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Functional Food Ingredients and Nutraceuticals Processing Technologies Edited by

John Shi, Ph.D.

Boca Raton London New York

CRC is an imprint of the Taylor & Francis Group, an informa business

© 2007 by Taylor & Francis Group, LLC

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487‑2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid‑free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number‑10: 0‑8493‑2441‑6 (Hardcover) International Standard Book Number‑13: 978‑0‑8493‑2441‑3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the conse‑ quences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978‑750‑8400. CCC is a not‑for‑profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging‑in‑Publication Data Functional food ingredients and nutraceuticals : processing technologies / edited by John Shi. p. cm. ‑‑ (Functional foods & nutraceuticals series ; 9) Includes bibliographical references and index. ISBN 0‑8493‑2441‑6 (alk. paper) 1. Functional foods. I. Shi, John. II. Series. QP144.F85F85 2006 613.2‑‑dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

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Foreword The linkages between diet and health, and their economic implications, continue to be elucidated and to gain public acceptance as credible science in this fascinating field of study. Knowledge of the specific contributions that functional foods, bioactive compounds, and nutraceuticals make to our health has grown immensely over the past decade. So, too, has the desire of consumers to purchase and integrate these materials into healthful diets, and for mainstream retail chains to give these products lucrative shelf space increased. To effectively disseminate this knowledge and achieve broad public benefit from this work, it is essential that emerging factual information in the scientific literature be distilled, assembled, and consolidated into more concise communications, as presented here, that can be put into action throughout the food continuum. This ninth volume in the CRC Press Functional Foods and Nutraceuticals Series, edited by Dr. G. Mazza (AAFC-PARC, Summerland, BC), continues the work of the earlier volumes and moves farther along the continuum to address the most recent advances in food engineering and processing. These advances hold the greatest promise for the immediate future to achieve stable, high-volume production of functional food and nutraceutical products of defined and reliable composition, thereby fostering the health and economic benefits that could ensue. Dr. John Shi, an internationally wellknown scientist in this field, and coeditor of an earlier volume of the series, steps out independently to take the role of editor as well as contributing author on this volume. The team of contributing authors, representing five continents, reflects the international distribution of interest and expertise on this topic, and the essential widespread collaboration that is required to generate advances most effectively. In 16 chapters, this volume provides a sequential study of key factors in the preparation of functional foods and nutraceuticals, from the selection of sources through the extraction, purification, decontamination, packaging, and preservation of a variety of products in these categories. Where the earlier volumes focused on an array of products and their biochemical constituents, this volume is process oriented, although it touches on a variety of products as well as their characteristics and how individual properties influence the suitability of the processes described. The authors and editor have done a masterful job and are to be congratulated for their success in selecting the most significant issues and the most promising opportunities for presentation in this enlightening publication. The dissemination of the information this volume contains will accelerate the integration of these approaches and technologies into the production processes of tomorrow. This book will be an excellent resource for students of food science, life sciences, as well as food industry professionals interested in functional foods. Yvon Martel, Ph.D. A/Assistant Deputy Minister Agriculture and Agri-Food Canada

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Preface People from North America and the European community are committing more time and effort to have greater control over their health by exploring alternative or herbal medicines and natural health products for disease prevention and better health. There has been a growing interest in the role of special micronutrients (phytochemicals) on human health and well-being. The relationships among special food components, their physiological functionality, and health benefits have been revealed progressively in the recent years. Functional foods contain significant levels of natural extracts, concentrates, or natural ingredients that are extracted from natural sources. These can reduce current healthcare costs by health improvement and disease prevention. During the past decade, the trend of functional food consumption was consumer driven. It also serves the needs of the aging population that wants greater control over their health and well-being. This trend is expected to continue, and as a result, scientific information on all aspects of functional foods is vital to the advancement of this emerging sector. The increase in consumer demand for functional foods has prompted international health organizations and government agencies to develop specific guidelines for their production and use. The scientific community must, therefore, utilize modern technologies to ensure efficacy and safety in the manufacturing of functional foods. Manufacturers are always eager to fulfill the consumer’s desire for functional food products that could be used to promote health. In order to provide a better understanding and to disseminate the latest developments in this rapidly expanding field, this ninth volume of the CRC Press Functional Foods and Nutraceuticals Book Series, Functional Foods and Nutraceuticals: Processing Technologies, was developed. Sixteen chapters in this book cover a broad spectrum of functional foods from biological material, applications of engineering techniques in functional food production, process engineering and modeling, functional food bioavailability, to product quality. The emphasis is on (1) applications of engineering techniques such as high pressure, supercritical fluid, membrane, microencapsulation, and molecular distillation in the processing of functional foods; (2) stability of bioactive components and antioxidative properties during processing and shelf life; (3) improvement in bioavailability of bioactive components by physical and chemical methods; and (4) mechanisms of antioxidant action and clinical and epidemiological evidence of functionality. The contributing authors are international experts in their respective fields, and I am grateful to each and every one of them for their thoughtfulness in contributions to this book. This book will be of interest to a wide spectrum of professionals from food scientists and technologists, nutritionists, biochemists, and engineers to entrepreneurs worldwide. It will also serve as a unique reference for food scientists for the R&D departments of food companies that are working with functional foods

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and ingredients. Additionally, it will serve as a source of basic information for college and university students majoring in food science and technology, food processing, and engineering. Readers will obtain sound scientific knowledge of engineering techniques and the quality of functional foods and nutraceuticals. John Shi, Ph.D. Editor

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Series Editor’s Preface The Functional Foods and Nutraceuticals Book Series, launched in 1998, was developed to provide a timely and comprehensive treatment of the emerging science and technology of functional foods and nutraceuticals which are shown to play a role in preventing or delaying the onset of diseases, especially chronic diseases. The first eight volumes in the series, Functional Foods: Biochemical and Processing Aspects, Volumes 1 and 2; Herbs, Botanicals and Teas; Methods of Analysis for Functional Foods and Nutraceuticals; Handbook of Fermented Functional Foods; Handbook of Functional Dairy Products; Handbook of Functional Lipids; and Dictionary of Functional Foods and Nutraceuticals, have received broad acceptance by food, nutrition, and health professionals. The latest volume, Functional Foods and Nutraceuticals: Processing Technologies, edited by Dr. John Shi, addresses the most recent advances in processing technologies for functional food ingredients and nutraceuticals. Distinctive features of this book include in-depth treatments of the peer-reviewed literature on supercritical fluid extraction, pressurized low polarity water extraction, membrane separation, distillation, dehydration, food pasteurization, and sterilization with high pressure, microencapsulation of omega-3 fatty acids, and bioprocessing. Other topics addressed include microbial modeling for bioreactor design, biochemical reactions in supercritical fluids, modeling of supercritical fluid extraction of plant material, stability of lycopene during processing, functional foods packaging, fruits with high antioxidant activity, and biological antioxidation mechanisms quenching of peroxynitrite. The book contains 16 excellent chapters written by 32 international experts at the forefront of functional food and nutraceutical science and technology. It is hoped that the effort will be beneficial to process engineers; food, nutrition, and health practitioners; and students, researchers, and entrepreneurs in industry, government, and university laboratories. Earlier volumes in the series addressed a range of topics and include: Functional Foods: Biochemical and Processing Aspects, Volume 1, the first volume of the series, edited by G. Mazza, is a bestseller, and is devoted to functional food products from oats, wheat, rice, flaxseed, mustard, fruits, vegetables, fish, and dairy products. In Volume 2, edited by Drs. John Shi, G. Mazza, and Marc Le Maguer, the focus is on the latest developments in the chemistry, biochemistry, pharmacology, epidemiology, and engineering of tocopherols and tocotrienols from oil and cereal grain, isoflavones from soybeans and soy foods, flavonoids from berries and grapes, lycopene from tomatoes, limonene from citrus, phenolic diterpenes from rosemary and sage, organosulfur constitutes from garlic, phytochemicals from echinacea, pectin from fruit, and omega-3 fatty acids and docosahexaenoic acid from flaxseed and fish products.

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The book Herbs, Botanicals and Teas, edited by Drs. G. Mazza and Dave Oomah, provides an in-depth literature review of the scientific and technical information on the chemical, pharmacological, epidemiological, and clinical aspects of garlic, ginseng, Echinacea, ginger, fenugreek, St. John’s wort, ginkgo biloba, kava kava, goldenseal, saw palmetto, valerian, evening primrose, liquorice, bilberries and blueberries, and green and black teas. The book, which is superbly referenced, also contains chapters on international regulations and quality assurance and control for the herbal and tea industry. The volume Methods of Analysis for Functional Foods and Nutraceuticals, edited by Dr. W. Jeffrey Hurst, presents advanced methods of analysis for carotenoids, phytoestrogens, chlorophylls, anthocyanins, amino acids, fatty acids, flavonoids, water-soluble vitamins, and carbohydrates. The fifth volume of the series, Handbook of Fermented Functional Foods, edited by Dr. Edward R. Farnworth, provides a comprehensive, state-of-the-art treatment of the scientific and technological information on the production of fermented foods, the microorganisms involved, the changes in composition that occur during fermentation and, most importantly, the effects of these foods and their active ingredients on human health. The Handbook of Functional Dairy Products, edited by Drs. Colette Shortt and John O’Brien, contains outstanding chapters dealing with probiotic lactobacilli and bifidobacteria, lactose hydrolyzed products, trans-galactooligosaccharides as prebiotics, conjugated linoleic acid (CLA) and its antiatherogenic potential and inhibitory effects on chemically induced tumors, immuno-enhancing properties of milk components and probiotics, and calcium and iron fortification of dairy products. On lipids, we have the volume, Handbook of Functional Lipids, edited by Professor Casimir C. Akoh, which presents up-to-date information on all major scientific and technological aspects of functional lipids, including isolation, production, and concentration of functional lipids; lipids for food functionality; lipids with health and nutritional functionality; and the role of biotechnology for functional lipids. Finally, the Dictionary of Functional Foods and Nutraceuticals, written by N. A. Michael Eskin and Snait Tamir, is essentially a mini-encyclopedia that provides the reader with valuable and up-to-date information on the occurrence, chemistry, and biological activity/efficacy of 480 functional foods and nutraceuticals. The information is based solely on peer-reviewed literature, and it is presented alphabetically in a clear and concise manner. G. Mazza, Ph.D., FCIFST Series Editor

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Acknowledgments This book is a product of collaborative efforts, and a number of scientists gave their valuable time to review the manuscripts. The editor is indebted to their contributions and support that improved this book significantly. The reviewers include Dr. Ireneo Kikic (University of Trieste, Italy); Dr. Thomas Gamse (Graz University of Technology, Austria); Drs. Yukio Kakuda, Gauri S. Mittal, and Warrer Stiver (University of Guelph, Canada); Drs. Selma Guigard and Marleny Saldana (University of Alberta, Canada); Dr. Paulyn Appah (Food Development Centre, Manitoba, Canada); and Drs. Giuseppe Mazza, Christopher Young, and Lamin S. Kassama (Agriculture and Agri-Food Canada). The editor also would like to express appreciation to Dr. Yvon Martel (A/Assistant Deputy Minister, Agriculture and Agri-Food Canada) for his kind preparation of the foreword for this book; and to Drs. John Lynch (Science Director) and Maria Nazarowec-White (Program Coordinator) for their help and support. The editor also wishes to acknowledge the encouragement and help from Dr. Jerry King (Los Alamos National Laboratory, Chemistry Division, United States); Dr. Asbjørn Gildberg (Norwegian Institute of Fishery and Aquaculture, Norway); Drs. Amparo Chiralt and Pedro Fito (Polytechnic University of Valencia, Spain); Drs. Albert Ibarz and Joaquin Giner Segui (University of Lleida, Spain); Dr. Sam K. C. Chang (North Dakota State University, United States); Dr. James H. May (University of Hawaii at Manoa, United States); Dr. Yueming Jiang (South China Institute of Botany, Chinese Academy of Sciences, China); Dr. Eleanor Riemer, Ms. Susan Lee, and Ms. Patricia Roberson (CRC/Taylor & Francis) for their support and encouragement in the preparation of the book proposal and manuscript.

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Editor Dr. John Shi is a senior research scientist in the Federal Department of Agriculture and Agri-Food Canada, and is also an adjunct professor of food engineering, at the School of Engineering, University of Guelph. He is co-editor of two books, Functional Foods II – Biochemical and Processing Aspects, and Asian Functional Foods, published by CRC Press, now Taylor & Francis. He graduated from Zhejiang University, China, earned a Masters degree in 1985, and Ph.D. in 1994 from Polytechnic University of Valencia, Spain. Dr. Shi is an international editor of the Journal of Food Science and Nutrition and Nutraceuticals and Foods, and also a member of the editorial boards of the Journal of Medicinal Foods and Journal of Agriculture, Food and Environment. As a post-doctoral fellow, he conducted research at North Dakota State University, USA; and as visiting professor he conducted international collaborative research at the Norwegian Institute of Fishery and Aquaculture, Norway, and at Lleida University, Spain. He was keynote speaker at a number of international conferences in the United States, Canada, Japan, China, Korea, Thailand, Spain, and Colombia. He has published more than ninety research papers in international scientific journals and twenty book chapters. His current research interests focus on processing technologies to separate health-promoting components from natural products and to develop functional foods.

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Contributors Mary Ann Augustin, Ph.D. Science Manager Food Chemistry and Formulation Science Food Science Australia Victoria, Australia

Caye M. Drapcho, Ph.D. Associate Professor Department of Biosystems Engineering Clemson University Clemson, SC, United States

Alberto Bertucco, Ph.D. Professor Institute of Chemical Engineering University of Padova Padova, Italy

Hideo Etoh, Ph.D. Professor Faculty of Agriculture Shizuoka University Shizuoka, Japan

Mércia de Fátima M. Bettini, Ph.D. Technical Director of Flavor Tec - Aromas de Frutas Ltda Pindorama, Brazil

Maja Habulin, Ph.D. Associate Professor Faculty of Chemistry and Chemical Engineering University of Maribor Maribor, Slovenia

Juan Eduardo Cacace, M.S. Visiting Research Engineer Pacific Agri-Food Research Center Agriculture and Agri-Food Canada Summerland, BC, Canada Feng Chen, Ph.D. Associate Professor Department of Food Science and Human Nutrition Clemson University Clemson, SC, United States Louise Deschênes, Ph.D. Research Scientist Food Research and Development Centre Agriculture and Agri-Food Canada St. Hyacinthe, QC, Canada

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Yueming Jiang, Ph.D. Professor South China Institute of Botany Chinese Academy of Sciences Guangzhou, China Yukio Kakuda, Ph.D. Professor Department of Food Science University of Guelph Guelph, ON, Canada Lamin S. Kassama, Ph.D. Post-doctoral Research Fellow Guelph Food Research Centre Agriculture and Agri-Food Canada Guelph, ON, Canada

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ˇ Zeljko Knez, Ph.D. Professor Faculty of Chemistry and Chemical Engineering University of Maribor Maribor, Slovenia Aditya Kulkarni, M.S. Research Assistant Faculty of Agriculture Shizuoka University Shizuoka, Japan Ashwani Kumar, Ph.D. Research Scientist Institute for Chemical Process and Environmental Technology National Research Council of Canada Ottawa, ON, Canada Chiara G. Laudani, Ph.D. Associate Professor Faculty of Chemistry and Chemical Engineering University of Maribor Maribor, Slovenia Giuseppe Mazza, Ph.D., FCIFST Principal Research Scientist Pacific Agri-Food Research Center Agriculture and Agri-Food Canada Summerland, BC, Canada Gauri S. Mittal, Ph.D. Professor School of Engineering University of Guelph Guelph, ON, Canada Valérie Orsat, Ph.D. Research Engineer Bioresource Engineering Department Macdonald Campus of McGill University Ste-Anne de Bellevue, QC, Canada

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ˇ ˇ Ph.D. Mateja Primozic, Associate Professor Faculty of Chemistry and Chemical Engineering Laboratory for Separation Processes University of Maribor Maribor, Slovenia G. S. Vijaya Raghavan, Ph.D. Professor Bioresource Engineering Department Macdonald Campus of McGill University Ste-Anne de Bellevue, QC, Canada Luz Sanguansri, Ph.D. Research Scientist Food Science Australia Victoria, Australia John Shi, Ph.D. Research Scientist Guelph Food Research Centre Agriculture and Agri-Food Canada Guelph, ON, Canada Helena Sovová, Ph.D. Professor Institute of Chemical Process Fundamentals Academy of Sciences of the Czech Republic Prague, Czech Republic Sara Spilimbergo, Ph.D. Professor Department of Materials Engineering and Industrial Technologies Faculty of Engineering University of Trento Trento, Italy

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Terry H. Walker, Ph.D. Associate Professor Department of Biosystems Engineering Clemson University Clemson, SC, United States Shiow Y. Wang, Ph.D. Plant Physiologist/Biochemist Fruit Laboratory ARS-USDA Beltsville, MD, United States

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Xiaoqin Zhou, M.S. Professional Engineer Department of Chemical Engineering University of Waterloo Waterloo, ON, Canada

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Table of Contents PART I

Supercritical Fluid Extraction Technology

Chapter 1 Supercritical Fluid Technology for Extraction of Bioactive Components ...............3 John Shi, Lamin S. Kassama, and Yukio Kakuda Chapter 2 Solubility of Food Components and Product Recovery in the Supercritical Fluid Separation Process....................................................................45 John Shi and Xiaoqin Zhou Chapter 3 Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials .........................................................................................................75 Helena Sovová Chapter 4 Biochemical Reactions in Supercritical Fluids .....................................................111 ˇ ˇ ˇ Zeljko Knez, Chiara G. Laudani, Maja Habulin, Mateja Primozic

PART II

Pressurized Low Polarity Water Extraction, Membrane Separation, Distillation, and Dehydration Technologies

Chapter 5 Pressurized Low Polarity Water Extraction of Biologically Active Compounds from Plant Products ..........................................................................135 Juan Eduardo Cacace and Giuseppe Mazza Chapter 6 Purification of Orange Peel Oil and Oil Phase by Vacuum Distillation ..............157 Mércia de Fátima M. Bettini

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Chapter 7 Dehydration Technologies to Retain Bioactive Components ...............................173 Valérie Orsat and G. S. Vijaya Raghavan Chapter 8 Membrane Separation Technology in Processing Bioactive Components ...........193 Ashwani Kumar

PART III

Bioprocessing Technology

Chapter 9 Bioprocessing Technology for Production of Nutraceutical Compounds ............211 Terry H. Walker, Caye M. Drapcho, and Feng Chen Chapter 10 Microbial Modeling as Basis for Bioreactor Design for Nutraceutical Production .......................................................................................237 Caye M. Drapcho

PART IV

Preservation and Packaging Technologies

Chapter 11 Food Pasteurization and Sterilization with High Pressure ...................................269 Alberto Bertucco and Sara Spilimbergo Chapter 12 Microencapsulation and Delivery of Omega-3 Fatty Acids .................................297 Luz Sanguansri and Mary Ann Augustin Chapter 13 Packaging Technologies of Functional Foods.......................................................329 Louise Deschênes

PART V

Antioxidant Properties and Material

Chapter 14 Biological Antioxidation Mechanisms: Quenching of Peroxynitrite ...................341 Aditya Kulkarni and Hideo Etoh

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Chapter 15 Stability of Lycopene During Food Processing ....................................................353 John Shi, Yukio Kakuda, Yueming Jiang, and Gauri S. Mittal Chapter 16 Fruits with High Antioxidant Activity as Functional Foods ................................371 Shiow Y. Wang

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Supercritical Fluid Technology for Extraction of Bioactive Components John Shi and Lamin S. Kassama Agriculture and Agri-Food Canada

Yukio Kakuda University of Guelph

CONTENTS 1.1 1.2

Introduction ......................................................................................................4 Process Concept Schemes and Systems ..........................................................6 1.2.1 Process Scheme and System................................................................7 1.2.1.1 Single-Stage Extraction Process...........................................7 1.2.1.2 Multistage Extraction Process ..............................................9 1.2.2 Physicochemical Properties of Supercritical (CO2) Fluids ...............10 1.2.2.1 Phase Diagram ....................................................................10 1.2.2.2 Physical Properties..............................................................10 1.3 Applications in the Food Industry .................................................................12 1.3.1 Extraction of Bioactive Compounds..................................................12 1.3.2 Fractionation of Flavors and Fragrances ...........................................18 1.3.3 Cholesterol-Free Food Products ........................................................20 1.3.4 Separation of Spices and Essential Oils............................................21 1.3.5 Decaffeination of Coffee and Tea......................................................23 1.3.6 Fish Oil Concentration.......................................................................26 1.4. Factors Affecting Extraction Yield ................................................................27 1.4.1 Pressure ..............................................................................................27 1.4.2 Temperature........................................................................................28 1.4.3 Moisture Content of Raw Materials ..................................................29 1.4.4 Cosolvent............................................................................................30

3

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1.4.5 Particle Size........................................................................................32 1.4.6 Flow Rate ...........................................................................................32 1.4.7 Effect of Time on Yield .....................................................................34 1.5 Summary ........................................................................................................36 References................................................................................................................37

1.1 INTRODUCTION Some chemicals are toxic and if consumed can lead to cancer and other ailments of public concern. As a result, the conventional solvent extraction methods are viewed with suspicion with regards to their role in the manufacturing of functional foods. Extracts from natural sources are key elements in the manufacturing of functional foods. These are any food fortified with extracts from natural sources or nutraceutical. A new paradigm, supercritical extraction technology, was developed to extract bioactive components for use as supplements for functional foods. This technology uses supercritical fluids as the extracting solvent which has a significant attribute paramount to the food and pharmaceutical industry because it leaves no residue in the extract and is gaseous at ambient temperature. Extraction with organic solvents is a well-established technique for selective separation of specific constituents from plant products. Organic solvents with low boiling point such as ethyl acetate, methanol, dichloromethane, and so forth, have been successfully used to isolate bioactive components from hops, spices, oil seeds, and other plant products. The procedure is used to decaffeinate coffee and tea, and to remove nicotine from tobacco. The solvents used for this purpose must meet the legal requirements put in place to ensure food quality and safety, and these regulations vary from country to country. These requirements are: high degree of purity, chemical stability, inert (no reaction with food constituent), low boiling point, and no toxic effects. Criteria for these regulations are set by national and international bodies such as the US Food and Drug Administration (FDA); European Economic Commission (EEC) Codex committee; the Canadian Food Inspection Agency (CFIA); and FAO/WHO Codex Alimentarius Commission. Most countries have regulations stating which extraction solvents are generally regarded as safe (GRAS). For example, the Canadian food additive regulation (FDAR: B.10.045) for food chemical codex specification and the EEC directive (88/344/EEC) state the regulations regarding the use of extraction solvents in the production of foodstuffs and food ingredients within Canada and the EEC. Although only mandatory within Canada and the EEC, in general they are comparable to those in other countries. Public health, environmental, and safety issues are the major concerns in the use of organic solvents in food processing. The possibility of solvent residues remaining in the final product has been a growing concern to consumers, thus warranting stringent environmental regulations. The demand for ultrapure and highadded-value products is redirecting the focus of the food and pharmaceutical industries into seeking the development of new and clean technologies for their products. The supercritical fluid extraction (SFE) technology has provided an excellent alternative to the conventional organic solvent extraction methods. Although the

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Supercritical Fluid Technology for Extraction of Bioactive Components

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technology was known for more than 100 years, its application in the food and pharmaceutical industry began only three decades ago.1,2 Since that time, over 100 plants of different capacities have been built globally for extraction of desired solutes from solid materials.3 Supercritical extraction is a novel separation technique that utilizes the solvent properties of fluids near their thermodynamic critical points.4 A variety of processes involving extractions with supercritical fluids (SCF) have been developed and are regarded as a viable extraction technology that meets the food quality and safety requirements. The physicochemical properties of supercritical fluids, such as the density, diffusivity, viscosity, and dielectric constant can be controlled by varying the operating conditions of pressure and temperature or both in combination.1,3,5 Many supercritical fluids (carbon dioxide, ethane, propane, butane, pentane, ethylene, ammonia, sulfur dioxide, water, chlorodifluoromethane, etc.) are used in supercritical extraction processes. Sihvonen et al.,1 Brunner,3 and Rozzi and Singh6 recommended carbon dioxide (CO2) in their reports because of its favorable properties and the ease of changing selectivity by the addition of agents such as ethanol and other polar solvents. Supercritical carbon dioxide (SC-CO2) is the most desirable supercritical fluid for extracting natural products for foods and medicinal uses. Its characteristic traits are inert, nonflammable, noncorrosive, inexpensive, availability, odorless, tasteless, environmentally friendly, and GRAS status.7 Its near-ambient critical temperature makes it ideal for thermolabile natural products.8,9 Supercritical CO2 has the following advantageous attributes over other solvents: (a) it has a solvating power similar to organic liquid solvents and higher diffusivities, lower surface tension, and viscosity; (b) separation can be affected by simply changing the operating pressure or temperature to alter the solvating power of the solvent; and (c) modifying CO2 with a cosolvent can significantly augment the selective and separation power and in some cases extend its solvating powers to polar components. Supercritical carbon dioxide is being given a great deal more attention as an alternative to industrial solvents as a result of (a) increased governmental scrutiny and new regulations restricting the use of common industrial solvents such as chlorinated hydrocarbons; (b) its nontoxic and environmentally friendly attributes, given that it leaves no traces of solvent residue in food; (c) sharp increase in energy cost, which increased the cost of traditional energy-intensive separation technique, such as distillation; (d) carbon dioxide is cheap, safe to use, recyclable, and has minimum disposal cost required; (e) stringent pollution-control legislation prompting industries to seek alternative means of waste treatment and utilization; and (f) increased performance demands on materials, which traditional processing techniques cannot meet. It must, however, be stated that commercial applications of the SFE technology remain limited to a few high-value products due to high capital investment,10,11 its novelty, and complex operating system. Adoption of the technology is on the rise as a result of advances in processing, equipment, and the realization of producing high-value products with high profitability.1,10 Supercritical fluid extraction is one of the most promising technologies being adopted in the chemical, food, pharmaceutical, and neutraceutical industries. Analytical techniques are superior to the conventional organic solvents when applied for qualitative and quantitative extraction

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of natural thermolabile products.12,13 Research efforts have proven that the SFE technologies could be successfully employed in the extraction and fractionation of fats and oils; purification of solid matrix; separation of tocopherols and antioxidants; removal of pesticide residue from herbs, medicines, and food products; detoxification of shellfish; and concentration of fermented broth, fruit juices, essential oils, spices, and coffee; and separation of caffeine, and so forth.3,14–16

1.2 PROCESS CONCEPT SCHEMES AND SYSTEMS The supercritical fluid extraction technology was conceptualized on the basis of obtaining pure extract without solvent residues which could be detrimental to consumers of food and pharmaceutical products. Extraction is an analytical process used to separate and isolate a targeted component from other substances. The success of the process is dependent on the distribution of the analyte between two phases, the separation and stationary phases.17 In the phase equilibrium between liquid and gas, the partition of the liquid phase increases with increasing pressure and decreases with increasing temperature. If the temperature and pressure are simultaneously increased, the transport properties of both liquid and gas increases and thus convergence occurs. When used under practical pressure and temperature conditions of 5 to 50 MPa and ambient to 300°C, respectively, the solubility properties of the supercritical fluid are greatly influenced by its density, diffusivity, and viscosity. The SC-CO2 is liquid-like and has a higher extraction flux than those obtained with organic liquids solvents. King et al.17 stated that at high CO2 densities, its solvent properties were similar to organic solvents like chloroform and acetone, and if intermediate compression were applied, it behaved like a nonpolar hydrocarbon such as n-pentane or diethyl ether. The separation phase occurs during the dynamic extraction period, while the stationary phase is the sample material loaded as a fixed-bed in the extraction column. Supercritical extraction (SCE) involves the use of compressed gases at or above their critical temperature (Tc) and pressure (Pc). It utilizes the ability of these special fluids to become excellent solvents to solvate certain solutes (bioactive components) from a solid matrix.6 The solute extraction stream from the sample matrix is directly proportional to the product of solubility and diffusivity in the supercritical medium. Hence the solute’s solubility increases with pressure, while its corresponding diffusivity is expected to decrease by two orders of magnitude. The solvent capacity is mainly the function of density and can be improved with the addition of a cosolvent, which modifies the density and polarity of the supercritical fluid, thus significantly increasing the yield. This technology has been successfully applied in the extraction of bioactive components (antioxidants, flavonoids, lycopene, essential oils, lectins, carotenoids, etc.) from a variety of biological materials such as hops, spices, tomato skins, and other raw or waste agricultural materials. The process requires intimate contact between the packed bed formed by a ground solid substratum (fixed-bed of extractable material) with a supercritical fluid.18 During the supercritical extraction process, the solid phase comprised of the solute and the insoluble residuum (matrix) is brought

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Supercritical Fluid Technology for Extraction of Bioactive Components

Back pressure valve

Pressure valve

CO2 outlet Separator

Extractor column

Heat exchanger

Heat exchanger

CO2 Pump

Cooling bath

7

Extracts outlet

CO2 Source

FIGURE 1.1 Schematic diagram of a typical single-stage supercritical fluid extraction system with CO2.

into contact with the fluid phase which is the solution of the solute in the supercritical fluid (solvent). The extracted material is then conveyed to a separation unit. The power of supercritical fluid extraction is linked to the solubility and phase equilibrium of substances in the compressed gas. The targeted bioactive component being extracted must be soluble in the supercritical fluid. Controlling the pressure and temperature of SCF varies the solubility and phase equilibria. The extraction of pure and high-value extract is accomplished without risk of environmental pollution or residual solvent contamination in the final product.

1.2.1 PROCESS SCHEME

AND

SYSTEM

The extraction with supercritical fluids is comparable to liquid-liquid solvent extraction even though, with supercritical extraction, compressed gas is used instead of organic solvents and the applied pressure is crucial. The supercritical fluid extraction process is governed by four key steps: extraction, expansion, separation, and solvent conditioning. The steps are accompanied by four generic primary components: extractor column (high-pressure vessel), pressure control valves, separator column, and pressure intensifier (pump) (Figure 1.1) for the recyclable solvent.3,5,17,19,20 The system has other built-in accessories, such as heat exchangers for providing a source of heating; condensers for condensing supercritical fluids to liquid; storage vessels; and a supercritical fluid source. Raw materials are usually ground and charged into a temperature-controlled extractor column forming a fixed bed, which is usually the case for batch and single-stage mode. 1.2.1.1 Single-Stage Extraction Process The supercritical fluid is fed at high pressure by means of a pump, which pressurizes the extraction tank and also circulates the supercritical medium throughout the system. Figure 1.2 shows an example of a typical single-stage supercritical extraction system. Once the SCF and the feed reach equilibrium in the extraction vessel, through the manipulation of pressure and temperature to achieve the ideal operating conditions, the extraction process proceeds. The mobile phase consisting of the SCCO2 fluid and the solubilized components are transferred to the separator where the

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Functional Food Ingredients and Nutraceuticals

Three stage separator columns

CO2 inlet

Extractor column

Pressure regulator

Dry test meter

Extracts

8

Ice water trap

CO2 pump

Co-solvent inlet

Mixer

CO-solvent pump

FIGURE 1.2 Schematic diagram of supercritical fluid extraction system used to fractionate bioactive components from plant matrix using supercritical carbon dioxide.

solvating power of the fluid is decreased by increasing the temperature or decreasing the pressure of the system. The extract precipitates in the separator while the supercritical fluid is either released or recycled back to the extractor. In the case where highly volatile components are being extracted, a multistage configuration may have to be employed as shown in Figure 1.2. As the solutions leave the extractor and flow to the first separation vessel via the pressure regulator, the pasty oleoresins settle to the bottom as they separate and can be collected, while the remaining solution goes to the second-stage separator where the fractionation of the volatile components occurs. For more sensitive products, the third stage of separation would be required for the complete isolation of pure volatile components. Saltzman et al.21 presented a design (Figure 1.2) where the solution flows through a heated valve and precipitates into a preweight U-tube in an ice-water bath. The glass wool at the U-tube exit traps the entrained solutes in the gas, while the gas flows through a dry-test meter which monitors the flow rate. Oszagyan et al.22 used a similar system as illustrated in Figure 1.2 to extract essential oil from Lavandula intermedia Emeric ex aloisel and herb of Thymus vulgaris L., and further fractionated volatile components (-Cymene, -Terpinene, Thymol, and Carvacrol) while Ozcan23 used it to fractionate volatile components from Turkish herbal tea (Salvia aucheri Bentham Var. canaescen Boiss and Heldr.). Duquesnoy et al.24 and Boutekedjiret et al.25 extracted and fractionated volatile compounds from plant materials using SCE with a similar multistage fractionation method. The processes described above are semibatch continuous, where the SCF flows in a continuous mode while the extractable solid feed is charged into the extraction vessel in batches. In a commercial processing plant, multiple extraction vessels are sequentially used to enhance process performance and output. Although the system is interrupted at the end of the extraction period, when the process is switched to a

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Supercritical Fluid Technology for Extraction of Bioactive Components

9

2 Stage separation vessels

Storage tank

3 Stage extraction columns

Co-solvent inlet

CO2 inlet Pu mps

FIGURE 1.3 Schematic diagram of commercial scale multistage supercritical fluid extraction system used to fractionate bioactive components.

prepared vessel for extraction, the unloading and/or loading of the spent vessels can be carried out while extraction is in progress, reducing the downtime and improving the production efficiency. 1.2.1.2 Multistage Extraction Process A semicontinuous approach on a commercial scale uses a multiple-stage extraction process which involves running the system concurrently by harnessing a series of extraction vessels in tandem as shown in Figure 1.3. In this system the process is not interrupted at the end of extraction period for each vessel, because the process is switched to the next prepared vessel by control valves for extraction while unloading or loading the spent vessels; although imperfect, continuity is attained. The primary extraction stages operate in a similar mode to the ones depicted in Figure 1.2 and Figure 1.3. The raffinate from the premier stage enters the first separation vessel while separation and fractionation of different compounds occurs based on their relative solubility. The options of cosolvent are available to enhance the solvent power of separation of specific components. This is effective for cases where more than one targeted component is to be extracted, giving the flexibility to vary the extraction parameters such as pressure and temperature to achieve different solubilities for different components being extracted at each stage of operation. Gamse26 suggested that highly soluble substances could be extracted at the initial stages at low SCF density, and by increasing the density in the subsequent stages remove the less soluble substances. The supercritical pressure, temperature, and flow rate at each stage could be controlled independently.

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1.2.2 PHYSICOCHEMICAL PROPERTIES FLUIDS

OF

SUPERCRITICAL (CO2)

The physicochemical properties of the supercritical fluids are crucial to the understanding of the process design calculation and modeling of the extraction process. Therefore, selectivity of solvents to discriminate solutes is a key property for the process engineer. Physical characteristics such as density and interfacial tension are important for separation to proceed; the density of the extract phase must be different from that of the raffinate phase and the interfacial properties influence coalescence, a step that must occur if the extract and raffinate phase are to separate. 1.2.2.1 Phase Diagram Supercritical state of fluid is influenced by temperature and pressure above the critical point. The critical point is the end of the vapor-liquid coexistence curve as shown on the pressure-temperature curve in Figure 1.4 where a single gaseous phase is generated. When pressure and temperature are further increased beyond this critical point, the fluid enters a supercritical state. At this state no phase transition will occur regardless of any increase in pressure or temperature nor will it transit to a liquid phase. Hence, diffusion and mass transfer during supercritical extraction are about two orders of magnitude greater than in the liquid state. 1.2.2.2 Physical Properties Substances that have similar polarities will be soluble in each other, but increasing deviation in polarity will make solubility increasingly difficult. Intermolecular polarities exist as a result of van der Waals forces, and, although solubility behaviors

Pressure (MPa)

Supercritical region Liquid phase PC Solid phase

Triple point

Gas phase Tc = 31.1°C Pc = 7.38 MPa TC

Temperature (°C)

FIGURE 1.4 Supercritical pressure-temperature diagram for carbon dioxide.

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11

TABLE 1.1 Comparison of Physical Properties of Supercritical CO2 at 20 MPa and 55°C with Some Selected Liquid Solvent at 25°C Properties

CO2

n-Hexane

Methylene Chloride

Methanol

Density (g/mL) Kinematic viscosity (m2/s) Diffusivity (m2/s) Cohesive energy density (δ(cal/cm∏))

0.75 1.0 6.0 × 109 10.8

0.66 4.45 4.0 × 109 7.24

1.33 3.09 2.9 × 109 9.93

0.79 6.91 1.8 × 109 14.28

Source: Modified from King et al.17

depend on the degree of intermolecular attraction between molecules, the discrimination between different types of polarities is also important. Substances dissolve in each other if their intermolecular forces are similar, or if the composite forces are made up in the same way. Properties such as the density, diffusivity, dielectrical constant, viscosity, and solubility are paramount to supercritical extraction process design. The dissolving power of SCF depends on its density and the mass transfer characteristic, and is superior due to its high diffusivity, low viscosity, and interfacial tension to liquid solvents. Although many different types of supercritical fluids are in existence and have many industrial applications, CO2 is the most desired for SCE of bioactive components. Table 1.1 shows some physical properties of compressed (20 MPa) supercritical CO2 at 55°C compared to condensed liquids commonly used as extraction solvents at 25°C. It should be noticed that supercritical CO2 exhibited similar density as those of the liquid solvents, while less viscous and highly diffusive. This fluidlike attribute of CO2 coupled with its ideal transport properties and other quality attributes outlined above make it a better choice over other solvents. The specific heat capacity (Cp) of CO2 rapidly increases as the critical point (31.1°C temperature, 7.37 MPa pressure, and 467.7 g/L flow rate) is approached. Like enthalpy and entropy, the heat capacity is a function of temperature, pressure, and density.5 Under constant temperature both the enthalpy and entropy of supercritical CO2 decreases with increased pressure and increases with temperature at constant pressure. The change in the specific heat as a result of varying the pressure and temperature is also dependent on density. For example, under constant temperature, specific heat increases with increased density up to a certain critical level. Above this critical level, any further increase of density decreases the specific heat. Sample matrix is an important parameter that influences solubility and mass transfer process during SCE. Properties such as particle shape and size distribution, porosity and pore size distributions, surface area, and moisture content influence solubility and mass transfer. The presence of water (moisture content) in the sample matrix during supercritical extraction also has an effect on the extraction outcome.

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Functional Food Ingredients and Nutraceuticals

1.3 APPLICATIONS IN THE FOOD INDUSTRY One of the most important trends in the food industry today is the demand for allnatural food ingredients that are free of chemical additives. Natural food antioxidants are derivatives of plant by-products. A quantum leap in supercritical fluid extraction technology was made by its applications in decaffeinating coffee, tea, and other bioactive (essential oils from spices) components used as ingredients in foods. Likewise, SCF extraction is used to extract flavor and fragrance components, highvalue compounds used in the food, pharmaceuticals, and neutraceutical industries. With this technology, extracts of natural nutrients could be utilized without the fear of organic solvent residues. A compendium of process parameters used for different product applications is listed in Table 1.2. Various processes of extracting bioactive components from agricultural materials have been used commercially. The major hurdles to overcome in SCE are to obtain extracted compounds with similar bioactivity as the synthetic compounds and also to maintain the required flavor, odor, and color components that may be detectable in the treated food product. Many extraction processes employ the following methods to extract bioactive components from plants (Labiatae family): solvent (polar and nonpolar) extraction and aqueous alkaline58; extraction of vegetable oils and mono/diglycerides59 by steam distillation and molecular distillation.23 These processes are limited by numerous disadvantages, because the solvents used are not very selective for the bioactive components. Consequently, the resulting extracts are not as pure as synthetic chemical compounds. The solvents used include hydrocarbons such as hexane, acetone, and methyl chloride which leave unwanted residues in the food products, which are prohibited in some instances by regulatory bodies for use in food as discussed earlier. Many researchers applied SCE technology for the extraction of bioactive components.3,56,60–62

1.3.1 EXTRACTION

OF

BIOACTIVE COMPOUNDS

Some bioactive components are phenolic or polyphenolic compounds of plant origins which interfere with the formation of free radicals, thus preventing the formation of hydroperoxides. However, during food processing, especially conventional thermal processing of food products, these bioactive components are lost. To restore these components necessitates fortification. Some of the more common bioactive compounds include lycopene, flavonoids, tocopherol, lecithin, ascorbic acid, citric acid, polyphenols, and so forth. Most of the separation procedures involve physical and chemical processes such as centrifugation, filtration, membrane separation, precipitation, chromatography, solvent extraction, crystallization, evaporation, molecular distillation, and SC-CO2. To overcome the separation problems encountered when producing soluble materials via such processing procedures requires conditions that may have detrimental effects on the nature of the extraction technique or the product stability. In order to achieve extract labile compounds, a rapid separation process may be needed to avoid any significant product loss. Most bioactive components used as food additives are in a concentrated form. Appropriate extraction procedures are consequently required

© 2007 by Taylor & Francis Group, LLC

Product Extracted

Raw Material

Sunflower oil Soybean oil

Sunflower seed Soybean

Corm germ oil Seed oil

Corn germ Avocado

Raw Material Pretreatment

Component Concentration (%)

Temp (°C)

Pressure (MPa)

Ground

40−50 32–35

Flaked, 20.1 0.38–5.1mm Dried, milled 23.4 Dried, ground (< 0.25 mm)

50

7.58–8.27

50 70

55.2 75.8

50 50 70–75 50

Cottonseed Paprika Peanuts Peanut hearts Rice bran Sorghum bran Sorghum germ Soybean Wheat bran Wheat germ

© 2007 by Taylor & Francis Group, LLC

CO2 Flow Rate Time (L/h) (h)

MC (%)

2.5

Recovery (%)

Cosolvent

Source

Ethanol

36

Cocero & Calvo27 Friedich et al.28

Ethanol

50 58.2

Ronyai et al.29 Friedich & Pryde30

900–1080

9.8

1140

24

3.5 3.4

55.2 55.2 68.9 68.9

8 7 8 11

8.6 5 9.0 2.8

30.8 7.2 48.0 42.6

70 78

65.5 66.2

6 11

6.3 6.1

19.2 5.0

70

68.9

7

9.4

16.8

50 50 50

55.2 55.2 55.2

8 5 6

11.4 11.4 10.1

19.4 4.0 7.0

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TABLE 1.2 Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2

Product Extracted (Essential oil) Limonene Carvone Anethole Eugenol and Caryophyllene Yaleranone Wheat germ oil

Licochalcone A Licochalcone B Defatted mustard Canola oil

Bergaptene

Raw Material

Raw Material Pretreatment

Component Concentration (%)

Sweet Dried ground orange Sieved (1, 0.7, Caraway 0.4, 0.2, 0.08 seed mm) Anise seed Cloves Spikenard Wheat germ Milled, 10.2 powder (20 µm diameter) Licorile Cut root Mustard Dried, fully 16 seed pressed (1mm) Canola seed Flaking (0.2–0.5 mm) cooking (90°C) Bergamot Peel dried 0.15 Citrus ground

© 2007 by Taylor & Francis Group, LLC

Temp (°C)

Pressure (MPa)

CO2 Flow Rate Time (L/h) (h)

23–40 9–10

60

35–50 13–41

0.12

2

35

12.7

360

0.5

40

30.4

300–400

3

45–70 41–62

180

3

40–60 8–10

2.6

2

MC (%)

Recovery (%)

Cosolvent

Source

6.7

60

Sovava et al.31

4.3–11.5

98.7

Ge et al.32

5% Ethanol 25–30

Ethanol

17–19

80

Taniguchi et al.33

44

Dunford and Temelli34

85

Poiana et al.35

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TABLE 1.2 (CONTINUED) Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2

Defatted & decholesterol of muscle Oil Bixin pigment Defatted & decholesterol beef Bixin pigment

Evening primrose oil Cardamom oil

Alfalfa

Leaf protein concentration Dehydrated Spray dried beef powder + air dried chunk Antarctic Freeze-dried 16.2 krill meal & ground Annatto Seed, 3.92 seed mm Fresh 19 frozen Ground beef Annatto Whole seed 1.3 seed (11.6 oil)

40

Evening primrose seed Cardamom seeds

Decholesterol beef tallow β-carotene

Crude beef tallow Sweet potato

Lanolin

Woolgrease

Dried, ground, 0.355 mm Freshly ground seeds, mesh

Freeze dried ground ( 0.25 mm)

© 2007 by Taylor & Francis Group, LLC

30– 70

300–360

90 70

Favati et al.36

1.4–3.2

87

Wehling et al.37

7.83

99

Yamaguchi et al.38 Chao et al.39

56-lipid 26-cholesterol

Chao et al.40

3

45–55 23–3927.6 300–600

80

24.5

51.4

3–4

50 60

34.5 31

1–2

35

31.

