proline is known to be resistant to degradation by digestive enzymes and this allows most proline-containing peptides to survive proteolytic attack en route in living body systems (Korhonen and Pihlanto 2006). Several other ACE inhibitors have a proline residue in the C-terminal position and it has been shown that Pro and Val in certain peptide sequences (such as Leu-Arg-Pro-Val-Ala-Ala-Glu, a peptide in lactoferrin) are essential for ACE inhibitory activity (Lee et al. 2006b). Glycine has also been implicated in the action of some immunomodulatory peptides (Tyr-Gly and Tyr-Gly-Gly), which have been shown to enhance the proliferation of human peripheral blood lymphocytes (Pihlanto-Leppälä 2002). As well, the imidazole ring in histidine or histidine-containing peptide is responsible for certain antioxidant properties where chelating and lipid radical-trapping abilities are exhibited (Je et al. 2005). Interestingly, some bioactive peptides are able to trigger two or more physiological roles in body systems, making these multifunctional bioactive peptides exhibit more than one bioactivity. Within the body systems, it is observed that bioactivity in one area often results in a beneficial effect in another. For example, the control of microbial hosts in the body often conduces into other benefits such as an enhancement in the body’s innate immunity. Thus, bioactivities and their effects on various parts of a living body system are interlinked. Also, since the symptoms of most diseases and ill-health conditions are often more than one, it follows that multifunctional peptides can be used to target some of these multiple pathological situations of some multisymptom illnesses simultaneously (Li  and Aluko 2010; Sistla 2013). Examples of multifunctional peptides and their mode of action are presented next.

19.3 Milk-derived multifunctional proteins and peptides 19.3.1 Lactoferrin and its hydrolysates Lactoferrin (LF) is a very potent protein identified in and obtained from secretory fluids, especially in human milk proteins (Sanchez et  al. 1992). It is a nonimmune globular and basic glycoprotein with molecular weight of about

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76–80 kDa and isoelectric point of 8.7. It consists of a single polypeptide chain of about 700 amino acids folded into two globular domains, the N- and C-lobes, which are connected to each other with a short α-helix (Baker and Baker 2005). Each of these two lobes contains two subdomains, with one glycosylation site and one iron-binding site. The difference in the degree of glycosylation is the reason for the variable molecular weight of human LF (Håkansson et al. 1995; Levay and Viljoen, 1995). Considered to be a member of the transferrin family, LF is a metal-binding protein. Each LF molecule is able to reversibly bind two ions of iron, zinc, copper, or other metals (Levay and Viljoen 1995). The binding clefts are found in each of the two protein domains. Each metal ion is bonded in the cleft in a hexadentate fashion with four bonds supplied by the polypeptide chain (i.e., two tyrosine residues, one histidine residue and one aspartate residue) and two from anions (carbonate or bicarbonate) (Nuijens et al. 1996) (see Figure 19.2). LF is absent from the milk of some species but is present in human milk at high quantities, especially in colostrum (7 g/L) (Sanchez et  al. 1992; Nuijens et  al. 1996; Atanasova and Ivanova 2010), signifying the key multifunctional roles played by this protein in infant human health (Sanchez et al. 1992). LF has attracted a huge research interest in recent years due to the wide spectrum of functions that have been ascribed to it. These multifunctionalities range from a role in the control of iron availability to immune system modulation. Specifically, LF has been shown to have very diverse functionalities such as antibacterial, antifungal, antiviral, antiparasitic, antitumor, anti-allergenic, anticancer, and immunomodulatory activities (Levay and Viljoen 1995; Baker and

Figure 19.2 Structure of recombinant human lactoferrin expressed in Aspergillus awamori.

Generated with Rasmol. PDB file based on 1b0l (http://dx.doi.org/10.2210/pdb1b0l/pdb) (Sun et al. 1999). For color detail, please see color plate section.

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Food-derived multifunctional bioactive proteins and peptides

Lactoferrin

Metal ions (bacterial substrate)

No substrate for bacterial growth after metal ion chelation

Figure 19.3 Antibacterial activity mechanism of LF. Essential metal ion substrates required for bacterial growth are chelated by LF.

Baker 2005; Baldi et al. 2005; Cecchini and Caputo 2009; Daley 2009; Kanwar et  al. 2009; Fernández-Musoles et  al. 2013; Korhonen and Marnila 2013; Lönnerdal 2013). In fact, well over 17 biological roles have been proposed for LF (Brock 2002). Interestingly, other peptide fractions from LF have also been shown to possess various multifunctional properties. The pepsin-mediated hydrolysate of LF, lactoferricin, has also been shown to possess anticancer, antitumor, and antimicrobial properties (Eliassen et  al. 2002; Korhonen and Pihlanto 2006). Lactoferricin is an amphipathic, cationic peptide and in humans consists of the fragment 1-47 of LF (Théolier et al. 2013). The multifunctional roles played by other LF hydrolysates have also been highlighted. A patent has actually been filed where LF hydrolysates are used to improve several physiological functions of the musculoskeletal system, including skeletal growth stimulation, bone resorption inhibition, chondrocyte proliferation stimulation, osteoblast proliferation stimulation, osteoclast development inhibition, and the treatment or prevention of skeletal, joint, or cartilage disorders (Cornish et al. 2007). Furthermore, a human LF–derived synthetic peptide with the cationic cluster Gly-Arg-ArgArg-Arg has been shown to be effective in preventing hepatitis B virus (HBV) infection. This peptide may constitute a nontoxic clinical approach in inhibiting HBV by neutralizing the viral particles (Florian et al. 2013). The various proposed physiological functions of LF is conferred by its most characteristic biochemical properties, namely, the ability to bind iron ions and its highly basic nature (Brock 2002). The antibacterial property of LF is well understood and attributable to two main mechanisms: metal ion sequestration and interaction with the outer bacterial membrane (the most dominant mechanism). The ability of LF to sequester free ions helps remove these essential metal ion substrates that may be required for bacterial growth (see Figure 19.3). Some of the bioactive functions of LF are closely linked to the peptide’s metal-ion chelation properties while others appear to be independent of ion binding (Farnaud and Evans 2003). Bovine LF has been shown to have strong antiviral activities against HIV, HSV, and poliovirus infections. However, it takes iron- and zincsaturated LF protein to exhibit this property (Berlutti et al. 2011; Florian et al. 2013). This was demonstrated by the fact that iron- and zinc-saturated bovine

Food-derived multifunctional bioactive proteins and peptides

Lactoferrin

Bacterial cell

LF binds to LPS and alters membrane permeability via peroxide formation

489

Cell lysis

Figure 19.4 Antimicrobial action of LF by cell envelope disruption. LPS, lipopolysaccharide.

LF inhibited HBV-DNA amplification in HepG2-infected cells whereas no such effect was observed for metal-free LF hydrolysates (Li et al. 2009). Most antimicrobial bioactive peptides act by penetrating and disrupting microbial membrane integrity or (depending on size and hydrophilicity) by translocating across the membrane and acting on internal targets (Hancock and Sahl 2006; Steinstraesser et al. 2011). Similarly, the second and most dominant mechanism for the antimicrobial action of LF is via interaction with the outer bacterial membrane (Odell et al. 1996). LF binds to an anionic site of the bacterial cell envelope (Farnaud and Evans 2003). Binding is achieved with the N-lobe of LF, the part containing the sequence for lactoferricin (Sojar et  al. 1998). The presence of oxidized iron assists the formation of peroxides, which, as free radicals, perforate holes in the cell envelope, thus affecting membrane permeability and leading to cell lysis (see Figure 19.4). LF prevents gastral infection by Helicobacter pylori through this mechanism (Wang et al. 2001). LF has also been shown to be bactericidal for Streptococcus mutans, Streptococcus pneumoniae, Escherichia coli, Vibrio cholerae, Pseudomonas aeroginosa, and Candida albicans (Arnold et al. 1980). LF and its hydrolysates are without a doubt multifunctional biomolecules. However, few clinical trials have been carried on the potency of LF. The actual mechanisms of action of the various functionalities are also not fully understood (Farnaud and Evans 2003; Berlutti et  al. 2011). New technologies and more structural information will help unravel this mystery. In infectious disease control, there is a threat to the treatment of several infective diseases due to the development of resistant mechanisms and the increase of antibiotic resistance of various pathogenic microbes (Wang et  al. 2001). New treatment strategies are therefore sought after. LF and its hydrolysates demonstrates variable bactericidal and/or bacteriostatic susceptibilities against a wide range of pathogenic microbial types including Gram-positive and Gram-negative microbes, rods and cocci, facultative anaerobes, and aerotolerant anaerobes (Arnold et al. 1980). That LF has a wide spectrum of functionalities with varied mechanisms shows its potential in nonantibiotic treatment strategies for various diseases.

490

Food-derived multifunctional bioactive proteins and peptides

19.3.2 Casein-derived phosphopeptides in milk Casein phosphopeptides (CPPs) are obtained from milk and are produced as a result of the hydrolytic action of trypsin on caseins. CPPs have the structure sequence –Ser (P)-Ser (P)-Ser (P)-Glu-Glu– and thus often contain a sequence of three phosphoseryl residues followed by two glutamic acid residues. The phosphate groups represent about of 30% of the phosphorus content in milk and the high negative charges conferred by the phosphate groups makes them resistant to further proteolysis (Meisel 1998; Clare and Swaisgood 2000). Thus, CPPs have been detected in ileostomy fluid, showing their ability to survive proteolytic attack during gastrointestinal passage in humans (Silva and Malcata 2005). Also, the phosphate groups on CPPs are exposed on the surface, creating a negatively charged polar and acidic domain for binding with divalent calcium ions (Fan et al. 2013). Several CPPs and their multifunctional roles such as anticariogenic properties, antihypertensive properties, immune enhancing effects, and cytomodulatory effects have been reported in the scientific literature (Mazzaoui et al. 2003; Meisel and FitzGerald 2003; Meisel 2004; Silva and Malcata 2005; Fan et  al. 2013). For example, CPPs have been shown to possess anticariogenic potential through their ability to stabilize amorphous calcium phosphate (ACP), which, once localized in dental plaque, could maintain a state of supersaturation reducing demineralization and enhancing remineralization (Rose 2000; Walker et al. 2006, 2009). The anticariogenic properties of milk have been attributed to the calcium and phosphate contents present in casein (Reynolds 1998) and the bioavailability of these components are enhanced when complexed with ACPs (Reynolds et al. 2003; Walker et al. 2006). The ability of CPP-fortified products to remineralize enamel subsurface lesions (early forms of tooth decay) and bone calcification has been demonstrated in vitro (Moany and Hegde 2012) and in human in situ studies (Walker et al. 2006, 2009) and clinical trials (Morgan et al. 2008). In fact, CPPs were the first bioactive peptide to be reported in literature where their role in enhancing vitamin D–independent bone calcification in infants with rickets was highlighted (Mellander, 1950). Currently, studies on CPPs are well advanced. There is a commercial product on the market under the trade name of Recaldent™ that has CPP-ACP complex as the active ingredient. CPP is also used in other products such as chewing gum, mouth rinses, and lozenges, based on their anticariogenic effects (Meisel 2001; Walker et al. 2009). Based on their mineral solubilization properties, other physiological benefits of CPPs include the prevention of anemia, hypertension, and osteoporosis (Korhonen and Pihlanto 2006). Another commercially available caseinophosphopeptide preparation, CPP-III, has immunoenhancing activity that can be used in enhancement of mucosal immunity (Otani et al. 2000). Recently, CPPs have been shown to have antigenotoxic potential, demonstrating their use in the making of formulations that can be used by workers exposed to low levels of radiation as an occupational hazard (Balaji Raja and

Food-derived multifunctional bioactive proteins and peptides

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Arunachalam 2011; Arunachalam et al. 2012). In these studies, CPP was produced by the action of trypsin on milk fermented by Lactobacillus acidophilusa and the radioprotective role of CPP was studied using albino mice and Catla catla fish (Balaji Raja and Arunachalam 2011). Cancer is a global burden. The search continues for safe and natural radioprotective agents with no side effect and that can be used in the control of radiation-induced cancers (Doll 1992; Arunachalam et al. 2012). CPPs therefore have potential to be used in the development of a new class of food-based peptide radioprotective nutraceuticals that can be used in the reduction and/or prevention of cancers caused by exposure to natural or low background radiation (Arunachalam et al. 2012).

19.3.3 Other milk peptides Chymotryptic peptides from α-S2 casein have demonstrated multifunctional properties. The peptides consist of α-S2 casein fractions 79–88 and f (148–161) with molecular weights of 1,205 and 1,718 Da, respectively (Srinivas and Prakash 2010). Purification and sequencing of these peptides yielded the sequence Gln–Lys–Ala–Leu–Asn–Glu–Ile–Asn–Gln–Phe (p10) and Thr–Lys–Lys–Thr–Lys– Leu–Thr–Glu–Glu–Glu–Lys–Asn–Arg–Leu (p14), and showed multifunctional properties including angiotensin-converting enzyme inhibition, prolyl endopeptidase inhibition, antioxidant, and antimicrobial activities (Sistla 2013). Structure–function relationship was observed in the mode of action of these peptides. Peptide p10 showed less ordered secondary structure, whereas p14 was a beta-rich peptide. Backbone flexibility differs for the two peptides and this could account for the differences in their mode of action. Thus, although both peptides had antimicrobial properties, the mechanisms of action were different. The antimicrobial properties of p10 were attributable to the cell depolarization while that of p14 was due to DNA binding mechanisms (Sistla 2013).

19.4 Egg-derived multifunctional proteins and peptides 19.4.1 Lysozyme Lysozyme has a molecular weight of 14.3 kDa and as a basic protein has an isoelectric point of 10.7 (Mine and Roy 2011). Being widely abundant in hen’s egg, lysozyme is an example of an intact protein that exerts antibacterial activities by acquiring new polymerization conformations in post-processing conditions such as heating (Ibrahim et  al. 1996). This antimicrobial potency of lysozyme is a combined effect of its muramidase activity as well as its cationic and hydrophobic properties (Pellegrini et al. 1997). Interestingly, research has shown that peptic hydrolysates of lysozyme display peculiar bacteriostatic activities independent of the intact protein enzymatic activity (Mine et al. 2004). Recently, bioactive peptides other than antimicrobial

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Food-derived multifunctional bioactive proteins and peptides

peptides from hen’s egg white lysozyme (HEWL) have been identified and antioxidant peptides from HEWL hydrolysates have been reported. The fractionated peptides had higher calmodulin-dependent phosphodiesterase (CaMPDE) inhibition activity and free radical scavenging activity than those of the hydrolysate (You et al. 2010). The peptides species responsible for the antioxidant properties were mainly from motifs of 13–23, 90–108, and 42–57 and also rich in acidic amino acids (Asp and Asn) and positively charged amino acids (Arg, His, and Lys) (You et al. 2010).

19.4.2 Phosvitin and ovalbumin Other researchers have also studied the multifunctional properties of egg yolk protein (phosvitin) hydrolysates (Xu et al. 2007). Other bioactive peptides from egg include ovolin, the tryptic digest of ovalbumin. This peptide has the structure Val-Tyr-Leu-Pro-Arg and has been shown to trigger anxiolytic-like (anxietyrelieving) activity, as shown in a plus-maze tests in mice (Oda et al. 2012). As well, peptide species such as ovokinin (Phe-Arg-Ala-Asp-His-Pro-Phe-Leu) and novokinin (Arg-Pro-Phe-His-Pro-Phe/Trp), which are, respectively, peptic and chymotryptic digest of ovalbumin, have been shown to be potent ACE inhibitors (Fujita et  al. 1995; Mine and Roy 2011). Hen egg proteins are therefore an important source of multifunctional peptides. The peptide-based antioxidants from egg proteins are attractive “natural” candidates for use in food and pharmaceutical products.

19.5 Plant protein-derived multifunctional peptides Beans, legumes, cereals, and pulse crops are highly nutritious food crops and are also abundant in bioactive proteins and peptides. Not only can bioactive peptides be isolated from legumes, but also, by-products of plant protein processing such as cakes could also act as a cheap source of proteins that can be hydrolyzed to release bioactive peptides (Agyei and Danquah 2011). Exploitation of the bioactive peptides from plant sources is therefore gaining research attention. Some multifunctional peptides of plant origin are covered next.

19.5.1 Limenin Limenin is a plant-based peptide that has been reported to have multifunctional properties. In their pioneering work, Wong and Ng (2006) employed chromatographic methods to isolate limenin, a 6.5 kDa peptide from shelf-beans (Phaseolus limensis) and the peptide exhibited antifungal potency, mitogenic activity toward mouse splenocytes, antiproliferative activity toward M1 and L1210 cells, HIV-1 reverse transcriptase inhibitory activity, and antibacterial activity. No research has been conducted on the exact mechanism of action of limenin. However, in the study conducted by Wong and Ng, N-terminal amino acid sequencing of

Food-derived multifunctional bioactive proteins and peptides

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limenin revealed a resemblance to those of other plant defensins. Thus, the antifungal mechanism of limenin could be comparable to that of plant defensins (Wong and Ng 2006).

19.5.2 Rapakinin Rapakinin is a peptide originally encrypted in rapeseed napin proteins and released by the action of subtilisins. It is made of the peptide sequence Arg–Ile– Tyr and acts as a potent agent with vasorelaxing, anti-hypertensive, and anorexigenic activities (Yamada et  al. 2010). Recently, rapakinin was shown to exhibit antiopioid activity after intracerebroventricular administration in mice (Yamada et al. 2011).

19.5.3 Rubisco-derived peptides Rubisco (i.e., ribulose-1,5-bisphosphate carboxylase/oxygenase) is the predominant protein in photosynthesizing plants and is responsible for carbon fixation. It is known to be the most abundant protein on the planet (Feller et al. 2008). Zhao et  al. (2008) have isolated two peptides, rubiscolin-6 (Tyr-Pro-Leu-AspLeu-Phe) and rubimetide (Met-Arg-Trp), from a pepsin–pancreatin digest of spinach Rubisco and found that these peptides have opioid, blood pressure– lowering and anxiolytic-like properties. The abundance of Rubisco in nature highlights its potential for the production of various multifunctional peptides that can be exploited in food and pharmaceutical products.

19.5.4 Soymorphin The soymorphins are a class of μ-opioid agonist peptides derived from the soybean β-conglycinin β–subunit. Soymorphins-5, -6, and -7 have the sequence Tyr-Pro-Phe-Val-Val, Tyr-Pro-Phe-Val-Val-Asn, and Tyr-Pro-Phe-Val-Val-AsnAla, respectively, and each have been shown to have multifunctional properties. Soymorphin-5 has anxiolytic-like and anorexic (satiety-controlling) properties. It is known to improve glucose and lipid metabolism and functions by activating the adiponectin system while ameliorating hyperglycemia after long-term oral administration of the peptide to diabetic mice (Yamada et  al. 2012). As well, soymorphin-5 has been shown to trigger anorexic responses upon oral administration and therefore decreases food intake in fasted mice. The peptide also suppresses the transit of food in the small intestines (Kaneko et al. 2010). This peptide therefore has a huge potential for use in functional foods designed for weight control. Multifunctional peptides have been obtained from other plant proteins such as zein from where ACE inhibitors and prolyl endopeptidase (PEP)– inhibiting peptides have been released via thermolysin treatment (Miyoshi et al. 1995). The sequence Leu-Arg-Pro, Leu-Ser-Pro, and Leu-Gln-Pro were shown to have ACE-inhibition properties, whereas Val-His-Leu-Pro-Pro-Pro showed PEP-inhibition properties (Miyoshi et al. 1995). PEP inhibition is an important

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Food-derived multifunctional bioactive proteins and peptides

property that is known to induce antiamnesic properties. Several of these peptides are therefore being exploited for future use as therapeutic drugs for amnesia (Miyoshi et al. 1995; Yanai et al. 2003; Sørensen et al. 2004; Wilson et al. 2011).

19.6 Perspectives in production Changes in the functional properties of proteins are often achieved via physical, chemical, or enzymatic treatments that alter protein conformation and structure (Li and Aluko 2010). Bioactive peptides are obtainable from protein biomolecules that in themselves do not trigger any physiological response but are only made “bioactive” post treatment. Owing to their biological roles, the development of specific bioprocesses for producing bioactive peptides is of paramount interest in human nutrition. Bioactive peptides are produced by a range of technologies; namely, “up–down degradation,” “bottom-up” construction, and “whole expression” or “extraction” (represented, respectively, pathways 1–3 in Figure 19.5). Each production route has its own merits and demerits. However, particular attention has been devoted to enzymatic hydrolysis. The enzyme-based process is particularly attractive because of the more moderate hydrolysis conditions, less or no undesirable side

Food proteins 1

Enzymatic hydrolysis (in vivo or in vitro)

Bioactive peptides

Solvent extraction methods

Microbial fermentation of proteins

Food processing operations

3

2

Wet peptide synthesis from amino acids

Peptide of interest

Expression in host cells via rDNA technology

Already known food bioactive peptide

Figure 19.5 Alternative routes for the production of bioactive peptides. Adapted from Danquah

and Agyei (2012).

Food-derived multifunctional bioactive proteins and peptides

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reactions and by-products, and the potential to produce high-quality products (Li and Aluko 2010; Agyei and Danquah 2011, 2012c). For the purpose of this discussion, a distinction ought to be made between “bioactive peptides” and “therapeutic peptides.” Here, the term “bioactive peptides” is restricted to food protein–derived peptides having biological activities. This is to the exclusion of chemically synthesized “therapeutic peptides,” often used in pharmaceutical products. It has been argued that the production of bioactive peptides by exploiting the proteolytic properties of microbial proteases is cost-effective, economical, and scalable (Korhonen and Pihlanto 2006; Agyei and Danquah 2011). Produced through this pathway, two major raw materials required are proteins and proteolytic enzymes (proteases). Food proteins are relatively cheap and abundant, thus their usage in the production of bioactive peptides will conduce to a reduction in production cost. With regard to the enzyme source, microbial proteases have several advantages over proteases from other sources. Microbial proteases are relatively cheap, more shelf-stable, easy to harvest and purify, and also exist in a wide variety that offers a range of enzymatic activities and specificities (Khalid and Marth 1990; Gobbetti et al. 1996; Ferrero 2001; Gupta et al. 2002; Agyei et al. 2013b). Owing to their long history of human use and GRAS (generally regarded as safe) status, lactic acid bacteria (LAB) proteases are among the widely used, especially for products intended for food and pharmaceutical consumption (Kaushik et al. 2009). Furthermore, the proteolytic system of the most lactobacilli species have been studied, including Lactobacillus casei (Khalid and Marth 1990; Kojic et al. 1991; Tsakalidou et al. 1999), Lactobacillus delbrueckii subsp. bulgaricus (Laloi et al. 1991), Lactobacillus sanfrancisco CB1 (Gobbetti et al. 1996), Lactobacillus helveticus (Martín-Hernández et al. 1994), and Lactobacillus delbrueckii subsp. lactis ACA-DC 178 and CRL 581 (Tsakalidou et  al. 1999; Espeche Turbay et  al. 2009). Proteases from these lactobacilli species include cell-envelope-associated proteinases (CEP), transport proteins, and a large number of intracellular peptidases such as endopeptidases, aminopeptidases, tripeptidases, and dipeptidases (Khalid and Marth 1990), all of which offer an extremely wide range of caseinolytic activities that are species and strain specific, thus increasing the combination of bioactive peptides that can be obtained from a single food protein. The prospects in application of lactobacilli proteases for the production of bioactive peptides have been a justification for proposing these enzymes as cheap raw materials for the industrial scale production of bioactive peptides (Agyei and Danquah 2011, 2012c; Danquah and Agyei 2012; Agyei et al. 2013b, 2013c). To this end, research is underway aimed at characterizing the growth and production of extracellular proteinases by some lesser known lactobacilli species such as Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830) (Agyei and Danquah 2012a, 2012b; Agyei et al. 2012d, 2013a).

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19.7 Bioprocess-based research challenges 19.7.1 Peptide production Despite the numerous routes for the production of bioactive peptides, there still exist some limitations to the production of large quantities of bioactive peptides that can satisfy growing and future market demands. Avenues of production such as transgenic, recombinant, or synthetic methods (Marx 2005) are costly and thus prohibitive for large-scale applications (Hancock and Sahl 2006). Furthermore, although a growing body of evidence suggests that bioactive peptides are produced naturally from dietary proteins during food processing or during gastrointestinal transit of the protein, it has also been shown that production through such uncontrolled routes may generate insufficient quantities essential to trigger the necessary physiological response in adult humans (Gauthier et al. 2006). Another challenge that is of much more concern is the fact that, from a process engineering perspective, there is currently no completely optimized bioprocess transferrable to a large-scale for the production of bioactive peptides. To date, there is no report that addresses the production of bioactive peptides from a bioprocess engineering and economics perspective in order to assess commercial viability. The lack of viable bioprocesses transferable to industrial scale is a major hindrance to the full exploitation of the physiological properties of these biomolecules. This setback also hinders the percolation of bioactive peptides into the consumer markets as whole isolated products. Although a number of bioactive peptides–containing food products (often dairy-based) are on the market, most of these products are prepared either by the use of soluble proteases or via microbial fermentation. The finished products therefore have to undergo extensive and often costly purification steps to isolate and enrich the bioactive peptide fractions. To avoid these time-consuming and often laborious steps, most dairy food processors prepare and market the product in the form of protein hydrolysates, which contain the active peptide together with other food components. When bioactive peptides are intended to be used as active pharmaceutical ingredients (APIs) they need to be isolated and purified before being formulated with other drug excipients. It follows, therefore, that the end use of a bioactive peptide determines the steps needed to be used in production, isolation, and purification. Currently existing methods in processing of bioactive peptides are prohibitive for large-scale applications, especially if peptides are intended for pharmaceutical applications (Korhonen and Pihlanto 2006; Agyei and Danquah 2011).

19.7.2 Fractionation and isolation of bioactive peptides During bioprocessing, the separation and purification stages represent a huge capital cost. It has been estimated that downstream separation and purification processes can account for up to 70% of the capital and operating costs (Brady

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et  al. 2008). Purification, fractionation, and enrichment of bioactive peptides from a hydrolysate mix are achieved by methods such as selective precipitation, membrane filtration, ion exchange, gel filtration technologies, ultrafiltrationnanofiltration, and liquid chromatography, or a sequential combination of these methods (Agyei and Danquah 2011; Muro et al. 2013). Selective precipitation of peptides is achieved by adjusting the pH to the isoelectric point or by the addition of a suitable precipitant such as organic solvent (ethanol, methanol, acetone), acids (trichloroacetic acid, sulphosalicyclic acid), or by the use of inorganic salts (ammonium sulphate, sodium sulpahte). Fractionation by precipitation is a cheap and easy technique but  since some of the chemicals used are harsh, it must be ensured they do not result in a compromise of the biological activity of the peptide being isolated. Liquid chromatographic techniques used for peptide purification include isoelectric focusing, size exclusion chromatography, reversed-phase liquid chromatography, and ion exchange chromatography. These methods are often used individually in cycles or in combination with other chromatographic methods sequentially. A number of research studies have employed chromatographic methods for the purification of bioactive peptides (Saito et  al. 1994; Gobbetti et  al. 2000; Ishiguro et  al. 2012; Mu and Sun 2012). Whereas most of these chromatographic methods are effective under laboratory scale, they are prohibitive for large-scale applications due to production cost and low productivity (Korhonen and Pihlanto 2006; Muro et al. 2013). Ultrafiltration and nanofiltration are two pressure-driven membrane-based processes that are increasingly being used in peptide purification. Membrane technology has a number of advantages over other techniques. The technology works without the addition of chemicals that could be toxic and render the product unfit for human consumption. It uses relatively low amounts of energy, resulting in reduced processing costs. Scale-up of membrane technology is relatively cheap and easy (Muro et al. 2013). Other researchers have used a combination of similar methods such as acid precipitation, diafiltration, and anion-exchange chromatography (Ellegård et  al. 1999) and electromembrane filtration (Bargeman et  al. 2002) in process-scale downstream purification of bioactive peptides. However, not much research work aimed at scaling up the purification technologies has been undertaken. This, coupled with the high cost of purification techniques, constitute the limiting factor to the industrial-scale commercialization of peptide-based products (Korhonen and Pihlanto 2006). The needed downstream purification techniques must be low in processing cost, easy to scale up, food-compatible, and must not have the potential to affect the structural integrity and/or the activity of the peptides. Thus, the development of commercially viable processes capable of upscaling bioactive peptide production and downstream purification is a worthwhile research endeavor.

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19.8 Conclusion Multifunctional bioactive peptides trigger inherent physiological responses that make them useful as therapeutic or prophylactic agents. Such biofunctionalities include antithrombotic, antihypertensive, immunomodulatory, anxiolytic, anorexic, and antioxidant properties—all of which are exploitable in therapeutic products and also for immunonutrition. These peptides are encrypted in food proteins but are inactive until released by an appropriate process such as during digestion in the gastrointestinal tract, via fermentation, or by the use of extracted proteases. The development of completely optimized bioprocesses transferable to large-scale operation for the production and purification of these important biomolecules will serve to enhance their percolation into the major consumer markets.

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Roy, F., J. I. Boye and B. K. Simpson. 2010. Bioactive proteins and peptides in pulse crops: Pea, chickpea and lentil. Food Research International. 43:432–442. Saito, Y., K. Wanezaki, A. Kawato and S. Imayasu. 1994. Structure and activity of angiotensin I converting enzyme inhibitory peptides from sake and sake lees. Biosci. Biotechnol. Biochem. 58:1767–1771. Sanchez, L., M. Calvo and J. H. Brock. 1992. Biological role of lactoferrin. Arch. Dis. Child. 67:657–661. Sheih, I. C., T.-K. Wu and T. J. Fang. 2009. Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresour. Technol. 100:3419–3425. Sienkiewicz-szłapka, E., B. Jarmołowska, S. Krawczuk, E. Kostyra, H. Kostyra and M. Iwan. 2009. Contents of agonistic and antagonistic opioid peptides in different cheese varieties. International Dairy Journal. 19:258–263. Silva, S. V. and F. X. Malcata. 2005. Caseins as source of bioactive peptides. International Dairy Journal. 15:1–15. Silva, S. V., A. Pihlanto and F. X. Malcata. 2006. Bioactive Peptides in Ovine and Caprine Cheeselike Systems Prepared with Proteases from Cynara cardunculus. J. Dairy Sci. 89: 3336–3344. Sistla, S. 2013. Structure-activity relationships of αs-casein peptides with multifunctional biological activities. Mol. Cell. Biochem. 384:29–38. Sojar, H. T., N. Hamada and R. J. Genco. 1998. Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin. FEBS Lett. 422:205–208. Sørensen, R., E. Kildal, L. Stepaniak, A. H. Pripp and T. Sørhaug. 2004. Screening for peptides from fish and cheese inhibitory to prolyl endopeptidase. Nahrung - Food. 48:53–56. Srinivas, S. and V. Prakash. 2010. Bioactive peptides from bovine milk α-casein: Isolation, characterization and multifunctional properties. International Journal of Peptide Research and Therapeutics. 16:7–15. Steinstraesser, L., U. Kraneburg, F. Jacobsen and S. Al-Benna. 2011. Host defense peptides and their antimicrobial-immunomodulatory duality. Immunobiology. 216:322–333. Sun, J., H. He and B. J. Xie. 2004. Novel Antioxidant Peptides from Fermented Mushroom Ganoderma lucidum. J. Agric. Food Chem. 52:6646–6652. Sun, X.-L., H. M. Baker, S. C. Shewry, G. B. Jameson and E. N. Baker. 1999. Structure of recombinant human lactoferrin expressed in Aspergillus awamori. Acta Crystallographica Section D. 55:403–407. Takahashi, M., S. Moriguchi, M. Yoshikawa and R. Sasaki. 1994. Isolation and characterization of oryzatensin: a novel bioactive peptide with ileum-contracting and immunomodulating activities derived from rice albumin. Biochem. Mol. Biol. Int. 33:1151–1158. Théolier, J., I. Fliss, J. Jean and R. Hammami. 2013. MilkAMP: a comprehensive database of antimicrobial peptides of dairy origin. Dairy Sci. & Technol.:1–13. Tsakalidou, E., R. Anastasiou, I. Vandenberghe, J. van Beeumen and G. Kalantzopoulos. 1999. Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein. Appl. Environ. Microbiol. 65:2035–2040. Walker, G., F. Cai, P. Shen, C. Reynolds, B. Ward, C. Fone, S. Honda, M. Koganei, M. Oda and E. Reynolds. 2006. Increased remineralization of tooth enamel by milk containing added casein phosphopeptide-amorphous calcium phosphate. J. Dairy Res. 73:74–78. Walker, G. D., F. Cai, P. Shen, D. L. Bailey, Y. Yuan, N. J. Cochrane, C. Reynolds and E. C. Reynolds. 2009. Consumption of milk with added casein phosphopeptide-amorphous calcium phosphate remineralizes enamel subsurface lesions in situ. Aust. Dent. J. 54:245–249. Wang, X., S. Hirmo, R. Willén and T. Wadström. 2001. Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BALB/cA mouse model. J. Med. Microbiol. 50:430–435.

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Wilson, J., M. Hayes and B. Carney. 2011. Angiotensin-I-converting enzyme and prolyl endopeptidase inhibitory peptides from natural sources with a focus on marine processing by-products. Food Chemistry. 129:235–244. Wong, J. H. and T. B. Ng. 2006. Limenin, a defensin-like peptide with multiple exploitable activities from shelf beans. J. Pept. Sci. 12:341–346. Xu, X., S. Katayama and Y. Mine. 2007. Antioxidant activity of tryptic digests of hen egg yolk phosvitin. J. Sci. Food Agric. 87:2604–2608. Yamada, Y., M. Iwasaki, H. Usui, K. Ohinata, E. D. Marczak, A. W. Lipkowski and M. Yoshikawa. 2010. Rapakinin, an anti-hypertensive peptide derived from rapeseed protein, dilates mesenteric artery of spontaneously hypertensive rats via the prostaglandin IP receptor followed by CCK1 receptor. Peptides. 31:909–914. Yamada, Y., A. Muraki, M. Oie, N. Kanegawa, A. Oda, Y. Sawashi, K. Kaneko, M. Yoshikawa, T.  Goto, N. Takahashi, T. Kawada and K. Ohinata. 2012. Soymorphin-5, a soy-derived muopioid peptide, decreases glucose and triglyceride levels through activating adiponectin and PPARalpha systems in diabetic KKAy mice. Am. J. Physiol. Endocrinol. Metab. 302:E433–440. Yamada, Y., K. Ohinata, A. W. Lipkowski and M. Yoshikawa. 2011. Rapakinin, Arg–Ile–Tyr, derived from rapeseed napin, shows anti-opioid activity via the prostaglandin IP receptor followed by the cholecystokinin CCK2 receptor in mice. Peptides. 32:281–285. Yamauchi, F. and K. Suetsuna. 1993. Immunological effects of dietary peptide derived from soybean protein. The Journal of Nutritional Biochemistry. 4:450–457. Yanai, T., Y. Suzuki and M. Sato. 2003. Prolyl endopeptidase inhibitory peptides in wine. Bioscience, Biotechnology and Biochemistry. 67:380–382. Yang, R., Z. Zhang, X. Pei, X. Han, J. Wang, L. Wang, Z. Long, X. Shen and Y. Li. 2009. Immunomodulatory effects of marine oligopeptide preparation from Chum Salmon (Oncorhynchus keta) in mice. Food Chemistry. 113:464–470. You, S.-J., C. C. Udenigwe, R. E. Aluko and J. Wu. 2010. Multifunctional peptides from egg white lysozyme. Food Research International. 43:848–855. Zhao, H., K. Ohinata and M. Yoshikawa. 2008. Rubimetide (Met-Arg-Trp) derived from Rubisco exhibits anxiolytic-like activity via the DP1 receptor in male ddY mice. Peptides. 29:629–632.

CHAPTER 20

Food-derived multifunctional bioactive proteins and peptides: Applications and recent advances Dominic Agyei1, Ravichandra Potumarthi1, and Michael K. Danquah1,2 1 2

Bioengineering Laboratory, Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia Department of Chemical Engineering, Curtin University of Technology, Sarawak, Malaysia

20.1 Applications of multifunctional peptides Owing to their multipurpose functionalities, bioactive peptides can play a significant role as leaders in the pharmaceutical, nutraceutical, and functional food industries as well as in the cosmetic industry. There is currently a booming market for bioactive peptides and a growing number of products are already on the market or under development by the food industries, exploiting the potential of food-derived bioactive peptides (Table 20.1).

20.1.1 Nutraceuticals and functional foods The potential for bioactive peptides to contribute to a healthier nutrition (e.g., by ingesting them with functional foods) has been widely discussed in the scientific community. Additionally, the discovery of bioactive peptides with potential health benefits has been the subject of growing commercial interest in the context of health-promoting functional foods. A number of reasons account for this trend. From a nutritional perspective, peptides are more bioavailable than their parent proteins or free amino acids (Hajirostamloo 2010). Also, due to the changes in the spatial three-dimensional structure of proteins upon hydrolysis, often the resulting peptides (especially those with low molecular weight) have been known to be less allergenic than their native proteins. This explains why milk protein hydrolysates are widely utilized in the formulation of hypoallergenic infant foods (Host and Halken 2004). The sales of dietary supplements and so-called functional foods and beverages are growing, suggesting an increasing belief in benefits of these “natural” approaches to disease prevention or management. Research continues to uncover novel bioactive peptides to reveal their possible functions and health benefits. In some

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

507

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Food-derived multifunctional bioactive proteins and peptides

Table 20.1 A description of some commercially available food-derived bioactive peptides. Bioactive peptide

Properties/ biological roles

Glycomacropeptide (GMP), κ-casein f(106–169) β-lactoglobulin fragments

Anticariogenic, antimicrobial, antithrombotic Reduction of blood pressure

Whey-derived peptides

Prevention of dental caries, influence the clotting of blood, antimicrobial properties Blood pressure reduction

Whey-derived peptides

αs1-casein f (1–9), αs1-caseinf (1–7), αs1-casein f (1–6) Caseinophosphopeptide

Casein-derived hydrolyzates Milk peptides

Product type; brand name

Manufacturer

Fresh cheese whey protein isolate; BioPURE-GMP

Davisco Foods International Inc., USA Davisco Foods International Inc., USA Davisco Foods International Inc., USA

Hydrolyzed whey protein isolate; BioZate Whey protein isolate; BioPURE-GMP

Blood pressure reduction

Fermented low-fat hard cheese; Festivo

Davisco Foods International Inc., USA MTT Agrifood Research, Finland

Aids mineral adsorption

Casein hydrolysates; Capolac® Sour milk; Calpis

Arla Foods Ingredients, Sweden Calpis, Co., Japan

Flavored milk drink, confectionary, or capsules; PRODIET F200/Lactium Casein-derived peptide; PeptoPro

Ingredia, France

Val-Pro-Pro, Ile-Pro-Pro; Blood pressure reduction derived from β-casein and κ-casein in sour milk as1-casein f (91–100) (Tyr- Reduction of stress effects Leu-Gly Tyr-Leu-Glu-GlnLeu-Leu-Arg) Casein-derived peptide Improves athletic performance and muscle recovery Whey-derived peptide Aids relaxation and sleep

Whey protein hydrolysate fortified with lactoferrin Whey protein extract

Available as commercial product

Helps reduce acne

Helps reduce symptoms of mild to moderate psoriasis Helps regulate blood sugar peaks after a meal Reduces inflammation and promotes healing in the digestive tract

Hydrolyzed whey protein isolate; Biozate

Whey-derived peptide; Vivinal Alpha

Lactoferrin-enriched whey protein hydrolysate; Praventin Whey protein extract; Dermylex

DSM Food Specialties, the Netherlands Borculo Domo Ingredients (BDI), the Netherlands DMV International, the Netherlands Advitech Inc., Canada

Casein hydrolysate; Insulvital

Wild Co., Germany

Milk peptides; Immunel

Wild Co., Germany

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Table 20.1 (Continued) Bioactive peptide

Properties/ biological roles

Available as commercial product Product type; brand name

Manufacturer

Supports and balances total immunity Reduction of stress effects

Milk peptides; Tegricel

Wild Co., Germany

Milk protein hydrolysate; Lactium®

Ingredia Nutritional, France

Helps sleep and memory

Whey protein isolate; α-lactalbumin

Caseinophosphopeptides

Anticariogenic

Caseinophosphopeptides

Helps mineral absorption

Caseinophosphopeptides

Anticariogenic

Caseinophosphopeptides

Anticariogenic

GC tooth mousse; water-based creme Caseinophosphopeptides; Kotsu Kotsu calcium Caseinophosphopeptides; Trident xtra care™ Caseinophosphopeptides; Recaldent™ Soy protein oligopeptides; HI-NUTE series whey proteins; Lacprodan® ALPHA-20

Davisco Foods International Inc., USA GC Europe N.V., Belgium Asahi Soft Drinks Co. Ltd., Japan Cadbury Adams, USA Cadbury Enterprises Pte. Ltd., Singapore Fuji Oil Co. Ltd., Japan Arla Foods Ingredients Group P/S, Denmark Metagenics Inc., USA

Milk peptides αs1-casein f(91–100) Tyr-Leu-Gly-Tyr-Leu-GluGln-Leu-Leu-Arg Alphalactalbumin

Soy protein oligopeptides Helps prevent obesity and muscle fatigue α-lactoalbumin (60 %) Helps reduce peptic ulcers

Fish peptides (Leu-Lys-Pro-Asn-Met)

Hypotensive

Bonito-derived peptide; Vasotensin®

Adapted from Korhonen and Pihlanto (2006); Carrasco-Castilla et al. (2012); Korhonen and Marnila (2013).

developed countries, the marketing of food products with health claims is a wellestablished industry. For example, in Japan, there is a booming industry for the production and marketing of Food for Specified Health Uses (FOSHU), or “foods containing ingredients with functions for health and officially approved to claim its physiological effects on the human body.” Under such context, the safety assessment of the foods as well as effectiveness for the desired health functionality is required before approval can be sought for the marketing of food as FOSHU. FOSHU approval operates under three schemes: (1) qualified FOSHU (where food with demonstrated functionality is approved, although no scientific and established mechanism of action has been demonstrated); (2) standardized FOSHU (where foods are approved following the establishment of standards and specifications with sufficient accumulation of scientific evidence); and (3) reduction-of-diseaserisk FOSHU (where food is approved when reduction-of-disease-risk is clinically and nutritionally established in an ingredient) (FOSHU 2013). Several reductionof-disease-risk FOSHU products in Japan contain peptide-based ingredients.

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20.1.2 Clinical trials: Prospects and challenges A number of clinical trials have been conducted for some food having protein hydrolysates or bioactive peptides as ingredients and components (Table 20.2). It is observed from the literature that a large proportion of the clinical studies done explored the blood pressure–lowering (antihypertensive) activities of bioactive peptides. Not much human studies have been done for other biofunctionalities such as antioxidant, anticancer, and immunolomodulating properties, to mention a few (Agyei and Danquah 2012). Furthermore, the dosage amount seems to vary for each experiment. As well, the states (whether in powdered, capsule, or liquid form) in which products are administered vary for each experiment. The establishment of the dosage needed for a food-derived peptide to trigger physiological response is an important information needed to help market a product based on health claims. Food components are known to have a chronic rather than an acute effect on health (Hartmann and Meisel 2007). As such, screening methods are needed for the measurement of both short- and long-term effects that follow the administration of certain food components that are proposed to promote good health (Hartmann and Meisel 2007). This will require a series of experiments at various levels, involving in vitro, animal models, and human studies. Valid bioindicators that represent an effect brought about by the bioactive food component must be developed and standardized. Such markers could be obtained in vivo (serum, fecal, urinary), in vitro (cytochemical), and also in situ (lesion samples) (Hartmann and Meisel 2007; Walker et al. 2009). Despite some advancement in this area, results from animal and human studies are quite contradictory. For example, whereas a number of research studies have demonstrated the multifunctional roles of CPP, studies conducted in connection with a European Union project (Caseinophosphopeptides: Nutraceutical/Functional Food Ingredients for Food and Pharmaceutical Applications) have concluded that CPPs does not enhance calcium absorption in the gut of humans (López-Huertas et al. 2006; Teucher et al. 2006; Hartmann and Meisel 2007). Another product with questionable biofunctionality is α-casozepine, released during trypsin digestion of αS1-casein. It is a decapeptide (is αS1-casein (f91-100)) with the sequence Tyr-Leu-Gly-Tyr-Leu-Glu-Asn-Leu-Leu-Arg (Messaoudi et al. 2005; Kim et al. 2006; Cicero et al. 2011). α-casozepine is known for its anxiolytic properties (Kim et  al. 2006) and products such as Lactium® have α-casozepine as ingredients and are marketed with this health claim. However, the European Food Safety Authority (EFSA) has issued a report about a milk protein hydrolysate product supplemented with 1.7% of α-casozepine (product name Lactium® and claimed to “helps to calm mind,” “mental state and performance,” and “stress”). The report stipulates that “…a cause and effect relationship has not been established between the consumption of ‘αS1-casein tryptic hydrolysate’ and alleviation of psychological stress” (EFSA 2011).

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Table 20.2 Examples of clinical trials/human studies. Population Product/ involved peptide in study

Duration

Dosage

Results (Bioactivity claimed)

Reference

30

Fermented milk containing tripeptides, IPP, and VPP

8 weeks

SBP and DBP ↓ vs. control (antihypertensive effects)

(Hata et al. 1996)

80

Powdered fermented milk containing tripeptides, IPP, and VPP Fermented milk containing tripeptides, IPP, and VPP Fermented milk containing tripeptides, IPP, and VPP Skim milk

4 weeks

2 ml/kg body wt (i.e. 0.033 mg Val-Pro-Pro and 0.025 mg Ile-Pro-Pro/kg body wt). 12 g of powdered fermented milk was consumed for 4 weeks 150 mL of product twice daily

SBP and DBP ↓ vs. control (antihypertensive effects)

(Aihara et al. 2005)

94

39

82

10

23

10 weeks

SBP and DBP ↓ vs. control (antihypertensive effects) 21 weeks 2.25 mg of Reduced SBP and Ile-Pro-Pro and DBP 2.55 to 3.75 mg (antihypertensive of Val-Pro-Pro effects) 8 weeks 2% solids-not-fat Reduced serum fortified skim milk triglycerides in the to the daily diet high-cholesterol subject Casein Consumption of Aids enamel phosphopeptides, 100 mL of CPP remineralization CPP mixed with fortified milk and bone milk; CPP has daily for calcification the structure 30 seconds for a (anticariogenic –Ser (P)-Ser (P)-Ser 15-day period properties); (P)-Glu-Glu# immunoenhancing (Recaldent) activity; antigenotoxic properties α-lactalbumin (a Two meals given Dosage per meal Increase in plasma whey protein) diet on two separate unspecified Trp-LNAA ratio rich in tryptophan 2-day periods and improvement in cognitive performance in high stress– vulnerable subjects

(Jauhiainen et al. 2005)

(Seppo et al. 2003)

(Buonopane et al. 1992)

(Walker et al. 2009)

(Markus et al. 2002)

SBP, systolic blood pressure; DBP, diastolic blood pressure; LNAA, large neutral amino acid; #, commercial product has CCP complexed with amorphous calcium phosphate as active ingredients.

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Food-derived multifunctional bioactive proteins and peptides

A number of causes can be attributed to the discrepancies in results of foodderived bioactive peptide functional studies. Most of these clinical studies are carried out by or at least in conjunction with industry and thus not all data on the composition and manufacturing of these peptides are made available (Jäkälä and Vapaatalo 2010). With this failure to give such vital information, a repeat experiment could lead to different results. With the need to comply with strict regulatory standards, in vivo and clinical studies aimed at elucidating the mode of action and potency of bioactive peptides is increasingly becoming expensive. To forestall this, researchers have resorted first to conducting in vitro experiments to identify potential bioactive peptides before committing to in vivo and human trials (Agyei and Danquah 2012). Other authors such as Foltz et al. (2010), however, have argued that it is inappropriate to use results of in vitro studies as justification to test the in vivo effects of bioactive peptides. This is because this approach largely neglects other challenges such as the low bioavailability of most peptides resulting from poor absorption, distribution, metabolism, and excretion (ADME). As such, in vivo efficacy of bioactive peptides can only be tested when these peptides have exhibited reasonable and physiologically relevant ADME profiles (Foltz et al. 2010).

20.2 Pharmaceutical products Proteins and peptides play several important roles in living body systems. Thus, the diverse physiological roles of peptides make them suitable candidates for the development of therapeutic agents (Agyei and Danquah 2011; Lax 2012). A considerably huge amount of scientific research data exists to demonstrate the therapeutic potential of bioactive peptides and these highlight the role food-derived peptides can play as active pharmaceutical ingredients (APIs) in therapeutic products. Due to a host of reasons such as heightened concerns with the side effects of small-molecule drugs and antibiotic resistance of several pathogenic microorganisms, there has been a search for a new generation of therapeutics that is safe and potent. Recently, there has been a surge of interest in peptide therapeutics within the pharmaceutical industry. It has been estimated that around 60 peptide drugs were approved and generated annual sales of around US $13 billion (about 1.5% of all drug products) in 2010. The numbers are increasing significantly with the current continuous annual growth rate between 7.5% and 10% (Lax 2012). Undoubtedly, most of these peptides are not food-derived; however, statistics show that the future of peptide-based therapeutics among consumers and regulatory bodies is bright. Not only do bioactive peptides have prospects in the treatment of certain single-symptom diseases, but they can also be used in the control of syndromic ill-health conditions that are accompanied by several disorders. For example, the involvement of dietary bioactive proteins and peptides in the control and possible treatment of autism spectrum disorders (ASD) has recently been reviewed (Siniscalco and Antonucci 2013). This prospect rests

Food-derived multifunctional bioactive proteins and peptides

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on the fact that most of the biochemical processes associated with ASD is addressed one way or the other by bioactive peptides. Some of these biochemical processes include oxidative stress; gastrointestinal impairments (intestinal increased permeability and dysbiosis); immune dysregulation; and immune activation of neuroglial cells (Siniscalco et al. 2012). Some properties of peptidebased therapeutics that highlights their use as APIs are: 1 Peptides are structurally diverse and have a wide spectrum of therapeutic action, low biodeposition in body tissues, and high biospecificity to targets (Agyei and Danquah 2011). Peptides are therefore effective in addressing a wide array of medical disorders including cancers and tumors, metabolic disorders, cardiovascular heath, and infectious diseases. 2 The acceptance of protein therapeutics by physicians, pharmaceutical corporations, and patients has increased over the past few decades. This increased acceptance is demonstrated by the fact that there is an over 20% probability of regulatory approval for peptide-based new chemical entities (NCEs), a rate that is double that of small molecules (Lax 2012). Also, for the past three decades in the United States alone, the number of peptide-based NCEs entering clinical study per year per decade has increased by about 1300% (from 1.2 per year in the 1970s, to 16.8 per year so far in the 2000s) (Reichert 2012). This increased acceptance has been partly attributed to the development of technological solutions to problems that hitherto affected the percolation of peptide therapeutics, namely, short half-life and difficulties in delivery (Saladin et al. 2009). Additionally, the use of peptide products is often unaccompanied by any side effects. Thus, side reactions are unanticipated or at best reduced for food-derived peptide drug candidates since food proteins and peptides have a long history of use and putatively are GRAS (Agyei and Danquah 2011). 3 Most peptides are composed of metabolically and allergenically tolerable amino acids. Thus, peptides are generally safe and nontoxic, considering their natural physiological roles in the body as peptide hormones, chemokines, and cytokines. For the most part, any known side effects with peptide drugs have often been related to dosage or local reactions at the injection site (Lax 2012). 4 Aside from their use as active ingredients, peptides also have the ability to be used as excipients in drug formulations for modification of biological activity and targeted delivery, or to aid transport across cellular membranes.