117.2 – 144.8 117.2 – 144.8

50

29.6

1.2

21.9

60

70

64.8

40–60

40

10

0.15–0.2

40

34.5

226.7250

94

48

41.4

840–1080

80

38

396–2880

4–8

Degnan et al.41

Soybean oil 20% v 1.7–5 < 8

97

Favati et al.42

3

85–95

Gopalakrishnan & Narayanan43

60–70

Chao et al.44

80

Spanos et al.45

4–6

15.5

10

CygnarowiczProvost et al.46

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Carotene lutein

Product Extracted

Raw Material

Phospholipid mixture

Soybean flakes

Tocopherol enrichment

Soybean flakes Rice bran Carrot

Carotene Essential oil

Star anise fruit

Protein

Spring mackerel Black pepper Eucalyptus leaves Ginger rhizomes Palm oil fiber

Essential oil Piperine Essential oil Essential oils gingeroles Triglycerides carotenoid

Raw Material Pretreatment

Component Concentration (%)

Ground, 5.134 mg/g thinly flakes, (0.1–0.25 mm)

Frozen puree (0.93mm) Dried, ground, particle size (0.57 mm) Freeze-dried, chopped Dried ground

Oven dried

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Pressure (MPa)

CO2 Flow Rate Time (L/h) (h)

MC (%)

Source

68.2

43.6–109

80

25

300

500 ppm

55

20.7

90

8–9

26.5

6.5

5.4

1.5

12.7

97

35

34.5

1080

15

1.6

87

40

9–15

2.55

9

18

Perakis et al.49

50

9

1.1

2.5

9.5

2.4

Porta et al.50

138

2

8.8

8.4

Catchpole et al.51

1.45

2.25

5

7

Franca and Meirele52

1.5 5.7

30

40 45– 55

30 20–30

10.8

Recovery (%)

Cosolvent

80

Air drying Dried ground

Temp (°C)

81

Ethanol (5% v)

Ethanol 10%

60-Soybean King et al.47 70-Rice bran 80-Wheat germ Vega et al.48

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TABLE 1.2 (CONTINUED) Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2

Essential oil vanillin Essential oil Capsaicine alkaloids Essential oil Antioxidants

Essential oil

Tomatoes Peppermint & Spearmint Vanilla pod Chili pepper

Dried skin ground Cut leaves

65 35

Dried ground

36

36

11

Dried ground

2

40

30

50

15

Coriander

Dried grounds 96 (0.4 mm) Sweet Thai Dried ground Tamarinds (mesh 40–70) Grape seed Wash dry 80 ground

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40–80 17–28

30

0.5

24–43 6–18

3.42

4–9

35–80 10–30

35–75 20–47

Cadoni et al.53

Chloroform 14–82

76

Barton et al.54

97 2

3

3

0.3

5

0.75

Catchpole et al.51

8.8

0.61

10

Anitescu et al.55

Ethanol

Luengthanaphol et al.56

2% Ethanol 77

Lee et al.57

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Lycopene -Carotene Essential oil Menthol

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when preparing them from their original matrices. Some compounds in the concentrated form are thermolabile, volatile, and prone to degradation when subject to intensive heat. Supercritical extraction with CO2 is the most viable method for food applications. Baysal et al.63 extracted lycopene and -carotene from tomato using SC-CO2. The processing conditions used were extraction pressure of 20, 25, and 30 MPa; temperatures of 35°C, 45°C, 55°C, and 65°C; resident time of 1, 2, and 3 h; and CO2 flow rate of 2, 4, and 8 kg/h. The best conditions for lycopene extraction were 2 h at a flow rate of 4 kg/h, pressure of 30 MPa, temperature of 55°C, and with the addition of 5% cosolvent (ethanol). They noted that if too much ethanol is used, it decreased the homogeneity of the extraction mixture, and reduced the separation efficiency. Brunner and Peter64 and Vega et al.48 corroborated this finding. Luengthanaphol et al.56 compared the SC-CO2 extraction to other methods and their effects on bioactivity. Their results compared to Tsuda et al.65 indicated the superiority of SC-CO2 coupled with cosolvent as shown in Table 1.3. Both studies show the superiority of antioxidant extraction with SC-CO2 and modifiers, although some disparity occurred which could have been caused by the variety used. Macias-Sanchez et al.61 extracted carotenoids and chlorophyll from Nannochloropsis gaditana and achieved the highest yield at 20 MPa and 60°C, the optimal pressure and temperature, respectively. Wang et al.66 also reported that the antioxidant activity of Bupleurum kaoi Liu fractionated with SC-CO2 gave the highest yield of phenol and the strongest antioxidant capacities.

1.3.2 FRACTIONATION

OF

FLAVORS

AND

FRAGRANCES

The extraction of compounds, for flavor and fragrances, by supercritical CO2 is paramount in the food industry. Mother Nature is a splendid synthesizer of flavors and fragrances in natural products. The cleaner and safer attributes make supercritical

TABLE 1.3 Yields and Activities of Antioxidants Extracted by Different Methods from Sweet Thai Tamarind Seed. SC-CO2 Extraction at 30 MPa and 80°C; Organic Solvent Extraction at Room Temperature PV after 24 h (meq/kg dry weight)

(-) Epicatechin Yield (mg/100 g dry weight ) Extraction SC-CO2 SC-CO2 + Cosolvent (10% Ethanol) Ethanol α-tocopherol

Luengthanaphol et al.56 Tsuda et al.65

Luengthanaphol et al.56

Tsuda et al.65

0.022 13

0.336 26

– 231

– ≈26

25

32

– 157

≈25

56

65

Source: Modified from Luengthanaphol et al. and Tsuda et al.

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19

TABLE 1.4 The Results of Gas Chromatography and Mass Spectrophotometer Analysis of Flavor Compounds Extracted by the Likens-Nickerson Extraction and the Supercritical CO2 Extraction Methods of Basmati Rice Likens-Nickerson Extraction Peak No.

Retention Time (min)

2 3 5 8 13 14 19 21 22 24

7.51 8.11 9.51 14.52 21.98 22.68 27.71 28.53 30.45 32.14

Hexamethyl disiloxane* Silicate anion tetramer* Dibutyl phthalate* Hexane cyclotrisiloxane* Pentanal Butan-2-one-3-Me Heptanol 2-Heptanone Octanol Octanal

28 30 31 34 35 37

35.14 36.12 37.34 40.22 42.77 43.82

2-Octanone Nonanal Decanol 2-Decenal Undecane Benzoic acid 2,5-bis (trimethylsiloxy) benzene*

39

46.28

Compound

SC-CO2 Peak No.

Retention Time (min)

Compound

12 13 19 20 21 23 29 30 31 32 34 37 40

22.11 22.56 28.32 29.27 30.92 32.26 35.19 35.44 36.13 37.42 40.31 42.52 45.11

Pentanal Butan-2-one-3-Me Heptanol 2-Heptanone Octanol Octanal 2-Octenal 2-Octanone Nonanal Decanol 2-Decenal Undecane Dodecane

44

45.24

Tetradecane

Compounds identified as artifacts. Source: Modified from Bhattacharjee et al.62

technology an ideal candidate for extracting such valuable and heat-sensitive products when compared to toxic organic solvents. The high-value-added natural products are good for use in soft drinks. An example is ginger extract which gives the pungency and flavor in ginger ale drinks. Bhattacharjee et al.62 compared the Likens-Nickerson extraction and the SCCO2 extraction methods on Basmati rice. The results showed that the SCE technique was superior and extracted the most flavor components that bore the closest resemblance to the original Basmati flavor (Table 1.4). The SC-CO2 technique extracted more flavor compounds and produced purer (resembles original basmati flavor) extracts than its counterpart (Liken-Nickerson method). Desired fragrances are isolated from concentrates extracted from flowers. The process consists of initial solvent extraction, usually with hexane, which yields an intermediate product called concrete. This product contains fragrances and other components like paraffin, fatty acids, fatty acids methyl ester, di-, and tri-terpenic

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compounds, pigments, and so forth. Postprocessing of the concrete can be done using SC-CO2 extraction. Reverchon67 used single-step SCE at a pressure of 8 MPa and temperature of 40°C followed by a two-stage separation procedure at a pressure of 8 MPa and temperature of –16°C at the first stage and a pressure of 1.5 MPa and a temperature of 0°C at the second stage. Under these optimum conditions the extracted volatile rose oil contains 50% 2-phenylethanol. When a cosolvent (ethanol) is mixed with SC-CO2, a yield of 50% to 60% was observed.68 Jasmine fragrance extracted at 12 MPa and 40°C gave results superior to those of other solvents. Sastry and Mukhopadhyay68 experienced increased yield of 45% to 53% with the use of cosolvents. Similarly, SC-CO2 has been used effectively to extract fragrances from orange, marigold, sandalwood, vetiver, and so forth.

1.3.3 CHOLESTEROL- FREE FOOD PRODUCTS Cholesterol is an unavoidable substance we require for the daily maintenance of our body. Lipoproteins are vehicles that transport cholesterol to various body tissues to be used, stored, or excreted, but if cholesterol is in excess it can lead to coronary heart problems (atherosclerosis). Low-density lipoprotein (LDL) termed “bad” cholesterol causes fat buildup in the arteries increasing the risk of heart disease. Highdensity lipoprotein (HDL) termed “good” cholesterol transports cholesterol back to the liver, where endogenous metabolism prevents cholesterol buildup and reduces the risk of heart disease. The indiscriminate consumption of saturated fats in our diet may raise the total LDL (> 100 mg/dL) level and decrease HDL (< 35 mg/dL) level, thus increasing the risk of heart disease.69 The recommended daily intake of cholesterol is about 300 mg.70 The correlation between serum cholesterol level and mortality rate of cardiovascular disease has been reported in many studies.69,71 Pork has cholesterol content of 30 to 450 mg/100 g, poultry 70 mg, fish 35 to 70 mg/100 g, and beef 65 to 331mg. One common source of cholesterol is from the consumption of fried fast-food products. The fast-food industry uses hydrogenated fats for their deep-fat frying processes because of its stability and high economic turnover. The hydrogenated fat is the potential source of trans fatty acids which are taken up by the fried foods during cooking (French fries, onion rings, chicken nuggets, etc.) and ultimately ingested by the consumer. Trans fats have been shown to increase LDL cholesterol levels and reduce HDL cholesterol levels, thus raising the risk of heart disease. Public health initiatives such as the National Cholesterol Education programs have raised consumer awareness, resulting in the advocation for healthy foods with low cholesterol. Thus, the food industry is under tremendous pressure to address this consumer concern. Supercritical fluid extraction is an emerging technology with great potential to revolutionize the oil/fat industry. Many researchers (Dunford and Temelli34; Temelli et al.34,72) reported the feasibility of supercritical fluid extraction of lipids from food without compromising their organoleptic quality. Chao et al.44 used a similar extractor configuration (Figure 1.3) as discussed earlier with three stage separations, to remove cholesterol. By decreasing the pressure at each stage sequentially from 17 to 11 to 4 MPa, they were able to achieve higher selectivity for cholesterol at the lower pressures. The results also showed that the fraction collected from the third

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TABLE 1.5 Solubility of Cholesterol in Supercritical CO2 under Different Operating Conditions Run No.

Pressure (MPa)

Temperature (°C)

Density of CO2 (g/L)

Solubility of Cholesterol (mg/L)

1 2 3 4 5 6 7 8

10 10 10 10 13 13 13 13

40 45 50 55 40 45 50 55

602.4 494.4 408.1 342.3 723.4 655.8 588.5 527.7

146.9 82.6 47.2 28.5 289.2 234.8 182.7 141.1

Source: Modified from Yeh et al.74

separator at 4 MPa contained concentrated cholesterol ranging from 272 to 433 mg/100 g lipid. Chao et al.40 used SC-CO2 at an operating pressure of 10 to 30 MPa and temperature range of 30°C to 50°C to reduce the cholesterol level in ground beef. Similarly, Hardardottir and Kinsella73 explored the removal of lipids and cholesterol from fish muscle with SC-CO2. They removed over 80% to 99% cholesterol using pressures of 14 to 35 MPa and temperatures of 40°C to 50°C. Although the authors noted limited effect on lipid/cholesterol yield with increased extraction pressure and temperature, increased extraction time from 3 to 9 h significantly increased the yield. Yeh et al.74 used eight operating conditions shown in Table 1.5 to optimize their process and observed that at 10.3 MPa and 55°C operating pressure and temperature, respectively, cholesterol level was reduced from 2867 mg/100 g to 14.1 mg/100 g. Supercritical-CO2 technology was used to fractionate milk fat, which is an excellent raw material with specific functionalities used in many products.75 Extracting cholesterol from anhydrous milk fat with SC-CO2 used in conjunction with adsorbents (silica gel) to maximize yield was demonstrated by Huber et al.76

1.3.4 SEPARATION

OF

SPICES

AND

ESSENTIAL OILS

Spices have strongly flavored or aromatic components that can be used in small quantities in food as a preservative or flavoring ingredient. Chili (capsicium species), ginger (Zingiber officinalis), and pepper (piper nigrum L) are classic pungent flavorines while ginger and chili have additional nutraceutical values. These products have high economic value in their concentrated form. The extraction of spices is usually carried out in two stages: stage one separates pungent oleoresins and the second stage the essential oil fractions. Essential oils are typically volatile terpenes and esters. Essential oils are concentrated pure plant extracts that have long been revered for their therapeutic applications and are derivatives from flowers, leaves,

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stems, berries, rinds, resins, or roots of plants. These are very important ingredients and food additives of high value. Catchpole et al.51 performed a detailed study on the extraction of spices and essential oils using SC-CO2, propane, and dimethyl ether. They reported ginger to be the easiest of all spices in terms of optimized yield relative to pressure and temperature, while capsaicin in chili could be extracted at moderate pressure and temperature, especially with the use of modifiers. Chili oil fraction contains fatty oil and carotenoids, and is speculated that the fatty oil acts as modifier for the capsaicins.77 The author extracted black pepper with much duress, because of its viscous characteristics, thus requiring higher pressure and moderate to high temperatures. The use of supercritical propane for extracting spices was reported by Illes et al.78 They found propane to adequately extract fatty oils, tocopherols, and carotenoids, but inadequate for capsaicins, while CO2 was adequate for capsaicinoids, fatty oils, and tocopherols, but not for carotenoids. Figure 1.5 shows the supercritical extraction of ginger with three extraction fluids (CO2, propane, and dimethyl ether).51 Propane gave the lowest yields while dimethyl ether gave the highest yield. They reported dimethyl ether to have mutual solubility with water. Ginger contains high amounts of volatiles, and CO2 extraction offers the advantage of dividing the extract into oleoresins and essential oil fractions by using a two-stage separation procedure with sequential pressure reduction. Similarly, if propane or dimethyl ether is used, considerable heating is required which ultimately results in thermal degradation, and a larger energy requirement in the form of cooling, depressurization, and boiling to recover the essential oils. In the case of ginger, the oxygenated fraction was much greater than the steam distilled oils and the gingerols in the oleoresin were extracted without decomposition. Oleoresins and piperine from pepper were extracted with insignificant losses although a longer processing time was required. Similar trends were observed for chili and pepper (Figure 1.6 and Figure 1.7). The extracts contained carotenoid pigments, and those obtained with SC-CO2 were bright red with pink residues, while those from propane and dimethyl ether were dark red. The extract obtained from chili with SCCO2 corroborated the results of other researchers5,79 in being viscous, pastry yellow, and semisolid, while those extracted with dimethyl ether were yellow/black and liquid at room temperature with a high quantity of water resulting in the dilution of the essential oil and piperine content. Nguyen et al.80 described the extraction of antioxidants from Labiatae herbs (rosemary, sage, oregano, and thyme) with SCCO2 at pressures in the vicinity of 50 MPa and temperatures ranging from 80°C to 100°C. The extracted oleoresin was precipitated into two fractions at various pressures and temperatures. The first fraction consisted of a green-brown, oil-soluble, heat-stable, resin containing less than 2% essential oil and exhibiting remarkable antioxidant properties. The second fraction was the essential oil containing more than 95 mL steam distilled oil/100 g. The use of SC-CO2 for the production of essential oils or oleoresins from spices is possible by selecting suitable pressures and temperatures. The oils extracted with supercritical technology were found to be superior in terms of their chemical composition and higher percentage of sesquiterpene compounds. Supercritical extraction with CO2 and hydrodistillation extraction methods were used to extract essential oil

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160

Yield (g/kg)

120 80 40 0 0

5

10 15 Solvent (kg/kg ginger )

CO2, 313 K

Propane, 323 K

20

25

Dimethylether, 313 K

FIGURE 1.5 Supercritical extraction yield of ginger using CO2, propane, and dimethyl ether (modified from Catchpole et al.51).

160

Yield (g/kg)

120 80 40 0 10

5

0

15

20

25

Solvent (kg/kg chili) CO2, 313 K

Propane, 323 K

Dimethylether, 313 K

FIGURE 1.6 Supercritical extraction yield of chili using CO2, propane, and dimethyl ether (modified from Catchpole et al.51).

(Juniperus communis L.)9 (Table 1.6). Oils obtained by SC-CO2 and hydrodistillation showed significant differences; the former was more selective and particularly efficient for the isolation of -thoujone and limonene. Anitescu et al.55 did a comparative analysis of coriander oil with supercritical CO2 and stream distillation (Table 1.7). They concluded that oils obtained by supercritical extraction gave a superior aroma compared to both the commercial and hydrodistillation extracted oils.

1.3.5 DECAFFEINATION

OF

COFFEE

AND

TEA

Caffeine (1,3,7-trimethylxanthine) is a bioactive plant component commonly found (since 1820s) in popular beverages such as teas (Camellia sinensis), coffees (Coffee Arabica, canephora, liberica), and soft drinks.81 Caffeine is a secondary metabolite, the product of nucleic acid catabolism, and belongs to the group of compounds

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200

Yiled (g/kg)

150 100 50 0 0

5

10

CO2, 313 K

15 20 25 Solvent (kg/kg pepper) Propane, 323 K

30

35

Dimethylether, 313 K

FIGURE 1.7 Supercritical extraction yield of pepper using CO2, propane, and dimethyl ether (modified from Catchpole et al.51).

TABLE 1.6 Essential Oil (Juniperus communis) Oil Extracted by Comparing Supercritical Extraction and Hydrodistillation (HD) Methods Pressure Temperature Dynamic time Modifier No. Compound 1 α-Thoujone 2 α-Pinene 3 Sabinene 4 Myrcene 5 3-Carene 6 Limonene 7 Terpinolene

(MPa) (°C) (min) (µL) RI* 928 943 972 987 1007 1020 1071

20 45 20 – 1 25.1 1.3 2.3 2.8 35.0 22.0 2.6

20 45 30 – 2 26.2 1.4 2.1 3.8 36.8 24.2 –

20 55 30 – 3 26.9 1.4 2.3 3.8 37.1 22.9 2.6

35 45 30 – 4 17.0 1.8 2.1 1.3 19.9 9.0 0.8

35 55 30 – 5 13.0 – 1.8 1.2 17.1 8.6 2.1

35. 55 30 80 6 22.0 1.9 1.9 2.4 29.1 15.6 2.7

35 55 30 400 7 4.0 22.4 34.6 4.2 2.6 6.1 2.7

HD

– 24.5 0.4 3.4 39.4 – 3.1

* Retention Index; Source: Modified from Pourmortazavi et al.9

known as purine alkaloids. Excessive ingestion of caffeine may cause certain health problems such as palpitations, gastrointestinal disturbance, anxiety, tremor, increased blood pressure, dizziness, and insomnia.82–85 The aroma and flavor coupled with the stimulant effects comes from caffeine. Coffee beans have about 2% to 3% while tea leaves have about 5% caffeine,82 depending on the variety and species. Decaffeinated coffee must contain less than 0.1% caffeine on a dry weight basis, as specified by EEC regulations. Therefore, decaffeination of coffee and tea poses significant challenges to both the producers and processors. The demand for decaffeinated coffee is high on the world market. It accounts for more than 20% of all coffee sales in the USA, with a 50% growing demand among the adult population.82 Research in

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TABLE 1.7 Results of Comparative Analysis of Essential (Coriander) Oil between Supercritical CO2 and Steam Distillation Processes

No.

Compounds

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

α-Thujene α-Pinene Camphene Sabinene β-Pinene Ββ-Myrcene ∆3-Carene Limonene 1,8-Cineole Linalol Camphor Menthol ρ-Cymen-8-ol Cis-Hex-3-enyl butyrate α-Terpineol β-Citronellol Neral Carvone Anethole Carvacrol Neryl acetate Geranyl acetate β-Caryophylene α-Humulene Eugenyl acetate β-Caryophylene Unidentified Compounds

Kovats RI

Comm Oil

Steam Distillation Oil

SC-CO2 Oil

928 936 951 975 980 990 1006 1030 1033 1103 1147 1174 1184 1186 1192 1226 1241 1245 1287 1299 1363 1382 1428 1463 1526 1594

Trace (Tr) 3.3 0.6 0.1 1.0 1.2 1.1 2.4 Tr 63.8 5.5 Tr Tr Tr 1.0 0.1 0.1 0.5 0.7 Tr 0.1 1.0 Tr Tr – Tr 3.8

Tr 2.3 0.4 0.3 0.3 0.8 0.3 2.3 0.1 62.8 5.6 0.1 0.1 0.1 0.9 0.3 0.1 1.0 0.4 0.1 0.1 1.8 2.1 0.3 Tr Tr 2.9

0.1 2.8 1.5 0.9 0.9 1.0 0.3 2.7 0.1 61.9 5.6 0.1 0.3 0.2 0.6 0.2 0.2 1.0 0.4 0.2 0.2 2.4 0.8 0.2 0.2 0.2 1.7

Source: From Anitescu et al.55

genetics engineering to produce transgenic tea and coffee plants deficient in caffeine is in progress.82,83,86 However, the consumption of genetically modified products is still contentious globally, and SCE technology gives the best option in combating these critical issues. The decaffeination of coffee and tea using SCE technology is among the first known commercial operation in the food industry. In the past, methylene chloride was used for decaffeination of coffee with one cycle of production lasting from 24 to 36 h and the end products usually contained toxic residues, thus posing more harm than the caffeine. Due to its suspected carcinogenic effect, the FDA placed regulations against methylene chloride use. However, decaffeination process with SC-CO2 fluid can be accomplished on green

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coffee or roasted coffee beans or tea leaves without deleterious effects on the flavor even after 10 h of processing, and many patents exist for such processes. The process requires charging the extraction vessel containing the coffee beans with CO2 at a pressure of 7 to 22 MPa and temperature of 31°C. The caffeine is dissolved in the SC-CO2 stream, which subsequently enters a washing tower or alternatively activated carbon scrubbers, distillation, recrystallization, or reverse osmosis are used in some instances to entrain caffeine. The method can strip coffee of its caffeine content (0.7% to 3%) by 71% to 97%.87 The caffeine recovered is sold for medicinal purposes and or for use in soft drinks. Peker et al.88 reported that soaking raw coffee beans in water prior to processing enhances the rate of decaffeination.

1.3.6 FISH OIL CONCENTRATION Fish oils are characterized by a high percentage of unsaturated straight-chain fatty acids ranging from C14 to C22 with one to six double bonds. They contain essential fatty acids (EFA) and polyunsaturated acids, grouped into omega-6 and -3 EFAs. The main sources of omega-3 (-3) are flaxseed, walnut, and marine plankton and fish. This review would focus on -3 oils derived from fish. Eicosapentaenoic acid (EPA) and decosahexaenoic acid (DHA) are predominant in fish oil, and have been reported to contribute to the prevention of atherosclerosis, heart attack, depression, and cancer if consumed in sufficient quantities.89 Fish oil derivatives in the form of -3 oils are in high demands as food additives. For example, Ocean Nutrition’s ME3TM Omega-3 powder is currently used in several breads. In the US, Wegman’s Food Markets, Rochester, NY, launched breads fortified with MEG omega-3 fats, two slices of which offers 80 to 90 mg of omega-3s.90 Encapsulated omega-3 fatty acids forms are available for fortified bakery products. Fish oils are processed as fatty acids or as methyl or ethyl esters which are more stable than the free acids form.91 Fatty acids are highly soluble in CO2 and as a result SC-CO2 extraction is a preferred method of fractionation. With this technology, it is possible to separate heat-sensitive compounds (ω-3 fatty acids) and avoid toxic solvent residues in the final product. The isolation and fractionation of ω-3 PUFA (polyunsaturated fatty acid) from fish, fish oil, and esters using SC-CO2 have been studied by several researchers.72,92,93 Eisenbach93 fractionated the ethyl esters from cold fish oil using SC-CO2 at a pressure of 15 MPa and an extracting temperature of 50°C. Alkio et al.94 produced EPA and DHA with 50% and 90% purity, respectively, from trans-esterified tuna oil using carbon dioxide. Temelli et al.72 obtained the highest yield of -3 fatty acids at 35 MPa and 35°C without denaturing the protein during SCE. They also compared solvent (hexane) extraction to SC-CO2 as shown in Table 1.8. A higher concentration of -3 was achieved with SC-CO2 (Table 1.8). At 25 MPa pressure and temperature from 40°C to 80°C, no significant effect on yield was observed in oil extraction from krill.38 Hardardotti and Kinsella73 did not see any yield change on the recovery of rainbow trout at operating pressure ranging from 13 to 35 MPa and temperature range of 40°C to 50°C.

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TABLE 1.8 Fatty Acid Composition of Fall Atlantic Mackerel Oil Concentrate Extracted with Hexane and SC-CO2 at Pressure 34.5 MPa and Temperature of 35°C Fractionated by Gas Chromatograph Fatty Acid

GC Retention Time (min)

Hexane Extract

SC-CO2 Extract

C16:0 C16:1 C18:0 C18:1 C18:3 C20:0 C20:1 C20:5 (EPA,ω-3) C22:5 C20:5 (DHA,ω-3) EPA+DHA (ω-3)

8.07 8.53 12.49 13.09 15.58 18.05 18.14 22.79 30.08 31.43

15.86 6.65 3.53 3.59 0.88 11.22 1.20 6.07 1.34 8.58 14.65

16.66 7.94 3.16 3.16 1.04 7.84 1.20 8.76 1.38 8.97 17.73

Source: Modified from Tamelli et al.72

1.4. FACTORS AFFECTING EXTRACTION YIELD Several bioactive components were extracted successfully by the SC-CO2 extraction method as outlined in the preceding sections. Optimization of yield is a function of various independent parameters. Process parameters such as solvent flow rate, resident time, moisture content, particle sizes, and particle size distribution in conjunction with supercritical pressures and temperatures are key parameters for achieving optimum results. Most of these parameters can have individual or combined effects on the extraction rate of a process; for example, the resident time can have an immense influence on the composition of the extracted compound.

1.4.1 PRESSURE Figure 1.8 is a typical extraction rate curve, and it is apparent that pressure significantly influences the rate of extraction, and likewise the extraction time. Extrapolating the normalized yield at the point where the yield curve become asymptotic gives significant different normalized yields of 15, 11, and 4% for pressures of 10, 9, and 8 MPa, respectively.8 Macias-Sanchez et al.61 observed similar trends in the SC-CO2 extraction of carotenoids and chlorophyll a from Nannochloropsis gaditana, although as pressure increased beyond a critical point the yields dropped as a result of increased density. Higher density causes a double effect: increases the solvation power and reduces the interaction between the fluids and matrix, thus decreasing the diffusion coefficient. Excessive pressure also increases the compactness of the sample matrix, thus reducing the pore sizes and apparently reducing the mass transport, which eventually diminishes the yield.95

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16

Yield (%)

12 8 4 0 0

100

200 8 MPa

300 400 Time (min) 9 MPa

500

600

700

10 MPa

FIGURE 1.8 The effect of pressure change on bioactive compound yield during supercritical extraction of essential oil from Juniperus oxycedrus on extraction rate at flow rate 1.5 kg/h and temperature 50°C (modified from Marongiu et al.8).

The selectivity of solute extraction is a function of extraction pressure, and if it increases, different solutes are extracted. D’Andrea et al.96 also found that with a working pressure of 25 MPa and temperature of 55°C, they attained optimum yield for the extraction of azadirachtin and 3-tigloylazadirachtol from neem seeds. Similarly, Tonthubthimthong et al.95 reported optimum yield at a pressure of 23 MPa and a temperature of 55°C.

1.4.2 TEMPERATURE Temperature is a parameter with significant influence on SFE; therefore manipulating it could have adverse implication on the process and yield. Figure 1.9 shows the general trend of increased extraction yield as temperature increases relative to the pressure. Tonthubthimthong et al.95 reported similar trends for extracting nimbin from neem seeds at 20 MPa and a CO2 flow rate of 0.62 mL/min, and found 35°C the optimum temperature for their process. Ge et al.32 indicated a temperature of 35°C gave the highest yield in the first 45 min of extraction, but during prolonged extraction from 45 to 120 min, the highest temperature condition was shown to produce the highest yield (Figure 1.9). Although many literature reports correlated increased temperature to yield,45 many others showed no particular trends as far as temperature was concerned.97 Some researchers reported yield was inversely proportional to temperature under 15 MPa. The combined effect of pressure and temperature on cholesterol extraction was studied by Chao et al.44 At a pressure-temperature setting of 34 MPa and 50°C, cholesterol yield of 160 mg/100 g was realized compared to 430 mg/100 g when the temperature drops to 40°C and 2.5 kg of CO2 was used. As the mass of CO2 increased, the yields decreased, but the lowest temperature still maintains the highest yield as shown in Figure 1.10. Also, under constant temperature, increases were achieved when pressure decreased. The results demonstrated that higher selectivity is possible at lower pressures and higher temperatures. Froning et al.98 corroborated this fact based on their experiment with lipid and cholesterol extraction from

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Yield (mg/100 g)

2600

2200

1800

1400 10

15

20

25 Pressure (MPa)

35°C

40°C

30

45°C

35

40

50°C

FIGURE 1.9 The effect of temperature change on bioactive compound yield during supercritical extraction of wheat germ on extraction, time 120 min, rate at flow rate 2.0 mL/min and sample size 5g (modified from Ge et al.32).

Cholesterol (mg/100 g)

500 400 300 200 100 0 0

5

10

15

20

25

Carbon dioxide (kg)

34.5 MPa/50°C 34.5 MPa/40°C

13.8 MPa/50°C 24.1 MPa/40°C

FIGURE 1.10 Supercritical carbon dioxide extraction of cholesterol in beef tallow, different pressures and temperatures (modified from Chao et al.44).

dehydrated chicken meat. The combination of pressure and temperature of 38.6 MPa and 55°C, respectively, yielded lipid (89%) and cholesterol (90%), while a pressure and temperature combination of 30.3 MPa and 45°C, respectively, produced much lower yield.

1.4.3 MOISTURE CONTENT

OF

RAW MATERIALS

Moisture content is a factor that influences extraction yield of bioactive compounds as shown in Table 1.9. A maxima yield of 1678 mg/100 g was achieved at 5% moisture content, and any further increase or decrease in moisture reduced that yield. Therefore, it is important to establish this magic number in order to maximize yield in the SCE process. The effect of sample pretreatment is crucial in attaining this

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TABLE 1.9 The Effect of Moisture Variation on Bioactive Compound Yield during Supercritical CO2 Extraction of Wheat Germ Water Content (% wet basis)

Yield (mg/100 g)

4 5 8 12

1470 1678 1352 1290

Source: Modified from Ge et al.32

objective. Water removal in most cases frees the internal pores and thus increases the mass transport intensity, because water in the sample matrix would inhibit the flow of SCF by changing surface tension and contact angles as a result of phase interaction between the three components (water, sample matrix, and SCF). For example, the higher the moisture content, the higher the probability for the formation of a thin film of water between the sample matrix and the SCF phase. Water has a small but finite solubility in SC-CO2, and as a result it can also be extracted with the targeted components, and its separation can then only be done at the end of the process.

1.4.4 COSOLVENT The use of cosolvent (entrainers) during SCF extraction is key to enhancing the extraction efficiency and cost effectiveness of the processes. Joslin et al.99 indicated two significant attributes of cosolvents: the interaction between the cosolvent and the solute (direct effect) and the cosolvent-solvent interactions (indirect effect). Cosolvents used in small doses (1% to 5% mol) in SCF can change the overall characteristics of the extraction fluid such as polarity, solvent strength, and specific interactions. These changes in turn can significantly alter the density and compressibility of the original SCF.5 Additionally, they can improve selectivity for desired components and facilitate selective fractional separations. Table 1.10 summarizes the results of different cosolvents (ethanol, methylene chloride, and methanol). Ethanol was found to have the greatest enhancement factor while methanol had the lowest.100 The solubility enhancement is the result of complex interaction between the -carotene, SC-CO2 , and cosolvent as indicated by Joslin et al.99 Temelli et al.101 observed an enhancement factor of 64, 63, and 29 by using ethanol as cosolvent to extract palmitic acid, stearic and behenic fatty acids, respectively. Baysal et al.63 used ethanol at different concentrations (5%, 10%, and 15%) to recover -carotene and lycopene from tomato paste. Although they observed that, with a high ethanol

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TABLE 1.10 The Effect of Fractionation of β-Carotene in Cosolvent in Supercritical Carbon Dioxide Mixtures at the Temperature of 70°C. Enhancement Factors were Determined Based on Density (17 mol–1) Pressure (MPa)

21.2 24.9 28.7 32.8 35.8 40.0 43.9

CO2 Density (mol–1) β-Carotene CO2 15.71 16.73 17.65 18.43 18.91 19.49 19.95

Yields (× 107)

1.95 3.33 6.23 10.00 12.50 19.10 25.4

β-Carotene CO2 + 1 wt % Ethanol (Enhancement factor = 4.7) 22.3 15.92 9.6 24.9 16.73 19.5 31.6 18.22 25.2 37.4 19.14 37.5 β-Carotene CO2 + 1 wt % methylene chloride (Enhancement factor = 3.5) 23.4 16.28 12.8 24.7 16.67 13.3 31.2 18.16 21.7 37.0 19.08 27.7 β-Carotene CO2 + 1 wt % Methanol (Enhancement factor = 2.1) 18.0 13.92 3.98 26.8 17.26 9.62 33.0 18.47 15.60 37.3 19.12 30.60 Source: Modified from Cygnarowicz et al.100

concentration, the extraction was hindered due to a decrease in the homogeneity of the extraction mixture, no statistically significant differences were found between the 10% and 15% concentrations. Quancheng et al.102 also investigated the effect of cosolvents on the extraction of tocopherols. They extracted tocopherols from rapeseed deodorizer distillate at 2%, 4%, and 6% with methanol, ethanol, and mixed

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ethanol as cosolvents, under operating conditions of 12 MPa and 60°C. The use of 4% cosolvent gave the best tocopherol yield followed by 2% and 6%, and the 2% and 6% methanol concentration gave the best yield of the three and the mixed ethanol the worst.

1.4.5 PARTICLE SIZE Particle size may have a significant impact on the flow behavior of SCF in the sample matrix. The mechanism of sample pretreatment, for example the methods of drying (air, oven, vacuum, or freeze drying), would influence particle sizes when subjected to attrition or size reduction. The sizes of particles, shapes, and their random layout (size distribution) would determine what goes through the medium and how fast. The layout would influence the type of pore, either open or blind pores, or their degree of interconnectedness. Process parameters such as pressure influence particle size distributions. Pressure tends to create compactedness,95 thus decreases the intergranular porosity resulting in increased solid density. The smaller the particle sizes, the larger is the surface area, and as a result bioactive components are released easily. However, Coelho et al.103 observed no significant effect of particle sizes on the extraction yield as a function of extraction time at a fixed flow rate. The oxygenated compounds increased from 81% to 85% as particle size decreased, as shown in Table 1.11. However, the findings of Ge et al.32 were contrary to Coelho et al.103 in their study on the effect of particle sizes on wheat germ (Table 1.12). Papamichail et al.104 extracted essential oil from celery with SC-CO2. They experienced increased yield (more oil released) as the particle sizes of the seed decreased and attributed that to the pretreatment milling and sieving. Maximum yield of 1838 mg/100 g was obtained with optimum particle size of 0.505 mm. Also, they observed that very fine and big particle sizes have low extraction yield probably due to higher resistances to mass transfer because of the compact tendency reflecting reduced pore sizes in finer particle sizes while less interactions with the supercritical fluids in the case for the latter. Likewise larger particles contain undamaged cell walls rendering then impervious,60 although some believed that higher SC-CO2 flow rate is capable of degrading protein structure to release the targeted bioactive components.105 Nelson106 revealed the need to break the lipoprotein matrix in order to release the embedded lipids in fish during SCE. Papamichail et al.104 reported that it was possible to extract more essential oil per kg of CO2 at a lower flow rate due to the intraparticle diffusion resistance. Therefore, a threshold level for each product has to be experimentally determined in pursuance to pilot plant or industrial processing.

1.4.6 FLOW RATE Coelho et al.103 presented three scenarios of 2.3, 1.5, and 0.85 kg/h flow rates as a function of extraction time and concluded the highest flow rate of 2.3 kg/h gave the higher rate of extraction of Foeniculum vulgare volatile oil with SC-CO2 at 9 MPa and 40°C. A similar trend was observed by Ge et al.32 who reported a flow rate of 3 mL/min yielded 1927 mg/100 g of bioactive component from wheat germ. Some

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TABLE 1.11 Comparative Supercritical CO2 and Hydrodistillation Extraction of Volatile Components from Fennel (Foeniculum vulgare) Fruits of Different Particle Sizes SCE CO2 (%)

HD (%) Volatile Components Canfene Sebinene Myrcene -Phellandrene Limonene -Terpinene Terpinolene Fenchone Estragol (E)-Anethole Piperitenone oxide Unknowns Waxes

Stalks

Fruits (0.5 mm)

Fruits (0.55 mm)

Fruits (0.35 mm)

0.2 0.1 1.2 2.0 2.1 Tr 0.5 15.8 18.9 42.5 0.2 4.8

0.2 0.2 1.4 2.2 3.6 0.1 0.6 16.8 20.9 42.2 0.2 3.4

0.2 0.2 1.4 2.2 3.5 Tr 0.6 16.2 21.0 42.5 0.3 5.5 0.6

0.1 0.2 1.3 1.9 3.1 Tr 0.6 17.1 21.9 44.6 0.3 0.3

Tr (Trace < 0.05)). Source: Modified from Coelho et al.103

TABLE 1.12 The Effect of Particle Sizes on Bioactive Compound Yield during Supercritical CO2 Extraction of Wheat Germ Sieving (mesh)

Particle Size (mm)

Yield (mg/100 g)

No grinding 20 30 40 60 80 100

2.1 0.86 0.51 0.40 0.22 0.18 0.13

1610 1710 1838 1550 1070 890 742

Source: Modified from Ge et al.32

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Yield (kg/kg feed)

0.18

0.12

0.06

0 0

50

100 150 Time (min) 1.1 kg/h

200

250

3 kg/h

FIGURE 1.11 The effect of flow rate on supercritical extraction of essential oil from celery at pressure 15 MPa and temperature 45°C (modified from Baysal et al.63).

of these facts were corroborated by Peker et al.88 in their experimental study on extraction rates of coffee beans with SC-CO2. They indicated the need for long extraction time in conditions where low flow rate is used. Figure 1.11 shows an apparent yield at high flow rate for extracting celery oil104 and Juniperus oxycedrus essential oil.8 Summarized in Table 1.13 are the results of Baysal et al.63 where a flow rate of 4 kg/h was identified as the optimum condition for attaining the highest yield. Similar trend was observed by Ferreira et al.18 for extracting essential oil from black pepper. They observed larger yield at 30 MPa using the upper-level flow rate (10.54 kg/s). When maximum solubility is attained, the highest CO2 flow rate would offer the highest recovery in extracting lipids from fish.72

1.4.7 EFFECT

OF

TIME

ON

YIELD

Several factors have direct or indirect implication on yield during SCE. Resident time is an important factor that influences yield and the economic viability of the process. Other factors such as temperature and pressure could have individual or

TABLE 1.13 Effect of Flow Rate on the Supercritical Extraction of Lycopene and -Carotene from Tomato Paste Flow Rate (kg/h)

Extraction Time (h)

Lycopene (%)

β-carotene (%)

2 4 8

4 2 1

14 22 20

30 43 34

Source: Baysal et al.63

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60

Content (%)

50 40 30 20 10 0 0

50

100 HM

150 200 Time (min) OM

250 HS

300

350

OS

Cumulative quantity (g)

(a)

1.5

1

0.5

0 0

50

100

150

200

250

300

350

Time (min) HM

OM

HS (b)

OS

Overall

FIGURE 1.12 Families of flavor compounds extracted from Santolina Insularis at different supercritical extraction times. HM: hydrocarbon monoterpenes; OM: Oxygenated monoterpenes; HS: Hydrocarbon sesquiterpenes; and OS: Oxygenated sesquiterpenes (modified from Cherchi et al.107).

combined effects. Cherchi et al.107 performed detailed analysis of flavor compounds in essential oil extracted from Santolina insularis by SC-CO2 extraction. They reported the change in concentration exhibited by the monoterpenes from 50% in the fraction collected after 30 min to 10% in the fraction collected after 240 min (Figure 1.12a) under optimum conditions of 9 MPa and 50°C and a two-stage separation. The first-stage separation was accomplished under 9 MPa and −12°C, while the final stage used 2 MPa and 15°C. The yield becomes asymptotic at 1.75% with increased extraction time while the rate decreases (Figure 1.12b). Hawthorne et al.108 studied the extraction rate of basil conducted at 30 MPa and 45°C for 10 min and identified 1,8-cineole, estragole, eugenol, and selinene. Yield in most cases

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was dependent on time. Tamelli et al.72 considered 3 to 4 h sufficient time to extract all extractable lipids from freeze-dried krill and 6 h for rainbow trout regardless of the conditions.

1.5 SUMMARY Supercritical carbon dioxide extraction has been shown to be a viable alternative to the conventional solvent extraction technique to extract bioactive components from agricultural materials. It offers a unique advantage of adding value to agricultural waste by extracting antioxidants and flavonoids (lycopene from tomato skin, essential oils, and flavor and fragrances, etc.), which are then used for the fortification of foods and other applications. Its drawbacks are the difficulties in extracting polar compounds and its susceptibility of extracting compounds from a complex matrix where the phase interaction with the intrinsic properties of the product inhibits its effectiveness. Many of these drawbacks can be ameliorated by using cosolvents. However, much investigation is required to understand the solvation effects on targeted bioactive components being extracted. Supercritical fluid extraction technology can be utilized to provide healthy snack foods, a problem that faces the snack food industry, with regard to fat/oil contents, which is becoming a greater public health concern. A lot of cost is incurred reformulating products such as French fries, onion rings, and other snack foods to eliminate trans fats and high cholesterol content. Defatting and decholesterol with SCF has been demonstrated as applicable to food products. Although most of the tests were conducted on dehydrated products, research has shown successful application of SC-CO2 on high moisture products where extraction could be accomplished without compromising the organoleptic characteristics. The SCF technology is available in the form of single-stage batch and could be upgraded to multistage semicontinuous batch coupled with a multiseparation process. Although much work was accomplished, it would not warrant complacency, since batch modes render the SCF technology cumbersome for certain industrial applications, and has been the drawback for broader commercial adaptation. However, the needs to improve the design into continuous modes are growing. One possibility is to integrate membrane technology into a supercritical process. Study on membrane and SC-CO2 has been attempted and needs an aggressive pursuance. This concept would make feasible the extraction of bioactive components from aqueous, less viscous, and pumpable substances in a continuous mode. Supercritical carbon dioxide extraction could only be cost effective under largescale production, which made it ideal for decaffeination of coffee, tea, and hops. Although it is expensive, it is much more economical when compared to conventional solvent extraction at high production scales. With improved processing conditions and reduced cost, SCE will become even more economical at low throughput.

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90. Ohr, L.M., Nutraceuticals and functional foods, Food Technology, 59, 63–65, 2005. 91. Espinosa, S., Diaz, S., and Brignole, E.A., Thermodynamic modeling and process optimization of supercritical fluid fractionation of fish oil fatty acid ethyl esters, Industrial and Engineering Research, 41, 1516–1527, 2002. 92. Dunford, N.T., Temelli, F., and LeBlanc, E., Supercritical CO2 extraction of oil and residual proteins from Atlantic Mackerel (Scomber Scombrus) as affected by moisture content, Journal of Food Science, 62, 289–294, 1997. 93. Eisenbach, W., Supercritical extraction, Physical Chemistry, 88, 882, 1984. 94. Alkio, M., Gonzalez, C., Jantii, M., and Altonen, O., Purification of polyunsaturated fatty acid ester from tuna oil with supercritical fluid chromatography, Journal of American Oil Chemists’ Society, 77, 315, 2000. 95. Tonthubthimthong, P., Chuapraset, S., Douglas, P., and Luewisutthicat, W., Supercritical CO 2 extraction of nimbin from neem seeds: an experimental study, Journal of Food Engineering, 47, 289–293, 2001. 96. D'Andrea, A., Ferri, D., Maccioni, O., van der Esch, S.A., and Vitali, F., Application of SCF technology to the extraction of active substances from Azadirachta indica (A. Juss) seeds, in Practice Oriented Results on the Use and Production of Neem Ingredients and Pheromones, anon. eds., Giessen, Druck and Graphic, 1994. 97. Gomez, A.M., Lopez, C.P., and de la Ossa, E.M., Recovery of grape seed oil by liquid and supercritical carbon dioxide extraction: a comparison with conventional solvent extraction, The Chemical Engineering Journal, 61, 227–231, 1996. 98. Froning, G.W., Fieman, F., Wehling, R.L., Cuppett, S.L., and Niemann, L., Supercritical carbon dioxide extraction of lipids and cholesterol from dehydrated chicken meat, Poultry Science, 73, 571–575, 1994. 99. Joslin, C.G., Gray, C.G., and Goldman, S., Solubility in supercritical fluids from the virial equation of state, Molecular Physics, 89, 489–503, 1996. 100. Cygnarowicz, M.L, Maxwell, R.J., and Seider, W.D., Equilibrium solubilities of -carotene in supercritical carbon dioxide, Fluid Phase Equilibria, 59, 57–71, 1990. 101. Tamelli, F. and Guclu-Ustundag, O., Solubility behaviour of ternary systems of lipids, cosolvents and supercritical carbon dioxide, unknown. 102. Quancheng, Z., Guihua, S., and Hong, J., Concentration of tocopherols by supercritical carbon dioxide with cosolvents, European Food Research and Technology, 219, 398–402, 2004. 103. Coelho, J.A.P., Pereira, A.P., Mendes, R.L., and Palavra, A.M.F., Supercritical carbon dioxide extraction of foeniculum vulgare volatile oil, Flavour and Fragrance Journal, 18, 316–319, 2003. 104. Papamichail, I., Loili, V., and Magoulas, K., Supercritical fluid extraction of celery seed oil, Journal of Supercritical Fluids, 18, 213–226, 2000. 105. Femenia, A., Garcia-Marin, M., Simal, S., Rossello, C., and Blasco, M., Effects of supercritical carbon dioxide (SC-CO2) oil extraction on the cell walls composition of Almond fruits, Journal of Agricultural and Food Chemistry, 49, 5828–5834, 2001. 106. Nelson, G.J., Isolation and purification of lipids from biological matrix, in Analysis of Fat, Oils and Lipoprotein, anon. ed., American Oil Chemists’ Society, Champaign, IL, 1991, 20–59. 107. Cherchi, G., Deidda, D., De Gioannis, B., Marongiu, B., Pompei, R., and Porcedda, S., Extraction of Santolina Insularis essential oil by supercritical carbon dioxide: influence of some process parameters and biological activity, Flavour and Fragrance Journal, 16, 35–43, 2001.

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108. Hawthorne, S.B., Riekkola, M.L., Serenius, K., Holm, Y., Hiltunen, R., and Hartonen, K., Comparison of hydrodistillation and supercritical-fluid extraction for the determination of essential oils in aromatic plants. Journal of Chromatography, 634, 297–308, 1993.