20.3 Dermopharmaceutical products Proteins and peptides play an active role in skin-care through a combination of several physiological activities (i.e., modulating cell proliferation, cell migration, cell and tissue inflammation, angiogenesis, and melanogenesis) (Fields et  al. 2009). Multifunctional bioactive peptides have great potential for use as active cosmetic and dermopharmaceutical ingredients due to their ability to stimulate some of these physiological responses. Interestingly, a number of commercial

514

Food-derived multifunctional bioactive proteins and peptides

patents exist that describe peptide sequences that are used as ingredients in dermatological products. The sequence X-Thr-Thr-Lys-Y, where X = lysine and Y = serine has been patented (US Patent No. 6620419), as useful in stimulating skin healing, skin hydration, and combating wrinkles and consequences of skin ageing (Lintner 2000). Another disclosed invention (US Patent No. 8071555) is a tetrapeptide with the amino acid sequence Pro-Glu-Glu-X (where X can be either lysine or isoleucine). This tetrapeptide is effective in controlling inflammatory skin disorders (Zhang et al. 2011).

20.4 Recent advances and emerging technologies 20.4.1 Foodomics and other “Omic” methodologies A number of advances have been in many “omic” techniques that offer high through-put and an integrated approach to studying food protein peptides. Foodomics is a term that has been coined to describe a broad knowledge of food, covering the assessment of their composition and impacts of (bio)technological processes involved in their production, time-dependent changes in food composition, and the impact of food consumption on human health (Picariello et  al. 2012). Thus, as a new global discipline, foodomics relies on the use of advanced omic tools including metabolomics, genomics, epigenomics, transcriptomics, and proteomics to address issues such as bioactivity, safety, quality, and food traceability, with the aim of improving consumer health, well-being, and confidence (Cifuentes 2013). Some of the foodomic tools that are of primary interest to the study and applications of food-protein-derived bioactive peptides are nutrigenomics, food proteomics, and food peptidomics. Food peptidomics seeks to unravel and also develop analytical strategies to study the peptidome of a given food product and establish the origin, evolution, organoleptic properties, and beneficial or adverse effects on human health (Carrasco-Castilla et al. 2012; Picariello et al. 2012). Advancement in “omic” techniques has been served greatly by the marriage of high-throughput mass-spectrometry-based methodologies (Mena and Albar 2013) and computational achievements in the development of new algorithms to analyse huge and bulky peptidomic data quickly and effectively. The proliferation of software programs, coupled with the use of tandem mass spectroscopic tools, provides a powerful and attractive tool for peptide quantification and characterization. Computational (in silico) peptidomics allow the prediction of bioactive peptides that can be obtained from a food protein of known sequence, before actual wetlaboratory production is undertaken. With the rapid discovery, characterization, and deposition of information on bioactive peptide in databases, it becomes increasingly possible to predict new peptides with improved bioactivities. This ‘bottom-up’ approach is captured in Figure  20.1 and can be adopted for the rational design of peptides with desired biofunctionalities. With this technology,

Food-derived multifunctional bioactive proteins and peptides

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6. Purification and activity testing of Peptides

5. Wet laboratory synthesis of desired peptides with conventional method (solidphase peptide synthesis)

4. In silico identification and characterization of peptide of interest

3. In silico digestion

2. From protein sequence databases, select protein substrates and enzyme(s) that will yield the peptide of interest

Figure 20.1 The production of bioactive peptide

via the in-silico approach.

1. Select peptide sequence with bioactivity of interest from literature

an online protein database can be consulted to select protein sequences that encrypt specific peptides of interest. This is followed by in silico digestion with the use of enzyme(s) sources of characteristic specificity that will release (but not cleave) the desired peptide. Characterization of the “soft” peptides produced can also be done in silico after which laboratory synthesis may follow if the predicted properties of the peptides obtained are satisfactory. This approach to bioactive peptide production offers several advantages over the hitherto conventional trial-and-error method in that it is less labor intensive, relatively cheaper, and gives an indication of the bioactivities of the peptides with a high level of accuracy even before synthesis of the peptides (Agyei and Danquah 2011). As well, the study of the secondary structure and predictions of the physicochemical and physiological properties of peptides can also be done in silico. An example of peptide database application that can be used to perform in  silico peptide studies is BIOPEP (www.uwm.edu.pl/biochemia/), which interlinks three databases of protein sequences, bioactive peptides, and proteolytic enzymes (Dziuba and Dziuba 2010). To date, there are 707 and 2,609 protein and peptide sequences and 25 proteolytic enzymes, with all three databases being regularly updated. Additionally, BIOPEP focuses on food-derived peptides

516

Food-derived multifunctional bioactive proteins and peptides

and also has inbuilt programs for the prediction of allergenic and toxic properties of peptides and can also be interfaced with global databases such as SWISS–PROT (Minkiewicz et al. 2008; Dziuba and Dziuba 2010). Other peptide sequence applications/databases are PepBank (http://pepbank.mgh.harvard.edu), BioPD (http://biopd.bjmu.edu.cn), SwePep (www.swepep.org), and EROP-Moscow (http://erop.inbi.ras.ru), among others. Some in-silico digestion databases such as PeptideCutter (http://web.expasy.org/peptide_cutter/), POPS (http://pops.csse.monash. edu.au/pops-cgi/index.php) and NeuroPred (http://neuroproteomics.scs.illinois.edu/ neuropred.html) are proteolyse prediction databases (Carrasco-Castilla et al. 2012). A number of other Web-based peptide libraries are also being created. MilkAMP is a new database that contains information (i.e., microbiological and physicochemical data) on antimicrobial peptides obtainable from dairy products. This database is freely available at http://milkampdb.org and is a useful tool that can aid in forecasting the development and use of biologically active peptides in the  pharmaceutical and food industries (Théolier et  al. 2013). Other authors have proposed the development of a Wiki-like food database that utilizes advance bioinformatic tools in food and nutritional research (Holton et al. 2013).

20.5 Quantitative structure activity relationship (QSAR) models Another area of advancement is the use of models. Although model prediction of the behavior of biological systems has been known for several decades, recent great advancements in computational methods have made it possible and relatively easy to model the activities of biologically active molecules and compounds in an accurate manner (Nantasenamat et al. 2010). An example of this is the quantitative structure activity relationship (QSAR), which works on the principle that the activity of a biological molecule can be predicted based on its physicochemical or structural properties such as electronic charge, hydrophobicity, and steric properties, resulting in multivariate data (Nakai and Li-Chan 1993; Pripp et al. 2005; Hansch et al. 1995). The quantitative structure–property relationships (QSPR) model, on the other hand, is used to study and correlate the structural features of a compound with its respective chemical properties (Le et al. 2012). The basic assumption in QSAR modelling is that the biological activity is related to the structural variation of the compounds, and this relationship can be modelled as a function of molecular structure (Carrasco-Castilla et al. 2012). Often, a mathematical expression can be used to describe the relationship between the amount of a substance and the level of physiological response it triggers. In QSAR models, accurate characterization of the physicochemical properties of amino acids in the peptide sequence is very important since the model relies

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on such descriptors to predict and express chemical structure relevant for activity. In their pioneering work, Hellberg et  al. (1987) established a system for describing the variation of amino acid sequences in peptides as three principal properties, also referred to as z-scores: hydrophilicity/hydrophobicity (z1), molecular size/bulkiness (z2), and electronic properties/charge (z3). These properties were obtained by principal components analysis of 29 physicochemical variables for the 20 natural amino acids (Kim and Li-Chan 2006). Other parameterization systems include the extended principal property scales (z′1, z′2, z′3), for both coded and noncoded amino acids (Jonsson et  al. 1989); amino acid side-chain descriptors that use isotropic surface area (ISA) and electronic charge index (ECI) (Collantes and Dunn 1995); molecular surface-weighted holistic invariant molecular (MS-WHIM) 3D-descriptors (Todeschini et al. 1994; Zaliani and Gancia 1999); MARCH-INSIDE (Markovian Chemicals In Silico Design) methodology, based on the Markov chain theory (Ramos de Armas et al. 2004); and principal components that score Vectors of Hydrophobic, Steric, and Electronic properties (VHSE) (Mei et al. 2005). Such scoring systems are useful strategies for developing peptide QSARs. The use of QSAR model systems replaces the “trial-and-error” approach of selecting molecules with biological properties, therefore saving time, resources, and cost involved in screening large numbers of plausible molecule candidates. QSAR models are widely utilized in drug discovery and are equally applied in food-derived bioactive peptide research because a strong structure–activity relationship exists in most peptides, making secondary structure and physicochemical properties of peptides important factors that determine the bioactivities triggered by food-derived peptides. QSAR of several food bioactive peptides has been successfully used to estimate the structure–activity relationship of antimicrobial, ACE-inhibitory (Norris et al. 2012; Gu and Wu 2013; Sagardia et al. 2013), and bitter-tasting peptides, as reviewed (Pripp et  al. 2005). As well, in-silico digestion and QSAR model prediction have been used to identify potent angiotensin I converting enzyme (ACE) inhibitory peptides from several common food proteins (Majumder and Wu 2010; Gu et al. 2011).

20.6 Research concerns and bottlenecks 20.6.1 Allergenicity A food allergy is an adverse health effect as a result of an immune response that occurs reproducibly after exposure to a given food protein (Boyce et al. 2010). Although peptides have been known to be less allergenic than their native proteins (Host and Halken 2004), it is also true that several native proteins act as precursors for both bioactive peptides and allergenic peptides, giving both toxic and nontoxic effects. For example, whereas cow’s milk is a rich source of bioactive proteins and peptides (Korhonen and Marnila 2013), it also acts as a

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storehouse for several protein- or peptide-based allergenic molecules, and constitutes the leading cause of food allergies in infants and young children under age 3 years (Koletzko et al. 2012; Ludman et al. 2013). All the constituents that are responsible for both pollen and food allergies are proteinaceous in nature. Some protein hydrolyses may retain part of the allergenicity of the native protein and thus can also be considered as allergens (Hartmann et al. 2007). Foodderived bioactive peptides must therefore be subjected to comprehensive safety assessment in order to rule out any cytotoxic and allergenic concerns that will render the products unsafe for human consumption. In many cases, such safety assessments are a requirement by food standards, for food labeling and advertisement purposes. Immune reactions of food allergens may be immunoglobulin (Ig)E mediated, non-IgE mediated, or mixed. Also, symptoms of food allergies are sometimes masked by other gastrointestinal problems such as dyspepsia and abdominal pain. Thus, appropriate diagnostic mechanisms are needed to clarify, for the average consumer, the difference between food allergies and food intolerance phenomena (which may be enzymatic, pharmacological, or toxin-mediated) (Hartmann et al. 2007; Koletzko et al. 2012). To this end, the diagnosis of food allergies in food proteins and its hydrolytic products have been reported in the findings of some studies (Boyce et  al. 2010; Waserman and Watson 2011; Koletzko et al. 2012). Additionally, since αs1 casein is the major allergen of cow’s milk, Elsayed et  al. (2004) have used synthetic peptides and derivatives from this protein to develop a sensitive technique for detecting masked cow-milk protein epitopes in processed food. Although their method was effective in showing latent allergenic properties, it is, however, laborious, tedious, and relatively expensive since it required pure peptide samples. Research is therefore needed to elucidate the allergenicity and antigenicity of bioactive peptides in a quick and robust manner, particularly when the peptides are obtained from allergenic food proteins.

20.6.2 Possibility of microbial resistance of peptides The possibility of pathogenic microbial strains developing resistance to bioactive peptides is a research concern that needs to be addressed. This is exemplified in the bioactivities of lactoferrin (LF). Owing to their antimicrobial potential, LF has been granted a GRAS status (notification number 67), to be used as an electrostatic spray to a concentration of less than 2% by weight, in the control of microbial contamination of raw meat (Taylor et al. 2004). However, it has also been reported that some pathogenic microorganisms show resistance or can develop resistance to LF, especially at certain physical conditions and metabolic states (Arnold et  al. 1981; Bortner et  al. 1989). It has therefore been suggested that the widespread use of LF or lactoferricin should be done with caution since it could lead to the development of resistant pathogens, which, in

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the case of LF or lactoferricin, also indicates the possibility of the pathogens to resist molecules of human innate immunity (Korhonen and Marnila 2013). On the other hand, other studies have shown conclusions that are contrary to this notion. For example, Chen et al. (2013) have demonstrated that several probiotic bacterial strains are resistant to LF and its hydrolysates. The probiotic strains included L. rhamnosus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus coryniformis, L. acidophilus, Bifidobacterium infantis, Bifidobacterium bifidum, and Pediococcus acidilactici. On the other hand, the growth of foodborne pathogens such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Enterococcus faecalis was inhibited by LF and its hydrolysates. It follows, therefore, that more experimental research is needed to clarify the discrepancies and evaluate the possibility and conditions that could favor microbial resistance of LF intended for food application.

20.6.3 Stability and bioavailability Multifunctional bioactive peptides, in order to exert any bioactive effects after oral ingestion, have to reach the target system in an active form. The challenge here is that proteins and peptides are highly susceptible to protease degradation en route to the body. Also, the low solubility and relatively bulky nature of multifunctional proteins and peptides leads to difficulty in transport and delivery across membranes. These leads to low stability that results in decreased oral bioavailability and short half-life (Agyei and Danquah 2011). Some multifunctional peptides such as LF have been shown to retain bioactivities and resist trypsin and chymotrypsin treatment in amounts comparable to duodenal contents in human infants and the presence of high concentrations of a specific trypsin inhibitor could account for this observation (Brines and Brock 1983). On the other hand, the presence of specific amino acid types is responsible for the ability of some peptides to resist proteolytic attacks. For example, it is known that (hydroxy)proline-containing peptides are generally resistant to degradation by digestive enzymes (Vermeirssen et al. 2004) so that the incorporation of proline groups in peptides help enhance their proteolytic stability. Although a number of simulated gastrointestinal digestion studies have been done (Hernández-Ledesma et al. 2004; Lo and Li-Chan 2005; Cinq-Mars et al. 2007; Escudero et al. 2010), most of these studies described ACE inhibitory peptides. Research information (on the other biofunctionalities) is therefore still scarce or and in some cases the results on the resistance of milk protein to enzymatic attack along the digestive tract are conflicting (Picariello et al. 2010). The mystery of peptide resistance to gastrointestinal digestion, intestinal absorption, and stability in blood needs to be unraveled. Thus, more detailed clinical studies are needed to establish the biological potency of multifunctional peptides that have proven to be effective in in vitro experiments.

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20.7 Conclusions and future projections Increasingly, there is a blurring of the boundaries between food and classic pharmaceutical products. Food products containing multifunctional peptides are a potent alternative to achieving therapeutic effects in human health by the use of nonpharmacological, food-derived biomolecules. The sequencing and prediction of the biological roles of peptides is escalating, owing to recent robust and highthroughput “omic” techniques. However, to be fully exploited, a number of research studies aimed at elucidating unknown toxicity and stability of peptides need to be undertaken. Once these bottlenecks are removed, we project that there will be an escalation of interest among consumers, researchers, and clinicians since multifunctional peptides constitute a more “natural” approach to disease prevention and management.

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SECTION III

Biochemistry and nutraceutical or healthrelated applications

CHAPTER 21

An overview of the molecular and cellular interactions of some bioactive compounds Amro Abd Al Fattah Amara Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Egypt

21.1 Introduction 21.1.1 Atoms, molecules, compounds, and macromolecules The biological system is rich with an endless number of molecules and macromolecules. Each has its own criteria. However, they can be classed into groups. Each group has similarities and differences. What are the criteria that govern these similarities and differences? Simply, it is their chemical structure. The structure is responsible for the function. Any of the biological system molecules and macromolecules is built from certain elements. However, oxygen, hydrogen, carbon and nitrogen are more representative. The name of a molecule is given for two or more atoms chemically bonded together. The new molecules react as one unit, such as O2. Their atoms bond with one another by chemical bonds through the interactions of the electrons in their outer shells. Compounds are structures that are bigger in size than molecules. Macromolecules are even larger, usually existing in polymeric form, such as protein, DNA, or polyhydroxyalkanoate. Polymers are composed of repeated homogenous or heterogeneous units, which are named homo or hetero polymers, respectively. Because they result from the biological system, special forms of chemical bonds are more common, such as covalent, ionic, and hydrogen bonds. Other natural forces exist such as van der Waal forces, which occur between two or more atoms existing in close proximity to one another (3–4 Å) (such as substrate and enzyme binding). Another force is the hydrophobic interaction, generated on the existence of the molecule or the macromolecules in water. Water-repelling molecules tend to  cluster in an aqueous environment, leading to association of nonpolar parts. Such a phenomenon could be found in phospholipid membrane, protein

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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(folding), and substrate/enzyme binding. Nonbond forces play an essential role in the biological system while they reveal the importance of water and its content of ions, charges, and molecules in the protein three-dimensional (3D) structure. One protein could exist in many 3D structures, particularly as a response to the  environment. Scientists have divided biological macromolecules into four major groups: proteins, nucleic acids, fats, and sugars. These four groups exist in polymeric form, but other forms of biopolymers exist. Biopolymers can be precisely classified into eight classes: nucleic acids, “polyamides” such as proteins, polysaccharides and cutin, organic polyoxoesters, inorganic polyesters with polyphosphate, polyisoprenoides such as natural rubber or Gutta-Percha, polyphenols such as lignin or humic acids and polythioesters (Steinbüchel 2001). The monomeric backbone of biopolymers and the macromolecules is important. One should note that the monomeric structure builds the 3D structure, which is responsible for function. Additionally, such biopolymer and macromolecules on degradation will be divided into their monomeric constituents. Monomeric structures in their single form (e.g., amino acids) are also important. For example, food that contains cysteine is better for you than that which contains cystine. The cell membrane transports cysteine 10 times faster than cystine. Our understanding of such properties will enable us to select the type of foods that will benefit us (Vasdev and Gill 2006; Amara 2010). None of the components in our biological system is functionless, but we still have limited knowledge. The role of the macromolecule structure/function and specificity is described in this chapter with a focus on proteins.

21.1.2 Understanding structure and function Bioactive compounds are built from the biosystem and therefore they are bioavailable. The biological system is sensitive to chemical structure, particularly enzymes. Other forms could also be very sensitive. Red blood cells (RBCs) could differentiate between O2 and CO2. The conditions and the structure draw the function of the RBCs—where they gain O2 and where they lose it, where they gain CO2 and where they lose it. Minor changes in RBCs can be responsible for serious disease; sickle cell anemia is an example. Analogues have a structure similar to certain compounds, with minor differences. Some analogues are more stable or even indegradable. Substrates are degraded by enzymes. Isopropyl-β-D-thio-galactosidase (IPTG) is a good example (Ko et al. 2003; Sambrook and Russell 2006). Analogues can show us that our biological system is governed by chemical and physical laws. Similar compounds to a certain substrate could interfere with its related enzymes and/or wrongly activate certain biopathway(s). Such a phenomenon could be helpful in research but might be harmful in real life. The biological system works mechanically, perfectly but blindly. It is like an intelligent computer, but even it could not observe that an executable file could interfere or damage it, like a virus. Understanding the effect of the structure on

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the function will let us know that improbable compounds could wrongly act as analogues and activate some pathways mistakenly. Alternatively, we can control their use to activate certain points in a certain pathway to overcome certain deficiencies or illnesses. In this case, the structure of both of the analogues and the activated part of the biological system, which is mostly proteins or enzymes, could be built and designed for the best, most effective result. Losing the natural structure activity will take us to the synthetic structures. Synthetic structures are made through chemical reactions, which during the industrial process might incorporate incorrect structures or isomers. The biological system guarantees 100% livo rotator structures. Additionally, incomplete synthesized forms could exist. What could such forms do for us? Such incompatible forms will lead to the activation of CYTP450, which might apparently look to be a positive point in our biological system, but in fact it is not. Activating the CYTP450 enzymes (usually with drugs) wake up the degradation machine in our bodies. No one can control the by-products of degradation. The degraded products could depend on the activated enzyme types and the existing chemical compounds, which are subjected to the degradation process as well as which compounds are activating the CYTP450 previously and which enzymes were activated. Different CYP450 enzymes have different degradation products for a single compound, which upon degradation could be converted to toxins and carcinogens. Alternately, bioproducts guarantee livo rotator compounds. Even some natural products are toxic, but we still have the opportunity to reduce their toxicity for propitiate use. In fact, most physiologically active products have toxicity at certain concentrations. The toxicity of certain natural compounds is an indication of the presence of active constituents. However, in certain concentrations they are safe for physiological activity, metabolism, or degradation. Bioactive compounds are built from the biosystem and therefore they are bioavailable. One should differentiate between low-molecular-weight bioactive compounds and high-molecular-weight or polymeric structures. High-molecular-weight molecules even have active safe physiological activity in their mother source but might become antigenic and induce the immune system if they entered our bodies without degradation to their fine monomeric forms. A foreign protein is an example. There is major movement toward natural active compounds, particularly those derived from edible plants. Compounds derived from edible plants proved historically to be safe for our health (Amara et al. 2008). They were subjected to open clinical trials and prove to be safe.

21.1.3 What are the criteria that enable classifying some compounds as safe and others as harmful? Safe compounds do their function with high selectivity without harming other components of the cells, can be degraded to nontoxic or harmful forms, and are homogenous with our biological systems (biocompatible). In contrast, unsafe compounds are either nonspecific or specific but could be degraded to harmful

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forms, are incompatible with our biological system, are toxic, and are mostly synthetic compounds. However, some natural compounds are harmful, but in low concentrations may be of use. Safe compounds also could be degraded to nonsafe forms under certain conditions, such as improbable activation of CYTP450 (described previously). Compounds, molecules, and macromolecules are changing their structure due to external or internal effects on their function. Change in the function could lead to problems and illnesses. Our understanding of the structure/function interaction and relationship will give us the chance to avoid problems, and to use such compounds more wisely. Not all the factors that affect structures/functions are due to physical and chemical effects. Heredity could cause changes in the DNA, which will give incorrect protein (protein folded to forms different from the correct native original structure) and can lead to incorrect functions.

21.2 Prokaryotic versus eukaryotic The prokaryotic system gives us simple and understandable examples, particularly those related to unicellular forms such as bacteria. Bacterial cells are unicellular, divided into two identical cells during its replication process. Any simple genetic change in the mother cell will be introduced to its offspring. Such change could be easily detected (often) if the new cells are compared with their parents. Mutants are usually detected and confirmed using different screening protocols. The screening protocol is usually based on phenotype differences. Changes that happened in the genes have phenotype, metabolism, or catabolic properties. Some products need more than one protein or enzyme such as antibiotics, which need many steps and many proteins to build their final structure. For that single amino acid or base pair, change might not be effective in the product or might only slow down or in contrast improve the product production rate. In modern genetics, there are many tools for detecting such changes. Modern genetics enables detecting a single base change. Base changes might not reflect changes on the protein level. If the base change did not change an amino acid, it will not be effective. Such base changes give silent mutation, which means that such change is still giving the same amino acids. However, changes in other parts such as the change in the promoter-binding site might change polymerase binding affinity and then the polymerization rate (increase or decrease). One base change may not affect the protein structure but might affect protein/DNA interaction. However, that is not all the probability concerning one base change. If we put the role of heredity into account, the base change in one parent might become two in his or her child. Sickle cell anemia is an example. Alternatively, such base change is in certain genes. Newly added change might elevate serious problems or even results in cancer (Knudson model) (Knudson 1971). (For more details, refer to Amara, 2013a, and the references within.)

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The reader might start to notice the complication of the structure/function process. The level of the complication will be bigger in the case of the multicultural creature because they contain chromosomes, alleles, and more complicated genetic forms. Most of the molecular and biochemical studies were done so far  on bacteria. The bacterial cells contain one copy of each type of genes. Recombinant strains technology has enabled the study of the human genes and the expression of some important proteins such as insulin and interferon. Recombinant E. coli enables gene and plasmid transfer, replication, and expression (Amara 2013b, 2013c).

21.3 lac operon From the many examples that could be given that match with our review aim and that describe the role of both macromolecules and bioactive compounds, lac operon genes, its regulatory genes, its substrate, and substrate analog could explain a lot. Surprisingly, an analog for allolactose (that is derived from lactose and that inactivate the repressor protein function on binding with it), the IPTG (Kercher et al. 1997) could activate the lac operone system without being used by β-galactosidase (Figure  21.1). Such an example proves that the protein is doing their work with high specificity and selectivity, and an analog, similar to its substrate, could confuse it or even confuse a complete unit of different steps such as lactose metabolism, transport, and regulation. This proves that the protein starts to work on specific criteria. They react chemically based mainly on the substrate structure, their own structures, and the environment. E. coli is able to differentiate between similar sugars such as lactose and glucose through the activity of the proteins produced from the lac operon and its regulatory genes, plus other structures such as promoters, operators, and so on (Figure 21.2). The lac operon and its transcription regulatory region are responsible for the transportation and metabolism of lactose in E. coli and some enteric bacteria. The lac operon contains three genes: lacZ encodes β-galactosidase, lacY encodes lac permease, and lacA encodes thiogalactoside trancacetylase (Wall 2011). The lac transcription regulator region contains one gene, lac I (encoded the repressor CH2OH O OH OH CH2OH OH O O OH

CH2OH OH

O O

CH2OH O S O OH OH OH OH

CH2

OH OH

OH OH

OH Lactose

OH Allolactose

Figure 21.1 The structure of lactose, allolactose, and IPTG.

OH

IPTG

CH3 C

H CH3

lac operon cAMP--CAP complex cAMP

lac regulatory region

CAP RNA polymerase bind tp cAMP--CAP complex

E.coli chromosomal segment

lacI Promoter

Repressor

Repressor binding site

lacO

RNA polymerase

lacZ

lacY Permease gene

B-Galactosidase gene

Promoter Repressor just leave the repressor binding site

RNA polymerase

lacA

E.coli chromosomal segment

Transacetylase gene

on

ti

ip

cr

s an

RNA polymerase

Tr

n

tio

sla

an Tr

Transacetylase

Allolactose Allolactose

Permease Ribosome β-Galactosidase

Lactose

Figure 21.2 The lac operon and its regulatory genes.

Amino acids

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gene). The lactose permease transports lactose into the cell. β-galactosidase, a cytoplasmic enzyme, subsequently cleaves lactose into glucose and galactose. The cell does not allow those enzymes to be expressed if there is no lactose or if a simpler and preferred sugar source is available in the medium, such as glucose. This could be done through an efficient regulation-specific control for the lac genes, depending on the availability of the substrate lactose to the bacterium. The lacZYA are co-transcribed into a single polycistronic mRNA molecule. Transcription of all genes starts with the binding of RNA polymerase to the promoter region (upstream of the genes). The cAMP-bound catabolic activator protein aids binding of the promoter. cAMP levels are low when intracellular glucose levels are high. Adenylate cyclase (the enzyme that catalyzes, forming cAMP) apparently senses the intracellular level of an unidentified intermediate in glucose catabolism. When glucose levels drop, cAMP levels rise and cAMP interacts with a protein called cAMP-receptor-protein to form a complex. The change increases its affinity to the lac operon adjacent to the RNA polymerase binding site. This binding facilitates transcription of lac operon by stimulating the binding of RNA polymerase to form a closed promoter complex. Then, the RNA polymerase proceeds to transcribe all the three genes (lacZYA) into mRNAs. The lac I gene coding for the repressor protein lies nearly upstream to the lac operon and is always expressed (constitutive). If lactose is missing from the growth medium, the repressor binds tightly to a short DNA sequence downstream of the promoter near the beginning of lacZ called the lac operator. The repressor binding to the operator interferes with the binding of the RNA polymerase to the promoter, and there is no transcription (or low level). When the cells are grown in the presence of lactose, a lactose metabolite called allolactose binds to the repressor. The repressor 3D configuration after binding to the allolactose will be changed and then becomes unable to bind to the operator. The RNA polymerase transcribes the lac genes, leading to higher levels of the encoded proteins (Kercher et al. 1997). From this example, we learn the role of the structure, the spontaneous, fluid, specific, selective, and cooperative of the enzyme(s) function and the factors that could confuse such a perfect system. Compounds entering our bodies through different roots can improve, disprove, inhibit, activate, and damage many processes in our bodies. However, natural bioactive compounds are the most abundant, which are compatible with our biological system if used wisely and selectively.

21.4 The structure of β-globin in sickle cell anemia β-globin is one of the most important macromolecules in our bodies. Four units of β-globin are working as one hemoglobin, which is responsible for the oxygen/ CO2 transport and exchange between the blood and the lungs and the rest of

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Figure 21.3 The 3D structure of β-globin. For color detail, please see color plate section.

Figure 21.4 Magnified part of the 3D structure of β-globin. For color detail, please see color plate section.

our bodies (Figures 21.3 and 21.4). The type and the order of the amino acid constituent of any protein and their order play significant roles in its 3D folding structure. These four subunits will interact with each other to enable oxygen and CO2 exchanges (with the same molecules). This should be due to the changes happening in their 3D structure based on their location (either in the blood vessels of the lung or in our body’s deep tissues). Sickle cell anemia is a genetic disorder in which amino acid number 7 changes from glutamic acid to valine in the β-globin (built from 147 amino acids), thus the overall structure of the RBCs will change from a circle to a sickle shape (Figures 21.5–21.8). This change in RBCs is not only due to the change in the structure of the β- globin; it is also due to a collective gathering for different effects such as the interaction between the β-globin molecules, O2, CO2, and the environment where they exist. Only in the presence of O2 will β-globin fold correctly and the RBCs will be in correct form. Valine, unlike glutamic acid, contains a nonpolar group, resulting in a hydrophobic “sticky” region interacting with neighboring molecules, producing the observed clumping. When oxygen is removed from the sickle cell hemoglobin, the β-globin molecule’s shape will be changed and combine with the other β-globin molecules. This causes the blood to clot and deprives vital organs

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Figure 21.5 The 3D structure of normal β-globin. For color detail, please see color plate section.

Figure 21.6 The 3D structure of sickle cell anemia β-globin. For color detail, please see color plate section.

Valine

Histidine

Leucine

Threonine

Proline

Glutamic acid

Figure 21.7 The 3D structure of eight amino acids in normal β-globin.

Glutamic acid

Lysine

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Valine

Histidine

Leucine

Threonine

Proline

Valine

Glutamic acid

Lysine

Figure 21.8 The 3D structure of eight amino acids in the mutant (Valine) β-globin.

from the blood supply. We should take into account similar reactions between large numbers of macromolecules in our bodies. Supplying our bodies with correct substrate will bypass some protein-folding problems. For that, patients with sickle cell anemia feel good in the presence of excess O2. Sickle cell anemia is a hereditary disease and is considered a degenerative disease. Each somatic cell in a patient with sickle cell anemia has the mutant gene that is responsible for the disease. That explains how our genetics is so important. One base change leads to serious disease. A wrong molecule could do a lot. Geneticsbased diseases cannot always be repaired. There is no clear solution. The only biological solution is to repair the DNA itself. While those types of diseases are  hereditary, they could be avoided if detected before the marriage stage. Bioactive molecules might give a solution, if complemented with the defect and substituted without causing another type of defect or deterioration. The antioxidant section of this chapter shows how vitamin C is used as a solution. We all have a natural defect gene for vitamin C production and we should also get it from exogenous sources (Amara 2010).

21.5 Direct reversal pathway for DNA repair Besides the biological and chemical effects on the compounds entered or produced in our biological system, physical effects could activate chemical reactions, particularly the DNA, which must be corrected. For that, each cell has different repairing pathways, checkpoints, apoptosis, and other mechanisms for controlling and repairing DNA change. DNA repair by direct reversal pathway is discussed here. In this pathway, the repair happens directly in a few steps (Sancar 2003). The reverse reaction starts when the photolyase enzyme is activated by energy absorption of blue or UV light (300–500 nm). For more details, refer to Lynch (2009) and Amara (2013a). This example gives us a hint

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Correct form Thymine UV Photolyase AND White light Thymine dimer

Figure 21.9 Direct reversal of DNA Incorrect form

damage.

of how a simple factor, such as UV light (photons), could change our DNA (Figure 21.9). Our DNA could be affected due to our lifestyle, such as excess use of cell phones and computers, or intake of unknown food additives such as pigments, flavors, or undesirable bioactive compounds. In fact, there are a large number of physical, chemical, and biological factors that could affect our DNA as well as other cell macromolecules. For some limit, bioactive compounds could play a significant role against such physical reactions. Mostly this type of reaction is translated to a chemical reaction due to the energy that induced such a reaction. The player is often the electron, which is responsible for oxidation-reduction reactions. Antioxidants play a central role in protecting us against such reactions (Amara 2013a).

21.6 Oxidants, free radicals, and antioxidants: The balance Oxidants and free radicals are structures opposite to antioxidants. They are in spontaneous competition. There is a certain balance required between them (Amara 2013a). Useful bioactive compounds become harmful ones if used in higher amounts. They affect negatively if one of them exceeds its limits. Free radicals and oxidants play a vital role in our bodies, particularly as a part of the immune system. They are responsible for controlling the daily exposure to pathogens, in cell signaling, apoptosis, etc. (Valko et al. 2004, 2005, 2006; Seifried et al. 2007; Amara 2010).

21.7 The “nutritional genomics” The relationship between nutrients and gene expression leads to the science of nutritional genomics, also called nutrigenomics. Of course, such compounds can react positively and support our biological system if used correctly. Mistakes in a gene lead to a modified protein with altered activity or dysfunction, which could cause disease. Such diseases are

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genetically based. The environment and the type of sublimated nutrients could cure disease. The food decreases the load on cell macromolecules by supplying it with similar gradients. Alternatively, supplying a gene’s missed product allows the complete pathway to take place. Exo sources will allow the cell to do more urgent biological activities (Amara 2010), as well as to reduce the total number of DNA/RNA/protein cycles, which could induce spontaneous mutation (Harman 1956; Amara 2010). (For more information about the types and the classes of the antioxidants, refer to Amara, 2010, and the references within.)

21.8 Food affects our genes Surprisingly for many, food affects genetics. Some ethnic groups prohibit some variants of food where they believe that it could have an effect on behavior (Amara 2012; Bakulski and Fallin 2014). Food could affect our genes directly, and a new branch of science named epigenetics was formed to study this. Epigenetics is characterized by alterations to the DNA molecule that affect gene expression but do not change the nucleotide sequence (McCulloch and Kunkel 2008). There are at least three known mechanisms: DNA methylation, histone modification, and genomic imprinting. Genetic variation between individuals is an important factor in specifying to which limit food could contain active constituents and affect our DNA (Siow et  al. 1995; Ambrosone et  al. 1999; Jindal 2008; Amara 2010). These SNP regions can influence either nutritional status or the renutrition process because renutrition becomes progressively more difficult with age. New names in molecular biology were realized such as genomics, proteomics, and metabolomics to map the changes happening due to the interaction or the response of biological systems with or without a particular nutrient (Hirono et al. 1995; Kunkel and Erie 2005).

21.9 Active antitumor compounds from medicinal plant Plants as a source for medicinal active compounds were used from the beginning of time and are still the most important source for different structures and formulae. Such useful compounds include digitoxin, rutin, papin, morphine, codeine, papaverine, atropine, scopolamine, quinine, quinidin, reserpine, ergotamin, ergnovine, cocaine, vincoleukoblastine, leurocristine, d-tubocurarine, protovertarines A and B, ephedrine, sparteine, physostigmine, pilocarpine, colchicine, and caffeine. In addition, crude drugs extracted from Digitalis purpurea leaves and Rauwolfia serpentina root and extracts from Podophyllum peltatum (podophyllin), Rhamnus

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purshiana (anthraquinones), Cassia species (anthraquinones), and Plantago species (mucilage) are widely used for their medicinal activity. Also, a novel chemical structure, isolated from plant sources, often prompts the chemist to a successful series of modified semisynthetic compounds (e.g., atropine to homatropine, reserpine to syrosingopine, morphine to N-allylnomorphine), which may have useful medicinal economic value (Farnsworth 1966; Cordell and Farnsworth 1976; Miyachi et al. 1987; Amara et al. 2008).

21.10 Structure of plant bioactive compounds 21.10.1 Monoterpenes Monoterpenes, such as allamandin isolated from Allamanda cathartica (Apocyanaceae), were found to contain iridoid lactone and having in vivo P-338 antileukemic (PS) activity (Kupchan et al. 1974; de la Mare 2012).

21.10.2 Sesquiterpenes Sesquiterpenes were isolated and showed antitumor and/or cytotoxicity activity (Lee et  al. 1975a; Blasko and Cordell 1988; Ben Sghaier et  al. 2011; Siriwan et al. 2011).

21.10.3 Miscellaneous sesquiterpenes Nonlactonic sesquiterpenes such as gossypol from the cotton seed showed a T/C of 150% at 10 mg/Kg in the PS system (Blasko and Cordell 1988).

21.10.4 Germacrenolides Lephantinin from Elephantopus mollis (Compositae), a germacrenolide member, has antitumor activity against PS and Walker 256 carcinosarcoma (WM) (Lee et al. 1975b).

21.10.5 Guaianolides and pseudoguaianolides Deoxyelephantopin from Elephantopus carolinianus (Compositae) was active in the WM system in vivo and Pseudoguaianolide fastigilin C, from Baileya spp. (Compositea), showed activity in the PS system in vivo (Lee et al. 1975; Pettit et al. 1975).

21.10.6 Elemanolides Vernolepin, an elemanalid dilactone isolated from Vernonia hymenolepis (compositea), shows KB activity and in vivo WM activity (Kupchan et al. 1968; Negrete et al. 1993; Koul et al. 2003).

21.10.7 Diterpenes Triptiolide and tiptolide from Triptergium wilfordii, a member of diterpines, have potent antileukemic activity. Bioactive diterpenoids isolated from Dilophus ligulatus have strong activity against NSCLCN6-L16 cells (Kupchan, 1974; Jolad et al. 1975; Molnar et al. 2006; Fronza et al. 2012).

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21.10.8 Simaroublide (quassinoids) Bruceatin, a simaroublide compound isolated from Brucea antidysenterica (Simaroubaceae), showed antitumor activity against PS, lE, LL, and B16 systems. Quassinoids (Kupchen 1975), also known as simaroublids, are a group of degraded triterpene lactone derivatives extracted from a plant family. Acetoxypicrasine B, a quassinoid, was isolated from the stem bark and leaves of Soulamea fraxinifobia (Blasko and Cordell 1988).

21.10.9 Triterpenes A chloroform fraction from the root and leaves of the pitcher plant, Sarracenia flava (Sarraceniaceae) was found to elicit activity against the PS leukemia in vivo (Kakpal 1974; Laszczyk 2009; Yeh et al. 2009).

21.10.10 Cucurbitacin The fruit juice of Ecballium elaterium (Cucurbitaceae) has yielded cucurbitacines, which are predicted to have cytotoxicity, possibly in vivo antitumor activity. Trewinine, a new cytotoxic cucurbitacin, was isolated from Trewia nudiflora (Blasko and Cordell 1988; Duangmano et al. 2012; Ishii et al. 2013).

21.10.11 Withanolides Withanolide Q, a steroidal lactone, was isolated from Withnia somnifera (Solanaceae) (Kirson et al. 1975). Withaferin A and six analogues were isolated from the aerial parts of Withania frutescens Pauq. and Winthania aristata Pauq. with aferin A and its 5-hydroxy-6-chloro derivative exhibited marked cytostatic activity against hela cells in culture (Blasko and Cordell 1988; Machin et al. 2010; Samadi et al. 2012).

21.10.12 Cardenolides The known cytotoxic cardenolide is cymarin, its toxicity in the KB system is extracted from Perquetine nigrescens (Asclepiadaceae) (Marks et al. 1975; Wang et al. 2007; Al-Ghoul et al. 2008).

21.10.13 Lignans Podophyllotoxin and several related aryltetralin lignans, deoxypodophyllo toxin, 3’-demthylpodophyllotxin, (1R,2R,3R)-desoxypodophyllo toxin-pelatin (Loike, 1984), and B-pelatatin, isolated from the plant families Berberidaceae and Cupressaceae, are cytostatic spindle poisons. Etoposide is the most active agent yet tested for the treatment of lung cancer. In addition, it has therapeutic value against AIDS-associated Kaposi sarcoma (Dewick and Jackson 1981; Blasko and Cordell 1988; Sakagami et al. 1991; Yoo and Porter 1993). Steganone, first isolated by Kupchan and coworkers, is an example of a dibenzocyclooctadiene-type lignan exerting antitumor activity (Blasko and Cordell 1988; Mulligan et al. 2012).

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21.10.14 Diterpene alkaloids Norerythrostachaldine is from Erythropheleum chlorostachys (Leguminosae) and its 3-B-di-acetate (Loder and Nearn 1975; Blasko and Cordell 1988). Two steroidal alkaloid glycosides were isolated from Nicotiana plumbaginifolia (Solanaceae), and are named Solaplumbin and Solaplubinin. The former alkaloid reduced tumor weight in the WM (Walker 256 carcinosarcoma) system by 87% at a dose of 15 mg/kg, whereas the latter alkaloid reduced tumor weight by 83% at 10 mg/kg and 89% at 20 mg/kg (Kang et al. 2008; Colombo et al. 2009).

21.10.15 Pyrrolizidine alkaloids Pyrrolizidine exerts remarkably diverse biological activities (Yan and Huxtable 1995). All of the active antitumor pyrrolizidine alkaloids contain an allylic alcohol function (e.g., supinine, heliotine, and its N-oxide, crispatine, fulvine, monocrotaline, and indicine N-oxide). Indicine N-oxide shows high- level activity against B16 melanoma, mammary xenograft, M5076 sarcoma, P388 leukemia, and Walker 256 carcinoma (Blasko and Cordell 1988; Cheng et al. 2013; Kostenko et al. 2013).

21.10.16 Isoquinoline alkaloids Thalicarpine, a potant antileukemic agent from Thalictrum minuselatum and Thalictrum dasycarpum, was synthesized in 10 steps via hernandaline and the synthesis of analogues was reported (Dutschewsha and Mallov 1966; Kupchan and Liepa 1974a, 1974b; Piyanuch et al. 2007; Wang and Yang 2008).

21.10.17 Benzo α phenanthridine alkaloids Benzo α phenanthridine alkaloids are widespread in nature. Two representatives, niadine and fugaronine, are highly active against the P388 lymphocytic leukemia system (Fish et al. 1975; Blasko and Cordell 1988; Lamoral-Theys et al. 2009).

21.10.18 Berberines The berberine alkaloids are closely related to the benzo α phenanthridines. Although berbeine showed in vitro activity, it was divided of in vivo activity in the Ehrich ascites test system in a dose range of 2.5–7.5 mg/kg/day (Shvarev and Tselin 1972; Blasko and Cordell 1988; Piyanuch et al. 2007).

21.10.19 Phenanthroidolizidine and phenanthroquinolizidine alkaloids Tylophorinicine, a new phenanthroindolizidine alkaloid, was isolated from the roots of Tylophora asthmatica and Pergularia pallide. Four alkaloids—tylohirsutinine, tylohisutinidine, diarylindolizidine derivative, and 13a-hydroxysepticine—were obtained from Tylophora hirsuta Wight. Diarylquinolizidine, Kayawongine, was isolated from Cissus rherifalina Planch. All previous compounds showed variable antitumor activity using different tumorigenic systems (Blasko and Cordell 1988).

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21.10.20 Taxus alkaloids The alkaloid taxol isolated from several Taxus species including T. brevifobia Nutt., T. baccata var. barroni Barron, and T. cuspidata, displays very good activity against the B16 melanoma and MX-1 mammary xenograft systems, and showed moderate activity against the L 1210, P388, and P1534 leukemia system, and CX-1 colon, and LX-1 lung xenografts. Taxol is effective by a mechanism different from that of any other known anticancer drug that usually acts on DNA, RNA, or protein synthesis. Taxol proved to be a mitotic inhibitor that promotes the assembly to depolymerize (Blasko and Cordell 1988; Sharma and Straubunger 1994; Gore et al. 1995; Langenfeld et al. 2013; Miao et al. 2012).

21.10.21 Acronycine Acronycine, an acridone alkaloid first isolated from the bark of Acronychia baueri, exerted a broad spectrum of in vivo antineoplastic activity (Blasko and Cordell 1988; Depauw et al. 2009; Nguyen et al. 2009).

21.10.22 Camptothecin alkaloids Camptothecin and related alkaloids are a well-established series of antitumor agents (Blasko and Cordell 1988; Pantazis et al. 1993a, 1993b, 1993c; Matsumoto et al. 1994).

21.10.23 Cephalotaxus alkaloids The active antitumor principal of Cephalotaxus harringtonia var. drupacea (Cephalotaceae) are harringtonine and homoharringtonine; the isolation, preparation, and pharmacological activity of Cephalotaxus alkaloids are available (Powell et al. 1972; Mikolajczak et al. 1972; Abdelkafi and Nay 2012; Langenfeld et al. 2013).

21.10.24 Bisindol alkaloids The clinically and commercially most important antitumor alkaloids are bisindole alkaloids such as vincaleukoblastine and leurocristine isolated from the leaves of Madagascan periwinkle and Catharanthus roseus, respectively. The successful clinical application of these compounds in the treatment of leukemia and Hodgkin’s disease has accorded special importance to the group of the alkaloids and their related derivatives (Brannon and Neuss 1975; Eckenrode et al. 1982; Harborne 1984; de Camargo 1991).

21.10.25 Maytanosinoids Maytasinne and related maytansine ester are potant antileukemic agents obtained from Maytenus ovatus and M. buchanaii (Celastraceae) (Kupchan et al. 1972, 1975).

21.10.26 Colchicine Colchicine, the major alkaloid of Colchicum autumnale L., and related compounds generally exert antimitotic properties, interfere with mitocrotubule-dependent cell functions, and irreversibly bind to tubulin (Blasko and Cordell 1988).

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21.10.27 Ellipticines The ellipticines comprise one of the most important groups of antitumor alkaloids. Ellipticine and 10- methoxyellipticine exert marked activity against the L1210, P388, and P1534 leukemias, the X5563 myloma, and Gardner lymphosarcoma (Blasko and Cordell 1988; Schwaller et al. 1990; Procházka et al. 2011; Stiborova et al. 2012).

21.10.28 Emetine Emetine, the major alkaloid of Cephaelis ipecacuanha Rich., and its 2,3-dehydro derivative are used clinically in the treatment of amebiasis. Emetine is effective against L1210 as well as P 388 leukemias, and thus the Emetine alkaloids have become a target of interest as potential antitumor agents (Blasko and Cordell 1988; Larsson et al. 2012; David et al. 2013).

21.10.29 Amaryllidaceae alkaloids Amaryllidaceae alkaloids such as ycorine type, which contain aphenanthridine moiety, display antitumor activity (Blasko and Cordell 1988).

21.10.30 Cis-aconitic acid Cis-aconitic acid occurs naturally in Aconitum, Achillea, and Equisetum species and is a potent inhibitor of 3,4-benzo-pyrene-induced carcinogenesis in mice by simultaneous subcutaneous administration (Kallistrators and Kallistrators 1975; Kallistrators 1975).

21.10.31 Crotepoxide Crotepoxide is a tumor inhibitor that showed activity against the LL system, and is originally isolated from the fruits of Croton macrostachys (Euphorbiaceae) (Kupchan et al. 1969).

21.10.32 Lapachol Lapachol was clinically evaluated where it has antitumor properties against the Walker 256 tumor system. The results were disappointing because an anticoagulant activity was a common side effect (Suffness and Douros 1980; Oliveira et al. 2012; Parrilha et al. 2012).

21.10.33 Flavonoids The flavonoids are all structurally derived from the parent substance flavone, which occurs as a white mealy farina on Primula plants, and all share a number of properties in common. The detection of a range of isoflavonoids in human urine is reported and their origin from lignans found in a range of foodstuffs (whole-grain products, various seeds, fruits, and berries) is discussed (Adlercreutz et al. 1993). Scutellaria baicalensis extracts are reported to have antitumor properties. Fourteen flavones were isolated from the acetone extract of scutellaria

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baicalensis roots. 5,7,2’-Trihydroxy flavone and 5,7,2’,3’-tetrahydroxyflavone have inhibitory effects on mouse skin tumor (Konoshima et al. 1992; Radhika et al. 2012).

21.11 Bioactive compounds not always friends One should highlight that not all of the bioactive compounds are useful. Some have harmful side effects. Some are tumor promoters and others are procarcinogens (Amara et al. 2008, 2013).

21.11.1 Carcinogenicity of antitumor drugs Second neoplasms can be developed as a complication of chemotherapy where malignancies were observed in patients receiving cytotoxic drugs for non-neoplastic diseases, as in patients suffering rheumatoid arthritis and glomerulonephrities (IARC 1981). Many cancer chemotherapeutic drugs carry some of very reactive moieties that are often associated with carcinogenicity in animals (Weisburger 1977). In 1982, the International Agency for Research on Cancer (IARC) classified a number of cytotoxic drugs as “carcinogenic” agents such as busulfan, cyclophosphamide, chlorombucil and melphan, actinomycine-D, adriamycin, and cisplastin (IARC 1982; Khalil et al. 1995; Chahwala and Hickman 1985).

21.11.2 Tumor promoters from plants Several Euphorbiaceae were studied for their irritant and/or tumor-promoting activity (Kinghorn and Evans 1975). Macrozamine, a known potent carcinogen, was isolated from the seed of Encephalartas altensteinii (Zamiaceae) (Watanabe et  al. 1975). Monocrotalin, a pyrolizidine alkaloid, and its major metabolite dehydroretonecine produced rhabdomyosarcomas in 51.6% of treated rats at the injection site (Allen et al. 1975; Schoental 1975).

21.12 Use of drug combinations There are several reasons to use drugs in combination rather than a single agent where the development of resistance to cancer drugs becomes a problem. Cancer is not made up of homogeneous cells; it is a mixture of heterogeneous cells that are genetically unstable and lead to mutation. Using a combination of drugs increases the chances that the mutant will not survive. In addition, active combinations decrease the cancer cells’ ability to repair damage and to delay or prevent the development of resistance (Devita et al. 1986; Hutson et al. 1992; Brumen et al. 1995).

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21.13 Screening for the presence of alkaloids and/or nitrogenous bases and flavonoids in plant extracts The existence of alkaloids and/or nitrogenous bases and flavonoids are an indication of the presence of an active physiological compound.

21.13.1 Test for alkaloids and/or nitrogenous bases (Harborne 1980) Extract 10 grams of the air-dried part of the plant, which should be grinded to powder with acidified water. Filtrate the acidified water after a propitiate extraction time. Add 5% ammonium hydroxide solution to the acidic filtrate to render it to alkaline and then extract the mixture with chloroform. Collect the chloroform layer in another container then evaporate to dryness and dissolve residue in 2 ml of 10% diluted hydrochloric acid and test with the following alkaloidal reagents: 1 Dragendorff’s reagents (solution a: 0.85 g bismuth subnitrate dissolved in a mixture of 10 ml glacial acetic acid and 40 ml water; solution b: 8 g potassium iodide dissolved in 20 ml water; color reagent: mix 5 ml solution a with 5 ml solution b, add 20 ml glacial acetic acid, complete to 100 ml with water). 2 Iodoplatinate’s reagent (solution a: 5% platinic chloride in water; solution b: 10% potassium iodide in water; color reagent: 5 ml solution a, 45 ml solution b, and 100 ml water were mixed). 3 Mayer’s reagent (1.355 g mercury iodide, 5.0 g potassium iodide in distilled water to make 100 ml). An orange, violet, or white precipitate obtained respectively indicates the presence of alkaloids and /or nitrogenous bases. Thin-layer chromatography was carried out to detect the presence of alkaloids in the chloroform plant extract using solvent system methanol:chloroform (1:9) as a running solution. Silica gel G60 Merck was used to prepare the chromatoplate (20 × 20 cm), which was activated at 110°C for 30 min. Two mg of each plant sample was dissolved in 0.5 ml chloroform, then 50 μl was spotted and the plate was immersed in the solvent system. After completing the run, the plates were taken out from the jar, allowed to dry, and then sprayed either with Dragendorff’s reagent or with Iodoplatinate’s reagent. Orange and violet colors were obtained, respectively showing the presence of alkaloids and/or nitrogenous bases. Mayer’s reagent will give a white precipitate.