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2

Solubility of Food Components and Product Recovery in the Supercritical Fluid Separation Process John Shi1 and Xiaoqin Zhou2 1 2

Agriculture and Agri-Food Canada University of Waterloo

CONTENTS 2.1 2.2 2.3

Introduction ....................................................................................................45 Solubility of Food Components in SFE ........................................................46 Factors Affecting Solubility in SFE-CO2 ......................................................48 2.3.1 Solvent Selectivity .............................................................................51 2.3.2 Polar and Nonpolar Solvents .............................................................52 2.3.3 Cosolvents-Modification of SFE Fluid Phase Behavior ...................53 2.3.4 Pressure Effects..................................................................................57 2.3.5 Effect of Water Content in Material ..................................................58 2.4 Solubility Prediction ......................................................................................60 2.4.1 Equations of State (EOS)...................................................................65 2.5 Summary ........................................................................................................67 References................................................................................................................68

2.1 INTRODUCTION Supercritical fluid extraction (SFE) has been used in the food industry since 1979,1 and has accrued 23 years of experience in the extraction of oils from plant materials. Initially, it was mainly used in the decaffeination of coffee and tea and the extraction of spices and hops. Those had to be large-scale processing operations in order to make them economically feasible and cost effective. However, with the development

45

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and advancements in processing conditions and equipments, the cost effectiveness improved allowing the small-scale productions of ultrahigh quality or high margin product using SFE for commercial distribution. The hottest area for supercritical extraction application is with nutraceuticals, as supercritical fluid extraction can be operated at mild conditions and will retain the bioactivity of components. Solubility is the amount of a substance dissolved by the solvent at thermodynamic equilibrium. For a multicomponent system (different components in the analyzed matrix and a mixture of solvents), the solubility of various components determines the selectivity and gives the opportunity of solving a separation problem. To design a supercritical fluid extraction process, the solubility of solutes in the supercritical fluid is fundamental. It influences the ideal operating conditions of the extractor and recovery unit. Also, it controls the minimum amount of supercritical fluid required to complete the extraction. In addition, the primary characteristics of a supercritical fluid which make it so attractive is the continuously changing solvating power. Since the solvating power is sensitive to temperature and pressure in the supercritical region, it can be finely adjusted by varying temperature and pressure. The goal of optimizing temperature and pressure of an SFE system is to maximize the solubility of solutes and increase their upper limit of yield. In past years, study has focused on the experimental solubility data in the supercritical region, solubility prediction, and improvement (modifying solvent). As the molecular structures of the food component in nutraceuticals become more complex, the interaction between solute, solvent, and the solubility of biocomponents in SCF become more complex. In the following sections, recent developments of the research of solubility in SFE related with food science are reviewed.

2.2 SOLUBILITY OF FOOD COMPONENTS IN SFE Because solubility is important to the process design of SFE, a lot of experimental work has been done on the measurements of solubilities of food component in SCF. Typical solubility behavior of a solid solute in SCF solvent is shown at Figure 2.1. Two convergence points are shown at PL and PU, and one minima (Pmin). At low pressure region (< Pmin), the solubility decreases as pressure increases, while in the region of pressure Pmin< P < PU, the solubility increases sharply with the increase of pressure. This region is usually observed in the near-critical and high compressed region of an SCF solvent. It indicates that in the supercritical fluid extraction, the solubility can be controlled by pressure. In other words, it is related to the state of the supercritical fluid. Hence, the prediction of solubility is usually based on the equation of state (EOS) of the solvent. The crossover points PL and PU are attracting some interest as a method of separating components with small difference in selectivity, such as isomers. For a multicomponent system, the crossover point of each component may not overlap, so there is a “crossover region” where most of the crossover points are located. At this point or in this region, the solubility of components are similar. Johnston et al.2 pointed out that the crossover point in fact is the turning point of the isotherm line of solubility, where

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T3

47

T2 T1

Mole fraction

T3 > T2 > T1

Pmin

PL

Pressure

PU

FIGURE 2.1 Solubility behavior of a solid solute in an SCF solvent.

∂ ( ln y2 ) =0 ∂T where y2 is the mole fraction of solute in gas phase and T is temperature. For one component, a slight increase in temperature at PU will cause the solubility to increase above the crossover pressure and decrease below the crossover pressure. The results are reversed at PL. Under these conditions a retrograde region is formed. For multicomponent systems, at the crossover point of one component, its solubility will not change with temperature, while others do, so the selectivity of those components increases. Through several cyclings of retrograde crystallization or solvation, the components are separated. The operation becomes similar to distillation and requires a temperature gradient. Chimowitz and Pennisi,3 and Foster et al.4 gave a detailed description of the operation in the crossover pressure region. Because fewer data are available in the critical region, this “distillation” operation runs more often in the upper crossover pressure than in lower crossover pressure. The other interesting observation is the sharp change of solubility with pressure. In view of the macroscopic thermodynamics, the influence of the pressure to the solubility can be explained by partial molar volume V2 directly ln φ2 =

1 RT

∫

P

V2 dp

0

where V2s is the saturate volume, f2 is the fugacity coefficient, and R is the gas constant. The partial molar volume is a differential quantity that describes the solution behavior at a particular pressure. Here, fugacity coefficient f2 is the pressure integral of partial molar volume (Equation 2.3). Using the equation above, the solubility behavior vs. pressure can be easily explained.

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When the pressure is much lower than the critical point, the partial molar volume is not a function of composition, so the equation is simplified to ∂ ( ln y2 ) V2s − V2 = ∂P RT At low pressure V2s << V2 hence the solubility decreases with pressure, and as the pressure increases V2s decreases more slowly than V2 . When the partial molar volume equals the saturate volume, the solubility reaches its minimum at a certain pressure. At high pressures, that are significantly above the critical pressure, V2 increases slowly; where V2 exceeds V2s, because of the repulsive force, the solubility will reach a maximum and decrease slowly with the increase of pressure. In this region, the solubility does not change much. The quickest increase of solubility occurs at a pressure corresponding to the minimum V2 . This theory explains the solubility behavior in the supercritical region (Figure 2.1), and is supported by some experimental data.5,6,7,8 Unfortunately, due to the lack of adequate experimental data for biosubstances, the practical application of the partial molar volume model is limited. So under these circumstances, the factors that influence solubility are usually analyzed phenomenologically. Biocompounds are usually present as a mixture in natural tissues and are often not used in their pure form, thus they are regarded as one pseudocomponent in research. Some researchers will measure the pure substance and others just measure the “oil solubility.” Staby and Mollerup9 published a list for fish oil related component from1970s. Bartle et al.10 compiled solubility data from 1980s, which included solubility in supercritical carbon dioxide (SC-CO2 ) of not only bioactive components but also other chemical components. Guclu-Ustundag and Temelli11 collected data on pure lipids in SC-CO2 from 1970 to 1999. Foster et al.4 collected some solubility data of various chemical substances in various supercritical fluids from 1968 to 1990. Recent solubility information for bioactive components from different sources is listed as Table 2.1.

2.3 FACTORS AFFECTING SOLUBILITY IN SFE-CO2 The most common fluid for SFE is supercritical carbon dioxide, because it is safe, cheap, nonflammable, and the critical state is easy to obtain (31°C, 7.8 MPa). This critical temperature is close to the room temperature and it preserves the bioactivity of biocompounds. In food processing and nutraceutical fields, it is approved as safe without declaration. In the liquid and supercritical states, CO2 behaves very similarly to hydrocarbon solvents with very low polarizability.12 In general, the extractability of compounds with supercritical CO2 depends on the occurrence of the individual functional groups in these compounds, their molecular weights, and polarity. The nonpolar, lipophilic solutes have the greatest solubility in SC-CO2, whereas the introduction of polar function groups will decrease the solubility. Extensive experiments have revealed the following13:

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TABLE 2.1 Publications on Solubility of Bioactive Components in Supercritical CO2 Substance

Temperature (°C)

Pressure (bar)

Resources

40, 50, 60 40–60 40–60

70–190 Singh et al.59 100–250 Yun et al.60 100–500 Murga et al.61

1993 1991 2004

Vanillic acid Echium, Borage, and Lunaria Seed Oils Essential oil from black pepper Fish oil Hazelnut oil Hippophae Rhamnoides L. seed oils Palm kernal oil Tomato seeds oil Lipids & Fatty acid (C6, C12, C16) and Fatty acids Triglyceride (C24, C36, C48) Lipids Triglycerides Triglycerides with acryl chain, Phospholipid and monoglyceride Fatty acids Lauric acid Lauric acid and oleic acid

40–60 10–55

100–500 Murga et al.61 60–300 Gaspar et al.62

2004 2003

Lauric, linoleic, myristic, oleic, palmitic, and stearic acid Methyl oleate, oleic acid, oleyl glycerols, oleyl Glycerol mixture, mono-, di, and trioleylglycerol Myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), and cetyl alcohol Perilla oil ( poly-unsaturated fatty acid) 2-ethyl-1-hexanol, 2ethylhexanoic acid 2-ethyl-1-hexanol+ 2ethylhexanoic acid Ethyl esters of C18:1, C20:3 and C22:6 Fish oil fatty acid ethyl esters

Sterols Phenolic compounds

Fatty acid esters

Cholesterol Cholesterol Syringic acid

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16–20 60–350 Ferreira et al.63 20, 40, 80, 120 60–650 Botch-Jensen et al.64 40–60 150–600 Ozkal et al.51 30–45 150–300 Yin et al.65

1993 1997 2005 2002

40–80 40–70 40–80

207–483 Hassan et al.66 108–245 Roy et al.49 25–300 Bharath et al.67

2000 1996 1993

40–80 40

100–300 Tilly et al.68 150–350 Hammam15

1990 1992

40 80

345–483 Norulaini et al.69 2004 276–483 Nik Norulaini et al.70 2004

30–60

130–400

Maheshwari et al.71

1992

50, 60

172–309

Nilsson et al.72

1991

35

81–218

Iwai et al.73

1991

40

200–400

Ikawa et al.74

1991

40, 50

68–180

Ghaziaskar et al.75

2003

40–100

138

Ghaziaskar et al.75

2003

40–100

90–250

Liong et al.76

1992

10, 40, 70

20–220

Staby et al.77

1993

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TABLE 2.1 (CONTINUED) Publications on Solubility of Bioactive Components in Supercritical CO2 Substance

Temperature (°C)

•

• • • • •

•

Resources

Artemisinin

37–65

100–270

Xing et al.78

2003

α-Carotene in SC-CO2 or Halocarbon β-Carotene

40–70

60–340

Hansen et al.79

2001

35–50

96–300

Sakaki80

1992

γ-Carotene

40–70

200–300

Cygnarowicz et al.38

1990

Caffeine, uracil, and erythromycin Capsaicin in SC-CO2 or Halocarbon Fat-soluble vitamins A, D, E, and K Limonene + linalool 40% limonene or 60% limonene Limonene + linalool

40–60

Burgos-Solorzano et al.81 Hansen et al.79

2004

35–70

Around 300 60–332

40, 60, 80

200–350

1997

41,42

70–100

Johannsen and Brunner82 Chafer et al.83

45, 55

69–111

Berna et al.84

2000

Nitroaromatics and adamantane 50–100

•

Pressure (bar)

100–450

2001

2001

Poly(α-hydroxybutyrate)

35–75

p-t-butylcalix[n] arenes

40–80

Lewin-Kretzschmar et al.85 122–355 Khosravi-Darani et al.86 150–300 Graham et al.87

Pure limonene and linalool

40, 50

69–111

Berna et al.84

Triacylglycerols

40, 60

172–310

Nilsson and Kudson88 1993

2002 2003 1998 2000

Nonpolar and slightly polar components with low molecular weight such as hydrocarbons and some typical lipophilic compounds can be extracted at lower pressure. Solvent power of SC-CO2 for low molecular weight compounds (<250) is high and decreases with increase of molecular weight. Organics with molecular weights greater than 400 are almost insoluble. SC-CO2 has high affinity with oxygenated organic compounds of medium molecular weight. Free fatty acids and their glycerides exhibit low solubilities. Pigments are even less soluble. Water has a low solubility (<0.5% w/w) at temperatures below 100°C. Compounds with strongly polar functional group such as –OH, COOH, are more difficult to extract. For instance, phenols with one carboxyl and three or more hydroxyl groups cannot be extracted by SC-CO2. When pressure increases, less volatile, higher molecular weight, and more polar component may be dissolved.

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•

51

The highly polar substances such as sugar and amino-acid need pressure up to 40 MPa to be extracted. Proteins, polysaccharides, and mineral salts are insoluble.

2.3.1 SOLVENT SELECTIVITY An important feature of an SCF solvent is that it can selectively dissolve more of a certain compound from a mixture of compounds having similar volatility but different chemical structures. Strictly speaking, the selectivity of a solvent is the ratio of partition coefficient (K2, K2) of two components (Equation 2.5). For a solid-fluid system, it is often approximated as the ratio of binary solubility (y2 , y2) in SC-CO2 (Equation 2.6). S=

y2 y2′

Two key factors that influence the selectivity are the volatilities of the solids and the strength of solute-solvent interactions in the supercritical phase. They are described in the following sections. Generally, for many high molecular weight compounds, the selectivity increases with pressure. For low molecular weight compounds, at low pressure, the selectivity attains a maximum, and then decreases with pressure, and a high level of selectivity is usually reached at higher temperatures. As a nonpolar solvent, SC-CO2 has high selectivity on lipids, due to strong solute-solvent interactions. A cosolvent may increase the selectivity of polar components, as it increases the solubility of components in the mixture at different folds. Usually the quantity of cosolvent is small, so the effect on selectivity is mainly due to the change of interaction between solute and solvent. This effect is limited, but if the partial pressure of the solute is changed by the cosolvent, the effect on selectivity is greater. Wong et al.14 showed that the selectivity of Terpinen-4-ol and other components increased at least twofold with water (Figure 2.2). A general rule is that the solubility increases with increasing pressure and decreasing molecular weight. Molecular weight mainly influences the vapor pressure of the components. High molecular weight substances are usually not volatile. Hence, increased molecular weight leads to decreased solubility of a homologous series in SC-CO2. Materials with molecular weights above 500 daltons have limited solubility. Low-to-medium molecular weight compounds such as aldehydes, ketones, esters, alcohols, and ethers are very soluble. Low molecular weight, nonpolar, aliphatic hydrocarbons with up to 20 carbons and small aromatic hydrocarbons are soluble. If the molecular weight and structure of two components are similar, a great deal more comprehensive analysis is required to determine selectivity. Hammam15 investigated different molecular weights of lipids and found that above 20 MPa, dilaurin was less soluble than trilaurin, reflecting its more polar characteristic, whereas below 20 MPa, the effect is opposite due to the lower molecular weight of dilaurin.

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Functional Food Ingredients and Nutraceuticals

60.0

% Composition

50.0

40.0 a-Pinene a-Terpinene 30.0

g-Terpine Terpinen-4-ol

20.0

10.0

0.0 Whole dried leaves

Rehydrated whole dried leaves

FIGURE 2.2 Recovery of target analytes from tea leaves. Comparison of whole dried leaves and rehydrated whole dried leaves (modified from Wong et al., 200114).

2.3.2 POLAR

AND

NONPOLAR SOLVENTS

Besides vapor pressure, solubility is strongly affected by the interaction of solvent and solute. A biocomponent usually has an aromatic group which has high polarity or other functional groups which have specific force such as electron donor-acceptor properties. Obviously, these compounds will have high solubility in polar solvent. Primary supercritical solvents are listed in Table 2.2. Organic solvents are usually used in polymer devaporization and petrol purification procedures.

TABLE 2.2 Critical Conditions for Some Primary Supercritical Solvents Substance Nonpolar Nonpolar Nonpolar Polar Polar Polar Polar Polar (weak) Polar (weak) Polar (weak) Polar (weak) Polar (weak)

Carbon dioxide Ethylene Xenon Acetone Ammonia Chlorotrifluoromethane Water Diethyl ether n-Pentane Propane Ethane Propylene

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Critical Temperature (K) 304.2 282.4 289.7 508.1 405.6 302 647.3 467.7 469.6 369.8 305.4 365

Critical Pressure (MPa) 7.38 5.03 5.84 4.7 11.3 3.92 22 3.64 3.37 4.24 4.88 4.62

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The polarizability of CO2 is 2.65 × 10–24 cm3, less than that of all the hydrocarbons other than methane.16 Under this condition the solubility of biocomponents (mostly hydrocarbons) in SC-CO2 will decrease several orders of magnitude below the ideal solubility of a solid in a liquid. Xenon possesses a polarizability of 4.01 × 10–24 cm3,17 which is slightly greater than CO2 and close to the polarizability of lower alkanes, such as ethane and propane which are used widely in the petroleum field. It is suitable for solvating some polymers whose molecular weight are approximately 100,000.18 Because of its convenient critical properties (Tc = 289.7K, Pc = 5.8 MPa), safety, and larger polarity, it may be well suited for extraction of bioactive components. However, xenon is much more expensive than CO2, so its application is limited. Halocarbons (CFCs) are very good solvents in SFE due to their high density, which is a good indication of strong solvating power. Their polarities are similar to diethyl ether,19 a solvent that is able to dissolve many solutes. Because of their effect on the ozonosphere, the use of chloro-fluoro-hydrocarbons is restricted by the Montreal Protocol. Recently, some modified halocarbons with zero ozone-depletion potential, such as difluoromethane (R-32), 1,1,1,2-tetra-fluoroethane (R-134a), and 1,1,1-trifluoroethane (R-143a) have been used for extracting biocomponents.19,20 The solubility of those polar biocomponents was increased by 10 to 20 times when compared to their solubility in SC-CO2 under the same conditions. However, the critical temperatures of these halocarbons range from 100°C to 70°C. The extraction of biocomponents has to proceed near or under supercritical conditions to avoid destroying the bioactivity. Polar solvents, such as acetone, have strong solvating powers, but due to the safety problems in food, they are not used. Generally, the common safe polar solvents are water, ethanol, and, sometimes, oil. They have high critical pressure and temperature, and are often used as a cosolvent.

2.3.3 COSOLVENTS-MODIFICATION OF SFE FLUID PHASE BEHAVIOR Adding a cosolvent (entrainer or modifier) will dramatically increase solubility. Usually, the amount of additive is 1–5 mol %. Adding 3.5 mol % methanol in SCCO2 will improve the solubility of cholesterol sevenfold compared to pure CO2 at 15 MPa21 and increase the solubility of 2-aminobenzoic acid sixfold. Chandra and Nair22 observed much higher extraction yields of carotene from carrots with the addition of hexane or chloroform to the SC-CO2, and this higher yield results from a combined effect of increased solubility and increased availability of the carotene by competing with sorption sites within the matrix. Table 2.3 lists some material systems that show the effect of cosolvent. Generally, the role of a cosolvent in SFE is to increase the polarity and solvent strength of the pure SC-CO2 while retaining its solubility sensitivity to temperature and pressure. A suitable cosolvent is one that as a liquid can normally dissolve the solutes. When considering safety, ethanol, ethyl acetate, and water are the best natural cosolvents for food-grade products and they are “Generally Recognized As Safe” (GRAS). Recently, some new compounds have come into use as entrainers, but they are cosolutes. They act as surfactants in SC-CO2 because of their ability to increase

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Cosolvents

Results

Cholesterol

(1.75–6.0 mol %) of acetone and hexane

Cotton seed

Ethanol or IPA

Fatty acids (palmitic acid and stearic acid and higher alcohols (cetyl alcohol and stearyl alcohol)

1–10 mol % ethanol and octane at 308.2K, 9.9–19.7 MPa

Glucose

Ethanol and water

Grape seeds Maize

Ethanol 5–30 wt % Methanol 500 µL

o- and m-Hydroxybenzoic acid

3.5 mol % acetone- or 3.5 mol % methanol-

Soybean lecithin

5 wt % ethanol or 10 wt % acetone

Besides SC-CO2, also check other primary supercritical systems and ethane-acetone produced the best solvate power Gossypol can be dissolved; Phosphorus content doubled Stearic acid: 4 mol % ethanol 10–15-fold; 4 mol % octane 3-fold Palmitic acid: 5 mol % ethanol 12–13-fold, 5 mol% octane 8–11-fold Stearyl alcohol: 5 mol % ethanol 4-fold, Mole % octane, 4–6-fold Cetyl alcohol: 3 mol % ethanol, 2–3-fold 3 mol % octane, 3-fold Glucose dissolved and ternary and quaternary phase diagram was obtained phenolic compounds, 25%, highest solubility Final yield doubled and comparable with organic solvent extraction Solubility was increased significantly; and exhibited a retrograde vaporization phenomenon and a common upper crossover pressure for each ternary system Made deoiling process possible at 170 bar

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Resources Foster et al., 199389

Kuk and Hron, 199490 Koga et al., 199691

Dohrn et al., 199392 Chafer et al., 200493 Ambrosino et al., 200494 Gurdial et al., 199395

Teberikler et al., 200126

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TABLE 2.3 Solubility Data and Cosolvent Effects

3.5 mol % cosolvent: acetone, ethanol, or methanol

Sunflower seed oil

Ethanol, up to 20%, 150 to 350 bars and 42 to 80°C

Xanthines and Cocoa Butter

Ethanol, 20–25wt %

Various binary, ternary, and quaternary systems

3.5 mol % cosolvent

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Cholesterol-CO2-Acetone (T = 35°C) increase 6–7-fold Cholesterol-CO2-Methanol (T = 35°C) 8–9 Stigmasterol-CO2-Ethanol (T=35°C) 6–9 Oil solubility in SC-CO2 greatly increases over the whole range of pressure and temperature conditions. Some phospholipids are coextracted at levels directly proportional to the added ethanol. A large amount of them was recovered in the ethanolic phase. Acidity of the extracted oil was lower than that without alcohol. Part of the free fatty acids was found in the ethanolic phase Targeted component got higher solubility (2fold), and selectivity; caffeine no change, Increased 3–7.2

Wong and Johnston, 198621

Cocero et al., 199696

Li and Hartland, 199697 Dobbs et al., 198716

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solubility. These include some modified sugars (peracylated sugars), which have hydroxyl groups, will help those philize hydroxylated compounds come into CO2.23 The effect of cosolvents is based on the interaction between solute-solvent or solvent-cosolvent. For the SFE process, there exists an extraction stage and a regeneration stage (recycle of the solvent). In the cosolvent-solute-solvent system, the interaction of solute and cosolvent is key in the extraction stage while the solventcosolvent interaction becomes important in the regeneration stage. In the extraction stage, the solubility of the solute in the cosolvent can be used to make the initial selection. In the regenerating stage, raising temperature at a constant pressure is preferred, so the solvent-cosolvent binary should have a wide two phase region at that temperature and pressure.24 The quantity of cosolvent is usually small, since large quantity may cause the critical points to move and may form multiphases.25 This is not expected in the operation. Due to lack of multicomponent phase equilibrium data, the mixture phase behavior at different ratios cannot be predicted. It is usually determined experimentally. Empirically, the quantities of cosolvent are controlled at about 5% of the weight. That requires a flexible cosolvent system to obtain the operation flexibility and efficiency. In addition, large quantities of cosolvent may coextract other components as byproducts, which is not desired. Teberikler et al.26 observed that 5% of the weight ethanol made the deoiling process for soybean lecithin possible at 17 MPa, whereas no oil was extracted when pure SC-CO2 was used at the same pressure. On the other hand, 5% of the weight ethanol did not resulted in any coextraction of phospholipids. However, when the ethanol was increased to 10% of the weight to accelerate the deoiling process, the phospholipids were coextracted. Recently, Yuan et al.27 pointed out that the Lennard-Jones energy parameter and dipole moment of cosolvent influenced the solute chemical potential significantly, which is related to solubility directly. However, that research was focused on qualitative analysis. If solubility is increased significantly by the addition of small amounts of cosolvent, the economics of existing or proposed processes through reduction in the pressure and/or recycle ratio could be improved.16 Furthermore, cosolvents could lead to more selective extraction of components which have similar volatility but different types of chemical forces, and for compounds which have extremely limited solubility in pure fluids, for example, biomolecules. Some detailed studies have shown that the cosolvent may work differently on different solutes. For example, Wong and Johnston21 found that in a sterol mixture, 3.5 mol % acetone will improve the solubility of cholesterol 5.7-fold, stigmaterol 1.8-fold, and almost no effect on ergosterol at 35°C and 12.3 MPa. Obviously the selectivity of cholesterol increases. Dobbs and Johnston28 observed the selectivity of benzoic acid-hexamethylbenzene increased from 1.9 to 2.5-fold with the addition of 3.5 mol % methanol, and the yield also increased. However, Brunner29 found that the selectivity always decreased with an increase in yield for hexadecanol-octadecane-CO2 system using acetone as cosolvent. Generally, if a pair of nonpolar solutes were chosen, a nonpolar cosolvent would increase each solubility about the same, thus causing little change in the selectivity.30 Similarly, a nonpolar cosolvent will not change the selectivity of a pair consisting of either a nonpolar and a polar solute or two polar solutes.

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3.0 Tr = T/Tc = 0.8

Reduced density

0.9 1.0

2.0

1.1 1.2 1.0

CP 1.55

0.0 0.1

1.0 Reduced pressure Pr = P/Pc

10.0

FIGURE 2.3 Variation of the reduced density (ρr) of a pure component near its critical point.

2.3.4 PRESSURE EFFECTS Cosolvents change the interaction of solute and solvent, and this phenomenon is often called the “chemical effect.” Another effect that significantly affects the solubility is the “physical effect” of temperature and pressure. In SFE, the solubilities are often extremely sensitive to pressure and temperature. That results in an important feature of SFE that allows the yield and the selectivity of the solvent to be finely tuned by temperature and pressure above the critical temperature. Usually, pressure is preferred as a control factor. The advantage of pressure control is that the solubility can be changed on a nearly instantaneous basis, whereas the temperature control suffers from thermal lag and the temperature gradients that limit the maximum useful speed of temperature increase, because the gradient elution requires the time for complete mixing of two liquid streams.31 Therefore, in most cases, the physical effect is usually referred to as the “pressure effect.” This extreme pressure sensitivity of solubility can be explained by its compressibility, which is expressed as a gradient of density over pressure. As the solubility parameters are proportional to the density of the solvent-SC-CO2, the sharp variance of density near the critical region (Figure 2.3) shows why solubility changes greatly at different temperatures and pressures. At the critical point, the slope of density-pressure is almost vertical, which means that the rate of change is close to infinite and results in extreme pressure sensitivity. To further understand the impact of pressure on the solubility, an enhancement factor E was derived E = y2

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6.5

6

Acridine Antracence Hexamethylbenzene

log E

5.5

5

4.5

4 16

17

18

19

20

21

22

Density (mol/L)

FIGURE 2.4 Enhancement factors at various SC-CO2 densities, modified from Dobbs and Johnston (1987).28

where P2sat is the solute saturated pressure. This equation removes the effect of pressure and focuses on the solvent-solute interaction in the SCF phase. For the sterols in SC-CO2, at 35°C and 20 MPa, the enhancement factors have almost the same value. In fact, the enhancement factor varies very little for many organic solids. Figure 2.4 shows the E values vary within a range of only 1.5 orders of magnitude for substances with a variety of polar functional groups, whose actual solubilities may vary by many orders of magnitude.28 These results indicate that in SFE, pressure is primarily responsible for the solubility, not the solute-solvent interaction. Furthermore, vapor pressures of the components in the mixture have significant impact on the selectivity. Vapor pressure measures the volatility of solutes. If the components in the mixture have similar chemical structures, their selectivity is mainly determined by their vapor pressure. A typical example is the components in a sterol mixture that contains three sterols: cholesterol, stigmaterol, and ergosterol. The CO2 is surprisingly more selective for cholesterol than ergosterol, despite the fact that both of them have very similar chemical structures. The solubilities of these two components differ by two orders of magnitude. The reason is that their vapor pressures are different. Although their vapor pressure is as low as 10-11 MPa, which is negligibly small, the selectivity follows the ratio of pressure. It should be mentioned that if an inert component is added to a mixture which does not strongly interact with the extracted component but is also volatile, that volatile component will decrease the solubility of the desired component as its partial pressure is decreased by the added volatile component. Water behaves in this manner with many biocomponents.

2.3.5 EFFECT

OF

WATER CONTENT

IN

MATERIAL

Many natural products contain water, and its solubility is close to oil, at normal temperature (30°C) and pressure (10 MPa or so). Wiebe and Gaddy32 measured the

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0.008

Solubility (g.water/L. CO2 S.T.P)

0.007 0.006 0.005 0.004 0.003 0.002 25°C 31.04°C(Tc) 50°C

0.001 0

0

100

200

300 400 500 Total pressure, atm

600

700

800

FIGURE 2.5 Composition of phase rich in carbon dioxide, modified from Wiebe and Gaddy (1941).32

vapor phase composition of carbon dioxide-water mixtures at various temperatures and pressures up to 70 MPa. Their measurements revealed the similar solubility behavior of the water-SC-CO2 system with the oil-SC-CO2 system (Figure 2.5). There was a minimum solubility at a certain pressure. Near this pressure, water solubility changes greatly, and at high pressures and above the supercritical region, water solubility increases slowly. King et al.33 obtained more mutual solubility data on water-SC-CO2 at lower temperatures (down to 15°C) and at lower pressures (a little over 20 MPa), which is the liquid and supercritical region, and their data agreed well with data from Wiebe32 in the overlapped region. According to their data, over the temperature range 15°C to 40°C and pressure range 5.1 to 20.3 MPa, the solubility of water in liquid and SC-CO2 varies from 2.2 × 10–3 to 5.8 × 10–3 mole fraction. It is close to the oil solubility and that may be the reason why water is extracted along with the oil. Further, as discussed above, if water comes into the SCF phase in large quantities, the partial pressure of oil in the SCF phase will decrease. That will influence the solubility of oil in SCF. The binary phase diagram is shown in Figure 2.6.33 The turning point D on Figure 2.6 corresponds to the minimum solubility. Dunford et al.34 observed that the solubility of Atlantic mackerel oil decreases significantly at moisture levels > 40.5%. However, for some proteins or other substances with hydroxyl groups, their water binding potential may help them dissolve in the SC-CO2, like a cosolvent. Due to its dual effects, the moisture content needs to be optimized. Dunford et al.35 reported an optimum moisture content of 10.2% for the extraction of oil and residual proteins from Atlantic mackerel. Figure 2.7 shows the typical effect of water on

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Pressure

Pressure

D

0

Water rich

CO2

1

0

1

Mole fraction of CO2

Mole fraction of CO2

(a)

(b)

FIGURE 2.6 Pressure-composition diagram for CO2/water system. a) Temperature below critical point of CO2. b) Temperature above critical point of CO2.

supercritical fluid extraction of oil. Figure 2.7 also shows that for the optimized moisture level, from C1 to C2 (for Atlantic mackerel, 0% to 26% of moisture), the solubility does not decrease significantly. Figure 2.7 proves again that the effect of pressure is much larger than that of solute-solvent interaction. If the extracted component has no or weak interaction with water, before the partial pressure of water changed significantly, water has almost no effect on the solubility and extract yield of oil. Snyder et al.36 observed that moisture level between 3% and 12% had little effect on the extraction of soybean oil at pressure of 1.7 MPa and a temperature of 50°C. In terms of an industrial process, the low sensitivity to water content has some advantages. The fact that in some regions the solubility is not sensitive to water content will reduce the need for a stronger drying process. Samples that do not require extensive drying will have lower drying cost, because it is usually an energy intensive process.

2.4 SOLUBILITY PREDICTION Obtaining solubility data requires a lot of experimental work, so the ability to predict solubility is important for design. The solubility can be predicted through properties of pure substances. Its foundation is built on the thermodynamics of solvent equilibrium state, which is described by phase equilibrium and chemical potential equilibrium. In the view of phase equilibrium, the fugacity of species i in the supercritical phase is equal to the fugacity of the pure solid. The compressed gas model37 is used to describe this equilibrium on the solid-gas interface

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Oil solubility

ymax

C1

C2

C3

Water content

FIGURE 2.7 Oil solubility vs. water concentration; plotted according to data of Dunford et al. (1997,1998).34,35

(

 Vi P − Pi sat Pi sat exp  RT  yi = Pφ i

)  

where the subscript i means the component i, and Vi is the volume of pure substance. The fugacity coefficient φ is calculated by equation of state (EOS). The most widely used EOS are the Peng-Robinson (P-R) equation and Redlich-Kwong (R-K) equation. They will be described in the next section. Cygnarowicz et al.38 used this method to estimate the solubility of β-carotene in supercritical carbon dioxide at temperature ranging 40°C to 70°C, and pressure ranging from 20 to 45 MPa, and the result agreed well with experimental data. Soave39 applied R-K EOS and Huron-Vidal mixing rules to calculate fugacity coefficient. This method contained two adjustable parameters and agreed well with the isothermal experimental data. In the view of chemical potential equilibrium, if the chemical potential of component i is the same in each phase, then the following equation is obtained to calculate the solubility

(

 Vi P − Pi vap  − ∆µ i   Pi vap exp  exp  kT  kT  yi = ρkT

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where ∆µ is excess chemical potential, ρ is density, and k is the Boltzmann constant. Calculating the excess chemical potential ∆µ is a great challenge. Goldman et al.40 developed a method based on the Redlich-Kwong equation of state and LennardJones intermolecular potential (Equation 2.11):  σ ij  12  σ ij  6  µ ij = 4 εij   −  r    r   where εij is the potential well depth for the interactions of component i, j and σij measure their range. They obtained an expression for chemical potential directly in terms of the intermolecular potentials at infinite dilution of components for ternary system ∆µ 2 = − log (1 − bρ) − γz ( x1 , x3 ) log (1 + bρ) + y ( x1 , x3 ) kT  bρ  bρ   + γ  log (1 + bρ) −   1 + bρ     1 − bρ where γ = 7.45 ( ε kT )

3/ 2

y ( x1 , x3 ) = 2 x1

σ12 3 σ 3 + 2 x3 233 − 1 3 σ σ

z ( x1 , x3 ) = 2 x1

σ12 3 σ 3 + 2 x3 233 − 1 3 σ σ

and ε and σ are the counterpart parameters of mixture, b is an adjustable parameter, and x is the mole fraction of each component. This model passed a simulation test and agreed well with the theory. The obvious advantage is that it can be used to directly demonstrate the cosolvent effect through solute chemical potential which is strongly dependent on the molecular interaction. In a view of solute property, Politzer et al.41 built relationships of molecular structure and solubility in SC-CO2 through surface potential. The logarithm solubility shows a linear relationship with the square of surface potential. Their work encompassed 22 solutes in CO2 at 308K and two pressures: 14 and 20 MPa. The methods above based on the thermodynamic equilibrium have a strong theoretical background. However, the inherent weakness is that they need extensive chemical physical data to characterize the solvent and solute. Therefore they are usually applied to simple structures where properties have been a fully investigated substance. That limits their application in the bioproducts, as those substances

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usually have complicated structures and only a limited amount of physical data available for calculation. Since solubility does not directly influence the mass transfer, sometimes a qualitative analysis is used. The solubility parameter concept is a good tool for representing the solubility level. It can be easily obtained from thermodynamic properties. Even if there is a lack of thermodynamic data, it still can be approximated by the UNIFAC model, which is based on summation of all the functional group contributions. The obvious advantage of this method is a simpler analysis that estimates the solubility based on the molecular structure. Giddings et al.42 obtained the correlation of solubility parameters d by extending the theory of the solubility power and using it to rapidly estimate a solubility level in the condensed solvent χ = V1 ( δ1 − δ 2 ) RT + χ s 2

δ = 1.25 Pc 0.5 ( ρr ,SF ρr .L ) where the subscript r stands for the reduced property and 1 is the solvent, 2 is the solute, χ is the total interaction parameter, and χs is the entropic interaction parameter. Fedors43 correlated chemical structure and solubility parameter. They collected many thermodynamic properties of typical atoms and function as group contributions. With this knowledge, the solubility parameter and the molar volume for both solvent and solute were estimated. In addition, a very simple correlation was obtained for temperature dependence of solubility parameter V  δ 22 = δ12  1   V2 

2.27

+

R V2

  V1  1.27  T1   − T2    V2  

It was suitable for both polar and nonpolar liquids. When |T1 - T2| < 150°C and both are at or below the normal boiling point of the liquid, this equation can be simplified as V  δ 2 = δ1  1   V2 

1.13

ρ  = δ1  2   ρ1 

1.13

It is built on the relationship between the solubility temperature dependency and the temperature dependency of density. When |T1 - T2| < 50°C, it can be further simplified as V2 = V1 1 + α (T2 − T1 )  δ 2 = δ1 1 + 1.13α (T1 − T2 ) 

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where α was the cubic coefficient of expansion, and the prediction accuracy was around 2%. To further utilize the advantage of solubility parameters, Prausnitz et al.7 built the relationship between solubility parameters and solubility, where ∆Hf is the change of enthalpy of fluid. It is valid for a nonpolar solute in a nonpolar solvent.

ln y2 =

∆H f  1 1  ν2 φ12 ( δ1 − δ 2 ) − − R  T f T  RT

2

Later, the reduced solubility parameter concept was developed by King and Friedrich44 ∆=

δ1 δ2

where δ1 and δ2 are the solubility parameters of solvent and solute, respectively. It described solubility parameters in a more general way, and even the relationship with partition coefficient can be built through the ∆ term. When plotting the data of various extraction systems, it showed apparent correlation between partition coefficient and reduced solubility parameter, which was confirmed in one equation, that was formalized by Giddings et al.42

(

) (

)

RT V2 δ 22 ln K normal K = 2 − ∆ ∆ where subscript “normal” means low-pressure reference state. This implied that the partition coefficients can be predicted by other well-known solutes under similar conditions if their reduced solubility parameters were calculated. All of the above correlations were based on thermodynamics and equation of state, so they have complicated forms. To simplify the calculations, the empirical models are still accepted by researchers. The empirical models of Chrastil45and Mitra and Wilson46 are based on the phenomenological method, which builds the correlation of solubility with their effect factors directly. One of most widely used empirical models was made by Chrastil.45 This is a simple empirical correlation. ln C2 = k ln ρ1 + a1 / T + a0 where a1 = ∆H R a0 = ln ( M 2 + kM 1 ) + q − k ln M 1

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where Mi is the molecular weight of component i, and k is an adjustable parameter. This correlation describes the fact that the solubility is proportional to the solvent density. Because it relates solubility with CO2 density directly and has high accuracy in prediction (4% error), it is used widely in the SC-CO2 extraction field.11,47 Some modification of this model was made by Del Valle and Aguilera48 (Equation 2.23), where C is the concentration of solute, but it did not improve the accuracy. However, it did successfully predict the solubility of vegetable oil (Equation 2.24) in the temperature range of 293K to 353K and pressure range of 20 to 80 MPa. It also showed that the solubility behavior of some bioactive components such as lycopene was very similar to vegetable oil49, and that their solubilities can be predicted by this equation C = exp( 40.361 − 18708 / T + 2186840 / T 2 ) × ρ10.724 ± 2.7 Adachi and Lu50 developed a five-adjustable-parameter equation (ki and ai are all adjustable parameters), however, the accuracy did not improve much. The determinant coefficient of regression for the solubility for hazelnut oil increased to 0.995 from 0.985 based on the prediction from the Chrastil equation.51

(

)

ln C2 = k1 + k2 ρ1 + k3ρ12 ln ρ1 + a0 + a1 / T As for the solubility of cosolvent, Walsh et al.52 gave an associated-perturbedanisotropic-chain theory, and obtained a complicated phase equilibrium calculation method. Usually, researchers choose suitable mixing rules for EOS to simplify calculation.

2.4.1 EQUATIONS

OF

STATE (EOS)

Equations of state are used to describe the thermodynamic states through the relationship of state parameters such as temperature, pressure, and volume. In SFE processing, EOS describes the behavior of solute being dissolved in supercritical fluids. The Peng-Robinson equation53 is usually used to describe a compressed gas. A general form of the Peng-Robinson equation is

P=

RT aα − V − b V (V + b ) + b (V − b )

where

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a = 0.45724

( RTc )2 Pc

(

)

α = 1 + m 1 − Tr   

2

m = 0.37464 + 1.54226ω − 0.26992ω 2 b = 0.07780

RTc Pc

(

)

ω = −1.000 − log P s Pc

Tr =0.7

For mixtures, the following mixing rule is used to calculate the fugacity coefficient.

a=

n

n

i =1

j =1

∑∑ y y a i

j ij

aij = ai a j 1 − kij + ( kij − k ji ) yi  Redlich-Kwong (R-K)54 is simplified to the following forms

a = 0.42747

( RTc )2 Pc

(

)

α = 1 + m 1 − Tr   

2

m = 0.48 + 1.57ω − 0.176ω 2 b = 0.08664

RTc Pc

(

ω = −1.000 − log P s Pc

)

Tr=0.7

Both P-R and R-K predict the state well in the compressed gas region, but P-R shows better prediction in the saturated liquid field. Although P-R and R-K are widely accepted by researchers, Schmitt and Reid55 pointed out that these kinds of equations of state are not appropriate for a solid solute system, as the essential assumptions are applicable for a liquid vapor system, which is considered the main reason that the prediction of P-R is not perfect over a wide pressure range even with optimized parameters. They gave a modified P-R equation accounting for the solid solute and obtained better prediction over a wide pressure range (from 5 to 45 MPa).

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Hartono et al.56 pointed out that a two-parameter equation Mohsen-Nia-ModdaressMansoori (MMM) was as accurate as the modified P-R equation for a biocomponent in a supercritical region. An alternative choice is the Virial equation of state. The Virial equation of state57 directly describes the multicomponent fluid mixture with molecular interactions. There is no adjustable parameter, and the solubility is obtained directly by the intermolecular potentials of the interaction of solvent, solute, and cosolvent. This method performs under SFE operation conditions with fourbody interaction level. Although the EOS above have good prediction performance, they require a lot of critical physical properties of solutes, which are not easily available and only few are available in the published literatures. Hence some empirical expressions are still widely used by researchers. For example, Saeki58 expressed an empirical equation based on power law and Maxwell thermodynamic relation. It has good agreement with some fluids such as neon, hydrogen, deuterium, and carbon monoxide in the supercritical state. Equations of state have a central role in supercritical fluids because they not only predict the solubility, but also give the qualitative phase behavior. Hence, more complicated theories and more multiparameter empirical methods continue to be developed to formulate the state more accurately. Although the present model accuracy is limited by its character, they can be used to build most of the known binary phase diagrams, which are the fundamentals of supercritical fluid extraction.

2.5 SUMMARY As discussed above, solubility determines the capacity of SFE, and measuring the solubility of targeted components in SC-CO2 will build a solid base for prediction and further process design. The measurement and prediction of solubility includes not only solubility in SC-CO2, but also the phase equilibrium data in multisolvent systems, as the modification of SC-CO2 will play a leading role in the future development which is based on the reality that a lot of bioactive components have low solubility in SC-CO2, and a small quantity of cosolvent can change the solubility dramatically. Cosolvents will decrease the pressures applied and further decrease the operation cost, but how much cosolvent is suitable, and how much the solubility will change, needs more attention. So far, many researchers studying cosolvents are at the qualitative stage and require many more experiments to determine the optimal conditions. The difficulty of prediction is that cosolvents will change the polarity and phase state of the SC-CO2 system. Few phase equilibrium data in or near the critical region are available, which makes the theory difficult to validate. So, developing an accurate and convenient EOS which can deal with multicomponents will be important. Through EOS, the solubility phase diagram can be determined, which is a design base of SFE. Furthermore, a lot of bioactive components are extracted together with byproducts. In order to improve the purity or decrease the untargeted component, the selectivity of SFE systems should be improved. Crossover pressure operation is a choice, which in fact is similar to distillation at the supercritical region. Its limitation is that usually there is not enough phase equilibrium data in the supercritical region, and temperature control is not as convenient as pressure control.

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Complex cosolvents can be an alternative choice. Some researchers have observed that at different operational stages, the extraction speeds of various components are different, so that the components in the extracted mixture will change. If the batch operation can be changed to a semibatch operation, the cosolvent can be added to the system at a suitable time to keep the selectivity at a high level. Optimizing the water content is also important. There are opportunities to decrease the pretreatment cost without change or even to increase the efficiency of SFE. To decrease the operation cost, pretreating tissues, breaking down the cell wall and releasing the oil, needs to be improved. Flaking and grinding are widely used methods because the cell breakage percentage is high. However, those are lab-scale methods. If applied to industry, decreasing the operation costs and increasing the percentage of cell breakage should be considered. In a word, to applied SFE technology, the solubility prediction and phase diagrams must be accurate to give a solid base for design, and the operation costs have to be decreased further.

REFERENCES 1. Rizvi, S.S.H., Benado, A.L., Zollweg, J.A., and Daniels, J.A., Supercritical fluid extraction: fundamental principles and modeling methods, Food Technology, June, 55–65, 1986. 2. Johnston, K.P., Lemert, R., Kim, S., and Wong, J., Multicomponent polar mixtures at supercritical fluid conditions, at AICHE, New York, NY, USA, 1987. 3. Chimowitz, E.H. and Pennisi, K.J., Process synthesis concepts for supercritical gas extraction in the crossover region, AIChE Journal, 32, 10, 1665–1676, 1986. 4. Foster, N.R., Gurdial, G.S., Yun, J.S.L., Liong, K.K., Tilly, K.D., Ting, S.S.T., Singh, H., and Lee, J.H., Significance of the crossover pressure in solid-supercritical fluid phase equilibria, Industrial and Engineering Chemistry Research, 30, 8, 1955–1964, 1991. 5. Eckert, C.A., Ziger, D.H., Johnston, K.P., and Ellison, T.S., The use of partial molar volume data to evaluate EOS for SCF mixtures, Fluid Phase Equilibria, 14, 167, 1983. 6. Eckert, C.A., Ziger, D.H., Johnston, K.P., and Kim, S., Solute partial molal volumes in supercritical fluids, Journal of Physical Chemistry, 90, 12, 2738–2746, 1986. 7. Prausnitz, J.M., Litchtenthaler, R.N., and Gomes de Azevedo, E., Molecular thermodynamics of fluid phase equilibria, Prentice Hall, Englewood Cliffs, NJ, 1986. 8. Patton, C.L. and Luks, K.D., Determination of partial molar volumes at critical end points, Fluid Phase Equilibria, 113, 1–2, 21–26, 1995. 9. Staby, A. and Mollerup, J., Separation of constituents of fish oil using supercritical fluids: a review of experimental solubility, extraction, and chromatographic data, Fluid Phase Equilibria, 91, 2, 349–386, 1993. 10. Bartle, K.D., Solubilities of compounds in supercritical carbon dioxide, Journal of Physical Chemistry Reference Data, 20, 4, 756, 1991. 11. Guclu-Ustundag, O. and Temelli, F., Correlating the solubility behavior of fatty acids, mono-, di-, and triglycerides, and fatty acid esters in supercritical carbon dioxide, Industrial and Engineering Chemistry Research, 39, 12, 4756–4766, 2000. 12. Hyatt, J.A., Liquid and supercritical carbon dioxide as organic solvents, Journal of Organic Chemistry, 49, 26, 5097–5101, 1984.