21.13.2 Tests for flavonoids (Harborne 1980) An ethanolic extract of the air-dried powder of each plant sample was exposed to magnesium metal turned up to 1 min, and then 3 ml of hydrochloric acid (1%) was added. A faint reddish color obtained shows the presence of flavonoids. One gram of the air-dried powder of each plant sample was treated with 5 ml 1% hydrochloric acid solution. The sample was shaken while adding 5 ml drop by drop of 5% sodium hydroxide until a yellow color was formed showing

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the presence of flavonoids. Thin-layer chromatography was carried out to detect the presence of flavonoids in an ethanolic extract of the air-dried powder of each plant sample using solvent system acetic acid:water (1:1). Silica gel G60 Merck was used to prepare the chromatoplate, which was activated at 110°C for 30 min. Two mg of each plant sample was dissolved in 0.5 ml chloroform, then 50 μl were spotted and the plate was immersed in the solvent system. After completing the run, the plate was taken out from the jar, allowed to dry, and, tested under a UV lamp at 224 nm and 366 nm, the appearance of fluorescence under the UV light shows the presence of flavonoids.

21.14 Protocol for in vitro detection of antioxidants This protocol is described here to show the reader that chemical compounds such as the pesticide lindane could activate aryle hydrocabon hydroxylase (AHH), one of the CYTP450 enzymes. Such an enzyme could degrade a benzo(α) pyrene, which is a procarcinogen. Such a reaction could happen due to our exposure to such toxic compounds.

21.14.1 AHH activity and antioxidant activity against H2O2 production by the lindane-induced mice hepatic microsomes Awney et al. (1997) used two protocols, AHH activity and antioxidant activity, to describe how to investigate the inhibitory effect of different plant extracts on aryl hydrocarbon B(a)p hydroxylase activity and the mediated H2O2 production (as a major source of DNA oxidative damage in cells) in mouse liver treated with lindane. However, such a protocol could be used to detect different types of antioxidants.

21.14.2 Experimental animals House up to five mice in a cage, according to National Institutes of Health guidelines, with free access to distill water and food. Chemicals: Lindane (1, 2, 3, 4, 5, 6-hexachlorocyclohexane) (99%), benzo (α) pyrene (99%), and nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) and catalase.

21.14.3 Preparation of mice hepatic microsomal fraction Prepare liver microsomes from mice injected intraperitoneally with organochlorine insecticide (e.g., Lindane) dissolved in corn oil (10 mg/kg) for 4 days. The control animals receive corn oil only. Then the mice were fasted overnight, starved after the last injection, and sacrificed by cervical dislocation. Open the abdominal cavities immediately and weigh the liver, then wash with cold 0.1 M phosphate buffer, pH 7.4. All the above operations were carried out at 4°C. The washed livers were minced with sterile scissors in 2 volumes of 0.25 M sucrose

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(2 ml/g wet liver), and homogenized with a Teflon piston using five strokes. The homogenate was centrifuged at a high speed for 20 min at 11,000 xg to remove intact cells, nuclei, and mitochondria. The supernatant was subsequently ultracentrifuged at 105,000 xg for 60 min to sediment the microsomal pellets decreased. The supernatant was discarded and the microsomal pellets in 0.25 M sucrose (3 ml/10 g liver) was resuspended and stored immediately in 1 ml aliquots at –70°C (Rayan et al. 1978).

21.15 Determination of AHH activity Aryl hydrocarbon hydroxylase activity can be determined by the method of Awney et  al. (1997) with some modifications. The reaction mixture, in a total volume of 1 ml, contained 50 pmoles of tris-chloride buffer, pH 7.4; 0.36 μmoles of NADPH; 3 μmoles of MgCl2/0.1 ml microsomes suspended in 0.1 M potassium phosphate buffer pH 7.4 (1 mg protein/ml). One hundred nmoles substrate, benzo (α) pyrene were added in 0.05 ml methanol to start the reaction. Plant extracts were added in 10 μg/ml. After 10 min of incubation at 37°C, the reaction was terminated by the addition of 1.0 ml cold acetone and the mixture was shaken with 3.0 ml hexane for 10 min to extract the derivatives of benzo (α) pyrene. A 1.0 ml aliquot of the organic layer was extracted with 2 ml 1 N NaOH and the fluorescence of the NaOH extract was measured immediately at 396 nm excitation and 522 nm emission by using a spectrofluorometer. The amount of enzyme is defined as the pmoles of phenolic product (equivalent to 3-hydroxybenzo (α) pyrene formed during the incubation)/mg protein/min (Awney et  al. 1997; Amara et al. 2008). The amount of the protein concentration in hepatic microsomes fraction was assayed by the method of Lowry et al. (1951) (or any protein quantification method). Bovine serum albumin can be used as a standard.

21.16 Determination of microsomal hydrogen peroxide The hydrogen peroxide formation during the metabolic activation of B (a) p in the presence of lindane-induced hepatic microsomes can be assayed by the method described by Hildebrandt et al. (1978). Plant extract was added to the reaction mixture of 10 μg/ml.

21.17 Conclusion Bioactive compounds have different structures and functions. They do their work mechanically but with high specificity and selectivity. They are in optimum natural structure (native). Minor change could lead to serious diseases such as sickle cell anemia. Such changes could happen through different routes.

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That which become built in our genetic material and which able to be transferred to our offspring is the most complicated one to repair, but the easiest one to be avoided (before marriage). Compounds from natural resources are usually safer than synthetic ones. Managing resources based on knowledge of structures and functions will save many efforts. Bioactive compounds from edible plants could save time, money, and give the best-expected results. In contrast, single unknown compounds could activate unknown biopathways. Structures in our bodies range from simple molecules, and compounds to more complicated ones, the macromolecules. Each has its own function. O2, which is a simple molecule, is essential for life. The different structures react with each other based on their chemical structure, orientation, and the environment where they exist. In the case of macromolecules, the structure will be more dynamic based on the type of monomeric constituents and the environments where they exist. Protein is the most complex macromolecules. Because of this, there are many different types and structures. One change in protein amino acids could lead to serious diseases.

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CHAPTER 22

Bioactive compounds as growth factors and 3D matrix materials in stem cell research Naveen Kumar Mekala1, Rama Raju Baadhe2, and Ravichandra Potumarthi3 1

Clinical Research Facility, Centre for Cellular and Molecular Biology (CSIR), Hyderabad, India Department of Biotechnology, National Institute of Technology, Warangal, India 3 Department of Chemical Engineering, Monash University, Clayton, Victoria, Australia 2

22.1 Introduction Stem cell research is a complex and very thrilling field that promises fantastic healing discoveries in several areas from cancer therapy to regeneration of various body parts. During this study use of biotic factors/molecules or growth factors has played an important role in the development of stem cell research (Wu et  al. 2004). These biologically active molecules have been administered into stem cells either to improve or maintain stem cell proliferation or to support controlled differentiation into more defined tissues. External factors influencing the growth and differentiation of stem cells would include, but are not limited to, extracellular matrix (ECM), soluble factors, pH, mechanical forces and mechanical properties of the cell environment, and direct contact with other cells (Mekala et al. 2012). Thus, in order to manipulate stem cell fate successfully, it is necessary to be able to identify the differentiation stage of the cells. Our chapter provides a detailed review concerning the use of biologically active molecules/compounds that interact with the stem cells and mediate their behavior and fate. Because cells are vulnerable to the higher concentration of bioactive molecules and/or growth factors, determining the appropriate concentration of each bioactive component is an essential step in the control of the differentiation steps (Mekala et  al. 2013). Along with stem cell differentiation studies we are also going to discuss the therapeutic benefits of biologically active molecules in the construction of three-dimensional (3D) artificial tissues and organs, helpful in regenerative medicine. As said above, the most widely used biotic compounds are various growth factors, which include cell differentiation factors, angiogenic factors, and other regulatory factors. These biotic factors may provide numerous cellular and Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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biochemical functions and these compounds may amend cell migration, proliferation, and attachment. These biotic compounds may be exogenously added or the cells may synthesize in response to physiological requirements. Literature also supports that the ECM of few stem cell types carries cytokines and growth factors and are potential modulators of cell functionality (Liu 2010). The most-studied growth-supporting biotic factors are multiple isoforms of transforming growth factor β (TGF-β), fibroblast growth factor (FGF), bone morphogenic proteins (BMPs), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), each with their own specific activity. Though the above biotic compounds are very essential, the optimal dose determination for varying physiological requirements is very difficult. Some of these biotic compounds have shown promising results in a variety of cell types both in vivo and in vitro.

22.2 Prominent growth factors 22.2.1 Transforming growth factor β (TGF-β) Transforming growth factors and its family, including activins and BMPs, have been widely applicable in the growth and maintenance of different organs, in which stem cells play significant roles (Watabe and Miyazono 2008; He et  al. 2013). It is clear from the literature that signals from the TGF family have shown to influence gene expression profiling of both embryonic and adult stem cells. Nodal signals are especially prone to have an important role in the preservation of ESC characteristics (Blank et al. 2008). The schematic in Figure 22.1 depicts the correlations of TGF signals in the specification of germ layers between in vivo and in vitro ESC differentiation systems. Ectoderm is differentiated from human and mouse ESCs in the absence of TGF signals, while primitive streak differentiation is persuaded by activin/nodal and BMPs; mesodermal differentiation occurs in the presence of BMPs and medium-range activin/nodal signals (Rodaway 2001). In this regard, it is clear that TGF family members are very significant during embryogenesis and somatic cell differentiation (Dreesen and Brivanlou 2007). Better understanding the roles of TGF in embryonic and somatic cell functionalities will support both basic research in the area of stem cells and their possible applications.

22.2.2 Fibroblast growth factor (FGF) Among the osteogenic supplements, fibroblast growth factor (FGF) is well recognized to have a crucial role in the cultivation of stem cell populations, as it assists ex vivo bone marrow stem cell (BMSC) expansion (Varkey et al. 2006). BMSCs include a population of multipotent mesenchymal stem cells (MSCs), which bridge the evolution from pluripotent embryonic stem cells to fully differentiated bone-forming cells (osteoblasts). Exposure of mesenchymal stem cells to

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BMPs

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Activin Skin Mesoderm Nueral

Activin

BMPs

Vascular/ Hematopoietic

BMPs ESCs

Primitive ectoderm

Posterior

Ectoderm Medium activin

Primitive streak Primitive endoderm

BMPs

Cardiac Skeletal

High activin

Activin Anterior

Liver Definitive ectoderm

Pancreas Others

Figure 22.1 Schematic of TGF members in stem cell multiplication and differentiation.

FGF positively selects adult stem cell populations (Bianchi et al. 2003; Maegawa et al. 2007) and stimulates bone regeneration and repair in vivo when the protein is directed in animal models (Nagai et al. 1995; Martin et al. 1997). Therefore, FGF could be an imperative addition for the cultivation of embryonic stem cells (ESCs) as well as when they are expanded for bone tissue repair.

22.2.3 Bone morphogenic proteins (BMPs) Numerous signaling pathways have been established to play the key role in stem cell fate determination. These pathways embrace those of the Wnt protein (Sato et  al. 2004), leukemia inhibitory factor (LIF) (Matsuda et  al. 1999), and bone morphogenetic proteins (BMPs) (Ying et  al. 2003). However, thorough understanding of the molecular mechanisms regulating the stem cell fate by these extracellular factors remains unknown. Bone morphogenic proteins are members of the TGF-β super family that have long been recognized to function in the development and regulation of a wide range of cell-based systems. These extracellular biotic compounds were originally isolated from bone extracts that induced ectopic cartilage and bone regeneration when implanted into muscle (Shimasaki et  al. 2004). However, bone morphogenetic proteins were also demonstrated to function in multiple developmental processes, including the induction of mesoderm during gastrulation, dorsoventral patterning within the neural tube, and hematopoiesis (Attisano and Wrana 2002). As expected from these complex in vivo functions, BMPs also play key roles in controlling stem cell fate choices during its differentiation. For example, BMPs can direct mesenchymal stem cells into chondrogenic as well as osteogenic cell lineages (Tiedemann et al. 2001). BMPs have also been shown to control the fate of neural crest stem cells (NCSCs) (Hyytiainen et  al. 2004). Furthermore, the latest discovery signifies the key role for BMPs in upholding embryonic stem (ES) cell self-renewal is reliable with the notion that this family of secreted factors have broad roles in regulating stem cell biology (Ying et al. 2003).

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22.2.4 Platelet-derived growth factor (PDGF) Platelet-derived growth factor is a glycol protein with a molecular weight of 17kDa, which are secreted by the platelets at the site of wound. Other than platelets, PDGF is also synthesized by endothelial cells and macrophages and promotes connective tissue healing and bone repair. PDGF regulates cell proliferation, migration, mitogenesis, angiogenesis, and ECM synthesis important in wound healing. This PDGF exists in four different forms with a highly conserved eight-cysteine homology domain. PDGF receptors are very vital in cell chemotaxis inhibition, protein synthesis, development of embryonic neuron fiber, and lung development. PDGF also demonstrate mitogenecity in vascular smooth muscle cells (VSMCs), which supports the angiogenesis in cardiovascular and neuronal systems.

22.2.5 Vascular endothelial growth factor (VEGF) Vascular endothelial growth factor is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo studies. Well-known in vitro activity of VEGF is the skill to endorse growth of vascular endothelial cells derived from arteries, veins, and lymphatics (Gille et al. 2001). It is made clear by the research that VEGF also promotes angiogenesis in 3D in vitro models (implants/scaffolds), inducing confluent microvascular endothelial cells to proliferate into collagen gels and form blood capillary-like structures (Carmeliet 2005). VEGF is also responsible for distinct angiogenic response in a variety of in vivo models such as chick chorioallantoic membrane, rabbit cornea, primate iris, and matrigel plug in mice (Nagy et al. 2002; Ferrara 2004). As per existing evidence, in addition to the above roles, VEGF can affect the movement of monocyte chemotaxis and increased production of B cells (Hattori et al. 2001). Subsequently, VEGF is reported to have hematopoietic effects (promotes RBC multiplication), inducing colonies by mature subsets of granulocyte macrophage progenitor cells (Gabrilovich et al. 1998). During bone tissue engineering, bone formation and angiogenesis are very closely associated and therapeutic angiogenesis is a novel approach that promotes new blood vessel formation by delivering angiogenic growth factors such as VEGF (Medha et al. 2011). Prior to efficient osteogenesis, there is a need for formation of new blood vessels that invade and mediate the oxygen and nutrient supply to osteoprogenitor cells (osteoblasts) (D’Amore 1999). It has been proved that MSCs plus VEGF led to more intense and homogenous vascularization of the defective regions and fastest resorption in the case of large bone defects (Medha et al. 2011).

22.3 Biotic scaffolds for tissue regeneration The composition of the 3D extracellular matrix is a complex mixture of structural and functional proteins, glycoproteins, glycosaminoglycans, and small molecules arranged in a unique, 3D tissue-specific architecture (Ingber 1991). Various biological components used in the fabrication of tissue engineering scaffolds are discussed below.

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22.3.1 Collagen Collagen is the abundant protein within the extracellular matrix of the mammalian system. More than 90% of the cell dry weight from most tissues and organs correspond to collagen. Still now, more than 20 different collagen types have been identified, each with an exclusive biologic function (Ottani et  al. 2002). Among collagen types, type I collagen is the chief structural protein present in tissues and can be found within both the animal and plant species. In the case of higher vertebrates, type I collagen is abundant in tendons and ligaments and provides the necessary strength to accommodate uniaxial and multiaxial mechanical loading. These tendons and/or ligaments can provide a convenient source of collagen for many medical device applications (Brown et  al. 2006). Bovine type I collagen is harvested from the Achilles tendon and is perhaps the most commonly used xenogeneic extracellular matrix component intended for therapeutic applications. Other than type I collagen, various alternative collagen types exist in the ECM of most tissues but in much lower quantities than type I. These collagen types provide dissimilar mechanical and physical properties to the ECM and add to the population of ligands that interact with the resident cell populations (Baldwin 1996). For example: •  Type IV collagen within the basement membrane of most vascular tissues and within tissues that contain an epithelial cell element. The ligand affinity of type IV collagen for endothelial cells is the cause for its use as a biocompatible coating for medical devices proposed to have a blood interface. •  Type III collagen within the submucosal tissue of preferred organs such as the urinary bladder, a site in which tissue elasticity and compliance are required for suitable function as opposed to the more rigid properties required of a tendon or ligament (Badylak 2004). •  Type VI collagen is a fairly small molecule that serves as a connecting unit between glycosaminoglycans and larger structural proteins such as type I collagen, thus providing a gel-like uniformity to the extracellular matrix.

22.3.2 Fibronectin Fibronectin is the second largest structural protein component next to collagen in quantity within the ECM. Fibronectin is a dimer of 250000MW subunits and survives both in soluble and tissue isoforms and contains ligands for adhesion of many cell types (Schwarzbauer 1991). The ECM of basement membranes, submucosal structures, and interstitial tissues all contain rich fibronectin. The cell pleasant characteristics of this protein have made it a smart substrate for in vitro cell culture and for use as a coating for synthetic scaffolds to promote host biocompatibility (Miyamoto et  al. 1998). Fibronectin is rich in amino acids Arg-Gly-Asp (RGD) subunit, a tripeptide that is imperative in cell adhesion via the α5β1 integrin. Fibronectin is also detected at an early stage within the developing embryos and it is vital for typical biologic development, particularly the growth of vascular structures. The significance of this molecule and its

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relations with other matrix components cannot be overstated with regard to cell–ECM communication (McPherson and Badylak 1998).

22.3.3 Laminin Laminin is a composite adhesion protein that exists in the extracellular matrix, especially within the basement membrane. This protein plays a vital role in premature embryonic development and is possibly the best studied of the ECM proteins found within embryonic bodies (Li et al. 2002). This trimeric cross-linked polypeptide sustains in abundant forms dependent upon the particular mixture of peptide chains (e.g., a1, b1, g1). The major role of laminin in the development and maintenance of vascular structures is mostly noteworthy when taking into account the ECM as a scaffold for tissue reconstruction (Ponce et al. 1999). The crucial role of the β-1 integrin chain in intervening hematopoietic stem cell interactions with fibronectin and laminin has been decisively recognized. Loss of the β-1 integrin receptors in mice results in intrapartum mortality. This protein emerges to be among the first and most important ECM factors in the course of cell and tissue differentiation (Werb et al. 1999). The definite role of laminin in tissue rebuilding when ECM is used as a scaffold for tissue and organ reconstruction in adults is unclear but its significance in developmental biology suggests that this molecule is vital for organized tissue development as opposed to scar tissue structure.

22.3.4 Glycosaminoglycans The 3D extracellular matrix contains a variety of mixtures of glycosaminoglycans (GAGs), depending on the tissue location of the ECM in the host, the age, and the microenvironment. The GAGs bind growth factors and cytokines, encourage water retention, and add to the gel properties of the ECM (Sohr and Engeland 2008). The heparin-binding properties of several cell surface receptors and of many growth factors (e.g., vascular endothelial cell growth factor, fibroblast growth factor) make the heparin-rich GAGs useful components of naturally happening substrates for cell growth (Zhou et al. 2002). The glycosaminoglycans of ECM include chondroitin sulfates A and B, heparin, heparan sulfate, and hyaluronic acid. Hyaluronic acid has been extensively investigated as a scaffold for tissue reconstruction and as a carrier for chosen cell populations in therapeutic tissue engineering. The amount of hyaluronic acid within ECM is at maximum in fetal and newborn tissues and therefore tends to be associated with desirable healing properties. The specific role, if any, of this GAG upon progenitor cell growth and differentiation during adult wound healing is unknown.

22.4 Conclusion Prospects of cell-based therapies depend on the prevention of in vivo teratoma/ tumor formation, in particular when using human stem cells (hSCs), such as induced pluripotent stem cells and embryonic stem cells. The fate of these hSCs

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in vivo is tightly regulated by various physiological agents that can effectively inhibit these anti-apoptotic factors, leading to selective and efficient proliferation and differentiation of stem cells. Taken together, this chapter provides the proof of concept that various biotic factors including growth factors, small molecules, and ECM components regulate the target pathway(s) for safe stem cell–based therapies.

References Attisano, L and J.L. Wrana. 2002. Signal transduction by the TGF-β super family. Science. 296(5573):1646–1647. Badylak, S.F. 2004. Xenogeneic extracellular matrix as a scaffold for tissue reconstruction. Transpl Immunol. 12:367–377. Baldwin, H.S. 1996. Early embryonic development. Cardiovasc Res. 31:E34–E45. Bianchi, G., A. Banfi and M. Mastrogiacomo. 2003. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res. 287:98–105. Blank, U., G. Karlsson and S. Karlsson. 2008. Signaling pathways governing stem-cell fate. Blood. 111(2):492–503. Bonewald, L.F. 1999. Regulation and regulatory activities of transforming growth factor beta. Crit Rev Eukaryotic Gene Expr. 9:33–44. Brown, B., K. Lindberg, J. Reing, D.B. Stolz and S.F. Badylak. 2006. The basement membrane component of biologic scaffolds derived from extracellular matrix. Tissue Eng, 12(3), 519–526. Carmeliet, P. 2005. VEGF as a key mediator of angiogenesis in cancer. Oncology. 69 (Suppl. 3):4–10. D’Amore, P.A. 1999. Angiogenesis. Sci Med. 6:44–53. Dreesen, O and A.H. Brivanlou. 2007. Signaling pathways in cancer and embryonic stem cells. Stem Cell Rev. 3(1):7–17. Entwistle, J., S. Zhang and B. Yang. 1995. Characterization of the murine gene encoding the hyaluronan receptor RHAMM. Gene. 163(2):233–238. Ferrara, N. 2004. Vascular Endothelial Growth Factor: Basic Science and Clinical Progress. Endocr Rev. 25(4):581–611. Gabrilovich, D., T. Ishida and T. Oyama.1998. Vascular endothelial growth factor inhibits the  development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood. 92:4150–66. Gille, H., J. Kowalski, B. Li, J. LeCouter, B. Moffat, T.F. Zioncheck and N. Ferrara. 2001. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2) A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem. 276(5):3222–3230. Hattori, K., S. Dias and B. Heissig.2001. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med. 193:1005–14. He, E., J.H. Cui and C. Li. 2013.The combined effects of transforming growth factor-β and basic fibroblast growth factor on the human degenerated nucleus pulposus cells in monolayer culture. Tissue Eng Regen Med. 10(3):146–54. Hodde, J.P., S.F. Badylak, A.O. Brightman and S.L. Voytik-Harbin. 1996. Glycosaminoglycan content of small intestinal submucosa: a bioscaffold for tissue replacement. Tissue Eng. 2:209–217 Hyytiainen, M., C. Penttinen and J. Keski-Oja. 2004. Latent TGF-β binding proteins: extracellular matrix association and roles in TGF-β activation. Critical Rev Clin Lab Sci. 41(3):233–264.

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Ingber, D. 1991. Extracellular matrix and cell shape: potential control points for inhibition of angiogenesis. J Cell Biochem. 47:236–241. Li, S., D. Harrison, S. Carbonetto, R. Fassler, N. Smyth and D. Edgar. 2002. Matrix assembly, regulation and survival functions of laminin and its receptors in embryonic stem cell differentiation. J Cell Biol.157:1279–1290. Liu, J. C. 2010. A novel strategy for engineering vascularized grafts in vitro. World J Stem Cell. 2(4):93. Maegawa, N., K. Kawamura and M. Hirose. 2007. Enhancement of ostoblastic differentiation of  mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2). J Tissue Eng Regen Med. 1:306–313. Martin, I., A. Muraglia and G. Campanile. 1997. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology. 138:4456–4462. Matsuda, T., T. Nakamura, K. Nakao, T. Arai, M. Katsuki, T. Heike and T. Yokota.1999. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. The EMBO journal. 18(15):4261–4269. McPherson, T.B and S.F. Badylak. 1998. Characterization of fibronectin derived from porcine small intestinal submucosa. Tissue Eng. 4:75–83. Medha, K., D.T. Hiteshkumar and S.K. Wasim. 2011. The Use of Growth Factors and Mesenchymal Stem Cells in Orthopaedics. Open Ortho J. 5(S2-M7):271–75. Mekala, N. K., R. R. Baadhe, S. R. Parcha and D.Y. Prameela. 2012. Osteoblast differentiation of  umbilical cord blood-derived mesenchymal stem cells and enhanced cell adhesion by fibronectin. Tissue Eng Regen Med. 9(5):259–264. Mekala, N. K., R. R. Baadhe, S. R. Parcha and D.Y. Prameela. 2013. Enhanced Proliferation and Osteogenic Differentiation of Human Umbilical Cord Blood Stem Cells by L-Ascorbic Acid, In Vitro. Current Stem Cell Res Ther. 8(2):156–162. Miyamoto, S., B.Z. Katz, R.M. Lafrenie and K.M. Yamada. 1998. Fibronectin and integrins in cell adhesion, signaling and morphogenesis. Ann NY Acad Sci. 857:119–129. Nagai, H., R. Tsukuda and H. Mayahara. 1995. Effects of basic fibroblast growth factor (bFGF) on bone formation in growing rats. Bone. 16:367–373. Nagy, J.A., E. Vasile and D. Feng.2002. Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis. J Exp Med. 196:1497–1506. Ottani, V., D. Martini, M. Franchi, A. Ruggeri and M. Raspanti. 2002. Hierarchical structures in fibrillar collagens. Micron. 33(7):587–596. Ponce, M., M. Nomizu, M.C. Delgado, Y. Kuratomi, M.P. Hoffman and S. Powell. 1999. Identification of endothelial cell binding sites on the laminin gamma-1 chain. Circ Res. 84: 688–694. Rodaway, A. 2001. Patient R Mesendoderm: an ancient germ layer?. Cell. 105:169–172. Sato, N., L. Meijer, L. Skaltsounis, P. Greengard and A.H. Brivanlou. 2004. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med.10:55–63. Schwarzbauer, J.E. 1991. Fibronectin: from gene to protein. Curr Opin Cell Biol. 3:786–791. Shimasaki, S., R.K. Moore, F. Otsuka and G.F. Erickson. 2004. The bone morphogenetic protein system in mammalian reproduction. Endocr Rev. 25(1):72–101. Sohr, S and K. Engeland. 2008. RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53. Cell Cycle. 7(21):3448–3460. Tiedemann, H., M. Asashima, H. Grunz and W. Knochel. 2001. Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis. Dev Growth Differ. 43(5):469–502. Varkey, M., C. Kucharski and T. Haque. 2006. In vitro osteogenic response of rat bone marrow stromal cells to bFGF and BMP-2 treatments. Clin Orthop Relat Res. 442:113–123.

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Watabe, T and K. Miyazono. 2008. Roles of TGF-β family signaling in stem cell renewal and differentiation. Cell Res.19(1):103–115. Werb, Z., T.H. Cu, J.L. Rinkenberger and L.M. Coussens. 1999. Matrixdegrading proteases and angiogenesis during development and tumor formation. Apmis. 107:11–18. Wu, X., S. Ding, Q. Ding, N. S. Gray and P.G. Schultz. 2004. Small molecules that induce cardiomyogenesis in embryonic stem cells. J Am Chem S. 126(6):1590–1591. Ying, Q. L., J. Nichols, I. Chambers and A. Smith. 2003. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell. 115(3):281–292. Zhou, R., X. Wu and O. Skalli. 2002. The hyaluronan receptor RHAMM/IHABP in astrocytoma cells: expression of a tumor-specific variant and association with microtubules. J Neuro-Oncol. 59(1):15–26.

CHAPTER 23

Phytosterols: Biological effects and mechanisms of hypocholesterolemic action Rafaela da Silva Marineli1, Cibele Priscila Busch Furlan1, Anne y Castro Marques1, Juliano Bicas2,3, Gláucia Maria Pastore3, and Mário Roberto Maróstica, Jr.1 1

Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil 2 Department of Chemistry, Biotechnology and Bioprocess Engineering, Federal University of São João del-Rei, Minas Gerais State, Brazil 3 Department of Food Science, Faculty of Food Engineering, University of Campinas, Campinas, São Paulo State, Brazil

23.1 Introduction Steroids are a class of lipids with a characteristic structure of three C6-rings and a C5-ring, which are commonly linked to methyl groups (positions 10 and 13) and an alkyl side chain (position 17) (Figure  23.1A). In the case of sterols, a hydroxyl group is present at position 3 in ring A (Figure 23.1B), rendering an amphiphilic property that is important for regulating the fluidity of eukaryotic cell membranes. These sterols are found in plants (phytosterols), animals (e.g., cholesterol), or fungi (e.g., ergosterol) (Figure 23.1C). Therefore, the term phytosterols refers to steroidal substances, structurally and physiologically similar to cholesterol, that are naturally present in foods of plant origin (Coultate 2002). These compounds form part of the human dietary intake and may have important biological effects on health improvement and health maintenance (Jong et al. 2003; Nagao and Yanagita 2008). Since 2009, our research group has been studying the biological effects of phytosterols in order to understand the mechanisms of action, particularly with regard to their hypocholesterolemic, antidiabetogenic, and antioxidant activities (Marineli et al. 2012; Marineli 2012; Marques 2013; Furlan et al. 2013). Obesity is currently a public health problem worldwide, and the use of phytosterols as dietary supplements can become an important tool in the reduction of comorbidities associated with excess fat mass (Kopelman 2000; Marques et al. 2012; Trigueros et al. 2013).

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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(A)

(B)

18 12 19 1 2

C 8

5 4

14

15

B

A 3

16

D

9 10

17

13

11

R

R

7 6

HO

(C)

R

Compound

R side chain

β-sitosterol

H

H

stigmasterol

HO

campesterol

cholesterol

ergosterol (double bond C7=C8 in ring B)

Figure 23.1 The basic carbon skeleton of common steroids (A) and sterols (B). Structure of the main phytosterols (β-sitosterol, stigmasterol, and campesterol), cholesterol, and ergosterol (C).

However, before phytosterols can be used as an adjunct in the treatment of obesity, it is necessary to better understand their role in metabolism. This review focuses on the main and most current scientific information on phytosterol biological effects in vivo, their metabolism, and their role in health.

23.2 Phytosterols 23.2.1 Definition, characterization, food sources, and dietary intake Phytosterols (i.e., plant sterols) are important constituents of plant cells. These compounds are derived from squalene, belonging to the triterpenes family. The compounds from this group contain 28 or 29 carbon atoms and one or two carbon-carbon double bonds, which contributes to the great

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Table 23.1 Phytosterols content in foods (mg.100–1 g). Food

Oils and fats Corn oil Sunflower oil Soybean oil Olive oil Palm oil Margarine Cereals Corn Wheat Rice Seeds and Nuts Nuts Sunflower seed

Total phytosterols (mg.100–1 g)*

509–1557 374–725 229–459 144–150 71–117 92–721 178 60–69 30

Food

Fruits Passion fruit Banana Apple Orange Vegetables Cauliflower Broccoli Lettuce Green olives Carrot

Total phytosterols (mg.100–1 g)*

44 17 13 7 40 39 38 35 16

127 300

*Sum of sitosterol, campesterol, stigmasterol, sitostanol, and campestanol. Adapted by Piironen et al. (2000) and Marangoni and Poli (2010).

variety of these structures in nature. So far, more than 250 compounds have  been identified. Among the most commonly found phytosterols are β-sitosterol (C-29), campesterol (C-28), and stigmasterol (C-29), which contribute up to 98% of the phytosterol dietary intake in the human diet (see Figure 23.1) (Ling and Jones 1995; Berger et al. 2004; Fernandez and Vega-Lopez 2005). Vegetable oil, seeds, nuts, and vegetables in general are the major dietary sources of phytosterols (Morton et al. 1995; Ryan et al. 2007). Plant sterols are present in foods in different forms (free, esterified to fatty acids, and linked to glycosides or phenolic acids), and its total content is generally calculated by the sum of all forms (Akihisa et al. 1991; Toivo et al. 2001). Table 23.1 shows the phytosterol content in some foods. The natural dietary intake is estimated at 100–400 mg/day of phytosterols for individuals from different countries; vegetarians certainly ingest higher amounts (Morton et al. 1995; Jiménez-Escrig et al. 2006; Hearty et al. 2009). Initially, phytosterols were used as pharmacological agents. As they are part of a normal diet, the idea to supplement these compounds to conventional foods has emerged. Thus, advances in the food industry have helped to improve their solubility, bioavailability, and incorporation in a wide variety of foods, either in free or esterified forms, such as margarine (especially), fruit juice, dairy and bakery products (St-Onge and Jones 2003), and chocolate (Graaf et al. 2002).

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23.2.2 Absorption, toxicity, and regulation Since 1912, several studies have investigated phytosterol absorption in animals. Earlier studies showed that sitosterol, stigmasterol, and ergosterol were not absorbed by rodents and rabbits (Schöfnheimer 1931; Ellis et al. 1912). With the development of new methods and the use of isotopes for evaluating the absorption of these compounds, other studies reported that phytosterols are absorbed in part, but the extent and the actual level of absorption is still controversial (Schöfnheimer 1931). However, the values observed tend to converge to a narrow range. Some researchers, for example, evaluated plant sterol absorption in various experimental models and found absorption values of 0% (rabbits), 4% (rats), and 6% (human), in relation to phytosterol intake (Pollak and Kritchevsky 1981). Salen et al. (1970) supplemented humans with sitosterol (240–320 mg), and estimated that the absorption of this compound at between 1.5 and 5%. The absorption rate of phytosterols varies depending on the side chain (R group in Figure 23.1C) size and degree of saturation. The larger molecules present increased hydrophobicity, which impairs their absorption, and saturated molecules are also less absorbed. These properties are illustrated in the data presented by Ostlund et al. (2002): The absorption levels of sitosterol and campesterol in humans were 0.512 ± 0.038% and 1.89 ± 0.27%, respectively, while their saturated counterparts (i.e., sitostanol and campestanol) were absorbed at 0.0441 ± 0.004% and 1.55 ± 0.017%, respectively. In addition, plant sterols are less absorbed than cholesterol, whose absorption is up to 60% of the total intake amount (Bosner et al. 1999; Ostlund et al. 2002; Brufau et al. 2008). In 2000, the Food and Drug Administration (FDA) recognized the use of phytosterols as safe for the general public, since they can be found in vegetable oil. Since then, several functional foods have been produced with the addition of these compounds (FDA 2000; Hicks et al. 2001). Studies confirm that phytosterols are well tolerated and have no adverse effect when used as a supplement (up to 9 g/day for 8 weeks) by healthy men and women (Davidson et al. 2001), probably because they are rapidly excreted in the bile, which makes intoxication by high doses nearly impossible (Coultate 2002; Lea and Hepburn 2006). Thus, as the phytosterols cannot be synthesized endogenously by humans, their circulating levels are low and dependent on diet and absorption efficiency.

23.2.3 Hypocholesterolemic effect Since 1950 (Pollak, 1953), many studies have described the ability of phytosterols to inhibit cholesterol absorption, thereby reducing blood cholesterol levels. This happens mainly by competition in the intestinal lumen, which was demonstrated in animal and human experiments. Therefore, phytosterols may act as an adjuvant in the prevention and treatment of cardiovascular diseases (Katan et al. 2003; Jong et al. 2003; Racette et al. 2010). Thus, a safe and well-tolerated supplementation of phytosterols may be useful in nonpharmacological management of hypercholesterolemia, to reduce low-density lipoprotein (LDL-c) levels

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more effectively. The effectiveness of their hypocholesterolemic action has been confirmed in healthy, hypercholesterolemic and type 2 diabetic subjects (Richelle et al. 2004; Lau et al. 2005; Bañuls et al. 2010). Pollak (1953) showed for the first time that plant sterols can reduce plasma cholesterol levels in humans significantly. Further investigations showed that feeding about 13 g of phytosterols per day for 3–5 weeks to 17 subjects resulted in a 20% reduction in blood cholesterol levels. The supplementation came from different food sources (Pollak and Kritchevsky 1981). Table  23.2 shows other important studies that demonstrate the phytosterols effectiveness as a hypocholesterolemic agent in vivo. Even though many different experimental and clinical trials have clearly demonstrated that phytosterols reduce LDL-c, other studies found no significant changes in serum high-density lipoprotein (HDL-c) and triglyceride levels (Quilez et  al. 2003; Brufau et  al. 2008). Moreover, it is unclear whether phytosterols have a positive effect on cardiovascular disease (Weingärtner et al. 2008).

23.2.4 Mechanism of action The human diet contains steroids from animal and vegetable origins, such as cholesterol and phytosterols (sitosterol), respectively. The route by which sterols enter in the enterocyte remains uncertain. Scientific evidence suggests that phytosterols affect cholesterol absorption in the intestinal lumen because their chemical structures are similar. Thus, when simultaneously present in the intestinal lumen, cholesterol and phytosterols compete for inclusion in the micelle structure. The more hydrophobic plant sterols are retained, causing a decrease in cholesterol absorption and its consequent elimination in the feces (Sanclemente et al. 2009). It is possible that this route includes passive diffusion through the micelleforming proteins or by Niemann-Pick C1-like 1 (NPC1L1), which is an intestine protein that absorbs dietary and biliary cholesterol (Allayee et al. 2000; Weingärtner et  al. 2011). Davis et  al. (2004) showed that expression of NPC1L1 in mice increased the levels of plasma cholesterol, when compared to the results obtained for NPC1L1 null mice. The presence of this enteric protein seems to be important in the absorption of phytosterols, as well as cholesterol. These results show an important role in the regulatory mechanism of uptake and absorption of steroidal structures by NPC1L1 (see Figure 23.2). Besides the NPC1L1, another transporter annexin 2–caveolin 1 (ANXA2CAV1) complex seems to work together with cholesteryl esters, so that they may be involved in the process of internalization of cholesteryl ester for membranes to the intestinal brush border. However, in some animals, such as rabbits, this mechanism ANXA2-CAV1 complex transfer does not occur in the same way cholesteryl ester is absorbed. For this reason, the function is still unclear. There is also evidence that other carriers, such as adenosine triphosphate binding cassette transporter (ABC) A1 and cluster of differentiation 36 (CD36) receptors, the class of receptor-type B1 or scavenger receptor class B member 1 (SCARB1),

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Table 23.2 Studies in vivo about biological effects of phytosterols. Compound

Conditions

Results

Reference

Phytosterols

Healthy subjects (n = 26) fed with 8.1 g per day, for 2 weeks.

Plasma total cholesterol was reduced by approximately 28%.

Pollak (1953)

Sterols and stanols

Meta-analysis of 41 trials, with several fortified food products.

The optimal dose of sterols or stanols (2–2.5 g/day) promotes a 10% reduction in serum LDL-c levels, while higher doses provide only a small additional effect.

Katan et al. (2003)

Phytosterols

Hamsters (n = 130) fed with high-fat diet (30.0% of lipids and 0.12% of cholesterol) and phytosterols (0.24 to 2.84%) for 12 weeks.

The fat deposition in the hepatocytes and consequent liver pallor were inversely related to the phytosterol amount ingested by the animals.

Ntanios et al. (2003)

Phytosterols

Subjects with metabolic syndrome (n = 108) supplemented with 4 g/day, for 2 months, with a usual diet.

Reduction in total cholesterol (15.9%), LDL-c (20.3%), and triglycerides (19.1%); however, no differences were observed in HDL-c, glucose, C-reactive protein, fibrinogen levels, and blood pressure.

Sialvera et al. (2012)

Oyster mushroom (Pleurotus ostreatus)

Subjects (n = 20) with hyperlipidemia (triglycerides ≥ 5 .2 mmol/L and/or LDL-c ≥ 2.6 mmol/L) received one portion of soup containing 30 g dried oyster mushrooms, for 21 days.

Reduction in triglycerides (–0.44 mmol/L) and oxidized LDL levels (–7.2 U/mL); however, no effects on LDL-c and HDL-c levels were found. The authors attributed the beneficial effects to the presence of linoleic acid, ergosterol, and ergosta-derivatives.

Schneider et al. (2011)

Fermented milk enriched with plant sterols (FMPS)

Subjects (n = 1,048) with moderate hyperlipidemia using five or more doses of FMPS by week, for 4 months.

Reduction in total cholesterol (11.1%), LDL-c (13.2%) and triglyceride (5.2%) levels, and increase in HDL-c (7.2%) concentration. The improvement in the lipid profile was associated with FMPS supplementation and also with changes in lifestyle of patients.

Masana et al. (2012)

Naturally occurring phytosterols

Healthy volunteers (n = 85) were studied with regard to their dietary habits, using a validated food frequency questionnaire.

Higher phytosterol intake was associated with a higher consumption of fruits, nuts, vegetables, and vegetable oils. The increase of the regular intake of phytosterol-source foods is justified, with cholesterollowering benefits and prevention of cardiovascular disease.

Sanclemente et al. (2012)

Phytosterols: Biological effects and mechanisms of hypocholesterolemic action

C

Lymph

Enterocyt

Intestinal lumen

571

P

Nucleus NPC1L1

C

P C

P

ACAT2

CE

Cy

Micelle C

ABCG5 ABCG8

P

Nucleus

Golgi complex

Faeces

Figure 23.2 Possible mechanism of cholesterol and phytosterol absorption, as already described in the literature. Inside the enterocytes, cholesterol is esterified by acetyl-CoA acetyltransferase 2 (ACAT2) to cholesteryl ester and esterified cholesterol. Subsequently, the products are packaged in chylomicrons and are transported by the lymphatic system to the circulation and liver. C, cholesterol; CE, cholesteryl ester; Cy, chylomicrons; P, phytosterols. Adapted from Sanclemente et al. (2009).

Bloodstream

Cy C

P ABCG1

Enterocyt

C Nucleus

P

C

ABCG5

C ABCG8

P Golgi

C

P

P Intestinal lumen

Faeces

Figure 23.3 Cholesterol absorption in the intestine and potential mechanism of action of phytosterols in reducing cholesterol absorption. ABCG5 and ABCG8 proteins provide dynamic transport, taking the phytosterols to the Golgi apparatus of enterocytes. These carriers appear to have an affinity for plant sterols, although they also carry cholesterol. Back to the intestinal lumen, the ABCA1 transporter protein may also participate in this process, but it is still unclear. C, cholesterol; Cy, chylomicrons; P, phytosterols. Adapted from Patel (2008).

ABCB1, among others, may be associated with the transport mechanism of sterols (Calpe-Berdiel et al. 2009). Inside the enterocytes, cholesterol is esterified by acetyl-CoA acetyltransferase 2 (ACAT2) to cholesteryl ester. Subsequently, the products are packaged in chylomicrons and transported by the lymphatic system to the circulation and liver (Temel et al. 2003). The transport mechanism of phytosterols in the enterocytes is controlled by ABCG5 and ABCG8 proteins (see Figure 23.3). These proteins provide a dynamic transport, taking the phytosterols to the Golgi apparatus of the enterocytes.

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Phytosterols: Biological effects and mechanisms of hypocholesterolemic action

These carriers appear to have an affinity for plant sterols, although they also carry cholesterol. Back in the intestinal lumen, the ABCA1 transporter protein may also participate in this process, but it is still unclear (Sabeva et al. 2011). Thus, less than 5% of dietary phytosterols are in fact absorbed by the enterocytes and packaged in chylomicrons; however, up to 60% of dietary cholesterol is absorbed and carried by the same mechanism (Brufau et al. 2008). According to Plat and Mensink’s (2002) hypothesis, which is based on animal experimentation, phytosterols may act by increasing the expression of ABCA1 converting liver X receptor (LXR) agonist. The receptor LXR has a central role in the transcription control of lipids metabolism. Once activated, it is involved in uptake, efflux, transport, and excretion of cholesterol (Zelcer and Tontonoz 2006). On the other hand, Plösch et al. (2006) showed that activation of ABCG5 and ABCG8 with LXR did not alter phytosterols and cholesterol absorption in mice. Moreover, overexpression of ABCG5 and ABCG8 altered the transport of cholesterol in the liver, but not in the gut, showing that transporters ABCG have distinct roles depending on the organ (Wu et al. 2004). Autosomal recessive disorders caused by the ABC proteins gene mutation are responsible for rare phytosterolemia or sitosterolemia. These changes create a phytosterol and cholesterol excessive absorption (Igel et  al. 2003). Moreover, polymorphism emergence in the ABCG8 gene alters the plasma concentration of phytosterols in healthy animals, which reinforces the role of ABCG8 in regulating the absorption of nonsteroidal cholesterol (Berger et al. 2004). Conversely, the overexpression of ABCG5 and ABCG8 reduced the intestinal cholesterol absorption in mice (Yu et al. 2002). In the liver, phytosterols, as well as cholesterol, may have different destinations, including (i) transportation to peripheral tissues by very low-density lipoprotein (VLDL-c) and low-density lipoprotein (LDL-c); (ii) conversion into bile salts; or (iii) transportation to the gallbladder to be excreted. In peripheral tissues, ABCA1 transporter may also participate in the transport process, delivering cholesterol to HDL-c, which carries it to the liver (Allayee et al. 2000). In addition to the cellular carrier, Nomaguchi et  al. (2011) demonstrated the effect of phytosterols derived from Aloe vera in obese mice. The authors suggested that phytosterols act as ligands of α and γ peroxisome proliferator activated receptors (PPARs), modifying the expression of genes involved in the transport of fatty acids, fatty acid oxidation, and ketogenesis in the liver of  animals. They also demonstrated that the genetic alteration induced by treatment with phytosterols is related to lipid transport, lipogenesis, gluconeogenesis, and signaling of PPARs. According to Hernández-Mijares et  al. (2011), the absorption of sterols in healthy subjects and subjects with metabolic syndrome is distinct. Healthy individuals absorb greater amounts of cholesterol from the diet and, therefore, higher amounts of phytosterols. In contrast, subjects with metabolic syndrome have a lower absorptive capacity for both. Additionally, cholesterol production

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is increased in patients with insulin resistance, which complicates the resolution of dyslipidemia through phytosterols. In other words, phytosterol supplementation is beneficial, especially for people with high intestinal absorption of cholesterol and low endogenous cholesterol synthesis. Although some mechanisms of action and transport proteins are reported in the literature, more studies of genetic polymorphisms and cellular signaling should be developed in this area to obtain more accurate results (Demonty et al. 2009). This information is critically important to ensure the development of foods fortified with phytosterols, which results in positive effects on population health.

23.3 Other biological effects In addition to the hypocholesterolemic and antiatherosclerotic effects of these compounds, some in vivo and in vitro studies have shown that phytosterols, mainly β-sitosterol, exert other biological activities, such as (i) anticarcinogenic effects, like protection against colon, breast, and prostate cancers (Awad et al. 1998, 2001a, 2001b, 2008; Awad and Fink 2000; Bradford and Awad 2007); (ii) immunomodulatory and anti-inflammatory properties (Bouic et al. 1996, 2001; Navarro et al. 2001); (iii) antioxidant potential (Summanen et al. 2003; Moreno 2003; Vivancos and Moreno 2005; Fuhrman et al. 2007; Mannarino et al. 2009; Ferretti et al. 2010; Marineli et al. 2012); and (iv) antidiabetogenic effects (Wasan et al. 2003; Tanaka et al. 2006; Misawa et al. 2008). Regarding the antitumoral effects, Awad et al. (2001b), for example, demonstrated the reduction of metastasis in vitro human cells of breast cancer by phytosterols. Ifere et al. (2009) observed reduction in mitosis and tumor suppression and an increase in apoptosis in prostate cancer cells. According Bradford and Awad (2000), phytosterols increase the turnover of sphingomyelin, ceramide formation and activation of LXRs, which reduces cell cycle progression, inhibits cell proliferation, and activates apoptosis in neoplastic cells. Another important biological effect attributed to these compounds is immunomodulation. It is speculated that phytosterols, when incorporated into tissues, alter the fluidity of cell membranes, affecting the biosynthesis of leukotrienes, prostaglandins, and eicosanoids, and therefore acting as an anti-inflammatory. As previously mentioned, phytosterols are also ligands for LXRs, which play a regulatory role in the immune system (Calpe-Berdiel et al. 2007). Devaraj et al. (2011) observed a reduction in serum levels of interleukins 1β and 6, both pro-inflammatory in healthy subjects who received orange juice fortified with phytosterols. Gabay et al. (2010) investigated the effect of stigmasterol prevention of osteoarthritis, and they observed a reduction of pro-inflammatory cytokines by nuclear factor kappa B (NF-κB) inhibition. However, more studies are needed to elucidate the mechanisms involved in regulating the immune system.

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Homma et al. (2003) found that the significant LDL-c plasmatic and apolipoprotein B (ApoB) reduction in subjects supplemented with phytosterols (2 g/ day) is associated with the reduction of oxidized LDL. Other studies in humans have confirmed that the changes in lipoproteins after dietary supplementation with this compound are related to the reduction of lipid peroxidation markers (Mannarino et al. 2009). The intake of dairy products enriched with phytosterols was evaluated for 6 weeks in hypercholesterolemic individuals. The results showed a reduction of LDL-c and total cholesterol without changing HDL-c and triglyceride levels. The intake of phytosterols was associated with significant reduction in plasma 8-isoprostane. This compound is a very specific indicator for the evaluation of the degree of oxidative stress in vivo, which suggests a possible antioxidant property. The protective effect of phytosterols intake against oxidative stress, related to peroxidation of lipoproteins, has also been reported in studies with animal models (Summanen et al. 2003; Fuhrman et al. 2007). Some researchers have investigated the interactions between the phytosterols (β-sitosterol, campesterol, and stigmasterol) and lipoproteins isolated from normolipemic subjects in vitro, and their antioxidant effect and susceptibility to lipid peroxidation induced by copper ions. The authors discovered that phytosterols exert a protective effect against LDL-c peroxidation, through its inhibition, and prevent physical and chemical changes caused by this lipoprotein (Ferretti et al. 2010). Van Rensburg et al. (2000) reported that low concentrations of β-sitosterol and other sterols reduced levels of thiobarbituric acid reactive substances (TBARS), secondary products of lipid peroxidation, while high concentrations of β-sitosterol promoted the increase of this marker in vitro. Previous studies suggested that β-sitosterol may protect against oxidative stress through the modulation of antioxidant enzymes and free radical production. This is consistent with the decrease observed in O2– and H2O2 levels after β-sitosterol treatment (Vivancos and Moreno 2005; Moreno 2003). Our research group recently found that phytosterol supplementation (2%) for 9 weeks in healthy Sprague-Dawley rats affects plasma and hepatic lipid peroxidation products directly and induces a significant reduction of these products, suggesting that phytosterols could act as an antioxidant in vivo system (Marineli 2012). However, the potential effect of phytosterol consumption in these biomarkers is still new and little studied. Despite their beneficial health effects, some studies have indicated that phytosterols, as well as cholesterol, are also susceptible to oxidation. The oxidation, in turn, generates sterol oxidation products, known by their toxic effects (Hovenkamp et al. 2008; O’Callaghan et al. 2013). Barriuso et al. (2012) analyzed campesterol, stigmasterol, and β-sitosterol degradation during 360 minutes at high temperature (180°C). Higher levels of total campesterol and β-sitosterol oxides, when compared to those achieved by cholesterol and stigmasterol derivatives throughout the whole heating process, were detected. In view of this result, it is necessary to establish whether the use of phytosterols as

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a supplement and in fortification of foods is actually harmless to consumers’ health (Kritchevsky and Chen 2005). Recent studies have investigated and identified the antihyperglycemic effects of phytosterols derived from Aloe vera in diabetic animals. These studies suggest that these compounds have a long-term control over blood glucose, and could be useful for the treatment of diabetes mellitus type 2 (Tanaka et al. 2006). Other authors also demonstrated the beneficial effects of phytosterols from Aloe vera in hyperglycemia and in reduction of visceral fat accumulation after administration of these compounds (25 mg/kg/day) for 44 days in Zucker diabetic fatty rats (Misawa et al. 2008). Nomaguchi et al. (2011) corroborated these findings: They also found a significant reduction in body weight gain and adipose tissue in obese mice. This suggests that phytosterols may play a positive role in the prevention of visceral obesity and also contribute to the control of metabolic syndrome. However, the mechanisms of this effect are still unclear. In the same context, Wasan et al. (2003) evaluated the phytostanol intake for 30 days in lean and obese rats. They found no change in body weight and glucose, insulin, and leptin concentrations. Also, there was no change in glucose tolerance in lean and normoglycemic rats, but there was a significant improvement of this parameter in the obese group after treatment. However, another study found no difference in weight gain, food intake, glucose tolerance test, and insulin sensitivity, both in the progression or regression after treatment with 2% phytosterols in hyperlipidic diet in mice (Calpe-Berdiel et al. 2008). In an experiment conducted by our research group with Swiss mice receiving a high-fat diet, the supplementation with 2% of phytosterols, for 9 weeks, did not reduce weight gain and blood glucose (Marques et al. 2012). On the other hand, in another experiment, we also found that 2% of phytosterol supplementation for 9 weeks in healthy Sprague-Dawley rats reduced plasma glucose concentration and improved the homeostasis model assessment of β-cell function (HOMA-β) (Marineli 2012). In a third experiment, Sprague-Dawley rats received a high-fat diet for 8 weeks. All animals that received 2% of phytosterol showed a reduction of retroperitoneal and epididymal adipose tissues (50%), and gain less weight and consume less food compared to control fat animals, but blood glucose does not change (Furlan et al. 2012). The different animal models and different diets influence the results directly. Human studies are needed to confirm these findings and to investigate its long-term effects.