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13. Brunner, G., Stofftrennung mit ueberkritischen gasen (gasextraktion), Chemie-Ingenieur-Technik 59, 1, 12–22, 1987. 14. Wong, V., Wyllie, S.G., Cornwell, C.P., and Tronson, D., Supercritical fluid extraction (SFE) of monoterpenes from the leaves of Melaleuca alternifolia (tea tree), Molecules, 6, 92–103, 2001. 15. Hammam, H., Solubilities of pure lipids in supercritical carbon dioxide, The Journal of Supercritical Fluids, 5, 101–106, 1992. 16. Dobbs, J.M., Wong, J.M., Lahiere, R.J., and Johnston, K.P., Modification of supercritical fluid phase behavior using polar cosolvents, Industrial and Engineering Chemistry Research, 26, 56–65, 1987. 17. Castellan, G.W., Physical chemistry, 2nd ed., Addison-Wesley, Reading, MA, 1964. 18. Krukonis, V.J., McHugh, M.A., and Seckner, A.J., Xenon as a supercritical solvent, Journal of Physical Chemistry, 88, 2687–2689, 1984. 19. Abbott, A.P., Eardley, C.A., and Scheirer, J.E., CO2/HFC 134a mixtures: alternatives for supercritical fluid extraction, Green Chemistry, April, 63–65, 2000. 20. Abbott, A.P., Corr, S., Durling, N.E., and Hope, E.G., Solubility of substituted aromatic hydrocarbons in supercritical difluoromethane, Journal of Chemical and Engineering Data, 47, 4, 900–905, 2002. 21. Wong, J.M. and Johnston, K.P., Solubilization of biomolecules in carbon dioxide based supercritical fluids, Biotechnology Progress, 2, 1, 29–39, 1986. 22. Chandra, A. and Nair, M.G., Supercritical fluid carbon dioxide extraction of - and carotene from carrot (Daucus carota L.), Phytochemical Analysis, 8, 5, 244–246, 1997. 23. Raveendran, P. and Wallen, S.L., Exploring CO2-philicity: effects of stepwise fluorination, Journal of Physical Chemistry B, 107, 6, 1473–1477, 2003. 24. Sunol, A.K., Hagh, B., and Chen, S., Entrainer selection in supercritical extraction, in Process Technology Proceedings, Vol. 3, Elsevier, Amsterdam, 1985. 25. Rowlinson, J.S. and Richardson, M.J., Solubility of solids in compressed gases, in Advances in Chemistry and Physics, edited by I. Prigogine, 2nd ed., Interscience Publishers, New York, 1959. 26. Teberikler, L., Koseoglu, S., and Akgerman, A., Selective extraction of phosphatidylcholine from lecithin by supercritical carbon dioxide/ethanol mixture, Journal of the American Oil Chemists’ Society, 78, 2, 115–119, 2001. 27. Yuan, H., Gao, G.T., and Zeng, X.C., Effects of the co-solvent energy parameter and dipolar strength on solute residual chemical potential, Fluid Phase Equilibria, 138, 1–2, 61–68, 1997. 28. Dobbs, J.M. and Johnston, K.P., Selectivities in pure and mixed supercritical fluid solvents, Industrial and Engineering Chemistry Research, 26, 7, 1476–1482, 1987. 29. Brunner, G., Selectivity of supercritical compounds and entrainers with respect to model substances, Separation Science and Technology, 10, 2–3, 289–298, 1983 . 30. Dobbs, J.M., Wong, J.M., and Johnston, K.P., Nonpolar co-solvents for solubility enhancement in supercritical fluid carbon dioxide, Journal of Chemical and Engineering Data, 31, 3, 303–308, 1986. 31. Smith, R.D., Udseth, H.R., and Wright, B.W., Micro-scale methods for characterization of supercritical fluid extraction and fraction processes, in Supercritical Fluid Technology, edited by Penninger, J.M.L., Radosz, M., McHugh, M.A., and Krukonis, V.J., Elsevier, Amsterdam, 1985. 32. Wiebe, R. and Gaddy, V.L., Vapor phase composition of carbon dioxide-water mixtures at various temperatures and at pressures to 700 atmospheres, Journal of the American Chemical Society, 63, 475–477, 1941.

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Functional Food Ingredients and Nutraceuticals 33. King, M.B., Mubarak, A., Kim, J.D., and Bott, T.R., The mutual solubilities of water with supercritical and liquid carbon dioxide, The Journal of Supercritical Fluids, 5, 296–302, 1992. 34. Dunford, N.T., Goto, M., and Temelli, F., Modeling of oil extraction with supercritical CO2 from Atlantic mackerel (Scomber scombrus) at different moisture contents, Journal of Supercritical Fluids, 13, 303–309, 1998. 35. Dunford, N.T., Temelli, F., and Leblanc, E., Supercritical CO2 extraction of oil and residual proteins from Atlantic mackerel ( Scomber scombrus) as affected by moisture content, Journal of Food Science, 62, 2, 289–294, 1997. 36. Snyder, J.M., Friedrich, J.P., and Christianson, D.D., Effect of moisture and particle size on the extractability of oils from seeds with supercritical CO2 , Journal of the American Oil Chemists’ Society, 61, 12, 1851–1856, 1984. 37. Prausnitz, J.M., Molecular thermodynamics of fluid phase equilibria, Prentice Hall, Englewood Cliffs, NJ, 1969. 38. Cygnarowicz, M.L., Maxwell, R.J., and Seider, W.D., Equilibrium solubilities of -carotene in supercritical carbon dioxide, Fluid Phase Equilibria, 59, 57–71, 1990. 39. Soave, G., A simple model for the supercritical extraction of solids, Journal of Supercritical Fluids, 19, 19–24, 2000. 40. Goldman, S., Gray, C.G., Li, W., Tomberli, B., and Joslin, C.G., Predicting solubilities in supercritical fluids, Journal of Physical Chemistry, 100, 7246–7249, 1996. 41. Politzer, P., Murray, J.S., Lane, P., and Brinck, T., Relationship between solute molecular properties and solubility in supercritical CO2, Journal of Physical Chemistry, 97, 729–732, 1993. 42. Giddings, J.C., Myers, M.N., McLaren, L., and Keller, R.A., High pressure gas chromatography of non-volatile species, Science, 162, 67–73, 1968. 43. Fedors, R.F., A method for estimating both the solubility parameters and molar volume of liquids, Polymer Engineering and Science, 14, 2, 147–154, 1974. 44. King, J.W. and Friedrich, J.P., Quantitative correlations between solute molecular structure and solubility in supercritical fluids, Journal of Chromatograhy, 517, 449–458, 1990. 45. Chrastil, J., Solubility of solids and liquids in supercritical gases, Journal of Physical Chemistry, 86, 3016–3021, 1982. 46. Mitra, S. and Wilson, N.K., Empirical method to predict solubility in supercritical fluids, Journal of Chromatographic Science, 29, 7, 305–309, 1991. 47. Gómez-Prieto, M.S., Caja, M.M., and Santa-María, G., Solubility in supercritical carbon dioxide of the predominant carotenes of tomato skin, Journal of the American Oil Chemists’ Society, 79, 9, 897–902, 2002. 48. Del Valle, J.M. and Aguilera, J.M., Improved equation for predicting the solubility of vegetable oils in supercritical CO2, Industrial and Engineering Chemistry Research, 27, 8, 1551–1553, 1988. 49. Roy, B.C., Goto, M., and Hirose, T., Temperature and pressure effects on supercritical CO2 extraction of tomato seed oil, International Journal of Food Science and Technology, 31, 2, 137–141, 1996. 50. Adachi, Y. and Lu, B.C.Y., Supercritical fluid extraction with carbon dioxide and ethylene, Fluid Phase Equilibria, 14 , 147, 156, 1983. 51. Ozkal, S.G., Salgin, U., and Yener, M.E., Supercritical carbon dioxide extraction of hazelnut oil, Journal of Food Engineering, 69, 2, 217–223, 2005. 52. Walsh, J.M., Ikonomou, G.D., and Donohue, M.D., Supercritical phase behavior: the entrainer effect, Fluid Phase Equilibria, 33, 3, 295–314, 1987.

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53. Peng, D. and Robinson, D.A., A New Two-Constant equation of state, Industrial and Engineering Chemistry Fundamentals, 15, 59–64, 1976. 54. Redlich, O. and Kwong, J.N.S., The thermodynamics of solutions, V, an equation of state: fugacities of gaseous solutions, Chemical Review, 44, 233–244, 1949. 55. Schmitt, W.J. and Reid, R.C., Solubility of monofunctional organic solids in chemically diverse supercritical fluids, Journal of Chemical and Engineering Data, 31, 204–212, 1986. 56. Hartono, R., Mansoori, G.A., and Suwono, A., Prediction of solubility of biomolecules in supercritical solvents, Chemical Engineering Science, 56, 24, 6949–6958, 2001. 57. Joslin, C.G., Gray, C.G, Goldman, S., Tomberli, B., and Li, W., Solubilities in supercritical fluids from the virial equation of state, Molecular Physics, 89, 2, 489–503, 1996. 58. Saeki, S., Empirical equation of state for supercritical fluids, Journal of Supercritical Fluids 8, 1, 30–45, 1995. 59. Singh, H., Yun, S.L.J., Macnaughton, S.J., Tomasko, D.L., and Foster, N.R., Solubility of cholesterol in supercritical ethane and binary gas mixtures containing ethane, Industrial and Engineering Chemistry Research, 32, 11, 2841–2848, 1993. 60. Yun, S.L.J., Liong, K., Keat, G., Gurdev S., and Foster, N.R., Solubility of cholesterol in supercritical carbon dioxide, Industrial and Engineering Chemistry Research, 30, 11, 2476–2482, 1991. 61. Murga, R., Sanz, M.T., Beltran, S., and Cabezas, J.L., Solubility of syringic and vanillic acids in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 49, 4, 779–782, 2004. 62. Gaspar, F., Lu, T., Marriott, R., Mellor, S., Watkinson, C., Al-Duri, B., Santos, R., and Seville, J., Solubility of echium, borage, and lunaria seed oils in compressed CO2, Journal of Chemical and Engineering Data, 48, 1, 107–109, 2003. 63. Ferreira, S.R.S., Meireles, M.A.A., and Cabral, F.A., Extraction of essential oil of black pepper with liquid carbon dioxide, Journal of Food Engineering, 20, 2, 121–133, 1993. 64. Botch-Jensen, C. and Mollerup, J., Phase equilibria of fish oil in sub- and supercritical carbon dioxide, Fluid Phase Equilibria, 138, 1–2, 179–211, 1997. 65. Yin, J.Z., Liu, R.J., Ding, X., and Yang, B., Experimental study on solubility of Hippophae Rhamnoides L. seed oils in supercritical carbon dioxide, Huagong Xiandai/Modern Chemical Industry 22, Suppl., 111–113, 2002. 66. Hassan, M.N., Rahman, N.N.A., Ibrahim, M.H., and Omar, A.K.M., Simple fractionation through the supercritical carbon dioxide extraction of palm kernel oil, Separation and Purification Technology, 19, 1–2, 113–120, 2000. 67. Bharath, R., Yamane, S., Inomata, H., Adschiri, T., and Arai, K., Phase equilibria of supercritical CO2 - fatty oil component binary systems, Fluid Phase Equilibria, 83, pt 2, 183–192, 1993. 68. Tilly, K.D., Chaplin, R.P., and Foster, N.R., Supercritical fluid extraction of the triglycerides present in vegetable oils, Separation Science and Technology, 25, 4, 357–367, 1990. 69. Norulaini, N.A., Zaidul, I.S., Anuar, O., Azlan, A., and Omar, A.K., Supercritical reduction of lauric acid in palm kernel oil (PKO) to produce cocoa butter equivalent (CBE) fat, Journal of Chemical Engineering of Japan, 37, 2, 194–203, 2004. 70. Nik Norulaini, N.A., Md Zaidul, I.S., Anuar, O., and Mohd Omar, A.K., Supercritical enhancement for separation of lauric acid and oleic acid in palm kernel oil (PKO), Separation and Purification Technology, 35, 1, 55–60, 2004.

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Functional Food Ingredients and Nutraceuticals 71. Maheshwari, P., Nikolov, Z.L., White, T.M., and Hartel, R., Solubility of fatty acids in supercritical carbon dioxide, Journal of the American Oil Chemists’ Society, 69, 11, 1069–1076, 1992. 72. Nilsson, W.B., Gauglitz, E.J., and Hudson, J.K., Solubilities of methyl oleate, oleic acid, oleyl glycerols, and oleyl glycerol mixtures in supercritical carbon dioxide, Journal of the American Oil Chemists’ Society, 68, 2, 87–91, 1991. 73. Iwai, Y., Fukuda, T., Koga, Y., and Arai, Y., Solubilities of myristic acid, palmitic acid, and cetyl alcohol in supercritical carbon dioxide at 35°C, Journal of Chemical and Engineering Data, 36, 4, 430–432, 1991. 74. Ikawa, N., Nagase, Y., Imanishi, N., and Fukuzato, R., Separation of polyunsaturated fatty acid by supercritical fluid extraction and chromatography, KOBELCO Technology Review, 12, 40–43, 1991. 75. Ghaziaskar, H.S., Eskandari, H., and Daneshfar, A., Solubility of 2-Ethyl-1-hexanol, 2-Ethylhexanoic acid, and their mixtures in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 48, 2, 236–240, 2003. 76. Liong, K., Keat, F., Neil R., and Ting, S.S.T., Solubility of fatty acid esters in supercritical carbon dioxide, Industrial and Engineering Chemistry Research, 31, 1, 400–404, 1992. 77. Staby, A., Forskov, T., and Mollerup, J., Phase equilibria of fish oil fatty acid ethyl esters and sub- and supercritical CO2, Fluid Phase Equilibria, 87, 2, 309–340, 1993. 78. Xing, H.B., Yang, Y.W., Su, B.G., Huang, M., and Ren, Q.L., Solubility of artemisinin in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 48, 2, 330–332, 2003. 79. Hansen, B.N., Harvey, A.H., Coelho, J.A.P., Palavra, A.M.F., and Bruno, T.J., Solubility of capsaicin and β-carotene in supercritical carbon dioxide and in halocarbons, Journal of Chemical and Engineering Data, 46, 5, 1054–1058, 2001. 80. Sakaki, K., Solubility of β-carotene in dense carbon dioxide and nitrous oxide from 308 to 323K and from 9.6 to 30 MPa, Journal of Chemical and Engineering Data, 37, 2, 249–251, 1992. 81. Burgos-Solorzano, G.I., Brennecke, J.F., and Stadtherr, M.A., Solubility measurements and modeling of molecules of biological and pharmaceutical interest with supercritical CO2, Fluid Phase Equilibria, 220, 1, 57–69, 2004. 82. Johannsen, M. and Brunner, G., Solubilities of the fat-soluble vitamins A, D, E, and K in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 42, 1, 106–111, 1997. 83. Chafer, A., Berna, A., Monton, J.B., and Mulet, A., High pressure solubility data of the system limonene + linalool + CO2, Journal of Chemical and Engineering Data, 46, 5, 1145–1148, 2001. 84. Berna, A., Chafer, A., and Monton, J.B., Solubilities of essential oil components of orange in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 45, 5, 724–727, 2000. 85. Lewin-Kretzschmar, U., Blasberg, L., and Harting, P., Solubility of nitroaromatics and adamantane in pure and surfactant-modified compressed gases, Tenside, Surfactants, Detergents, 39, 1, 29–35, 2002. 86. Khosravi-Darani, K., Vasheghani-Farahani, E., Yamini, Y., and Bahramifar, N., Solubility of poly (α-hydroxybutyrate) in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 48, 4, 860–863, 2003.

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87. Graham, B.F., Lagalante, A.F., Bruno, T.J., Harrowfield, J.M., and Trengove, R.D., Solubility of p-t-butylcalixarenes in supercritical carbon dioxide: a comparison of static and dynamic measurement techniques, Fluid Phase Equilibria, 150, 151, 829–838, 1998. 88. Nilsson, W.B. and Hudson, J.K., Solubility of simple and mixed triacylglycerols in supercritical CO2, Journal of American Oil Chemists’ Society, 70, 8, 749–754, 1993. 89. Foster, N.R., Singh, H., Yun, S.L.J., Tomasko, D.L., and Macnaughton, S.J., Polar and nonpolar cosolvent effects on the solubility of cholesterol in supercritical fluids, Industrial and Engineering Chemistry Research, 32, 11, 2849–2853, 1093. 90. Kuk, M.S. and Sr. Hron, R.J., Supercritical carbon dioxide extraction of cottonseed with co-solvents, Journal of the American Oil Chemists’ Society, 71, 12, 1353–1356, 1994. 91. Koga, Y., Iwai, Y., Hata, Y., Yamamoto, M., and Arai, Y., Influence of cosolvent on solubilities of fatty acids and higher alcohols in supercritical carbon dioxide, Fluid Phase Equilibria, 125, 1–2, 115–128, 1996. 92. Dohrn, R., Bunz, A.P., Devlieghere, F., and Thelen, D., Experimental measurements of phase equilibria for ternary and quaternary systems of glucose, water, CO2 and ethanol with a novel apparatus, Fluid Phase Equilibria, 83, pt 2, 149–158, 1993. 93. Chafer, A., Fornari, T., Berna, A., and Stateva, R.P., Solubility of quercetin in supercritical CO2 + ethanol as a modifier: Measurements and thermodynamic modelling, Journal of Supercritical Fluids, 32, 1–3, 89–96, 2004. 94. Ambrosino, P., Galvano, F., Fogliano, V., Logrieco, A., Fresa, R., and Ritieni, A., Supercritical fluid extraction of Beauvericin from maize, Talanta, 62, 3, 523–530, 2004. 95. Gurdial, G.S., Macnaughton, S.J., Tomasko, D.L., and Foster, N.R., Influence of chemical modifiers on the solubility of o- and m-hydroxybenzoic acid in supercritical CO2, Industrial and Engineering Chemistry Research, 32, 7, 1488–1497, 1993. 96. Cocero, M.J. and Calvo, L., Supercritical fluid extraction of sunflower seed oil with CO2-ethanol mixtures, JAOCS, 73, 11, 1573–1578, 1996. 97. Li, S. and Hartland, S., New industrial process for extracting cocoa butter and xanthines with supercritical carbon dioxide, JAOCS, 73, 4, 423–429, 1996.

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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials Helena Sovová Academy of Sciences of the Czech Republic

CONTENTS 3.1 3.2 3.3

3.4

3.5

3.6

3.7

Supercritical Fluid Extraction from Plant Materials .....................................76 Extraction Bed and the Concept of Broken and Intact Cells .......................78 Mass Balance and Model Equations for Plug Flow .....................................79 3.3.1 Mass Balance for Solvent and Matrix...............................................79 3.3.2 Mass Balance for Solute ....................................................................80 3.3.3 Time Constants and Dimensionless Model Equations......................82 SFE of Oils from Seeds .................................................................................85 3.4.1 Oil Solubility in SC-CO2 ...................................................................85 3.4.2 Model for Vegetable Oil Extraction................................................... 87 3.4.3 Equilibrium-Controlled Period ..........................................................89 3.4.4 Diffusion-Controlled Period...............................................................90 3.4.5 Approximate Solution for Both Periods............................................90 SFE of Essential Oils.....................................................................................92 3.5.1 Solute-Matrix Interaction...................................................................92 3.5.2 Simulation of Essential Oil Extraction..............................................92 3.5.3 Two Extraction Periods......................................................................92 SFE for Combined Equilibrium.....................................................................94 3.6.1 Equilibrium According to Perrut et al. ..............................................94 3.6.2 Simulation of SFE with Combined Equilibrium...............................96 3.6.3 Two Extraction Periods......................................................................96 Flow Patterns..................................................................................................96 3.7.1 Axial Dispersion ................................................................................96 3.7.2 Models for Differential Extraction Bed (Ideal Mixer)......................98 3.7.3 Natural Convection ............................................................................98 3.7.4 Channeling and Scale Up of Extractor............................................100

75

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3.8

Preliminary Evaluation of Extraction Curves..............................................101 3.8.1 Splitting Extraction Curve into Two Sections.................................101 3.8.2 Equilibrium-Controlled Period ........................................................103 3.8.3 Diffusion-Controlled Period.............................................................103 3.9 Conclusions ..................................................................................................106 Acknowledgment ...................................................................................................106 References..............................................................................................................107

3.1 SUPERCRITICAL FLUID EXTRACTION FROM PLANT MATERIALS A substance is in a supercritical state when both its pressure and temperature are higher than its critical pressure and temperature. Supercritical fluids, due to their liquid-like density and gas-like viscosity, are very well suited for extraction of valuable substances from plant materials. The most frequently used supercritical solvent is supercritical carbon dioxide (SC-CO2) because of the following characteristic attributes: Strong dependence of solvent properties on pressure and temperature, enabling wide control of solvent power in a broad range of operating conditions Easy separation of extract from the solvent by depressurization provides a distinct separation of solute from solvent since CO2 is gas at ambient conditions High diffusivity of solutes in supercritical fluids and, consequently, high mass transfer rates No traces of organic solvents in extracts Thermo-labile components are not exposed to high temperatures (critical point temperature and pressure of CO2 are 31.1C and 74 bars, respectively) No degradation of residual botanic material that can be used as foodstuff, feedstuff, or as a source of substances insoluble in supercritical solvent The main drawback of supercritical fluid extraction (SFE) is higher capital cost of the necessary high-pressure extraction equipment, compared to conventional extraction. Supercritical CO2 is the solvent of choice in the SFE of bioactive components from plant materials. Since CO2 is an environmentally friendly compound, it presents an alternative to hazardous traditional solvents. Though its solvent power for nonpolar compounds is lower than that of many organic solvents and it is even lower for the polar compounds (this disadvantage can be partially overcome by the addition of a polar modifier to the solvent), benefits of SC-CO2 prevail. Besides the advantages mentioned above it is also inflammable, nonexplosive, cheap, and easily accessible. About 100 industrial plant materials using SC-CO2 are onstream worldwide, most of them processing natural products for the food industry, nutraceuticals, or phytopharmaceuticals.

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In a typical extraction procedure, botanic material is pretreated by drying and grinding to form small particles. The extractor is filled with the particles, heated to the desired temperature, and pressurized with solvent. The solvent is then passed through an extraction bed at constant flow rate until the material is depleted or until the extraction rate drops below an economically acceptable value. Thus, the extraction can be carried out in a semicontinuous mode. The CO2 is recycled between the extractor and separator that is operated at a lower pressure corresponding to lower solubility where the solute precipitates, while gaseous CO2 passes to a condenser. When pressure is increased by a high-pressure pump, the supercritical solvent enters the extractor again. Temperature control is necessary in all parts of the high-pressure equipment. The capacity of industrial extractors ranges from hundreds of liters to several cubic meters. The extraction unit usually consists of two or more extractors; while one extractor is depressurized and refilled with fresh material, the solvent is passed through the other extractor under pressure and thus a continuous flow of extract to the separator is achieved. Extraction units are usually equipped with a series of separators operating at different pressures and temperatures, which enables partial fractionation of extract according to different solubility of its components. As the aim of industrial-scale SFE is to isolate a desired compound from a matrix at minimum cost and with minimum ballast substances coextracted, a careful optimization of extraction pressure, temperature, time, and solvent flow rate is necessary (besides optimization of conditions in separators). The solvent is usually fully saturated with solute in the initial extraction period. Analytical SFE, on the other hand, operates extractors of small capacity (a few cm3) and aims to extract soluble substances from matrix quantitatively as fast as possible. The extract is usually collected in a trap containing a liquid solvent, and then injected in a chromatograph or other analytical equipment. Different liquid modifiers are added to SC-CO2, and relatively high solvent flow rate, pressure, and temperature are set up in order to accelerate the extraction process. Fluid phase concentration usually does not reach saturation values because of the high solvent flow rates. As total solvent consumption is very low due to the small size of the extracted sample, the solvent is not recycled. The size of laboratory equipment ideal for SFE studies usually ranges from tens of cm3 to several liters. It is used to study the relationship between operating conditions (pressure, temperature, solvent flow rate, and extraction time, and eventually modifier concentration in the solvent) and material pretreatment on one side and extraction yield and extract composition on the other side, and also to optimize SFE. To scale up the process, detailed knowledge is required. The knowledge based on laboratory experiments is condensed in mathematical models, which are again tested by comparison with a wide range of experimental data. Thus, the knowledge of the process is gradually improved as a result of interaction between experimental work and theory.

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Functional Food Ingredients and Nutraceuticals  y = y (H, t) Q

h=H

V

h=0

y=0

 Q

FIGURE 3.1 Schematic drawing of extractor, extraction bed of volume V and height H with · solvent flow rate Q and fluid phase concentration y.

3.2 EXTRACTION BED AND THE CONCEPT OF BROKEN AND INTACT CELLS The extractor is a high-pressure cylindrical vessel usually vertically positioned, as shown schematically in Figure 3.1. The solvent flows through a fixed bed formed by solid particles and is gradually saturated with extracted substances. Let us assume for the sake of simplicity that only one component (or pseudocomponent consisting of more substances that behave similarly) is extracted. Solid phase thus consists of the solute and insoluble residuum called matrix, and fluid phase is a solution of the solute in the solvent. Different models for SFE based on solute mass balance for solid and fluid phase are available in literature. The mass balance is in the form of partial or ordinary differential equations and contains a term for mass flux from solid to fluid phase, which again is a function of equilibrium relationship between the solid and fluid phase. Solution to the equations yields time-dependent concentration profiles, and the resulting fluid phase concentration at extractor outlet serves for calculation of extraction yield. Different models for SFE from plant materials are based on different assumptions on phase equilibrium, mass transfer in solid phase, and flow pattern. The existence of different models is justified by differences in many aspects of extraction process like the type of plant tissue and its permeability, solute location in the tissue, extent of solute-matrix interaction, flow distribution, and flow direction in the extraction bed. The underlying practical problem is how to select the proper model to describe the concrete extraction process as closely as possible. In this chapter we discuss the main aspects of SFE from plant materials utilizing a generalized model1 enabling us to refer to different types of phase equilibrium,

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flow pattern, and mass transfer. The model belongs to a group of SFE models based on the concept of broken and intact cells of disintegrated plant tissue.2–5 The concept results from a well-known fact that SFE from plant materials can be split into two periods. The faster extraction in the first period is controlled mainly by phase equilibrium, while the slower extraction in the second period is controlled mainly by intraparticle diffusion. During grinding of the botanic material, outer layers of the formed particles are mechanically damaged. Reverchon and coworkers3–5 published scanning electron microscope images of disintegrated plant materials, showing that the particle surface consists solely of broken cells. (The term “cell” in SFE models is not used in the same context as in botany; it refers to a cavity containing solute.) According to the model, the solute in the broken cells becomes easily accessible for extraction. Its diffusion to the bulk of fluid phase is fast due to favorable transport properties of supercritical solvents. Walls of intact cells in the particle core, on the other hand, are not easily permeable and the solute diffusion from the core is therefore orders of magnitude slower than the diffusion from broken cells. Thus, the first extraction period corresponds to the extraction from broken cells, and the solute from intact cells is extracted in the second period. Reverchon and co-workers assumed that broken cells may form a monolayer on a particle surface and by surface image they were able to estimate the total volume of broken cells in particles. They also assumed that the broken cells, in contrast to the cells forming the particle core, were initially completely filled with solute. Such assumptions correspond well to the extraction from materials where the solute is not evenly distributed. For example, in the leaves of some plant materials essential oil is concentrated on the leaf surface in the glandular trichomes.6 Ground seeds or roots, however, have more or less homogeneous solute distribution in particles. In order to simulate sufficiently large amounts of easily accessible solute in homogeneous particles, a deeper region of broken cells consisting of many layers must be assumed in the model. The model with homogeneous initial solute distribution in a particle,1 where the fraction of particle volume filled with broken cells, r, equals the fraction of solute located initially in broken cells, is discussed below. For r = 0, all solute is initially closed in intact cells and the model is identical with conventional SFE models that do not distinguish between broken and intact cells; for r = 1, all solute is easily accessible and the model is identical to the models for extraction from a particle surface.

3.3 MASS BALANCE AND MODEL EQUATIONS FOR PLUG FLOW 3.3.1 MASS BALANCE

FOR

SOLVENT

AND

MATRIX

The mass of solvent, M, and the mass of matrix, N, in the extraction bed are N = V (1 − ε ) ρs

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where V is the extraction bed volume, ε is the void fraction, ρs is the solid phase density, and ρf is the solvent density. The matrix consists of broken cells of mass rN and intact cells of mass (1 - r)N. Solvent-to-matrix mass ratio in the extraction bed is γ=

ερ f M = N (1 − ε ) ρs

(3.2)

Let us assume that the solvent flow through the extraction bed is uniform, with no axial mixing (plug flow). The solvent residence time in the extraction bed, the time of contact with the botanic material, is tr =

M γ H = = Q q u

(3.3)

· where u is the interstitial velocity, Q is the mass flow rate, q· is the specific mass flow rate, and H is the bed height.

3.3.2 MASS BALANCE

FOR

SOLUTE

It is convenient to express solute concentrations as solute-to-matrix and solute-tosolvent mass ratios x and y, respectively. In particles we further distinguish the mean concentration in a particle core consisting of intact cells, xc, and the concentration in broken cells, xb. Solute mass balance is

Nxu =

N (1 − r ) H

H

∫

xc ( h, t ) dh +

0

Nr H

H

∫

xb ( h, t ) dh +

0

M H

H

∫ y ( h, t ) dh + 0

(3.4)

t

∫

+ Q y ( h = H , t )dt 0

where xu is the concentration in untreated botanic material, the first term on the right-hand side stands for mass of solute in the intact cells, the second term for mass of solute in broken cells, the third term is for solute in fluid phase inside the extraction bed, and the fourth term is the mass of extract, E t

∫

E = eN = Q y ( h = H , t ) dt 0

© 2007 by Taylor & Francis Group, LLC

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Solvent starts flowing out from the extractor at t = 0. To examine the relationship between concentrations and mass transfer rate, the mass balance is written in its differential form for unit bed volume as ρs (1 − ε ) (1 − r )

∂xc ∂x ∂y   ∂y + ρs (1 − ε ) r b + ρ f ε  + u  = 0  ∂h  ∂t ∂t ∂t

(3.6)

together with mass balance for broken cells and a particle core that are ρs (1 − ε ) r

∂xb = jc − j f ∂t

ρs (1 − ε ) (1 − r )

∂xc = − jc ∂t

(3.7)

(3.8)

where jf is the mass flux from broken cells to fluid phase and jc is the mass flux from intact cells to broken cells, per unit volume of extraction bed. Mass flux jf is driven by difference between concentrations in the bulk fluid phase and at a particle surface, y+

(

j f = k f a0 ρ f y + − y

)

(3.9)

where kfa0 is the volumetric fluid phase mass transfer coefficient. As mass transfer resistance in broken cells is neglected in the model, solid phase concentration in the whole region of broken cells is assumed to be in equilibrium with y+ y + = y + ( xb )

(3.10)

Specific surface area a0 is the surface area per unit volume of the extraction bed. It is expressed with help of l, the characteristic particle dimension defined as particle volume divided by particle surface a0 = (1 − ε )

particle surface 1− ε = particle volume l

(3.11)

Mass flux jc is driven by concentration difference between intact and broken cells: jc = kc ac ρs ( xc − xb )

(3.12)

The specific surface area of particle cores, ac, is calculated according to the following relationship:

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ac = (1 − ε ) =

core surface corre volume = (1 − ε ) particle volume particle volume

core surface = core voolume

(1 − ε ) (1 − r ) lc (3.13)

where lc is the characteristic particle core dimension defined as the core volume divided by core surface. As the mass transfer resistance in fluid phase is very low, part of the solute is transferred from the broken cells to the solvent during extractor pressurization. Therefore, at t = 0 the concentration in the broken cells, xb0, is lower than xu, while the fluid phase concentration, y0, is higher than zero. As these concentrations were established in the period of “static extraction” without solvent flow, they are independent of axial coordinate h. Due to the low mass transfer resistance we further assume that equilibrium between the broken cells and the fluid phase is established at t = 0. On the other hand, the period of pressurization is too short for the concentration in the intact cells to change significantly. Thus, the initial and boundary conditions are assumed as follows xu − xb 0 =

γ y0 r

y0 = y + ( xb 0 ) xc 0 = xu y ( h = 0, t ) = 0

(3.14)

Equation 3.6 through Equation 3.10 and Equation 3.12 and Equation 3.14 represent a complete mathematical model for concentration profiles in the extraction bed with solvent plug flow. The equations are integrated numerically and extraction yield, e, is calculated from the outlet fluid phase concentration y(h = H, t) according to Equation 3.5. To fit the model to the experimental data, it is not necessary to rely solely on the numerical solution; a preliminary analysis of the extraction data based on time constants is possible.

3.3.3 TIME CONSTANTS

AND

DIMENSIONLESS MODEL EQUATIONS

Each individual process component has its own time constant, independent of the · others. Thus, the solvent flow is characterized by a mean residence time tr = M/Q (see Equation 3.3). The external mass transfer time is

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tf =

ε ε l = k f a0 1 − ε k f

83

(3.15)

and the intraparticle diffusion time is tc =

(1 − ε ) (1 − r ) = kc ac

lc kc

(3.16)

Finally, the equilibrium extraction time te, which is the time necessary to extract all solute that was initially in the broken cells when the external mass transfer resistance is negligible, is represented by te =

rxu y0 q

(3.17)

If the mean residence time tr is regarded as a characteristic time, then the extraction time t and the time constants can be expressed as the dimensionless time τ and dimensionless model parameters, respectively, as follows τ=

Θf =

t tr

tf lqε = t r k f γ (1 − ε )

Θc =

t c lc q = t r kc γ

Γ=

t r γy0 = t e rxu

(3.18)

Dimensionless parameters Θf and Θc are the external and intraparticle mass transfer resistances, respectively, and Γ compares the amount of solute in fluid phase at equilibrium at t = 0 with the amount of solute present originally in the broken cells. Introducing these parameters and dimensionless variables Xb = xb/xu, Xc = xc/xu, Y = y/y0, z = h/H, the following dimensionless model equations are constituted after rearrangement τ

∫

e xu = r Γ Y ( z = 1, τ ) d τ 0

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∂Y ∂Y + = Jf ∂τ ∂z

(3.6a)

∂Xb 1 − r = J c − ΓJ f ∂τ r

(3.7a)

∂Xc = − Jc ∂τ

(3.8a)

Jf =

(

1 Y+ −Y Θf

)

Y + = Y + ( Xb ) Jc =

1 ( X c − Xb ) Θc

(3.9a)

(3.10a)

(3.12a)

Xb0 = 1 − Γ , Y0 = 1 Xc0 = 1 Y ( z = 0, τ ) = 0

(3.14a)

The dimensionless extraction yield, e/xu, is in the range 0 to 1. Different algorithms for solution of partial differential equations Equation 3.6a through Equation 3.8a are available. We have chosen to transform the partial differential equations to 3n ordinary differential equations written for a series of n identical ideal mixers of dimensionless height 1/n, followed by their integration using the Runge-Kutta method. The complete set of model equations for n mixers is τ

∫

e xu = r Γ Yn d τ

(3.5b)

0

dY j + n (Y j − Y j −1 ) = J fj dτ

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dXbj 1 − r J cj − J fj = dτ r

(3.7b)

dXcj = − J cj dτ

(3.8b)

J fj =

(

1 Y j+ − Y j Θf

)

Y j+ = Y j+ ( Xbj ) J cj =

1 ( Xcj − Xbj ) Θc

(3.9b)

(3.10b)

(3.12b)

Xb 0 j = Xb 0 = 1 − Γ Y0 j = Y0 = 1 Xc 0 j = Xc 0 = 1 Y j−1 ( τ ) = 0 for j = 1,2,...,n

(3.14b)

The higher n is, the closer the model simulates plug flow. The applications to SFE of different substances are discussed below.

3.4 SFE OF OILS FROM SEEDS 3.4.1 OIL SOLUBILITY

IN

SC-CO2

Vegetable oils in seeds serve as a store for energy for future plant use, and their mass fractions in seeds are therefore high, typically 10 to 50% wt. Common vegetable oils are composed of triglycerides of unsaturated and saturated fatty acids. In the extraction of C16-C18 vegetable oils, the oil can be regarded as a pseudocomponent, where its solubility in SC-CO2 is predicted according to the equation

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(

c = ρ10f .724 exp − 18708 T + 40.361 + 2186840 T 2

)

± 2.7

(3.19)

developed by del Valle and Aguilera.7 The symbols are c (kg/m3) for the solubility, ρf (g/cm3) for the density of pure CO2, and T (K) for the temperature. The equation, based on solubility data for soybean oil, sunflower oil, cottonseed oil, and corn oil, was validated for temperatures ranging from 20°C to 80°C, the pressures between 15 and 89 MPa, and solubility below 100 kg/m3. Several solubility isotherms calculated according to the equation are depicted in Figure 3.2 in terms of solute-tosolvent mass fraction ys = c/ρf. According to Equation 3.19, the solubility increases with increasing solvent density, and, at constant solvent density, also with increasing temperature. A crossover region is obvious at lowest pressures where the solubility values decrease at constant pressure with increasing temperature. It is a result of a rapid drop in solvent density with increasing temperature near the critical point. Equation 3.19 has been successfully applied to many vegetable oils where C18 fatty acids are dominant. Only the solubility of vegetable oils rich in free fatty acids is underestimated because the free fatty acids are more soluble in CO2 than their triglycerides. Lee et al.9 examined the equilibrium relationship y+ = y(xb) for oil extracted with CO2 from flaked canola seeds at 55C and 36 MPa. They prepared samples of partially extracted flakes with oil concentration ranging from 0.2–0.7 g/g oil-free seed and measured the concentration of oil in the solvent phase in equilibrium with seeds for each sample. The concentration was 0.011 ± 0.001 g/g CO2, which is close to ys = 0.012 g/g CO2 calculated according to Equation 3.19, for all samples. Thus, 0.1 20°C 0.08

40°C

ys , kg/kg

60°C 0.06

80°C

0.04

0.02

0

15

25

35

45 55 Pressure, MPa

65

75

85

FIGURE 3.2 Solubility of C18 vegetable oils in dense CO2 in dependence on pressure and temperature according to del Valle and Aguilera7; CO2 density calculated with Altunin and Gadetskii equation.8

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TABLE 3.1 Calculation of Dimensionless Model Parameters for Extraction of a Solute from Spherical Particles; Distribution Coefficient K Is Valid in the Case of Solute-Matrix Interaction Physico-Chemical Parameters xu d, mm ε γ q,· kg/kg min r K kf, m/s kc, m/s

Dimensionless Model Parameters

0.025 0.60 0.40 0.50 0.125 0.60 0.24 1.11 · 10–3 2.56 · 10–5

r 0.60 Γ 0.1667 Θf 0.25 Θc 12.0 – K 1.20 — — Initial concentrations xb0 0.02083 y0 0.005

Equations — Equation Equation Equation Equation — — Equation Equation

3.18 3.11 and Equation 3.18 3.13 and Equation 3.18 3.28

3.14 and Equation 3.27 3.37

over the range of seed oil concentrations used, the seed tissue has no physical or chemical affinity to oil and acts merely as an inert substrate. The equilibrium concentration of oil in the solvent phase is therefore assumed to be constant as long as any oil is present in the broken cells y+ = ys for xb > 0, y+ = 0 for xb = 0

(3.20)

We shall further use the term “constant solubility” for equilibrium described by Equation 3.20. Dimensionless form of the equation for j-th mixer is Y j+ = 1 for Xbj > 0 , Y j+ = 0 for Xbj = 0

3.4.2 MODEL

FOR

(3.21)

VEGETABLE OIL EXTRACTION

To illustrate the dependence of concentration profiles and the extraction curve on the type of phase equilibrium, we have chosen a basic set of model parameters, r = 0.6, Γ = 0.167, Θf = 0.25, and Θc = 12 (see Table 3.1). To simulate SFE of vegetable oils with plug flow, model Equation 3.5b through Equation 3.9b plus Equation 3.12b and Equation 3.14b were numerically integrated together with Equation 3.21 using the basic set of parameters and n = 40; the results are shown in Figure 3.3. The depicted solid phase concentration is calculated as mean concentration in particle X = rXb + (1 - r)Xc. The concentration profiles in both phases gradually shift to the right-hand side of the figure, in the direction of solvent flow. As long as the fluid phase concentration at solvent outlet remains constant, the extraction curve e/xu versus τ is a straight line. When the extraction of solute from broken cells is complete, mass transfer from particles continues much slower and concentration profiles flatten; the slope of the extraction curve decreases proportionally with the decrease in

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Y

0.6 0.4 0.2 0

0

0.2

0.4

0.6

0.8

1

0.6

0.8

1

z 1 0.8

X

0.6 0.4 0.2 0

0

0.2

0.4

z 1 0.8

e/xu

0.6 0.4 0.2 0

0

10

20 t

30

40

FIGURE 3.3 Simulated concentration profiles and extraction curve for plug flow and constant solubility. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, n = 40. Fluid phase concentration profiles Y(z) and solid phase concentration profiles X(z) shifting to the right and decreasing: (_______) solution of complete model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40. Extraction yield e/xu: (_______) solution of complete model equations; (+ + + +) approximate sections of extraction curve according to Equation 3.24 and Equation 3.25.

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89

outlet fluid phase concentration. The transition from equilibrium-controlled period to diffusion-controlled period occurs at extraction yield higher than the yield of easily accessible solute rxu . The reason for the increase is that a certain amount of solute from the intact cells is transferred into the broken cells and becomes easily accessible while the solute deposited initially in broken cells is transferred into the fluid phase.

3.4.3 EQUILIBRIUM-CONTROLLED PERIOD Several researchers assume constant solubility and plug flow in order to model the first period of oil extraction with SC-CO2. Lee et al.9 solved the mass balance equations for oil extraction from rape seed flakes numerically and adjusted external mass transfer coefficient kfa0 to experimental data with the result that kfa0 was proportional to u0.54. Brunner10 pointed out that oil extraction is a steady-state process as long as any easily accessible oil remains at the solvent inlet, where it is depleted first. (It can be shown that the steady-state extraction lasts until τ = τ1 = Θf(1 – Γ)/Γ. Thus, in our example depicted in Figure 3.3, τ1 = 1.25.) During the steady-state extraction, both fluid phase concentration profile and extraction rate are constant and the mean driving force in the extraction bed ys – ymean is related to measurable quantities ys and y(h = H) according to ys − ymean =

y (h = H ) ys ln ys − y ( h = H )

(3.22)

Taking into account that in a steady-state period ∂y/∂t = 0, volumetric mass transfer · coefficient kfa0 can be determined from mass flux Vkfa0ρf (ys – ymean) = Qy(h = H). Ferreira et al.11 applied this procedure to evaluate the external mass transfer coefficient with experimental data for black pepper with SC-CO2 at 15–30 MPa and 30C –50C. The botanic material was ground to a mean size of approximately 0.1 mm, and the interstitial solvent velocity, u, was in the range of 0.2–0.9 mm/s. Mass transfer coefficient kf , increasing with increasing interstitial velocity, was found to be independent of pressure and temperature and its value was between 3 × 10–4 and 9 × 10–4 m/s. In terms of the present model, Equation 3.22 corresponds to a steady-state fluid phase concentration profile  z  Y = 1 − exp  −  Θ f 

(3.23)

(depicted in Figure 3.3a) for τ = 1, and the dimensionless extraction yield is based on the following relationship

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  1 e = Γr τ 1 − exp  − xu  Θf 

   

(3.24)

The exponential term for Θf < 0.22 is lower than 0.01. For these values of Θf , the solvent flowing out from the extraction bed is practically saturated with solute. After exhaustion of the easily accessible solute from particles at the solvent inlet, that is, when xb(z = 0) becomes zero, the fluid phase concentration profile starts shifting toward the solvent outlet and the overall extraction rate gradually slows down.

3.4.4 DIFFUSION-CONTROLLED PERIOD In the second part of the extraction curve the fluid phase concentration is much lower than the solute solubility in the solvent. If the assumption on equilibrium at a particle surface and constant solubility holds, the solute that arrives from the particle core to its surface dissolves immediately in the solvent and the concentration in broken cells becomes practically zero. The driving force for internal diffusion according to Equation 3.12 is then equal to xc and the extraction yield in the second period is approximated by the following equation e  τ − τc  = 1 − (1 − r ) exp  −  xu Θ c 

(3.25)

where the shift in time, τc > 0, is related to the fact that the driving force for solute transfer from the intact to broken cells was decreased in the first extraction period by the nonzero concentration in the broken cells. As shown in Figure 3.3, the extraction yield calculated using the complete model equations and the yield according to Equation 3.25 with τc adjusted to 3.75 overlap in the second extraction period.

3.4.5 APPROXIMATE SOLUTION

FOR

BOTH PERIODS

An approximate model with plug flow based on the concept of broken and intact cells was derived by Sovová2 as an extension of Lack’s model. The model simulates both concentration profiles and extraction yield, which is given as   1 e = Γr τ 1 − exp  − xu  Θf 

 Θf   for τ ≤ τ m = Γ 

(

)

   exp  τ − τ m Θc  − 1 + r e  = r Γτ − Θ f exp  r ΓΘc ln − 1  xu r     for

© 2007 by Taylor & Francis Group, LLC

  Θ f    

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τ < τn =

91

 Θf  1  + Θ c ln  1 − r + r exp  ,  r ΓΘ c   Γ 

 e  1    τ − τ m    − 1 expp  − = 1 − r ΓΘ c ln 1 + (1 − r )  exp     r ΓΘ c    Θ c   xu   for τ ≥ τn

(3.26)

The steady-state extraction period according to Equation 3.26 finishes when τ = τm. Then the fluid phase concentration profile starts shifting through the extraction bed. At time τ = τn the last easily accessible solute is transferred from the broken cells to fluid phase and the extraction is further controlled solely by intraparticle diffusion. A model2 was derived for γ 0 where the solute accumulation in the fluid phase was not taken into account. Therefore a discrepancy exists between extraction curves calculated according to Equation 3.26 and those obtained by numerical solution to the complete model equations, as illustrated in Figure 3.4 for the basic set of parameters r = 0.6, Γ = 0.167, Θf = 0.25, and Θc = 12. To simulate precisely the extraction curve calculated with the complete model, the approximate parameters would have to be adjusted to r = 0.697, Γ = 0.211, Θf = 0.917, and Θc = 11.6. Thus, the approximate model is useful to smooth the experimental data and obtain the first estimation of model parameters, which, however, should be refined by further calculations. As the approximate model does not involve solute-matrix interaction, it should not be applied for the system where solute interacts with matrix. 1 0.8

e/xu

0.6 0.4 0.2 0

0

10

20 τ

30

40

FIGURE 3.4 Comparison of extraction curves calculated for constant solubility, plug flow, and model parameters r = 0.6, Γ = 0.167, Θf = 0.25, Θc= 12, and n = 40. (_______) solution of complete model equations; (+ + + +) approximate model2 with analytical solution.

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3.5 SFE OF ESSENTIAL OILS 3.5.1 SOLUTE-MATRIX INTERACTION Essential oils as mixtures of volatile and predominantly low-polar compounds are more soluble in SC-CO2 than vegetable oils. Their main components, terpenes and oxygenated terpenes, are fully miscible with SC-CO2 at temperatures of 40°C –50°C and pressures of 10–20 MPa, as Reverchon states in a comprehensive review on SFE of essential oils.12 Nevertheless, when essential oils are extracted with SC-CO2 from leaves or other botanic materials where their concentrations are only a few percent or less, their equilibrium fluid phase concentration is much lower than that measured in absence of tissue matrix. For example, Goto et al.13 observed that the equilibrium fluid phase concentration of menthol, a major component in peppermint essential oil, during SFE from peppermint leaves, was two orders of magnitude lower than the solubility of pure menthol in SC-CO2. To explain how the matrix affects the essential oil equilibrium, the authors assumed that the essential oil is adsorbed on the lipids present in the leaves and thus the experimental equilibrium concentration is represented by an adsorption isotherm. To describe the solute-matrix interaction they applied the simplest and the most frequently used relationship for SFE, linear equilibrium with partition coefficient K y + = Kxb

(3.27)

Its dimensionless form for j-th mixer is Y j+ = KXbj , K = 1 (1 − Γ )

3.5.2 SIMULATION

OF

(3.28)

ESSENTIAL OIL EXTRACTION

Model Equation 3.5b through Equation 3.9b plus Equation 3.12b and Equation 3.14b together with Equation 3.28 were numerically integrated for the basic set of model parameters and n = 40. Due to the linear equilibrium, completely different concentration profiles from vegetable oil extraction were obtained, as shown in Figure 3.5. As the solid phase concentration gradually decreases during the extraction process, the equilibrium concentration in fluid phase at a particle surface also decreases likewise the driving force y+ - y. As a result, the concentration profiles corresponding to solute-matrix interaction are flatter and the extraction becomes slower than in the case without solute-matrix interaction.

3.5.3 TWO EXTRACTION PERIODS The first and second extraction periods cannot be completely separated as it was in the case without solute-matrix interaction. On one hand, the solute diffusing from the intact to broken cells during the first period increases the concentration in broken cells and therefore also the equilibrium fluid phase concentration. On the other hand, as the concentration in the broken cells is directly proportional to the fluid phase

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1 0.8

Y

0.6 0.4 0.2 0

0

0.2

0.4

0.6

0.8

1

0.6

0.8

1

z 1 0.8

X

0.6 0.4 0.2 0

0

0.2

0.4 z

1 0.8

e/xu

0.6 0.4 0.2 0

0

10

20 τ

30

40

FIGURE 3.5 Simulated concentration profiles and extraction curve for linear equilibrium and plug flow. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, and n = 40. a) Decreasing fluid phase concentration profiles Y(z) and solid phase concentration profiles X(z): (_______) solution of complete model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40. Extraction yield e/xu: (_______) solution of complete model equations; (+ + + +) approximate sections of extraction curve according to Equation 3.24 and Equation 3.29.

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concentration at a particle surface, it is neither zero nor constant in the second extraction period. Nevertheless, at least the initial slope of the extraction curve can be estimated according to Equation 3.24 as in the case of constant solubility (Figure 3.5). The extraction in the second extraction period is slower than that derived for constant solubility with Equation 3.25, because the nonzero concentration xb reduces the driving force. An acceptable estimate for extraction yield in the second period is given in the following equation1  τ − τc e = 1 − (1 − r ) exp  − xu  Θ c 1 + (1 − r ) K ΓΘ c r 

(

)

    

(3.29)

The agreement of Equation 3.29 with the results obtained by solution of complete model equations for the basic set of model parameters is shown in Figure 3.5.

3.6 SFE FOR COMBINED EQUILIBRIUM 3.6.1 EQUILIBRIUM ACCORDING

TO

PERRUT

ET AL.

A versatile SFE model would combine the constant solubility and linear equilibrium in one expression. Two papers independently solving this task were published almost simultaneously. Goto et al.14 suggested the BET adsorption isotherm which is often used to describe adsorption equilibria. The BET isotherm simulates a smooth increase in equilibrium fluid phase concentration with increasing solid phase concentration, from y+ determined solely by solute-matrix interaction at lowest solid phase concentrations to y+ asymptotically approaching ys at high solid phase concentrations. The other equation, proposed by Perrut et al.15 as a simplified description of phase equilibrium during SFE from plant materials, defines the equilibrium fluid phase concentration as a discontinuous function of solid phase concentration as depicted in Figure 3.6 y ys

b

a

c

KXf d Xf

X1

FIGURE 3.6 Combined equilibrium relationship with constant solubility for high solid phase concentrations and with linear equilibrium for low solid phase concentrations according to Perrut et al.15

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y + = ys for xb > xt

y + = Kxb for xb ≤ xt ,

95

(3.30)

where ys > Kxb. The sudden fall in equilibrium concentration y+ at xb = xt can be explained as follows. Matrix has certain capacity of interacting with solute, as expressed by transition concentration xt. As long as the solid phase concentration is less than or equal to xt, all solute in solid phase interacts with matrix and the equilibrium fluid phase concentration is determined by the distribution coefficient K. When the solid phase concentration is higher, part of solute remains free, unbound to the matrix, and its equilibrium fluid phase concentration equals solute solubility. According to this simplified model, when SFE starts at xb0 > xt, solid phase concentration gradually decreases to xt and when it reaches this value, the equilibrium fluid phase concentration at the particle surface falls from ys to Kxt, below the fluid phase concentration in the diffusion layer close to the surface. Mass transfer from the broken cells to fluid phase is interrupted until the fluid phase concentration drops below y = Kxt. In the meantime, the solute is only washed out

(

j f = k f a0 ρ f y + − y

)

for xb ≠ xt or xb = xt , y < Kxt , j f = 0 otherwise. (3.31)

Equation 3.30 includes both equilibrium relationships mentioned above: for xt = 0 it is identical with Equation 3.20 for constant solubility, and for xt > xb0 it describes linear equilibrium according to Equation 3.27. It seems that any solute can occur both in free form and bound to the matrix, depending on its solid phase concentration and on matrix and solvent properties. Indeed, Perrut et al.15 observed the combined equilibrium during SFE of vegetable oil from seed, where constant equilibrium would be expected. When the extraction with plug flow and combined equilibrium (Equation 3.30) starts in section “a” of the equilibrium curve (Figure 3.6), the whole period of solute extraction from the broken cells is governed by constant solubility and the solutematrix-interaction does not influence the course of the extraction. When the extraction starts in section “b,” the outlet concentration firstly approaches the solubility, then the equilibrium value at transition concentration xt, and finally the intraparticle diffusion-controlled extraction follows. When the extraction starts in section “c,” the amount of free solute present in the extraction bed at t = 0 is not sufficient to saturate the solvent and thus the initial outlet concentration is lower than solubility; as soon as the initial solution is washed out of the extractor, only the solute interacting with matrix is extracted. Finally, when the extraction starts in section “d,” all solute interacts with matrix, as described in Section 3.5. In the dimensionless model for a series of mixers, Equation 3.30 reads as follows Y j+ = 1 for Xbj > Xt , Y j+ = KXbj for Xbj ≤ Xt

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– – where Xt = xt/xu and K = Kxu/y0. Section “a” is characterized by 1 – Γ ≥ 1/K, section “b” by Xt < 1 – Γ < 1/ K , section “c” by Xt = 1 – Γ and K < 1 (1 − Γ ) , and section “d” by Xt ≥ 1 – Γ and K = 1 (1 − Γ ) .

3.6.2 SIMULATION

OF

SFE

WITH

COMBINED EQUILIBRIUM

Model Equation 3.5b through Equation 3.9b plus Equation 3.12b and Equation 3.14b, together with Equation 3.32 were numerically integrated with the basic set of the same parameter values and number of mixers as in the case of constant equilibrium (r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, and n = 40), using two more – equilibrium parameters Xt = 0.48 and K = 0.5. Thus, the extraction starts in section “b.” The results shown in Figure 3.7 apparently indicate an extraction with constant equilibrium where the second, diffusion-controlled period starts as soon as the dimensionless solid phase concentration in the broken cells drops to Xt. Further analysis, however, will show that the shape of the extraction curve starting in section “b” is different from that of constant equilibrium.

3.6.3 TWO EXTRACTION PERIODS The first extraction period consists of two sections of different slopes. If the external mass transfer resistance is neglected for the sake of simplicity, the shape of the extraction curve in the first extraction period is calculated as1 e 1 − Γ − Xt = Γr τ for τ ≤ τ1 = 1 + xu Γ 1 − KXt

(

e = Γr  τ1 + KXt ( τ − τ1 )  for τ >τ1 xu

) (3.33)

In the second, diffusion-controlled period, the solute interacting with matrix in broken cells is practically in equilibrium with the solute in fluid phase, and the extraction curve is approximately described by Equation 3.29 (unless Xt = 0, when Equation 3.25 should be applied). As Figure 3.7 shows, the agreement of estimated extraction curve sections with complete extraction curve is good.

3.7 FLOW PATTERNS 3.7.1 AXIAL DISPERSION In the previous sections we have shown how phase equilibrium and diffusion resistance affect the shapes of concentration profiles and the extraction curve. Another aspect that should be taken into account and which is often omitted is the flow pattern in the extraction bed. To express deviations from plug flow, Goodarznia and Eikani16 inserted an axial diffusion term into the differential mass balance equation

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1 0.8

Y

0.6 0.4 0.2 0

0

0.2

0.4

0.6

0.8

1

0.6

0.8

1

z

1 0.8

X

0.6 0.4 0.2 0

0

0.2

0.4 z

1 0.8

e/xu

0.6 0.4 0.2 0

0

20

40 τ

60

80

FIGURE 3.7 Simulated concentration profiles and extraction curve for plug flow and com– bined equilibrium. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, Xt = 0.48, K = _______ 0.5 concentration profiles X(z) shifting to the right and decreasing: ( ) solution of complete model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40. Extraction yield e/xu: (_______) solution of complete model equations; (- - - -) approximate equilibrium-controlled section according to Equation 3.33; (+ + + +) approximate diffusion-controlled section according to Equation 3.29.

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ρs (1 − ε ) (1 − r )

 ∂y ∂xc ∂x ∂2 y  ∂y − Dax 2  = 0 + ρs (1 − ε ) r b + ρ f ε  + u ∂h ∂t ∂t ∂h   ∂t (3.34)

where Dax is the axial dispersion coefficient. Reis-Vasco et al.6 applied Equation 3.34 on SC-CO2 extraction of mint oil from leaves and found out that Equation 3.34 yields a better representation of experimental extraction curves than the plug flow model. The axial dispersion values adjusted to fit experimental data were decreasing with increasing flow rate and were about one order of magnitude larger than those obtained from the correlations found in literature. Nevertheless, the authors regarded the introduction of an additional adjustable parameter into the model as too high a cost for slightly better accuracy and therefore selected the plug flow model as the most useful one. Another possibility to express axial dispersion in extraction bed is decreasing the number of mixers in the model Equation 3.5b through Equation 3.14b. Though the changes in the degree of axial dispersion are not continuous as according to Equation 3.34, it is possible to cover a wide range of flow patterns from plug flow for n ∞ up to ideal mixer for n = 1, which is discussed in the next section.

3.7.2 MODELS

FOR

DIFFERENTIAL EXTRACTION BED (IDEAL MIXER)

The models published for SFE from plant materials by Goto et al.13,14 consider a differential extraction bed where no concentration gradient is assumed. Thus, the differential extractor is described by ordinary differential equations for an ideal mixer (Equation 3.6b through Equation 3.8b for n = 1). Such models are simpler than the plug flow models and some of them have analytical solutions. They would be ideal for SFE application in short extraction beds or when strong axial dispersion exists in fluid phase. Compared to plug flow, the extraction in an ideal mixer is slower in the first extraction period due to the lower driving force (Figure 3.8). The following equation

y=

y+ 1+ Θf

and

Γr τ e = xu 1 + Θ f

(3.35)

holds for the initial steady-state period. The effect of external mass transfer resistance on outlet concentration and the extraction rate is more pronounced than in the case of plug flow (Equation 3.24).

3.7.3 NATURAL CONVECTION The best extractor performance is achieved with a plug flow. Unfortunately, supercritical fluids are extremely prone to natural convection, due to their low kinetic viscosity. Such convection can be induced by solvent density differences caused by the differences in local solute concentrations. The occurrence of natural convection

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1 0.8

e/xu

0.6 y + = ys

0.4 0.2 0

0

10

20 τ

30

40

1 0.8

e/xu

0.6 y+ = Kxb

0.4 0.2 0

0

10

20

30 τ

40

50

60

1 0.8

e/xu

0.6 y+ = ys for xb > xt y+ = Kxb for xb ≤ xt

0.4 0.2 0

0

20

40 τ

60

80

FIGURE 3.8 Effect of flow pattern on extraction curves for three types of equilibrium. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, n = 40, and for combined equilibrium Xt – = 0.48 and K = 0.5. (_______) plug flow; (– – – –) ideal mixer; (- - - - -) two parallel flows with unc = 0.75u.

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depends also on the direction of solvent flow through the extraction bed, as Beutler et al.17,18 discovered. They observed this phenomenon in SC-CO2 extraction of different solutes, such as black pepper oleoresin or fat, from botanical matrix. When the flow was from the top to the bottom, the fat extraction yield was twice as high as for the opposite flow direction. The authors explained this phenomenon by higher density of solute-laden CO2 than that of pure solvent; the solute-laden solvent can be more easily withdrawn from the bottom of the extractor than from its top. The better withdrawal of solute leads to higher concentration gradients in the solid phase and thus to a higher mass transfer driving force. Barton et al.19 observed the effect of flow direction when they extracted peppermint oil with dense CO2. Though the extraction yields by downflow CO2 reached 90 to 98% of those extracted by steam distillation, the yield of peppermint oil in the CO2 upflow mode was only 76%, even at higher solvent treatments and extraction times. Also, Sovová et al.20 who extracted grape seed oil with SC-CO2 observed retardation in extraction when the flow direction was switched to the upflow mode; the effect was more pronounced for lower interstitial velocities. Equation 3.26 was used to simulate experimental data. While a simple plug flow model was adequate for SFE in downflow mode, the natural convection in upflow mode was simulated assuming parallel flows of different velocities in the extraction bed. The simplest application of this approach is splitting the bed into halves with interstitial velocities u + unc and u – unc. The resulting extraction curves calculated for the basic set of model parameters with unc = 0.75u are compared with the results of the simple plug flow in Figure 3.8; the effect of different velocities is most pronounced for constant solubility. The existence of natural convection in a fixed bed where supercritical solvent flows slowly in the direction to the top was confirmed by Dams21 and Stüber et al.22, who dissolved in dense CO2 solid substances from porous particles. Dams observed the natural convection indicated by flattening of solid phase concentration profiles. Stüber et al. measured extraction curves for a wide range of extraction conditions; to simulate the results they applied the model for a differential bed and included the effects of gravity-opposing flow (upflow) and gravity-assisting flow (downflow) on extraction rate into correlations for external mass transfer coefficient.

3.7.4 CHANNELING

AND

SCALE UP

OF

EXTRACTOR

Another unfavorable type of flow pattern occurring in extraction beds is channeling. When the extraction bed is inhomogeneous, the solvent flows preferably through the paths of minimum hydraulic resistance and extracts the solute from their vicinity. Most of the solute in the extraction bed has very limited contact with the flowing solvent and as a result the extraction may even be retarded by orders of magnitude. With an increase in the cross section of the extractor the probability of inhomogeneous particle distribution and the tendency of channeling increase. This tendency depends also on the properties of extracted material; it is large for example when extracted particles are sticky or when they are too small and the extraction bed has therefore large hydraulic resistance. Berna et al.23 published experimental results on SC-CO2 extraction of orange peel essential oil from different cultivars and on

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different scales, showing a large extraction retardation on production scale compared to the pilot scale. When a bed of inert particles of diatomaceous earth was placed in the extractor as solvent flow distributor, the difference between extraction yields measured in a small and a large extractor was significantly reduced. We have shown1 that the model for a differential extraction bed can simulate SFE in channeling extractor, when the external volumetric mass transfer coefficient kfa0 is adjusted to unrealistically low values, corresponding to the difficult solute approach from distant particles to flow channels. Prediction of scale-up effects is, however, difficult because there is no rule for predicting the extent of channeling in a larger-scale extraction bed. On the other hand, if the channeling is suppressed by proper measures, and flow pattern in the larger extractor is similar to that in the smaller extractor, the model with equilibrium and mass transfer parameters adjusted to the data measured in the smaller extractor is able to predict well the large-scale extraction.

3.8 PRELIMINARY EVALUATION OF EXTRACTION CURVES 3.8.1 SPLITTING EXTRACTION CURVE

INTO

TWO SECTIONS

Before the SFE is simulated using the complete model equations, model parameters should be roughly estimated from the shapes of extraction curves. The first condition for measurement of “readable” extraction curves is to suppress deviations from plug flow and particularly the channeling. This can be achieved relatively easily in a small-scale extractor, especially when the gravity-assisting solvent flow direction is chosen. In the case without solute-matrix interaction, the equilibrium- and diffusioncontrolled sections of extraction curves are obvious at first sight (Figure 3.3). In the case of solute-matrix interaction the differentiation is more difficult but possible especially when at least two extraction curves measured at identical conditions except for residence times are available. The extraction of easily accessible solute should be close to equilibrium extraction, which is possible due to excellent transport properties of supercritical solvents. Let us assume that the second experiment will be performed at identical solvent flow rate and with twofold solid charge in the extractor. (It is better to maintain the flow rate constant, as its change would affect not only the residence time but also the external mass transfer coefficient.) In our example, extraction yields e were calculated first for model parameters given in Table 3.1 and then for the same parameters except for a doubled residence time. The obtained extraction curves e(q) and e(t) are compared in Figure 3.9. The slopes of e(q) are in the initial, equilibrium-controlled extraction period practically equal to the equilibrium fluid phase concentration until about 11 g/(kg matrix) is extracted. The curves stop overlapping in the following, transition, period controlled by both equilibrium and intraparticle diffusion. Finally, when approximately 21 g/(kg matrix) has been extracted and the extraction is controlled almost solely by intraparticle diffusion, the shapes of both curves e(t) are almost identical, as can be proved by shifting the first extraction curve by a proper time interval as indicated in Figure 3.9. After identification of equilibrium-

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e, g/kg

20

10

0

0

10

20 q, kg/kg

30

40

30

e, g/kg

20

10

0

0

50

100

150 t, min

200

250

300

FIGURE 3.9 Estimation of model parameters from extraction curves e(q) and e(t) measured close to plug flow and with almost saturated solvent in the first extraction period. Solute matrix interaction (linear equilibrium) is assumed. (_______) simulated extraction curve for residence time tr = 4 min, model parameters according to Table 3.1; (- - - -) the same curve shifted by 36 min to the right; (– – – –) simulated extraction curve for residence time tr = 8 min (q· = 0.0625 min-1) and other physico-chemical parameters unchanged; (+ + + +) equilibrium model Equation 3.36.

and diffusion-controlled extraction curve sections, each section can be evaluated independently according to different relationships. It should be taken into account that the models simulate the extraction of one compound or one pseudocompound. When two or more solutes with completely different solubilities and comparable contents in botanic material are extracted and when the only experimental data available is the extraction curve measured for total extract, the curve should be simulated as a superposition of extraction curves for individual components and not as an extraction curve for a single compound.

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3.8.2 EQUILIBRIUM-CONTROLLED PERIOD The equilibrium parameters are estimated by fitting approximate model equations to the first, equilibrium-controlled part, of the extraction curve. The equations given in Sections 3.4.3 and 3.6.3 as functions of dimensionless parameters can be rearranged for Θf → 0 to e = qys = ys qt

(3.36)

for the case without solute-matrix interaction, and to , y0 = e = qy0 = y0 qt

Kxu 1+ Kγ r

(3.37)

for solute-matrix interaction described by linear equilibrium. For combined equilibrium according to Equation 3.30, depicted in Figure 3.6, the shape of the first section of extraction curve depends on where on the equilibrium curve the extraction starts, as we discussed in Section 3.6.1. Equation 3.36 should be applied for the extraction starting in section “a,” Equation 3.37 for the extraction starting in “d,” and the following equation for t ≤ t1 = e = qys = ys qt

r ( xu − xt ) − γKxt , q ( ys − Kxt )

e = q  ys t1 + Kxt ( t − t1 )  for t > t1.

(3.38)

for the extraction starting in section “b.” From the slopes of the curve we can read equilibrium characteristics ys and Kxt, and from t1 we can estimate xt, after r is estimated from the diffusion-controlled part of the extraction curve. The equation for the extraction starting in section “c” is similar to Equation 3.38 but its initial slope e/q is lower than ys. More details on modeling the extraction with combined equilibrium are given elsewhere.1

3.8.3 DIFFUSION-CONTROLLED PERIOD The intraparticle diffusion time is much larger than the time constants for other phenomena affecting SFE and thus the diffusion characteristics can be obtained easily from the second extraction period data. Extraction yield in this period is according to Equation 3.25 and Equation 3.29 approximated by  1− r  t  for y + = Kxb1 . e = xu 1 − C1 exp  −   , C2 = t c for y + = ys , C2 = t c +   C Kq 2   (3.39)

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The content of extractable solute in botanic material, xu, and constants C1, C2 are determined by fitting Equation 3.37 to the experimental extraction curve. The fact that the shape of the extraction curve in the second period is known has practical impact in analytical SFE as it enables the prediction of analyte content in extracted sample, Nxu, from several (at least three) measurements in the second period. If the amount of extract is determined in this period at three time points with the same time intervals, the initial amount of solute in the extraction bed is calculated after rearrangement of Equation 3.39 as

E∞ = Nxu = m1 +

 ( e2 − e1 )2  m22 = N  e1 +  2 e2 − e1 − e3  m2 − m 3 

(3.40)

where m1 = E(t1) is the amount of extract in the first measurement, m2 = E(t2) - E(t1) is the increase in extract in the first time interval and m3 = E(t3) - E(t2) is the increase in extract in the second interval. Nowadays, with computers, a direct fitting of Equation 3.39 to more than three experimental points is recommended instead of the three-point extrapolation, which is extremely sensitive to experimental error. To express the relation between intraparticle diffusion time (tc), effective intraparticle diffusivity (De), and particle size and shape, Reverchon24 used the results of Villermaux25 who examined approximate relationships between diffusion times and particles of different shapes and found the following relationship tc = µ

l2 De

(3.41)

where µ is equal to 3/5, 1/2, and 1/3 for spheres, cylinders, and slabs, respectively, and l is the characteristic particle dimension. Though the model applied by Reverchon and Villermaux does not take into account the existence of broken cells and easily accessible solute in particles, it can be easily applied to the second extraction period when the characteristic particle core dimension, lc, is substituted for l: tc = µ

lc2 De

(3.42)

As Equation 3.16 holds simultaneously, the solid phase mass transfer coefficient is approximately kc =

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De µlc

(3.43)

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Particles are usually assumed to be of spherical shape. Characteristic dimension for spherical particles of diameter d is equal to d/6 and their core characteristic dimension is d (1 − r ) 6

13

lc =

(3.44)

Inserting Equation 3.44 in Equation 3.42 we obtain the intraparticle diffusion time for spherical particles

tc =

d 2 (1 − r ) 60 De

2 3

(3.45)

Equation 3.45 written for r = 0 and modified for simultaneous external mass transfer resistance was applied in SFE from plant materials several times, for example, by del Valle et al.26 Reverchon24 demonstrates with experimental data for essential oil extraction from ground leaves of different particle sizes the importance of correct modeling of particle shape. The internal diffusion time was less dependent on particle size than according to Equation 3.45 derived for spherical particles. When it was taken into account that the extracted particles had a form of slabs whose characteristic dimension is less dependent on particle size and more on the slab thickness, which is not affected by grinding, a good agreement of model calculations with experimental extraction curves was achieved. Other published models are based on the assumption that the intraparticle mass transfer coefficient is related to solute diffusion in particle pores; mass balance equations then include the rate of solute transfer from solid phase to the pores or solid-fluid equilibrium at pore walls, and the diffusion in pores. For example, Goto et al.13 assumed adsorption equilibrium according to Equation 3.30 on the pore surface. Diffusion in the pores filled with solvent is also involved in shrinking core models (Roy et al.27 and Akgün et al.28). These models are based on the assumption that as the solute in spherical particle is depleted, its concentration in solid phase remains unchanged but the volume of the solid phase containing the solute, which is regarded as a particle core, is diminishing; the intraparticle mass transfer resistance increases as the solute has to diffuse through increasing length of pores between the shrinking core and particle surface. Shrinking core models do not require any knowledge of equilibrium equation y+ = y+(x) as the equilibrium solid phase concentration at a particle core surface remains constant. Whatever models are used to simulate intraparticle diffusion, all of them, after some simplification, lead to the equation for extraction yield in the form of Equation 3.39.

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3.9 CONCLUSIONS Supercritical extraction of botanic materials is characterized, in contrast to other extraction processes, by the combination of special properties of supercritical fluids (liquid-like density, very low kinematic viscosity, and good transport processes) and extracted plant materials (low permeability of intact dry plant tissue in contrast to the easily accessible solute in broken cells close to the particle surface). The process of supercritical fluid extraction can be split into two periods, the first one governed by phase equilibrium where solute is extracted from broken cells, and the second one controlled by intraparticle diffusion where the solute is extracted from intact plant tissue in the particle core. Using mathematical model with broken and intact cells, we have analyzed the effects of three phenomena — phase equilibrium, mass transfer, and flow pattern — on the extraction process. A simple experimental design enables us to distinguish the extraction periods and estimate phase equilibrium and mass transfer parameters separately. Solvent flow pattern significantly affects extraction yields. Scale up of the extraction process is usually connected with unfavorable changes in flow pattern, accompanied by a decrease in extractor performance. These changes are related to the inhomogeneous structure of the extraction bed formed by solid particles. Though the changes in flow pattern are not fully predictable, they can be largely suppressed when appropriate measures including the selection of solvent flow direction are taken.

ACKNOWLEDGMENT Financial support of project AS CR No. K4040110 is gratefully acknowledged.

GLOSSARY a0 c d Dax De e E h H j k K l M n N q q

Specific particle surface, m2/m3 Fluid phase concentration, kg/m3 Particle size, m Axial dispersion coefficient, m2/s Effective diffusivity in particle core, m2/s (=E/N) extraction yield, kg/(kg matrix) Extract, kg Axial coordinate, m Extraction bed height, m Flux across interface, kg/m3 s Mass transfer coefficient, m/s Distribution coefficient (equilibrium parameter) defined by Equation 3.27 Characteristic particle dimension, m Solvent in extraction bed, kg Number of mixers Insoluble solid phase (matrix), kg (=Q/N) specific solvent amount, kg/kg (= Q /N) specific solvent flow rate, kg/kg s

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Q Q r t te tr T u V xb xc xt

107

Solvent passed through extraction bed, kg Solvent flow rate, kg/s Grinding efficiency (fraction of broken cells in plant tissue) Extraction time, s (=tr/Γ) equilibrium extraction time, s Mean residence time, s Temperature, K Interstitial velocity, m/s Extraction bed volume, m3 Concentration in broken cells, kg/(kg matrix) Mean concentration in intact cells (particle core), kg/(kg matrix) Transition concentration (equilibrium parameter) defined by Equation 3.30, kg/(kg matrix) Solute in untreated material, kg/(kg matrix) (=x/xu) dimensionless solid phase concentration Fluid phase concentration, kg/(kg solvent) Solubility (equilibrium parameter), kg/(kg solvent) Equilibrium fluid phase concentration, kg/(kg solvent) (y/y0) dimensionless fluid phase concentration Dimensionless axial coordinate

xu X y ys y+ Y z

Greek letters

γ Γ ε Θ ρ τ

Solvent-to-matrix ratio in extraction bed, kg/kg (=γy0/(rxu)) initial free solute ratio, kg/kg Void fraction Dimensionless mass transfer resistance defined by Equation 3.18 Density, (kg solvent or matrix)/(m3 fluid or solid phase) (= t/tr) dimensionless time

Subscripts 0 c f s

Initial conditions Particle core Fluid phase Solid phase

REFERENCES 1. Sovová, H., Mathematical model for supercritical fluid extraction of natural products and extraction curve evaluation, J. Supercrit. Fluids, 33, 35, 2005. 2. Sovová, H., Rate of the vegetable oil extraction with supercritical CO2: I. modelling of extraction curves, Chem. Eng. Sci., 49, 409, 1994. 3. Marrone, C. et al., Almond oil extraction by supercritical CO2: experiments and modelling, Chem. Eng. Sci., 53, 3711, 1998.

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4. Reverchon, E. et al., Supercritical fractional extraction of fennel seed oil and essential oil: experiments and mathematical modeling, Ind. Eng. Chem. Res., 38, 3069, 1999. 5. Reverchon, E. and Marrone, C., Modeling and simulation of the supercritical CO2 extraction of vegetable oils, J. Supercrit. Fluids, 19, 161, 2001. 6. Reis-Vasco, E.M.C. et al., Mathematical modelling and simulation of pennyroyal essential oil supercritical extraction, Chem. Eng. Sci., 55, 2917, 2000. 7. del Valle, J.M. and Aguilera, J.M., An improved equation for predicting the solubility of vegetable oils in supercritical CO2, Ind. Eng. Chem. Res., 27, 1551, 1988. 8. Angus, S., Armstrong, B., and de Reuck, K.M., International Thermodynamic Tables of Fluid State, CO2, Pergamon Press, Oxford, 1976, p. 37. 9. Lee, A.K.K., Bulley, N.R., and Meisen, A., Modelling of supercritical CO2 extraction of canola oilseed in fixed beds, J. Am. Oil Chem. Soc., 63, 921, 1986. 10. Brunner, G., Mass transfer from solid material in gas extraction, Ber. Bunsenges. Phys. Chem., 88, 887, 1984. 11. Ferreira, S.R.S. et al., Supercritical fluid extraction of black pepper (Piper nigrum L.) essential oil, J. Supercrit. Fluids, 14, 235, 1999. 12. Reverchon, E., Supercritical fluid extraction and fractionation of essential oils and related products, J. Supercrit. Fluids, 10, 1, 1997. 13. Goto, M., Sato, M., and Hirose, T., Extraction of peppermint oil by supercritical carbon dioxide, J. Chem. Eng. Japan, 26, 401, 1993. 14. Goto, M. et al., Modeling supercritical fluid extraction process involving solute-solid interaction, J. Chem. Eng. Japan, 31, 171, 1998. 15. Perrut, M., Clavier, J.Y., Poletto, M., and Reverchon, E., Mathematical Modeling of sunflower seed extraction by supercritical CO2, Ind. Eng. Chem. Res., 36, 430, 1997. 16. Goodarznia, I. and Eikani, M.H., Supercritical carbon dioxide extraction of essential oils: modeling and simulation, Chem. Eng. Sci., 53, 1387, 1998. 17. Beutler, H.J. et al., Einflu der Lösungsmittelführung auf den Hochdruck-ExtraktionsProze, Chem. Ing. Tech., 60, 773, 1988. 18. Beutler, H.J., Lenhard, U., and Lürken, F., Erfahrungen mit der CO2-Hochdruckextraktion auf dem Gebiet der Fettextraktion, Fat Sci. Technol., 90, 550, 1988. 19. Barton, P., Hughes, R.E., Jr., Hussein, M.M., Supercritical carbon dioxide extraction of peppermint and spearmint, J. Supercrit. Fluids, 5, 157, 1992. 20. Sovová, H., Kuera, J., and Jeû, J., Rate of vegetable oil extraction with supercritical CO2 : II. extraction of grape oil, Chem. Eng. Sci., 49, 415, 1994. 21. Dams, A., Stoffübergang bei der überkritischen Extraktion im Festbett, Chem. Ing. Tech., 61, 712, 1989. 22. Stüber, F. et al., Supercritical fluid extraction of packed beds: external mass transfer in upflow and downflow operation, Ind. Eng. Chem. Res., 35, 3618, 1996. 23. Berna, A. et al., Supercritical CO2 extraction of essential oil from orange peel: effect of the height of the bed, J. Supercrit. Fluids, 18, 224, 2000. 24. Reverchon E., Mathematical modeling of supercritical extraction of sage oil, AICHE J., 42, 1765, 1996. 25. Villermaux, J., Chemical engineering approach to dynamic modelling of linear chromatography, J. Chromatogr., 406, 11, 1987. 26. del Valle, J.M., Napolitano, P., and Fuentes, N., Estimation of relevant mass transfer parameters for the extraction of packed substrate beds using supercritical fluids, Ind. Eng. Chem. Res., 39, 4720, 2000. 27. Roy, B.C., Goto, M., and Hirose, T., Extraction of ginger oil with supercritical carbon dioxide: experiments and modeling, Ind. Eng. Chem. Res., 35, 607, 1996.

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28. Akgün, M., Akgün, N.A., and Dincer, S., Extraction and modeling of lavender flower essential oil using supercritical carbon dioxide, Ind. Eng. Chem. Res., 39, 473, 2000.

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4

Biochemical Reactions in Supercritical Fluids ˇ Zeljko Knez, Chiara G. Laudani, Maja Habulin, ˇ ˇ Mateja Primozic University of Maribor

CONTENTS 4.1 4.2 4.3

Introduction ..................................................................................................111 Enzyme Catalysis in Nonconventional Media ............................................114 Enzyme Catalysis in Supercritical Fluids (SCFs) .......................................117 4.3.1 Enzyme Stability in Supercritical Fluids.........................................118 4.3.2 Effect of Pressure.............................................................................119 4.3.3 Number of Pressurization-Depressurization Steps ..........................121 4.3.4 Effect of Temperature ......................................................................122 4.3.5 Effect of Water Activity ...................................................................122 4.3.6 Enzyme Reactors..............................................................................124 4.3.6.1 Process Schemes and Downstream Processing Schemes..........................................................124 4.3.7 Processing Costs ..............................................................................126 4.4 Conclusion....................................................................................................126 References..............................................................................................................127

4.1 INTRODUCTION Enzymatic catalysis has gained considerable attention in recent years as an efficient tool for synthesis of natural products, pharmaceuticals, fine chemicals, and food ingredients. The production of fine chemicals results in the generation of considerable volumes of waste, as the syntheses generally include a number of steps. The yield of each of these steps is usually 60% to 90%, but 10% is not unusual. Based on these data we can conclude that typically 1 kg of end product leads to the generation of 15 kg of wastes or more. Most generated wastes are solvents and by-products from solvents and intermediates. Therefore, ideally several reactions should be performed in water or in supercritical fluids.

111

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The high selectivity and mild reaction conditions of enzymatic transformations make them an alternative to the synthesis of complex bioactive compounds, which are often difficult to obtain by standard chemical routes. However, the majority of organic compounds are not highly soluble in water, which was traditionally perceived as the only suitable reaction medium for the application of biocatalysts. The realization that most enzymes can function perfectly well under nearly anhydrous conditions and, in addition, display a number of useful properties, for example, highly enhanced stability and different selectivity, has dramatically widened the scope of their application to organic synthesis. Another great attraction of using organic solvents rather than water as a reaction solvent is the ability to perform synthetic transformations with relatively inexpensive hydrolytic enzymes. Generally, in vivo, the synthetic and hydrolytic pathways are catalyzed by different enzymes. However, elimination of water from the reaction mixture enables the “reversal” of hydrolytic enzymes and thus avoids the use of the expensive cofactors or activated substrates that are required for their synthetic counterparts. Water is the most common solvent for biochemical reactions but in a biotechnological perspective, there are a lot of advantages of conducting enzymatic conversions in monophasic organic solvents as opposed to water.1 Some are listed below: • • • •

High solubility of most organic (nonpolar) compounds in nonaqueous media Ability to carry out new reactions impossible in water because of kinetic or thermodynamic restrictions Reduction of water-dependent side reactions Insolubility of enzymes in organic media, which allows their easy recovery and reuse

However, the use of organic solvents can be problematic because of their toxicity and flammability, and also there are increasing environmental concerns. As a result, supercritical fluids (SCFs) have attracted much attention in recent years as an alternative to organic solvents for enzymatic reactions. The present research activity will be focused on the development of selective methods for the production of polyfunctional molecules by enzymatic reactions in supercritical carbon dioxide (SC-CO2). Among all the possible SCFs, carbon dioxide is largely used. The use of SC-CO2 instead of organic solvents in biocatalysis presents several additional advantages. It performs mainly as a lipophilic solvent, nontoxic, nonflammable, and cheap. Supercritical CO2 has been most frequently used as a medium for biotrasformations: its critical pressure (73.8 MPa) is “acceptable” and temperature (31.1°C) is consistent with the use of enzymes and labile solutes. It has the GRAS (Generally Regarded As Safe) status. In addition, its “naturalness” is greatly appreciated by the food and healthcare related industries. The use of SCFs as solvents for enzymatic transformations is a relatively new area of research, which is expected to expand in the future. Close to the critical point, small changes in temperature or pressure can produce large changes in density and solvation ability of SCFs. This attribute of SCFs can be fruitfully exploited and integrated in biotransformation and downstream

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processing steps in a single bioreactor. Beyond the critical point, both phases are indistinguishable, and the fluid is monophasic and occupies all the vessel volume. It can be described as a dense gas or an expanded liquid. Generally, SCFs exhibit liquid-like density and therefore have good solvating power, but they retain gas-like compressibility. Consequently, it is possible to control their solvating power by changing the pressure and/or temperature, with a continuous transition from a good to poor solvent. Moreover, low viscosity and high diffusion coefficients of these fluids enhance mass transport and reaction kinetics. These unique properties of SCFs enable one to design efficient integrated processes by coupling an enzymatic reaction with subsequent fractionation and product recovery steps. Molecules in the supercritical (SC) phase are not uniformly distributed in space, but the solvent molecules aggregate around the solute through solvent-solute intermolecular interactions forming clusters, where the aggregated molecules are in dynamic equilibrium with free solvent molecules. Thus, the solvation depends strongly on the density of the SCFs and differs from that in the liquid solution or gas phase.2 When catalytic reactions are performed in SCFs, the outcome of the reactions can be affected in a number of ways. In general, replacement of conventional liquid solvents by SCFs can increase the rate and tune the selectivity of reactions for the following reasons:3 •

•

•

Rapid diffusion of solutes or weakening of the solvation around reacting species facilitates the reactions and sometimes changes the reaction pathway Local clustering of solutes or solvents resulting in an appreciable increase in the local concentration of substrate (and catalyst) causes acceleration of the reaction Reduction or increase in the cage effect often affects the reaction performance of rapid chemical transformations such as radical reactions

In addition to these benefits, SCF chemistry, as a reaction media it poses economical, technical, environmental, and health advantages. The high volatility of CO2 allows it to be completely and easily removed from the product, resulting in an overall “solvent-free” reaction. By using SC-CO2 , an integrated production process can be performed, because it can act as solvent for the reaction and also as a separation medium. The variable solvating power of SC-CO2 and other SCFs facilitates the integration of biocatalytic and downstream processing steps in a single robust bioreactor. The main drawback of SC-CO2 is that it has limited solvating power with respect to polar compounds. This is a serious limitation for biotechnological applications where most natural molecules of interest (e.g., alkaloids, carotenoids, phenols, proteins, and sugars) are only sparingly soluble in SC-CO2. In this case, a polar cosolvent or so-called entrainer, such as acetone, ethanol, methanol, or water, is added in order to increase the polarity of the medium and to solubilize the target solute via the formation of hydrogen bonds. Typically, cosolvents are added to the SCF at moderate concentrations of less than 10 mol %.4 In a batch reactor, the cosolvent can be added directly into the reactor prior to pressurization, whereas in a continuous process, the addition should be made to the CO2 inflow via a liquid pump to deliver a constant flow rate at the operating pressure. However, the use of

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another component in the system further increases the complexity and may also complicate downstream processing. Moreover, the solubility enhancement effect of a cosolvent is usually limited in the case of very polar compounds. Two alternative methods have been developed for some specific cases. To solubilize polyols (e.g., glycerol and sugars) has been proposed5 to form a hydrophobic complex between the polyol and phenylboronic acid (PBAC), which is much more soluble in the SC phase. This method was used to perform the esterification of glycerol and sugar with oleic acid in SC-CO2. Another method involves the adsorption of polar substrates onto a solid hydrophilic support such as silica gel. Compared with the former, this approach is more general because it is not necessary to have two vicinal hydroxyl groups in the substrate molecule.5 In addition, recent advances in the understanding of the chemical properties of materials that are soluble in CO2 have permitted the development of novel surfactants that allow dissolution of both hydrophilic and hydrophobic materials in CO2. The use of SCFs decreases the mass transfer limitations because of the high diffusivity of reactants in the SC medium, the low surface tension, and because of the relatively low viscosity of the mixture. The Schmidt number, Sc = η/ρ⋅D (where η is dynamic viscosity, D is diffusivity, and ρ is density), for CO2 at 200 bars is 45 times lower than for water at 1 bar and 20°C. High diffusivity of SCF and low surface tension lead to reduced internal mass transfer limitations for heterogeneous chemical or biochemical catalysis. One of the main advantages of the use of dense gases as a solvent for enzymecatalyzed reactions is the simple downstream processing. The physico-chemical properties of dense gases are determined by their pressure and temperature, and are especially sensitive near their critical point. By reducing the solvent-power of a dense gas in several stages, fractionation of the product and unreacted reactants is possible. Fractionation is also possible by extracting the mixture, usually with the same dense gas as used in the reaction, but under different process conditions. In all downstream processing schemes, various particle formation techniques or chromatographic techniques can be integrated.

4.2 ENZYME CATALYSIS IN NONCONVENTIONAL MEDIA The breakthrough of biocatalysis in nonaqueous media started in the early 1980s.6–10 Nowadays, it is well established that many enzymes, such as lipases, can remain active and stable in pure organic solvents. Changing the hydrophobicity of predominantly aqueous media through addition of organic solvents causes the hydrophobic effects, which keep the hydrophobic residues buried in the core of the proteins and folded in an aqueous environment where enzymes are kinetically trapped in their native structure in organic solvents.11–14 In addition, in the media of low water content, enzyme inactivation, caused by hydrolysis of peptide bonds and deamidation of asparagine and glutamine residues, is reduced.11 Indeed, many times enzymes are more stable in organic solvents than in water. For instance, increased thermal stability in dry organic solvents with substantial increase in enzyme half-life at 100°C

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TABLE 4.1 Advantages of Biocatalysis in Nonaqueous Media Increased substrate solubility Simplified recovery of biocatalyst Shift to synthetic reactions Mild reaction conditions and minimization of side-reactions Environmentally benign catalyst High selectivity Simplified work-up of products Avoids microbial contaminations

compared to water has been observed.9,15 There are a number of advantages of using enzymes in organic solvents. These are listed in Table 4.1. Many substrates that are insoluble in water can be dissolved by organic solvents. Enzymes are often insoluble, an advantage that simplifies their recovery and reuse many times and is economically very important. A change from an aqueous environment can favor a shift in the equilibrium, enabling synthetic reactions to be achieved with hydrolytic enzymes. Moreover, the unique selectivity and activity of enzymatic reactions is achieved under mild reaction conditions. The enzyme instability in harsher reaction conditions is common in industrial processes, which can, of course, be a disadvantage. The search for enzymes from various extremophilic organisms may, however, result in biocatalysts that can withstand more extreme conditions.16,17 Furthermore, enzymes are environmentally benign and are, unlike metal catalysts, completely degraded in nature. Although some enzymes do display perfect selectivity, many others can receive a broad range of unnatural substrates still with high chemo-, regio- or enantioselectivity. Additionally, the use of an organic solvent often simplifies work-up procedures and avoids microbial contamination of the reaction medium.11,18 The conventional notion that enzymes are only active in aqueous media has long been discarded, thanks to the numerous studies documenting enzyme activities in nonaqueous media, including pure organic solvents and supercritical fluids (SCFs). Enzymatic reactions in nonaqueous solvents offer new possibilities for producing useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules, biopolymers, peptides and proteins, modified fats and oils, structured lipids, and flavor esters). According to conventional notion, enzymes are active only in water. Historically, enzymatic catalysis has been carried out primarily in aqueous systems. Although water is a poor solvent for preparative organic chemistry, it is the unique specificity of enzymes that drew the interest of chemists who were seeking highly selective catalytic agents. Experiments to place enzymes in systems other than aqueous media date back to the end of the nineteenth century.19–23 Initial studies considered the addition of small quantities of water-miscible organic solvents like ethanol or acetone to aqueous enzyme solutions ensuring high availability of water to retain the catalytic activity of enzymes. Then, the biphasic mixtures (aqueous

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enzyme solution emulsified in a water-immiscible solvent such as isooctane or heptane) were used, in which the substrates from the organic phase diffuse into the aqueous phase, that undergoes enzymatic reaction and the products diffuse back. The size of water droplets may be reduced to facilitate mass transfer resulting in the formation of microemulsions or reverse micelles, whose stabilization is achieved by adding surfactants.24,25 Developments in using enzymes in nearly nonaqueous solvents containing traces of water have stimulated research in achieving various kinds of enzymatic transformations.26–34 Enzymatic reactions in nonaqueous solvents offer numerous possibilities for the biotechnological production of useful chemicals using reactions that are not feasible in aqueous media. These reactions include chiral synthesis or resolution;35–38 production of high-value pharmaceutical substances;39–43 modification of fats and oils;44 synthesis of flavor esters and food additives;45–51 production of biodegradable polymers,52 peptides, proteins, and sugar-based polymers.53 In nonaqueous solvents, hydrolytic enzymes could undergo synthetic reactions while they also exhibit altered selectivities,11 pH memory,10,15,54 increased activity and stability at elevated temperatures,9,55 regio-, enantio- and stereoselectivity,44,56 and may also be affected by their water activity.57 Currently, there is a considerable interest in the use of enzymes (particularly lipases, esterases, and proteinases) as catalysts in organic synthesis.18,44,56,58–62 Five major technological advances are believed to have significantly influenced the industry for adopting enzymatic biotransformations:63 (1) the development of large-scale downstream processing techniques for the release of intracellular enzymes from microorganisms; (2) improved screening methods for novel biocatalysts;64–68 (3) the development of immobilized enzymes; (4) biocatalysis in organic media; and most recently, (5) recombinant-DNA (r-DNA) technology to produce enzymes at a reasonable cost. There seems to be no agreement as to why the biocatalysis in organic media could not have taken off earlier.69–71 Perhaps the traditional belief that most enzymes are incompatible with most organic syntheses in nonaqueous media, which also poses a psychological hurdle. Also, until recently, there was no demand for enantiopure compounds, and hence, no enzymes needed to be used. The establishment of industrial processes,72,73 and the realization that most enzymes can function well in organic solvents,9,10,74,75 have heightened interest in the use of enzymes. Also, the need for enantiomerically pure drugs is driving the demand for enzymatic processes. This combined with the discovery of strikingly new properties of enzymes in organic solvents has led to the establishment of organic phase enzyme processes in industry.56,60 Enzymes occupy a unique position in synthetic chemistry due to their high selectivities and rapid catalysis under ambient reaction conditions. Nevertheless, synthetic chemists have been reluctant to employ enzymes as catalysts, because most organic compounds are water insoluble, and the removal of water is tedious and expensive. The fact that enzymes are stable, and in some cases, improve their high specificity in near anhydrous media, has dramatically changed the prospects of employing enzymes in synthetic organic chemistry. The problems that arise for most biotransformations are low solubility of reactants and products, and limited stability of biocatalysts. Carrying out reactions in an aqueous-organic two-phase system

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would be a solution to overcome the first problem. This is not always possible due to the limited stability of enzymes at liquid-liquid interface or in organic solvents. Hence, other approaches are necessary. These include addition of complexing agents such as dimethylated cyclodextrins or adsorbing materials like Amberlite™ XAD7 resins (Eli Lilly, Indianapolis, IN, USA), use of membrane-stabilized interface (Sepracor™, Marlborough, MA, USA) and continuous extraction of reaction products (Forshungs-zentrum-Julich, Julich, Germany). The catalyst’s stability can be increased using a variety of methods including the addition of antioxidants (e.g., dithiothreitol), immobilization, cross-linking, separation from deactivating reagents, variation of reaction conditions, and by genetic engineering.76 Solvent systems used as the reaction media for enzymatic catalysis may be categorized as: (1) aqueous; (2) water: water-miscible (monophasic aqueous-organic system); (3) water: water-immiscible (biphasic aqueous-organic system); (4) nonaqueous (monophasic organic system); (5) anhydrous; (6) supercritical fluids; (7) reversed micelles; (8) solvent-free systems; (9) gas phase; and (10) ionic liquids.