23.4 Conclusion Based on the above, phytosterol consumption is recognized to promote positive biological activities, such as hypocholesterolemic effect, and other functionalities are under investigation, such as the antioxidant and antiobesity effects. Considering their nontoxic character and the benefits associated with their consumption,

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research related to their addition to food and their use as a functional food have been encouraged. However, little is known about their mechanisms of action and recommended doses. Additional studies investigating relevant clinical endpoints are needed before a diet supplemented with phytosterols can be recommended for the prevention of cardiovascular diseases.

Acknowledgments This work was supported by Fundação de Amparo à Pesquisa do Estado de São  Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work does not present conflicts of interest. None of the authors had any financial or personal interest in any company or organization sponsoring the research.

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CHAPTER 24

Overview of the role of food bioactive compounds as complementary therapy for celiac disease Antonio Cilla1, Laia Alemany1, Juan Antonio Giménez2, and José Moisés Laparra3 1

Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia, Valencia, Spain Department of Chemistry and Biodynamics of Food, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland 3 Microbial Ecology and Nutrition Research Group, Institute of Agrochemistry and Food Technology, National Research Council (IATA-CSIC), Valencia, Spain 2

24.1 Introduction Celiac disease (CD) is a chronic enteropathy of immune origin triggered by gluten (e.g., gliadins) and some other cereal-related proteins in genetically predisposed individuals. It appears primarily as a pediatric disorder associated to alterations of intestinal architecture with villous atrophy and crypt hyperplasia. However, CD is also being increasingly diagnosed in adults. Although the disease is strongly associated with Human Leukocyte Antigen (HLA) genes, encoding the HLA-DQ2.5, HLA-DQ2.2, and HLA-DQ8 heterodimers, these genetic factors only explain part of the prevalence of the disease within the general population (Greco et al. 2002; Bahia et al. 2010). Epidemiological studies evidenced the complex interplay between genetic and environmental factors and gluten intake, this one not being the only triggering factor of the disease. Milk-feeding type and incidence of infections also influence the gut ecosystem and are some other factors influencing the risk of developing CD (Sandberg-Bennich et al. 2002; De Palma et al. 2012). Studies in children and adult populations revealed a direct association between alterations in the gut microbiota composition of subjects with symptomatic and untreated CD and patients under a gluten-free diet in relation to microbiota composition in healthy subjects (Sanz et al. 2011). Increasing data started to appear reinforcing the need to maintain adequate macro- and micronutrient intake as well as vitamins and naturally occurring prebiotic fructans that appear deficient in the gluten-free diet (GFD), as it is the only current therapy for CD patients (Wild et al. 2010; Botero-López et al. 2011). Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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Table 24.1 Current adjuvant therapeutic strategies to the gluten-free diet. Strategy

Intervention

Biotechnology Immunization Genetic

Microbial enzymes (prolyl endopeptidases and other glutenases) Vaccines to induce tolerance to gluten-derived peptides Selection of genetically engineered barley, rye, and wheat varieties by transamidation of gliadin Use of zonulin inhibitors for decreasing intestinal permeability Down-regulation of the adaptive immune responses and immune cell-targeted therapies Probiotics, prebiotics

Intestinal permeability Immunomodulatory Nutritional

Bioactive food-derived components exert modulatory effects on inflammatory processes and maturation or function of immune cells that could help control intestinal cell homeostasis and physiology (Bassaganya-Riera and Hontecillas 2006; Maynard and Weaver 2011; Ferretti et al. 2012). Therefore, there is a need to identify modifiable factors that contribute to CD risk and pathogenesis in order to make progress in developing complementary strategies to the existing ones (Mukherjee et al. 2012) to improve the quality of life of patients and/or to prevent the disease from developing in at-risk populations (Table 24.1). The clinical relevance of potential nutritional strategies based on the use of bioactive food-derived components is still inferential and needs to be established whether they only can control the symptoms and/or control the severity of the immune response(s) conferring oral tolerance to gluten.

24.2 Gliadins-mediated alterations in intestinal epithelium Significant progress has been made in identifying the mechanisms of pathogenesis of CD, especially those related to the activation of specific or adaptive immunity (Schuppan et  al. 2009). However, knowledge of the effects of gliadin on enterocytes and innate immunity is more limited (Lammers et al. 2008; Olivares et al. 2012). Gluten is a mixture of proteins (gliadins and glutenins) that form part of the cereal flour where different isoforms of gliadins (α/β- ,γ- ϖ5- ,ϖ2 -) and glutenins can represent up to 80% of the total protein content. The high proportion of proline and glutamine residues in gliadins give them a particular “left-handed helical conformation” favoring the accumulation of immunogenic and/or cytotoxic peptides (Hausch et  al. 2002; Laparra and Sanz 2010a). Ex vivo studies identified dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I, located in the vesicles of the brush border membrane of enterocytes, which constrains degradation of gliadins (Hausch et al. 2002). Notably, amino acid sequences with

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tyrosine-containing groups show the potential to trigger activation of T cells (Cornell and Wills-Johnson 2001). One of the first biological events resulting from the toxicity of the gliadins in  enterocytes is the relocation of different molecular components (zonulin, occludin, cadherin, and claudins) in the intercellular junctions leading to impaired intestinal permeability. These events favor the interaction of occludin with the extrinsic cell death receptors and activation of apoptosis of enterocytes according to their degree of differentiation, thereby flattening intestinal microvilli and decreasing cell polarization that causes deficient absorption of nutrients in CD patients. The possible mechanisms underlying the gliadins-mediated cytotoxic effects to intestinal cells include the production of pro-inflammatory cytokines such as TNF-α and IL-1β, which activate NFκB (Viatour et al. 2005). These events take place through the calcium (Ca+2)-dependent mediated activation of protein kinase C, causing disruption of the cellular cytoskeleton and actin polymerization processes triggered by interaction of gliadin-derived peptides with specific receptors on the membrane of intestinal cells as chemotactic receptor CXCR3 (Lammers et al. 2008). In addition, gliadins cause an abnormal modulation of the endocannabinoid system (Battista et al. 2013) and upregulation of intracellular oxidative stress in dendritic cells (DC) in a non-disease-specific manner, together with the tissue transglutaminates (TG2)-mediated down-regulation of PPAR-γ (Luciani et al. 2010). Recent data indicate that impaired activity of PPAR receptors can exacerbate autoimmune diseases (Lovett-Racke et al. 2004, Dubuquoy et al. 2006). In this context, it has also been described as the important regulatory effects of α-amylase/trypsin inhibitors like CM3 and 0.19, which commonly appear in complexes with ϖ-gliadins, as strong activators of “Toll-like receptor” (TLR)-4 not only in enterocytes, but also immune competent cells boosting the inflammatory action of gliadins (Junker et al. 2012). Inflammatory processes in the intestinal epithelium are accompanied by the degradation of connective tissue components by the action of zinc-dependent metalloproteinases (MMP) -1, -3, -9, and -12. Together with alterations described in the architecture of the intestinal epithelium, they allow access of gliadinsderived peptides to the intercellular space, promoting the sub- (CD4+) and intra-epithelial (CD8+) infiltration and maturation of lymphocytes to a cytotoxic immune response (Schuppan et  al. 2009). In the activation of this adaptive immune response it is important the TG2-mediated anchor of gliadin peptides are together with other proteins of the extracellular matrix, promoting their interaction with HLA-DQ molecules of antigen presenting cells and subsequently activation of CD4+ and CD8+ T cells (Skovbjerg et al. 2008). In addition to immunogenic peptides, serine-containing peptides from gliadins exert major cytotoxic effects affecting intestinal cell structure and functions. These events are also associated with stimulation of innate immune response(s) characterized by high IL-15 synthesis and infiltration of lymphocytes (α/β- or

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γ/δ-TCR), which are functionally different from the Th1 CD4+ in the intestinal epithelium. IL-15 plays a critical role inducing the expression of molecules of the nonclassical MHC-I (MICA) and the NKG2D receptor in infiltrating lymphocytes. Data from studies using transgenic mice expressing the human MHC class II molecule HLA-DQ8 revealed the important role of retinoic acid production, which is primarily produced by a subset of mucosal dendritic cells for the induction of oral tolerance, in the presence of high levels of IL-15 favoring the  production of IL-12-family cytokines, which induce pro-inflammatory Th1- and Th17-cell responses (Maynard and Weaver 2011). This could be particularly important because it has been described as having a dual role in the pathogenesis of the disease for gliadin-specific Th17 cells. They produce proinflammatory cytokines (such as IL-17, IFNγ, IL-21), mucosa-protective IL-22, and regulatory TGFβ, which actively modulates IL-17A production by T cells in the celiac mucosa (Fernández et  al. 2011). Data from a macaque model for celiac disease demonstrated the declined capability of intestinal T cells to secrete IL-17 and IL-22, which occurs in parallel to the gradual apparition of tissue damage (Xu et al. 2013). The production of IL-22 by intestinal innate lymphoid cells, which express the transcription factor retinoic acid–related orphan receptor-γt, has been linked to the necessary activation of the aryl hydrocarbon receptor (Ahr) (Lee et al. 2011). At same time, lack of AhR is also associated with increased intestinal colonization by the Bacteroides spp. (Li et al. 2011) population that has been found in CD patients with a more severe disease (Sánchez et al. 2012). Oxidative stress can be an additional mechanism for gliadin toxicity (Luciani et  al. 2010). The increased concentration of lipid hydroperoxydes identified in  small intestinal biopsies and peripheral blood of children affected by CD (Stojiljkovic et  al. 2012) may drive adaptive immune maturation to cytotoxic reactivity and compromise the physiological functionality of intestinal epithelium, rendering it more susceptible to the continuous gliadin insult and dysregulation of intestinal homeostasis caused by up-regulation of MICA/B genes (Allegretti et al. 2013).

24.3 Gluten-free diet (GFD) and nutrition A strict GFD throughout the entire life is the only effective treatment for CD patients, eliminating trigger activity on T cells. Notably, gluten is used widely by the food industry because of its attractive nutritional features and potential to be used in different technological processes. Epidemiological data indicate that dietary intake of trace levels of gluten do not explain the high number of patients with high serological markers and those in which no mucosal healing occurs despite undergoing a strict gluten-free diet, suggesting cross-contamination or  voluntary transgressions of a GFD. However, cereals provide some other

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Table 24.2 Concentration of bioactive components in whole wheat–based cereals (Fardet et al. 2008). Component

mg/100 g grain

Phytic acid Ferulic acid Alkylresorcinols Vitamin E Folates Iron Manganese Zinc Selenium Copper

906 mg 10–198 mg 28–142 mg 1.4 mg 87 µg 3.2 mg 3.1 mg 2.6 mg 0.5–74.6 µg 369 µg

components with nutritional value, making it necessary that a GFD provide all the nutrients required for the patient according to age and the evolutionary stage and severity of the disease. Whole grains are rich sources of numerous ingredients recognized as bioactive components such as fermentable carbohydrates including dietary fiber, resistant starch, and oligosaccharides (Slavin 2003), proteins, minerals, vitamins, and antioxidants (Ötles and Cagindi 2006). Wheat contains diverse bioactive compounds (Table 24.2) and shows antioxidant features due to the high phenolic compounds, principally ferulic, alkylresorcinol and hydroxycinnamic acids (Belodrajdic and Bird 2013). Today there is still an open debate about the dietary advantages and harmlessness of oats versus wheat, barley, and rye to CD when those are proposed as substitutes to increase fiber and protein intake and compliance with the diet (Richman 2012). Thus, the best way to correct the absence of gluten within the diet could be to recommend a higher consumption of fruits, vegetables, and legumes. Data from human studies reported vitamin and trace element deficiencies in patients ascribed to a GFD, based on the fact that many cereal grains contain significant concentrations of several minerals (iron, manganese, and zinc), vitamins B1 (thiamine) and B2 (riboflavin), niacin, pantothenic acid, and folate (McKevith 2004; Angiolini and Collar 2010). In comparison with wheat, oats also provide higher amounts of calcium, iron, vitamin B1, and vitamin B2. Vitamin E is another antioxidant present in whole grains with an important function in the protection of cell membranes from oxidative damage (Fardet et al. 2008), which cannot be ruled out since neurologic impairment because of the deficiency of vitamin E and copper have also been reported in CD patients (Henri-Bhargava et al. 2008). In the last few years, the number studies have increased investigating the use of cereals other than common wheat and pseudocereals to make modern and innovative baked goods (Angioloni and Collar 2010; Bergamo et  al. 2011). However, scarce data support the safety features of these products to CD patients

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(Di Cagno et al. 2004; Bergamo et al. 2011). These studies showed that particular bread formulation with starters that possess high proteolytic capacity on gluten and the use of gluten-free flours from millet and buckwheat (Di Cagno et al. 2004; Bergamo et al. 2011) and tef and quinoa (Bergamo et al. 2011) did not cause an immunological response to celiac patients. Nutritional evaluation on CD patients over 12 years and direct family members who live with them also revealed a surprising tendency to be overweight and obese in those patients that do not display classic symptoms (Tucker et al. 2012). This observation could be of relevant importance because CD has been reported in 4–13% of patients suffering nonalcoholic steatosis (NASH), although the pathogenesis of liver disease in CD patients remains unclear (Abenavoli et al. 2013). In view of the aforementioned CD patients represents a population for whom nutrient requirements and avoidance of intestinal insults preventing mucosal integrity parallel and very often show deficiencies. These observations point out the need for nutrition support and surveillance to optimize compliance with the diet to maintain physiological processes at the intestinal level and prevent injury and illness.

24.4 Role of pre- and probiotics in celiac disease Prebiotics are nondigestible food ingredients, mostly oligosaccharides, which beneficially affect the host by stimulating growth and/or activity in specific intestinal bacteria. Fermentation of prebiotics leads to the production of short-chain fatty acids (SCFA) able to modulate inflammation by inhibition (IL-2 and IFNγ) or induction (IL-10) of regulatory cytokine production (Cavaglieri et al. 2003). Compounds such as propionate and butyrate also stimulate mucin production, required for epithelial protection because of the decreased differentiation of intestinal cells toward the secretory goblet cell lineage and the subsequent reduced thickness of the mucus layer in CD patients (Capuano et  al. 2011). SCFA can also modulate inflammation by activation of free fatty acid receptor 2 (FFA2, also known as GPR43) (Soldavini and Kaunitz 2013) or adaptive immune response(s) by binding to leukocyte receptors (GPR41 and GPR43), supressing its proliferation and cytokine production by Th1-lymphocytes (Cavaglieri et al. 2003). It has also been shown that prebiotics (e.g., Sialyl lactose and Raftilose P95) also promote anti-inflammatory effects via activation of PPAR-γ (Zenhom et  al. 2011). However, it cannot be ruled out that administration of galactooligosaccharides (GOS)/inulin to newborn animals can favor greater bacterial translocation transiently, although, without negative effects on the health of animals (Barrat et al. 2008). Thus, current data support that prebiotic-mediated positive effects to CD appear to be directly influenced by the type of prebiotic structure and bacterial species present in the intestine (Commane et al. 2005).

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In the few last years, a direct association between intestinal dysbiosis and the severity of inflammatory response(s) in CD patients has been well established. It has been described a decreased bacterial diversity, with increased numbers of  the Bacteroides-Prevotella and E. coli group and the Bacteroides fragilis group in comparison to healthy individuals (Nadal et al. 2007; Collado et al. 2009). Besides, the proportion of beneficial Lactobacillus spp. and Bifidobacterium spp. is decreased in CD patients. The possible mechanisms underlying the beneficial effects caused by these bacterial populations appear derived from their immunomodulatory features reducing pro-inflammatory cytokine production (D’Arienzo et  al. 2008; Laparra et  al. 2012) and/or the Th1/Th2 cell balance (Isolauri et al. 2001), capacity to eliminate immunogenic peptides (Laparra and Sanz 2010a), improvement of intestinal permeability, and restoration of the intestinal ecosystem. Animal studies have demonstrated the probiotic strainspecific influence on innate immune responses via their interaction with TLRs and antigen presenting cells (Tomosada et al. 2013) and better preservation of proteome patterns in jejunal sections in gliadins-fed animals (Olivares et  al. 2012). The immunomodulatory potential of probiotic strains has led to their proposed use as coadjutants in vaccines because of their boosting effect in T (CD4+)-cells response (D’Arienzo et al. 2008).

24.5 Micronutrients (iron, zinc, and selenium) Epidemiological data from various countries about the nutritional adequacy of the GFD reveal micronutrient deficiencies (iron, Fe; zinc, Zn; and selenium, Se) in CD patients, although in some cases there can be found contrasting results (Martin et al. 2013; Shepherd and Gibson 2013). These deficiencies usually associate with impaired immune functions and aggravate the severity of the disease (Wild et al. 2010; Abu Daya et al. 2013). It becomes difficult to study the relationship of Fe with any outcome when inflammation is present because pro-inflammatory cytokines and related mediators influence T-cell functions (Bemelmans et al. 1994), dietary Fe assimilation, and systemic Fe homeostasis (Young et al. 2009). Fe is important in immune and inflammatory response(s) influencing neutrophil activation, macrophage effector functions, and Th1/Th2 patterns; however, the effects of increasing total Fe intake on markers of inflammation has been indicated but poorly studied (Schumann et al. 2005; Hodgson et al. 2007). Alterations in Fe homeostasis, leading to ferropenic anemia, are most common extraintestinal manifestations of CD and only recent data indicate that administration of B. longum CECT 7347 to newborn rats sensitized with interferon-γ positively contributed to restore alterations of hemoglobin levels caused by gliadins (Laparra et al. 2013). In CD patients, the exchangeable Zn pool seems to be related to impaired gut function (Tran et al. 2011). Zn deficiency increases susceptibility to bacterial and

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viral infections that can generate Th1-protective responses, which have been investigated as a possible factor contributing to CD risk. In addition to positive effects on immune response(s), adequate control of Zn homeostasis could modulate the influx of calcium (Ca+) and influence downstream signals triggered by the interaction of gliadins with the CXCR3 receptor (Lammers et  al. 2008; Laparra and Sanz 2010b). Thus, it could inhibit the Ca-induced activation of TG2, preventing villous atrophy (Stenberg et al. 2008). Dietary zinc also contributes to maintain the cell redox balance through various direct or indirect regulation of redox signaling (Cortese et al. 2008; Oteiza 2012). Findings about oxidative stress, altered redox signaling, and associated cell/tissue dysfunction in cell and animal models promoted by gliadins highlight the potential relevant role of zinc in the preservation of gliadin-induced alterations in physiological cell redox homeostasis and metabolic function. However, nutritional intervention strategies in this sense have not been investigated. The essential trace mineral Se is of fundamental importance because it is a characteristic component of antioxidant proteins such as glutathione peroxidases, thioredoxin reductases, and iodothyronine deiodinase families. An adequate function of these proteins is required for the thyroid function and preservation of intestinal integrity (Stazi and Trinti 2008). Selenium seems to play an important role in immunity by affecting innate, T-cell-mediated and adaptive antibody responses, leading to dysregulation of the balanced host response (Maggini et al. 2007). For example, Se deficiency may aggravate interleukin (IL)-15 increased activation of effector mechanisms of epithelial damage by stimulating T-helper 1 cytokine proliferation and production and intraepithelial lymphocyte cytotoxicity by protecting these lymphocytes from apoptosis (Stazi and Trinti 2008).

24.6 Vitamins (A, C, and E), antioxidants (phytochemicals), and fatty acids Prospective food intake studies revealed that vitamin deficiencies are implied at different levels of immunity where vitamins A, C, and E play particular roles in skin and intestinal barriers (Shepherd and Gibson 2013). Proteome analyses of biopsies from CD patients (Simula et al. 2010) demonstrated a reduced abundance of retinol binding protein. This observation could be explained by vitamin A deficiency in these patients. It contributes to the normal antibody-mediated Th2 response suppressing IL-12, TNF-a, and IFN-g production of Th1 lymphocytes by interaction with retinoic acid receptors (Halevy et al. 1994). Nutritional deficiency of vitamin A is associated with diminished phagocytic and oxidative burst activity of macrophages activated during inflammation (Ramakrishnan et  al. 2004) and a reduced number and activity of natural killer (NK) cells (Dawson et al. 1999). An additional important aspect that should not be ruled out is that inflammatory conditions in the gut and extensive mucosal damage

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could aggravate the severity of CD due to adverse reactivity to a dietary protein instead of favoring immune tolerance in the gut (Maynard and Weaver 2011). Significantly increased levels of oxidized proteins and lipids have been demonstrated higher in CD patients with active disease, whereas retinol and tocopherol plasma concentrations were significantly lower than in patients under a GFD (Hozyasz et al. 2003). Oxidized lipoproteins activate mast cells, causing an increased production of inflammatory cytokines, suggesting that vitamin E–mediated prevention of oxidation of LDL may offer some benefit to patients with active disease (Odetti et al. 1998). A recent study demonstrated in the small-bowel mucosal biopsy organ culture system in CD patients that co-administration of vitamin C prevented the production of nitrites, interferon-α/γ, TNF-α, and IL-6, although increased the expression of IL-15, triggered by gliadins, suggesting that vitamin C supplementation might be beneficial for celiac patients (Bernardo et al. 2012). Also it has been attributed a significant role for ascorbate in inflammation and protection against oxidative stress since it has been found at high concentrations in leukocytes suppressing the production of Th-1-type cytokine IFN-γ in peripheral blood mononuclear cells (Jenny et al. 2011). At the intestinal level, increased levels of reactive oxygen species (ROS) found in celiac duodenum samples have been associated with sustained TG2 activation (Luciani et al. 2009). The aforementioned data indicate that mucosal damage evoked by gliadins or even in asymptomatic patients and a redox imbalance persists in CD, and is associated with decreased paraoxonase (PON) activities such as PON1 (Ferretti et al. 2012), and PON3 (Rothen et al. 2007). PON1 is mainly synthesized by the liver; however, in vitro studies suggested that the intestine could also represent a source for increased concentrations of PON1 in the systemic circulation (Kruit et  al. 2006), contributing to modulate extraintestinal manifestations of the disease. The modifications of PON1 activities in CD patients have been associated with increased anti-TG antibody levels and mucosal damage (Marsh 2-3) (Ferretti et al. 2012). The intake of dietary antioxidants (carotenoids such as lycopene and polyphenols, among others, curcumin, epigallocatechin gallate, resveratrol, and quercetin) may help target signal transduction cascades leading to the activation of the NFκB, interferon regulatory factor (IRF)-1 and member 1 of the signal transducer and activators of the transcription family (STAT-1α) potentially mediated by inhibition of iNOS and COX-2 expression as evidenced in in vitro studies (De Stefano et al. 2007; Calder et  al. 2009). These antioxidant compounds could modulate nrf-2 activation, inhibiting the expression of pro-inflammatory cytokines, chemokines, cell adhesion molecules, and matrix MMPs (Surh et  al. 2008; Kim et  al. 2010) representing nontoxic agents to decrease the severity of the disease. Increasing evidence has also led to propose conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids (PUFA) as important nutrients for modulating mucosal immunity in patients with inflammatory bowel disease (BassaganyaRiera and Hontecillas 2006). It has been reported that c9-t11-CLA influences

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transcription factor activity and inhibits the maturation of dendritic cells obtained from HLA-DQ8 transgenic mice by activation of detoxifying and antioxidant enzymes γglutamylcysteine (γGCL), NAD(P)H-quinone oxidoreductase (NQO1), and heme oxygensae-1 (HO-1) (Bergamo et al. 2008). In addition, CLA was shown as effective in preventing gliadin-induced depletion of intestinal defenses caused by down-regulation of the NRF-2 cytoprotective pathway (Bergamo et al. 2011). From a critical review of the current literature it could be hypothesized that a mechanism of action through modulation of cytosolic phospholipase A2 activity has been implied in activation of intraepithelial cytotoxic T lymphocytes of CD patients (Tang et al. 2009). However, it still remains unclear if these effects are mediated by the formation of bioactive eicosanoids and their effects on nuclear receptors or changes in membrane lipid composition.

24.7 Conclusions CD is an autoimmune enteropathy conditioned not only by genetic determinants and gluten exposure, but also several different environmental factors. The gliadinsmediated mucosal damage to intestinal epithelium has a profound impact and is an important contribution to the onset of the disease. Proteome studies revealed important alterations in expression patterns of proteins responsible for cell homeostasis and oxidative stress as well as particular receptors that can be stimulated by biologically active food-derived components in the detrimental effects caused by gliadins to intestinal cells. It can be established that an association between nutrients with important functional activities beyond its exclusive nutritional value can control inflammation and maturation or function of immune cells. Altogether, findings indicate that bioactive food-derived components could help nutritional intervention strategies adjuvant to the pharmacological ones to preserve intestinal cell homeostasis and oxidative stress and, thereby, severity of the disease or increase the threshold of tolerance to dietary gluten. However, human studies are required to confirm that CD patients and populations at risk can benefit from these strategies, only evaluated preclinically to date.

Acknowledgments This work was supported by Consolider Fun-C-Food (No. CSD2007-00063) from the Spanish Ministry of Economy and Competitiveness and Project REFRESH (FP7-REGPOT-2010-1-264105) Unlocking the potential of the Institute of Animal Reproduction and Food Research for strengthening integration with the European Research Area and Region Development. Project financed in the area of “Research Potential” of the 7th Framework Programme.

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CHAPTER 25

Bioactive lipid components from ruminant milk and meat: The new face of human health Malgorzata Szumacher-Strabel1, Mohamed El-Sherbiny1,2, Adam Cieslak1, Joanna Szczechowiak1, and Hanna Winiarska3 1

Poznan University of Life Sciences, Department of Animal Nutrition and Feed Management, Poland National Research Centre, Department of Dairy Sciences, Dokki, Cairo, Egypt 3 Poznan University of Medical Sciences, Department of Pharmacology, Poland 2

25.1 Introduction: Bioactive components from ruminant milk and meat: Overview and characteristics Products of animal origin, including milk and meat, are a major source of food for humans in many regions of the world, and thus a rich source of compounds with biological activity. In addition to basic functions, they have others that allow assigning these products to so-called functional foods. There is substantial evidence that milk and meat contains bioactive compounds that benefit human health by reducing disease risk. These compounds include, among others, fatty acids, peptides, and oligosaccharides. Peptides that are derived mainly from milk proteins act as growth factors, antimicrobial factors, antihypertensive agents, immune regulatory factors, and mineral carriers, while oligosaccharides have anti-inflammatory and anti-infective properties. This chapter mainly discusses milk and meat fatty acids as bioactive components. Monounsaturated and polyunsaturated fatty acids, as well as conjugated isomers of unsaturated fatty acids, have a wide range of beneficial effects, including reduction in susceptibility to atherosclerosis, cancer, and diabetes, particularly type II diabetes. The challenge for unsaturated fatty acid compounds is to increase their level in milk and meat (e.g., define mechanisms to intensify their production in the rumen and tissues). At the nutrition level, there are four ways for increasing their level in animal products: (1) supplementation of animal’s diets with phytochemicals, (2) supplementation of animal’s diets with sources of unsaturated fatty acids, (3) modulation of dietary forage-to-concentrate ratio, and (4) use of different feeding systems and strategies. The challenge is to improve Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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processes (e.g., rumen biohydrogenation [BH] and mammary gland de-novo synthesis) and effects of the methods listed above. The discussion over the possibility of improving fatty acid composition of milk and meat should also take into account genetic differences within and between species and breeds. These differences are, for example, due to different feed components and hence differences in rumen microflora and enzyme activity produced by them. Increasing the concentration of biologically active compounds in the products may increase health-promoting food intake and thus may ensure health and well-being.

25.2 Rumen metabolism as a source of bioactive lipids: Current knowledge Rumen, one of the most mysterious organs in animal biology, occupies a large part of the ruminant digestive system. It could be classified as the first fermentor ever discovered by humans. The complexity of rumen composition leads it to act as a “bioreactor” producing dozens of bioactive compounds. This part of the chapter mainly discusses some of the processes occurring in ruminants, mainly connected with the presence of bioactive lipid compounds, as well as discusses briefly the rumen microbial population, focusing on microorganisms involved in these processes.

25.2.1 Biohydrogenation and lipolysis: The key to newly discovered bioactive lipid compounds Rumen metabolism has been well studied in the last century. Very early results highlighted two of the most important reactions occurring in the rumen (i.e., lipolysis and biohydrogenation). These reactions, or in other words “microbial activities,” can produce some fatty acid intermediates, which can be described as bioactive compounds.

Lipolysis Rumen lipolysis or hydrolysis is known as the first metabolic process affecting dietary lipids and result in the release of free fatty acids (FFA) from esters found in triacylglycerides, phospholipids, and glycolipids in addition to glycerol and galactose, which are later converted to volatile fatty acids. According to Bauchart et  al. (1990), up to 85% of the dietary unprotected lipids are hydrolyzed. However, lipolysis was observed to be more efficient in the case of diets rich in fiber when compared to low-fiber diets. This hypothesis is based on the greater enzymatic activity of lipase when diets rich in fiber and nonacid pH are administered (Gerson et al. 1983). Lipolysis is an important step before initiating the other process called biohydrogenation and any decrease of unsaturated fatty acid levels that reach the

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duodenum could be due to reduced rumen lipolysis. Lipolysis and biohydrogenation appear to be the processes used by microorganisms to protect themselves from the toxic effects of unsaturated fatty acids (Dehority 2003). After ingestion, dietary esterified lipids are hydrolyzed to FFA and glycerol as well as small amounts of mono- and diglycerides by microbial lipases, which are extracellular enzymes assembled in small beads (Jenkins 1993). The number of microorganisms capable of hydrolyzing esters is low and their activity is highly specific (Henderson 1971; Henderson and Hodgkiss 1973; Fay et  al. 1990). Various strains of Butyrivibrio fibrisolvens and Anaerovibrio lipolytica are capable of hydrolyzing the ester bonds, but B. fibrisolvens lipase hydrolyzes phospholipids, A. lipolytica hydrolyzes only tri- and di-glyceride, and their rates of hydrolysis differ. Lipase activity also occurs in ciliate protozoa, but not in fungi (Dehority 2003), although their contribution is lower than that of bacteria. FFA may also arise from hydrolysis of plant galactolipids and phospholipids catalyzed by several bacterial galactosidases and phospholipases (e.g., phospholipase A and phospholipase C), produced by rumen microbes (Jenkins 1993). The half-life of free unsaturated fatty acids is relatively short due to their rapid hydrogenation by rumen microbes into the corresponding saturated configuration. BH has been estimated at 600 and 900 g/kg of PUFA, and it only contributes to a limited extent to the recycling of metabolic hydrogen, as only 1–2% of it is used for this purpose (Czerkawski and Clapperton 1984). Harvatine and Allen (2006) completed an in vivo experiment with lactating dairy cows to determine rates of FA biohydrogenation of fat supplements with different degrees of unsaturation, and developed a kinetic model of ruminal biohydrogenation. Based on their results, they showed that passage rates of C16:0, C18:0, and total C18 carbon FA linearly decreased as UFA increased, while the trans C18:1 fractional passage rate was affected quadratically with a maximum rate for the intermediate treatment. Increasing UFA increased the extent of C18:2 and C18:3 biohydrogenation, and decreased the extent of C18:1 and trans C18:1 biohydrogenation.

Biohydrogenation Biohydrogenation, commonly abbreviated as “BH,” was initially suggested by Banks and Hilditch in 1931, who founded their suggestion on a comparison between tissue lipids or “tallows” of both ruminants and nonruminants. They observed a lower saturation rate of tissue lipids from nonruminants when compared to the ruminants. They also suggested that this difference can be due to an activity occurring only in ruminant tissues. This suggestion has been recently amended that this activity or biohydrogenation occurs in the digestive system of ruminants, mainly in the rumen. Biohydrogenation is the second step in the processes carried out by rumen microorganisms on dietary lipids; BH is a chemical process mainly occurring in the rumen as a result of UFA toxicity to the biohydrogenating rumen bacterium, especially Butyrivibrio fibrisolvens (Maia et al.

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2010). The harmful effects of fatty acids on microbial cells, connected with altered permeability of cell membranes and disturbances in the functioning of the sodium-potassium pump, result in the inhibition of cell growth and metabolism (reduced synthesis of microbial protein). This consequently limits the amount of unsaturated fatty acids (UFA) released at pH 2.5–3.5 in the abomasum, subsequently reaching the small intestine for absorption. One of the very early research that helped to understand and discover biohydrogenation was conducted by Reiser in 1951, who suggested that the formation of trans fatty acid intermediates when incubating linseed oil (a source of linolenic acid) in the rumen fluid can provide evidence for ruminal biohydrogenation. Several researchers followed that experiment to investigate biohydrogenation of polyunsaturated fatty acid sources. For instance, Shorland et al. (1955) indicated that stearic acid is the end product of biohydrogenation of linolenic acid and they also clarified that during this transformation some C18 fatty acid intermediates were produced. High stearic acid content was also observed by Garton et al. (1958) as a result of fatty acid hydrogenation. In his papers (1959 and 1960), Wright indicated the main agent responsible for rumen biohydrogenation, as he clarified that bacteria, along with rumen protozoa, play an important role in the biohydrogenation process. Wood et al. (1963) confirmed the results reported by Shorland et al. (1955); their earlier results showed that about 45% of the 14C-labeled linoleic remaining in the rumen after 48 hours of incubation were totally saturated, 30–50% were hydrogenated to C18 intermediates, while a small part remained as linoleic acid. Ward et  al. (1964) also investigated the biohydrogenation of C18 fatty acids using 14C-labelled oleic, linoleic, and linolenic acids. They reported that over 90% of the linoleic acid was totally converted to stearic acid in the rumen fluid. Additionally, partial accumulation of trans C18:1 isomers was also observed. The study conducted by Wilde and Dawson (1966) was also one of the first that illustrated the full metabolic pathways of α-linolenic acid by rumen microorganisms showing the production of several intermediates ending up with the stearic acid. Kepler et al. (1966) and Kepler and Tove (1967) performed one of the earlier studies using the B. fibrisolvens, in which they concluded that linoleic acid is first isomerized to cis-9, trans-11 CLA, and then hydrogenated to a mixture of trans C18:1. Research conducted by Dawson and Kemp (1969), Girard and Hawke (1978), and Singh and Hawke (1979) was directed to study the role of rumen protozoa in the biohydrogenation process. They suggested that the presence of rumen protozoa has no effect on biohydrogenation and any contribution could be due to the activity of ingested bacteria. Nowadays, it is more evident that ciliate protozoa are the microbes influencing fatty acid metabolism in the rumen, as they effectively accumulate unsaturated fatty acids (Devillard et al. 2006; Cieślak et al. 2009) and a small number of bacterial species with high biohydrogenating activity. Butyrivibrio fibrisolvens is a rather diverse group of Gram-positive bacteria, all of which metabolize linoleic acid at many times the rate of other ruminal species (Maia et al. 2007).

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25.2.2 Rumen populations responsible for rumen lipolysis and biohydrogenation The rumen is one of the most complex and unique environments, its microbial populations comprise 1010 bacteria, 107 protozoa, 106 fungi and yeasts per ml of rumen fluid, at a temperature of 38–39 °C and a normal range of pH between 5.9 and 6.7 (Buccioni et al. 2012). Rumen microbial populations are closely correlated and interact in their roles. Nutrition strategies and digestion principles in ruminants are mainly based on the microbial populations that are not only involved in feed digestion and degradation, but their activities are also the main reasons for the presence of hundreds of intermediates and bioactive compounds in ruminant products. The last century witnessed hundreds of studies conducted by nutritionists, microbiologists, and chemists trying to modulate and understand the different pathways occurring in the rumen microbial system in order to make it more efficient.

Rumen bacteria The rumen bacterial population is the main agent responsible for the physiological development of the rumen, in addition to its important role in the digestion and conversion of different diets (mainly plants) into a wide range of edible animal products. From the first day after birth, some rumen bacteria can be identified a long time before the maturation of the rumen (Jami et al. 2013). The bacterial population present in the rumen was distinguished according to Stewart et  al. (1997) into 26 species based on Gram-negative and -positive rumen bacterium clarifying both major and minor fermentation products. Studies conducted by Dawson and Kemp (1969) showed that rumen bacteria play an important role in the lipolysis process when compared to other rumen populations. Lipases produced by Anaerovibrio lipolytica species were widely investigated (Henderson 1971; Henderson and Hodgkiss 1973; Prins et al. 1975). Anaerovibrio lipolytica seems to exhibit some disability to hydrolyze galactolipids and phospholipids despite the abundant presence of this species in grazing animals (107/ml). However, Rattray and Craig (2007) suggested that this species plays other roles in glycerol fermentation, especially in the case of triacylglycerols. Butyrivibrio spp. also showed different lipase activities (phospholipase A, phospholipase C, lysophospholipase, and phosphodiesterase), whereas no effect was observed on triacylglycerols. Maia et al. (2007) explained the toxic effect of polyunsaturated fatty acids on different bacterial species and this explanation provides evidence that the two properties are found simultaneously in the bacterial species, lipase releases free fatty acids (toxic to bacterial species), that will later be removed by biohydrogenation. Butyrivibrio fibrisolvens was the first identified species that performs C18:2 fatty acid biohydrogenation, forming several intermediates as conjugated linoleic acids and trans-11 C18:1 (Kepler et al. 1966). Kemp et al. (1975) and Hazlewood et al. (1976) later identified other bacteria that were capable of biohydrogenating fatty acids. Most isolates converted linoleic acid only as far as C18:1, mainly

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trans-11 C18:1, and these were termed group A by Kemp et al. Bacteria carrying out stearate formation (group B) were identified as Fusocillus spp. (Kemp et al. 1975), a genus description that, like the cultures themselves, has not survived being a misnomer. Cis-9, trans-11 C18:2 is usually the predominant conjugated linoleic acid (CLA) isomer found in the rumen and in milk, but many others are present, with trans-9, trans-11 C18:2 usually more abundant than the others (Shingfield et  al. 2003; Palmquist et  al. 2005; Chilliard et  al. 2007). There are, however, times when the trans-10, cis-12 isomer becomes a major intermediate, which proved to occur mainly by using high-starch feeding or by fish or vegetable oil dietary supplementation (Bauman and Griinari 2001; Shingfield and Griinari 2007). High concentrations of trans-10 C18:1 are found in the digesta and consequently in the FA flowing to animal tissues (Daniel et al. 2004; Shingfield and Griinari 2007; Szumacher-Strabel et al. 2009). Under these circumstances, milk fat depression occurs, with other consequences to the animal, including lower intake and decreased fiber digestion (Bauman and Griinari 2001). Some experiments conducted using post-ruminal infusion indicated that trans-10, cis-12 C18:2 exert antilipogenic effects in dairy cattle (Baumgard et al. 2000; Lock et al. 2006). Recent studies suggest that it may actually be trans-10 C18:1 rather than trans-10, cis-12 CLA that decreases mammary lipogenesis (Shingfield et al. 2009). It is important, therefore, to understand how these isomers are formed. There are, in addition, other possible pathways of linoleic acid metabolism, including hydration and chain elongation or shortening, which may increase in importance depending on the diet. In all these aspects of fatty acid metabolism it is important to understand the role of different microbial species.

Rumen protozoa Larger organisms (5–250 μm long) present in the rumen have been designated at various times as protozoa. Up to half of the rumen microbial biomass may be protozoan (Williams and Coleman 1992) and approximately three-quarters of the microbial fatty acids present in the rumen may be present in protozoa (Keeney 1970). The largest, most obvious, and most important protozoa are the ciliates, of which there are two groups, both in the subclass Trichostomatia: the holotrich protozoa and the entodiniomorphs. Earlier studies (Katz and Keeney 1966; Viviani 1970; Emmanuel 1974) showed that mixed protozoa from the sheep rumen contained at least two to three times more unsaturated fatty acids than bacteria. These unsaturated fatty acids included CLA and trans-11 C18:1 showed to be more abundant in protozoa than in bacteria. The most likely explanation is that protozoa incorporate CLA and trans-11 C18:1 formed by ingested bacteria. Some ciliate protozoa showed to be selectively retained within the rumen by a migration mechanism that depends on some compounds in the rumen system or “chemotaxis” (Abe et  al. 1981; Ankrah et al. 1990). As a consequence, protozoa reaching the duodenum are

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proportionally lower compared to the expected if they were to flow with the rest of ruminal digesta (Weller and Pilgrim 1974). The flow of microbial nitrogen at the duodenum of steers was recently shown to be 12–15% protozoal in origin, whereas in terms of fatty acid flow, protozoa accounted for between 30 and 43% of the CLA and 40% of the trans-11 C18:1 reaching the duodenum (Yanez-Ruiz et al. 2006). The contribution of protozoa to the flows of C16:0 and C18:0 to the duodenum was less than 20 and 10%, respectively. Thus, protozoa do not themselves produce CLA and trans-11 C18:1 by their own metabolism; nevertheless, they might be expected to have a significant influence on CLA and trans-11 C18:1 available to the host animal. However, the status of PUFA flow in general remains unclear. It has been known for a long time that protozoal lipids contain proportionally more UFA than the bacterial fraction (Katz and Keeney 1966; Harfoot and Hazlewood 1997; Cieslak et., 2009). Recently, it was established that these UFA include CLA and vaccenic acid (VA) (Devillard et al. 2006; Cieślak et al. 2009), further increasing the possible significance of protozoa in the delivery of healthpromoting FA from the rumen. Different protozoal species had different compositions, with larger species including Ophryoscolex caudatus containing more than 10 times higher concentrations of CLA and VA than some small species, such as Entodinium nannelum (Devillard et  al. 2006). Isotricha prostoma, a large species and the only holotrich examined, had low concentrations of CLA and VA. In incubations with fractionated ruminal digesta, linoleic acid metabolism was very similar in strained ruminal fluid and its derived bacterial fraction, while its mixed protozoal fraction exhibited a much lower activity. The opposite direction of the reaction, namely desaturation, also did not occur in the protozoal fraction. Radioactivity from 14C stearate was not incorporated into CLA or VA by protozoa. Thus, the protozoa are rich in CLA and VA, yet they appear not to synthesize these two FA from LA or stearate, confirming the opinion of Dawson and Kemp (1969). It might be argued that the high UFA content of protozoa results from the ingestion of plant particles, especially chloroplasts (Wright 1959; Stern et  al. 1977). Huws et  al. (2009) showed recently that the engulfment of chloroplasts is a major contributor to the high linolenic acid (LNA) concentration of protozoa. This cannot explain the high concentration of CLA and VA in protozoa, as these FA are absent from the plant material. Biohydrogenating activity must be involved. Our investigations suggest that the most likely explanation is that protozoa preferentially incorporate CLA and VA formed by ingested bacteria. The lower conversion to stearate perhaps occurs because the bacteria responsible for conversion of monoenoic acids to SFA are more vulnerable to protozoal digestive activities. A simpler explanation is that the reactions occurring at the early stages of biohydrogenation are much more active than the last one, and that if both are decreased by, say, 95%, by protozoal digestive activities, this may leave the remaining enzymatic activity sufficient to form, for example, CLA and VA

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from LA, but not enough to form significant amounts of stearate. These findings imply that the availability of PUFA (including CLA) and VA for absorption by the host animal could depend more on the flow of protozoa rather than bacteria from the rumen.

25.2.3 The de-novo synthesis in ruminant tissue Endogenous synthesis, lipogenesis, and de-novo synthesis are similar terms that describe a number of processes occurring in the intestinal mucosal cells, liver, adipose tissue, and mammary glands of ruminants. These reactions lead to several fatty acid intermediates that can act as bioactive lipid compounds (Laliotis et al. 2010). In this section we illustrate the de-novo synthesis in lactating ruminants as an important source of newly discovered lipid bioactive compounds. Milk fatty acids are mainly derived from two main sources: de-novo synthesis in the mammary epithelial cells and the fatty acids from the diet (absorbed digested lipids) or from body fat mobilization (Chilliard et al. 2000). Under normal conditions, both short-chain (4–8 carbons) and medium-chain (10–14 carbons) fatty acids are de novo synthesized in the mammary gland, whereas long-chain fatty acids (more than 16 carbons) are derived from the uptake of circulation; in contrast, the C16:0 fatty acid is derived from both sources (Cozma et al. 2013). Lately, some proof showed that both milk C15:0 and C17:0 fatty acids are also endogenously synthesized from propionate in ruminant tissues, in addition to the odd- and branched-chain fatty acids synthesized by rumen bacteria (Vlaeminck et al. 2006). In lactating ruminant mammary epithelial cells, the main precursors for the de-novo fatty acid synthesis are both acetate and butyrate, which is converted later to β-hydroxybutyrate in the rumen wall (Jensen 2002). Acetate is produced by rumen fermentation of carbohydrates; it is first converted to acetyl-CoA by the acyl-CoA synthetase enzyme, initially using acetyl-CoA in the prolongation of newly synthesized fatty acids, adding two carbons to a growing fatty acyl chain, each time derived from malonyl-CoA produced from acetate and catalyzed using the acetyl-CoA carboxylase enzyme (Chilliard et al. 2000; Chilliard and Ferlay 2004; Shingfield et al. 2010). It has also been clarified in some in-vitro studies that the longer-chain fatty acids and/ or saturation is more affected by the inhibitory effects of acetyl-CoA carboxylase, explaining the decrease of some medium-chain fatty acids in milk fat (Chilliard et al. 2000). We mentioned previously that fatty acids (absorbed digested lipids) from the diet constitute an important source of fatty-acid synthesis in ruminants, which refers to fatty acids absorbed from dietary and microbial fatty acids transferred to the bloodstream, where they are released from circulating lipoproteins by lipoprotein lipase or nonesterified fatty acids bound to albumin that originate from the digestive tract or mobilized fat (Bauman and Griinari 2001; Clegg et  al. 2001). These fatty acids are absorbed in the duodenum and then esterified

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in absorbent cells involved in the assembly of low-density lipoproteins that pass to the blood (Vernon and Flint 1988). Fatty acids derived from the mobilization of body fat are another source of milk fatty acids (10% of milk FA). The role of mobilized fatty acids is clearer in negative energy balance (Bauman and Griinari 2003). Fatty acid uptake from the circulation facilitates the formation of triacylglycerides through glycerol esterification; it consists mainly of esterified tri-hydric alcohol glycerol with three attached long-chain fatty acids in three positions (sn-1, sn-2, and sn-3) (Chilliard and Ferlay 2004). It is mainly synthesized in the endoplasmic reticulum of mammary epithelial cells. The attached fatty acids are not randomly assigned on the glycerol molecule; C8:0, C10:0, and C12:0 are mostly esterified and placed on the second position (sn-2), C18:0 could mostly be found in the first position (sn-1), while C4:0, C6:0, and oleic acid are more typically placed in the third position (sn-3). Some other fatty acids, such as C16:0, are equally placed in the first and second positions (Jensen 2002). However, about 60% of the fatty acids positioned at sn-1 and sn-2 are mediumand long-chain fatty acids (≤C18 carbons), 24% of fatty acids at sn-2 is oleic acid, while a large proportion of short-chain fatty acids and oleic acid are positioned at sn-3 (Bernard et al. 2008). This distribution of fatty acids in the sn-3 position plays an important role affecting physical properties of milk fat, represented in a lower melting point when compared to the cow normal temperature for a higher fluidity (Timmen and Patton 1988). Desaturation and elongation processes are performed in the mammary tissues; however, long-chain fatty acids are mostly desaturated in the mammary secretory cells, while performed fatty acids cannot undergo elongation within the mammary gland (Chilliard and Ferlay 2000). Mammary secretory cells contain the enzyme Δ9-desaturase that acts by adding a cis-9-double bond on the FA chain (Shingfield et al. 2008). Δ9-desaturase activity in the ruminant mammary gland is assumed to occur as a mechanism to ensure the liquidity of milk for efficient utilization by the offspring (Timmen and Patton 1988). In this respect, the mammary gland transforms C18:0 into cis-9 C18:1 and contributes 60–80% of the entire amount of oleic acid secreted in milk (Glasser et al. 2007; Shingfield et al. 2013). Likewise, the activity of Δ9-desaturase is estimated to contribute to 90% of cis-9 C14:1 and 50% of cis-9 C16:1 in milk fat (Mosley and McGuire 2007). The other FAs shorter than 18 carbon chain length, such as C10:0, C12:0, C14:0, C15:0, and C17:0, can also be used as substrates for Δ9-desaturase (Shingfield et al. 2010). Moreover, it is estimated that 25% of the vaccenic acid (trans-11 C18:1) formed in the rumen is desaturated in the mammary gland to rumenic acid (cis-9, trans-11 C18:2), the main isomer of conjugated linoleic acid in milk (Mosley et  al. 2006). Mammary endogenous synthesis from trans-11 C18:1 is responsible for 70–95% of the milk cis-9, trans-11 C18:2 (Shingfield et al. 2013).

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25.3 Bioactive lipid components in ruminant products as affected by feed modulation: Recent updates 25.3.1 The effect of different feed additives on bioactive lipid components The different impacts of the discovered lipid bioactive compounds on human health are the main cause for the dynamic revolution observed in the last decade in ruminant nutrition research. These bioactive lipid components, especially fatty acids (FA), present in the milk fat and muscles of ruminants were shown to be affected by different modulations on the rumen diets. This part of the chapter focuses mainly on highlighting the different effects of feed on bioactive components in ruminant products that have been recently found.

25.3.2 The effect of plant secondary metabolites on bioactive lipid components A growing interest in using secondary plant metabolites, such as essential oils, saponins, or tannins, in ruminant nutrition has been recently observed, particularly in Europe, after the European Union prohibited the use of antibiotic growth promoters in livestock production (EU Directive No. 1831/2003/CEE) (OJEU, 2003). Accordingly, a number of studies were conducted to evaluate the potential of plant secondary metabolites to modulate rumen microorganism fermentation, increasing feed efficiency of ruminants (Szumacher-Strabel and Cieslak 2012; Bodas et  al. 2012) and modifying the fatty acid composition of meat (Jeronimo et al. 2010) and milk (Benchaar et al. 2012). Among secondary plant metabolites, essential oils, tannins, and saponins draw particular attention because of their potential ability to modify biohydrogenation of unsaturated fatty acids associated with changes in rumen ecology (Benchaar and Greathead 2011).

25.3.3 Tannins and fatty acid profile in ruminant products Tannins are an interesting example of bioactive molecules derived from plants, with promising beneficial opportunities for use in animal nutrition. However, tannins were widely known to exert antinutritional effects on ruminants by forming complexes with dietary proteins (Bodas et al. 2012). Despite this general concept tannins have been increasingly studied in the last decade for their positive impact on the quality of ruminant products (Khiaosa-Ard et al. 2009; Vasta et  al. 2010; Toral et  al. 2013). Tannins showed some inhibitory activity against rumen bacteria, with this inhibition potentially due to the direct interaction with enzymes involved in the biohydrogenation pathway, which can inhibit proliferation of rumen microorganisms (Szumacher-Strabel et al. 2011; Cieslak et al. 2012; Bodas et al. 2012). One of the main goals for ruminant nutritionists nowadays is the manipulation of different pathways of ruminal biohydrogenation, resulting in a higher

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polyunsaturated fatty acid (PUFA) content in the rumen, especially long-chain PUFA, which can be later absorbed in the intestine, improving the fatty acid profile in ruminant products. There are limited numbers of studies on the use of tannins and their impact on the profile of fatty acids in the rumen ecosystem, and their results are often inconsistent and contradictory. The in-vitro study conducted by Vasta et al. (2009a) showed that three different sources of tannins that is, Ceratonia siliqua, Accacia cyanophylla, and Schinopsis lorentzii added at 0.6 and 1 mg/ml buffered ruminal fluid, decreased the rumen biohydrogenation of C18 fatty acids, especially the last step where vaccenic acid (trans-11 C18:1) is converted to stearic acid (C18:0). Khiaosa-Ard et al. (2009) noted that incubating condensed tannins from Acacia mearnsii (7.9% of DM) with the rumen fluid led to a different profile in the effluent compared with the control diet, with trans-11 C18:1 being considerably increased instead of the saturated form C18:0. Unfortunately, these results are in disagreement with in-vivo studies described by Benchaar and Chouinard (2009) and Dschaak et al. (2011), where milk fatty acid composition was not influenced by quebracho-condensed tannins added at 150 g/d and 3% of DM, respectively. Based on the above results, interpretation of the findings obtained from in-vitro experiments should be more precise and verified in vivo. Despite the fact that similar results were obtained from other in vivo studies (Vasta et al. 2009b, 2010), it is still difficult to draw a clear and unequivocal conclusion concerning the impact of tannins on product quality. The results recorded in those experiments show a higher accumulation of VA, cis-9, trans11 C18:2 (CLA) and total PUFA in the meat of lambs fed a concentrated diet with tannins when compared with lambs fed a herbage diet with tannins (Vasta et al. 2009b). This may indicate that not only the type and dose of tannins but also the basal diet composition can result in changes in the fatty acid profile (Toral et al. 2013). Results from the study of Toral et al. (2013) indicated the effect of adding a high level (20 g/kg of DM) of quebracho tannins in a diet rich in linoleic acid on dairy ewes. In that study significant differences in trans-11 C18:1 and cis-9, trans-11 C18:2 were observed transiently on day 3, which according to the authors, cannot be useful to beneficially modify the fatty acid profile, especially over a long time frame. In the formation of biologically active isomers of conjugated linoleic acid, a big importance is also played by the activity of Δ9-desaturase, which is involved in the endogenous synthesis of CLA in ruminant tissues (Buccioni et al. 2012). Rana et al. (2012) supplemented the goat diet with tannins from Terminalia chebula (3.1 g/kg body weight). In that study the authors observed a 47% increase in Δ9-desaturase activity, which resulted in an increase of total CLA content (58.73%) in muscles. Vasta et  al. (2009b, 2009c) suggested that desaturased activity is sensitive to dietary compounds and for this reason the expression of Δ9-desaturase mRNA and/or protein can increase when tannins are added to the

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diet. In their study Rana et al. also observed a significant reduction in saturated fatty acid content by about 25% and an increase in vaccenic acid content by about 83% in muscles, which can be explained by changes in the bacterial population involved in the last step of biohydrogenation.

25.3.4 Essential oils and fatty acid profile in ruminant products Essential oils are aromatic compounds containing functional groups (e.g., phenols or terpenoids) that exhibit antimicrobial activity (Benchaar and Greathead 2011). The mechanism of action for these substances is based on high affinity to bacterial cell membranes (especially Gram-positive bacteria), resulting in altered transport across the membranes, failing, disrupting, and inactivating enzymes (Bodas et al. 2012). Due to antimicrobial properties essential oils are increasingly commonly introduced to livestock diets to reduce methane emissions and improve milk production as well as utilize feed ingredients (Szumacher-Strabel and Cieslak 2012). Information on the impact of essential oils on meat and milk lipid profiles is very limited, and most recent studies concern in vivo experiments on different ruminant species. Hristov et  al. (2013) administered to dairy cows leaves of Origanum vulgare at three different levels of 250 g/day, 500 g/d, and 750 g/d, but the milk fatty acid composition was not affected by these additives. A similar effect was also reported by Benchaar et  al. (2012) in a study on dairy cows receiving eugenol (50 mg/kg DMI) in high- and low-concentrate total mixed rations. In view of the well-known antimicrobial properties of this plant secondary metabolites, it would be expected to change ruminal biohydrogenation; however, the above-mentioned studies have shown no change in the fatty acid profile, probably because the rumen microflora is able to adapt to the essential oils, mainly attributable with the degradation or neutralization of these compounds (Malecky and Broudiscou 2009), or on the development of mechanisms resistant to these substances (Bodas et al. 2012). Nevertheless, Vasta et al. (2013) and Boutoial et  al. (2013) reported that supplementation of the essential oil from Artemisia herba alba (400 ppm of DM) and distilled rosemary extract (10% and 20%), respectively, affect the fatty acid profile. In the first study meat lamb that received Artemisia herba alba had higher contents of vaccenic, cis-9, trans-11 C18:2, and linoleic acids than the control group. The latter study showed that an addition of both 10% and 20% of rosemary extract to the goats’ diet increased the percentage of C18:2 and polyunsaturated fatty acids in milk.

25.3.5 Saponins and fatty acid profile in ruminant products Saponins comprise numerous groups of glycosides, found in angiosperms. Saponins have a capacity to form sustained complexes with cholesterol in cell membranes, causing destruction and lysis of cells (Bodas et  al. 2012). Many researchers pointed to the fact that the effects of saponin administration depend

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on many factors, including the time of application (Wina et al. 2006), saponin source, and levels and diet composition (Makkar et  al. 2007). The potentially advantageous action of saponins in the rumen, antibacterial, defaunating, and antimethanogenic effects are indicated by the results of studies conducted in recent years (Nasri and Ben Salem 2012; Hassanat et  al. 2013; Szczechowiak et al. 2013). However, information on the effect of saponins on rumen biohydrogenation is very limited. Two groups of bacteria are involved in the conversion of dietary unsaturated fatty acids: group A (mostly Gram-positive bacteria such as Butyrivibrio fibrisolvens), which converse polyunsaturated fatty acid to vaccenic acids, and group B (mostly Gram-negative bacteria) carrying out the next step of biohydrogenation (Buccioni et  al. 2012). In 1994, Wallace et  al. reported that saponins are not affected in the same way by all species of bacteria. The authors observed that growth of Prevotella ruminicola was stimulated, whereas that of Butyrivibrio firisolvens and Streptococcus bovis was inhibited. Based on the recorded results it was expected that ruminal biohydrogenation can be modulated by an addition of saponins. Nasri et  al. (2011) found that supplementation of Barbarine lambs with saponins from Quillaja saponaria (30, 60, 90 mg/kg DMI) increases the content of polyunsaturated fatty acids in lamb meat, which can be explained by the reduction of biohydrogenation of PUFA in the rumen. In a similar study conducted by Brogna et al. (2011), in which Quillaja saponaria was also added (30, 60, 90 ppm), the concentration of C20:4 was higher in the meat of lambs receiving 60 ppm than in the control groups. That author also reported a reduced concentration of C14:1 cis-9 and its desaturation index, which may indicate that saponins influence (indirectly or directly) ∆9-desaturase activity.

25.3.6 The effect of oils on bioactive lipid components Due to the fact that the milk fatty acid composition is highly dependent on the source, form of lipids, incorporation rate, and the type of basal diet (Shingfield et  al. 2010), recent studies have been focused on the use of vegetable oil or vegetable oil pellets and animal fats, mainly fish oil, as additives to the animals’ ration. It is believed that an increase of lipid content in the ration can bring many benefits, including increased energy value of the ration. This is particularly important for high-producing animals through reduced risk of acidosis in the case of feeding diets with large contents of concentrate as well as changes in the profile and amount of fatty acids in the products obtained from animals. Milk fatty acid composition depends largely on the type of feed taken together with the fatty acids and fatty acid metabolism in the rumen and mammary glands (Jenkins et al. 2008). One of the methods to increase the content of CLA and n3 fatty acids in products derived from ruminants is connected with the introduction of vegetable oils or oilseeds in feed rations. Shingfield et al. (2010) explained the effect of oil on

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dairy cow milk fat proportion by increasing rumen outflow of C18:0 or cis-9 C18:1 and conversion of C18:0 to cis-9 C18:1 in mammary glands via Δ9-desaturase. The most common oils used in the investigations on the effect on the fatty acid profile in ruminants are sunflower oil and soybean oil (rich in linoleic acid; cis-9, cis-12 C18:2), rapeseed oil (rich in oleic acid; cis-9 C18:1), and linseed oil (rich in linolenic acid; cis-9, cis-12, cis-15 C18:3) (Stauffer, 2005). Due to the fact that the oils differ in their fatty acid composition, a different impact on the content of CLA in ruminant products could be expected. The results obtained so far proved that oils rich in linoleic acid are very effective in modification of the fatty acid profile (Gómez-Cortés et  al. 2011; Halmemies-Beauchet-Filleau et  al. 2011; Martínez Marín et al. 2012; Varadyova et al. 2013). Incremental amounts of sunflower oil (17, 34, 51 g/kg of DM) were investigated as potential factors enhancing the milk fatty acid profile in a high-concentrate diet (20:80 forage:concentrate ratio). The addition of 34 and 51 g/kg of DM caused the highest increases of trans-11 C18:1 and cis-9, trans-11 C18:2 (Gómez-Cortés et  al. 2011). Interesting results were obtained by Varadyova et al. (2013) in batch culture experiments, where a high concentrate and fiber diet were supplemented with sunflower oil (35 g/kg DM) with a combination of fumaric or maleic acid (8 mmol/L). In that study the concentration of trans-11 C18:1 acid increased for both diets, supplemented with sunflower oil and both organic acids. It was also observed that this combination can be useful in decreasing ruminal biohydrogenation of oleic acid. We also need to mention here a study conducted by Tsiplakou and Zervas (2013), in which the goat diet was supplemented with a combination of soybean (55.5 g/d) and fish oil (11.1 g/d). This strategy significantly increased the concentration of trans-11 C18:1, cis-9, trans-11 C18:2, trans-10, cis-12 C18:2, C20:5 n3 (EPA), and C22:6 n3 (DHA) in both blood plasma and milk.

25.4 The effect of farming systems and different feeding strategies on bioactive lipid components Animal nutrition, especially ruminant nutrition, is an important industry nowadays based on different and diverse types of raising systems and farm designs, in addition to different feeding strategies, normally variable and affected by many factors (countries, feed availability, objective of animal raising, etc.). In this part we mainly discuss different effects of farming systems and feeding strategies on bioactive lipid components in ruminant products.

25.4.1 Could feeding systems and strategies affect bioactive lipid components in ruminant products? Farming systems are highly related with different feeding strategies used by breeders. That is why farming systems are interchangeable, serving mainly the purpose and goal of the raising process and creating a wide range of systems that

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needs a whole book to be listed. This situation is due to designation of farming system to several factors such as genotype, reproduction, health characteristics, agro-climatic conditions, and socioeconomic environment (Zervas and Tsiplakou 2011). Generally, feeding strategies in different farming systems are based on three branches: grazing feeding, integrated farming systems, and industrial farming systems. These general branches are now the origin for several feeding strategies developed in the last century. In each of these farming systems specific farming strategies are applied, mainly based on the two sources of ruminant nutrition: forage and concentrates. Forage and concentrates are associated and complementary, with forage being considered more important for ruminant nutrition, despite its low content of basic nutrients that can lower the competition compared to concentrate. For this reason the use of both sources is inevitable. In recent years several scientific studies highlighted and confirmed the diverse effect of farming systems and feeding strategies on different production variables, giving some indication that not only feed additives can modulate the composition of ruminant products, but also farming systems and feeding strategies could play an important role. In milk, various effects of different feeding strategies on the fatty acids present in ruminant products can be found (e.g., a higher level of unsaturated fatty acids could be observed in milk from ruminants fed on pasture-based diets) when compared to diets consisting of total mixed rations with roughage based on maize (Slots et al. 2009). Some other evidence showed that feeding strategies based on pasture grazing increase milk microcomponents (fatty acids and vitamins) and volatile compounds (flavors and terpenes), which consequently increase such milk desirability for consumers due to its nutritional and health impacts (Morand-Fehr et al. 2007). Yayota et al. (2013) confirmed their hypothesis after evaluating milk samples from nine different farms using grass silage (reed canary grass), six other farms using maize silage, and finally four farms using by-products (soybean curd residue or brewer grains). The proposed results showed a stable milk composition (milk protein, fat, and lactose) among different feeding strategies in addition to negligible changes in milk yield between different feeding systems. However, significant differences were found in the fatty acid composition, with greater contents of fatty acids less than C16 in addition to the saturated fatty acids in milk samples obtained from farms that used grass silage and maize silage. In contrast, contents of fatty acids of more than C18, monounsaturated fatty acids, and polyunsaturated fatty acids were significantly higher in samples from farms based on by-products feeding compared to the farm that used grass silage, confirming the effect of feeding systems and feeding strategies on milk fatty acid structure and characteristics. A similar study was conducted by Borreani et al. (2013), aiming at identification of different effects of intensive dairy farming systems in Italy on the milk fatty acid profile. That study was performed on 20 dairy farms that used five different feeding strategies. Three of these systems were based on corn silage

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with supplementation of different levels of commercial concentrate (a, high level of commercial concentrate; b, low level of commercial concentrate; c, without commercial concentrate), in the other two systems corn silage was replaced with grass or legume silages (d) and fresh herbage (e). The results showed a noticeable variation in the milk fatty acids profile, the C16:0 and saturated fatty acid concentrations were found to be higher in milk samples from the dairy farms with the (a) and (e) systems, respectively, while the lowest concentration was observed in farm (b). Additionally, samples from farms (d) and (e) had the lowest n6/n3 ratio when compared to the other farms. Other studies totally confirmed the effect of different feeding strategies on the fatty acid proportion in milk. For example, a study conducted by Slots et al. (2009) showed a high concentration of milk alpha linolenic acid and polyunsaturated fatty acids in feeding systems that used high cereals, pasture, and grass silage, as well as a noted increase in milk linoleic acid, monounsaturated fatty acids, and a high ratio of linoleic and alpha linolenic acids. Some studies provided different results when two different feeding systems (grazing and corn silage–alfalfa hay mix) with the same fish oil supplements were used (AbuGhazaleh et al. 2007). Milk yield and protein yield were higher in milk from dairy cattle under the corn silage-alfalfa hay mix system when compared to other cows under the alfalfa grass pasture diet system. Likewise, a higher milk cis-9, trans-11 CLA level was observed in the corn silage–alfalfa hay mix system when compared to the alfalfa grass pasture diet system. Similar results were obtained from studies on meat, of which some are presented in Table 25.1.

25.4.2 Forage-to-concentrate ratio and type: An old strategy with new expectations Feeding systems and strategies are mainly based on different levels and ratios of forage (F) and concentrate (C); several studies were conducted mainly to illustrate the effect of different F:C ratios. Table 25.2 presents some of these studies. These results undoubtedly confirm that different forage-to-concentrate ratios could interfere and affect rumen biohydrogenation and the produced milk fatty acids. Forage levels and types may be the main factors responsible for these effects. Some other studies (Yoder et  al. 2013; Halmemies-Beauchet-Filleau et al. 2013) also showed that forage quality and forage conservation methods can affect fatty acid contents in milk, especially bioactive lipid compounds.

25.4.3 Effect of improved/enriched milk and meat on “western” disease prevention Obesity and resulting health problems such as type 2 diabetes mellitus (T2DM), atherosclerosis, and cardiovascular diseases are becoming a pandemic in developing and industrialized countries. Diabetes, especially T2DM, is one of the most widespread chronic diseases. This global tendency is a consequence of an aging population, growth of population size, urbanization, and high prevalence of

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Table 25.1 Some studies illustrating the effect of feeding strategies on meat fatty acid levels. Study

Goals of the study

Treatments

Effect on meat fatty acids

Dervishi et al. (2010)

Effect of different feeding systems on the fatty acid composition, conjugated linoleic acid, and some relative gene expressions in the semitendinosus muscle of light lambs

The used feeding systems were

High concentrations of vaccenic acid and conjugated linoleic acid, in addition to a lower oleic acid and n6/n3 ratio when compared to the other diets

1. Grazing alfalfa 2. Grazing alfalfa + concentrate ad libitum 3. Indoor-kept lambs with grazing ewes 4. Drylot (indoors dry unified system for the ewes and concentrate ad libitum for lambs) Atti and Mahouachi (2009)

Comparing the effect of different rearing systems (indoors vs. pasture) and nitrogen source (soya bean vs. faba bean) on growth, tissue composition, meat quality, and fatty acid composition of fat-tailed lambs

Lambs were divided into four groups

FA profile and meat characteristics were similar for all dietary treatments

1. Grazing lambs receiving faba bean 2. Grazing lambs receiving soya bean 3. Stall lambs receiving faba bean 4. Stall lambs receiving soya bean Joy et al. (2008)

The effect of feeding management (grazing- or concentrate-feeding) on carcass composition, wholesale cut and tissue percentages, intramuscular fatty acids, and noncarcass components of Churra Tensina light lambs

Lambs were divided into two systems

1. Indoors-kept lambs 2. Grazing lambs

Fatty acid contents were not affected by treatments, although the linoleic acid and linolenic acid ratio was lower in grazing lambs

Table 25.2 Examples of different studies highlighting the role of the F:C ratio on fatty acids. Study conducted by

Goals of the study

Treatments

Important results

Neveu et al. (2013)

The effects of supplementation of extruded flaxseed and the manipulation of forage to concentrate ratio on the performance and milk FA composition of lactating dairy cows

(a) 60:40 F:C ratio

Diets containing extruded flaxseed or low forage reduced the concentrations of saturated fatty acids and increased levels of monounsaturated fatty acids and polyunsaturated fatty acids. Extruded flaxseed supplementation increased milk fat alpha linolenic acid content by 100% and conjugated linoleic acid by 54%.

(b) 60:40 F:C ratio + extruded flaxseed

(c) 40:60 F:C ratio (d) 40:60 F:C ratio + extruded flaxseed Benchaar et al. (2012)

Kargar et al. (2012)

The effects of a low- or a high-concentrate diet with eugenol on feed intake, digestion, ruminal fermentation, microbial populations, milk production, and milk FA profile of lactating cows.

The effect of supplementing yellow grease at two F:C ratios and to compare yellow grease to hydrogenated palm oil at a lower F:C ratio on rumen fermentation parameters and milk fatty acid profile and composition.

(a) High concentrate diet + eugenol

The ratio of trans-11 18:1 to trans-10 18:1 was not affected by dietary treatments, indicating no changes in the FA biohydrogenation pathway in the rumen.

(b) Low concentrate diet + eugenol

Levels of C16:0, cis-9 cis-12 cis-15 C18:3, and cis-9 trans-11 C18:2 were higher in milk from cows fed lowconcentrate diets.

(a) F:C of 34:66 (control)

Increasing F:C ratio from 34:66 to 45:55 increased milk short-chain FA contents without affecting total milk fat conjugated linoleic acid.

(b) F:C of 34:66 + 2% hydrogenated palm oil

Increasing total milk fat conjugated linoleic acid level without affecting desaturase indices when adding yellow grease in comparison to the hydrogenated palm oil.

(c) F:C of 34:66 + 2% yellow grease (d) F:C of 45:55 + 2% yellow grease

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obesity and sedentary lifestyle (Shaw et al. 2010). It is predicted that diabetes will eventually be the seventh leading cause of death in the world (WHO 2012). The principal cause of increased morbidity and mortality among diabetic patients are atherosclerosis and the associated cardiovascular diseases (e.g., myocardial infarction, stroke, and peripheral vascular disease) (Seshasai et  al. 2011). Moreover, this increased risk for certain macro- and microvascular complications that are typically associated with diabetes has been also reported in people with prediabetes, an intermediate stage between glucose concentrations within reference intervals and hyperglycemia that meets current criteria for diagnosis of diabetes (Gong et al. 2009). According to the American Diabetes Association (ADA; 2010), prediabetes can be diagnosed on the basis of either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). The majority of persons with prediabetes progress to overt diabetes in the long run (Saaristo et al. 2010; Shaw et al. 2010). Increased prevalence of both prediabetes and type 2 diabetes results in increased disability, decreased quality of life, and enormous healthcare costs (Zhang et al. 2010). Therefore, if successful, early intervention to delay or prevent the development of diabetes and its vascular complications offers potential benefit to individuals, the healthcare system, and society. Several studies have demonstrated beneficial effects of lifestyle intervention in preventing the development of T2DM in prediabetic populations. Lifestyle modification, which improves insulin sensitivity and β-cell function, should serve as the cornerstone of treatment focusing on diet, 7% weight loss, and 150 minutes per week of moderate physical activity (Saaristo et al. 2010; Tuomilehto et al. 2001). In the Finnish Diabetes Prevention Study and Diabetes Prevention Program (DPP), lifestyle modification led to a 58% relative risk reduction in the incidence of T2DM (Tuomilehto et al. 2001; Knowler et al. 2002). Moreover, these effects were sustained in long-term follow-up. Lifestyle intervention has been found to be a highly effective, safe, and cost-effective method for the prevention of diabetes in high-risk persons, the benefit of which can extend for many years (Ramachandran and Snehalatha 2011). However, it should be emphasized that the impressive results of lifestyle intervention have been obtained predominantly from clinical trials conducted at academic centers, involving frequent clinic visits and multidisciplinary teams. Most importantly, the services were offered at no cost to the study participants. Thus, it is difficult to achieve similar success rates in routine clinical practice in the community. Given the increasing prevalence of obesity, it would be advantageous to identify potential therapeutic nutrients/functional foods to improve glucose and lipid metabolism within the context of obesity. The naturally CLA-enriched ruminant food products, such as ruminant milk and meat, may have potential as a therapeutic nutrient with respect to dyslipidemia, insulin resistance, and atherosclerosis. CLA isomers have been proposed as an antidiabetic and inhibiting atherosclerosis development agents based on improvement in glucose tolerance,

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Bioactive lipid components from ruminant milk and meat

insulin sensitivity, and lipid metabolism seen in several animal models (Moloney et al. 2007; Noto et al. 2007; Ryder et al. 2001) However, the effect of CLA on body composition and lipid and glucose metabolism is controversial and seems to be isomer-, dose-, time-, and species-dependent. It should be noted that the majority of the evidence originates from the studies based on synthetic CLA isomers. Unfortunately, up to now only a few intervention studies using natural enriched CLA products were carried out. However, the results of these trials are disappointing. No effects on body composition lipids and carbohydrate metabolism were observed both in normal-weight (Brown et al. 2011; Tricon et al. 2006) and obese subjects (Desroches et al. 2005). This discrepancy between animal and human studies may be due to the differences in the concentration and source of CLA administered, time of treatment, metabolic status, and age of investigated population. The CLA-enriched diet in C57BL/6J mice is a well-characterized model of obesity, dyslipidemia and impaired insulin peripheral action, induced insulin resistance, and hyperlipidemia. In this animal model of metabolic abnormalities the trans-10, cis-12 CLA was found to reduce fat mass and increase the lean mass but significantly contributed to insulin resistance and liver steatosis, whereas cis-9, trans-11 isomer prevented insulin resistance (Halade et  al. 2009). Similarly, in a study with apoE mice consuming cis-9, trans-11 CLA or trans-10, cis-12 CLA, results suggested that cis-9, trans-11 CLA exerted antidiabetic effects, whereas trans-10, cis-12 CLA asserted prodiabetic effects (Navarro et al. 2010). On the other hand, Ryder et al. (2001) have proved that diet supplemented with CLA mixture (cis-9, trans-11: trans-10, cis-12 CLA, 50:50) normalized impaired glucose tolerance and corrected hyperinsulinemia in the prediabetic Zucker diabetic fatty fa/fa (ZDF) rats after 14 days of treatment. Treatment with a CLA mixture was associated with elevation of adipocyte protein2 mRNA expression. This protein contains a peroxisome proliferator-activated receptor γ response element (PPARE), so it can be speculated that CLA isomers are a ligand for PPARγ, thus expressing a mechanism of action similar to thiazolidinediones, drugs used in the therapy of T2DM. Another mechanism in which CLA isomers could express beneficial antidiabetic effects is a positive influence on glucose uptake by skeletal muscles. In in-vitro study of acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 CLA isomers was associated with stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. The mechanism of these effects is isomer-specific. Both isomers stimulate phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while only trans-10, cis-12 CLA acts via AMP-activated protein kinase (AMPK) (Mohankumar et al. 2013). It is likely that the opposite effects of CLA isomers on insulin and glucose metabolism reflect the different metabolic consequences of the antiobese effect

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of trans-10, cis-12 CLA between animal models. In the ZDF rats hyperglycemia is secondary to obesity. Therefore, the antiobesity action of trans-10, cis-12 CLA would have partially accounted for improved glucose metabolism, while in the C57BL/6J mouse the antiobesity action of trans-10, cis-12 CLA results rather in  lipodystrophy and consequent hyperlipidemia that probably induces the prediabetic state. The results of human studies on the effects of diet enriched in natural or synthetic CLA isomers on health are inconclusive. In some studies no effects of CLA supplementation (Shadman et al. 2013) or consumption of CLA-enriched products (Naumann et al. 2006, Raff et al. 2008) on glucose and insulin has been observed. In others, supplementation with a CLA mixture has shown beneficial effects on insulin resistance in healthy male subjects (Eyjolfson et al. 2004) and type 2 diabetic subjects (Belury et  al. 2003). It should also be noted that in a cross-sectional study an inverse association between the cis-9, trans-11 CLA content in adipose tissue and diabetes risk was observed and it seems to be consistent with the hypothesis that CLA may be involved in insulin effects regulation (Castro-Webb et al. 2012). Atherosclerosis, the most important contributory factor in diabetes-related cardiovascular diseases, can be considered as a chronic, low-grade inflammatory process. One of the key early events in the pathogenesis of atherosclerosis is the activation and adhesion of circulating monocytes to the endothelium followed by transmigration into the subepithelial space (Packard and Libby 2008). Some evidence suggests that CLA modulates the macrophage pro-inflammatory phenotype, with suppression of pro-inflammatory genes such as vascular cell adhesion protein 1 (VCAM-1), matrix metalloproteinase (MMP)-9, platelet endothelial cell adhesion molecule (PECAM)-1, and pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, and IL-6 (Lee et al. 2009; Toomey et al. 2006) exerting an atheroprotective effect. In the work of Mitchell et al. (2012) atherosclerosis (measured directly by lesion area) was significantly reduced with trans-10, cis-12 CLA alone and mixed isomer CLA supplementation in apoE mice. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that trans-10, cis-12 CLA rapidly changes plasma and  liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis. The results of analysis studies concerning dietary supplementation with CLA on body composition are conflicting. In some studies the reduction of body fat mass in humans (Chen et al. 2012; Gaullier et al. 2007; Watras et al. 2007) and in animals (Kloss et al. 2005) has been shown, whereas in others no effects on body composition (Onakpoya et  al. 2012; Brown et  al. 2011) has been noted. Moreover, it is known that the effects of CLA on adipogenesis and lipid metabolism are isomer-specific. Specifically, the trans-10, cis-12 CLA isomer seems to be responsible for CLA’s antiobesity effects. The trans-10, cis-12

620

Bioactive lipid components from ruminant milk and meat

CLA affects preadipocytes from both animals and humans. This CLA isomer reduces triglyceride (TG) content in differentiating 3t3-L1 preadipocytes and this effect is reversible and dependent on the time of treatment (Evans et al. 2001). Study of the cis-9, trans-11 CLA action on 3t3-L1 preadipocytes showed anti-inflammatory properties of this isomer via inhibition of tumor necrosis factor α (TNF α) –induced down-regulation of insulin receptor substrate 1 (IRS-1) and GLUT4 mRNA expression (Moloney et al. 2007). The trans-10, cis-12 CLA isomer reduces stearoyl-CoA desaturase-1 (SCD-1) gene expression, and SCD-1 is responsible for desaturation of saturated acids into monosaturated fatty acids (MUFA) (Choi et al. 2000). The trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, decreases simultaneously de novo fatty acid synthesis and TG estrification in cultures of human preadipocytes. CLA decrease adipocytes differentiation by decreasing nuclear receptor PPARγ (peroxisome proliferator-activated receptor-type gamma) expression or activity (Evans et al. 2002). Study on molecular mechanisms involved in metabolic effects exerted by CLA has proved that isomers of CLA (cis-9, trans-11 CLA vs. trans-10, cis-12 CLA) have opposite effects on lipid metabolism and insulin resistance. In ob/ ob C57BL/J6 mice the cis-9, trans-11 CLA isomer improved lipid metabolism and this effect was associated with down-regulation of hepatic sterol regulatory element-binding protein (SREBP)-1c, whereas feeding with trans-10, cis12 CLA isomer inhibited weight gain and reduced adipose tissue mass but induced the prodiabetic state resulting from hyperlipemia (Roche et al. 2002). These findings concurs with the Ryder et al. (2001) study, which showed that feeding a blend of the two CLA isomers (mentioned above) improved lipid metabolism in ZDF rats, but the cis-9, trans-11 CLA isomer improved TG metabolism also when given alone. Data obtained from clinical studies demonstrated that CLA supplementation may improve the plasma triacylglicerol (TG) level in healthy humans (Noone et  al. 2002), CLA may also induce weight loss and decrease serum leptin levels in subjects with type 2 diabetes (Belury et al. 2003).

25.5 Conclusion The evidence from animal studies is promising but extrapolation from animal to human studies is difficult due to the differences in the amount of CLA. Moreover, in most of the studies the mixture of synthetic CLA isomers was used and more research is needed to determine the isomer-specific mechanisms of action and efficacy of naturally CLA-enriched products as antiobesity, antidiabetic, and atheroprotective agents. It has to be noted that the long-term effects of CLA are completely unknown; furthermore, the optimal dose, type of isomer, and timing of therapy in humans remain unclear.

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WHO. 2012 Fact Sheet No 312 Diabetes. http//www.who.int/mediacenre/factsheets/fs312/en/. Wilde, P.F. and R.M.C. Dawson. 1966. The biohydrogenation of alpha-linolenic and oleic acid by rumen micro-organisms. Biochem. J. 98:469–475. Williams, A.G. and G.S. Coleman. 1992. The Rumen Protozoa. Springer-Verlag, New York, NY, USA, pp. 441. Wina, E., S. Muetzel and K. Becker. 2006. The dynamics of major fibrolytic microbes and enzyme activity in the rumen in response to short- and ling-term feeding of Sapindus rarak saponins. J. Appl. Microbiol. 100:114–122. Wood, R.D., M.C. Bell, R.B. Grainger and R.A. Teekel. 1963. Metabolism of labelled linoleic-l-14C acid in the sheep rumen. J. Nutr. 79:62–68. Wright, D.E. 1959. Hydrogenation of lipids by rumen protozoa. Nature 184:875–876. Wright, D.E. 1960. Hydrogenation of chloroplast lipids by rumen bacteria. Nature 185:546–547. Yanez-Ruiz, D.R., N.D. Scollan, R.J. Merry and C.J. Newbold. 2006. Contribution of rumen protozoa to duodenal flow of nitrogen, conjugated linoleic acid and vaccenic acid in steers fed silages differing in their water-soluble carbohydrate content. Br. J. Nutr. 96:861–869. Yayota, M., M. Tsukamoto, Y. Yamada and S. Ohtani. 2013. Milk composition and flavor under different feeding systems: A survey of dairy farms. J. Dairy Sci. 96:5174–5183. Yoder, P. S., N.R. St-Pierre, K.M. Daniels, K.M. O’Diam and W.P. Weiss. 2013. Effects of short-term variation in forage quality and forage to concentrate ratio on lactating dairy cows. J. Dairy Sci. 96:6596–6609. Zervas, G. and E. Tsiplakou. 2011. The effect of feeding systems on the characteristics of products from small ruminants. Small Ruminant. Res.101:140–149. Zhang, P, X. Zhang, J. Brown, D. Vistisen, R. Sicree, J. Shaw and G. Nichols. 2010. Global healthcare expenditure on diabetes for 2010 and 2030. Diabetes Res. Clin. Pract. 87:293–301.

CHAPTER 26

The milk fat globule membrane: A potential source of health-promoting glycans Sarah A. Ross1,2, Jonathan A. Lane1, Michelle Kilcoyne2, Lokesh Joshi2, and Rita M. Hickey1 1

Teagasc Food Research Centre, Cork, Ireland Glycoscience Group, Biosciences Research Building, National University of Ireland, Galway, Ireland

2

26.1 Introduction The mammalian milk glycome has recently gained increased interest due to health-promoting properties that have been associated with both free and conjugated glycans. Research in this area has mainly focused on the structural and functional characterization of human milk oligosaccharides (HMO) and glycoproteins. These molecules are highly abundant in human milk and therefore can be obtained in significant quantities for both structural and biological characterization. For example, mature human milk contains 5–15 g/L of free oligosaccharides while human colostrum contains 50 g/L (Kunz et al. 1999; Bode 2006; Kunz and Rudloff 2008). Initially, the majority of researchers focused on characterizing the prebiotic activities of HMO (György et al. 1954; Ward et al. 2006; LoCascio et al. 2007). However, the fact that 90% of all HMO were found intact in infant feces suggested that these compounds had additional effects once consumed (Bode 2009). Consequently, researchers discovered that HMO had other health-promoting properties such as the ability to promote development of the immature immune system, regulate immune responses, contribute toward brain development, and protect against infection (Wang et al. 2003; Newburg et al. 2005; Bode 2006, 2012; Kunz and Rudloff 2006; Kavanaugh et al. 2013; Lane et al. 2013). Indeed, HMO have protective effects against both viral and bacterial infection as they resemble the complementary receptors present on human cells and can act as decoy receptors, thus preventing adhesion and subsequent colonization and infection (Sharon and Ofek 2000). For example, Coppa et al. (2006) demonstrated that HMO, including 6′-sialyllactose and

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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3′-fucosyllactose, can inhibit the adhesion of Salmonella fyris, Vibrio cholerae, and enteropathogenic Escherichia coli serotype O119 to the human colonic adenocarcinoma intestinal epithelial cell line, Caco-2. In addition, the authors reported inhibition of E. coli and V. cholerae adhesion to Caco-2 cells by the neutral high-molecular-weight glycoconjugate fraction. The ability of HMO to reduce bacterial colonization has also been demonstrated in in vivo trials. Mysore et al. (1999) demonstrated that Helicobacter pylori colonization in rhesus monkeys treated with the milk oligosaccharide 3′-sialyllactose was dramatically reduced. Furthermore, there is clear evidence that breast-fed infants acquire less diseases or less severe infections, such as diarrhoeal diseases, when compared to formula-fed infants (Duffy et al. 1986; Newburg et al. 1998; Morrow et al. 2004), a property that has been associated with the protective nature of HMO. The immune-regulatory activities of HMO have been widely reported. For example, roles for HMO in lymphocyte maturation and cytokine production (Velupillai and Harn 1994; Eiwegger et al. 2004) have been demonstrated. Interestingly, the levels of sialic acid, a negatively charged sugar that caps the terminus of many HMO structures, on brain-related glycoproteins and gangliosides have been linked to improved cognitive function and learning ability. The beneficial role of HMO in brain development has been demonstrated by Wang et al. (2003), who reported increased concentrations of sialylated gangliosides in the brains of breast-fed infants compared to formula-fed infants. Human milk glycoconjugates have also demonstrated health-promoting properties similar to those associated with free milk oligosaccharides. For example, human milk gangliosides have been shown to bind and neutralize bacterial toxins including H. pylori vacuolating toxin (Wada et al. 2010) and cholera toxin (Iwamori et al. 2008). Additionally, the human milk gangliosides GM1 and GM3 have demonstrated an ability to prevent binding of enterotoxigenic E. coli to Caco-2 cells in vitro (Idota and Kawakami 1995) and human milk fat globule membrane (MFGM) mucin has prevented rotavirus-associated gastroenteritis in a mouse model (Yolken et al. 1992). Schroten et al. (1992) also reported that S-fimbriated E. coli adherence to buccal epithelial cells can be inhibited by human MFGM. This activity was attributed to the presence of heavily glycosylated mucins. Furthermore, human milk mucins have been shown to inhibit Salmonella enterica serovar Typhimurium invasion of human Caco-2 and FHs 74 Int (human small intestine) cells (Liu et al. 2012) while Newburg et al. (1998) demonstrated the human milk glycoprotein lactadherin (LDH) provides protection to infants from rotavirus infection. These studies highlight the important role HMO and glycoconjugates play in the early stages of life. Although human milk is clearly a source of biologically active oligosaccharides and glycoconjugates, the difficulty is that it is not readily available in large enough quantities to be a commercially viable source. Consequently, research has begun to focus on domestic animal milks as an alternative source of functional oligosaccharides and glycoconjugates (O’Riordan et al. 2014). The use of

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mature domestic animal milks as a source of free milk oligosaccharides has proven challenging as only trace quantities of oligosaccharides are present in these milks. For example, the oligosaccharide content of bovine, ovine, and caprine milk is 0.03–0.06 g/L, 0.02–0.04 g/L, and 0.25–0.30 g/L, respectively (Urashima et al. 2001; Lane et al. 2010). This has led researchers to focus on fractions from dairy sources, which are rich in glycoconjugates, as an alternative to free oligosaccharides as they may demonstrate similar biological activity. For example, buttermilk is a sustainable source of MFGM-associated glycosylated ingredients. As a by-product of butter production, buttermilk is low in cost and is available in large quantities. In the United States, 610 million kg of butter is produced annually, resulting in the production of 35.4 million kg of buttermilk, which is condensed or evaporated (Jiménez-Flores and Brisson 2008). These figures highlight the potential of buttermilk, and in particular buttermilk derived from bovine milk, as a sustainable source of functional food ingredients. Furthermore, bovine MFGMassociated ingredients have demonstrated health-promoting activities similar to those of HMO (e.g., prevention of pathogen colonization in in vitro and in vivo) (Sanchez-Juanes et al. 2009; Sprong et al. 2012). Considering this, our review focuses on the composition and functional properties of mammalian MFGM, with particular attention paid to human and bovine MFGM, and the important roles glycosylated MFGM components could play as nutraceuticals such as providing protection against infection by enteric pathogens for immune-compromised individuals including newborns and older adults.

26.2 MFGM composition The MFGM is composed of lipids and proteins that surround and stabilize milk fat droplets (Cebo et al. 2010). These fat droplets form within the endoplasmic reticulum (ER) of the mammary gland epithelial cells (Zaczek and Keenan 1990) and move toward the apical membrane. The droplets then bud into the lumen of the alveolar epithelial cells and as they do so, become surrounded by the apical membrane, which forms the main part of the MFGM (Mather and Keenan 1998). The average size of the milk fat globule (MFG) differs between species. For example, bovine MFGs have been found to be slightly larger than those of goat milk (4.2 and 3.7 μm, respectively) (Guri et al. 2012). In the absence of MFGM, the lipid droplets in milk would exist as aggregates rather than in a dispersed form (Spitsberg 2005). For instance, if the MFGM is disrupted by physical agitation such as churning, butter is formed due to lipid aggregation while the aqueous phase of the MFGM forms the buttermilk as a by-product (Spitsberg 2005; Harrison 2006). MFGM consists of three parts. The first is an inner monolayer derived from the ER, which surrounds the lipid droplet. This monolayer is surrounded by a proteinaceous coat that is surrounded by an outer bilayer (Keenan and Mather

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The milk fat globule membrane: A potential source of health-promoting

2002; Evers et al. 2008). Thus, the MFGM is composed of a trilayer structure (Abrahamse et al. 2012), with the outer bilayer originating from the apical membrane (Mather and Keenan 1998; Reinhardt and Lippolis 2006). When the fat globule is extruded into the alveolar epithelial cell lumen, a 10–20 nm space exists between the inner monolayer and the nascent outer bilayer (Wooding 1971; Heid and Keenan 2005). It is within this space that the proteinaceous coat is formed and that the cytoplasmic organelles can be found (Wooding 1971; Heid and Keenan 2005). Interestingly, studies have shown that the proteinaceous coat of the MFGM of human and bovine milk have similar carbohydrate and amino acid compositions (Freudenstein et al. 1979). Cytoplasmic crescents often form in the outer bilayer due to the trapping of cytoplasm between the two layers. This occurs in the milk of many mammals (Huston and Patton 1990; Dewettinck et al. 2008). The size of the crescents can vary and studies by Evers et al. (2008) have shown that these crescents can reach a volume exceeding that of the fat globule itself. Both the inner and outer layers of the MFGM are  composed of polar lipids and proteins. The bilayer contains peripheral and transmembrane proteins as well as lipid components such as cholesterol. In addition, many of the lipids and proteins are heavily glycosylated (Dewettinck et al. 2008). MFGM is heterogenous, with some areas of the membrane containing proteins that are evenly distributed, while other areas lack proteins (Robenek et al. 2006). In addition, distinct domains of the MFGM are characterized by different shapes such as ridges and elongated or rounded bumps (Robenek et al. 2006). The external surface of human and horse MFGM is rough due to the presence of mucin 1 (MUC1)-containing filaments whereas the absence of these filaments on the surface of the MFG of mammals such as sheep, cows, and goats result in a smooth surface (El-Loly 2011). These filaments extend outwards from the human MFGM to a distance of 0.5 μm, with some extending as far as 1 μm, and have been shown to bind to E. coli, neutralizing the threat of bacterial disease (Buchheim et al. 1988). In addition, portions of the outer bilayer appear to be absent in some MFG (Evers et al. 2008), as visualized where neither lipophilic dyes nor the lectin wheat germ agglutinin (WGA) fluorescent probes, which bind to the carbohydrate residues sialic acid, were incorporated in some areas of human, bovine, and ovine MFGs. Fong et al. (2007) recently reported on the concentrations of MFGM in bovine milk and found 3.6 ± 0.3 g/L of MFGM in cream, with the protein and lipid fractions making up an estimated 22.3 ± 1.5% and 71.8 ± 1.7%, respectively. The polar lipids found in MFGM are glycerophospholipids—such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol—and sphingolipids—such as sphingomyelin (SM). There are also small quantities of glycosphingolipids in the form of lactosylceramide (LacCer), glucosylceramide, and gangliosides (Christie et al. 1987; Sprong et al. 2002). MFGM lipids are arranged asymmetrically with SM, phosphatidylcholine (which contains the

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635

polar choline) and the glycolipids present on the outside of the globule membrane, while phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are found inside the membrane (Dewettinck et al. 2008). Milk glycolipids are almost exclusively located in the outer layer of the MFGM (Georgi et al. 2013). MFGM proteins contribute 1–2% of the total protein content in milk (Riccio, 2004). These proteins, like the lipids, are arranged asymmetrically in the membrane. Such proteins include adipophilin (ADPH), which can be found in the inner monolayer as it has a high affinity for triglycerides, and xanthine oxidoreductase (XOR), which is found between the inner monolayer and outer bilayer. Butyrophilin (BTN) is a transmembrane protein found in the outer bilayer with its cytoplasmic tail in the proteinaceous coat (Heid and Keenan 2005; Dewettinck et al. 2008). It is thought that these three proteins (ADPH, XOR, and BTN) interact to form a complex that connects the inner and outer membrane and aids in the binding of the bilayer to the MFG during the extrusion of the globule from the alveolar epithelial cell (Mather and Keenan 1998; Mather 2000; Cavaletto et al. 2004). An overview of the MFGM structure is given in Figure 26.1. Many different functions are associated with MFGM proteins including protein synthesis/folding, transport, and metabolism. Other functions include cell signalling and membrane/protein trafficking and have been attributed to almost half of the bovine MFGM proteins (Reinhardt and Lippolis 2006). Differences exist in the type and abundance of proteins in the MFGM of different species (Mather 2000; Cavaletto et al. 2004; Fong et al. 2007; Zamora et al. 2009; Cebo et al. 2010). Although the proteins of human and bovine MFGM differ, six of the minor bovine MFGM proteins are homologous to the human MFGM proteins (Fong et al. 2007).

PP3 XOR BTN ADPH Lipid core

LDH

Gly colipids (neutral, acidic)

CD36 MUC1 CLUS

CD59

Figure 26.1 Structure of the MFGM. The sizes of the membrane components are not proportional. The MFGM consists of an inner phospholipid monolayer and outer bilayer derived from the endoplasmic reticulum and apical membrane, respectively. A proteinaceous coat exists between both layers. Glycoproteins present in the MFGM include XOR (xanthine oxidoreductase), CLUS (clusterin), BTN (butyrophilin), CD36 (cluster of differentiation 36), MUC1 (mucin 1), CD59, LDH (lactadherin), and PP3. Adipophilin (ADPH) is not a glycoprotein; however, it is involved in MFGM formation with XOR and BTN. Neutral and acidic glycolipids are also present in the MFGM.

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It is evident that bovine MFGM is a rich source of proteins and lipids, many of which are glycosylated. Indeed, MFGM contains lipids and proteins in a 1:1 ratio (Spitsberg 2005) and their diverse glycosylation could indicate that these milk components may possess many health-promoting properties. Overall, bovine MFGM glycoproteins differ from their human counterparts in their glycosylation. For instance, bovine MFGM contains primarily core-1 O-linked oligosaccharides while those of human MFGM are predominantly core-2-type structures. Furthermore, human MFGM N-linked oligosaccharides contain terminal fucose (Fuc) residues, which are not present in that of bovine MFGM (Wilson et al. 2008). In addition, bovine MFGM glycoproteins contain a high degree of N-linked oligosaccharides terminating in N-acetylgalactosamine (GalNAc), in the form of N, N’-diacetyllactosamine (LacdiNAc, GalNAc-β-(1 → 4)-GlcNAc). For example, the oligosaccharide chains of bovine BTN and cluster of differentiation 36 (CD36) contain 37% and 28% LacdiNAc, respectively (Sato et al. 1993). Similarities also exist between bovine and human glycoprotein oligosaccharides. For example, both contain bi-, tri-, and tetra-antennary complex N-linked structures that are sialylated (Wilson et al. 2008). In the next section, we discuss the structure and function of MFGM glycolipids and glycoproteins and highlight the potential of these bioactives for inclusion in functional foods.

26.3 Glycoconjugates of the MFGM 26.3.1 Glycolipids Approximately 70% of the total glycolipid content of bovine milk is found to be associated with the MFGM (Newburg and Chaturvedi 1992). These glycolipids can be divided into two groups, neutral and acidic (or gangliosides) (Jensen 2002), and are summarized in Table 26.1. The neutral glycolipids of bovine MFGM are composed mainly of LacCer (65%) and its precursor glucosylceramide (35%), both of which contain nonhydroxylated fatty acids. The neutral glycolipids of human MFGM are mainly galactosylceramide, glucosylceramide, and LacCer and 20% of the fatty acid residues of these glycolipids are hydroxylated (Newburg and Chaturvedi 1992). Additionally, ovine and human MFGM contain globotriosylceramide and globotetraosylceramide but bovine MFGM does not (Zancada et al. 2010). The neutral glycolipids consist of one or more carbohydrate residues linked to the lipid moiety ceramide (a sphingosine linked to a fatty acid through an amide linkage). For example, galactosylceramide consists of a galactose (Gal) residue connected to a ceramide via a glycosidic linkage while addition of a β-linked Gal to a hydroxyl group of the glucose (Glc) residue forms LacCer (Schnaar et al. 2009). Acidic glycolipids (gangliosides) are composed of a ceramide attached via a glycosidic linkage to an oligosaccharide with at least one sialic acid and various other residues attached (Khatun et al. 2013). Approximately 90% of gangliosides in milk are associated with the MFGM (Colarow et al. 2003). The gangliosides of mammalian MFGM consist of

Group

Neutral glycolipid

Neutral glycolipid

Neutral glycolipid

Ganglioside

Ganglioside

Ganglioside

Ganglioside

Ganglioside

Ganglioside

Ganglioside

Ganglioside

Glycolipid

Lactosylceramide (LacCer)

Glucosylceramide

Galactosylceramide

Monosialoganglioside 1 (GM1)

Monosialoganglioside 2 (GM2)

Monosialoganglioside 3 (GM3)

Disialoganglioside 1A (GD1A)

Disialoganglioside 1B (GD1B)

Disialoganglioside 2 (GD2)

Disialoganglioside 3 (GD3)

Trisialoganglioside (GT)

3 sialic acid residues

2 sialic acid residues

2 sialic acid residues

2 sialic acid residues

2 sialic acid residues

1 sialic acid residue

1 sialic acid residue

1 sialic acid residue

Gal residue connected to ceramide

Glc residue connected to ceramide

Glc and Gal residues connected to ceramide

Glycosylation

Unknown

Possible protection of neonatal gut E. coli binding Antiadhesive in vitro (bacterial and viral)

Unknown

Unknown

Unknown

Antiadhesive in vitro (bacterial and viral)

Antiadhesive in vitro (viral)

Antiadhesive in vitro (bacterial) Bacterial toxin binding

E. coli binding

E. coli binding

E. coli

Bioactivity

Table 26.1 Overview of the mammalian MFGM glycolipids and their properties.