4.3 ENZYME CATALYSIS IN SUPERCRITICAL FLUIDS (SCFs) During the last decade, the tremendous potential of enzymes as a practical catalyst for chemical processes in nonaqueous environments has been well recognized. The use of biocatalysts in organic solvents offers many advantages over using pure water. Among these media, SCFs, such as carbon dioxide (SC-CO2), exhibit properties similar to organic solvents, but with the additional capacity of enhancing transport phenomena (due to their high diffusivities) and facilitate reaction products separation by tuning solvent power, which makes them more attractive to be used as “greendesigner” solvents.77,78 The interest of using biocatalysts in SC-CO2 has been growing rapidly in recent years, mainly in industrial and pilot plant applications.79 The growing interest in SCF technology results from the attractive possibilities offered by this technique: processing at moderate, usually ambient temperatures, use of nontoxic, nonflammable, and environmentally acceptable solvents (usually pressurized CO2) and easy-to-change solvent power (not possible with conventional organics). For example, SCFs at temperatures and pressures slightly above the critical points (e.g., 31°C and 73.8 MPa for CO2), exhibit unique combined properties: liquid-like density (and hence solvent power) and high compressibility, very low viscosity, and high diffusivity. The first two properties make the solvent power controllable by changing pressure and/or temperature, while low viscosity and high diffusivity markedly enhance mass transport phenomena and hence kinetics of a process. However, enzymes are not soluble in SCFs, therefore enzymatic catalysis in SCFs would always be heterogeneous. The use of enzymes as catalysts in nonaqueous media has been described in scientific literature since the middle of the 1980s.10 Organic media offer certain advantages over aqueous media: stabilization of enzymes, dissolution of hydrophobic compounds, and the feasibility of shifting thermodynamic equilibrium toward the synthesis of esters and amides (e.g., in the case of hydrolytic enzymes). Certain

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SCFs, such as CO2, may prove an interesting alternative inasmuch as they exhibit properties similar to organic solvents and their solvent power, which is dependent on the specific density (and hence easily controllable by regulation of pressure and temperature), may be advantageously used in the recovery of products. This opens the way for an integrated production of product recovery processes. The use of SCFs as media for enzymatic reaction was first proposed in the middle of the 1980s by Randolph et al.,80 Hammond et al.,81 and Nakamura et al.82 The aim of the initial studies was to demonstrate that enzymes are active in SCCO2. Randolph et al.80 showed that alkaline phosphatase and cholesterol oxidase are active in SC-CO2; Hammond et al.81 demonstrated the same for polyphenyl oxidase; and Nakamura et al.82 carried out successful transesterification reactions using lipase. Successively, Randolph et al.83 examined the effect of aggregation of cholesterol on cholesterol oxidase activity and found that the addition of cosolvents (entrainers) promotes aggregation and thus resulted in an increase in reaction rate proportional to the degree of aggregation. Recently, the benefit of using SCFs for enzymatic reactions has been demonstrated by Mori and Okahata84 and Kamat et al.85,86 and Chaudhary et al.,87 for example, for improved reaction rates, control of selectivities by pressure, and so forth. Some examples of enzymatic reactions are shown in Figure 4.1 and Figure 4.2. Most of the research published to date dealt with two problems: (1) conformation and stability in the supercritical environment (mainly CO2) and the effect of pressure on reaction rate; (2) the effect of water/moisture content on the activity of enzymes.

4.3.1 ENZYME STABILITY

IN

SUPERCRITICAL FLUIDS

The use of enzymes in supercritical fluids is full of potential problems. For example, the number of parameters that influences the stability of the enzymes increases dramatically when using supercritical fluid. This is the reason why, up to now, no prediction could be made on whether enzymes are stable under supercritical conditions or if the equivalent of even higher activity and selectivity is available compared OH O

.

OH O

OH O OEt

Pseudomonas SP

OH

OEt +

SC CO2

FIGURE 4.1 Reaction scheme of the enantioselective hydrolysis of HPAE in SC-CO2.88

R R1

+ H

O

O

OH C

CH3 C

O

C

CH3

Pseudomonas SP SC CO 2

+

C R

1

H

OH

R

OH

R

C R

1

+ CH3

O C

OH

H

FIGURE 4.2 Reaction scheme of the enzymatic esterification of secondary alcohols in SC-CO2.89

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with the reactions in organic solvents. In the following sections the influence of parameters on the enzyme stability will be discussed. Early investigations80–82 demonstrated that certain enzymes are active in SCCO2. Randolph et al.83 first studied the conformation of several spin-labeled variants of cholesterol oxidase in SC-CO2 and concluded that these proteins were not influenced by the SC-CO2 environment; a similar result has recently been obtained for lipase.90 In contrast, Kasche et al.91 reported that α-chymotrypsin, trypsin, and penicillin amidase were partially denaturated by SC-CO2 and suggested that the decompression process led to their denaturation, but no in situ measurements were conducted to substantiate this suggestion. Most recently Zagrobelny and Bright92 carried out a more detailed examination of the same problem. The conformation of trypsin in SC-CO2 was studied at the pressure range of 5 to 25 MPa and monitored the conformational changes of trypsin in situ as a function of pressure. Their results clearly demonstrate that: (1) significant changes in protein conformation can be induced by SC solvents; (2) most of the conformational changes occur during compression; (3) the native trypsin conformation is only slightly more stable than the unfolded form. Performance characteristics, specific rates of conversion, and yield factors are essential for rating any technological process. On the whole, enzymatic reactions in SC-CO2 proceed at rates similar to those of organic solvents such as n-hexane93,94 and cyclohexane.90 As well as ensuring similar rates of processing and enzyme stability, the SC technology offers important advantages over organic solvent technology, such as ecological friendliness and product fractionation, which can easily be linked with direct micronization and crystallization from SC-CO2 by fluid expansion. In addition, CO2 does not usually oxidize substrates and products, allowing the process to be operated at a temperature of 40°C. Although many enzymes are stable in SCFs, one should pay considerable attention to finding the correct reaction conditions for each substrate/enzyme/SCF system. Although successful reactions have been reported with Subtilisin Carlsberg protease and Candida lipases in SCCO2, there is also evidence for their instability85,95,96 or the existence of a narrow pressure range of activity.97,98 These enzymes are fairly stable in other SCFs such as fluoroform, ethylene, ethane, propane, and sulphur hexafluoride.85 Immobilized Mucor miehei lipase appears to be very stable in SC-CO2; it is a monomeric enzyme with three stabilizing disulfide bonds,99 which may play a role in maintaining its activity in SC-CO2. Nevertheless, one of the most important advantages of using the SCFs as reaction media is that upstream processing after reaction can be greatly simplified as the technique is easily combined with other unit operations.

4.3.2 EFFECT

OF

PRESSURE

The influence of the system pressure on the stability of enzymes is not so significant up to 30 MPa. This is of great advantage because it means that the solvent-power of the supercritical fluid can be adjusted for reaction performance. On the one hand, the solubility of substances increases with higher pressures because of a higher fluid density and this is essential to bring the initial products in the reactor and remove the end products from the reactor. On the other hand, higher pressure normally

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results in higher reaction rates. Therefore a pressure increase is in most cases positive for enzymatic reactions. An isothermal change of pressure of SCFs may change the reaction rate by changing either solubility or the reaction rate constant. A pressure increase improves solubility, thereby increasing production rates, and this effect is most pronounced in the near-critical region. Certain enzymes show considerable apparent pressure activation.100 Pressure can modify the catalytic behavior of enzymes by changing, for example, the rate-limiting step101 or modulating the selectivity of the enzyme.102 If an enzyme is stable in a supercritical fluid, its stability is usually not influenced by pressure ranges of up to 30 MPa. Conversely, reaction rates may be influenced by pressure. In most cases a pressure increase acts positively for enzymatic reactions or there are no changes in the reaction rates. Pressure-induced deactivation of enzymes takes place mostly at pressure exceeding 150 MPa. Reversible pressure denaturation mostly occurs at pressures below 300 MPa, and higher pressures are required to cause irreversible denaturation.103 The effect of pressure on the reaction rate constant has not yet been determined, but the effect on the overall production rate has been examined in several papers. Erickson et al.104 carried out transesterification of triglycerides, using lipase from Rhizopus arrhizius. The reactants were trilaurin and palmitic acid and the pressure ranged from 10 to 30 MPa. A strong negative effect of pressure increased the rate of palmitic acid incorporation into triglyceride was detected, especially in the nearcritical region. The interesterification of trilaurin and myristic acid, catalyzed by lipase, was investigated by Miller et al.90 in the pressure range of 6 to 11 MPa. The interesterification rate and the overall rate (based on total trilaurin conversion) increased with increase in pressure; however, the interesterification rate increased much more rapidly than the overall rate, indicating that the selectivity of the reaction for interesterification over hydrolysis improved at higher pressures. The operational stability of enzymes in SC-CO2 is of crucial importance from the point of view of application. Miller et al.90 measured the interesterification rate over 80 h of continuous operation and observed no loss of activity of lipase. Cholesterol oxidase is stable at 10 MPa and 35°C for at least 50 h.83 Pressure has also been found to have little effect on the stability of lipase from Mucor miehei in the range from 13 to 18 MPa, causing only 10% loss of activity93 after six days at 40°C, unlike temperature, which contributed to a 20% loss at 60°C. Additionally, in some cases a negative effect of pressure on the catalytic activity of biocatalysts in compressed gases may be observed. The catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide, and tert-amyl alcohol at constant temperature and pressure up to 30 MPa and fixed enzyme hydration was lowered.105 In near-critical fluids an increase in pressure of only 20 MPa caused a sixfold decrease in the catalytic efficiency of subtilisin in CO2. In SC-CO2 the formation of carbamates are responsible for lower enzymatic activity in this medium. Carbamates are the product of the reaction between basic free amino groups in the enzymes and acidic SC-CO2.95 On the other hand, lysozyme lipase unfolded and partially oligomerized in moist SC-CO2 at 80°C and its denaturation was not caused by interaction with SC-CO2 but by heating the protein in the presence of water, as found by Weder.106

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One of the advantages of using SCFs as enzymatic reaction media is separation of products from the reaction mixture by changing the pressure of the supercritical fluid. With respect to the facts mentioned previously, the solvent power of the SCF can be adjusted for running reactions, and products can be easily removed from the reactor. When the lipid-coated lipase was employed in SC-CHF3 the enzyme activity107 could be switched on and off by adjusting the pressure or temperature of the SC-CHF3. The effect of pressure on the extent of conversion and product composition of the enzyme-catalyzed hydrolysis of canola oil in SC-CO2 was investigated using lipase from Mucor miehei immobilized on macroporous anionic resin.108 A conversion of 63% to 67% (triglyceride disappearance) was obtained at 24 to 38 MPa. Monoglyceride production was favored at 24 MPa. The amount of product obtained was higher at 24 to 38 MPa due to its enhanced solubility in SC-CO2.

4.3.3 NUMBER

OF

PRESSURIZATION-DEPRESSURIZATION STEPS

The influence of pressurization-depressurization steps in batch reactors on the enzyme activity is of importance to many researchers.109 Pressurizing an enzyme does not play an important role, but depressurization is usually the step that influences residual enzyme activity. The supercritical fluid permeates through the enzymes by diffusion, a process that is relatively slow. After a certain time the enzymes are saturated with the SCFs. If the expansion is too fast and the fluid diffuses out of the enzyme slowly, this causes a higher fluid pressure in the enzyme than in the system. The pressure difference results in cell-cracking, where the cell membranes are broken by the resulting pressure inside the cells. This causes the unfolding of the enzymes and the loss in tertiary structure, which is required for their activity and selectivity. Experiments have shown that depressurization from supercritical CO2 conditions is much smoother than entering the two-phase region and expanding the gas of the fluid.110 This is caused by a change in density which is continuous in supercritical conditions. The expanding liquid CO2 causes evaporation of the fluid accompanied by a large change in density, and this volume expansion causes the unfolding of the enzyme. Depressurization is important when using the benefit of SCFs for simple downstream processing. In this case, by operating a cascade of depressurizations (with a possible change in temperature), product fractionation can be achieved.111 In order to have successful industrial applications, enzymes as biocatalysts must retain their activity for a considerable period of time. The activity of the lipase from Candida antarctica for the production of isoamyl acetate in SC-CO2 was studied by Romero et al.111 in a tubular reactor. The yield of isoamyl acetate was 100% for 30 days, and then slowly decreased. Habulin et al.112 found similar results with immobilized Rhizomucor miehie lipase, reporting a 4% decrease in conversion after one month of treatment. Cholesterol oxidase from different sources can exhibit different stabilities in SCCO2.83 The cholesterol oxidase from Gloecysticum retained its activity for three days and the one from Streptomyces sp. for only one hour. In some cases the half-life of

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the biocatalysts can be increased by adjusting pressure.113 The half-life of α-chymotrypsin increased when the pressure increased from 8 to 15 MPa.

4.3.4 EFFECT

OF

TEMPERATURE

The effect of temperature is much more significant than the pressure effect. For enzyme stability, a temperature increase above certain levels, depending on the enzyme sources, results in the deactivation of the enzyme. This limitation places the temperature limits on the extraction process. Normally, at higher pressure levels an increase in temperature results in higher solubilities of substances in SCFs because the increase in the vapor pressure of the compounds to be dissolved overcomes the reduction in density. Reaction rate also increases at higher temperatures, although enzyme deactivation at higher temperatures may occur. At the moment no correlation between temperature and the stability of the different types of enzymes is available. Lipase from Aspergillus niger was incubated in SC-CO2 at 30 MPa and different temperatures.114 Its residual activity was optimal at 323K. At higher temperatures a rapid decrease in activity was observed. This thermal deactivation was connected to changes in the water distribution in the system. In microaqueous media, including SCFs, thermal stability of biocatalysts can also be improved. Reaction rate for subtilisin protease-catalyzed reactions increased by 80% in SC-CO2.115 The optimal temperature for esterification between n-butyric acid and isoamyl alcohol, catalyzed by porcine pancreas lipase, increased from 313K at atmospheric pressure to 323K in near-critical propane.116 The improved stability of the lipase in the low-water-content environment is a consequence of a well-known fact that reactions, which may be responsible for the denaturation of enzymes, are hydrolytic and therefore require water.117 Thermal activation and deactivation (energy of activation and deactivation enthalpy, respectively) may be determined from the Arrhenius diagram. The ratio between the amounts of inactive and active forms of the enzyme at a temperature, where the greatest initial reaction rate is observed, is expressed with a deactivation constant. If the activation and deactivation enthalpy values are high this indicates that enzyme activity is very dependent on temperature.

4.3.5 EFFECT

OF

WATER ACTIVITY

Water concentration in the reaction system is one of the most important factors that influence activity of an enzyme. Water is crucial to enzymes and affects enzyme action in various ways: by influencing enzyme structure via noncovalent binding and disruption of the hydrogen bonds; facilitating reagent diffusion; and influencing the reaction equilibrium.118 The subtilisin Carlsberg catalyzed transesterification of N-acetyl phenylalanine methyl ester,119 N-acetyl phenylalanine ethyl ester,120 N-trifluoroacetyl phenylalanine methyl ester,121 and N-trifluoroacetyl phenylalanine ethyl ester122 was studied in SC-CO2. The water content of the reaction affected the reactivity of the system; for

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the transesterification of the methyl esters with ethanol the optimum concentration of water was determined to be about 0.74 M, while during the transesterification of the ethyl esters123 with methanol it was about 1.3 M. Use of an enzyme in pure SC-CO2 may lead to removal of the water, which is included or bonded to the enzyme. The quantity of the removed water is temperature and pressure depended and if too high may lead to enzyme denaturation and loss of activity. The solubility of water in SC-CO2 can be calculated by Chrastil equation124 a  c = ρk ⋅ exp  + b  T  where c is the solubility (g/L), ρ is the CO2 density (g/L), and T the temperature (K). The calculated water parameters are k = 1.549, a = −2826.4, and b = –0.807. Water is important in the supercritical fluid because water-saturated CO2 causes the inhibition of enzymes and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Enzymes require a specific amount of water to maintain their active conformation. Enzyme stability generally decreases with increasing water concentration, whereas their activities require some water to be present. Therefore, the water content has to be optimized to find the best balance between enzyme life and activity. If water acts as a substrate for an enzymatic reaction, the optimal parameters for continuous reaction require among other things enough moisture to compensate for complete reaction and sufficient enzyme moisture for losses due to water solubility in SC-CO2.125,126 However, if the water concentration in the supercritical medium is too high or if it is a product of the reaction, the increase in moisture may cause enzyme deactivation. Not surprisingly, all studies published so far have pointed out the strong influence of moisture on enzymatic activity and reaction rates. Optimum water content in the support was estimated at 10 wt %, irrespective of the operating conditions,94 but this value may be contested in view of other reports.127 To prevent dehydration of the enzyme, the fluid in contact with the protein must contain water. The most hydrophilic hydrocarbons (e.g., hexane) dissolve 0.01% water, but SC-CO2 may dissolve as much as 0.3% to 0.35% water. However, it is not the solubility of water itself but the partition of water between the enzymatic support and the solvent (SC-CO2), which matters. Marty et al.94 carried out an extensive analysis of the partition of water between the enzymatic support and SC-CO2 as a function of pressure and temperature. They found that increasing temperature and pressure had a negative effect on the adsorption of water by the support. This is opposite to the results obtained by gas adsorption, which suggests that the solvation effects predominate over the vapor pressure effects. The same authors also extensively studied the influence of ethanol (entrainer) content in SC-CO2 and found that ethanol has a strong “drying” effect on the enzyme support; indeed, the more hydrophilic the fluid, the more pronounced is the dehydration of the enzymes. Increasing water content, above the optimum level, adversely affects the overall performance. This appears to be related to hydrophilic hindrance of the hydrophobic substrate on its way to the

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active sites on the enzyme, and eventually makes the thermodynamic equilibrium less favorable.128 Chulalaksananukul et al.129 measured the residual activity of lipase from Mucor miehei after a day in SC-CO2 at a temperature range of 40°C to 100°C and at various water concentrations. As the temperature increased, the enzyme molecule at first unfolded reversibly and then underwent one or more of the following reactions: formation of incorrect or scrambled structures, cleavage of disulfide bonds, deamination of trypsine residues, and hydrolysis of peptide bonds. Each process requires water and is therefore accelerated with increasing water concentration.

4.3.6 ENZYME REACTORS 4.3.6.1 Process Schemes and Downstream Processing Schemes Batch-stirred tank, extractive semibatch, recirculating batch, semicontinuous flow, continuous packed-bed, and continuous-membrane reactors have used dense gases as solvents. 4.3.6.1.1 Batch-Stirred-Tank Reactor Batch-stirred-tank reactors are usually used for screening enzymatic reactions in dense gases.130,131 The design of the system is shown in Figure 4.3. Initially, the substrates are pumped into the reactor and then the enzyme-preparation is added. Finally, dry gas is pumped into the reactor, up to the desired pressure. The initial concentration of the reactant must never exceed its solubility limit in the gas.

T = const.

PI

P

2 Gas 1

FIGURE 4.3 Design of experimental batch-stirred-tank apparatus for synthesis under high pressure. 1 magnetic stirrer and heater; 2 reactor; P high-pressure pump; PI pressure indicator.

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PI

PI

HPP

PI

TIR

TIR H

Gas

3

FI

HPP HPP 4

1

2

FIGURE 4.4 Design of experimental continuous packed-bed apparatus for synthesis under supercritical conditions. 1, 2 substrates; 3 reactor; 4 separation column; HPP high-pressure pump; PI pressure indicator; TIR temperature indicator and regulator; H heat exchanger; FI flow indicator.

4.3.6.1.2 Continuous Packed-Bed Reactor A continuous packed-bed reactor is presented in Figure 4.4. The pump delivers a high-pressure gas into the system. The substrates are pumped into the system, using a liquid pump. The initial concentration of the reactant must never exceed its solubility limit in the gas. Supercritical CO2 is depressurized through the expansion valves into separator column 4, where the product and the unreacted substrates are recovered. The gas phase is finally vented into the atmosphere after measuring the flow rate with a rotameter. The gas can be condensed and recycled when used on a pilot or industrial scale. 4.3.6.1.3 Continuous High-Pressure Enzyme Membrane Reactor In a continuous high-pressure enzyme membrane reactor (Figure 4.5), a 35-mmdiameter membrane is placed between two sintered plates and fitted in the reactor. A measured amount of the catalyst (hydrated enzyme preparation) is put in the reactor, which is electrically heated, with a heating jacket, to constant temperature. The substrates and the gas are pumped into the membrane reactor with the highpressure pump. The products and unreacted reactants are collected in the separator. The catalyst remains in the reactor (behind the membrane). Continuous processes in particular are favored in the industry because they are cost efficient and the reactors can be kept relatively smaller in size.132,133 This reduction in size reduces both costs and safety problems of the high-pressure equipment needed for SC reactions.

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TIR PI

P

PI

H

4

FI

TIR

Gas P

P

3 2

1

1

FIGURE 4.5 High-pressure continuous stirred-tank membrane reactor. 1 substrates; 2 magnetic stirrer and heater; 3 reactor with membrane; 4 separation column; P high-pressure pump; TIR temperature indicator and regulator; PI pressure indicator; FI flow indicator.

4.3.7 PROCESSING COSTS Economic evaluations were made for the enzyme-catalyzed production of oleyl oleate in a high-pressure batch-stirred-tank reactor (HP BSTR) and in a high-pressure continuously operated reactor (HP COR). It was assumed that the reaction mixture was completely precipitated from CO2. The production costs are strongly dependent on the solubility of the substrates in dense gas, the enzyme lifetime, and the productivity of the biocatalyst. The study shows that the return on investment would take less than one year.109

4.4 CONCLUSION The application of SCFs as reaction media for enzymatic synthesis has several advantages, such as the higher initial reaction rates, higher conversion, possible separation of products from unreacted substrates, over solvent-free, or solvent systems (where water or organic solvents are used). Owing to the lower mass transfer limitations and mild (temperature) reaction conditions, at first the reactions which were performed in nonaqueous systems will be transposed to supercritical media. An additional benefit of using SCFs as reaction media is that they give simple and ecologically safe (no heat and solvent pollution) recovery of products. However, for some specific reactions, solvent-free systems are preferred because of their higher yields. The main area of development should be related to the hydrolysis of glycerides, transesterification, esterification, and interesterification reactions. As lipases have high and stable activity in SC-CO2 (even at high temperatures) even greater and more intensive developments are expected in the future. Only a few papers on the separation and synthesis of chiral compounds have been published so far. Because

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enzymes have high selectivity, and owing to the great importance of enantioselective synthesis or enantiomeric resolution in the pharmaceutical industry, this area of research is expected to have substantial growth.

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117. Mattiasson, B. and Aldercreutz, P., Tailoring the microenvironment of enzymes in water-poor systems, Trends in Biotechnol., 9, 394, 1991. 118. Krishna, H.S., Developments and trends in enzyme catalysis in nonconventional media, Biotechnol. Adv., 20, 239, 2002. 119. Aaltonen, O., Enzymatic catalysis, in Chemical Synthesis using Supercritical Fluids Jessop, P.G. and Leiner, W., Eds., Wiley-VCH, Weinheim, 1999. 120. Turner, C. et al., Lipase-catalyzed reactions in organic and supercritical solvents: application to fat-soluble vitamin determination in milk powder and infant formula, Enzyme Microb. Tech., 29, 111, 2001. 121. Rezaei, K. and Temelli, F., On-line extraction-reaction of canola oil using immobilized lipase in supercritical CO2, J. Supercrit. Fluid, 19, 263, 2001. 122. Schmitt-Rozieres, M., Deyris, V., and Comeau, L.C., Enrichment of polyunsaturated fatty acids from sardine cannery effluents by enzymatic selective esterification, J. Am. Oil. Chem. Soc., 77, 329, 2000. 123. Smallridge, A.J., Trewhella, M.A., and Wang, Z., The enzyme-catalysed stereoselective transesterification of phenylalanine derivatives in supercritical carbon dioxide, Aust. J. Chem., 55, 259, 2002. 124. Chrastil, J., Solubility of solids and liquids in supercritical gases, J. Phys. Chem., 86, 3016, 1982. 125. Hampson, J.W. and Foglia, T.A., Effect of moisture content on immobilized lipasecatalyzed triacylglycerol hydrolysis under supercritical carbon dioxide flow in a tubular fixed-bed reactor, J. Am. Oil. Chem. Soc., 76, 777, 1999. 126. Martinez, J.L., Rezaei, K., and Temelli, F., Effect of water on canola oil hydrolysis in an online extraction-reaction system using supercritical CO2, Ind. Eng. Chem. Res., 41, 6475, 2002. ˇ The influence of water on the synthesis of n-butyl oleate 127. Leitgeb, M. and Knez, Z., by immobized Mucor miehei lipase, J. Am. Oil. Chem. Soc., 67, 775, 1990. 128. Basheer, S., Mogi, K., and Nakajima, M., Surfactant-modified lipase for the catalysis of the interesterification of triglycerides and fatty acids, Biotechnol. Bioeng., 45, 187, 1995. 129. Chulalaksananukul W., Condoret J.S., and Combes D., Geranyl acetate synthesis by lipase-catalyzed transesterification in supercritical carbon dioxide, Enzyme Microb. Technol., 15, 691, 1993. ˇ Habulin, M., and Krmelj, V., Enzyme catalyzed reactions in dense gases, 130. Knez, Z., J. Supercrit. Fluid, 14, 17, 1998. ˇ and Habulin, M., Compressed gases as alternative enzymatic-reaction sol131. Knez, Z. vents: a short review, J. Supercrit. Fluid, 23, 29, 2002. 132. Hitzler, M.G. et al., Selective catalytic hydrogenation of organic compounds in supercritical fluids as a continuous process, Organic Process Res. Dev., 2, 137, 1998. 133. Tundo, P., Continuous Flow Methods in Organic Synthesis, Ellis Horwood, Chichester, 1991.

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5

Pressurized Low Polarity Water Extraction of Biologically Active Compounds from Plant Products Juan Eduardo Cacace and Giuseppe Mazza Agriculture and Agri-Food Canada

CONTENTS 5.1 5.2

Introduction ..................................................................................................135 Pressurized Low Polarity Water Extraction Process ...................................136 5.2.1 Basic Concepts.................................................................................136 5.2.2 Equipment ........................................................................................138 5.3 Applications of PLPW Extraction ...............................................................142 5.3.1 Effect of the Extraction Temperature and Pressure ........................142 5.3.2 Fractionation of Compounds of Different Polarity .........................147 5.4 Modeling of PLPW Extraction of Bioactives from Plant Materials...........150 5.5 Conclusions ..................................................................................................152 References..............................................................................................................152

5.1 INTRODUCTION Plants synthesize many classes of organic chemical compounds ranging from simple structures to complex molecules as part of their normal metabolic processes. These compounds are broadly characterized as: (1) primary metabolites which encompass substances such as nucleic acids, proteins, lipids, and polysaccharides that are the fundamental biologically active chemical units of living plant cells, and (2) secondary metabolites which typically have larger, more complex chemical architectures that incorporate one or more primary metabolites into their structures. Various types

135

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of secondary metabolites synthesized by plants are commonly referred to as phytochemicals and include carotenoids, phenolics, alkaloids, terpenes, sterols, saponins, nitrogen-containing compounds, and organosulfur compounds.1 The most studied of the phytochemicals are the phenolics and carotenoids. Phenolics are compounds possessing one or more aromatic rings with one or more hydroxyl groups and generally are categorized as phenolic acids, flavonoids, stilbenes, coumarins, tannins, isoflavones and lignans. The flavonoids are further classified as flavonols, flavones, flavanols (catechins), flavanones, and anthocyanins. It is known that many phytochemicals can significantly affect human metabolism and health, and therefore, there is considerable interest in extraction of these compounds for their incorporation into functional foods, nutraceuticals, and pharmaceutical products. Also, certain classes of phytochemicals (terpenes and pyrazines) are useful for the production of flavor and fragrances and for incorporation into topical preparations. Phytochemicals typically are not soluble in water under ambient conditions due to their organic nature and the preponderance of nonionic bonds in their architectures. However, they are readily soluble in various organic solvents such as aliphatic alcohols, hexanes, dioxanes, acids, ethers, methylene chloride, trichloroethylene, and acetonitrile. Numerous methods are known for extracting phytochemicals from plant materials; most are based on sequential extraction processes incorporating one or more organic solvents in combination with washing steps.2–7 Some methods teach the use of alkali or alkaline solvents in combination with organic solvents for increased extraction efficiency.8–10 Starting plant materials are usually physically disrupted by means of grinding, shredding, chopping, pulverizing, compressing, or macerating in order to improve extraction efficiency. Phytochemical extracts have to be further processed to remove all trace of the organic solvents, and/or to separate and purify individual phytochemicals. More complex techniques such as hydrodistillation, pressurized solvent extraction, microwave-assisted extraction, supercritical CO2 extraction, and pressurized low polarity water (PLPW) extraction have been used in an attempt to reduce the use of organic solvents and increase the efficiency of the extraction process.2,5,11–13

5.2 PRESSURIZED LOW POLARITY WATER EXTRACTION PROCESS 5.2.1 BASIC CONCEPTS Pressurized low polarity water (PLPW) extraction, also known as subcritical water extraction, is a technology that modifies the properties of water by increasing the temperature up to 374ºC and keeping the pressure high enough to maintain the water in the liquid state to improve its extraction ability. It is known that the physical and chemical properties of water within sealed systems can be manipulated by concurrently controlling the temperature and pressure, whereby the water remains in the liquid state even though its temperature may be significantly increased above its atmospheric boiling point of 100ºC. In this condition, pressurized low polarity water can be maintained in the liquid form up to a temperature of 374ºC and a pressure

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Pressurized Low Polarity Water Extraction

1000

°C

4.5

70

3

0

50

3

m

kg/ 400

m

/ kg

1.5

60 0

kg /

3.5

3.0

4.0

0

kg

m3

3

300

3

/m

8

kg

/m

kg

0 90

2.5

kg / m3

2.0

95 0

/m

00

°C

20

0°C

T

3

500

1000

2000

40 0°C

0

=0 ° 20 C ° 40 C ° 60 C ° 80 C 1 0 °C 0°C

10000

137

50

10.0

360°C 340°C 320°C 300°C 280°C 260°C

1.0

Pressure (bar)

100

Density = 1000 kg/m 3 Entropy = 0.5 kj/kg K

200

240°C 220°C 200°C 180°C 160°C 140°C

= 90%

80%

70%

60%

50%

40%

30%

T = 100°C

Quality

Qu

alit y

1.0

20%

=1

0%

120°C

80°C

60°C

0.1

0

1000 Enthalpy (kj/kg)

2000

FIGURE 5.1 Pressure-enthalpy chart of water (adapted from Haar et al.14).

of 22.1 MPa (221 bars) after which it becomes supercritical water. The low polarity water can be considered in a region from normal atmospheric pressure and room temperature to 22 MPa and 374ºC (Figure 5.1). The polarity, viscosity, surface tension, and dissociation constant of subcritical water are significantly lower compared to water at ambient temperature and pressure conditions, thereby significantly altering its chemical properties to approximate those of organic solvents. Thus, increases of water temperature from 25ºC to 200ºC, for instance, would decrease its dielectric constant from 79 to 35, reaching values similar to those for ethanol (24) or methanol (33). A pressure of 5 MPa (50 bars) would be high enough to keep the

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water in the liquid phase during this increase of temperature (Figure 5.1). A higher extraction pressure would be detrimental for the process because of a slight increase in the dielectric constant and a considerable increase in the cost of the equipment. At 25ºC, the dielectric constant of water increases from about 79 to 93 when the pressure increases from 10 to 600 MPa (100 to 6000 bars).14 The polarity of pure water would be comparable to water-methanol or water-acetonitrile mixtures at 25ºC.15 Consequently, pressurized low polarity water can easily solubilize organic compounds such as phytochemicals, which are normally insoluble in ambient water.16 Extraction using PLPW has been compared with and reported to be superior to conventional extraction techniques including hydrodistillation,17–20 solid-liquid extraction,13,18,21 and supercritical CO2 extraction.22,23 Some of the benefits that have been mentioned include higher selectivity, cleanliness, speed, and cost savings of both raw material and energy. The heat advantage of PLPW extraction over hydrodistillation has been reported at 20 times per kg of water, although 10 kg more water would be used.24 While methods based on sequential extraction processes with organic solvents are useful for extraction and purification of small quantities of phytochemicals for research purposes, they are difficult to scale up to commercial volumes because of problems associated with cost and safe removal and recovery of the organic solvent from the extracts and spent plant materials. Furthermore, the types and concentrations of organic solvents must be carefully selected in order to avoid structural changes to the target phytochemicals during extraction that may adversely affect one or more of their desirable physical, chemical, or biological properties.

5.2.2 EQUIPMENT The most commonly used equipment in PLPW systems (Figure 5.2) consists of storage tank (T) for pure water connected to a high-pressure pump (P), which is connected to a valve (V1) and to a heating coil (HC) housed within a temperaturecontrolled chamber (O). The pressure in the line is displayed by a pressure gauge installed outside the oven. The heating coil is connected to an extraction vessel (EV), which is also mounted inside the temperature-controlled chamber. The chamber is equipped with programmable temperature controller and recorder (TC). The extraction vessel is connected to a cooling coil (CC), which in turn is connected to the inlet of a pressure regulation valve or backpressure regulator (BPR). The pressure regulation valve is connected to a collection vessel (CV). The collection vessel can be changed periodically to provide a plurality of collection volumes, thereby separating and individually collecting multiple eluant fractions. A source plant material is loaded into the extraction vessel and it is maintained inside the vessel during extraction by fitting adequate frits in the inlet and outlet of the vessel. Water is pumped from the storage tank into the extraction vessel until a desired in-line pressure is achieved, usually in the range of about 4 to 7 MPa. Liquid chromatography (LC) pumps and gas chromatography (GC) ovens have been used most frequently for pumping and heating the water (Table 5.1). The pressurized water within the extraction vessel is then heated by raising the temperature within the extraction chamber while in-line pressure is maintained at a

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V1 TC P

CC T

T

BPR HC

O

EV CV

FIGURE 5.2 Diagram of pressurized low polarity water extractor: T, water tank; P, water pump; HC, heating coil; TC, temperature controller; EV, extraction vessel; O, oven; CC, cooling coil; BPR, backpressure regulator; CV, collection vial.

desired level by the pressure regulation valve. Stainless steel tubing coils of various lengths have been used as heating and cooling elements. Varied pressure regulators have been used from very simple home tubing restrictors to automatic backpressure regulators (Table 5.1). Self-contained equipments have been also used for pressurized liquid or accelerated liquid extraction. Flow-control devices taking action on the pump achieve precise flow rates of subcritical water through the extraction vessel. The temperature within the chamber is set and maintained by an automatic controller during an extraction procedure. Components of the system between the pump and the pressure regulation valve must be connected by tubing resistant to high pressure (8 to 10 MPa) required for PLPW extraction. Extraction of bioactives using PLPW has been usually performed using pure water. Water may be further processed by distillation or filtration, and optionally, could be purged with nitrogen to remove all dissolved oxygen prior to its use. Purified water typically has a pH in the range of 5.9 to 6.2. However, if so desired, the pH of purified water can be adjusted into a range of 3.5 to 9.5 with acids or bases, prior to its use, to enhance solubilization and extraction of various phytochemicals. Extraction of bioactives from flaxmeal was optimized by modifying the pH of subcritical water. Thus, buffered water at either high or low pH was used to improve extraction of bioactives.25 Yields of proteins and the lignan secoisolariciresinol diglucoside (SDG) were maximized at alkaline pH 9. The pH was the factor that defined the equilibrium yields of SDG irrespective of the temperature. At pHs 4 and 9, extraction of SDG from flaxmeal at 160ºC and 190ºC reached the same equilibrium yield (Figure 5.3). It is known that alkaline pH helps to solubilize protein.26,27 At the same time, pH could have helped to extract lignans by breaking phenolic-protein interaction and thus releasing the lignans from the plant matrix. Alkaline pH may

© 2007 by Taylor & Francis Group, LLC

Targeted phytochemicals Essential oils Essential oils Essential oils Oxygenates with AO4 activity Fragrance and flavor compounds Anthocyanins Phenolics from whole flaxseed and flaxseed meal

1

Cell

Pump

80 × 3 ID2 mm Frit 2 µm 150 × 11 ID mm (14 mL) Frit 2 µm (10.4 mL) Frit 0.5 µm

LC3 Pump (0.5–3.0 mL/min) LC pump (2.0 mL/min) LC pump (1–3 mL/min) Isco 100 D (1 mL/min) LC pump

50 × 9.4 ID mm (3.47 mL) 0.5 µm and 2 µm frits (10.4 mL) 14.8 ID mm (55 mL) 100 100 100 200

× × × ×

7 0 ID mm 9.4 ID mm 19.3ID mm 7.7 ID mm

(24 mL/min) LC pump (0.3–4 mL/min)

Heater Coil/oven coil/oven 3 m × 0.76 mm ID coil/oven 3 m coil/oven 1 m coil/oven Coil/oven 3 m × 0.76 mm ID coil/oven

Backpressure regulator; 2 Inside diameter; 3 Liquid chromatoraphy; 4 antioxidant

© 2007 by Taylor & Francis Group, LLC

Cooler

BPR1

Coil/cooling recirculation bath (25°C) 1 m coil/cooling recirculation bath (25°C) 1 m coil/ice water bath

Home variable restrictor (2–20 MPa)

40 cm coil/water bath (25°C) Coil/cooling water bath 1 m × 0.76 mm ID coil/water bath (room temperature)

Reference 13 2,17,18,21

Pressure control valve (2–9 MPa) BPR (6–7 MPa) 11 cm to 100 µm restrictor Micro metering valve (4 MPa) BPR (5.2 MPa cartridge)

43,46 23,31 24,22 48 40,25

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TABLE 5.1 Components of PLPW Extraction Systems Used for Extraction of Plant Bioactives

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141

25

119

20

95

15

71

Yield (%)

SDG (mg/g meal)

Pressurized Low Polarity Water Extraction

48

10 160°C pH 4 160°C pH 9 190°C pH 4 190°C pH 9

5

24

0

0 0

100

200

300

400

500

Time or volume (min or mL)

FIGURE 5.3 Pressurized low polarity water extraction of SDG from flaxmeal at 160ºC and 190ºC and pH 4 and 9.

have also hydrolyze d complex polymeric phenolics, reducing them to more available and easily extracted compounds. Whatever mechanisms pH affected, high pH raised SDG and protein yields. Thus, pH effect overcame the increase of solubility and yield obtained by temperature. Carbohydrate extraction from flaxmeal was improved using PLPW at pH 4.25 Also, high-temperature pressurized liquid extraction (80°C to 100°C) using acidified water was as effective as acidified 60% methanol in extracting anthocyanins from grape skins.28 Another modification of water to improve its extraction efficiency has been the addition of sulfur dioxide. Sulfured PLPW (containing 1400 g/mL sodium metabisulfite) extractions of grape skin phenolics over a temperature range of 100ºC to 160°C were compared with conventional hot water or aqueous 60% (v/v) methanol extractions (50°C, 1 h). The PLPW extracts from modified sulfured water had higher levels of total anthocyanins and total phenolics than extracts that used pure water. Furthermore, subcritical water and subcritical sulfured water extracts had comparable or higher levels of anthocyanins than extracts obtained using conventional hot water or 60% methanol.29 A modification of the heating system in the subcritical water extraction could be the replacement of the oven with a water heater interconnected between the pump and the extraction vessel. In such a system the extraction vessel would be provided with a jacket or a surface heater, thereby maintaining the temperature of pressurized low polarity water flowing through the extraction vessel. The jacket may be integral to the extraction vessel, or alternatively, be mountable onto the exterior surface of the vessel.30

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5.3 APPLICATIONS OF PLPW EXTRACTION PLPW has been applied to extract a variety of biochemicals from a wide range of plant species (Table 5.2). It has been used to extract antioxidants from rosemary31 and Taiwan yams;32 anthocyanins from red grape skin;28,29 ginsenosides from American ginseng,33 catechins and epicatechin from tea leaves and grape seeds,34 oil from cedarwood;35 essential oil from oregano,17 anthraquinones (antibacterial, antiviral, and anticancer compounds) from roots of Morinda citrifolia,36 tanshinone I and IIA from Salvia miltiorrhiza used in Chinese medicine;37 flavones, anilines, and phenols from orange peels;38 kava lactones from kava roots;39 lignans from whole flaxseed40 and flaxseed meal,25 and saponins from cow cockle (Saponaria vaccaria) seed.41 Plant material for the extraction can include homogenous samples; or mixtures of whole plant parts such as seeds, flowers, leaves, stems, and roots; and also, with source plant materials disrupted and processed by methods including one or more of grinding, shredding, chopping, pulverizing, compressing, and macerating. The biomass can be fresh hydrated plant materials or plant materials may be dehydrated prior to extraction, or alternatively, processing by one or more of the methods described above prior to extraction. The plant materials may be packed into an extraction vessel either alone or in combination with inert physical substrates such as glass wool, glass beads, resin beads, silica sand, stainless steel wire cloth, and other like substrates whereby the inert substrates maintain spacing and distribution of the source plant materials throughout the vessel during the course of the extraction procedure thereby facilitating mass transfer while preventing migration and packing of the plant material against the outlet frits.30 Recently, Ho et al.25 showed that extraction of lignans, proteins, and carbohydrates with PLPW was positively affected by the addition of glass beads to flaxseed meal for packing the cell. The overall lignan yield increased from 10% to 50% at all temperatures tested with copacking the meal with glass beads.