Unknown

3’-sialyllactose contributes to antiadhesion (viral)

Unknown

Unknown

Unknown

3’-sialyllactose contributes to antiadhesion (viral)

Unknown

Unknown

Unknown

Unknown

Unknown

Function of glycan

Jensen 2002 Sanchez-Juanes et al. 2009 Idota and Kawakami 1995 Salcedo et al. 2013 Iskarpatyoti et al. 2012 Jensen 2002

Jensen 2002

Jensen 2002

Jensen 2002

Jensen 2002 Idota and Kawakami 1995 Salcedo et al. 2013 Iskarpatyoti et al. 2012

Jensen 2002 Portelli et al. 1998

Jensen 2002 Idota and Kawakami 1995 Salcedo et al. 2013 Otnaess et al. 1983

Schnaar et al. 2009 Zancada et al. 2010

Schnaar et al. 2009 Zancada et al. 2010

Schnaar et al. 2009 Sanchez-Juanes et al. 2009 Zancada et al. 2010

References

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The milk fat globule membrane: A potential source of health-promoting

monosialoganglioside (GM) 1, GM2, GM3, disialoganglioside (GD) 1A, GD1B, GD2, GD3, and trisialoganglioside GT (Jensen 2002). These structures are mainly composed of long-chain fatty acids but have also been found to contain medium, monounsaturated, and polyunsaturated fatty acids. Most research performed to date has concentrated on human and bovine glycolipids but some analyses have been carried out on other mammalian milk glycolipids. For instance, Zancada et al. (2010) determined the neutral glycosphingolipid content of ovine whole milk. The profile was found to be similar to that of human milk neutral glycosphingolipids. In addition, Iwamori et al. (2008) demonstrated that gangliosides belonging to the biosynthetic pathway of the ganglio series of gangliosides for neural tissues, including GD2 and GD1b, were absent in human and bovine milk but present in goat milk. Interestingly, the overall concentration of glycolipids in human milk decreases during the transition from colostrum to mature milk, with GD3 being the major ganglioside found in colostrum (Uchiyama et al. 2011; Georgi et al. 2013), which could suggest a role in protecting the newborn from infection. Additionally, ganglioside concentrations were found to change in the murine mammary gland over the course of lactation with the concentration of acidic glycosphingolipids reducing over this time. Furthermore, it was found that GD1a was the predominant ganglioside found in the MFGM of colostrum (Momoeda et al. 1995). LacCer and GD3 from bovine whole milk and MFGM was shown to bind to four strains of enterotoxigenic E. coli (Sanchez-Juanes et al. 2009). These binding activities may play a role in prevention of bacterial adherence to human gastrointestinal cells. The glycoconjugates act as soluble receptors and prevent bacterial binding to host cell receptors. Interestingly, the glycolipids isolated from whole milk demonstrated a greater binding capacity than those isolated from MFGM, which was most likely due to conformational differences as a result of the difference in fatty acid composition (Sanchez-Juanes et al. 2009). Human milk GM1, GM3, and GD3 inhibit adhesion of pathogenic bacteria such as enterotoxigenic E. coli, Campylobacter jejuni, and Listeria monocytogenes to human intestinal Caco-2 cells (Idota and Kawakami 1995; Salcedo et al. 2013). Ovine milk neutral glycosphingolipids have also demonstrated binding ability to certain EPEC and UPEC strains (Zancada et al. 2010). Milk glycolipids also confer protection to the host by binding to bacterial toxins and preventing outcomes such as cell membrane disruption, inhibition of protein synthesis, fever, and diarrhea. Human milk GM1 had inhibitory activity against cholera toxin in vivo in a rabbit intestine model and against E. coli heatlabile enterotoxin in vitro (Otnaess et al. 1983). In addition, the bovine brain gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT have been shown to neutralize the vacuolating cytotoxin (VacA) of H. pylori. Internalization of the toxin by human gastric cells was inhibited in the presence of GM1. Interestingly, lyso-gangliosides (lyso-GM1, lyso-GM2, and lyso-GM3), which lack fatty acids, bound VacA while 3’-sialyllactose, a carbohydrate moiety of GM3, alone did not

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inhibit the activity of VacA. These results suggest that the carbohydrate portion of the gangliosides alone cannot contribute to VacA inhibition but is an important overall factor in neutralization of the toxin (Wada et al. 2010). Although the gangliosides mentioned in the latter study were brain-derived, the same effect may be observed while using the carbohydrate moiety of milk-derived gangliosides. Further studies are required to determine the value of milk-derived glycolipids. Protection of the host from viral infection has also been linked to milk glycolipids. For example, human milk GM3 and GD3 inhibit adhesion of reovirus to L cells (murine areolar and adipose cells) and HeLa cells (human cervical epithelial cells) (Iskarpatyoti et al. 2012). In this study, GM3 and GD3 were preincubated with reovirus before inoculation of the cell line with the virus-ganglioside mixture and infectivity was subsequently determined by immunofluorescence. In addition, 3’-sialyllactose inhibited infection of reovirus with L cells and HeLa cells, when preincubated with reovirus. However, the antiadhesive capability was lower than that of GM3 and GD3, indicating the carbohydrates play a role in viral inhibition but that the molecular conformation of membrane-bound ganglioside may be an important factor in the antiviral activity of the carbohydrate (Iskarpatyoti et al. 2012). Furthermore, human milk GM2 binds respiratory syncytial virus and inhibits adhesion of the virus to Hep-2 (human larynx) cells (Portelli et al. 1998). These studies highlight the potential use of MFGM glycolipids, however, it is obvious that further studies are required to determine the antiviral activity associated with these compounds. Indeed, the limited knowledge on the in vivo bioactive potential of glycolipids associated with MFGM warrants further investigation to realize their commercial potential.

26.3.2 Glycoproteins More information exists regarding the glycoprotein fraction of MFGM compared to the glycolipid fraction. Some of the most researched glycosylated proteins of the MFGM are the mucins, BTN, CD36, and LDH (PAS6/7). The “PAS” name given to the MFGM glycoproteins relates to their positive reaction with periodic acid-schiff (PAS) stain as a result of their glycosylation. An overview of the major MFGM glycoproteins is presented in Table 26.2, while their structural properties and beneficial effects are discussed individually below.

26.3.2.1 Mucins Mucins of the MFGM are type 1 integral-membrane proteins (Jonckheere et al. 2012). They contain a variable number of tandem repeat (VNTR) domains that are rich in the amino acids serine, threonine, and proline. The hydroxyl groups of serine and threonine function as glycosylation sites while the proline confers rigidity to the structure of the protein and also extends its structure from the cell surface (Patton et al. 1995; Moran et al. 2011). The extent of glycosylation of each protein differs and depends on the number of repeats in the VNTR. Thus, mucins are polymorphic proteins in species such as human and cow (Huott et al.

Glycosylation

Predominantly O-linked glycosylation, N-linked also present Bovine MUC1: 5 potential N-linked glycosylation sites (Asn161, Asn201, Asn241, Asn384, Asn460) Bovine and human MUC1 residues sialic acid, Man, Gal, Fuc, GalNAc, GlcNAc

Bovine: No O-linked glycosylation sites, 2 N-linked glycosylation sites at Asn 55 and Asn 215 Bovine N-linked oligosaccharides, LacdiNAc at Asn55, Fuc

Bovine PAS6: 1 O-linked (Ser9) and 2 N-linked glycosylation sites (Asn41 and Asn209) Bovine PAS6 residues: Man, Fuc, Gal, GalNAc, GlcNAc, Sialic acid Bovine PAS7: 1 O-linked (Thr16) and 1 N-linked (Asn41) glycosylation site Bovine PAS7 residues: Man, Fuc, Gal, GalNAc, GlcNAc, Neu5Ac Human LDH: 5 N-linked glycosylation sites

Glycoprotein

Mucin 1

BTN1A1 (PAS5)

Lactadherin (PAS6, PAS7, Milk fat globule EGF-8)

Cell adhesion (human) Anti-infective (viral and bacterial) (human)

Milk secretion Multiple sclerosis regulation

Anti-infective (bacterial and viral)

Bioactivity

Table 26.2 Overview of the mammalian MFGM glycoproteins and their properties.

Human milk LDH sialic acid plays a role in viral anti-infectivity Resistance to digestion

Unknown

Fuc inhibits binding of Norwalk virus to intestinal cells in vitro Sialic acid, Fuc, Man prevent E. coli binding to Caco-2 cells in vitro Lewis x blocks HIV attachment Resistance to digestion

Importance of glycans

Hvarregaard et al. 1996 Seok et al. 2001 Picariello et al. 2008 Taylor et al. 1997 Newberg et al. 1998 Quaranta et al. 2001 El-Loly 2011 Yolken et al. 1992 Peterson et al. 1998

Sato et al. 1995 Mather 2000 O’Riordan et al. 2014 Ogg et al. 2004 Robenek et al. 2006 Mana et al. 2004 Stefferl et al. 2000

Pallesen et al. 2001 Wilson et al. 2008 Liu et al. 2005 Mather, 2000 Shimizu and Yamauchi 1982 Schroten et al. 1992 Kvistgaard et al. 2004 Liu et al. 2012 Parker et al. 2010 Ruvoen-Clouet et al. 2006 Saeland et al. 2009 Peterson et al. 1998

References

Antimicrobial through nitric oxide and hydrogen peroxide production

Bovine residues: Man, Gal, Fuc, GalNAc, GlcNAc, Sialic acid, LacNAc, LacdiNAc Bovine LP28: 2 O-linked (Thr16 and Thr86) and 1 N-linked (Asn77) glycosylation site Bovine LP18: 1 O-linked (Thr86) and 1 N-linked (Asn77) glycosylation site Caprine LP: 1 O-linked and 1 N-linked glycosylation site

Caprine: O-linked glycosylation at Thr207, Thr1069, and Thr1071 O-linked glycosylation containing sialic acid Human: N-linked glycosylation

Unknown

Human residues: Fuc, Man, Gal, GlcNAc. High abundance of biantennary fucosylated glycans

Proteose peptone component 3 (Lactophorin)

Xanthine oxidoreductase

Carbonic anhydrase VI

Clusterin, Apolipoprotein J

Unknown

Unknown

Charlwood et al. 2002

Karhumaa et al. 2001 Kitade et al. 2003

Unknown

Possible protective role in neonatal gut

Inagaki, Nakaya et al. 2010b Girardet et al. 1995 Coddeville et al. 1998 Lister et al. 1998 Girardet et al. 1993 Innocente et al. 1998 Inagaki, et al. 2010a Campagna et al. 2004 Shida et al. 1994

Berglund et al. 1996 Greenwalt et al. 1992

References

Cebo et al. 2010 Picariello et al. 2008 Stevens et al. 2000 Hancock et al. 2002 Martin et al. 2004

Heat stability of LP16 MARK3 hybridoma mitosis E. coli heat-labile enterotoxin binding

Unknown Resistance to digestion

Importance of glycans

Unknown

Antilipase activity Emulsifying agent Rotaviral replication inhibitor Antibacterial

Unknown Unknown

Unknown Bovine: 8 N-linked glycosylation sites Bovine residues: Man, Gal, Fuc, GalNAc and GlcNAc

CD59 CD36 (GP88, GPIIIb, PASIV, FAT, glycoprotein IV)

Bioactivity

Glycosylation

Glycoprotein

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1995). Other features that are characteristic of the membrane-spanning mucins are a cytoplasmic C-terminus and one membrane-spanning domain (Pallesen et al. 2007). On average human milk contains 729 ± 75 μg/mL of mucin glycoprotein (Peterson et al. 1998). The levels of mucins in milk samples are dynamic and change depending on the stage of lactation. For example, MUC1 and mucin 15 (MUC15) concentration increased 7.7- and 7.4-fold, respectively, in bovine milk samples taken 7 days post-parturition compared to colostrum (Reinhardt and Lippolis 2008). Degradation of the protein is avoided due to the high degree of glycosylation of the proteins (Peterson et al. 1998). The three main mucins that have been characterized in bovine MFGM are MUC1, MUC15, and mucin X (MUCX) (Pallesen et al. 2001, 2007; Liu et al. 2005). There are similarities between the mucins of different species (e.g., bovine and human MUC1 share 76–93% amino acid similarity at the cytoplasmic and transmembrane domains, respectively). Interestingly, the least conserved identity occurs at the VNTR domains (Sando et al. 2009). Differences also exist, for instance, fucosylated oligosaccharides are found on human mucins but fucosylation is absent in bovine mucins (Wilson et al. 2008). Mucins are highly O-glycosylated proteins, as shown by high levels of binding of the lectin Jacalin, which prefers binding to Gal residues, to mucins such as MUC1 and MUCX (Liu et al. 2005), and contain N-linked oligosaccharides to a lesser extent. The N-linked glycosylation of MUC1 has been demonstrated by successful cleavage of N-linked oligosaccharides from MUC1 using the enzyme PNGase F (Pallesen et al. 2001). The five major bovine MUC1 isoforms vary in molecular mass, ranging from 156 to 193 kDa (Huott et al. 1995; Pallesen et al. 2001), and account for 1.21% (w/w) of the total bovine MFGM proteins (Kvistgaard et al. 2004). MUC1 is extensively glycosylated, with bovine and human MUC1 glycans contributing ≥50% of the total molecular mass of the protein (Huott et al. 1995; Patton et al. 1995; Pallesen et al. 2001). It is mainly O-glycosylated but five potential N-glycosylation sites have been identified for bovine MUC1 (Pallesen et al. 2001). One of the most common carbohydrate residues associated with the MUC1 glycoprotein is sialic acid, followed by Gal. Sialic acid accounts for approximately 30% (molar %) of the total carbohydrate content of the bovine glycoprotein (Huott et al. 1995; Patton et al. 1995; Pallesen et al. 2001) and 11% (molar %) of the total carbohydrate content of the human milk glycoprotein (Shimizu and Yamauchi, 1982; Patton et al. 1995). Other carbohydrate residues associated with bovine and human MUC1 include Gal, mannose (Man), N-acetylglucosamine (GlcNAc) and GalNAc, while fucosylation of N-linked oligosaccharides has been detected at low levels (Shimizu and Yamauchi 1982; Pallesen et al. 2001; Liu et al. 2005; Sando et al. 2009). In contrast to MUC1, MUCX is more loosely associated with the MFGM, leading to its presence in milk fractions such as skim milk. It contains fewer N-linked oligosaccharides compared to MUC1 (Liu et al. 2005). The use of Morrisey’s silver staining method (which stains glycoproteins more rapidly when

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compared to nonglycosylated proteins) demonstrated that bovine MUCX stained more rapidly than bovine MUC1, indicating that MUCX may have a higher carbohydrate content compared to MUC1 (Liu et al. 2005). A mucin termed mucin 4 has also been purified from human MFGM (Liu et al. 2012), but it is likely that this mucin is MUCX (Zhang et al. 2005). MUC15 has been identified in mammalian milks including bovine, human, ovine, and caprine milk (Pallesen et al. 2008), and it makes up 1.5% (w/w) of the total bovine MFGM protein content. It too is highly O-glycosylated, and N-glycosylated to a lesser extent (Pallesen et al. 2007). Fifteen potential N-linked glycosylation sites have been identified for this glycoprotein (Pallesen et al. 2002). It has a molecular weight of 94 kDa in bovine MFGM, 65% of which is attributed to the carbohydrate content of the glycoprotein (Pallesen et al. 2007), and approximately 150 kDa in human MFGM, which implies the presence of even greater quantities of carbohydrate. Human and bovine MUC15 display 59% and 87% similarity in the extracellular and cytoplasmic/transmembrane domains, respectively (Pallesen et al. 2008). Fuc, GalNAc, GlcNAc, Gal, Man, and sialic acid are present in the ratio 1:4:6:5:4:5. The O-linked oligosaccharides of bovine MFGM are largely mucin core-1-type structures, with the extended O-linked structures containing high concentrations of GlcNAc, which implies long structures (Pallesen et al. 2007). Glycosylation patterns of mucins are subject to change depending on several factors including location in the host and infection or disease status. For example, prolonged infection with H. pylori is associated with increased sialic acid and decreased Fuc levels of mucin. The glycosylation returns to normal after elimination of the bacteria from the system (Ota et al. 1998). The protection mucins confer through inhibition of pathogenic infection has been well documented (Schroten et al. 1992; Yolken et al. 1992). MUC1 binds fimbriated E. coli, preventing the bacteria from binding to buccal epithelial cells (Schroten et al. 1992). Bovine MUC1 also displays antiviral properties and has been shown to decrease neuraminidase-sensitive rotavirus infection in MA104 cells (embryonic monkey kidney cells) (Kvistgaard et al. 2004). The human mucins, MUC1 and MUC4, have antiadhesive and anti-invasive activity against S. enterica serovar Typhimurium (Liu et al. 2012). The oligosaccharides are known to contribute to the antiadherent activities associated with the mucins. Binding of Norwalk virus to gut epithelial cells was shown to be inhibited by α(1 → 2)-linked Fuc of MUC1 from human milk (Ruvoen-Clouet et al. 2006). Sialic acid on MUC1 from bovine milk has been shown to play a role in prevention of adhesion of bacteria to Caco-2 cells in vitro (Parker et al. 2010) and mucins also play a role in protection against human immunodeficiency virus (HIV). The Lewis x moieties of human milk MUC1 bind to dendritic cell receptors and block HIV attachment to the cell, preventing transmission of HIV to T cells (Saeland et al. 2009). However, further in vivo studies involving the mucins of MFGM are required to demonstrate the efficacy of the effects described above.

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26.3.3 BTN BTN in milk is often called BTN1A1 to differentiate it from the other BTN genes, of which there are seven (Rhodes et al. 2001). Bovine BTN1A1 is an acidic and hydrophobic protein (Ishii et al. 1995) that contains 526 amino acids (Jack and Mather 1990). It is a member of the immunoglobulin superfamily (Gardinier et al. 1992; Banghart et al. 1998), containing two immunoglobulin-like domains and a cytoplasmic C terminal tail (Robenek et al. 2006); in human milk, it is expressed predominantly as protein variants of approximately 66 kD (Quaranta et al. 2001). It is a type 1 transmembrane protein (Jack and Mather 1990) found in a number of membranes such as the plasma membrane of insect cells (Banghart et al. 1998) and the apical membrane of epithelial cells of the mammary glands (Robenek et al. 2006), as well as the MFGM. While associated with the MFGM, it undergoes little proteolysis during and after secretion of milk (Banghart et al. 1998). Studies by Ishii et al. (1995) demonstrated that BTN1A1 associates with XOR through its C terminal domain or, more specifically, through its B30.2 domain. The interaction between BTN1A1 and XOR is deemed important as it stabilizes XOR binding to the MFGM (Jeong et al. 2009). It has been suggested that the BTN1A1-XOR complex associates with ADPH, further aiding in the formation of complete MFGM (Mather and Keenan 1998). BTN differs slightly between species (e.g., bovine BTN has a molecular mass of approximately 64 kDa while those of horse, goat and camel are approximately 70 kDa, 67 kDa, and 63 kDa, respectively) (Cebo and Martin 2012; Cebo et al. 2012). The differences in molecular mass are directly associated with glycosylation levels. BTN1A1 makes up 20–40% of the total protein content of bovine MFGM (Banghart et al. 1998). In human milk, BTN was found to be 41 ± 3 μg/ mL (Peterson et al. 1998). Interestingly, in humans there is no common trend in BTN concentrations as lactation progresses, which is highlighted by the fact that BTN levels in human milk decrease in some women and increase in others over the course of lactation (Peterson et al. 1998). In contrast, BTN in bovine milk increases 3.2-fold in milk samples taken on day 7 compared to colostrum samples (Reinhardt and Lippolis 2008). Bovine BTN contains three potential N-linked glycosylation sites, two in the N terminal domain and one in the C terminal domain, but does not contain O-linked oligosaccharides (Valivullah and Keenan 1989). The N-linked oligosaccharides are found only at the two N terminal sites at Asn55 and Asn215 (Sato et al. 1995). The glycoprotein displays site-specific glycosylation (e.g., LacdiNAc is present in approximately 50% of the oligosaccharide structures present at Asn55). In contrast, this moiety is not found at Asn215 (Sato et al. 1995). BTN displays a variety of functions, although little is known of the role glycosylation plays in these functions. Sequencing of human and bovine BTN identified 84% sequence similarity and revealed a possible receptor function by sequence correlation (Taylor et al. 1996). BTN also plays an important role

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in regulation of milk secretion (Ogg et al. 2004) and has been found on the inner monolayer of the MFGM. A second model of milk lipid secretion has been proposed where BTN1A1–BTN1A1 interactions between the proteins of the two layers can aid in formation of the complete MFGM (Robenek et al. 2006). It is through such interactions that the milk lipid droplets become coated in membrane and are eventually extruded from the cell (Jack and Mather 1990; Jeong et al. 2009). BTN has been associated with a variety of health benefits. For instance, bovine MFGM BTN has been linked to the protection and amelioration of symptoms of experimental autoimmune encephalomyelitis (EAE) in mice—the animal model for multiple sclerosis (Mana et al. 2004). However, conflicting reports exist as BTN has also been shown to induce EAE (Stefferl et al. 2000). To date, BTN has not been shown to possess anti-infective activities and the importance of the oligosaccharides and the role they play in bioactivity has yet to be established.

26.3.4 LDH LDH, also known as PAS6/7, together with BTN is one of the most abundant glycoproteins of the human MFGM (Quaranta et al. 2001). All mammalian MFGM LDH contain two epidermal growth factor (EGF)–like domains in the amino terminus. The second EGF-domain contains an Arg-Gly-Asp (RGD) sequence motif. In contrast to all other mammals, human MFGM LDH contains only the second EGF domain (Kvistgaard et al. 2004). LDH also contains a sequence in the C terminus that is homologous to the C1 and C2 domains of the blood-clotting factors V and VIII (Andersen et al. 1997; Silvestre et al. 2005). Human MFGM LDH is found at a concentration of 93 ± 10 μg/mL (Peterson et al. 1998) with a molecular mass of 46 kDa (Liu and Newburg 2013). Bovine milk LDH consists of two variants known as PAS6 and PAS7, which have molecular masses of 50 and 47 kDa, respectively. PAS6 and PAS7 share a similar polypeptide core but differ in their glycosylation (Hvarregaard et al. 1996). LDH shows a high level of structural variation between different species. Goat and ovine LDH consist of a single polypeptide chain of approximately 54 and 56 kDa, respectively, whereas bovine LDH consists of two polypeptide chains. Camel milk LDH is also found as two variants of 49 and 55 kDa (Cebo et al. 2010; Cebo and Martin 2012) while equine milk contains four variants of LDH, ranging from 45 to 60 kDa (Cebo et al. 2012). Multiple isoforms of LDH have been identified in human MFGM and three truncated forms of the protein were also identified (Cavaletto et al. 1999). The levels of LDH in human milk decrease over the course of lactation and human milk contains significantly higher levels of LDH in early compared to late lactation (Peterson et al. 1998). In bovine milk, it was found that LDH levels remain relatively unchanged in samples taken 1 week post parturition compared to colostrum (Reinhardt and Lippolis 2008). Dickow et al. (2011) found that LDH association with the MFGM

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is affected by temperature, in particular, cooling Holstein-Friesian milk to 4 °C increased the association. Human milk LDH contains five N-linked glycosylation sites in total (Picariello et al. 2008). In bovine milk, the PAS6 variant of the protein contains two N-linked glycosylation sites at Asn41 and Asn209 and one O-linked glycosylation site at Ser9. PAS7 contains one N-linked glycosylation site at Asn41 and one O-linked glycosylation site at Thr16 (Hvarregaard et al. 1996). PAS6 and 7 contain approximately 10.6% and 16.5% (w/w) carbohydrate, respectively (Seok et al. 2001). The oligosaccharides of the bovine glycoprotein variants have both Fuc and sialic acid in the form of N-acetylneuraminic acid (Neu5Ac). Man was present in greater concentrations in PAS6 when compared to PAS7, which correlates with a greater number of N-linked oligosaccharides present (i.e., two N-linked sites for PAS6 compared to one for PAS7) (Hvarregaard et al. 1996; Seok et al. 2001). LDH has displayed a number of biological activities. For example, human milk LDH plays a role in cell adhesion in an RGD-dependent manner (Taylor et al. 1997). It has also been shown that it mediates vascular endothelial growth factor (VEGF)–dependent angiogenesis both in vitro and in vivo (Silvestre et al. 2005). In addition, recombinant LDH suppresses both apoptosis and inflammation associated with ischemic stroke in the rat model (Cheyuo et al. 2012). LDH is resistant to degradation in the infant stomach, most likely due to its high degree of glycosylation (Peterson et al. 1998). Human MFGM LDH has been shown to protect breast-fed infants from rotavirus infection, acting as a homologue for the rotavirus receptor and binding to the virus, thus preventing it from binding host cells (Newburg et al. 1998). It also provides protection from other infectious diseases such as bacterial-associated diarrhea (Quaranta et al. 2001; El-Loly 2011). The sialic acid content of human milk LDH plays an important role in its antirotavirus activity as removal of sialic acid by chemical hydrolysis reduces the binding of LDH to the virus (Yolken et al. 1992). Interestingly, bovine LDH does not possess these antirotavirus properties, which is most likely due to differences in glycosylation (Kvistgaard et al. 2004).

26.3.5 CD59 CD59, also known as Protectin, is an 18-20 kDa GPI-linked glycoprotein found on cell membranes (Watson et al. 2006) and has been shown to be present in human MFGM due to its GPI-linked anchor (Hakulinen and Meri 1995). It is also found in the human central nervous system (Akatsu et al. 1997) and leukocytes and epithelial cells (Nose et al. 1990). To date, only limited research has focused on the CD59 content in other mammalian milks. In a study by Hakulinen and Meri (1995), CD59 was purified from human milk with an average concentration of 1.75 μg/mL. Others have demonstrated that the levels of the protein remain stable throughout lactation in human (Bjorge et al. 1996) and bovine (Reinhardt and Lippolis 2008) milk.

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While limited research has been conducted on the glycosylation of milk CD59, structural information does exist for CD59 isolated from other sources. For example, human erythrocyte CD59 contains eight potential O-linked and one N-linked glycosylation site (Rudd et al. 1997) at Asn18 (Ninomiya et al. 1992). Wheeler et al. (2002) demonstrated that the glycosylation of GPI-anchored and soluble CD59 are differentially glycosylated, likely due to differing lengths of exposure to glycosyltransferases in the Golgi. While little information is available on the role of milk-derived CD59, the roles of CD59 isolated from other areas have been defined. CD59 is involved in the regulation of T cell responses (Longhi et al. 2006) and increases the killing activity of natural killer cells, and a murine variant of CD59 has demonstrated a possible role in B cell proliferation in the humoral response (Kimberley et al. 2007). It is also involved in lymphocyte activation (BohanaKashtan et al. 2004) and functions in cell death. It has been demonstrated that cross-linking of CD59 expressed on T cells, with antibodies, induces apoptosis (Monleon et al. 2000). It is likely that milk-derived CD59 could also possess similar functions in aiding the immune response. The most wellstudied function of CD59 is its role in inhibition of the formation of the membrane attack complex in the complement cascade, where it prevents the C9 molecules from incorporating into the C5b-8 complex (Morgan 1999; Morgan et al. 2006). Menu et al. (1994) demonstrated that the N-linked glycosylation of human recombinant CD59 is required for efficient CD58-dependent T cell responses. Conflicting reports exist on the role the oligosaccharides play in complement inhibition by CD59. Ninomiya et al. (1992) showed PNGase F treatment of CD59 resulted in a reduction in complement-inhibitory activity, which demonstrated the importance of N-linked glycosylation in this activity. In contrast, other studies have reported that N-linked oligosaccharides do not appear to play a role in complement cascade inhibition (Rother et al. 1996; Rushmere et al. 1997). Further studies are required to determine the functions of milk CD59 and the activities ascribed to its glycosylation.

26.3.6 CD36 CD36 is a 78 kDa glycoprotein that has been found in bovine, human (Greenwalt et al. 1992; Spitsberg, 2005), camel (Saadaoui et al. 2013), caprine, ovine, and equine (Cebo and Martin 2012) MFGM. It forms 2–5% of the total protein content of bovine MFGM (Rasmussen et al. 1998) and has been found at a concentration of 50 mg/g of MFGM proteins in bovine milk (Chatterton et al. 2013). CD36 has four free sulfhydryl groups at the cytoplasmic tail (Spitsberg et al. 1995) and contains at least one intrachain disulphide bridge, which is necessary for its correct transport and processing (Rasmussen et al. 1998). CD36 gene expression increases during lactation (Bionaz and Loor 2008) but protein expression remains unchanged in day 7 bovine milk compared to colostrum (Reinhardt

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and Lippolis 2008). However, little is known about how lactation influences the attached oligosaccharides. Bovine MFGM CD36 contains approximately 24% (w/w) carbohydrate content and eight N-linked glycosylation sites have been characterized for the glycoprotein, namely Asn78, Asn101, Asn171, Asn204, Asn234, Asn246, Asn320, and Asn416 (Berglund et al. 1996). The oligosaccharides are highmannose, hybrid, and complex type, including bi-, tri-, and tetra-antennary structures (Nakata et al. 1993). The oligosaccharides are fucosylated and include GalNAc (i.e., the LacdiNAc moiety is present) and more than one oligosaccharide type can be found at each site (Berglund et al. 1996). The high degree of glycosylation most likely confers resistance to the membranebound protein from degradation (Greenwalt et al. 1992). However, to date few biological activities have been attributed to the glycosylation of this protein.

26.3.7 Proteose peptone component 3 Proteose peptone component 3 (PP3), also known as lactophorin (LP), is a 28 kDa phosphoglycoprotein (Zhu and Damodaran 2011). It is a substrate of the milk enzyme plasmin that is proteolytically degraded to form a second glycopeptide (18 kDa) called lactophorin 18 (LP18). PP3 has been detected in caprine (Lister et al. 1998), camel (El-Hatmi et al. 2007), bovine, and ovine milk but has not been identified in human milk (Sørensen et al. 1997). It is present at a high concentration in bovine milk at 300 mg/L (Sørensen and Petersen 1993). El-Hatmi et al. (2007) reported that camel milk PP3 concentration varies from 2.7 to 6.8 g/L during the first eight days of lactation. Girardet et al. (1995) demonstrated that bovine MFGM PP3 contains two O-linked glycosylation sites at Thr16 and Thr86 and one N-linked glycosylation site at Asn77 while LP18 only contains one of the O-linked (Thr86) and the N-linked glycosylation site (Girardet et al. 1995; Inagaki et al. 2010b). Three neutral oligosaccharides have been identified for the O-linked glycosylation site of PP3, namely GalNAc (Tn antigen), T antigen, and Gal-β-(1 → 4)-GlcNAcβ(1 → 6)-[Gal-β-(13)-]GalNAc (extended mucin core type-2) (Coddeville et al. 1998). The N-linked structures have been identified as bi-, tri-, and tetra-antennary, mono-sialylated oligosaccharides containing N-acetyllactosamine (LacNAc) and LacdiNAc, which also may or may not be fucosylated (Inagaki et al. 2010b). Furthermore, caprine MFGM PP3 was found to contain one N-linked and one O-linked glycosylation site (Lister et al. 1998). There is debate as to whether PP3 is a membrane-bound protein or whether it is loosely associated with MFGM. It has been found in the bovine MFGM fraction but this soluble glycoprotein has also been sourced from bovine whey (Sørensen et al. 1997). Interestingly, Fong et al. (2007) hypothesized that the presence of PP3 in the bovine MFGM could be due to the binding of the protein to the membrane during processing. The incorporation of free milk proteins into the MFGM

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during milk processing has been demonstrated previously; however, more studies are required to determine if the presence of PP3 in the MFGM is due to this phenomenon. PP3 has demonstrated antilipase activity in milk (Girardet et al. 1993), which likely functions to protect the MFG from spontaneous lipolysis (Sørensen et al. 1997). Stable emulsifying properties have also been identified for the glycoprotein in bovine milk (Innocente et al. 1998), which could be an important factor in milk processing. A component of LP28 called lactophorin 16 (LP16), thought to be formed due to proteolytic degradation of LP28, was shown to be a potent inhibitor of the replication of human rotavirus and was shown to play a role in prevention of human rotavirus infection in mice. Although the importance of the glycosylation in this activity has yet to be established, the authors predicted that glycosylation may play an important role as oligosaccharides confer heat stability and LP16 is remarkably heat-resistant (Inagaki et al. 2010a). In addition, a synthetic peptide consisting of a portion of the C terminus of LP28 demonstrated antibacterial activities against pathogenic Staphylococcus aureus, Salmonella St Paul, and Pseudomonas aeruginosa. This demonstrates the potential of the C terminal peptide backbone of milk-derived LP28 glycoprotein for protection against pathogenic infection (Campagna et al. 2004). Furthermore, PP3 derived from bovine, ovine, and caprine milk has been linked to mitosis—a PP3-containing fraction of the mammalian milks increased mitosis of MARK3 hybrid cells (murine myeloma–B cell fusion). Interestingly, desialylation of the PP3 fraction lead to decreased activity, indicating the glycosylation plays a role in mitotic stimulation. It was hypothesized that the negative charge attributed by the carboxyl group of the sialic acid residues could enhance stability and solubility of the proteins (Mati et al. 1993). Shida et al. (1994) also demonstrated the ability of glycoproteins sourced from the proteose peptone fraction from bovine milk to bind E. coli heat-labile enterotoxin, demonstrating a possible protective function against bacterial-associated disease. Removal of the carbohydrates from these glycoproteins resulted in an inability to bind the toxin, highlighting the important role the oligosaccharides play in this activity.

26.3.8 XOR XOR has been identified in mammalian milks including camel (Baghiani et al. 2003), human (Godber et al. 2005), goat (Cebo et al. 2010), bovine, sheep, and horse (Cebo and Martin 2012). Bovine XOR is found as a peripheral protein on the inner monolayer of the MFGM (Heid and Keenan 2005; Robenek et al. 2006). It exists as a 300 kDa homodimer with each subunit consisting of a molybdenum cofactor, two Fe2S2 redox centers, and one FAD redox center. In contrast, human milk XOR lacks molybdenum cofactors and is deficient in Fe2S2 centers (Godber et al. 2005). XOR exists in the cell as two interconvertible forms: xanthine oxidase (XO), which reduces molecular oxygen with the generation of superoxide leading to purine degradation, and xanthine dehydrogenase (XDH), which

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reduces NAD+ without generation of the reactive oxygen species superoxide (Chen et al. 2012). XDH is the major form that is found in the cell, and can be converted to XO by proteolysis or alternatively by oxidation of sulfhydryl residues (Enroth et al. 2000). Whether or not XOR is a glycosylated protein remains unclear. Indeed, glycosylation of this protein could be dependent on its source. Human XO has been found to be N-glycosylated (Picariello et al. 2008) and goat MFGM XO has recently been shown to contain sialic acid in an O-linked oligosaccharide. O-linked glycosylation sites have been identified at Thr207, Thr1069, and Thr1071 in the caprine XO sequence (Cebo et al. 2010). However, to date the glycosylation of bovine MFGM XOR has not been confirmed and it has even been suggested that bovine XOR is not glycosylated (Mather 2000). XOR has been isolated from sheep’s milk at a concentration of 22.6 ± 3.3 mg/L (Benboubetra et al. 2004) and bovine MFGM XOR has been shown to be present at a concentration of 16-33 mg/g (Chatterton et al. 2013). Human milk XOR levels in term milk decreased significantly over the course of lactation. However, pre-term milk only had a slight decrease in XOR levels over time (Molinari et al. 2013). Interestingly, in both cases, the XOR levels are higher in colostrum compared to mature milk, indicating the important function it serves in the neonatal gut. In contrast, bovine milk levels of XDH are 2.6-fold higher in MFGM samples taken 7 days post parturition compared to colostrum samples (Reinhardt and Lippolis 2008). XOR has demonstrated antimicrobial properties. Human milk XOR inhibits E. coli and Salmonella enteritidis growth in vitro through nitric oxide production (Stevens et al. 2000). This activity has been further validated by Hancock et al. (2002), who demonstrated that nitric oxide produced by bovine and human MFGM XOR can protect against E. coli infection in vitro. In addition, hydrogen peroxide produced by bovine XOR MFGM can lead to protection from S. aureus infection (Martin et al. 2004). Unfortunately, none of these studies have linked the anti-infective activity of XOR to its glycosylation, the role of which has yet to be fully understood.

26.3.9 Carbonic anhydrase The 40 kDa glycoprotein carbonic anhydrase (CA) has been identified in the human MFGM (Quaranta et al. 2001) as well as in bovine milk (Ichihara et al. 2003). CA VI is the CA variant that is found in MFGM and is a member of a family of zinc-containing enzymes (Karhumaa et al. 2001). It is the only member that is secreted, and has been identified in the saliva or salivary glands and the milk of mammals including that of sheep, human, cow, and dog (Fernley et al. 1989; Parkkila et al. 1990; Karhumaa et al. 2001; Ogawa et al. 2002). Both human and bovine colostral milk contain higher levels of CA VI compared to mature milk. Interestingly, the saliva of newborn infants contains lower concentrations of CA VI compared to adult saliva, most likely due to the slower saliva secretion rate of newborns compared to adults. As the human gastrointestinal tract (GIT) is supplied with CA VI through saliva, the high levels of CA VI in human and

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bovine colostrum could be an example of an evolutionary measure to ensure supply of adequate levels of CA VI to newborns (Karhumaa et al. 2001; Kitade et al. 2003). The exact physiological role of CA VI in the GIT has yet to be established; however, the increased levels of CA VI in colostrum suggest a regulatory or protective role in the neonatal gut. Bovine milk CA VI levels decrease over the length of lactation. Studies by Nishita et al. (2007) demonstrated that the colostral concentration of CA VI is on average 119 ng/mL while levels in mature milk decrease to 7.9 ± 12.1 ng/mL. The glycosylated structures of milk CA VI are not well established. However, CA VI isolated from bovine submaxillary gland and parotid gland (Hooper et al. 1995; Jiang et al. 1996) have been shown to have different glycosylation depending on their site of post-translational modification. This is due to the presence of different glycosyltransferases in each of the sites of isolation. Bovine CA VI isolated from submaxillary gland and parotid gland contain oligosaccharides modified with sulfated and nonsulfated GalNAc, respectively (Hooper et al. 1995). The protein isolated from bovine submaxillary gland contains two possible N-linked glycosylation sites (Jiang et al. 1996). In addition, human- and ovine-secreted CA VI possesses three potential N-linked sites, two of which are glycosylated (Aldred et al. 1991; Thatcher et al. 1998). Studies on the glycosylation of mammalian milk CA VI are required in order to fully understand the structure and function of the protein including its glycosylation pattern.

26.3.10 Clusterin Clusterin, also known as apolipoprotein J, is a 70 kDa (Kounnas et al. 1995) glycoprotein composed of an α- and β-chain connected by disulfide links. Human serum clusterin α- and β-chains can contain 0–30% and 27–30% carbohydrate (w/w), respectively (Kapron et al. 1997). Thus, there are many isoforms of these chains with differing extents of glycosylation. Charlwood et al. (2002) first established the presence of human MFGM clusterin and, since then, it has been shown that the human MFGM contains higher levels of the protein when compared to bovine MFGM (Hettinga et al. 2011). The protein can be found in many fluid types including breast milk, seminal fluid, and urine, as well as many cells such as certain cells of the heart, brain, and stomach (Rosenberg and Silkensen 1995). Its levels are regulated by the endocytic receptor glycoprotein 330 (gp330), which causes endocytosis and subsequent degradation of clusterin (Kounnas et al. 1995). Human serum clusterin contains three N-linked glycosylation sites in each subunit, while it does not possess any O-linked glycans (Kapron et al. 1997). It has been demonstrated that human MFGM clusterin contains 10 possible N-linked glycans, with an abundance of biantennary fucosylated structures. The glycans contain Gal, Man, sialic acid, and GlcNAc (Charlwood et al. 2002). No evidence to date exists on bovine milk clusterin glycosylation. It is known that levels of bovine milk clusterin are not static over the course of lactation. MFGM clusterin levels decline in day 7 MFGM samples when compared to colostrum MFGM (Reinhardt and Lippolis 2008).

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The role of milk clusterin has yet to be established; however, the functions of clusterin from other sources have been investigated. For example, human seminal clusterin plays a role in male fertility (O’Bryan et al. 1990), human serum clusterin plays a role in inhibition of the complement system (Hochgrebe et al. 1999), and human clusterin also acts as an extracellular chaperone protein, targeting proteins for degradation (Hammad et al. 1997). Human serum clusterin can bind S. aureus in vitro (Partridge et al. 1996). However, the clinical relevance of this interaction has not been established. It is possible that MFGM-derived clusterin could also display similar bacterial binding and thus could act as a decoy receptor in vivo. Further studies on the glycosylation, and the glycosylation-dependent health promoting properties, of milk-derived clusterin are required.. Deglycosylation of the human serum clusterin has led to increased binding to ligands compared to the wild-type protein, indicating the oligosaccharides could hinder the optimal binding of the protein to various ligands. However, it is likely that the glycosylation of clusterin mediates the binding to carbohydrate receptors, as demonstrated when Gal significantly inhibited binding of wild-type clusterin to liver cells (Stewart et al. 2007). It is likely that the glycosylation of clusterin sourced from MFGM may behave in a similar manner to human serum clusterin.

26.4 Commercial potential Over the past number of decades, manufacturers have attempted to improve foods such as infant formula by adding health-promoting ingredients including long-chain fatty acids, inulin, and fructo- and galacto-oligosaccharides. More recently, manufacturers have recognized domestic animal milks as a potential source of such ingredients as they are readily available and relatively low in cost. Bovine milk is seen as one of the most attractive sources as it is produced in high quantities in countries all over the world and consumed by millions of individuals daily. This nutritional food is also an ideal source of biologically active MFGM glycoconjugates, which can be easily harvested for use as functional food ingredients (Haug et al. 2007). Indeed, the isolation and purification of bovine MFGM proteins has already successfully been established at laboratory scale for CD36 (Greenwalt 1993), PAS6/7 (Kim et al. 1992), ADPH, BTN (Nielsen et al. 1999), MUC1 (Sando et al. 2009), and XOR (Sanders et al. 1997). In addition the laboratory-scale extraction of human and camel MFGM XOR (Sanders et al. 1997) and human protectin (Hakulinen and Meri 1995) has been successful. Interestingly, the purification of the whole bovine MFGM fraction has also been established (Vanderghem et al. 2008). The use of intact MFGM containing all the membrane-bound and associated proteins may be advantageous in that the full complement of glycolipids and glycoproteins would be available to confer their beneficial properties.

The milk fat globule membrane: A potential source of health-promoting

Step 1: Separation

Raw milk

Cream Step 2: Washing of cream 2–3 wash steps

Step 3: MFGM release -Churn -Freeze-thaw

Skim milk

Washing solution Washed cream

Discarded washing solution

Butter

Buttermilk *GC

Butter serum Step 4: MFGM collection -Ultracentrifugation -Microfiltration -Freeze-drying

653

Butter oil

*GC

M FGM

Figure 26.2 Outline of MFGM processing. Isolation of MFGM consists of four steps: (1) separation of raw milk into cream and skim milk; (2) washing of cream to remove serum proteins; (3) release of MFGM through methods including churning or freeze-thaw; and (4) collection of MFGM through the use of ultracentrifugation, microfiltration, or freeze-drying. *GC, glycoconjugate fraction.

MFGM can be purified from whole milk through a four-stage process (Singh 2006), as illustrated in Figure 26.2. This process involves separation of fat globules, washing of cream, MFGM release from globules, and MFGM collection. In more detail, the first step involves separation of the fat globules from whole milk, which is achieved by centrifugation or the use of a cream separator. Next, the cream is washed in buffers such as deionized water or sucrose solutions, a requirement for removal of free milk components such as caseins and whey proteins (Dewettinck et al. 2008). It has been demonstrated that the use of large wash volumes, while keeping washing steps to a minimum, maximizes the yield of MFGM components. Additionally, the use of deionized water as the washing buffer negates the need for salt removal from the purified MFGM (Le et al. 2009b). MFGM is separated from the triglyceride core through agitation, churning, or freeze–thaw (Dewettinck et al. 2008). It enters the aqueous phase and can be collected from buttermilk or butter serum by methods such as microfiltration (Le et al. 2013), ultracentrifugation, or freeze-drying. In the past, the large-scale production of MFGM was limited by the need to further develop isolation technologies. However, recent advancements in such technologies suggest that whole MFGM is likely to become a common functional food ingredient in the future. An example of such advancement is the use of microfiltration to remove micellar caseins, which are of a similar size to MFGM components (Singh 2006).

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This method involves the treatment of buttermilk with sodium citrate, which disrupts the casein micelles, and subsequent microfiltration through a 0.1 μm membrane produces an MFGM fraction that contains a high ratio of MFGM proteins (70% of the total protein fraction) compared to skim milk proteins (6% caseins and 24% whey) (Corredig et al. 2003). Furthermore, Morin et al. (2007a) demonstrated that the use of skim milk ultrafiltrate to wash cream prior to buttermilk generation can increase the ratio of MFGM proteins to skim milk proteins, although there was still some loss of MFGM components. Interestingly, microfiltration of buttermilk produced from washed cream displayed a two-fold increase in permeation flow. Therefore, washing followed by filtration proves to be another viable option for MFGM isolation (Morin et al. 2007a). Indeed, a number of patents exist describing whole MFGM isolation (e.g., WO 2001035760 A1, bulk preparation of MFGM) and application (e.g., EP 2509429 A1, use of MFGM components in infant formula by Morinaga). Moreover, methodologies for the isolation of particular components of MFGM have also been patented (e.g., US 6800739 B2, a method developed by Davisco for isolation of MFGM glycoproteins from bovine milk). These patents have led many dairy processing companies to isolate glycosylated ingredients from MFGM for use as functional food ingredients. Such companies include Synlait, Arla Foods, and Büllinger SA, who produce Lipidex, Lacprodan MFGM-10/Lacprodan PL20, and INPULSE, respectively. Lipidex contains many biologically active ingredients including phospholipids (5–7%), gangliosides (0.45%), and sialic acid (0.45%). Lacprodan MFGM-10 contains a number of oligosaccharides and glycoconjugates including sialylated structures, lactoferrin, and IgG as well as MUC15 (0.69%) (6.86 μg/mg of total protein) (Pallesen et al. 2007), LDH (0.17%), and MUC1 (0.32%) (Kvistgaard et al. 2004). This product has demonstrated health-promoting activities both in vitro and in vivo. For instance, Lacprodan MFGM-10 inhibited the infection of rotavirus in human intestinal cells in vitro (Kvistgaard et al. 2004) and increased oral levels of probiotic Lactobacillus gasseri in infants compared to nonsupplemented infant formula (Vestman et al. 2013). Health-promoting properties have also been associated with Lacprodan PL20. Indeed, clinical trials demonstrated that fortification of a drink with Lacprodan PL20 led to reduced stress levels in patients when compared to the nonfortified drink. This clinical trial and other health benefits of Lacprodan PL20 are outlined in a review by Burling and Graverholt (2008). Interestingly, Lacprodan MFGM-10 and Lacprodan PL20 have also been identified as ideal sources of milk polar lipids as outlined in the patent application (US 20130071446 A1). Here, these products have been identified as sources of milk polar lipids for inclusion in infant formulae to increase brain development and improve cognitive function in infants. INPULSE is an MFGM-enriched product with scientifically proven health-promoting activity. In fact, when incorporated into milk and fed to healthy children for 4 months, the numbers of short febrile episodes were significantly decreased compared to a control group

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655

(Veereman-Wauters et al. 2012). Although this was attributed to the phospholipid fraction of MFGM, this study highlights the potential use of MFGM in everyday products as a health-promoting supplement. Some issues in MFGM isolation still require resolution. For example, there is a need to establish milk processing conditions that do not result in a loss of activity and/or concentration of MFGM bioactive compounds. Conditions that adversely affect MFGM isolation include the incorporation of air bubbles into milk by milking machinery, which destabilizes the MFGs and causes changes to the MFGM. Cooling, heating, and pressurizing of milk can lead to a loss of both glycoproteins and glycosphingolipids (Evers 2004). Pasteurization of cream can lead to an increase in whey protein binding to MFGM and spray drying can decrease the phospholipid content of MFGM (Morin et al. 2007b). Although washing of cream can be used to produce an MFGM fraction free of contaminants, such as free milk proteins, caseins, and lactose, it can also increase the loss of loosely bound proteins from MFGM when compared to the integral proteins. Therefore, to maximize MFGM recovery without compromising composition, further investigation is required to determine the exact methods of pre- and post-churning processing which minimize loss of MFGM bioactivity. Additionally, carbohydrate analysis after processing is necessary in order to establish the method that best preserves glycosylation structure and function to capitalize fully on the beneficial health-promoting activities they possess.

26.5 Future perspectives The bioactivities demonstrated by MFGM glycoconjugates highlight their potential use as functional ingredients that could be incorporated into an array of products. Glycosylated MFGM components show enormous potential in acting as decoy receptors that bind pathogens and prevent adhesion to host cells. The pathogens are then flushed out of the body through the natural defenses, leaving the host unharmed (Sharon and Ofek 2000). MFGM glycoconjugates could also be used in the prevention of impending bacterial infection. Their use as supplements in food and beverages could increase the resistance of consumers against the possible threat of infection. Furthermore, MFGM glycosylation could be exploited by immunocompromised patients to fight infection and some in vivo studies suggest they may ameliorate the symptoms of previous infection (Mouricout et al. 1990). There are a number of products in which MFGM glycoconjugates could be incorporated, including infant formula, which currently lacks some protective ingredients that are present in maternal breast milk. Other food matrices that could be supplemented with these bioactives include water-based beverages and cereals (Sørensen et al. 1997). Indeed, laboratory-based research has already begun to investigate the possibility of including MFGM in foods such as yogurt (Le et al. 2009a).

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A novel approach to produce new bioactives from MFGM may be to ferment MFGM-containing milk fractions. Lactobacillus helveticus strains are widely used for production of dairy products such as yogurt and cheese and grow well on dairy sources (Matar et al. 1996), and could be used to produce fermented MFGM products. Their glycolytic activity could successfully hydrolyze MFGM glycans to produce free oligosaccharides (Adamberg et al. 2005) and they possess proteolytic and peptidase activities (Matar et al. 2001) which could result in the production of bioactive glycopeptides. Indeed, the production of bioactives with health-promoting benefits, such as anticancer (Rachid et al. 2006), immunomodulatory (Meena et al. 2008), and antihypertension (Aihara et al. 2005) activities by fermentation with L. helveticus, is well documented. Although research to date has mainly focused on MFGM glycoconjugates and their health-promoting activities in the prevention of infection and disease, MFGM could also be used for other health-promoting properties. For example, it shows potential to act as a delivery system for drugs and to enhance their intestinal absorption (Sato et al. 1994). MFGM may also be used as an alternative source of phospholipids for liposome preparation to prevent the gastric digestion of protein-based prescriptions. A recent study has shown that it can protect lactoferrin from hydrolysis in vitro (Liu et al. 2013). Ultimately, there are many possible uses of MFGM for improved human health applications.

26.6 Conclusions It is our belief that MFGM has been undervalued in the food industry; however, this perspective has changed in recent years as the knowledge of MFGM composition and bioactivities has increased significantly. The potential it holds commercially as a functional ingredient is being uncovered due to the increased number of studies that have characterized its physical and chemical properties, particularly those of the glycoconjugates. In order to capitalize on all the health-promoting bioactivities MFGM offers, further studies are required to determine the most suitable methods of MFGM isolation and purification that would most preserve its integrity and bioactivities. Further work is required to fully characterize the physical properties of MFGM glycoconjugates and their health-promoting roles in order to truly understand the potential this milk component holds.

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CHAPTER 27

Seaweed and milk derived bioactive peptides and small molecules in functional foods and cosmeceuticals Maria Hayes, Melani García-García, Ciarán Fitzgerald, and Tomas Lafarga Food BioSciences Department, Teagasc Food Research Centre, Dublin, Ireland

27.1 Introduction Marine resources including seaweeds, microalgae, and microorganisms produce structurally diverse bioactive compounds including carbohydrates, polyunsaturated fatty acids (PUFAs), pigments, antioxidants, proteins, small molecules including terpenoids, bioactive peptides, and others. These compounds are known to possess a myriad of beneficial bioactivities and are often produced as a result of the harsh environment in which marine plants and animals must live and survive. In addition, mammalian milk proteins are a valuable source of amino acids but also have known biological properties that can be attributed to the native protein molecules as well as to inherent, bioactive peptides encrypted within the parent protein. For example, the first milk produced by mammals, known as colostrum, and which is often referred to as “beestings,” is rich in bioactive and antimicrobial peptides. The first antimicrobial peptides derived from the milk protein casein were identified by Hill et al. (Lahov and Regelson 1996; Hayes et al. 2006), who isolated the antibacterial glycopeptides, known as casecidins. Isracidin αs1-casein f (1-23) [αs1-CN f(1-23), where f(1-23) refers to amino acids 1 to 23 of the peptide], a positively charged antimicrobial peptide with the primary amino acid structure R1PKHPIKHQGLPQEVLNENLLRF23, was shown to have a broad spectrum of activity against Gram-positive bacteria (Lahov and Regelson 1996). Bioactive peptides with benefits beyond antimicrobial action can also be generated from dairy proteins through the use of enzyme hydrolysis, fermentation using lactic acid bacteria (LAB), and food processing techniques. Bioactive peptides are defined as peptide sequences consisting of between 2 and 30 amino acids in length that are encrypted within the parent protein but which impart a

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

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health and/or beneficial effect once released. Milk-derived bioactive peptides may also be used in the treatment and prevention of diseases associated with metabolic syndrome (Ricci-Cabello et al. 2011). Indeed, consumption of dietary seaweeds has also been suggested to reduce the incidence of high blood pressure and obesity associated with metabolic syndrome (Teas et al. 2009). Functional foods can be defined as a food/food ingredient that imparts a beneficial health benefit to the consumer that goes above and beyond basic human nutrition. According to the Academy of Nutrition and Dietetics, all foods provide some level of physiological function but the term functional foods is reserved for fortified, enriched, or enhanced foods that have a potentially beneficial effect on health when consumed as part of a varied diet, on a regular basis, at effective levels based on significant standards of evidence (Crowe and Francis 2013). The market for functional foods is estimated to have a net worth of $130 billion by the year 2015 (Crowe and Francis 2013). In addition, an ever-expanding market for skin care products exists and both seaweed and dairy peptides and small molecules can be used for cosmetic use and as cosmeceuticals (Agatonovic-Kustrin and Morton 2013). Today there are over 400 suppliers of cosmeceutical ingredients (Brandt et al. 2011). Extracts from both raw material sources can be used as excipients in the formulation of products or for their bioactive and therapeutic benefits. Indeed, human skin is very similar in terms of structure to seaweeds and many compounds found in seaweeds can be beneficial to human skin, as discussed later. Cosmeceuticals are different from cosmetics and drugs, as cosmeceuticals affect the structure or function of the skin and have drug-like effects but are marketed using skin appearance– based claims (Agatonovic-Kustrin and Morton 2013; Lupo 2005). There is increasing interest in the use of algal- and dairy-derived peptides for cosmeceutical as well as functional food use. This chapter discusses the supply of seaweed and dairy proteins that can be used for the generation of bioactive peptides and small molecules with both functional food and cosmeceutical applications. It discusses the sustainability of seaweed supply in different countries and the bioactivities of small molecules and novel, bioactive peptides that can be derived from both seaweed and dairy proteins. Functional foods and cosmeceutical products that contain bioactive peptides and other small molecules that are currently on the market are also discussed.

27.2 Seaweeds rich in protein with potential for use in peptide generation 27.2.1 Edible seaweeds and trends in seaweed consumption Edible seaweeds including the brown algae or phaeophyta, the green algae or chlorophyta, and red algae or rhodophyta have a long history of safe use in

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the human diet (Fleurence 1999). Over 145 species of seaweed are used worldwide in food. In particular, seaweeds are consumed in large quantities in Japan, China, and Korea (Černá 2011). In Japan, over 20 different species of red, green, and brown seaweeds are included in meals (Zava and Zava 2011). The three most popular seaweeds eaten in Japan are Porphyra species (Nori), Undaria species (Wakame), and Laminaria species (Kombu). Indeed, there is a problem with overconsumption of iodine in Japan as seaweed is served in approximately 21% of Japanese meals and 20–38% of the Japanese population between the ages of 40 and 79 consume seaweed more than five times per week (Yoshinga et al. 2001). Consumption in other countries is not as popular but there is a history of use in Europe in Ireland, Denmark, and Wales. In Ireland, before the arrival of the potato, seaweed was used as a fertilizer and food. From the Brehon Code Law that existed in Ireland, we know that the Celts used seaweeds as a food and placed a high value on it. Dillisk (also known as Dulse) is mentioned: According to the Brehon Law, seaside arable land was enhanced in value by having rocks on its sea-border producing this plant, and there was a penalty for consuming the dillesk belonging to another without leave. (Joyce 1906)

Today in Nova Scotia- and Maine, dried Palmaria palmata (Dulse) is often served as a salty cocktail snack. In Wales, laver bread is made and consumed. This bread is manufactured using the red alga Porphyra umbilicalis (McHugh 2003). According to an article published by Fleurence et  al. in 2012, edible seaweed consumption in Europe is 70 tons per year compared to 97,000 tons per year in Japan. However, interest in the use of seaweed extracts and bioactives for use as ingredients in functional food products has grown recently and niche markets exist for seaweed products. In addition, the use of seaweed in molecular gastronomy has grown recently. In an article by Ole Mouritsen (2012) that describes seaweed use in gastronomy, he states that algal cuisine is sustainable, as the ocean is one of the last resorts for mankind to exploit in order to obtain food to feed the hungry.

27.2.2 Seaweeds rich in protein and nitrogen content The protein content of seaweeds ranges from 5 to 47% based on dry weight (Černá 2011). Generally, green and red seaweeds contain higher protein content of between 10 and 47% of the dry weight (DW) compared to brown seaweeds, which contain 5–15% protein DW. Table 27.1 shows the percentage of protein present in a number of seaweeds, some of which are consumed as sea vegetables (FAO 2002) or have other uses. The protein content of seaweed is often assessed by measuring the nitrogen content and multiplying it by different conversion factors. In seaweeds, the nitrogento-protein factor ranges from 3.75 to 5.72 (Lorenco et  al. 2002; Černá 2011).

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Table 27.1 Percentage protein present in different seaweed species. Seaweed name

Red seaweeds Gelidiella acerosa Gracilaria birdiae Gracilaria domingensis Gracilaria folifera Gracilaria changgi Gracilaria salicornia Palmaria palmata Porphyra species Porphyra species Porphyra vietnamensis Green seaweeds Codium reediae Codium tomentosum Enteromorpha compressa Enteromorpha intestinalis Ulva fasciata Ulva fasciata Ulva lactuca Ulva reticulata Ulva reticulata Brown seaweeds Dictyota acutiloba Laminaria species Padina pavonica Hizikia fusiforme Sargassum polycystum Sargassum vulgare Undaria pinnatifida

Protien content (%) dry matter

Method of analysis used

Reference

31.1 7.1 6.2 7 34.5 5.9 18.3 31.3 44 16.5

Biuret Bradford Bradford Biuret Kjeldahl Lowry Kjeldahl Kjeldahl Kjeldahl Lowry

Manivannan et al., (2009) Gressler et al., (2010) Gressler et al., (2010) Manivannan et al., (2008) Norziah and Ching (2000) McDermid and Stuercke (2003) Galland-Irmouli et al., (1999) Dawczynski et al., (2007) Marsham et al., (2007) McDermid and Stuercke (2003)

7 6.1 12.3 15.2 16.4 8.8 6.3 7.1 20 13.5

Lowry Biuret Biuret Kjeldahl Biuret Lowry kjeldahl kjeldahl Biuret Biuret

McDermid and Stuercke (2003) Manivannan et al., (2008) Manivannan et al., (2009) Akköz et al., (2011) Manivannan et al., (2009) McDermid and Stuercke (2003) Ramos et al., (2000) Wong and Cheung (2000) Shanmugam and Palpandi (2008) Manivannan et al., (2009)

12 7.5 13.6 11.6 5.4 16.3 19.8

Lowry Kjeldahl Biuret Kjeldahl Kjeldahl Kjeldahl Kjeldahl

McDermid and Stuercke (2003) Dawczynski et al., (2007) Manivannan et al., (2009) Dawczynski et al., (2007) Matanjun et al., (2009) Ramos et al., (2000) Dawczynski et al., (2007)

Protein content expressed in terms of percentage (%) dry matter of seaweeds that are consumed as sea vegetables globally.

This may lead to an overestimation of the protein content in seaweeds. However, nitrogen is an important component of DNA and ATP. All of the essential and nonessential amino acids are present in seaweeds. In red seaweeds, essential amino acids represent almost half of the total amino acid content. Indeed, Wong and Cheung (2000) found essential amino acid levels of between 42 and 48% in red and green seaweeds (Černá 2011). The quality of seaweed proteins has been compared to that found in soybean, casein and beef (Matanjun et al. 2009).

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27.3 Seaweed sustainability in different coutries harvesting of seaweeds Seaweed harvesting and farming and maintaining a sustainable supply of the raw seaweed product are important issues for consideration if seaweed is to be used successfully as a food and/or cosmeceutical ingredient. Mechanical seaweed harvesting, licensing of aquaculture sites for seaweed culture, and conservation of seaweed beds are important issues. In Ireland few companies can mechanically harvest seaweed but in Norway and France seaweed is extensively harvested. In France seaweed is harvested using a machine called a Scoubido and this machine is especially suitable for harvesting seaweeds from rocky shorelines. In Norway boats use a rake to drag the seashore for seaweed and the harvesting industry is regulated on a grid-type system whereby a square of the ocean is left fallow for 5 years to ensure regeneration of the seaweed (Walsh and Watson 2009). There is a tradition of seaweed farming in eastern Africa, especially in Tanzania and Zanzibar. In Tanzania, seaweed harvesting began in the 1930s. Seaweed was harvested from the wild for export to France, the United States, and Denmark (Mshigeni 1973). However, wildstock of seaweeds were wiped out and the first experiments on farming seaweeds imported from the Philippines— Kappaphycus alvarezii (cottonii) and Eucheuma denticulatum (spinosum)—were carried out in the 1980s (Msuya 2011). A peg and line or off-bottom method of farming seaweed is used in these countries in shallow intertidal areas. Seaweed farming has both economic and social benefits in Africa. The world market currently requires more K. alvarezii (cottonii) as when this seaweed is exported it is used to extract carrageenan, which is used as an emulsifier and stabilizer in the food, pharma, and cosmetics industry (Msuya 2011). In North America and Canada, the brown seaweed Ascophyllum nodosum (rockweed) is the most important commercial seaweed in Canada and it is the dominant perennial brown seaweed in the intertidal zone along the Atlantic coastline of the Maritimes where it forms extensive beds. Commercial exploitation of Rockweed along the coastal areas of Nova Scotia began in the late 1950s when it was used as raw material for sodium alginate and “kelp” meal. Today this seaweed is used as a fertilizer and as an animal feed supplement. Traditionally the harvest of rockweed in the Maritime provinces of Canada was an open fishery with no limit on the number of harvesters, their area of operation, or levels of exploitation. After 1959, the provincial government issued a few exclusive-purchasing licenses in southwestern Nova Scotia. The majority of the resource was totally open to harvest and the level of exploitation was generally low, with a few areas of concentrated harvest in southwestern Nova Scotia (Sharp 1986). Since 1995, a precautionary approach was taken and a research and monitoring program was introduced in New Brunswick to ensure sustainability of

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the seaweed supply and to ensure that harvesting by hand or mechanically did not damage the ecosystem. An alternative to wild harvesting of seaweeds is aquaculture. However, expansive aquaculture of seaweeds can have negative implications for the environment. For example, in 2008, the world’s largest green-tide occurred in the Yellow Sea in China. The hypothesized cause was the expansion of Porphyra yezoensis aquaculture along the Jiangsu coastline and the reoccurrence of a green-tide in 2009 was predicted. The responsible species was Ulva prolifera from P. yezoensis aquaculture rafts (Liu et al. 2010).

27.4 Bioactive peptides from macroalgal and milk sources and heart health 27.4.1 ACE-I and renin inhibitory peptides Bioactive peptides can impart positive health effects on a number of human regulatory systems through their action on specific enzymes including angiotensin-converting enzyme (ACE-I), renin, platelet-activating factor acetylhydrolase (PAF-AH), dipeptidyl peptidase IV (DPP-IV), and others. The enzymes ACE-I and renin play an important role in the control of high blood pressure and this is one of the main factors related to the incidence of stroke in humans. These enzymes function as part of the renin–angiotensin–aldosterone system (RAAS) and control blood pressure and saltwater balance. This system is shown in Figure 27.1. Several ACE-I inhibitory peptides derived from food proteins have been isolated from casein, sour milk, and plant sources such as soy (Sato et al. 2002). Suetsuana et  al. (2000) and later Sato et  al. (2002) reported the isolation of ACE-I inhibitory peptides from the brown seaweed Wakame (Undaria pinnatifida) in 2002. These peptides were identified by amino acid composition analysis, sequence analysis, and liquid chromatography–mass spectrometry (LC-MS) as Val-Tyr (IC50 35.2 μM), Ile-Tyr (6.1 μM), Ala-Trp (18.8 μM), Phe-Tyr (42.3 μM), Val-Trp (3.3 μM), Ile-Trp (1.5 μM), and Leu-Trp (23.6 μM) and were resistant to gastrointestinal protease digestion in vitro. Furthermore, each peptide was found to have an antihypertensive effect after a single oral administration in spontaneously hypertensive rats (SHRs) when administered at a dose of 1mg/Kg body weight (Sato et al. 2002). In addition, Sheih et al. (2009) isolated and characterized a novel ACE inhibitory peptide from algal protein waste. The ACE-I and antihypertensive IAPG isolated from a microalga was also reported by Murray and FitzGerald (2007). ACE-I inhibition may also be due to the presence of small molecules known as phlorotannins that occur mainly in the brown algae. Phlorotannins are phenolic compounds formed by the polymerization of phloroglucinol and are defined as 1, 3, 5-trihydroxybenzene monomer units. They are biosynthesized through the acetate-malonate pathway, are highly hydrophilic, and range in size between

Seaweed and milk derived bioactive peptides and small molecules

* Angiotensinogen Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-...

Prorenin

675

ACE-I

Angiotensin (1-5)

Angiotensin (1-7) Asp-Arg-Val-Tyr-Ile-His-Pro

Asp-Arg-Val-Tyr-Ile

ACE-I INHIBITION

Renin

Angiotensin I RENIN INHIBITION

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Endopeptidase

ACE-II

ACE-II Prolylcarboxylpeptidase

Bradykinin Asp-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Angiotensin (1-9)

Angiotensin (1-7)*

Kallidin

Asp-Arg-Val-Tyr-Ile-His-Pro

Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His

ACE-I

ACE-I

MAS

Inactive Metabolites Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Angiotensin III Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Aminopeptidase A

Angiotensin IV Val-Tyr-Ile-His-Pro-Phe

Aminopeptidase N

ACE-I INHIBITION

RECEPTOR BLOCKERS

ALDOSTERONE INHIBITION

AT1

AT2

AT4

Aldosterone

Figure 27.1 The renin–angiotensin–aldosterone system. The vascular renin–angiotensin– aldosterone system (RAAS). Expression of vascular Renin is low and may result from circulating renin, which is taken up at the prorenin receptors of the vascular cell. ACE hydrolyzes angiotensin I to angiotensin II, both in the extracellular and intracellular space. Renin is the rate-limiting enzyme in the process.

126 and 650,000 Daltons (Wijesekara and Kim 2010). The phlorotannin phlorofucofuroeckol A isolated from the brown seaweed Ecklonia kurome had an ACE-I inhibitory IC50 value of 12.74 uM compared to the positive control Captopril© (IC50 0.05 μg/ml) when tested. Harnedy and FitzGerald (2013) reported the isolation of ACE-I inhibitory fractions from the red seaweed Porphyra species but the sequences of the ACE-I inhibitory peptides were not reported. The enzyme renin [EC 3.4.15.23] is the rate-limiting enzyme in the production of all angiotensin peptides. Renin conversion of angiotensinogen to Ang I (Figure 27.1) is the rate-limiting step in Ang II formation and Renin has long been considered the optimal target for RAAS inhibition. ACE-I inhibitors do not influence non-ACE pathways such as chymase, while AT 1 receptor inhibition

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increases circulating concentrations of angiotensin peptides. Previous studies found that renin inhibition by the nonpeptide renin inhibitor Aliskiren® reduced atherosclerosis in mice (Lu et al. 2008). Renin inhibitory peptides were identified previously from pea protein sources. In 2010, Li and Aluko (2010) identified the renin inhibitory dipeptide with the amino acid sequence IR. More recently, the tridecapeptide Ile-Arg-Leu-IleIle-Val-Leu-Met-Pro-Ile-Leu-Met-Ala (IRLIIVLMPILMA) was isolated from the red seaweed Palmaria palmata (Fitzgerald et al. 2012). This peptide was generated through hydrolysis of the P. palmaria protein using the enzyme papain. This peptide had an IC50 value of 3.344 mM (+/–0.31) compared to the positive control. The hydrolysate from which this peptide was generated was assessed for its renin inhibitory ability in bread (Fitzgerald et al. 2013). It was observed that the addition of 4% P. palmata protein hydrolysate to the wheat bread control did not affect the texture or sensory properties of the bread to a large degree. Furthermore, when compared to the control, the bread containing the hydrolysate retained renin inhibitory bioactivity after the baking process. This study highlighted that baked products may be a suitable delivery vehicle for bioactive compounds (Fitzgerald et al. 2013). The vascular inflammatory role of platelet-activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized nonesterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis. Work carried out by Fitzgerald et al. (2013) demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The peptide NIGK isolated from a papain hydrolysate of P. palmata protein had a PAF-AH IC50 value of 2.32 mM compared to the positive control (Fitzgerald et al. 2013). Renin inhibitory peptides were isolated previously from buckwheat proteins but few renin or PAF-AH inhibitory peptides have been isolated to date from seaweeds and this research area warrants further investigation as seaweed proteins contain all the essential amino acids and amino acids containing bulky side chains, which are thought to play a role in the inhibition of ACE-I and renin. Milk proteins are a known source of bioactive peptides and these peptides are encrypted in both casein (α-, β-, γ-casein) and whey proteins (β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulins, lactoferrin, proteose-peptone fractions). Indeed, dairy products are among the best precursors of bioactive peptides and are the most studied source of bioactive peptides to date (RicciCabello, 2011; Hartmann et  al. 2007; Hernández-Ledesma et  al. 2011; Hayes et al. 2007). Over the last three decades a number of ACE-I inhibitory peptides were isolated, identified, and characterized from milk protein sources. The most well-known ACE-I inhibitory peptides are the β-casein-derived lactotripeptides Val-Pro-Pro and Ile-Pro-Pro f(84-86) and f(74-76).These were identified by Nakamura et al. from the fermented milk drink known today as Calpis©, which is made using the strains Lactobacillus helveticus and Saccharomyces cerevisiae. Initial

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studies using randomized controlled trials in prehypertensive and hypertensive subjects were published and several antihypertensive products appeared on the market. However, more recent studies carried out from 2009 until 2011 (Wuerzner et al. 2009; Engberink et al. 2008; van Mierlo et al. 2009) found that the effect of lactotripeptides on blood pressure did not have the desired beneficial blood pressure–lowering effects and no statistically significant differences in blood pressure were observed between the groups that consumed the peptides and the placebo-consuming group (Ricci-Cabello et al. 2011). The concentration of peptide consumed along with the initial blood pressure need to be taken into consideration in future trials. To date, renin inhibitory or PAF-AH inhibitory peptides have not been isolated from dairy sources.

27.5 Marine molecules for the treatment of diabetes and obesity Marine molecules including peptides could be used in functional food formulations for the prevention of diabetes and obesity. Dipeptidyl-peptidase IV (DPP IV/CD26) is a regulatory protease and also works as a binding protein. Important DPP IV substrates include neuropeptides such as neuropeptide Y or endomorphin, circulating peptide hormones such as peptide YY, growth hormone–releasing hormone, glucagon-like peptides (GLP)-1 and -2, and gastric inhibitory polypeptide, as well as paracrine chemokines such as RANTES (regulated on activation normal T-cell expressed and secreted), stromal cell–derived factor, eotaxin, and macrophage-derived chemokine. The clinical uses of selective DPP IV inhibitors or DPP IV-resistant analogues, especially for the insulinotropic hormone GLP-1, were tested and were found to enhance insulin secretion and to improve glucose tolerance in diabetic animals (Mentlein 1999). Thus, DPP IV appears to be a major physiological regulator for some regulatory peptides, neuropeptides, circulating hormones, and chemokines. DPP IV also hydrolyzes incretin hormones. This hydrolysis results in an increased and prolonged insulin response. In 2013, Harnedy and FitzGerald (2013) assessed the contribution of protein fractions and proteolytic enzyme preparations from Palmaria palmata to in vitro cardioprotective, antidiabetic, and antioxidant activity. Aqueous, alkaline, and combined aqueous and alkaline P. palmata protein fractions were hydrolyzed with the food-grade proteolytic preparations Alcalase 2.4 L, Flavourzyme 500 L, and Corolase PP. The hydrolysates had ACE-I and DPP IV inhibitory activity with IC50 values in the range 0.19–0.78 and 1.65–4.60 mg mL−1, respectively. The oxygen radical absorbance capacity (ORAC) and ferric-reducing antioxidant power (FRAP) values ranged from 45.17 to 467.54 and from 1.06 to 21.59 μmol trolox equivalents/g, respectively. Furthermore, hydrolysates (1 mg mL−1) were shown to inhibit renin within the range 0–50%. In general, Alcalase 2.4 L and Corolase PP hydrolysates of aqueous

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protein displayed the highest in-vitro activity. The results indicate that protein fractions generated had significant effects on in-vitro biofunctional activity. This study demonstrated the potential of P. palmata protein hydrolysates as multifunctional, functional food ingredients for the prevention and control of hypertension and type 2 diabetes. Furthermore, the molecule fucoxanthin, which is an orange-colored pigment found in seaweeds and contributes more than 10% of the estimated total production of carotenoids in nature, is thought to play a role in the prevention of diabetes. Insulin resistance in peripheral tissue is one of the major pathogenic characteristics of type 2 diabetes. The results of work carried out by Hayoto et al. (2007; see also Wang et al. 2014) found that there was a reduction in the plasma insulin level in KK-Ay mice fed 0.2% Fucoxanthin, compared to control mice. Seaweed proteins and peptides are also thought to play a role in the prevention of diabetes and obesity. The compound astaxanthin, a xanthophyll carotenoid found in marine algae such as Hematococcus pluvialis and Chlorella species, is also thought to play a role in cardioprotection and prevention of diabetes. Arunkumar et al. (2012) investigated the mechanisms underlying the insulin sensitivity effects of astaxanthin in a nongenetic insulin-resistant animal model. The results showed that astaxanthin improved insulin sensitivity by activating post-receptor insulin signalling, that is, enhancing the auto-phosphorylation of Insulin Receptor-b (IR-b), IRS-1 associated PI3-kinase step, phospho-Akt/Akt ratio, and GLUT-4 translocation in skeletal muscle (Arunkumar et al. 2012). Obesity is defined as abnormal body weight and can be assessed by measuring the circumference of the waist and by calculating the body mass index (BMI) of an individual. Persons with a BMI greater or equal to 30Kg/m2 are considered obese and the BMI of an individual is calculated as kilogram of body weight/ height (m2) (Henda and Bordenave-Juchereau 2013). Obesity is often the first indicator of metabolic syndrome. Astaxanthin and fucoxanthin are also both thought to play a role in the prevention of obesity (Arunkumar et  al. 2012). Furthermore, seaweed-derived bioactive peptides or cryptides may also have potential use as appetite-suppressive molecules in the prevention and/or treatment of obesity syndrome. An important and promising aspect in the fight against obesity is the study of anorexigenic gut hormones, such as cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1), that are produced by enteroendocrine cells in the presence of nutrients in the gastrointestinal tract. Bioactive peptides, which can be released from food protein, can mimic the effect of CCK and have an influence on satiety. Such peptides could be used as satiating ingredients in the development of new functional foods for the prevention and treatment of obesity. A diet rich in dairy and calcium (Ca) has been variably associated with improvements in body composition and decreased risk of type 2 diabetes (Jones et al. 2013). Jones and colleagues (2013) wanted to assess if this was true and found that a diet rich in dairy and calcium was not

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associated with greater weight loss than control. Modest increases in plasma PYY concentrations with increased dairy/Ca intake, however, did contribute to enhanced sensations of satisfaction and reduced dietary fat intake during energy restriction (Jones et al. 2013).

27.6 Bioavailability of bioactive peptides for food use Following consumption, the gastrointestinal epithelium acts as both a physical and a biochemical barrier to absorption of food-derived bioactive peptides and drugs. The physical barrier is represented by the impermeable gastrointestinal epithelium and the biochemical barrier consists of enzymatic peptidases (Renukuntla et al. 2013). In order for a bioactive peptide to be delivered to its active site it is necessary to fully understand these barriers. The gastrointestinal (GI) tract (Figure  27.2) has site-specific absorption based on the peptide/drug consumed and based on regional differences in pH, enzyme activity, thickness of mucosa, and residence time and surface area. The pH of the GI tract varies from 1 to 7. The bioavailability of protein and peptides depends on their ability to cross the intestinal mucosa and reach the systemic circulation. Transport across the intestinal epithelium may be either active or passive and the mechanisms of transport depend on the physicochemical properties of the INTESTINAL EPITHELIUM

Carrier mediated transport C E L L

Passive transport GI Tract

ATP Active transport

C Y T O P L A S M

Paracellular transport Transcellular transport B L O O D

Transporter

Figure 27.2 Intestinal transport system of peptides. Transport across the intestinal epithelium may be either active or passive and the mechanisms of transport depend on the physicochemical properties of the peptide and on the length of the peptide in question. Active transport of peptides involves movement from low to high concentrations by transmembrane proteins and energy in the form of ATP is used. Passive transport involves diffusion of drug molecules in the direction of the concentration gradient. Carrier-mediated transport involves the movement of molecules via transporters. Detailed understanding of the structural features of a bioactive peptide is needed to target these transports for efficient delivery of the peptide to the target sites.

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peptide and on the length of the peptide in question. Active transport of peptides involves movement from low to high concentrations by transmembrane proteins and energy in the form of adenosine triphosphate (ATP) is used. Passive transport involves diffusion of drug molecules in the direction of the concentration gradient. Carrier-mediated transport involves the movement of molecules via transporters (Renukuntla et al. 2013). Detailed understanding of the structural features of a bioactive peptide is needed to target these transport systems for efficient delivery of the peptide to the target sites.

27.7 Cosmeceutical development Cosmeceuticals are cosmetics that deliver a biologic activity in support of cosmetic claims to provide beneficial topical actions (Zhang et al. 2009). More than 25 different peptides are found in several skin care products in the United States alone and many peptides are currently in development worldwide (Zhang et al. 2009). Peptides are important in the modulation of cell proliferation, cell migration, imflammation, angiogenesis, melanogenesis, and protein synthesis. Peptides used in cosmeceuticals consist of natural L-amino acids that are easily broken down over time to their amino acid constituents. Issues such as reproducible activity, stability and safety, formulation and delivery are important considerations when speaking about the use of peptides in cosmeceuticals (Zhang et al. 2009). Table 27.2 shows a selection of peptides that are found in cosmeceuticals on the market today. Cosmeceutical peptides can improve the appearance of aging skin. Table 27.2 Peptides found in cosmeceutical products as active ingredients. Company

Peptide Name

Bioactivity/Action

Product

Source

Sederma

Dipeptide-2

Eyeliss

Rapeseed

Sederma Sederma

Palmitoyl tetrapeptide-7 Palmitoyl pentapeptide-3

Matrixyl 3000, Rigin Matrixyl

IgG/matrikine Procollagen

Sederma Sederma Pentapharm Pentapharm

Palmitoyl oligopeptide Palmitoyl oligopeptide Oligopeptide-20 Pentapeptide-3

Biopeptide-CL Biopeptide-EL Pepha-timp Vialox

Collagen Elastin TIMP-2 Not known

Atrium

Acetylpeptide-1

Melitane

MSH agonist

Atrium

Nonapeptide-1

Melanostatine

Lipotec

Hexapeptide-10

Lymph drainage via ACE inhibition Elasticity via IL6 reduction Collagen stimulation via signalling Retinoic acid like activity Increases collagen and HA MMP inhibitor via TIMP Botox-like via acetylcholine receptor Melanin increase via MSH regulation Tryosinase activation inhibition Increases cell proliferation and laminin V

MSH antagonist Laminin

Serilesine

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There are a number of cosmeceutical peptides such as signal peptides, carrier peptides, and neurotransmitter-inhibiting peptides that mimic the actions of Botox. In general, cosmeceutical peptides activate wound healing mechanisms that can activate fibroblasts in response to fragmented chains of elastin and collagen. Cosmeceutical peptides can increase collagen production to improve skin appearance, giving smoother, younger-looking skin (Lupo 2005; Zhang et al. 2009). Recent research indicates that metabolism, gene expression, and aging intersect at a molecular level and molecular biology plays a key role in cosmeceutical development. Products are now developed to target molecular targets (Prakash and Majeed 2009). To understand the role of cosmeceutical peptides in skin care and as cosmeceutical ingredients, we must first look at the structure of skin.

27.8 Skin structure Skin consists of three layers: the epidermis, the dermis, and the hypodermis (Pierard 1999). A basement membrane rich in extracellular matrix proteins (ECM) including collagen, epilugrin, laminin, fibronectin, elastins, heparin sulphate proteoglycans, and nidogen separate the epidermis from the dermis layers (Zhang et al. 2009). The dermis provides a support matrix for extensive vascular and nerve networks. The dermis is composed of ECM collagen of which there are 16 different types. Type I collagen is the primary constituent of the dermis. The dermis also contains approximately 10% type III collagen. Both type I and type III collagen are fibrillar and rod-shaped (Engel et al. 2003). ECM provides support and influences cellular behavior through signalling by matrix components to cells through cell-membrane receptors (Zhang et al. 2009). Signalling molecules are produced by proteolysis of ECM to produce soluble peptides known as matrikines (Maquart et  al. 2004). Matrikines are divided into two classes: natural matrikines, which signal directly from the extracellular matrix, and cryptic matrikines, which require proteolysis to release the ligand from the ECM protein. In 2004 it was discovered that ECM components contained domains that can interact with and activate receptors with intrinsic tyrosine kinase activity and this results in cell responses including proliferation, migration, differentiation, and dedifferentiation (Tran et al. 2004).

27.9 Peptides in cosmeceuticals Peptides derived from collagen that are known ingredients in cosmeceutical products include the peptide derived from procollagen I with the amino acid sequence KTTKS, a subfragment of procollagen I (residue 197-241). This peptide is capable of stimulating collagen production and fibronectin in a dose- and

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time-dependent manner (Katayama et al. 1993). It is found in a product produced by Sederma. In addition to collagen, elastin is found in the ECM. Elastin has the peptide sequence VGVAPG in the hydrophobic region. Elastokines promote cell cycle progression and chemotactic activity and matrix metalloproteases (MMP) up-regulation activities. VGVAPG is an elastin peptide with these bioactivities. It stimulates wound healing and is found in a Sederma product known as Bio-peptide-EL. Shorter peptides such as GHK, which was isolated from human plasma and is known for its copper binding activities, is also used in wound healing preparations in dermatology. Marine-derived peptides are also used as cosmeceutical ingredients.

27.9.1 Mechanism of action of cosmeceutical peptides Marine-derived peptides can act as enzyme inhibitors, neurotransmitter inhibitors, or carrier peptides in Cosmeceuticals (Gorouhi and Maibach 2009). Enzyme inhibitory peptides directly or indirectly inhibit an enzyme and a number of enzyme inhibitory peptides have been identified in relation to functional food development including ACE-I inhibitory peptides. Soy and rice protein–derived peptides and proteins are frequently used as antiaging, anti-UVA/UVB agents. Rice peptides known as Colhibin® inhibits MMP activity and induces the hyaluronase synthase 2 gene in keratinocytes. Antiaging and hair-conditioning products often contain this peptide formulation (Sim et al. 2007). A number of ACE-I inhibitory peptides were identified from marine algal sources to date (Hayes et al. 2012; Harnedy and FitzGerald 2013) that could play a role in cosmeceutical products. Previously, the ACE-I inhibitory peptide VW derived from rapeseed, which reduces blood pressure when delivered orally was marketed as a cosmeceutical ingredient as Dipeptide-2 by Sederma and is used in conjunction with a second peptide hesperidin to protect vasculature, prevent inflammation, and oxidation. It is marketed as Eyeliss and helps promote lymph drainage through ACE-I inhibition (Zhang et al. 2009). Renin inhibitory peptides could also play a role in lymph drainage as they are important in the control of blood pressure regulation and saltwater balance in the body. Neurotransmitter inhibitor peptides inhibit acetylcholine release at the neuromuscular junction and have curare-like effects (Gorouhi and Maibach 2009). As mentioned previously, a number of ACE-I inhibitory peptides were isolated previously from dairy sources including the lactotripeptides IPP and VPP. It is well known that Botox acts on peripheral cholinergic neurons where they selectively proteolyse synaptosome-associated proteins (Gorouhi and Maibach 2009). Peptides such as Tripeptide-3 (Syn(R)-Ake), which mimics the effect of the natural peptide Waglerin 1, derived from Tropidolaemus wagleri, is able to reduce the frequency of innervated muscle cell contractions by 82% after 2 hours of treatment (Gorouhi and Maibach 2009) in a similar manner to Botox.

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Carrier peptides act as transporters of important substances such as trace elements including copper (Cu) and Magnesium (Mn), which are required for wound healing and enzymatic processes. Penetrating and membrane transduction peptides accelerate delivery of bioactive molecules in the skin. Arginine-rich peptides are known to transport various proteins (Hou et al. 2007).

27.9.2 Tyrosinase inhibitors from macroalgae as cosmeceuticals: Phlorotannins According to the World Health Organization (WHO), the incidence of nonmelanoma and melanoma skin cancers has increased over the past decade. Two to 3 million nonmelanoma skin cancers and 132,000 melanoma skin cancers occur globally each year (WHO 2001). Melanin is an effective absorber of light and is able to dissipate over 99.9% of absorbed UV radiation (Meredith and Reisz 2004). It is thought that the protection of skin cells from Ultraviolet B (UVB) radiation damage reduces the risk of cancer. Furthermore, though exposure to UV radiation is associated with increased risk of malignant melanoma, studies have shown a lower incidence of skin cancer in individuals with more concentrated melanin (Ebanks et al. 2009). Melanin is a natural, tyrosine-derived pigment produced by a specialized group of cells known as melanocytes, which are controlled by the enzyme tyrosinase that also catalyses the rate-limiting step of the biosynthetic pathway of melanin (Ebanks et al. 2009). Tyrosinase is a well-known copper-containing enzyme widely distributed in microorganisms, plants, and animals (Chang 2012). This enzyme is also involved in the detoxification of host plant defensive phenols, the scerotization of insect cuticles, and the synthesis of amino acid–based antibiotics (Chang 2012). Tyrosinase inhibitors are used widely in dermatological treatments, and are also applied in cosmetic treatments. Moreover, the inhibition of tyrosinase activity or its production can prevent melanogenesis and darkening of the skin (Chang 2012). As mentioned previously, marine brown algae (phaeophyta) or seaweeds accumulate a variety of phloroglucinol-based polyphenols, which are known as phlorotannins (Kang et al. 2004). These phlorotannins consist of phloroglucinol units linked to each other in various ways, and these could be involved in the control of pigmentation in plants and other organisms through inhibition of tyrosinase activity. They have potential to be used as functional ingredients in the cosmeceutical industry. Figure 27.3 outlines an extraction process that may be employed to generate phlorotannin-rich extracts from brown macroalgae. In some brown algae, phlorotannins comprise 20% of the dry tissue weight. As they can have up to eight interconnected rings of phloroglucinol (phenol rings), they are more potent free radical scavengers than other polyphenols and have strong antioxidant activities. The phenol rings act as electron traps to scavenge peroxy and hydroxyl radicals along with superoxide anions (Tierney et al. 2010).

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1. Seaweed 2. 200 mg sample 3. Remove lipids (fats) from the seaweed using ® ASE method 4. Vortex and repeat step 3 four times 5. Centrifugate at 13.000 rpm for 7 minutes 6. Pool the supernatantthe liquid portion 7. Dry under nitrogen (N2) 8. Add 7:3 acetonewater to dried samples and add ascorbic acid (0,3 %) 9. Vortex repeat, pool fraction and dry

Phlorotannins extracts

Figure 27.3 Procedure used to extract phlorotannins from brown seaweeds. Schematic representation of the steps required to isolate phlorotannin-containing extracts from brown seaweeds. Adapted from Koivikko et al. (2008).

27.10 Marine ingredients and skin conditions including dermatitis In Asia, fair skin is desired by many and tyrosinase inhibitors in skin-whitening products are the number one seller in many Asian countries. Most skin or mucosal diseases result from inflammation caused by inflammatory agents such as bacteria, fungi, or viruses. The most common skin diseases include eczema, psoriasis, and dermatitis including contact dermatitis, atopic and seborrheic dermatitis. Inflammation results from accumulation of reactive oxygen species (ROS) which play a role in photo-aging as they can cause oxidative damage of cellular DNA and proteins causing aging of skin and skin cancers (Tierney et al. 2010; Agatonivic-Kustrin et al. 2013). Fibrillar collagen is degraded as a result of up-regulation of MMPs due to oxidation and photo damage. Carotenoids, such as fucoxantin and neoxanthin, found in red algae can act as direct quenchers of ROS (Agatonivic-Kustrin et al. 2013). These carotenoids are lipophilic and accumulate in compartments like membranes or lipoproteins from where they can readily react with peroxyl radicals and protect cellular mechanisms (Stahl and Seis 2005). As mentioned in Chapter 21, bioactive compounds can play a preventative role in inflammatory diseases. Atopic dermatitis (AD) is an inflammatory skin disorder associated with a family history of allergy and can occur at any age but particularly affects children. Phlorotannins from brown algae have known anti-inflammatory and hyaluronidase inhibitory activities (Thomas and Kim 2013). Phloroglucinol along with five other phlorotannins generated from the brown algae Eisenia bicyclis and Ecklonia kurome were tested for their hyaluronidase inhibitory activities. It is reported that, in vitro, these phlorotannins

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had a stronger inhibitory effect on hyaluronidase activity. This suggests that polyphenolic, marine-derived compounds could be used as anti-inflammatory substances in cosmeceuticals to treat atopic dermatitis (Shibata et  al. 2002; Thomas and Kim 2013). Recently Artym and Zimecki (2013) carried out a review of milk-derived proteins and peptides in clinical trials. One study mentioned in this review was a multicenter, double-blind, randomized trial of an α-lactalbumin (LA)–enriched and symbiotic-supplemented infant formula that was tested for safety, tolerance, and prevention of atopic dermatitis (Rozé et  al. 2012). The infants fed the experimental formula exhibited less crying and agitation as well as less frequent manifestation of atopic dermatitis. Growth was similar in infants fed experimental and standard (Rozé et al. 2012).

27.11 Market and regulation of cosmeceutical products The Food and Drug Administration (FDA) does not recognize the term “cosmeceutical,” although it is used in the industry to refer to cosmetic products that have medicinal or drug-like benefits (Brandt et al. 2011). As they are neither pharmaceuticals nor cosmetics claims made by manufacturers for cosmeceuticals are investigated by the Federal Trade Commission whose function is to look at advertised claims of pharmaceutical properties for scientific validation. There are over 400 cosmeceutical manufacturers worldwide including Procter & Gamble, Estée Lauder, Avon, and L’Oréal and skin care cosmeceuticals account for 80% of the total U.S. and European cosmeceutical market (Brandt et al. 2011). Cosmeceuticals are not regulated as such in the European Union, United States, or Japan. In the EU most are considered cosmetics; in the United States, most are seen as drugs that probably have not been approved by the FDA. In Japan, they are regulated as quasi-drugs. A recognized, legal definition of a cosmeceutical is not known at present, which is not the case for cosmetics and drugs (Sharma 2011). In the United States, a report titled “Classification and Regulation of Cosmetics and Drugs: A Legal Overview and Alternatives for Legislative Change” suggested the inclusion of a third category of cosmeceuticals that would include sunscreens and other products that fell between drugs and cosmetics. In the United States, the Food, Drug and Cosmetic Act governs products that are either in the category of “drugs” or “cosmetics.” The introduction of this third category met with resistance from companies heavily invested in cosmeceuticals (Brandt et al. 2011). In 2005, the estimated value of antiaging cosmeceuticals was U.S. $150 million. This was mostly driven by the Baby Boomer generation (Kumar 2005). Economies such as China, Brazil, and the Russian Federation as well as India are the anticipated growth markets for cosmeceuticals (Brandt et al. 2011).

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27.12 Market and regulation of functional foods European regulations on nutrition and health claims became effective on January 19, 2007, and each EU member state now has a common regulation allowing health claims on foods. The standards set out by the European Food Safety Authority (EFSA) are considered the “gold standard” with regard to regulation and toughness regarding review (Hayes 2012). Bioactive component use is regulated under the General Food Law (178/2002/EC), which assigns legal responsibility for the safety of products to the operator. Article 2 of the General Food Law states that food means any substance or product intended to be, or reasonably expected to be, ingested by humans excluding (a) feed, (b) live animals, (c) plants prior to harvesting, (d) medicinal products within Directive 2004/27/EC, (e) cosmetics within Directive 76/768/EC, tobacco, narcotics, or residues and contaminants. If considered a food the product is categorized as one of the following: novel food (Directive 258/1997/EC); food for particular nutritional use, covering dietetic foods (Directive 89/398/EC); food additives (Directive 89/107/EC); food supplements (Directive 2002/46/EC); or flavorings (Directive 91/71/EC completing Directive 88/388/EC). If making a health claim, the claim relates to any claim that “states, suggests or implies that a relationship exists between a food category, a food or one of its constituents and health”. Specific conditions and requirements for use of health claims can be found in Chapter IV of Regulation 1924/2006/EC. In Japan the Ministry of Health, Labour and Welfare uses the Foods of Specified Health Use (FOSHU) as a regulatory system to approve the statements made on functional food labels and in China there are 27 categories of product-specific health claims that are related to reduction of disease risk or are function related (Hayes 2012).

27.13 Conclusion The functional foods and cosmeceutical markets have grown significantly in recent years and as a result the landscape of research into this area continues to develop. The marine environment provides a myriad of protein-rich resources from which bioactive peptides and small molecules can be isolated and purified. This review details examples of where known peptides and small molecules are of use in these markets. Extensive research should continue to improve the bioavailability of these compounds and new applications of bioactive peptides may emerge in time. Marine proteins as an alternative to plant and animal proteins is an area with great potential, especially if these proteins are targeted at key consumer groups including older adults, infant nutrition, and sports professionals. Some research has been conducted to date in this area. For example, ingestion of the hydrolyzed marine protein supplement NutriPeptinTM, a processed protein supplement with potentially beneficial amino acid composition, together with a

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PROCHO beverage, improved ergogenic effects on mean power performance in cyclists (Vegge et al. 2012). However, issues regarding cost of production and scale will need to be overcome before marine proteins are used in everyday food products.

Acknowledgments The authors would like to acknowledge the NutraMara Programme. NutraMara is carried out under the Sea Change Strategy with the support of the Marine Institute and the Department of Agriculture, Food and the Marine, funded under National Development Plan 2007–2013.

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Index

3D matrix materials collagen 559 fibronectin 559–60 glycosaminoglycans 560 laminin 560 stem cell research 558–60 AAS see androgenic anabolic steroids ACE inhibitors see angiotensin I converting enzyme inhibitors acetoxypicrasine B: 540 acid hydrolysis, glycosides 306 acidophiles 251–2 applications 246 biomolecules 246 enzymes 246, 252, 253 acidothermophiles 256–7 acronycine 542 acyl2SGL mimetics antitubercular vaccines 363–5 structure 365 Aegle marmelos (L.) Correa 164–9 history and distribution 164 medicinal uses 167–9 phytochemistry 167 plant description 167 age-related diseases free-radical reactions 317 glycosides 303, 317 ajmalicine 183 structure 184 ajmaline 183 structure 184 alcoholic and phenolic glycosides 317–18 aldehyde glycosides 319 alkali hydrolysis, glycosides 306 alkaliphiles 251 applications 246

biomolecules 246 enzymes 246, 251, 252 alkaloids antimicrobial 131 screening 545 alkylresorcinols cereals 113–14 health benefits 114 properties 114 structure 114 allamandin 539 allergenicity, bioactive peptides 517–18 allergies, antiallergic compounds 214–15 Amaryllidaceae alkaloids 543 AMF see arbuscular mycorrhizal fungi amygdalin 321 biosynthesis 319 androgenic anabolic steroids (AAS) 417 angiotensin I converting enzyme (ACE-I) inhibitors 486, 674–7 fungal 215 heart health 674–7 proline 486 anthocyanin acylation 468–9 anthocyanin biosynthesis 464–70 anthocyanin modification 467–9 basic flavonoid upstream pathway 464–6, 469–70 transcriptional regulation 469–70 anthraquinone glycosides 319–22 anti-inflammatory properties, phytosterols 573 antiallergic compounds, fungal 214–15 antibacterial compounds see also antimicrobial… bioactive peptides 487–8 biotechnology-based 448–51 lactic acid bacteria (LAB) 452–3

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

693

694

Index

antibacterial compounds (cont’d) lactoferrin (LF) 487–8 novel strategies 448–9 Streptomyces sp. 449 terpene derivatives 450 anticancer compounds see also antitumor compounds alternative sources/techniques 446–7 bacterial metabolites 443–6 biotechnology-based 435, 440–7 fungal metabolites 441–3 structure 435 anticariogenic activity, casein phosphopeptides (CPPs) 490 antifungals antimicrobial compounds 452–3 Bacillus sp. 453 biotechnology-based 452–3 antigenotoxic potential, casein phosphopeptides (CPPs) 490–1 antihypercholesterolemic compounds, fungal 204–7 antihypercholesterolemic phytosterols 417–18 antihyperglycemic compounds, fungal 213–14 antihyperglycemic effects, phytosterols 575 antihyperlipidemic compounds, fungal 204–7 antimicrobial compounds antibacterial compounds 448–51 antifungals 452–3 bioactive peptides 487–8 biotechnology-based 447–53 fungal 211–12 lactoferrin (LF) 487–8 antimicrobial phytochemicals 128–33 alkaloids 131 efflux pumps 133 mode of action 131–3 phenolic compounds 129, 130 quorum sensing (QS) 133 terpenes 129–31 antimicrobials from medicinal plants 123–41 bioactive compounds 125 biodiversity conservation 135–6 drying process 134 extraction systems 134–5 historical aspect 123–4 junipers 127 plant extracts 125 prioritization 135–6 production schematic 135

research initiatives 126–8 source identification 133–4 threatened medicinal plants (TMPs) 136 antioxidant activity Cantharellus sp. 202–3 Coccoloba uvifera components 151–9 D-pinitol 386–90 fungal bioactive compounds 195–204 Ganoderma sp. 203–4 Grifola gargal 200 Pleurotus sp. 200–3 polyphenols 67, 76–8 steroids 422 antioxidant compounds bacterial metabolites 439–40 biotechnology-based 434–40 fungal metabolites 435, 437–9 Graphislactone A 435, 437 isopestacin 435, 437 modes of action 434 pressurized hot water extraction (PHWE) 76–8 structure 435 antioxidants chemical treatment 47–8 fresh-cut fruits 47–8 protocol for in vitro detection 546–7 antioxidants, oxidants and free radicals: the balance 537 antioxidants (phytochemicals), celiac disease (CD) 591 antitubercular vaccines, acyl2SGL mimetics 363–5 antitumor compounds see also anticancer compounds C-phycocyanin (C-PC), anticancerous activity 293 carcinogenicity 544 casein phosphopeptides (CPPs) 490–1 Cibacron blue affinity eluted protein (CBAEP) 209 Fomes fomentarius 210 fungal 208–10 Ganoderma sp. 208–9 medicinal plants 538–9 Paecilomyces sp. 209 Polyporaceae family 208–9 antitumoral activity brassinosteroids (BRs) 421 ergosterol 419–20

Index phytosterols 573 steroids 419–22 antiviral compounds fungal 212–13 Ganoderma sp. 212–13 aphenanthridine 543 apple fruit (Malus domestica) by-products 26–7 characteristics 42–3 chemical treatment 47–8 dietary fiber 27 modified atmosphere packaging (MAP) 50–1, 53–5 phenolic compounds 27 variability of phytochemicals 42–3 arabinoxylans beer technological impacts 106 bread technological impacts 105–6 cereals 104–7 dietary fiber 107 ester bonds 105 starch technological impacts 106–7 structure 104–5 technological impacts 105–6 arbuscular mycorrhizal fungi (AMF) 225–37 agronomic potential 230–2 Argentina 231 biocompounds 227–32 Brazil 231–2 dark septate endophytes (DSE) 228–9, 231, 234–6 ectomycorrhizas and biocompounds 232–4 medicinal plants 236–7 Mediterranean ecosystems 231 mycorrhizal networks 232 plant fungal endophytes 226–7 symbiosis of plant roots 228–30 arbutin, biosynthesis 318 Argentina, arbuscular mycorrhizal fungi (AMF) 231 aristolochene 170, 171 Aristolochia indica L. Aristolochiaceae 169–70 history and distribution 169 medicinal uses 170 phytochemistry 170, 171 plant description 170 Artemisia annua, medicinal properties 124 artemisinic acid 381–6 antiaging targets 381–6 molecular targets, predicting 381–6 PharmMapper 382–5

695

structure 381–2 virtual screening (VS) 381–6 artichoke (Cynara scolymus L.) 16–17 by-products 16–17 phenolic compounds 16–17 aryl hydrocabon hydroxylase (AHH) antioxidants detection 546–7 determination of activity 547 ascorbic acid see also vitamin C modified atmosphere packaging (MAP) 54–5 oranges 54–5 role 38 sources 38 ASD see autism spectrum disorders astaxanthin, obesity prevention 678 α-atlantone 181 atmosphere composition effect, fresh-cut fruits 48–55 atoms, molecules, compounds, macromolecules, polymers 527–8 aurapten 167, 168 autism spectrum disorders (ASD), bioactive peptides treatment 512–13 avenanthramides, cereals 113 β-globin sickle cell anemia 533–6 structure 533–6 β-glucans cereals 107–10 dietary fiber 107–9 fat replacers 109 food applications 108–10 health benefits 109–10 properties 108–10 water-binding capacity 109 Bacillus sp., antibacterial compounds 453 bacterial metabolites anticancer compounds 443–6 antioxidant compounds 439–40 cyanobacteria 444 marine actinomycetes 445 Streptomyces sp. 445 baicalein 179 structure 179 baliospermin 172 Baliospermum montanum (willd.) Muell-arg (Euphorbiaceae) 170–2 history and distribution 170–1 medicinal uses 172

696

Index

Baliospermum montanum (willd.) Muell-arg (Euphorbiaceae) (cont’d) phytochemistry 172 plant description 171 basic flavonoid upstream pathway anthocyanin biosynthesis 464–6, 469–70 grapes 464–6, 469–70 beer technological impacts, arabinoxylans 106 beet (Beta vulgaris L.) by-products 22–4 flavonoids 23 phenolic compounds 22–3 beneficial health effects, vegetables and fruit products 4–5 benzo α phenanthridine alkaloids 541 benzo(1,2,3)thiadiazole-7-carbothioic acid s-methyl ester (BTH), elicitation 472–3 berberine 173–4 berberines 541 Berberis asiatica, medicinal properties 137 Bergenia sp., medicinal properties 128 berries anthocyanin biosynthesis 464–70 anthocyanin modification 467–9 metabolic engineering 463–75 resistance to pathogens and pests 470–5 secondary metabolites 463–75 BH see biohydrogenation BHA see butylated hydroxyanisole BHT see butylated hydroxytoluene bile acids, steroids 415–16 bioactive lipid components from ruminants milk and meat 599–620 biohydrogenation 603–6, 608–10 biohydrogenation (BH) 601–2 characteristics 599–600 conjugated linoleic acid (CLA) 617–20 diabetes 614–17 farming systems effects 612–20 feed additives effects 608 feed modulation effects 608–12 feeding systems effects 612–20 lipolysis 600–1, 603–6 obesity 614–20 oils effects 611–12 plant secondary metabolites effects 608 rumen metabolism as a source 600–7 sources 600–7 ‘western’ disease prevention 614–20

bioactive peptides 483–98 see also bioactive proteins allergenicity 517–18 angiotensin I converting enzyme (ACE-I) inhibitors 486, 674–7 autism spectrum disorders (ASD) treatment 512–13 bioavailability 519, 679–80 BioPD databases 516 BIOPEP databases 515–16 bioprocess-based research challenges 496–7 casein phosphopeptides (CPPs) 490–1 chymotryptic peptides 491 clinical trials 510–12 commercially available food-derived 507–9 computational (in silico) peptidomics 514–16 defining 669–70 dermopharmaceutical products 513–14 egg-derived multifunctional proteins and peptides 491–2 emerging technologies 514–16 EROP-Moscow databases 516 European Food Safety Authority (EFSA) 510–12 food allergens 517–18 food-derived multifunctional proteins and peptides 507–20 Food for Specified Health Uses (FOSHU) 509 foodomics 514–16 fractionation 496–7 future projections 520 heart health 674–7 in silico (computational) peptidomics 514–16 isolation 496–7 lactoferrin (LF) 486–9, 518–19 microbial proteases used in production 495 microbial resistance 518–19 milk protein-derived peptides 485, 486–91 MilkAMP database 516 multifunctional food peptides 485–91, 507–20 nanofiltration 497 NeuroPred databases 516 nutraceuticals 507–9 ‘omic’ methodologies 514–16 overview 483–5