5.3.1 EFFECT

OF THE

EXTRACTION TEMPERATURE

AND

PRESSURE

The extraction of bioactives from plant material with the PLPW process is clearly affected by the temperature. Thus, SDG, the major lignan in flaxseeds, along with two other phenolic compounds, p-coumaric acid glucoside and ferulic acid glucoside, were extracted with varied success at different temperatures in a PLPW system. Extraction yields of lignans and other phenolic compounds from flaxseed increased from 10 % at 100ºC up to approximately 90% at 140ºC to 160ºC.40 Temperature also affected PLPW extraction of lignan SDG from flaxmeal. Yield of flaxmeal SDG increased from 30% at 130ºC to a maximum of almost 100% at 190ºC.25 In PLPW extraction of phenolic compounds from grape seeds at 150ºC, catechin increased by 32% and epicatechin increased by 99% over the recovery at 50ºC extraction.42 Saponin yields of PLPW extracts increased with temperature and time. While only 33.2% (w/w) of total saponins was extracted at 125°C in 3 h, 60.2% (w/w) was recovered at 175°C in the first 15 min extraction from cow cockle ground seeds.41 The temperature has such a remarkable effect on the extraction and composition of the final extract that a study of temperature effect is a prerequisite in any new PLPW

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extraction research. The effect of temperature on extraction of bioactives using PLPW has also been reported in extractions of lactones from kava root,39 phenolics from grape skin,28 and essential oils from peppermint and savory,23 fennel,21 rosemary,24,31 thyme;43 clove,44 Origanum micranthum,45 Rosa canina,46 and Origanum onites.47 It is clear from these and others studies (Table 5.2) that temperature has to reach a minimum value for the water to gain the properties required to extract a given bioactive. Below the required temperature the extraction does not occur or results in a very low yield. A very good example of the effect of temperature on compound yield is the extraction of volatile compounds from peppermint (Figure 5.4). Four groups of compounds were extracted: the first included carvone, pulegone, and piperitone; the second eucalyptol, menthone, neomenthol, and menthol; the third menthyl acetate; and the fourth β-caryophyllene.23 Only one line per group has been plotted in Figure 5.4 to simplify the plot. As can be observed, extraction yields increased with temperature from values as low as zero to as high as 100%, depending on the group of compounds evaluated. At temperatures below 50ºC, only compounds of the first two groups were extracted, and yields varied depending on the extraction time. Menthyl acetate and β-caryophyllene were not extracted or their yields were very low because their solubility in water at those temperatures was likely very low. In order to begin extraction of menthyl acetate and β-caryophyllene, temperatures above 50ºC and 150ºC had to be used. Increasing the extraction time did not considerably increase the yield of these compounds when the extraction temperature was kept below 50ºC and 150ºC, respectively. Yield points represented in Figure 5.4 are yields of 15 min extraction at each temperature. Thus 80% yield at 175ºC was calculated by adding yields of five extractions of 15 min each. However at 50ºC, menthyl acetate yield after 15 min extraction was 1% or 2%; if the extraction time were increased five times, the yield would be at most 5% to 10%. Effect of temperature has been attributed to an increase on the solubility of the compounds.24 It is also possible that the temperature affects the extraction by breaking interactions between the analytes and the plant matrix.42 Because the extraction yields increase with temperature and also temperature has to reach a minimum value for the bioactives to be extracted with water, the extraction temperature must be selected for the targeted compounds. There is an optimal temperature at which the yield is maximized for every bioactive. Ideally this temperature would be related to the modified properties of the water required for the extraction in the PLPW system, which in turn are linked to the properties of the bioactive that has to be extracted. Thus, the most efficient extraction of the lignan SDG and other phenolics from flaxseed occurred in the temperature range from 140ºC to 160ºC40 and from flaxmeal at 190ºC.25 Recommended temperatures for PLPW extraction of other phytochemicals are: 150ºC for phenolics compounds from grape seeds;42 120ºC for anthocyanins from berries;48 100ºC for volatile flavor and fragrance compounds from Rosa canina,46 and 150ºC for essential oils from Achillea monocephala.49 Most of these extraction temperatures are relatively high, and may be detrimental when heat sensitive bioactives are present in the raw material. Thus, at 160ºC the total SDG measured in flaxseed extracts, solvent wash, and extracted seed residues

© 2007 by Taylor & Francis Group, LLC

Target Phytochemicals

Plant Material

Temperature/Pressure

Major Compounds

Eucalyptol, linalool, terpinen-4-ol, αterpineol, geraniol, ETMCb Essential oils Laurel 50ºC to 200ºC (150ºC)/5 MPa 1,8-cineole, 2 unidentified peaks Essential oils Clove 125ºC, 250ºC/2.4, 5, 10, 17 MPa Eugenol, eugenyl acetate Essential oils Thyme 100ºC, 125ºC, 150ºC, 175ºC (150ºC)/2, 6, 9 MPa α-pinene, p-cymene, γ-terpinene, limonene, E-3-caren-2-ol, thymol, carvacrol, caryophyllene Essential oils Oregano 100ºC–175ºC (150ºC) Thymol Essential oils Achillea monocephala 100ºC, 125ºC, 150ºC, 175ºC/6 MPa 1,8-cineole,camphor, α-campholenal, borneol, terpinen-4-ol and manymore Essential oils Origanum micranthum 100ºC, 125ºC, 150ºC, 175ºC/4 to 8 MPa α-terpineol, linalool, borneol, terpinen-4ol, and many more Essential oils Fennel 50ºC to 200ºC (150ºC)/2 MPa α-pinene, mircene, limonene, camphor, phelandrene, anethol Phenolic antioxidants Sage 70ºC, 100ºC or 150ºC/10 MPa Rosmarinic acid, carnosol, carnosic acid, methyl carnosate Antioxidant compounds Rosemary leaves 25°C, 100°C, 150°C, 200°C and 100°C, 150°C, Scutellarein,rosmanol, genkwanin, 200°Cc/6 MPa carnosol, carnosic acid, NI 1 Fragrance and flavor Peppermint 150°C, 175°C and 50°C, 100°C, 125°C, 150°C, Carvone,pulegone, eucalyptol, menthone, compounds 175°C, 200°Cc/6 MPa neomenthol, menthol, menthyl acetate Fragrance and flavor Savory 100°C, 150°C, 175°C and 50°C, 100°C, 125°C, p-cymene, thymol, carvacrol, linalool, compounds 150°C, 175ºC, 200ºCc/6 MPa borneol, thymoquinone Fragrance and flavors Rosemary 125ºC, 150ºC, 175ºC α-pinene, limonene, camphene, camphor, 1,8-cineole, borneol Essential oils

Marjoram

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100ºC – 175ºC (150ºC)a/2–20 MPa

Reference 13 18 44, 22 43

17 49 45 21 50 31 23 23 24

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TABLE 5.2 Applications of PLPW Technology to the Extraction of Plant Bioactives

Rosa canina

50ºC, 100ºC, 150°C/2.5, 5, 7.5 MPa

Eldelberry, raspberry, bilberry, chokeberry

110°C–160°C (100°C–120°C)

Anthocyanins and phenolics Phenolics/lignans

Grape skin

100°C to 160°C by 10°C

Whole flaxseed

100°C, 120°C, 140°C, 160°C/5 MPa

Lignans, proteins

Flaxseed meal

130°C, 160°C, 190°C/5 MPa

Saponins Kava lactones

Cow cockle Kava root

125°C, 175°C/5 MPa 25°C, 50°C, 100°C to 200°C by 25°C/6–7 MPa

a

Benzaldehyde, benzyl alcohol, phenylethyl 46 alcohol, eicosane, and more Phenolic acids, ellagic acid, catechin, 48 epicatechin, resveratrol, quercetin, kaempferol, anthocyanins Monoglucoside and acylated anthocyanins 29 SDG d, p-coumaric acid glucoside, ferulic acid glucoside SDGd proteins, carbohydrates Saponins, cyclopeptides Dihydrokavain, kavain, desmethoxyyangonin, yangonin, dihydromethysticin

40 25 41 39

Selected temperature in brackets; b ethenyl-α,α-4trimethyl-3-(1-methylethenyl)-cyclohexanemethanol; c sequential equal time extractions at listed temperatures of one sample load; d SDG Secoisolariciresinol diglucoside.

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Fragrance and flavor compounds Anthocyanins

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100

Yield (%)

80

60

40

Carvone Menthol Menthyl acetate β-caryophyllene

20

0 50

100 150 Temperature (ºC)

200

FIGURE 5.4 Effect of temperature on pressurized low polarity water extraction of volatile compounds from peppermint (adapted from Kubátová et al.23).

was lower than the amount achieved at 140ºC; and there was a lower SDG percentage in the seed residue at 160ºC, which may be attributed to degradation of phenolics. At temperatures higher than 160ºC degradation of SDG also occurred in PLPW extractions of bioactives from flaxmeal. However, the detrimental effect of temperature could be alleviated when a high rate of extraction removes most of the bioactives in a relatively short time. Thus, SDG yields of 96% to 98% were reached at temperature as high as 190ºC when the rate of the extraction was increased by the increase of pH to 9.25 Equilibrium time and maximum recovery were reached in less than 100 min in extractions avoiding degradation of SDG. Elevated temperature increases the extraction rate, which in turn reduces the volume of water required for the extraction and the time to reach maximum recovery. The increase of the extraction temperature increased the extraction rate of lignans from flaxmeal, thus the volume of water required for the extraction and the time to reach maximum recovery were reduced. A raise of temperature from 160ºC to 190ºC reduced the extraction time by half.25 Increased extraction rates produced by temperature raises have been reported for the extraction of kava lactones from kava roots39 and flavor and fragrance compounds from savory and peppermint,23 rosemary,24 and clove.44 However, for some plant material and targeted compounds the raise in temperature cannot be unlimited. The combination of high temperatures and long time of extraction must be avoided when temperature sensitive bioactives are being extracted. Ju and Howard28 reported that extraction temperatures higher than 100°C resulted in anthocyanin degradation in pressurized liquid extraction, which was especially marked at 140°C. Reductions from 24% to 40% of the total anthocyanins content have been reported with pure and modified PLPW in the range of temperatures from 110ºC to 160°C.29 It has been demonstrated that most phenolic compounds react easily at high

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temperature (65ºC) when they are in contact with air. However, when higher temperatures are applied under nitrogen atmosphere, there are no degradations, since the degradation process for phenolic compounds is an oxidative process requiring the presence of oxygen.42 At high temperatures, dark brown PLPW extracts with strong burning smell and increased viscosity have been found in extractions from parsley,30 flaxmeal,25 Origanum micranthum,45 oregano,17 and leaves and flowers of Achillea monocephala.49 This has been attributed to the presence of browning reaction products such as furfural, acetylfuran, and 5-methylfurfural in the extracts of flowers of Achillea monocephala at 175ºC.49 However, the degradation in PLPW extractions is lower than the effects measured at atmospheric pressure extractions even at lower temperatures. Thus, PLPW allows for the use of temperatures higher than those used in conventional extraction techniques, probably because PLPW requires shorter extraction times and an oxygen-reduced environment. The effect of pressure in PLPW extraction of bioactives from diverse plant material has also been reported. Rovio et al.44 studied the effect of four pressures at two temperatures in PLPW extraction of flavor and fragrance from clove. No significant differences were found for the recoveries of eugenol and eugenyl acetate from clove at 25, 50, 100, and 175 kg/cm2 (2.4, 4.9, 9.8, and 17.2 MPa). Similar responses have been reported on PLPW extractions from Rosa canina,46 oregano,17 Origanum micranthum,45 and marjoram.13 These results are in agreement with the change of water dielectric constant with an increase of pressure. As it has been mentioned above, an increase of pressure of 590 MPa (from 10 to 600 MPa at 25°C) results in a small increase of dielectric constant from 79 to 93.14

5.3.2 FRACTIONATION

OF

COMPOUNDS

OF

DIFFERENT POLARITY

The extraction performance of a compound in a PLPW system is related to the polarity of the compound. Extraction yields of major phenolics present in flaxseed were not markedly different, indicating that they have a similar polarity. This suggests that fractionation of the major phenolic compounds in flaxseed using a PLPW system may not be viable. However, fractionation of phenolic compounds has been achieved in black currants and parsley.30 A sequential-temperature extraction of frozen black currant particles was performed in a PLPW system at temperatures from 80ºC to 240ºC. The total phenolic concentration of the extracts decreased from 80ºC to 120ºC and the yield of the extraction increased until it reached a plateau. However, when the temperature was further increased up to 240ºC, the concentration and the yield increased continuously with the temperature (Figure 5.5). The concentration of anthocyanins decreased continuously to zero and the yield increased to reach equilibrium and remained at those values even after further temperature increase up to 240ºC. HPLC chromatograms of samples collected at 80ºC, 120ºC, and 200ºC (Figure 5.6) indicate that high polarity compounds were extracted at the initial lower temperatures, and their content in the extraction cell decreased with further extraction. Although the identification of the compounds being extracted was not pursued, the increase in yield at the highest temperatures could be attributed to the extraction of newly generated high polarity compounds by hydrolysis of low polarity polymeric compounds or to the extraction of different molecules that

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1000

5000

Concentration (mg/L)

4000

TPhen Anthoc Yield (mg/100 g)

600

3000

TPhen Anthoc

400

2000

200

1000

0

Yield (mg/100 g)

Concentration (mg/L)

800

0 50

100

150 200 Temperature (ºC)

300

250

FIGURE 5.5 Total phenolic and anthocyanic yield and extract concentration in pressurized low polarity water sequential-temperature extractions from frozen black currant at 80ºC to 240ºC.

(C) 200°C

(B) 120°C

(A) 80°C

0

10

20

30 40 50 Retention time (min)

60

70

80

FIGURE 5.6 HPLC chromatograms at 280 nm of frozen black currant extracts collected at 80ºC, 120ºC, and 200ºC in pressurized low polarity water sequential-temperature extractions.

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otherwise would not be extracted. Extractions of those compounds are indicated by the appearance of new peaks at the beginning of the 280 nm chromatograms of the extracts collected at 200ºC and 240ºC (Figure 5.6). On the other hand, anthocyanins were extracted continuously and completely with extractions at 80ºC, 120ºC, and 200ºC and no new compounds that absorb at 525 nm were extracted with further increase of temperature. These observations clearly suggest that by suitably controlling the extraction temperature and the collection of the extracted fractions, PLPW extraction can be used for the recovery or fractionation of compounds of different polarity. Extractions of phenolics from grape seeds using PLPW42 and antioxidant compounds from rosemary produced similar results.31 In these studies, depending upon the temperature used, there were large differences in both the identity and recovery rate of the phenolic compounds extracted. In grape seed extraction at 150ºC, catechin increased by 32% and epicatechin increased by 99% over the recovery from the 50ºC extraction. There were also some compounds that were not detected in the extracts produced at 50ºC and 100ºC but which were detected in the extracts obtained at 150ºC. It was considered that the use of higher extraction temperature helped in the breaking of bonds between the analytes and the matrix, increasing yields of some compounds or extracting compounds that otherwise would not be extracted. In rosemary extractions, when water was heated to 200ºC, the dielectric constant of water was reduced and the solubilities of less polar compounds increased by several orders of magnitude, changing the composition of the extracts.31 Similarly, Palma et al.42 reported that the behavior of the analytes depended on the matrix, and the analytes in grape seeds were more strongly bonded to the matrix than in grape skin. Therefore, for grape skins shorter extraction times and lower temperatures would result in significant increase of yield and fractionation of phenolics.42 The temperature of the water affects the extraction in two ways: first by changing the dielectric constant of the water and thus the solubility of targeted compounds, and second by breaking the interactions between the analytes and the matrix. Production of essential oils richer in oxygenated fragrance compounds of more value and with less contaminant monoterpenes and sesquiterpenes from peppermint can be produced by adequate selection of extraction temperature (Figure 5.4). Oxygenated compounds extract at substantially lower water temperatures than nonoxygenated compounds.23 Extraction at 50ºC to 100ºC will result in extracts richer on valuable oxygenated compounds with a low fraction (< 5%) of terpenes such as pcymene or β-caryophyllene. Similarly, desirable carnosol-rich rosemary extracts can be produced by PLPW extraction of rosemary leaves at 100ºC. Furthermore, by using such a procedure it is possible to obtain enriched extracts with very high antioxidant activity.31 Also, continuous PLPW extraction of oil from marjoram resulted in extracts richer in odoriferous and hence more valuable oxygenated compounds than hydrodistilled oils, which contained 11 to 22 times larger amounts of monoterpenes.13 Therefore, extracts produced by PLPW extractions can give a higher-quality and higher-value product with more intense characteristic natural aroma.

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5.4 MODELING OF PLPW EXTRACTION OF BIOACTIVES FROM PLANT MATERIALS Two simple models have been applied to describe the extraction profiles obtained with PLPW extraction.51 The first of these models is based on the thermodynamic distribution coefficient (KD), which assumes that analyte release from the matrix is rapid compared to elution; and the second model is a two-site kinetic model which assumes that the extraction rate is limited by the analyte release rate from the matrix, and is not limited by the thermodynamic (KD) partition that occurs during elution. The two models are defined by the following equations51 Thermodynamic model: Sa (1– ) Sb Sa 0 S = + S0  KDm  S0  (Vb − Va )d + 1  

(5.1)

ST = 1 − [ Fe− k 1t ] − [(1 − F )e− k 2 t ] S0

(5.2)

Kinetic model:

Sa: cumulative mass of the analyte extracted after volume Va (mL) Sb: cumulative mass of the analyte extracted after volume Vb S0: initial total mass of analyte in the matrix. Sb /S0 and Sa /S0: cumulative fraction of the analyte extracted by the fluid of the volume Vb and Va ST: mass of the analyte removed by the extraction fluid after time t KD: distribution coefficient; concentration in matrix/concentration in fluid F: fraction of the analyte released quickly k1 and k2: first order rate constant (mL–1) for the quickly and slowly released fractions d: density of extraction fluid at given condition (g/mL) m: mass of the extracted sample (g) The kinetic model does not include solvent volume, but relies solely on extraction time. Therefore, doubling the extractant flow rate should have little effect on the extraction efficiency per unit time if the extraction efficiency is controlled by the kinetics of the initial desorption step (assuming the other extraction parameters remain constant). On the contrary, the thermodynamic model is only dependent on volume of extractant used. Therefore, the mechanism of thermodynamic elution and desorption kinetic can be compared simply by changing the flow rate in PLPW extraction. If the concentration of bioactive compounds increases proportionally with increase in flow rate at certain extraction time, the extraction mechanism can be

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explained by the thermodynamic model. However, if the increase of flow rate has no significant effect on the extraction of the bioactive compounds, with the other extraction parameters kept constant, the extraction mechanism can be modeled by kinetic diffusion.40,51 In this case, plots for several flow rates of the extracted amount or yield of the analyte as a function of the extraction time must lie on the same line indicating no effect of the flow rate. The mechanism of control and therefore the model valid for PLPW extraction may be different depending on the raw material, the targeted analyte, and extraction conditions. Thus, phenolic extractions from flaxseed performed at flow rates from 1 to 4 mL/min were affected by the flow rate, which indicates that the mass transfer of the solute from the surface of the solid into the bulk of the water regulated most of the extraction process in a similar way to the thermodynamic model. However, there was no difference in the extraction rate among phenolics extractions performed at 1, 0.5, and 0.3 mL/min, which were not affected by the flow rate. Thus, extractions at low flow rates would have been controlled by the diffusion in the seeds, as the two-site kinetic model establishes above. It has been suggested that at low flow rates (<1 mL/min), Biot number may have increased to values higher than 50 to 100; in this condition the effect of the boundary layer is negligible: the extraction would be controlled by the diffusion inside the seeds and not show any effect of the flow rate. Also, no effect of the flow rate has been found in extractions of SDG, proteins, and carbohydrates from flaxmeal.40 The data from PLPW extraction for savory, presented in Figure 5.7, clearly show that the extraction rates of essential oils had an increase proportional to the water

100

Yield (%)

80

60

40 0.25 mL/min 0.5 mL/min 1 mL/min 2 mL/min 4 mL/min

20

0 0

10

20

30 Time (min)

40

50

60

FIGURE 5.7 Effect of flow rate on thymol yields in pressurized low polarity water extractions from savory at 100ºC (adapted from Kubátová et al.51).

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flow rate, and thus the thermodynamic elution of analytes from the matrix is the prevailing mechanism as evidenced by the fact that extraction rates increased proportionally with the water flow rate. This is also confirmed by the fact that simple removal calculations based on a single KD (for each essential oil compound) give good fits to experimental data for flow rates from 0.25 to 4 mL /min.51 The increased recovery rates directly proportional to extraction flow rates show that high water flow rates can be used to shorten PLPW extraction times without increasing the amounts of fluid needed to achieve a high recovery.51

5.5 CONCLUSIONS Pressurized low polarity water (PLPW) extraction, also known as subcritical water extraction, is a highly promising “green” technology for the extraction and fractionation of biologically active compounds for use as functional food ingredients and nutraceuticals. PLPW has been applied to extract a variety of phytochemicals from a wide range of plant species including antioxidants from rosemary and yams, anthocyanins from berries and red grape skin, ginsenosides from American ginseng, catechins and epicatechin from tea leaves and grape seeds, essential oil from oregano, kava lactones from kava roots, lignans from flaxseed and flaxseed meal, and saponins from cow cockle (Saponaria vaccaria) seed. PLPW extraction has been compared and reported to be superior to conventional extraction techniques including solidliquid extraction,18,21 hydrodistillation, and supercritical CO2 extraction. Key benefits include higher selectivity, cleanliness, speed, and cost savings of both raw material and energy.

REFERENCES 1. Liu, R.H., Potential synergy of phytochemicals in cancer prevention: mechanism of action, J. Nutr., 134, 3479S–3485S, 2004. 2. Luque de Castro, M.D., Jimenez Carmona, M.M., and Fernandez Perez, V., Towards more rational techniques for the isolation of valuable essential oils from plants, Trends in Analyt. Chem., 18(11), 708–716, 1999. 3. Mazza, G., Cacace, J.E., and Kay, C.D., Methods of analysis for anthocyanins in plants and biological fluids, J. AOAC Intl., 87(1), 129–145, 2004. 4. Gertenbach, D.D., Solid-liquid extraction technologies for manufacturing nutraceuticals, in Functional Foods: Biochemical and Processing Aspects, Volume 2, Eds. John Shi, G. Mazza, and Marc Le Maguer, CRC Press, Boca Raton, FL, 2002, pp. 331–366. 5. Zhang, R., Xu, Y., and Shi, Y., The extracting technology of flavonoids compounds, Food Mach., (1), 21–22, 2003. 6. Garcia Viguera, C., Zafrilla, P., and Tomas Barberan, F.A., The use of acetone as an extraction solvent for anthocyanins from strawberry fruit, Phytochem. Anal., 9(6), 274–277, 1998. 7. Kahkonen, M.P., Hopia, A.I., and Heinonen, M., Berry phenolics and their antioxidant activity, J. Agric. Food Chem., 49(8), 4076–4082, 2001.

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8. Amen Chen, C., Pakdel, H., and Roy, C., Separation of phenols from Eucalyptus wood tar, Biomass Bioenergy, 13(1–2), 25–37, 1997. 9. Dix, B. and Marutzky, R., Tannin extracts from spruce and pine barks, J. Applied Polymer Sci., Applied Polymer Symposium 40, 91–100, 1984. 10. Eliasson, C., Kamal Eldin, A., Andersson, R., and Aman, P., High performance liquid chromatographic analysis of secoisolariciresinol diglucoside and hydroxycinnamic acid glucosides in flaxseed by alkaline extraction, J. Chromatog. A., 1012(2), 151–159, 2003. 11. King, J.W., Advances in critical fluid technology for food processing, Food Sci. Tech. Today, 14(4), 186–191, 2000. 12. Kaufmann, B. and Christen, P., Recent extraction techniques for natural products: microwave-assisted extraction and pressurised solvent extraction, Phytochem. Anal., 13(2), 105–113, 2002. 13. Jimenez Carmona, M.M., Ubera, J.L., and Luque de Castro, M.D., Comparison of continuous subcritical water extraction and hydrodistillation of marjoram essential oil, J. Chromatog. A., 855(2), 625–632, 1999. 14. Haar, L., Gallagher, J.S., and Kell, G.S., NBC/NRC Steam Tables: Thermodynamic and Transport Properties and Computer Programs for Vapor and Liquid States of Water in SI Units, Hemisphere Publishing Corporation, Washington, 1984. 15. Yang, Y., Belghasi, M., Lagadec, A., Miller, D.J. and Hawthorne, S.B., Elution of organic solutes from different polarity sorbents using subcritical water, J. Chromatog. A., 810, 149–159, 1998. 16. Miller, D.J. and Hawthorne, S.B., Solubility of liquid organic flavor and fragrance compounds in subcritical (hot/liquid) water from 298K to 473K, J. Chem. Eng. Data, 45, 315–318, 2000. 17. Soto Ayala, R. and Luque de Castro, M.D., Continuous subcritical water extraction as a useful tool for isolation of edible essential oils, Food Chem., 75(1), 109–113, 2001. 18. Fernandez Perez, V., Jimenez Carmona, M.M. and Luque de Castro, M.D., An approach to the static-dynamic subcritical water extraction of laurel essential oil: comparison with conventional techniques, Analyst, 125(3), 481–485, 2000. 19. Hawthorne, S.B., Grabanski, C.B., Martin, E., and Miller, D.J., Comparisons of Soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction for environmental solids: recovery, selectivity and effects on sample matrix, J. Chromatog. A., 892(1–2), 421–433, 2000. 20. Jimenez Carmona, M.M. and Luque de Castro, M.D., Isolation of eucalyptus essential oil for GC-MS analysis by extraction with subcritical water, Chromatographia, 50(9/10), 578–582, 1999. 21. Gamiz Gracia, L. and Luque de Castro, M.D., Continuous subcritical water extraction of medicinal plant essential oil: comparison with conventional techniques, Talanta, 51(6), 1179–1185, 2000. 22. Clifford, A.A., Basile, A., and Al Saidi, S.H.R., A comparison of the extraction of clove buds with supercritical carbon dioxide and superheated water, Fresenius' J. Analyt. Chem., 364(7), 635–637, 1999. 23. Kubátová, A., Lagadec, A.J.M., Miller, D.J., and Hawthorne, S.B., Selective extraction of oxygenates from savory and peppermint using subcritical water, Flavour Fragrance J., 16(1), 64–73, 2001. 24. Basile, A, Jimenez Carmona, M.M., and Clifford, A.A., Extraction of rosemary by superheated water, J. Agric. Food Chem., 46(12), 5204–5209, 1998.

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25. Ho, C.H.L., Cacace, J.E., and Mazza, G., Extraction of lignans from flaxseed meal with pressurized low polarity hot water, LWT-Food Science and Technology (in press), 2006. 26. Wanasundara, P.K.J.P.D. and Shahidi, F., Removal of flaxseed mucilage by chemical and enzymatic treatments, Food Chem., 59, 47–55, 1997. 27. Oomah, B.D., Mazza, G., and Cui, W., Optimization of protein extraction from flaxseed meal, Food Res. Intl., 27(4), 355–361, 1994. 28. Ju, Z.Y. and Howard, L.R., Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin, J. Agric. Food Chem., 51(18), 5207–5213, 2003. 29. Ju, Z.Y. and Howard, L.R., Subcritical water and sulfured water extraction of anthocyanins and other phenolics from dried red grape skin, J. Food Sci., 70, 270–276, 2005. 30. Mazza, G. and Cacace, J.E., Extraction of phytochemicals, U.S. Patent pending, 2006. 31. Ibañez, E., Kubátová, A., Señoráns, F.J., Cavero, S., Reglero, G., and Hawthorne, S.B., Subcritical water extraction of antioxidant compounds from rosemary plants, J. Agric. Food Chem., 51(2), 375–382, 2003. 32. Chen, P.Y., Tu, Y.X., Wu, C.T., Jong, T.T., and Chang, C.M.J., Continuous hot pressurized solvent extraction of 1,1-diphenyl-2-picrylhydrazyl free radical scavenging compounds from Taiwan yams (Dioscorea alata)., J. Agric. Food Chem., 52, 1945–1949, 2004. 33. Choi, M.P.K., Chan, K.K.C., Leung, H.W., and Huie, C.W., Pressurized liquid extraction of active ingredients (ginsenosides) from medicinal plants using non-ionic surfactant solutions, J. Chromatog. A., 983, 153–162, 2003. 34. Piñeiro, Z., Palma, M., and Barroso, C.G., Determination of catechins by means of extraction with pressurized liquids, J. Chromatog. A., 1026, 19–23, 2004. 35. Eller, F.J. and Taylor, S.L., Pressurized fluids for extraction of cedarwood oil from Juniperus virginianna, J. Agric. Food Chem., 52, 2335–2338, 2004. 36. Shotipruk, A., Kiatsongserm, J., Pavassnt, P., Goto, M., and Sasaki, M., Pressurized hot water extraction of anthraquinones from the roots of Morinda citrifolia, Biotechnol. Prog., 20, 1872–1875, 2004. 37. Ong, E.S. and Len, S.M., Evaluation of pressurized liquid extraction and pressurized hot water extraction for Tanshinone I and IIA in salvia miltiorrhiza using LC and LC-ESI-MS, J. Chromatog. Sci., 42, 211–216, 2004. 38. Lamm, L.J. and Yang, Y., Off-line coupling of subcritical water extraction with subcritical water chromatography via a sorbent trap and thermal desorption, Anal. Chem., 2237–2242, 2003. 39. Kubátová, A., Miller, D.J., and Hawthorne, S.B, Comparison of subcritical water and organic solvents for extracting kava lactones from kava root, J. Chromatog. A., 923, 187–194, 2001. 40. Cacace, J.E. and Mazza, G., Pressurized low polarity water extraction of lignans from whole flaxseed, J. Food Eng., in press, 2005. www.elsevier.com/locate/jfoodeng 41. Guclu-Ustundag, O. and Mazza, G., Pressurized low polarity water extraction of saponins from cow cockle (Saponaria Vaccaria) seed, AAFC-PARC internal report, Summerland, BC, Canada, 2006. 42. Palma, M., Piñeiro, Z., and Barroso, C.G., Stability of phenolic compounds during extraction with superheated solvents, J. Chromatog. A., 921, 169–174, 2001. 43. Ozel, M.Z., Gogus, F., and Lewis, A.C., Subcritical water extraction of essential oils from Thymbra spicata, Food Chem., 82(3), 381–386, 2003.

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44. Rovio, S., Hartonen, K., Holm, Y., Hiltunen, R., and Riekkola, M.L., Extraction of clove using pressurized hot water, Flav. Frag. J., 14(6), 399–404, 1999. 45. Gogus, F., Ozel, M.Z., and Lewis, A.C., Superheated water extraction of essential oils of Origanum micranthum, J. Chromatog. Sci., 43(2), 87–91, 2005. 46. Ozel, M.Z. and Clifford, A.A., Superheated water extraction of fragrance compounds from Rosa canina, Flav. Frag. J., 19(4), 354–359, 2004. 47. Ozel, M.Z. and Kaymaz, H., Superheated water extraction, steam distillation and Soxhlet extraction of essential oils of Origanum onites, Analyt. Bioanalyt. Chem., 379(7–8), 1127–1133, 2004. 48. King, J.W., Grabiel, R.D., and Wightman, J.D., Subcritical water extraction of anthocyanins from fruit berry substrates, Proc. 6th Intl. Sym. Supercritical Fl., tome 1, 409–418, 2003. 49. Gogus, F., Ozel, M.Z., and Lewis, A.C., Extraction of essential oils of leaves and flowers of Achillea monocephala by superheated water, Flav. Frag. J., In press, 2006. 50. Ollanketo, M., Peltoketo, A., Hartonen, K., Hiltunen, R., and Riekkola, M.L., Extraction of sage (Salvia officinalis L.) by pressurized hot water and conventional methods: antioxidant activity of the extracts, Eur. Food Res. Tech. 215(2), 158–163, 2002. 51. Kubátová, A., Jansen, B., Vaudoisot, J.F., and Hawthorne, S.B., Thermodynamic and kinetic models for the extraction of essential oil from savory and polycyclic aromatic hydrocarbons from soil with hot (subcritical) water and supercritical CO2, J. Chromatog. A., 975(1), 175–188, 2002.

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Purification of Orange Peel Oil and Oil Phase by Vacuum Distillation Mércia de Fátima M. Bettini Flavor Tec – Aromas de Frutas Ltd, Brazil

CONTENTS 6.1

Introduction.................................................................................................157 6.1.1 Steam Distillation.............................................................................158 6.1.2 Simple Distillation and Fractional Distillation................................158 6.1.3 Vacuum Fractional Distillation ........................................................159 6.1.3.1 Significant Reduction of Thermal Hazard .......................159 6.1.3.2 Greater Fractionation Efficiency ......................................160 6.1.3.3 Enhanced Purity of Distillate ...........................................160 6.2 Industrial Scale Purification of Orange Peel Oil.........................................160 6.2.1 Citrus Industry..................................................................................160 6.2.2 Sweet Orange Oil.............................................................................161 6.2.2.1 Purification of Orange Peel Oil........................................161 6.2.2.2 Method of Deterpenation — Vacuum Fractional Distillation .......................................................162 6.2.2.3 Folded Oils........................................................................162 6.2.2.4 Uses and Applications ......................................................163 6.2.2.5 Experimental Results ........................................................163 6.3 Conclusions ..................................................................................................170 References..............................................................................................................172

6.1

INTRODUCTION

Distillation is a process of heating a substance until the most volatile constituents change into the vapor phase, and then cooling the vapors to recover the constituents in liquid form by condensation. The main purpose of distillation is to separate a mixture into individual components by taking advantage of their different level of volatilities. Distillation is one of the main methods of extracting essential oils from plants. The percentage of each constituent in the vapor phase usually depends on

157

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its vapor pressure at a certain temperature. The principle of vacuum distillation may be applied to substances such as oils that would be damaged by overheating by the conventional method [1]. There are different methods of distillation, depending on the desired product; some of these processes are described below.

6.1.1 STEAM DISTILLATION Steam distillation is one of the common methods of distillation. Although steam distillation appears to be the best method for extracting essential oils, supercritical CO2 extraction is becoming quite popular. Most aromatherapists believe that essential oils are only effective when extracted by steam distillation and all other essential oils should be labeled with their extraction methods, that is, cold pressed oils, concretes, absolutes, and so forth. These preparations should not be confused with “true” essential oils. In the steam distillation process the material to be extracted is carefully gathered and placed into a copper or stainless steel vat, known as the distillation chamber. Steam is then generated by heating (preferably low heat) to build pressure within the distillation chamber causing the glands of the plant to rupture and release its essence which is then carried by the steam vapor. The steamy vapor is cooled and condensed in a coil, which is usually submerged in refrigerated cold water. The water and essential oil separate and the essential oils usually stay on top of the water due to their lower densities. The water and essential oil are sent to a collection chamber where the oil is easily skimmed off the top of the distillated water. The distillated water may contain many low water soluble components of the plant. These by-products of the distillation process are known as “hydrosol” and can be used for skin care, usually for children and the elderly. When subjecting a plant material to heat or steam as in the distillation process, the physicochemical properties of the material may be altered. The thermolabil components are degraded and here the information given by some authors with regard to the essential oils may be flawed. To achieve effective aromatherapeutic benefits, it is important to recognize that the extracted essential oils are not always in the same form when they were in the plant. Therefore, when subjecting plant materials to heat or steam as in the distillation process, the chemical structures within the essential oil after distillation are different than those found in the original plant material. For instance, chamomile is converted to the chemical azulene, which is not found in the plant itself but is produced during distillation [2]. When designing equipment for high vacuum process, parameters such as pressures, pipe sizes, and vessel sizes, must be adequate to avoid excessive vapor speed and pressure drops. The entire system must be free of leaks [3], and compensation for the excess pressure drop (2 or 3 mm Hg) across the entire system must be incorporated into the design process.

6.1.2 SIMPLE DISTILLATION

AND

FRACTIONAL DISTILLATION

If one desires to get a pure compound, the distillation process has to be repeated many times and this will make it a cumbersome process. In practice, the vapors are

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condensed and revaporized in fractionating columns connected sequentially in series. Equilibrium between liquid and vapor presumably occur on each plate. However, it is not likely that equilibrium is reached on each plate, therefore, it is imperative to calculate the plate efficiency, an important design factor. The column is packed with an inert material to promote contact between the liquid and the vapor. One of the most important design factors in packed columns is to prevent channeling — a condition where the vapors and liquid flow in opposite directions without coming into contact. The number of theoretical plates is determined under a specific set of operating conditions during the construction of the column. The efficiency of design is referred to as the Height Equivalent per Theoretical Plate (HETP). When all the vapors that go up through the column condense and flow back down, the returning liquid is called the reflux. The total reflux consists of the total product received from the outlet when no distillate is produced. As soon as the product is drawn from the condensed vapors, the amount of liquid returning through the column is reduced and produces changes to the operating conditions of the column. If all the condensed vapors are drawn off, then there will be no downflow to wash the upcoming vapors and in effect the column would stop functioning altogether and become just a long pipe. However, most columns would retain some amount of reflux and, for practical considerations, there would be some heat losses through radiation and convection along the column [3].

6.1.3 VACUUM FRACTIONAL DISTILLATION Vacuum distillation is a method of distilling substances at temperatures below their normal boiling point (standard atmospheric pressure). By reducing the pressure, a much lower distillation temperature will be required. High vacuum distillation may be used for certain classes of chemical compounds that decompose, polymerize, react, or are destroyed by conventional distillation methods. Low cost per pound and high throughput may be obtained on certain groups of compounds such as vitamins, epoxy resins, highly concentrated pure fatty acids, plasticizers, fatty acid nitrogen compounds, and a host of other heat-sensitive materials which may require only deodorizing and decolorizing. The pharmaceutical industry, in their continuing search for new products with high and intermediate molecular weights, finds that it is still an invaluable commercial practice to separate products by molecular weight from excess reactants and catalysts. 6.1.3.1 Significant Reduction of Thermal Hazard High vacuum distillation is a safe process to separate mixtures of organic or silicon compounds, most of which cannot withstand prolonged heating above 250ºC without excessive structural change or decomposition. As opposed to conventional atmospheric and pressure packed distillation towers, the high vacuum process utilizes the heat of condensation as a source of radiant heat emission to heat the surface film on the evaporator. With short residence time and lower distilling temperatures, thermal hazard to the organic material is greatly reduced.

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6.1.3.2 Greater Fractionation Efficiency The best approach to a mechanical method of creating a uniform film thickness with a fast-moving liquid is to feed the liquid to the center of a heated spinning disc. By centrifugal action, the material spreads as a film across the heated disc that intimately faces a large condensing surface. The lighter compound evaporates and condenses in a fraction of a second. The heavier residues that are not evaporated slide off the outer edge of the disc into a concentric residue collector and are discharged. The degree of separation is a function of the molecular weight differences of the distilled mixture. The greater the difference in the molecular weights, the purer the distillate. The closer the molecular weights of the mixture are, the less efficient is the fractionation process, resulting in the need for successive runs of the distillate. 6.1.3.3 Enhanced Purity of Distillate Purity of the distillate also depends on the film thickness. Controlling positive pressure and supply to the heated evaporator surface will usually provide a uniform film throughout the distillation. The absence of air molecules in the high vacuum distillation column permits most of the distilling molecules to reach the condenser with relatively few molecules returning to the liquid film surface in the evaporator. Experimental results show a relationship between the molecular weight and distillation temperature for a broad range of different materials. There is no predictable temperature at which distillation may occur; condensation begins whenever a sufficient temperature difference occurs between the evaporator and condenser. There is an infinite number of sets of operating conditions for every material that is fed through the system. Since pressure is constant, the only variables are flow rate (governing film thickness) and evaporator temperature [4].

6.2 INDUSTRIAL SCALE PURIFICATION OF ORANGE PEEL OIL The orange peel oil and essences are produced by the orange juice industry, as will be explained in Sections 6.2.1, citrus industry and 6.2.2.1, purification of orange peel oil. With the objective of preparing products rich in aromatic compounds, compounds that are very important for flavor, the deterpenation process on those oils and essences will be discussed in Sections 6.2.2.2 and 6.2.2.3.

6.2.1 CITRUS INDUSTRY Citrus juice processing generates a large amount of wastes with almost 50% of the fruit weight ending up as waste in the form of peel, segment, membranes, rags, and seeds. Besides that, during juice concentration, volatile constituents contained in the vapor phase are condensed by the aroma recovery systems. The depletion of these flavoring components results in decreased juice quality. Unless these compounds

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are reincorporated back into the juice, the nutritional value will be diminished. The aroma recovery system consists of two separate phases: essence oil (oil phase) and aqueous essences (water phase). The average yield of these products is very low. All these products are rich in aldehydes, esters, and other special volatile compounds. As the storage and transportation of these products are expensive, the evaluation of the concentration processes is of great interest [5]. It is very important that the special light fractions and the concentration of flavoring volatile compounds from the oil phase and peel oil be obtained by vacuum fractional distillation.

6.2.2 SWEET ORANGE OIL Expressed sweet orange oil is primarily used for flavoring beverages, soft drinks, ice cream, sweets, pharmaceutical preparations, and also perfumes. It contains limonene, minor amounts of other monoterpene hydrocarbons, linalol terpineol, aliphatic alcohols, and aldehydes such as octanal, nonanal, decanal, traces of the sesquiterpenic aldehydes and alpha and beta sinensal, methyl anthranilate, coumarins, and waxes. For flavoring foods and beverages, the so-called concentrated terpeneless oils or folded oils are preferred and are commercially available. 6.2.2.1 Purification of Orange Peel Oil Sweet orange oil is expressed from the fresh peels of ripe citrus fruitsaurantium L.var.dulcis (Citrus sinensis Os beck) (fam. Rutaceae). The tree probably originated from the Far East between the Himalayas and Southwest China. It is now cultivated in the United States (California, Texas, and Florida), throughout the Mediterranean region (southern Italy, Sicily, Spain, Algeria, Tunisia, Morocco, Israel, and Cyprus), South Africa, Brazil, and the West Indies. The main producers of the equipment that expresses oil are the United States and Cyprus, whereas the hand-pressed equipment is produced in Guinea [6]. Many investigators have pointed out that the quality of citrus oils is dependent upon many factors such as soil type, weather, method of extraction, and maturity of the fruit. Citrus oils are located in the oval, balloon-shaped oil sacs or vesicles located in the outer ring or flavedo of the fruit. Winton and Winton [1] described the exact location of these oil sacs in their discussion of the microscopic structure of the flavedo of the orange. To extract the oil from the peel of citrus fruits, the oil sacs must be ruptured by either pressure or rasping. The methods used in Florida were investigated by von Loesecke and Pulley [6] and they revealed that extraction method had an effect on the quality of the oil [7]. Cold-pressed citrus oils may be further processed to remove all or part of the terpenes and sesquiterpenes. The resulting products are known as terpenefree, terpeneless, or sesquiterpeneless oils. For products with low sesquiterpene concentrations, the term terpeneless is generaly used. Citrus oils from which terpenes have been removed are also called folded oils as the remaining flavorful oxygenated compounds are more concentrated. The degree of concentration is often calculated

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from the ratio of the principal constituent in the concentrated oil to that in the prime oil from which it was made. 6.2.2.2 Method of Deterpenation — Vacuum Fractional Distillation The bulk of terpeneless oils are produced by fractional distillation under high vacuum (1 to 2 mm Hg) so that the oil boils at a relatively low temperature to ensure minimum damage to the heat sensitive constituents during the long distillation period. At these pressures the boiling points of the constituents may be too low for efficient separation, but as the terpenes are rarely separated individually, this is usually a design problem, and reflux balance can be readily solved if necessary. The aromatic vapors rise through a fractionating column that enables fractions at defined boiling ranges to be separated and collected. Depending on the nature of the starting material and the desired character of the end product, the various fractions may be reblended as necessary and any unwanted fractions rejected [8]. 6.2.2.3 Folded Oils Folded orange oil is the concentrated product obtained from the winterized oil of fresh oranges. The process used will remove unneeded materials while retaining the highly aromatic components. Folded oils are several times more potent than the original oil and maintain the best fragrance. The two primary commercial varieties of folded orange oil are 5-fold and 10-fold. The higher the fold (distillation) the more concentrated is the finished product. Stable emulsions are more easily formed with folded oils since they required less oil to produce the desired flavor and fragrance levels [9]. The oil is primarily used as a flavoring agent for both foods, beverages, and pharmaceutical products, and is 10 times stronger than the usual orange oil. Concentrated fractions with high contents of fragrant compounds such as ethylbutyrate, valencene, aldehyde C 10, and others, can be obtained. Their smell and taste, that come from the original esters and aldehydes of the raw materials, are very good. Cold-pressed orange and grapefruit oils contain about 90% d-limonene. D-limonene is a hydrocarbon and has the following structure CH3

H2C

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CH3

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D-limonene boils at 178ºC, and it is generally thought that d-limonene contributes very little to citrus flavor. However the oil flavor and taste can be varied by adjusting the d-limonene concentration. In general, the term five-fold oil is used when starting with 5 lb of cold-pressed orange oil and distilling 4 lb; the remaining 1 lb is referred to as five-fold oil. The folding of oils is surrounded by secrecy and mysticism and various flavor houses have developed their own methods. Some use solvent extraction, usually about 70% ethanol followed by chilling to separate the extracted oil. Since d-limonene boils at about 27ºC and 2 mm Hg, and reliable mechanical vacuum pumps are available, it is possible to fractionate d-limonene at 5 to 10 mm Hg absolute pressure. This is probably the most common method used today. There are some low boiling point esters and aldehydes that are distilled with d-limonene, and their separation and recovery is presently the subject of research. 6.2.2.4 Uses and Applications Folded orange oils are used as a food ingredient and also as a flavor enhancer in beverages. They can be incorporated into new and existing formulations to enhance fragance and color. A wide array of industrial applications from general purpose cleaning to air fresheners have been enhanced by the addition of folded orange oils [10]. Original citrus oils are characterized by the presence of large percentages of terpenes (C10/H16) and smaller amounts of sesquiterpenes (C15/H24). Both of them, original and folded oils, carry oxygenated compounds comprising alcohols, aldehydes, ketones, acids, and esters, which are responsible for the characteristic odor and flavor profiles. The terpenoid composition of various original citrus oils is similar, and their principal component is d-limonene. The terpenes possess little intrinsic odor or flavor value but it would be incorrect to say that they have no flavoring effect. An original citrus oil from which the terpenes have been removed is significantly flatter and lacks the characteristic freshness associated with a complete peel oil [8]. When part of its terpenes is removed, folded oils will be obtained. 6.2.2.5 Experimental Results Figure 6.1 illustrates the industrial production of essences, as oil and water phases. In one of the stages of the evaporator, there is a column where the volatile compounds, the oil and water phase, are evaporated and recovered in a tank. The Essence Recovery System is connected to TASTE evaporators (Thermally Short Time Evaporator) which have the first juice stage in the first evaporator effect (that is, the first stage is the hottest effect or boiler steam effect). The essence-bearing vapors are “boiled-off” with the water vapors in the first stage. The vapors are condensed in the shell side of the second stage (second effect). The essence-bearing vapors rise to the top of the shell side of the second stage and through a series of baffles in the tube nest to remove as much condensable water vapor as possible. The noncondensable portion of these vapors is removed through the vent line at the top

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Back pres regulator & strainer Product & noncondensable cooler

Ammonia surge drum

Noncondensable Water out Dual pressure Relief valve

Overhead condenser

Reﬂux chamber

Sporian UMC-DA-5 Liquid level control

Matering valve

Barometric isolation valve Oﬀ

Atm steam

Water out valve Column vacuum gauge

On

Hot water to hot well

No 2 T/N vacuum Essence vacuum gauge

Bottom dump

NH3 liquid out

2 T/N

NH3 liquid in

Scrubber

Stripping cal & reboiler

Condenser

Essence recovery column assy

Flow meter 500 Gal cold wat tank

Ammonia surge drum

Essence storage tank

Water To hot well

Botton seal tank

Essence valve

Water to baro cond Hot well

Steam header To essence vacuum gauge

FIGURE 6.1 Recovery system of water phase plus oil phase in the orange juice industry.