Index PepBank databases 516 PeptideCutter databases 516 pharmaceutical products 512–14 plant protein-derived multifunctional peptides 492–4 POPS databases 516 precipitation 497 production 494–7 proline 486 purification 497 quantitative structure activity relationship (QSAR) modelling 516–17 rapakinin 493 recent advances 514–16 renin inhibitory peptides 674–7 research challenges 496–7 Rubisco-derived peptides 493 seaweed 674–7 sources 484–5 soymorphins 493–4 stability 519 SwePep databases 516 SWISS–PROT databases 516 ultrafiltration 497 bioactive proteins see also bioactive peptides autism spectrum disorders (ASD) treatment 512–13 clinical trials 510–12 dermopharmaceutical products 513–14 egg-derived multifunctional proteins and peptides 491–2 lysozyme 491–2 ovalbumin 492 pharmaceutical products 512–14 phosvitin 492 bioavailability bioactive peptides 519 bioactive peptides, food use 679–80 fungal steroids 402–3 mycosteroids 402–3 phytosterols 400–1 steroids 400–3 zoosteroids 401–2 biocatalyst immobilization, glycosides 309–11 biodiversity conservation antimicrobials from medicinal plants 135–6 Himalayan region 135–6 threatened medicinal plants (TMPs) 136

697

biohydrogenation (BH) bioactive lipid components 601–2, 603–6, 608–10 rumen metabolism 601–2, 603–6, 608–10 rumen populations responsible 603–6 BioPD databases, bioactive peptides 516 BIOPEP databases, bioactive peptides 515–16 bioprocess design, glycosides 308–11 biotechnology-based bioactive compounds 433–54 anticancer compounds 435, 440–7 antimicrobial compounds 447–53 antioxidant compounds 434–40 biotic scaffolds for tissue regeneration, stem cell research 558–60 bisindol alkaloids 542 BMPs see bone morphogenic proteins bone morphogenic proteins (BMPs), stem cell research 557 brassinosteroids (BRs), antitumoral activity 421 Brazil, arbuscular mycorrhizal fungi (AMF) 231–2 bread technological impacts, arabinoxylans 105–6 BRs see brassinosteroids bruceatin 540 BTH see benzo(1,2,3)thiadiazole-7-carbothioic acid s-methyl ester BTN see butyrophilin bufadienolides, biosynthesis 325–6 butylated hydroxyanisole (BHA), limitation 152 butylated hydroxytoluene (BHT), limitation 152 butyrophilin (BTN) 635–6, 640, 644–5 by-products fruit 24–30 phytochemicals in 3 from processing vegetable and fruit products 5–11 vegetable 11–24 C. elegans model D-pinitol, predicting molecular targets 386–90 protein homologues identification 387–90 C-PC see C-phycocyanin C-phycocyanin (C-PC) 283–93 anticancerous activity 293 applications 290–3

698

Index

C-phycocyanin (C-PC) (cont’d) characterization 289–90 cosmetics additive 291 diagnostic applications 291–2 food additive 291 future prospects 293 heterotrophic production 286–7 isolation 287–8 mixotrophic production 286 as a natural dye 290–1 nutraceutical applications 292–3 organisms producing 285 pharmaceutical applications 292–3 photoautotrophic production 286 production 284–90 purification 289–90 recombinant production 287–90 CA see carbonic anhydrase Calotropis procera, medicinal properties 127 camptothecin alkaloids 542 cancer see also anticancer compounds; antitumor compounds carcinogenicity of antitumor compounds 544 resistance to cancer drugs, drug combinations 544 skin cancer 683 tumor promoters, plant 544 Cantharellus sp., antioxidant activity 202–3 carbonic anhydrase (CA) 650–1 carcinogenicity of antitumor compounds 544 cardenolides 540 biosynthesis 323–5 cardiac glycosides 322–6 bufadienolides 325–6 cardenolides 323–5 carotenoids carrot (Daucus carota L.) 17–19 characteristics 38 citrus fruit by-products 24–6 structure 10–11 tomato (Solanum lycopersicum L.) 13–16 tropical fruits 29–30 carrot (Daucus carota L.) by-products 17–19 carotenoids 17–19 lycopene 18 phenolic compounds 17–19

casein phosphopeptides (CPPs) anticariogenic activity 490 antigenotoxic potential 490–1 milk protein-derived peptides 490–1 CBAEP see Cibacron blue affinity eluted protein CD see celiac disease CD36 647–8 CD59 646–7 celiac disease (CD) 583–92 antioxidants (phytochemicals) 591 conjugated linoleic acid (CLA) 591–2 fatty acids 591–2 gliadins-mediated alterations in intestinal epithelium 584–6 gluten-free diet (GFD) 583–4 iron 589–90 micronutrients 589–90 nonalcoholic steatosis (NASH) 588 phytochemicals (antioxidants) 591 polyunsaturated fatty acids (PUFA) 591–2 pre- and probiotics 588–9 selenium 589–90 vitamins A, C, and E 590–1 zinc 589–90 cellulase, flavonoid glycosides 332–3 cephalotaxus alkaloids 542 cereals 103–15 see also celiac disease (CD) alkylresorcinols 113–14 arabinoxylans 104–7 avenanthramides 113 β-glucans 107–10 bioactive components 586–8 bioactive compounds 104–14 dietary fiber 107–12 phenolic acids 112–13 phenolic compounds 112–14 resistant starch (RS) 110–12 wheat-based 587 whole-grain food, defining 104 Chelidonium majus, antimicrobial properties 127 chemical treatment antioxidants 47–8 apple fruit (Malus domestica) 47–8 color preservation 47 fresh-cut fruits 40–1, 47–8 chitosan, elicitation 474–5

Index cholesterol antihypercholesterolemic compounds 204–7 antihypercholesterolemic phytosterols 417–18 hypocholesterolemic action, phytosterols 565–76 cholesterol absorption effects, phytosterols 416 chrysin 179 structure 179 chymotryptic peptides 491 Cibacron blue affinity eluted protein (CBAEP), antitumor compounds 209 cis-aconitic acid 543 citrus fruit by-products 24–6 carotenoids 24–6 dietary fiber 24–5 flavonoids 25–6 limonoids 26 phenolic compounds 24–5 CLA see conjugated linoleic acid CLEAs see cross-linked enzyme aggregates clinical trials bioactive peptides 510–12 European Food Safety Authority (EFSA) 510 protein hydrolysates 510–12 clusterin 651–2 Coccoloba uvifera, antioxidant activity of components 151–9 antioxidant properties 156–7 chelation of metal ions Cu2+ and Fe2+ 154–5, 158 DPPH free radical scavenging assay 154, 157–8 ferric-reducing power assay 155, 158–9 in vitro antioxidant properties 154–5 materials and methods 153 proximal composition 155–6 total titratable acid (TTA) 153, 156 Trolox equivalent antioxidant capacity 154, 157 colchicine 542 collagen 3D matrix material 559 stem cell research 559 color preservation chemical treatment 47 fresh-cut fruits 47

699

commercially available food-derived bioactive peptides 507–9 compounds, macromolecules, atoms, molecules, polymers 527–8 computational (in silico) peptidomics, bioactive peptides 514–16 condensed tannins, pressurized hot water extraction (PHWE) 88–9 conjugated linoleic acid (CLA) bioactive lipid components from ruminants milk and meat 617–20 celiac disease (CD) 591–2 metabolic effects 617–20 connective tissue degradation, gliadins-mediated alterations in intestinal epithelium 585 consumer demand 3 continuous-flow stirred tank reactor (CSTR), glycosides 309, 311 cortisone 415 structure 336, 416 Coscinium fenestratum (Gaertn.) Coleb. (Menispermaceae) 173–5 description 173 distribution 173 history 173 medicinal uses 174–5 phytochemistry 173–4 cosmeceuticals 670 development 680–1 market size 685 mechanism of action 682–3 peptides 680, 681–3 regulation 685 tyrosinase inhibitors 683–4 cosmetics additive, C-phycocyanin (C-PC) 291 coumarate, structure 185 CPPs see casein phosphopeptides cross-linked enzyme aggregates (CLEAs), glycosides 310 crotepoxide 543 CSTR see continuous-flow stirred tank reactor cucurbitacin 540 cyanobacteria, bacterial metabolites 444 cyanogenic glycosides 319, 321 cymarin 540 cynaropicrin, structure 181 cytokines role, gliadins-mediated alterations in intestinal epithelium 585

700

Index

D-pinitol antioxidant activity 386–90 molecular targets, predicting 386–90 dark septate endophytes (DSE), arbuscular mycorrhizal fungi (AMF) 228–9, 231, 234–6 de-novo synthesis of fatty acids in ruminant tissue 606–7 Decalepis hamiltonii Wight & Arn. (Asclepediaceae) 175–7 history and distribution 175 medicinal uses 176–7 phytochemical constituents 176 plant description 175–6 deoxyelephantopin 539 deoxynojirimycin 355–7 structure 356 dermatitis 684–5 dermopharmaceutical products, bioactive peptides and proteins 513–14 DHT see dihydrotestosterone diabetes antihyperglycemic fungal compounds 213–14 bioactive lipid components from ruminants milk and meat 614–17 marine molecules for treating 677–9 dietary fiber apple fruit (Malus domestica) 27 arabinoxylans 107 β-glucans 107–9 cereals 107–12 citrus fruit by-products 24–5 onion (Allium cepa L.) 20 resistant starch (RS) 110–12 soluble 110–12 soluble/insoluble 10–11 dihydrotestosterone (DHT), structure 416 diosgenin, structure 336 diterpene alkaloids 541 diterpenes 539 DNA methylation, mechanism by which food affects genes 538 DNA repair by direct reversal pathway 536–7 drug combinations, resistance to cancer drugs 544 drying process, antimicrobials from medicinal plants 134 DSE see dark septate endophytes

ectomycorrhizas and biocompounds, arbuscular mycorrhizal fungi (AMF) 232–4 efflux pumps, antimicrobial mode of action 133 EFSA see European Food Safety Authority egg-derived multifunctional proteins and peptides 491–2 lysozyme 491–2 ovalbumin 492 phosvitin 492 elemanolides 539 elicitation benzo(1,2,3)thiadiazole-7-carbothioic acid s-methyl ester (BTH) 472–3 chitosan 474–5 fruits 463, 471–5 harpin 474 metabolic engineering 463, 471–5 methyl jasmonate (MeJ) 473 secondary metabolites enhanced production 471–5 ellipticines 543 emetine 543 empirical equations, polyphenols solubility measurement 72–3 endophytes, plant fungal 226–7 enzymatic hydrolysis, glycosides 306–11 enzyme aggregates, cross-linked enzyme aggregates (CLEAs) 310 enzyme-resistant starch see resistant starch (RS) enzymes deglycosylation 306–7 extremophiles 245–55 flavonoid glycosides 331–3 ergosterol, antitumoral activity 419–20 EROP-Moscow databases, bioactive peptides 516 essential oils polyphenols 81 pressurized hot water extraction (PHWE) 81 ruminant products 610 ester bonds, arabinoxylans 105 etoposide 540 EU directives, vegetable and fruit processing 7–8 Eugenia singampattiana bedd. (Myrtaceae) 177–8 history and distribution 177 medicinal uses 178

Index phytochemistry 178 plant description 177 European Food Safety Authority (EFSA) bioactive peptides 510 clinical trials 510 extraction and pretreatment systems, vegetable and fruit by-products 30–2 extraction systems, antimicrobials from medicinal plants 134–5 extremophiles 245–59 acidophiles 246, 251–2 acidothermophiles 256–7 alkaliphiles 246, 251 biomolecules 245–6 enzymes 245–55 haloalkaliphiles 256, 258 halophiles 246, 252–5 halophilic alkalithermophiles 258 metallophiles 246, 256 piezophiles 246, 255 polyextremophiles 256–9 psychrophiles 246, 249–50 radiophiles 246, 256 thermophiles 246–8 farming systems effects, bioactive lipid components from ruminants milk and meat 612–20 fat replacers, β-glucans 109 fatty acid profiles, ruminant products 608–12 fatty acids see also bioactive lipid components from ruminants milk and meat celiac disease (CD) 591–2 de-novo synthesis in ruminant tissue 606–7 feeding strategies effects 614–16 FBR see fluidized-bed reactor feeding systems effects, bioactive lipid components from ruminants milk and meat 612–20 ferulate, structure 185 FGF see fibroblast growth factor fiber see dietary fiber fibroblast growth factor (FGF), stem cell research 556–7 fibronectin 3D matrix material 559–60 stem cell research 559–60 flavones, lettuce (Lactuca sativa L.) 12–13

701

flavonoid glycosides 327–35 biocatalysis 331–3 biologic activity 334–5 cellulase 332–3 classification 327–30 enzymes 331–3 hydrolases 333 laccase 332 lipases 331 microbial transformation 333–4 neurodegenerative diseases 334 peroxidase 332 production 331 structure 327–30 transferases 332 flavonoid upstream pathway, anthocyanin biosynthesis 464–6, 469–70 flavonoids 543–4 beet (Beta vulgaris L.) 23 biocatalysis 331–3 characteristics 37–8 citrus fruit by-products 25–6 classification 327–30 isoquercetin 330, 335 naringenin 307–8, 335 naringin 306–8, 313, 316, 328, 330, 333, 335 onion (Allium cepa L.) 19–21 pressurized hot water extraction (PHWE) 81–8 production 331 prunin 307–8, 330, 335 quercetin 335 rutin 335 screening 545–6 structure 66, 327–30 flavonols lettuce (Lactuca sativa L.) 12–13 onion (Allium cepa L.) 19–21 fluidized-bed reactor (FBR), glycosides 309, 311 Fomes fomentarius, antitumor compounds 210 food affecting genes, DNA methylation, histone modification, genomic imprinting 538 food allergens, bioactive peptides 517–18 food-derived multifunctional bioactive proteins and peptides 507–20 Food for Specified Health Uses (FOSHU), bioactive peptides 509 food losses 5–8

702

Index

food peptides, multifunctional see multifunctional food peptides foodomics, bioactive peptides 514–16 foods, functional see functional foods FOSHU see Food for Specified Health Uses free radicals, oxidants and antioxidants: the balance 537 fresh-cut fruits 37–56 abiotic stress 41 antioxidants 47–8 apple fruit (Malus domestica) 42–3 atmosphere composition effect 48–55 chemical treatment 40–1, 47–8 color preservation 47 cutting effect 45 modified atmosphere packaging (MAP) 48–52, 54–5 operations 38, 39 oranges 44–5, 49–52, 54–5 peaches 42 phytochemical content, factors affecting 39–41 polyphenol oxidase (PPO) 47–8 preservation methods 40–1 quality, factors affecting 39–41 quality variability 41–2 raw material 41–5 strawberries 43–4 temperature effect 48–55 washing-disinfection 40, 46–7 fruit by-products 24–30 apple fruit (Malus domestica) 26–7 citrus 24–6 grapes 27–9 tropical fruits 29–30 fruit processing EU directives 7–8 flow diagram 7–8 fruits see also berries; fresh-cut fruits; grapes resistance to pathogens and pests 463, 470–5 fucoxanthin diabetes treatment 678 obesity prevention 678 seaweed 678 fugaronine 541 function/structure interaction, bioactive compounds 528–30 functional foods defining 670 market size 670, 686

nutraceuticals 507–9 regulation 686 fungal bioactive compounds 195–216 see also arbuscular mycorrhizal fungi (AMF) angiotensin I converting enzyme (ACE-I) inhibitors 215 antiallergic compounds 214–15 antihypercholesterolemic compounds 204–7 antihyperglycemic compounds 213–14 antihyperlipidemic compounds 204–7 antimicrobial compounds 211–12 antioxidant activity 195–204 antitumor compounds 208–10 antitumoral activity 419–22 antiviral compounds 212–13 bioactive compounds/biological activity 195–216 Cantharellus sp. 202–3 Cibacron blue affinity eluted protein (CBAEP) 209 Fomes fomentarius 210 Ganoderma sp. 203–4, 208–9, 212–13 Grifola gargal 200 overview 196–9 Paecilomyces sp. 209 Pleurotus sp. 200–3 Polyporaceae family 208–9 statins 204–7 fungal metabolites anticancer compounds 441–3 antioxidant compounds 435, 437–9 fermentation processes 438–9 Graphislactone A 435, 437 isopestacin 435, 437 podophyllotoxin (PDT) 442 solid state fermentation (SSF) 438 taxol 441–2 fungal steroids antitumoral activity 419–22 bioavailability 402–3 future directions, virtual screening (VS) 390–1 future perspectives, milk fat globule membrane (MFGM) 655–6 future projections, bioactive peptides 520 future prospects C-phycocyanin (C-PC) 293 medicinal plants 139–41 future research, pressurized hot water extraction (PHWE) 90

Index Ganoderma sp. antioxidant activity 203–4 antitumor compounds 208–9 antiviral compounds 212–13 gas chromatography, steroids analysis 412–13 gene ontology (GO) analysis, D-pinitol, predicting molecular targets 387 genetic diversity studies, medicinal plants 138 genomic imprinting, mechanism by which food affects genes 538 germacrenolides 539 GFD see gluten-free diet Ginkgo biloba, medicinal properties 124 ginsenosides, production 336–7 gliadins-mediated alterations in intestinal epithelium celiac disease (CD) 584–6 connective tissue degradation 585 cytokines role 585 oxidative stress 586 serine-containing peptides role 585–6 gluten-free diet (GFD) adjuvant therapeutic strategies 583–4 celiac disease (CD) 583–4 nutrition 586–8 glycans see milk fat globule membrane glycoconjugates 632–3, 636–52 butyrophilin (BTN) 635–6, 640, 644–5 carbonic anhydrase (CA) 650–1 CD36 647–8 CD59 646–7 clusterin 651–2 glycolipids 636–9 glycoproteins 639, 640–1 lactadherin (LDH) 632, 635, 639, 640, 645–6 milk fat globule membrane (MFGM) 632–3, 636–52 mucins 639–43 proteose peptone component 3 (PP3) 648–9 xanthine oxidoreductase (XOR) 635, 644, 649–50 glycolipids 636–9 glycoproteins 639, 640–1 glycosaminoglycans 3D matrix material 560 stem cell research 560 glycosides 303–37 see also flavonoid glycosides acid hydrolysis 306 age-related diseases 303, 317

703

alcoholic and phenolic glycosides 317–18 aldehyde glycosides 319 alkali hydrolysis 306 amygdalin 319, 321 anthraquinone glycosides 319–22 applications 303, 313–17 arbutin 318 biocatalyst immobilization 309–11 biologic activity 313–17 bioprocess design 308–11 bufadienolides 325–6 cardenolides 323–5 cardiac glycosides 322–6 case studies 317–37 chemical properties 311–13 classification 304–6 continuous-flow stirred tank reactor (CSTR) 309, 311 cross-linked enzyme aggregates (CLEAs) 310 cyanogenic glycosides 319, 321 deglycosylation 306–7 enzymatic hydrolysis 306–11 enzymes used for deglycosylation 306–7 flavonoid glycosides 327–35 fluidized-bed reactor (FBR) 309, 311 hydrogels 309 hydrolysis 306–11 identification 311–13 neurodegenerative diseases 317, 334 pharmacological applications 303, 313–17 plug-flow reactor (PFR) 309, 311 production 308–11 reactor types 308–11 salicin 317–18 saponins 335–7 structure 304–6 sulphur glycosides 327, 328 tannins 326–7 therapeutic applications 303, 313–17 thermoreactive water-soluble polymers 309 vanillin 176, 319, 320 gossypol 539 grapes anthocyanin acylation 468–9 anthocyanin biosynthesis 464–70 anthocyanin modification 467–9 basic flavonoid upstream pathway 464–6, 469–70 by-products 27–9

704

Index

grapes (cont’d) metabolic engineering 464–70 phenolic compounds 27–8 polyphenols 27–8 resistance to pathogens and pests 470–5 resveratrol 27–9 Graphislactone A 435, 437 Grifola gargal antioxidant activity 200 fungal bioactive compounds 200 growth factors bone morphogenic proteins (BMPs) 557 fibroblast growth factor (FGF) 556–7 platelet-derived growth factor (PDGF) 558 stem cell research 555–61 transforming growth factor β (TGF-β) 557 vascular endothelial growth factor (VEGF) 558 guaianolides 539 Habenaria edgeworthii, medicinal properties 137–8 haloalkaliphiles 256, 258 halophiles 252–5 applications 246 biomolecules 246 enzymes 246, 254–5 halophilic alkalithermophiles 258 harpin, elicitation 474 harringtonine 542 heart health angiotensin I converting enzyme (ACE-I) inhibitors 674–7 bioactive peptides 674–7 renin inhibitory peptides 674–7 herbal medicines see antimicrobials from medicinal plants; medicinal plants Himalayan region biodiversity conservation 135–6 medicinal plants 135–6 Hippophae rhamnoides, medicinal properties 128 histone modification, mechanism by which food affects genes 538 HMO see human milk oligosaccharides homoharringtonine 542 human milk oligosaccharides (HMO), milk fat globule membrane (MFGM) 631–3 hydrogels, glycosides 309 hydrolases, flavonoid glycosides 333

hyphenated techniques, steroids analysis 414–15 hypocholesterolemic action, phytosterols 565–76 immunomodulation, phytosterols 573 imperatorin 167, 168 in silico (computational) peptidomics, bioactive peptides 514–16 in vitro protocols, medicinal plants 137–8 India, Western Gats region 163–86 Aegle marmelos (L.) Correa 164–9 Aristolochia indica L. Aristolochiaceae 169–70 Baliospermum montanum (willd.) Muell-arg (Euphorbiaceae) 170–2 bioactive compounds and endangered medicinal plants 163–86 biodiversity 161–4 Coscinium fenestratum (Gaertn.) Coleb. (Menispermaceae) 173–5 Decalepis hamiltonii Wight & Arn. (Asclepediaceae) 175–7 Eugenia singampattiana bedd. (Myrtaceae) 177–8 Oroxylum indicum (l.) Benth.Ex kurz (bignoniaceae) 179–80 Pterocarpus santalinus L. (Fabaceae) 180–2 Rauvolfia serpentina (L.) Benth.Ex kurz (apocyanaceae) 182–4 Trichopus zeylanicus gaertn. (Dioscoreaceae) 185–6 infant formula, milk fat globule membrane (MFGM) 652, 654, 655 inflammatory response regulation, steroids 418–19 insoluble/soluble dietary fiber 10–11 institutional setup, medicinal plants 139, 141 iridoid lactone 539 iron, celiac disease (CD) 589–90 ishwarol 170, 171 iso-lariciresinol, structure 181 isopestacin 435, 437 isoprenoids 269–79 acetyl-CoA metabolism 274–5 applications 270 biological activities 270 biosynthesis 271–2 diacylglycerol pyrophosphatase (DPP1) 276 farnesyl diphosphate synthase (ERG20) 273, 274

Index geranylgeranyl diphosphate synthase (BTS1) 276 HMG-CoA reductase 272–3 lipid phosphate phosphatase (LPP1) 276 MEP pathway 271, 278 metabolic channelling 277 metabolic engineering 272–4 mevalonate (MEV) pathway 271, 272–4 microbial production 269–79 modeling approaches 278–9 protein engineering interventions 277 simulation approaches 278–9 squalene synthase (ERG9) 273, 274 uptake control (UPC2) 275–6 isoquercetin 335 structure 330 isoquinoline alkaloids 541 jatrorrhizine 173–4 junipers, medicinal properties 127 LAB see lactic acid bacteria lac operon 531–3 regulatory genes 531–3 laccase, flavonoid glycosides 332 lactadherin (LDH) 632, 635, 639, 640, 645–6 lactic acid bacteria (LAB), antibacterial compounds 452–3 lactoferrin (LF) 486–9 antibacterial activity mechanism 487–8 antimicrobial action 487–8 bioactive peptides 518–19 hydrolysates 486–9 microbial resistance 518–19 physiological functions 487–8 structure 487 laminin 3D matrix material 560 stem cell research 560 lapachol 543 LBVS see ligand-based virtual screening LDH see lactadherin lephantinin 539 lettuce (Lactuca sativa L.) by-products 11–13 flavones 12–13 flavonols 12–13 phenolic compounds 11–13 polyphenols 11–12 leurocristine 542 LF see lactoferrin

705

ligand-based virtual screening (LBVS) 372 lignans 540 structure 66 limonene, structure 178 limonoids, citrus fruit by-products 26 linoleic acid see conjugated linoleic acid (CLA) lipases, flavonoid glycosides 331 lipid peroxidation in foods 151 lipid phosphate phosphatase (LPP1), isoprenoids 276 lipids see also bioactive lipid components from ruminants milk and meat antihyperlipidemic compounds 204–7 lipogenesis, ruminant tissue 606–7 lipolysis rumen metabolism 600–1 rumen populations responsible 603–6 lipoproteins effects, phytosterols 574 liquid chromatography, steroids analysis 413–14 losses, food 5–8 lovastatin 204–7 lupeol 176, 181 lycopene carrot (Daucus carota L.) 18 structure 11 tomato (Solanum lycopersicum L.) 13–16 lysozyme, bioactive protein 491–2 macroalgae phlorotannins 683–4 tyrosinase inhibitors 683–4 macromolecules, atoms, molecules, compounds, polymers 527–8 MAP see modified atmosphere packaging marine actinomycetes, bacterial metabolites 445 marine ingredients/molecules see also seaweed diabetes and obesity treatment 677–9 skin conditions 684–5 market size cosmeceuticals 685 functional foods 670, 686 marmin 167, 168 marmisin 167, 168 maytanosinoids 542 maytansine ester 542 maytasinne 542

706

Index

medicinal plants see also antimicrobials from medicinal plants active ingredients, biogeographical and ecological influences 136–7 antitumor compounds 538–9 Artemisia annua 124 Berberis asiatica 137 Bergenia sp. 128 Calotropis procera 127 Chelidonium majus 127 future prospects 139–41 genetic diversity studies 138 Ginkgo biloba 124 Habenaria edgeworthii 137–8 Himalayan region 135–6 Hippophae rhamnoides 128 in vitro protocols 137–8 institutional setup 139, 141 National Medicinal Plant Board 139 optimization of procedures 138–9 prioritization 140–1 quality standards and specifications 139 regulatory policies 139, 141 Sanguisorba officinalis 127 source of antimicrobials 124–5 Taxus sp. 124 traditional knowledge application 140 Tussilago farfara 127 Valeriana jatamansi 137 vis-à-vis traditional medicine 126 Mediterranean ecosystems, arbuscular mycorrhizal fungi (AMF) 231 MeJ see methyl jasmonate MEP pathway isoprenoids 271, 278 metabolic engineering 271, 278 metabolic engineering 463–75 anthocyanin biosynthesis 464–70, 469–70 basic flavonoid upstream pathway 464–6, 469–70 berries 463–75 elicitation 463, 471–5 grapes 464–6 isoprenoids 272–4 MEP pathway 271, 278 mevalonate (MEV) pathway 272–4 resistance to pathogens and pests 470–5 secondary metabolites 463–75

metabolic syndrome absorptive capacity effect 572–3 milk protein-derived peptides 670 phytosterols 575 seaweed 670 metallophiles 256, 257 applications 246 biomolecules 246 enzymes 246 methyl jasmonate (MeJ), elicitation 473 mevalonate (MEV) pathway isoprenoids biosynthesis 271, 272–4 metabolic engineering 272–4 steroids biosynthesis 405, 406, 407 MFGM see milk fat globule membrane microbial proteases used in production of bioactive peptides 495 microbial resistance bioactive peptides 518–19 lactoferrin (LF) 518–19 micronutrients, celiac disease (CD) 589–90 microsomal hydrogen peroxide determination 547 milk fat globule membrane (MFGM) 631–56 butyrophilin (BTN) 635–6, 640, 644–5 carbonic anhydrase (CA) 650–1 CD36 647–8 CD59 646–7 clusterin 651–2 commercial potential 652–5 composition 633–6 future perspectives 655–6 glycoconjugates 632–3, 636–52 glycolipids 636–9 glycoproteins 639, 640–1 human milk oligosaccharides (HMO) 631–3 infant formula 652, 654, 655 isolation 652–5 lactadherin (LDH) 632, 635, 639, 640, 645–6 mucins 639–43 processing 652–5 proteose peptone component 3 (PP3) 648–9 xanthine oxidoreductase (XOR) 635, 644, 649–50 milk protein-derived peptides 484 anticariogenic activity 490 bioactivities/influences 485

Index casein phosphopeptides (CPPs) 490–1 lactoferrin (LF) and its hydrolysates 486–9 metabolic syndrome 670 multifunctional 486–91 nutraceuticals 507–9 MilkAMP database, bioactive peptides 516 modified atmosphere packaging (MAP) apple fruit (Malus domestica) 50–1, 53–5 ascorbic acid 54–5 fresh-cut fruits 48–52, 54–5 oranges 49–52, 54–5 strawberries 50, 52–3 vitamin C 54–5 molecules, compounds, macromolecules, atoms, polymers 527–8 monoterpenes 539 montanin 172 mucins 639–43 multifunctional food peptides 485–91, 507–20 mycolyltransesterase enzymes 358–60 mycolyltransesterase inhibitors 360–2 mycorrhizal fungi see arbuscular mycorrhizal fungi (AMF) mycorrhizal networks, arbuscular mycorrhizal fungi (AMF) 232 mycosteroids antitumoral activity 419–22 bioavailability 402–3 nanofiltration, bioactive peptides 497 naringenin 307–8, 335 naringin 313, 316, 328, 333, 335 hydrolysis 306–8 structure 330 NASH see nonalcoholic steatosis National Medicinal Plant Board, medicinal plants, policies and strategies 139 neurodegenerative diseases flavonoid glycosides 334 glycosides 317 NeuroPred databases, bioactive peptides 516 niadine 541 nitrogenous bases, screening 545 nonalcoholic steatosis (NASH), celiac disease (CD) 588 nonlactonic sesquiterpenes 539 norerythrostachaldine 541 nutraceutical applications, C-phycocyanin (C-PC) 292–3

707

nutraceuticals bioactive peptides 507–9 Food for Specified Health Uses (FOSHU) 509 functional foods 507–9 milk protein-derived peptides 507–9 nutritional genomics (nutrigenomics) 537–8 obesity bioactive lipid components from ruminants milk and meat 614–20 marine molecules for treating 677–9 obesity prevention astaxanthin 678 fucoxanthin 678 ‘omic’ methodologies, bioactive peptides 514–16 onion (Allium cepa L.) by-products 19–21 dietary fiber 20 flavonoids 19–21 flavonols 19–21 oranges ascorbic acid 54–5 fresh-cut fruits 44–5, 49–52, 54–5 modified atmosphere packaging (MAP) 49–52, 54–5 phenolic compounds 44–5 vitamin C 44–5, 54–5 oroxylin 179 structure 179 Oroxylum indicum (l.) Benth.Ex kurz (bignoniaceae) 179–80 history and distribution 179 medicinal uses 179–80 phytochemical constituents 179 plant description 179 ovalbumin, bioactive protein 492 oxidants, free radicals, and antioxidants: the balance 537 oxidative metabolism 151 PAA see peracetic acid Paecilomyces sp., antitumor compounds 209 pathogen and pest resistance see resistance to pathogens and pests PDGF see platelet-derived growth factor PDT see podophyllotoxin peaches fresh-cut fruits 42 variability of phytochemicals 42

708

Index

PepBank databases, bioactive peptides 516 PeptideCutter databases, bioactive peptides 516 peptides, bioactive see bioactive peptides peracetic acid (PAA), washing-disinfection 46 peroxidase, flavonoid glycosides 332 pest and pathogen resistance see resistance to pathogens and pests PFR see plug-flow reactor PharmMapper advanced options 384–6 artemisinic acid 382–5 basic options 382–3 virtual screening (VS) 382–5 phenanthroidolizidine 541 phenanthroquinolizidine alkaloids 541 phenolic acids cereals 112–13 pressurized hot water extraction (PHWE) 81–8 structure 66 phenolic compounds see also polyphenols alkylresorcinols 113–14 antimicrobial 129, 130 antioxidant properties 113 apple fruit (Malus domestica) 27 artichoke (Cynara scolymus L.) 16–17 avenanthramides 113 beet (Beta vulgaris L.) 22–3 carrot (Daucus carota L.) 17–19 cereals 112–14 characteristics 37–8 citrus fruit by-products 24–5 grapes 27–8 lettuce (Lactuca sativa L.) 11–13 oranges 44–5 phenolic acids 66, 81–8, 112–13 potato (Solanum tuberosum L.) 21–2 tropical fruits 29–30 phlorotannins macroalgae 683–4 seaweed 683–4 tyrosinase inhibitors 683–4 phosvitin, bioactive protein 492 PHWE see pressurized hot water extraction phycobiliproteins 283–93 phycobilisomes 283–4, 288–90, 292 phycocyanins 283 see also C-phycocyanin (C-PC)

phytochemicals 3–11 beneficial health effects 4–5 from by-products of vegetable and fruit processing 5–11 defining 4 fresh-cut fruits 39–41 in by-products 3 phytochemicals (antioxidants), celiac disease (CD) 591 phytostanols 417–18, 575 phytosterols 565–76 see also steroids absorption 568 anti-inflammatory properties 573 antihypercholesterolemic 417–18 antihyperglycemic effects 575 antitumoral activity 419–22, 573 bioavailability 400–1 biological effects 565–76 biosynthesis 403–5 brassinosteroids (BRs) 421 characterization 566–7 cholesterol absorption effects 416, 568–73 defining 566–7 dietary intake 566–7 emulsifiers 417 food sources 566–7 hypocholesterolemic action 565–76 hypocholesterolemic effect 568–73 immunomodulation 573 lipoproteins effects 574 mechanism of action 568–73 metabolic syndrome 575 phytostanols 417–18, 575 regulation 568 safety 568, 574–5 sources, food 566–7 structure 565–6 toxicity 568 piezophiles 255 applications 246 biomolecules 246 enzymes 246, 255 piperidine-based trehalase inhibitors 355–7 plant bioactive compounds, structures 539–44 plant fungal endophytes, arbuscular mycorrhizal fungi (AMF) 226–7

Index plant protein-derived multifunctional peptides 492–4 rapakinin 493 Rubisco-derived peptides 493 soymorphins 493–4 plant secondary metabolites effects, bioactive lipid components from ruminants milk and meat 608 plant tumor promoters 544 plants, medicinal see medicinal plants platelet-derived growth factor (PDGF), stem cell research 558 Pleurotus sp., antioxidant activity 200–3 plug-flow reactor (PFR), glycosides 309, 311 podophyllotoxin 540 podophyllotoxin (PDT) 442 polyextremophiles 256–9 polyhydroxylated pyrrolizidine alkaloids 352–5 polymers, macromolecules, atoms, molecules, compounds 527–8 polyphenol oxidase (PPO), fresh-cut fruits 47–8 polyphenols see also phenolic compounds antioxidant activity 67, 76–8 bioactivity 77–8 classification 9–10, 64–7 defining 64 empirical equations for measuring solubility 72–3 essential oils 81 grapes 27–8 lettuce (Lactuca sativa L.) 11–12 major 9–10 pressurized hot water extraction (PHWE) 67–90 properties 11–12, 64–7 references for PHWE 82–8 selective extraction 76 solubility in pressurized hot water 71–4 sources 64–6 structure 9–10 thermal degradation 71–2 thermodynamic models for measuring solubility 73–4 tomato (Solanum lycopersicum L.) 14–15 Polyporaceae family, antitumor compounds 208–9 polyunsaturated fatty acids (PUFA), celiac disease (CD) 591–2

709

POPS databases, bioactive peptides 516 potato (Solanum tuberosum L.) by-products 21–2 phenolic compounds 21–2 PP3 see proteose peptone component 3 PPO see polyphenol oxidase prebiotics, celiac disease (CD) 588–9 pressurized hot water extraction (PHWE) advantages 70 antioxidant compounds 76–8 bioactivity effects 77–8 condensed tannins 88–9 equipment 79–81 essential oils 81 extraction mechanism 69–70 flavonoids 81–8 future research 90 modes of operation 79–81 optimizing 74–9 phenolic acids 81–8 plant polyphenols 71–90 polyphenols 67–90 proanthocyanidins 88–9 references for polyphenols 82–8 selective extraction 76 system 67–70 temperature effect 77–9 pretreatment and extraction systems, vegetable and fruit by-products 30–2 proanthocyanidins, pressurized hot water extraction (PHWE) 88–9 probiotics, celiac disease (CD) 588–9 progesterone, structure 336 prokaryotic vs eukaryotic systems 530–1 proline, angiotensin I converting enzyme (ACE-I) inhibitors 486 proteases used in production of bioactive peptides 495 proteins, bioactive see bioactive proteins proteose peptone component 3 (PP3) 648–9 protoberberine 173–4 prunin 307–8, 335 structure 330 pseudoguaianolides 539 psychrophiles 249–50 applications 246 biomolecules 246 enzymes 246, 249–50 Pterocarpus santalinus L. (Fabaceae) 180–2 history and distribution 180 medicinal uses 181–2

710

Index

Pterocarpus santalinus L. (Fabaceae) (cont’d) phytochemistry 181 plant description 181 PUFA see polyunsaturated fatty acids pyrrolidine-based trehalase inhibitors 355–7 pyrrolizidine alkaloids 541 QS see quorum sensing QSAR modelling see quantitative structure activity relationship modelling quality standards and specifications medicinal plants 139 World Health Organization (WHO) 139 quantitative structure activity relationship (QSAR) modelling bioactive peptides 516–17 integrating virtual screening 378 steps 378–80 structural-based virtual screening (SBVS) 378–80 quassinoids (simaroublide) 540 quercetin 335 quorum sensing (QS), antimicrobial mode of action 133 radiophiles 256 applications 246 biomolecules 246 enzymes 246 rapakinin, bioactive peptides 493 Rauvolfia serpentina (L.) Benth.Ex kurz (apocyanaceae) 182–4 history and distribution 182 medicinal uses 183 phytochemistry 183 plant description 182 regulatory policies, medicinal plants 139, 141 renin inhibitory peptides, heart health 674–7 reserpine 183 structure 184 resistance to cancer drugs, drug combinations 544 resistance to pathogens and pests bioactive peptides 518–19 chitosan 474–5 elicitation 463, 471–5 harpin 474 lactoferrin (LF) 518–19 metabolic engineering 470–5 methyl jasmonate (MeJ) 473

resistant starch (RS) cereals 110–12 dietary fiber 110–12 health benefits 110–11 technological impacts 111–12 resveratrol, grapes 27–9 reverse docking approach for predicting biological activity of compounds 381 RS see resistant starch Rubisco-derived peptides, bioactive peptides 493 rumen metabolism biohydrogenation (BH) 601–2, 603–6, 608–10 lipolysis 600–1, 603–6 rumen bacteria 603–4 rumen protozoa 604–6 source of bioactive lipid components 600–1 ruminant products see also bioactive lipid components from ruminants milk and meat essential oils 610 fatty acid profiles 608–12 saponins 610–11 tannins 608–10 ruminant tissue de-novo synthesis of fatty acids 606–7 lipogenesis 606–7 rutin 335 safe/unsafe compounds, criteria for classifying 529–30 salicin, biosynthesis 317–18 Sanguisorba officinalis, antimicrobial properties 127 saponification, steroids 411 saponins 335–7 ginsenosides 336–7 ruminant products 610–11 structure 335–6 savinin, structure 181 SBVS see structural-based virtual screening (SBVS) scopoletin 167, 168 screening alkaloids 545 flavonoids 545–6 nitrogenous bases 545 screening, virtual see structural-based virtual screening (SBVS); virtual screening (VS)

Index seaweed 669–78, 683–4 bioactive peptides 674–7 diabetes treatment 677–9 edible 670–2 farming 673–4 fucoxanthin 678 harvesting 673–4 metabolic syndrome 670 obesity treatment 677–9 phlorotannins 683–4 protein content 671–2 sustainability 673–4 tyrosinase inhibitors 683–4 secondary metabolites elicitation, enhanced production via 471–5 metabolic engineering 463–75 plant secondary metabolites effects, bioactive lipid components from ruminants milk and meat 608 selective extraction polyphenols 76 pressurized hot water extraction (PHWE) 76 selenium, celiac disease (CD) 589–90 serine-containing peptides role, gliadins-mediated alterations in intestinal epithelium 585–6 serpentine 183 structure 184 serpentinine 183 structure 184 sesquiterpenes 539 sickle cell anemia, β-globin 533–6 simaroublide (quassinoids) 540 simaroublids 540 sinigrin 327, 328 skimmianine 167, 168 skin conditions see also cosmeceuticals cancer 683 dermatitis 684–5 marine ingredients 684–5 skin structure 681 solaplubinin 541 solaplumbin 541 solid state fermentation (SSF) 438 soluble/insoluble dietary fiber 10–11 solvent extraction, vegetable and fruit by-products 31 source identification, antimicrobials from medicinal plants 133–4

711

soymorphins, bioactive peptides 493–4 spectroscopy, steroids analysis 414 Spirulina platensis see C-phycocyanin (C-PC) squalene, structure 178 SSF see solid state fermentation starch technological impacts, arabinoxylans 106–7 statins fungal bioactive compounds 204–7 lovastatin 204–7 steganone 540 stem cell research 555–61 3D matrix materials 558–60 biotic scaffolds for tissue regeneration 558–60 bone morphogenic proteins (BMPs) 557 collagen 559 fibroblast growth factor (FGF) 556–7 fibronectin 559–60 glycosaminoglycans 560 growth factors 555–61 laminin 560 platelet-derived growth factor (PDGF) 558 transforming growth factor β (TGF-β) 556, 557 vascular endothelial growth factor (VEGF) 558 steroidogenesis 405, 408 steroids 395–424 see also phytosterols analytical methods 405–15, 422–3 analytical techniques 410 androgenic anabolic steroids (AAS) 417 animal steroids 401–2 antioxidant activity 422 antitumoral activity 419–22 bile acids 415–16 bioavailability 400–3 biological activity 415–22 biosynthesis 403–5 classification 396–9 cortisone 336, 415, 416 determination 412 dihydrotestosterone (DHT) 416 fungal steroids 402–3 gas chromatography 412–13 health effects 410 hyphenated techniques 414–15 inflammatory response regulation 418–19 liquid chromatography 413–14 mevalonate (MEV) pathway 405, 406, 407

712

Index

steroids (cont’d) mycosteroids 402–3 phytosterols bioavailability 400–1 plant steroids bioavailability 400–1 purification 411–12 sample preparation 409–15 saponification 411 spectroscopy 414 steroidogenesis 405, 408 structure 395–9 zoosteroids 401–2 stilbenes, structure 66 strawberries fresh-cut fruits 43–4 modified atmosphere packaging (MAP) 50, 52–3 variability of phytochemicals 43–4 Streptomyces sp. antibacterial compounds 449 bacterial metabolites 445 structural-based virtual screening (SBVS) 372–80 case studies 376–7 citation index of docking software 373 computational approaches 386–90 computational tools 373–5 databases 375 docking software 372–4 pitfalls 380 quantitative structure activity relationship (QSAR) modelling 378–80 web resources 375 structure/function interaction, bioactive compounds 528–30 sulphur glycosides 327, 328 supercritical fluid extraction, vegetable and fruit by-products 31 SwePep databases, bioactive peptides 516 SWISS–PROT databases, bioactive peptides 516 tannins biosynthesis 326 ruminant products 608–10 structure 327 taxol 441–2 mechanism of action 442, 542 precursor 446 structure 435

Taxus sp. alkaloids 542 medicinal properties 124 temperature effect, fresh-cut fruits 48–55 terpene derivatives, antibacterial compounds 450 terpenes, antimicrobial 129–31 terpenoids see isoprenoids testosterone dihydrotestosterone (DHT) 416 structure 336, 416 TGF-β see transforming growth factor β (TGF-β) thalicarpine 541 thermodynamic models, polyphenols solubility measurement 73–4 thermophiles 246–8 applications 246 biomolecules 246 chaperone proteins 247 drawback 247–8 enzymes 246, 247–8 structure 247 thermoreactive water-soluble polymers, glycosides 309 threatened medicinal plants (TMPs) 136 tiptolide 539 TMPs see threatened medicinal plants tomato (Solanum lycopersicum L.) by-products 13–16 carotenoids 13–16 lycopene 13–16 polyphenols 14–15 traditional knowledge application, medicinal plants 140 traditional medicine, vis-à-vis medicinal plants 126 transcriptional regulation, anthocyanin biosynthesis 469–70 transferases, flavonoid glycosides 332 transforming growth factor β (TGF-β), stem cell research 556, 557 trehalase inhibitors 346–57 deoxynojirimycin 355–7 piperidine-based 355–7 polyhydroxylated pyrrolizidine alkaloids 352–5 pyrrolidine-based 355–7 trehazolin 349–52 validoxylamines 346–9 trehalases 346

Index trehalose properties 345 structure 346 trehalose mimetics 345–65 trehalose-processing mycolyltransesterase enzymes 357–62 mycolyltransesterase enzymes 358–60 mycolyltransesterase inhibitors 360–2 trehalose-processing sulfotransferase, trehalose sulfation 362–3 trehalose sulfation, trehalose-processing sulfotransferase 362–3 trehazolin 349–52 trehazolin analogues 349–52 trewinine 540 Trichopus zeylanicus gaertn. (Dioscoreaceae) 185–6 history and distribution 185 medicinal uses 185–6 triptiolide 539 triterpenes 540 tropical fruits by-products 29–30 carotenoids 29–30 phenolic compounds 29–30 tuberculosis vaccines, acyl2SGL mimetics 363–5 tumor promoters, plant 544 Tussilago farfara, antimicrobial properties 127 tyrosinase inhibitors cosmeceuticals 683–4 macroalgae 683–4 phlorotannins 683–4 seaweed 683–4 ultrafiltration, bioactive peptides 497 unsafe/safe compounds, criteria for classifying 529–30 Valeriana jatamansi, medicinal properties 137 validoxylamines 346–9 vanillin biosynthesis 319 hydrolysis 320 structure 176 vascular endothelial growth factor (VEGF), stem cell research 558

713

vegetable and fruit by-products pretreatment and extraction systems 30–2 solvent extraction 31 supercritical fluid extraction 31 vegetable and fruit products beneficial health effects 4–5 by-products from processing 5–11 processed 4–5 vegetable by-products 11–24 artichoke (Cynara scolymus L.) 16–17 beet (Beta vulgaris L.) 22–4 carrot (Daucus carota L.) 17–19 lettuce (Lactuca sativa L.) 11–13 onion (Allium cepa L.) 19–21 potato (Solanum tuberosum L.) 21–2 tomato (Solanum lycopersicum L.) 13–16 vegetable processing EU directives 7–8 flow diagram 6–7 VEGF see vascular endothelial growth factor vernolepin 539 vincaleukoblastine 542 virtual screening (VS) 371–91 see also structural-based virtual screening (SBVS) artemisinic acid 381–6 computational approaches 373–5, 386–90 D-pinitol, predicting molecular targets 386–90 defining 371 future directions 390–1 ligand-based virtual screening (LBVS) 372 PharmMapper 382–5 reverse docking approach for predicting biological activity of compounds 381 structural-based virtual screening (SBVS) 372–80 vitamin A, celiac disease (CD) 590–1 vitamin C see also ascorbic acid celiac disease (CD) 590–1 modified atmosphere packaging (MAP) 54–5 oranges 44–5, 54–5 washing-disinfection 46–7 vitamin E, celiac disease (CD) 590–1 VS see virtual screening

714

Index

washing-disinfection fresh-cut fruits 40, 46–7 peracetic acid (PAA) 46 vitamin C 46–7 water-binding capacity, β-glucans 109 ‘western’ disease prevention, bioactive lipid components from ruminants milk and meat 614–20 wheat-based cereals, bioactive components 587 whole-grain food, defining 104 withaferin A: 540

withanolides 540 World Health Organization (WHO), quality standards and specifications, medicinal plants 139 xanthine oxidoreductase (XOR) 635, 644, 649–50 xanthotoxin 167, 168 zinc, celiac disease (CD) 589–90 zoosteroids, bioavailability 401–2

Figure 6.1 Coccoloba uvifera L. ripe fruit.

Biotechnology of Bioactive Compounds: Sources and Applications, First Edition. Edited by Vijai Kumar Gupta, Maria G. Tuohy, Mohtashim Lohani, and Anthonia O’Donovan. © 2015 John Wiley & Sons, Ltd. Published 2015 by John Wiley & Sons, Ltd.

H O

N H N H

O H N

H O

H N

O

H

O

(a)

N

N H

N

+ H

N

O

-

H

H

N H

O O

N

O

H

N H H

O + N

O

H

(c)

(b)

H

O

O O

O O

O

O O

O O O

(d)

(e)

Figure 7.9 Photograph of R. serpentina (a); chemical structures ajmaline (b), ajmalicine (c), reserpine (d), Serpentine (e), and Serpentinine (f).

(f)

Citation index

AutoDock DOCK GOLD Glide FlexX FRED LigandFit CDOCKER Surflex-Dock ParaDockS Molegro Virtual Docker Fleksy Hex

Figure 15.1 Citation index of various docking software (data collected until February 2013).

GLY125

SER38

PHE126 VAL124 Artemisinic Acid

GLY123

ALA40

LYS100 ASP42 ASNS H-Bonds Donor

MET86

Acceptor

Figure 15.6 Artemisinic acid docked in the active pocket of the heat shock protein 90. The active site residues are labelled and the surface is colored as per H-bond donor and acceptor residues.

Immune system process (3)

Cell adhesion (1)

Cell cycle (19)

Cell communication (73)

Metabolic process (89) Cellular process (74)

Generation of precursor metabolites and energy (2)

Developmental process (43)

Response to stimulus (3)

System process (16) Reproduction (4)

Transport (13)

Cellular component organization (1)

Apoptosis (7)

Figure 15.7 Pie chart showing various biological processes that might get affected by D-pinitol treatment. Number below each biological process represents total number of potential enzymes involved in respective biological processes and targeted by D-pinitol in C. elegans.

Gln51

Tyr7 Leu52

Tyr49 Pro53

Tyr63 Arg13

lle10

Gln64 Lys102 Ser65 Asp98 lle65 Cys101 Asp94

Asn66 Arg100

Glu97

Figure 15.8 Interaction of D-pinitol with GSTP, generally upregulated in the stress conditions. Amino acid residues in the binding cavity are labelled. Surface is generated around D-pinitol and colored on the basis of hydrogen bond donor/acceptor. Donors are colored in green and receptor acceptors in cyan. Hydrogen bonds are shown with dotted green lines.

Figure 19.2 Structure of recombinant human lactoferrin expressed in Aspergillus awamori. Generated with Rasmol. PDB file based on 1b0l (http://dx.doi.org/10.2210/pdb1b0l/pdb) (Sun et al. 1999).

Figure 21.3 The 3D structure of β-globin.

Figure 21.4 Magnified part of the 3D structure of β-globin.

Figure 21.5 The 3D structure of normal β-globin.

Figure 21.6 The 3D structure of sickle cell anemia β-globin.

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