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Barometric condenser

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of the tube, which is connected to the barometric condenser with a bypass for the essence system. This is also repeated in the next tube. When the essence system is “on” the noncondensable vapors are routed to the essence system through remote-controlled air-operated valves, and the vent line to the barometric condenser is shut off. The essence vapors enter the essence system at the vent condensers. The vent condenser condenses approximately 90% to 95% of the essence product. Cold water is used as a condensing medium. The essence product (condensed) flows downward through a single line to a dirt trap.There are two lines running up from the trap: one line is the essence product line running to the product chillers, the second line is the reflux line, which is used to control the strength of the essence. The amount of reflux flow is dependent on the desired strength of the water phase essence. The reflux rate is controlled only by the amount of product taken from the system. There is a constant amount of essence product generated by the overhead condenser, the essence product that does not continue through the system flows back to the tube nest as reflux. The essence product flows from the dirt trap to the double-circuit chiller condenser. This unit uses flooded NH3 refrigeration on the shell side. One circuit of the chiller-condenser is used to chill the essence product, and the other circuit is used to further condense the noncondensable vapors that rise from the vent condenser. The second circuit (noncondensable) of the chiller condenser is connected to the bottom of the refrigerated condenser. This condenser is a vertical shell and tube heat exchanger using flooded NH3 on the shell side. The noncondensable vapors flow through the tubes in a final condensing stage. The chilled product flows to the decanter assembly. The remaining noncondensable vapors flow upward and into the evaporator barometric condenser. The product lines from the bottom of the scrubber and the final condenser run to the decanter assembly located in the cold wall storage tank. The product line from the bottom of the scrubber to the decanter assembly has a flow meter located in the line. This flow meter measures product flow into the decanter assembly. The essence system product flow is created by vacuum generated by the evaporator barometric condenser. There is a continuous vacuum path from the evaporator tube to the evaporator barometric condenser, through each unit. The vacuum in the essence system is controlled by remote controlled valves [3]. The purification process of orange peel oil and orange essence oil at Flavor Tec is described by Figure 6.2. Peel oil, oil phase, and water phase, are by-products of the citrus processing plants. These products can be concentrated to obtain folded oils and folded essences (in the case of the oil phase and water phase), besides the special light fractions and terpenes. Figure 6.2 shows the flow diagram for the processing of raw materials at Flavor Tec. As shown, the light fractions and folded oils or essences, are processed by vacuum fractional distillation. As shown in the first step of Figure 6.2, the volatile compounds that will be the raw materials for the FTNF (From The Named Fruit) aromas, are obtained from the natural fruit in the citrus processing plant, as follow: the peel oil comes from the peel in a special process of washing, cleaning, centrifugation, and dewaxing. The

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Raw material (fruit) Volatile compounds - peel oil, oil phase, water phase Distillation/fractional process

Terpenes

Folded oils/folded essences

Light fractions

Selling

FTNF aromas Analytical evaluations

Shelf-life study

Juice addition Shelf-life study

FTNF = From the named fruit

FIGURE 6.2 Citrus oils concentration at Flavor Tec by distillation.

oil phase and water phase are also recovered by the citrus juice process, as shown in Figure 6.1. In the Flavor Tec process these raw materials feed the distillation equipment to produce the folded oils or folded essences, plus terpenes and light fractions by high vacuum fractional distillation (second and third steps in Figure 6.2). By blending the different percentages of the folded oils, folded essences, and light fractions, the FTNF aromas are produced at Flavor Tec. The percentage of each of the compounds, will change according to the needs of the final flavor, for example, freshness, sweetness, ripeness, and so forth. All the distilled products are evaluated by physicochemical and sensory analytical methods. After the analytical evaluations of all the obtained products are completed, their shelf life at room temperature and cold store will be studied. The shelf life of the FTNF aromas used in citrus juices will also be evaluated. Based on these evaluations it will be possible to guarantee the quality of the aromas, as well as their addition to the final feed. A typical industrial fractional vacuum distillation plant is shown in Figure 6.3. In order to increase the yield and quality of the final products at Flavor Tec, the distillator is continuously fed with raw materials 24 hours a day. Figure 6.4 shows the chromatographic profile of a 10-fold orange oil. Figure 6.5 shows the profile of the orange terpene obtained during the distillation process. The average yield of the terpenes on the process of a 10-fold oil is about 90% and about 80% on the process of a 5-fold oil. The purity grade of d-Limonene is a minimum of 96% by high-resolution gas chromatography (HRGC). The different

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FIGURE 6.3 Vacuum fractional distillation at Flavor Tec.

concentration of each component of single, 5-fold, 10-fold oils and terpene can be seen in Table 6.1. The values of the components as area percentage (area %) were analyzed by HRGC, using the following analytical conditions: Gas Chromatograph Shimadzu, 14BPFSC, 115/220 V AC (50/60 Hz) and 2 KVA max. capacity. Detector: Flame Ionization; T = 220ºC. Injector: Split / splitless (Split = 1:100);T = 200°C. Column: Capillary – Shimadzu, Type bonded phase. Phase: CBP1 (nonpolar). Size: 30m × 0.25 mm. Film thickness: 0.25 micron. Material: Fused silica. Temperature program = 40ºC to 190ºC (4ºC/min). The area percentage (HRGC) of each identified component of the orange essence oils, single, 5-fold, and 10-fold products, is listed in Table 6.2. There are more aromatic or fragrant components in the essence oil as compared to peel oil, as can be seen in Tables 6.1 and 6.2. It is therefore advisable to remember that the peel oil is obtained from the orange peel while the essence oil is from the concentration of the orange juice. The latter has fresher compounds than the former . Special attention might be given to the valencene content in the folded oil phases. This component is very important for the fruit taste characteristic. During the concentration process of the essence oil, special light fractions can be obtained, if processed under ideal conditions. Table 6.3 shows the concentration of one of these fractions, obtained from the essence oil (oil phase). It is a very light fraction because it is rich with top

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3 7

5

9

11 10 12

8

2 1 4

1 2 3 4 5 6 7 8 9 10 11 12

6

α - Pinene Sabinene Mircene + Octanal Phelandrene D – Limonene Nonanal Linalol Citronelal Decanal Neral Geranial Valencene

FIGURE 6.4 Chromatographic profile of 10-fold orange oil (HRGC).

note components, for example, ethyl-butyrate and octanal, as can be seen in Figure 6.6. Special attention might also be given to the high level of the light components, for example, hexanal and ethyl-butyrate, which are very important for the top notes of a flavor. The FTNF aromas are produced by blending the folded oils and oil phases plus the special light fractions, as show in Figure 6.2. At Flavor Tec, this blending is done with different fractions, as show in Figure 6.2. The FTNF aromas are produced by blending the folded oils and folded oil phases. It depends on the desired characteristics of the final flavor, for example, fruity, fresh, green, and so forth. As an example, Table 6.4 shows the concentration of some components of two different FTNF aromas called A and B. They are products of Flavor Tec, after concentration of the raw materials and blending with light fractions. The main differences between the aromas A and B are: A is fresher than B, as demonstrated by its ethyl-butyrate content, and B has more juice note, as shown in its valencene content. These special characteristics of each component will give unique flavor notes when added to the final food product.

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3 5

1 2

4

1 2 3 4 5 6 7 8

6

7 8

α-Pinene Sabinene Mircene + Octanal Phelandrene D-limonene Nonanal Linalol Citronelal

FIGURE 6.5 Chromatographic profile of orange terpene (d-limonene) (HRGC).

TABLE 6.1 Area Percentage by HRGC of the Identified Components of the Orange Peel Oils in Several Concentrations Evaluated Products (area %) Pick No.

Components

1 2 3 4 5 6 7 8 9 10 11 12

α-Pinene Sabinene Mircene + Octanal Phelandrene D-limonene Nonanal Linalol Citronelal Decanal Neral Geranial Valencene

© 2007 by Taylor & Francis Group, LLC

Single Oil 5-Fold Oil 10-Fold Oil Terpene 0.49 0.32 2.19 0.15 95.36 0.03 0.38 0.04 0.24 0.06 0.10 0.04

0.31 0.20 1.54 0.10 91.92 0.11 1.01 0.13 1.15 0.29 0.50 0.14

0.13 0.13 1.08 0.05 81.71 0.10 2.08 0.35 3.07 0.92 1.51 0.58

0.53 0.43 2.35 0.16 96.30 0.02 0.13 0.01 – – – –

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TABLE 6.2 Area Percentage by HRGC of Some Components of the Orange Essences or Orange Oil Phases in Several Concentrations Evaluated Products (area %) Pick No.

Components

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Hexanal Ethyl-butyrate T-2-hexanal α-Pinene Sabinene Mircene + Octanal Phelandrene D-limonene Nonanal Linalol Citronelal Decanal Neral Geranial Valencene

Single Oil Phase 5-Fold Oil Phase 10-Fold Oil Phase 0.01 0.03 0.01 0.38 0.34 2.17 0.08 94.57 0.10 0.70 0.01 0.30 0.11 0.10 0.23

0.29 0.14 0.02 0.93 0.34 1.46 0.09 85.75 0.09 2.13 0.13 1.21 0.54 0.49 2.31

0.71 0.33 0.06 2.10 0.67 2.30 0.12 68.20 0.14 2.78 0.57 3.70 2.04 1.00 4.08

TABLE 6.3 Area Percentage by HRGC of the Main Components of the Light Fraction of the Orange Oil Phase Pick No.

Components

Oil Phase Light Fraction (area %)

1 2 3 4 5 6 7 8

Hexanal Ethyl-butyrate T-2-Hexanal α-Pinene Sabinene Mircene + Octanal Phelandrene D-limonene

5.38 2.54 0.41 13.84 4.34 12.76 0.64 57.51

6.3 CONCLUSIONS Fractional distillation has been shown to be an efficient process. At high vacuum, it is a safer process from an operational point of view, but also much gentler on the raw materials, because of the short residence time and low distillation temperature. This results in better fragrant characteristics in the final products. To obtain topquality products, the raw materials, orange peel oil and oil phase, need to be well preserved from harvest until their use in the process.

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4 2 3

171

6

5 8 7

1 2 3 4 5 6 7 8

Hexanal Ethyl-butyrate T-2-hexanal α-Pinene Sabinene Mircene + Octanal Phelandrene D-Limonene

FIGURE 6.6 Chromatographic profile of special light fraction of orange oil phase (HRGC).

TABLE 6.4 FTNF Orange Aromas (A and B) HRGC Data Pick No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Components

Sample A (area %)

Acetaldehyde Hexanal Ethyl-butyrate T-2-hexanal α-Pinene Sabinene Mircene + Octanal Phelandrene D-limonene Nonanal Linalol Citronelal Decanal Neral Geranial Valencene

TR 0.27 0.18 0.10 1.23 0.60 2.98 0.17 87.36 0.05 1.02 0.12 1.38 0.40 0.52 0.53

© 2007 by Taylor & Francis Group, LLC

Sample B (area %) TR 0.10 0.05 0.05 0.74 0.28 1.91 0.09 86.08 0.10 1.70 0.17 1.80 0.54 0.80 1.17

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It is well known that single citrus oils and essences can come in large volumes from the industry. The storage and transportation costs of these products are high, which is another reason why more interest is been shown in the concentrated products. Research results have shown limonene to be a good candidate for human clinical chemoprevention trials. Figure 6.4, Figure 6.5, and Figure 6.6 with chromatograms and Table 6.1, Table 6.2, and Table 6.3 of the main components that were obtained from this work at Flavor Tec, show that the folded oils, essences, and the aromas, besides their fragrant components, also maintain good levels of dlimonene concentration when added to juice and other beverages. Although considerable differences have been observed with these preparations, wide acceptance by sensory panels has been achieved. It is very important to note that these folded oils and aromas are useful in both aspects: (1) as functional foods, because of the limonene content, and (2) the taste and fragrance, because of their fragrant compounds such as esters, aldehydes, and so forth. It is very important to bear in mind that the aroma is not a curative but will in general enhance food products.

REFERENCES 1. PE Chemical Engineering Review by Wagar Ahmad, PhD. Binary distillation weight and mole fractions, www.reviewpe.com/pe/samples/b6_7htm, (retrieved October 5, 2004). 2. Steam distillation. Infuse your life with excellence, www.serene-aromateraphy.de/ steamdistillation.html, (retrieved October 28, 2004). 3. Redd, J.B., Hendrix, D.L., and Hendrix, C.M.Jr., Process procedures, Quality Control Manual For Citrus Processing Plants, Ag Science, Inc., Auburndale, Florida vol II, section I, division B, 1992. 4. Myers Vacuum, Molecular distillation: the unique principle behind a unique process, www.myers-vacuum.com/mv1.html, (retrieved November 3, 2004). 5. Bettini, M.F.M., Orange concentrated essences by fractional distillation, presented at 25th IFU – Symposium, Stuttgart, Germany, May , 2004. 6. Formacek, V. and K.-H. Kubeczka, Orange oil, sweet, Essential Oils Analysis by Capillary Chromatography and Carbon-13 NMR Spectroscopy, John Wiley & Sons, New York, 185, 1982. 7. Kesterson, J.W., Hendrickson, R. and Braddock, R.J., Introduction, review of literature, Florida Citrus Oils, technical bulletin 749, Gainesville, FL, 3,1971. 8. Heath, H. B, London, England and Reineccius G., Dept. of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota, Flavor Chemistry and Technology, England, 1986. 9. Florida Chemical Company, Inc., Folded orange oil, www.d-limonene.com/ datasheets/foldedorangeoil.html, (retrieved October 6, 2004).

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7

Dehydration Technologies to Retain Bioactive Components Valérie Orsat and G. S. Vijaya Raghavan Macdonald Campus of McGill University

CONTENTS 7.1 Introduction ..................................................................................................173 7.2 Drying of Biological Materials....................................................................174 7.3 Heating of Biological Materials ..................................................................175 7.4 Quality Changes in Biological Materials ....................................................176 7.5 Artificial Drying...........................................................................................179 7.6 Convective Drying .......................................................................................180 7.7 Drum Drying ................................................................................................180 7.8 Spray Drying ................................................................................................181 7.9 Fluidized and Spouted Bed Drying .............................................................181 7.10 Freeze Drying...............................................................................................182 7.11 Vacuum Drying ............................................................................................183 7.12 Refractance Window Drying........................................................................183 7.13 Solar Energy in Drying................................................................................184 7.14 Combination Drying ....................................................................................184 7.15 Microwave-Vacuum Drying .........................................................................186 7.16 Heat Pump Hybrid Dryers ...........................................................................186 7.17 Conclusion....................................................................................................187 Summary ................................................................................................................187 Acknowledgment ...................................................................................................188 References..............................................................................................................188

7.1 INTRODUCTION The phenomenal growth in the use of phytochemicals in functional and nutraceutical foods forces the food industry to control the quality of those ingredients in terms of efficacy and safety of the ingredients claiming to be health promoting.1 The

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industry must ensure that there are governing guidelines during processing to ensure the nutritional and functional activity in the finished products. The choice of drying system depends upon the annual volume produced, the marketing pattern, the material to be dried, and the capacity and nature of existing facilities. Presently, most commercially available dryers are based on heated air (convection dryers); however, there has been substantial research aimed at adapting other technologies to agroproducts drying. Two general areas are involved: (1) improvement of heated air dryers to improve energy efficiency and eliminate overdrying (e.g., fluidized and spouted beds concepts), and (2) alternate modes of heat and mass transfer. Among the alternative modes are conduction drying, infrared drying, microwave drying, and heat pump drying. Although improving fuel efficiency is one of the main motivations for continued research, several other advantages may be sought. Among them are faster drying, better quality of dried products, and use of renewable energies; however, advances in one direction often require a trade-off somewhere else.

7.2 DRYING OF BIOLOGICAL MATERIALS The removal of moisture prevents the growth and reproduction of microorganisms which cause decay and minimizes many of the moisture driven deterioration reactions. Differences in relative humidity in the environment in which the food is kept can make differences in the rate of microorganism multiplication. Water activity is related to the relative humidity. Relative humidity, RH, is defined as the ratio of the partial pressure of water vapor in the air to the vapor pressure of pure water at the same temperature. Water activity, aw , is a property of solutions and is the ratio of vapor pressure of the solution compared with the vapor pressure of pure water at the same temperature. Under equilibrium conditions water activity equals RH/100. At the usual temperatures permitting microbial growth, most bacteria require a water activity in the range of about 0.90 to 1.00. Qualitatively, water activity is a measure of unbound, free water in a system available to support biological and chemical reactions. Water activity, not absolute water content, is what bacteria, enzymes, and chemical reactants encounter and is affected by at the microenvironmental level in food materials. Dehydration targets to lower the product’s aw to reach an equilibrium between product quality and product shelf life. The quality of the dried product and its cost are greatly influenced by the drying operation. The drying time, temperature, and water activity influence the quality of the final product. Low temperatures generally have a positive influence on the quality of biological materials, but require longer processing times, which have a detrimental effect on quality and a higher cost of operation. Low water activity retards or eliminates the growth of microorganisms, but results in higher lipid oxidation.2 Storage stability of a food product increases as the water activity decreases, and lipid-containing foods are susceptible to lipid oxidation at low water activities and must be stored in oxygen impermeable packages. In certain circumstances, there is poor color retention when the food is dried too rapidly. Other food materials have different drying and handling problems to which targeted solutions must be developed.2

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Drying is a process of simultaneous heat and mass transfer for moisture removal. External heat evaporates the surface moisture, while the internal moisture can be forced to the surface and then evaporated or it can be evaporated internally and then transported to the surface. The transfer of heat depends on the air temperature, air humidity, air flow rate, pressure, surface area, physical nature of the material, its composition, and the process by which the heat is transferred to the material whether it be by conduction, convection, or radiation. To speed up the heat and mass transfer in the fruits, vegetables, or plant materials to be dehydrated, they are cut into small pieces or thin layers. Increased surface area provides more contact with the heating medium and more surface from which moisture can escape. The greater the temperature gradient between the heating medium and the food, the greater will be the rate of heat transfer into the food, which provides the driving force for moisture removal. The hotter the air, the more moisture it will be able to carry away from the drying product. In convective drying, the drying rate is generally high at the beginning of the process since convective drying depends largely on the concentration of moisture at the surface. Afterward, the drying rate falls rapidly with gradual reduction of moisture. With thermal treatment under convection, the limitation resides in the fact that in order for the center of the material to reach the desired temperature, the surface may have to be overheated, leading to losses in product quality. This is where dielectric heating can benefit with its volumetric heating where selective absorption of energy by the water molecules takes place inside the product.

7.3 HEATING OF BIOLOGICAL MATERIALS In convection heating, the heat transfer occurs between the surface of a material and its surrounding fluid. The rate of transfer then depends on the properties of the fluid and the fluid flow. The characteristics of the fluid such as density, specific heat, and so forth, are expressed in the convective heat transfer coefficient which also takes into account the geometrical shape of the object undergoing heating. In conduction heating, the heat transfer mode involves energy transfer from regions of higher to lower temperatures by contact between matter. The rate of heat transfer is then proportional to the temperature gradient from the hotter region to the colder region and the capacity of the material to conduct the heat. Electroheat is defined as the branch of science and technology dealing with the transformation of electrical energy into thermal energy for a given purpose.3 Electrotechnology applications range over the entire electromagnetic spectrum, with ultraviolet radiation and x-rays in the high end (>109 MHz); infrared radiation (107 MHz); microwaves (915 to 22,125 MHz); radio frequency (0.5 to 900 MHz) and induction and resistance heating in the lower frequency range. Research interest in agrifood applications lies mostly in high-frequency applications with dielectric, induction, and resistance heating. High-frequency electromagnetic heat transfer, in the microwave and radio frequency range, can be considered as heat arising from the oscillation of molecular dipoles (microwave) and the movement of ionic constituents (radio frequency) in response to an alternating electric field. Unlike conventional energy, this energy is

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absorbed throughout the volume of the wet material. The evaporation of moisture within the material results in an increase of the internal pressure which can rapidly drive out the moisture from the interior of the solid. Microwave cooking/heating is the most rapidly expanding meal preparation technology in the world. Its effect on the functional activity of phytochemicals is not well defined and more research is needed in this area. Processing as a whole affects the content, activity, and availability of biological compounds, hence there is a need to better understand the mechanisms affecting the nutraceutical stability of functional foods. The degree to which a given material responds to an electromagnetic field depends on the frequency and intensity of the field and on the characteristics of the material itself. The electrical basis of interaction is described in terms of the dielectric properties: dielectric constant and dielectric loss factor. These, respectively, represent the proportion of impinging energy that can penetrate the material, and the amount of energy that can be absorbed. The dielectric properties of materials are of major importance in the consideration of high-frequency or microwave applications with biological materials. Fundamental studies aimed at characterizing the dielectric responses of complex biological materials over a wide range of frequencies, temperatures, and moisture contents are still required to be undertaken. Resistance or ohmic heating is the term used to describe direct electrical resistance of food products. The process allows the rapid, uniform heating for highquality processing of liquid food products. Direct resistance heating uses the fact that metallic materials, insulators, and foodstuffs are highly conductive at main frequencies so that the source energy is dissipated ohmically. In such applications, the material comes in direct contact with the electrodes. Induction heating on the other hand has not had many applications with biological materials but has rather been used in the metal industry. The majority of applicators for induction are based on coils wrapped around the workpiece. The current flowing in the coil establishes an axial magnetic flux which in turn sets up a circumferential current in the material, the depth of which depends on the operating frequency. The flowing current is responsible for imparting electrical energy to the material.4

7.4 QUALITY CHANGES IN BIOLOGICAL MATERIALS Conditions in drying and thermal processing influence greatly the quality of processed biological materials. Browning reactions occur in the presence of high heat with lower moisture causing decrease in nutritional value, textural changes, and overall quality. Color loss, shrinkage, denaturation, loss of aroma and flavor, vitamin loss, and textural changes are important quality changes affected by thermal processing. Degradation of nutrients, color, texture, and so forth during dehydration can be described by a first order model of the form: X = Xo exp (-kt), for constant processing conditions.5 Thermal processing has often been demonstrated to have a negative effect on the nutrient content. As the temperature of the product increases, the reaction rate constant is increased. As such, a low-temperature drying process results in lower nutrient degradation. Hence, a longer constant-rate drying period

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should be favored for quality retention. The drying process of choice must target to minimize chemical degradation reactions since loss of nutrient can be viewed as the decomposition of a particular chemical compound. Importance has to be given to the appearance and functional properties of the dried products.6 In a study conducted by Nindo et al.,7 it was demonstrated that the drying method significantly affects the antioxidant activity of dried asparagus. The drying methods studied were tray drying, spouted bed drying, microwave spouted bed, refractance window, and freeze drying; freeze drying and refractance window obtained the highest retention of total antioxidant activity. In the case of folate, its stability in dried food is mediated by the content of ascorbic acid. During thermal processing, levels of folate and ascorbic acids may both decrease depending on the intensity of the process. In a study conducted by Stralsjo et al.,8 folate retention during drying of rosehip berries was determined. Folate degradation was shown to be influenced by drying time and drying temperature. The duration of drying appears most important at higher water activity (greater than 0.75). The major chemical components of chamomile were studied after the flowers were dried by different drying methods, namely, sun drying, shade drying, and oven drying at 40°C.9 The drying methods studied had no effect on the different components present; however, the drying methods had a significant effect on the proportion of the various components and their retention. Shade drying yielded the highest oil yield, whereas oven drying was better suited for concentrating particular components such as isobutyl and isobutyrate since oven drying vaporized some of the more volatile components. In order to optimize practices for thermal processing and drying of biological materials, extensive research has been carried out around the world on various drying methods, such as fluidized bed drying, spouted bed drying, particulate medium conduction drying, flash drying, freeze drying, spray drying, drum drying, tunnel convective drying, tray drying, microwave drying, radio frequency drying, infrared drying, electric field drying, superheated steam drying, osmotic dehydration drying, conveyor dryers, impingement drying, solar drying, rotary drying, and so forth.10 All drying methods have been developed to optimize the drying of target materials, where not all drying processes are suitable for all types of biological materials.11 The quality of biological materials is so specific that drying of each product should be studied on an individual basis. In the case of biomaterials, their phytochemicals’ content and quality must be maintained during drying as research is demonstrating their ability to provide health benefits.1 Phytochemicals are very reactive and their content varies depending on plant cultivar, ripeness at harvest, storage conditions, and level of processing. Production of vegetal ingredients must ensure the preservation of their inherent quality attributes. In general, the concentration of bioactive compounds will steadily decrease during processing. The development of processing methods to minimize the loss of bioactive components is imperative.12 When processing plant materials, a variety of changes will occur to the antioxidants present. Changes in the functionality of the antioxidants depend on the

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energy transfer, oxygen availability, temperature, food composition, time, and light exposure.13 Retention of chlorophyll and ascorbic acid from cabbage samples air dried (60°C to 85°C) followed by microwave vacuum drying is higher than in samples air dried only. It is recognized that rapid drying can retain more chlorophyll and nutrients in the dried product.14 In drying with hot air, the processing time and temperature are typically long and high. The material’s natural protective layer is destroyed, exposing the antioxidants to the free access of air with rapid lipid oxidation which in turn destroys the functionality of antioxidants. Lipid oxidation and antioxidant losses can be reduced by short drying times, low temperatures, and low pressures.13 A study was conducted by Piga et al.15 on the influence of drying air temperature on the phenolic compounds and antioxidant activity of dried plums. High temperature drying destroyed anthocyanins and significantly reduced flavonoids and ascorbic acids. The analysis is, however, more complex when studying the functional properties of the dried foods since during processing the polyphenols in their intermediate stage of oxidation may have a greater antioxidant power than initially, and high temperature treatments may cause the formation of new compounds, or degradation components that have a higher antioxidant activity. In the case of thermodegradable vitamin C in wild-rose fruits (Cinebasti fructus), Jinescu et al.16 confirmed that the presence of a large amount of polyhydroxilic acids such as malic and citric acid improves the stability of the vitamin C during drying. Furthermore, it was determined that by allowing a greater maximum allowable moisture content of the dried product, lower process temperatures and longer drying times ensured the maximum retention of vitamin C. Similar results were obtained by da Silva et al.17 for hot air drying of camu-camu slices (Myrciaria dubia). The tree fruit berries were dried in a vertical tray dryer. The highest ascorbic acid retention of 78% was obtained with the lowest drying temperature of 50°C and highest final moisture content of 10% (wet basis). A predictive model was developed by Ramallo and Mascheroni18 for predicting vitamin C retention during hot air drying of pineapple slices. Excellent predictive agreement was obtained with maximal retention of ascorbic acid when the drying temperature was kept at the lowest temperature tested of 45°C. In a study on processing of tea leaves, it was clearly demonstrated that type and intensity of processing causes a significant effect on their total polyphenolic content and their antimutagenic activity. In the case of tea leaves, low intensity processing is recommended.19 There is no doubt that food processing, and specifically heating, affects the properties of processed products. However, it does not always have a detrimental effect. In the case of lycopene in the processing of tomatoes, the lycopene degradation occurs through isomerization and oxidation. Heat induced isomerization converts the all-trans lycopene to its cis form which has lower stability. However, lycopene absorption as a health promoting nutrient, is higher in the cis-isomeric form than in the unprocessed trans form.20 When considering a processing method, a good balance must be maintained between the stability and quality of the product and its functionality as a nutritious and health-promoting food ingredient.

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Some other interesting plant components are also favored by a thermal treatment. This is the case of taxol, a plant-based cancer chemotherapeutic agent used in the treatment of certain cancers. In a thin layer drying study of taxus clippings, it was demonstrated that the lowest yields of the plant component were obtained when drying took place at the lowest temperature setting of 30°C, while the highest yield was obtained with the highest temperature setting studied of 60°C.21

7.5 ARTIFICIAL DRYING Several types of dryers and drying methods, each better suited for a particular situation, are commercially used to remove moisture from a wide variety of food products and biomaterials. While sun drying of crops is still practiced for certain fruits such as plums, apricots, grapes, and dates, atmospheric dehydration processes are used for apples, plums, herbs, and several vegetables; continuous processes such as tunnel, belt through, fluidized bed, and foam-mat drying are mainly used for vegetables. Spray drying is suitable for fruit juice concentrates, and vacuum dehydration processes are useful for low moisture-high sugar fruits like peaches, pears, and apricots. The selection of a particular dryer or drying method depends on the type of raw material and its properties (Table 7.1), the desired characteristics of dried product, and the restrictions on the operating conditions and costs.

TABLE 7.1 Common Dryer Types for Liquid and Solid Foods Dryer Type

Usual Food Type

Air convection dryers Kiln Cabinet, tray or pan Tunnel Continuous conveyor belt Belt trough Air lift Fluidized bed Spray Drum or roller dryers Atmospheric Vacuum Vacuum shelf Vacuum belt Freeze dryers

Pieces Pieces Pieces, slurries, liquids Pieces Pieces, slurries, liquids Pieces Small pieces, granules Small pieces, granules Liquid, slurries Pieces and slurries Pieces, slurries, liquids Pieces, slurries, liquids Pieces, slurries, liquids Slurries, liquids Pieces, liquids

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There are three basic types of drying process: mainly, sun and solar drying; atmospheric drying, including batch or continuous (kiln, tower, and cabinet dryers, tunnel, belt, belt trough, fluidized bed, explosion puff, foam mat, spray, drum and microwave); and subatmospheric dehydration (vacuum shelf/belt/drum and freeze dryers).

7.6 CONVECTIVE DRYING Convective drying consists of passing heated air through layers of product. It can be conducted with tray or cabinet dryers, where perforated trays hold thin layers of materials. Tunnel dryers are similar to cabinet dryers, where trays of material are stacked and moved through a tunnel. For a continuous process, conveyor belts are used. A study conducted by Mendez et al.22 investigated the effect of convective hot air drying under constant conditions on garlic drying kinetics and quality. Garlic slices were dried in a conventional hot air tunnel, under constant conditions. Three air temperatures were compared (40°C, 50°C, and 60°C), in combination with two air velocities (1 and 2 m/s). Allicin and pyruvic acid contents were measured as an indicator of the effect of the process on garlic quality. The allicin content decreased with rising drying air temperature, and moderate temperatures (40°C and 50°C) allowed better allicin retention than did the higher temperature (60°C). In the case of air-dried bay leaves, no significant loss of quality was experienced when drying the leaves at 60°C.23 The level of thermal sensitivity is product dependent. The effect of drying temperature on the quality of dried Echinacea purpurea was determined in a study by Stuart and Wills.24 An increase in process temperature from 40°C to 70°C had a significant effect on the concentration of cichoric acid, whereas temperature had no effect on the concentration of alkylamides. Larrauri et al.25 studied the effect of drying temperatures (60°C, 100°C, and 140°C) on the polyphenol content and antioxidant activity of dried red grape pomace. When drying at higher temperatures, a significant reduction in total extractable polyphenols and condensed tannins was observed along with a reduction in total antioxidant activity. Drying at the lower temperature of 60°C did not significantly affect the functionality of the dried grape pomace.

7.7 DRUM DRYING In drum drying, heat is transferred to the material by conduction with heated revolving drums (Figure 7.1). The dried product is flaked off the drum with a scraper. Drum dryers have high drying rates and high energy efficiency. Typically, the temperature of the process is approaching the boiling point of water and the process time is in the order of seconds. This process is adequate for viscous or pureed foods that can withstand high temperatures for short time.26 A study was conducted by Hsu et al.27 to determine the antioxidant activity of yams dried by various methods, including drum drying. In their study, freeze drying, hot air drying (60°C) and drum drying (95°C) all reduced the antioxidant activity

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Liquid feed deposited between heated rotating drums Knife

Dry product

FIGURE 7.1 Schematic of drum drying process.

of the dried yam when compared to fresh yam. Drum drying, with its high temperature, had the highest reduction of total antioxidant activity.

7.8 SPRAY DRYING Spray drying is the transformation of a fluid into a dry particulate by spraying in a hot medium. Hot air spray drying takes place at temperatures between 150°C and 200°C. The dried product may take the shape of a powder, granules, or agglomerates depending on the physical and chemical properties of the fluid and the operating conditions of the spray drying process. Andrade and Flores28 studied the spray drying of roselle extract to exploit its importance as a source of pectin, anthocyanin, and ascorbic acid. Operating factors of importance are the feed rate, the inlet drying air temperature, and the pressure of the compressed air at the nozzle. Not all materials can be spray dried. In some cases the material requires a carrier, such as maltodextrine, to favor agglomeration and provide stability for the components the drying process wants to retain. In spray drying, as with other drying processes, the degradation of the phytocomponents follows a first order kinetic.29

7.9 FLUIDIZED AND SPOUTED BED DRYING Fluidized bed drying involves the fluidizing of particles in a flowing gas stream, typically heated air. With the fluidized motion of the particles in heated air, drying occurs rapidly. Fluidized bed drying offers a tight control of process and product temperatures necessary for processing heat sensitive plant and food products.30 An alternative to spray or fluidized bed drying for fluids has been investigated by Souza and Oliveira31 with the spouting of a bed of inert material with warm air and spraying the fluid to coat the material with a thin film which fragments into a powder as the material collides in the spouted bed (Figure 7.2). The advantage of this process is its simplicity and low cost while with the proper operating parameters, it can provide high quality products with low degradation. Souza et al.32 demonstrated that spouted bed drying produced end products with high concentration of active substances such

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07 06

150 mm

08

Caption 01 - Blower 02 - Valves 03 - Air heater 04 - Flow meter 05 - Spouted bed 06 - Atomizer 07 - Pressure taps 08 - Thermocouples

05

08 01

02

03

07 04

FIGURE 7.2 Schematic diagram of spouted bed dryer as tested by Souza et al.32

as total flavonoids and total tannins. An increase in the drying temperature increases the degradation of the flavonoid compounds.33

7.10 FREEZE DRYING Freeze drying operates at low temperatures and low pressures which can help reduce quality degradation. Under vacuum, frozen water changes directly into vapor, a process known as sublimation, yielding particular dried product characteristics. The food pieces are frozen and vacuum is applied in the airtight drying chamber. Thermal energy is then applied to the food pieces (by heated plates or microwaves) to promote the sublimation process. In freeze drying, the food remains at a low temperature yielding high retention of nutrients. Batungwanayo et al.34 studied freeze drying as a drying method to preserve and concentrate allicin in garlic. Cloves of fresh garlic were freeze dried either whole, or cut into halves and slices. Both freeze drying temperature and size of garlic samples had an important effect on drying kinetics. The allicin content of whole garlic cloves decreased with an increase of freezedrying temperature. However, for garlic slices the effect of the temperature was not significant. Excellent retention of allicin was experienced with whole garlic samples freeze dried at a temperature of 20°C. A freeze drying study of medicinal herbs demonstrated that the quality of freeze dried product was slightly lower than fresh material, but the quality remained considerably higher than for oven-dried samples at 35°C to 40°C.35

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To preserve higher levels of alkylamides from Echinacea purpurea, freeze drying was found to be the best drying method when compared with vacuum microwave drying, which was in turn a better drying method than air drying at 70°C for drying roots. In the case of leaves drying, air drying at 50°C was the best process for optimal retention of alkylamides.36

7.11 VACUUM DRYING In vacuum drying, the product is heated by steam, conduction (hot oil, heated elements, etc.), or radiation, while under low pressure. This drying process may offer product specific advantages such as low-temperature drying and low oxidation. Kutovoy et al.37 studied the vacuum drying of grape by-products. When dried under vacuum (500 mm Hg) at a temperature below 50°C, up to 95% of nutritious ingredients, vitamins, ferments, and other biologically active components were preserved.

7.12 REFRACTANCE WINDOW DRYING Refractance Window™ drying is a novel drying method patented by MCD Technologies Inc. (http://www.mcdtechnologiesinc.com). The process uses circulating water at 95°C to 97°C to carry heat to dehydrating materials which circulate in a thin layer on a conveyor belt (Figure 7.3). The drying process is similar to drum drying, except that the product temperature is much lower, between 70°C and 85°C compared to 120°C to 150°C with drum drying. Abonyi et al.38 conducted a study to determine the carotene losses in carrots dried using Refractance Window, freeze drying, and drum drying. Carotene losses were slightly, but not significantly, higher for Refractance Window than for freeze drying, whereas drum dried products experienced a considerable carotene loss caused by the more intense heating treatment. Product removal

Exhaust air

Moist air extraction

Cooling water

Blower Air Product application T

Cooling water out Cooling water in

T

Water heater

Mylar conveyor belt

Hot water pump T - Temperature sensor

FIGURE 7.3 Schematic diagram of Refractance Window™ System.

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7.13 SOLAR ENERGY IN DRYING In most tropical countries, a commercial dryer is too expensive to consider its purchase. In countries where harvesting time occurs at the beginning of the dry season, the most popular method of drying is exposure to the sun. Crops are often left to dry in the field before harvesting. In some countries various crops are dried on scaffolds or inverted latticework cones. At the village level, probably the most common practice is to spread the harvested product on the ground on a specially prepared area (e.g., matting, sacking, or concrete) exposed to the sun. An alternative that is being encouraged in hot, dry countries of Asia and Africa is solar drying. Solar heat is trapped with a solar collector constructed from an aluminum sheet painted black.39 The collector may be fixed to the drying bin in such a way that an air space exists between it and the bin wall. Energy absorbed by the collector heats the ventilating air by a few degrees as it is forced through the air space. Various solar energy dryers have been designed around the world. AbdelRehim and Fahmy40 developed a photovoltaic dryer with dual packed beds for drying medicinal herbs. Their dryer is equipped with electrical heaters powered by a photovoltaic system. Flat-type solar collector dryers have been designed in Poland with corrugated absorbers equipped with a backup coal-fired air heater. The solar dryer is equipped with variable speed fans to modulate the air flow according to transient changes in solar irradiance and heat output.41 With solar energy, like any other drying process, the temperature and duration of exposure to light have to be monitored to retain the functional properties specific to the products being dried.

7.14 COMBINATION DRYING Rapid drying of plant materials has always been problematic due to the high sensitivity of such products to heat. Indeed thermal treatments may have detrimental effects on quality characteristics such as color, flavor, texture, and nutritional value. For this reason, traditional methods of drying require modification and improvement. Combination of processes can favor complementary process benefits. The traditional methods of drying high-quality thermosensitive materials are sun drying (inexpensive, slow, and unpredictable), low temperature drying (slow), and freeze drying (expensive, high-quality products). The potential of microwaves to reduce drying time while preserving quality has been studied and demonstrated for a number of agrifood commodities.42,43 The combination of osmotic dehydration for preconcentration, with microwave drying, implemented with convective air has been investigated for a variety of fruits.43–46 The osmotic preconcentration can remove 50% of the initial weight of the fruits with dry sugar or concentrated syrup. Since the cost of drying depends on the amount of moisture removed, predehydration can significantly reduce the cost of dried material production. It has been demonstrated by Pan et al.47 that osmotic pretreatments can, in certain cases, preserve the quality of subsequently dried products with respect to nutrient retention, compared to direct drying. Microwaves further offer a great potential for energy savings (in comparison

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to freeze drying) due to the speed of drying and the direct coupling of the energy with the material. In a study conducted by Marcotte et al.,46 blueberries and cranberries were dried using a hybrid technology consisting of pretreatment, osmotic dehydration, and convective drying. The performance of this hybrid technology was evaluated in terms of quality attributes such as color, taste, rehydration ratio, and water activity as well as sugar and anthocyanin content. The most significant loss of anthocyanins took place during osmotic dehydration for both berries. This was due to intensive mass exchange between berries and the osmotic syrup. Convective drying at low temperatures gave dried berries of high quality. Nindo et al.7 studied the combined microwave and spouted bed drying of asparagus and compared the quality of the dried product with refractance window-dried, tray-dried, freeze-dried, and spouted-bed-dried products. Total antioxidant activity of the dried asparagus after refractance window and freeze drying was significantly higher than after tray drying, spouted-bed-drying, and combined microwave and spouted bed drying. When comparing only methods using heated air, it is the microwave spouted-bed-drying asparagus which retained the highest total antioxidant activity. Refractance window drying retained the highest level of ascorbic acid. Hybrid drying of strawberries was studied by Venkatachalapathy and Raghavan.44 Berries were dried to 15% moisture content (wet basis) using a procedure involving sample preparation, osmotic dehydration, and microwave-assisted drying leading to a dried berry that was comparable in quality to a freeze-dried berry in a much shorter time. Convection drying took 9 to 10 hours (Figure 7.4), compared to 2 to 3 hours for microwave-convection dried berries.44

Convection with treat 0.2 W/g Convection no treat

Moisture, % (dry basis)

1200 1000

0.1 W/g 0.3 W/g

800 600 400 200 0 0

90

180

270 360 450 Time, min

540

630

720

FIGURE 7.4 Drying strawberries by convection alone and by microwave-convective drying at three power levels.44

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7.15 MICROWAVE-VACUUM DRYING Further drying improvements can be obtained by using subatmospheric pressures. Water evaporation takes place at lower temperatures under vacuum, and hence the product processing temperature can be significantly lower, offering higher product quality. Many comparisons have been made between microwave-vacuum drying and other systems, mainly focusing on hot air and freeze drying. In a comparative study conducted by Popovich et al.,48 a detailed analysis of ginsenosides was conducted on ginseng dried with different methods. Different drying methods influence the retention of specific ginsenosides which all have distinct bioactivity. Freeze drying of ginseng proved to have greater Rg1 and Re retention, whereas, Rb1 and Rd ginsenosides were found in greater amounts in microwave-vacuum-dried ginseng. In a comparative study conducted by Sunjka et al.,43 microwave-vacuum drying of cranberries exhibited enhanced characteristics when compared to microwaveconvective drying. Drying performance results (defined as mass of evaporated water per unit of supplied energy) showed that microwave-vacuum drying is more energy efficient than microwave convective. Microwave-vacuum drying was compared with freeze drying, air drying, and a combination drying method of air drying and microwave-vacuum drying for preserving the antioxidant functional properties of Saskatoon berries.49 Drying yielded a considerable loss of anthocyanin. The best retention was obtained with freeze drying followed by microwave-vacuum drying, combination drying, and air drying (75°C).

7.16 HEAT PUMP HYBRID DRYERS Heat pump dryers have many advantages compared to the conventional air drying systems, such as improved product quality, reduced energy consumption. and reduced environmental incidence. An electrically driven heat pump shares the advantage of low temperature drying and manifests the same desirable characteristics of resistance heat, yet is considerably more energy efficient.50,51 The simplest drying method is to blow heated air over the moist material and to vent the moist air to atmosphere. All the latent heat carried in the water vapor is lost to the atmosphere. In the case of a heat pump, the moisture in the exhaust is condensed at the evaporator and the latent heat is recovered to the circulating airstream via the condenser.52 The heat pump, in a closed cycle, reclaims heat and uses it at a cost equal to that of operating the compressor motor. In a study conducted by Sosle et al.,53 various agriproducts such as apple, banana, onion, and potato rings were heat pump dried from 85% wet basis to 10% to 12% wet basis. The products obtained by the process were of exceptionally high quality. The color degradation was negligible and the flavor was free from any processinduced aberrations. The low temperature drying achieved in the equipment was less destructive toward the cell walls and led to a gradual, gentle collapse of the cells, which recovered their turgor remarkably upon rehydration.

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Novel applications of heat pump drying now include radio frequency54 or microwave energy.55 The ability of microwaves to selectively heat areas with higher dielectric loss factors is very important in this application. Unlike other conventional systems, dielectric drying has an inherent ability to “moisture level” a given product; that is, more power is applied to regions with higher moisture content than to regions with lower moisture content. The use of dielectric heating provides an efficient supply of energy required to speed up the process of moisture rejection from the material undergoing drying. This component overcomes some of the heat and mass transfer drawbacks of convective air drying.

7.17 CONCLUSION There exist a great number of processing choices for the dehydration of biomaterials and foods. The impact of each drying system is product specific in terms of end product quality and functionality of its components. The choice in processing method will depend on the intended end use of the product. Any given process can be detrimental to a certain product, with a significant reduction in the retention of its bioactive ingredients or their activity level. On the other hand, the exact same process can be suited to yield higher levels of health promoting bioactive compounds during the processing of a different plant material. The mechanisms of product degradation and bioactivity are intimately related to the processing conditions, principally time and temperature, and their control.

SUMMARY Drying is one of the oldest methods of food conservation practiced worldwide. The removal of moisture prevents the growth and reproduction of decay-causing microorganisms and minimizes many of the moisture driven deterioration reactions. Drying offers a substantial reduction in weight and volume thus minimizing storage and transportation costs and enabling storage of the product at ambient temperature for extended periods of time. The sharp rise in energy costs has favored a growing interest in the development of new or improved drying technologies. Traditionally, dehydration involves the application of heat to vaporize water combined with some means of removing the water vapor at the surface of the fruit or vegetable products. Drying is a combined heat and mass transfer operation for which energy is supplied. Several types of dryers and drying methods, each better suited for a particular situation, are commercially used to remove moisture from a wide variety of plant products including fruit and vegetables. This chapter presents them along with the most recent developments in drying research as they apply to product quality deterioration during dehydration. The appearance and functional properties of dried products are important in the choice of a drying process. The remaining antioxidant and functional activities of dried products promote their nutraceutical marketability.

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ACKNOWLEDGMENT The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada and Le Fonds Québéçois de la Recherche sur la Nature et les Technologies, for the research carried out in their laboratory.

REFERENCES 1. Dillard, C.J. and German, J.B., Phytochemicals: nutraceuticals and human health, J. Sci. Food Agric., 80(12), 1744–1756, 2000. 2. Okos, M.R., Narsimhan, G., Singh, R.K., and Weitnauer, A.C., Food dehydration, in Handbook of Food Engineering, Heldman and Lund, Eds., Marcel Dekker, New York, pp. 437–562, 1992. 3. Risman, P., Terminology and notation of microwave power and electromagnetic energy, J. Microwave Power Electromagn. Energy, 26(4), 1991. 4. Metaxas, A.C., Foundations of Electroheat: A Unified Approach, John Wiley and Sons, New York, 1996. 5. Van Loey, A.M., Smout, C., Indrawati, and Hendrickx, M.E., Kinetic data for biochemical and microbiological processes during thermal processing, in Engineering Properties of Foods, 3rd ed., M.A. Rao, S.S.H. Rizvi, and A.K. Datta, Eds., CRC Taylor & Francis, Boca Raton, FL, chap 13, pp. 611–643, 2005. 6. Prothon, F., Ahrne L., and Sjoholm, I., Mechanisms and prevention of plant tissue collapse during dehydration: a critical review, Critic. Rev. Food Sci. Nutr., 43(4), 447–479, 2003. 7. Nindo, C.I., Sun, T., Wang, S.W., Tang, J., and Powers, J.R., Evaluation of drying technologies for the retention of physical quality and antioxidants in asparagus, Lebensmittel-Wissenschaft und –Technologie, 36(5), 507–516, 2003. 8. Stralsjo, L., Alklint, C., Olsson, M.E., and Sjoholm, I., Total folate content and retention in Rosehyps (Rosa ssp.) after drying, J. Agric. Food Chem., 51(15), 4291–4295, 2003. 9. Omidbaigi, R., Sefidkon, F., and Kazemi F., Influence of drying methods on the essential oil content and composition of Roman chamomile, Flavr. Fragr. J., 19, 196–198, 2004. 10. Mujumdar, A.S., Ed., Handbook of Industrial Drying, 2nd ed., two vols., Marcel Dekker, New York, 1995. 11. Sadykov, R.A., Pobedimsky, D.G., and Bakhtiyarov, F.R., Drying of bioactive products: inactivation kinetics, Dry. Technol., 15(10), 2401–2420, 1997. 12. Tamura, Y., and Nishibe, S., Changes in the concentrations of bioreactive compounds in plantain leaves, J. Agric. Food Chem., 50(9), 2514–2518, 2002. 13. Pokorny, J. and Schmidt, S., The impact of food processing in phytochemicals: the case of antioxidants, in Phytochemical Functional Foods, CRC Press, Woodhead Publishing England, chap. 15, 2003. 14. Yanyang, X., Min, Z., Mujumdar, A.S., Le-Qun, Z., and Jin-Cai, S., Studies on hot air and microwave vacuum drying of wild cabbage, Dry. Technol., 22(9), 2201–2209, 2004. 15. Piga, A., Del Caro, A., and Corda, G., From plums to prunes: influence of drying parameters on polyphenols and antioxidant activity, J. of Agric. & Food Chem., 51(12), 3675–3681, 2003.

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16. Jinescu, G., Woinaroschy, A., Trebencu, C.-E., and Ionescu, E., Vitamin C degradation during the drying process of the granulated wild-rose extract, in Drying 2004: Proc. 14th Int. Dry. Symp., M.A. Silva and M.C.S. Rocha, Eds., State University of Campinas, São Paulo, Brazil, pp. 1128–1133, 2004. 17. da Silva, M.A., Arevalo-Pinedo, R. and Kieckbusch, T.G., Ascorbic acid thermal degradation during hot air drying of camu-camu (Myrciaria Dubia (H.B.K.) Mc Vaugh) slices at different air temperatures, in Drying 2004: Proc. 14th Int. Dry. Symp., M.A. Silva and M.C.S. Rocha Eds., State University of Campinas, pp. 1898–1905, 2004. 18. Ramallo, L.A. and Mascheroni, R.H., Prediction and determination of ascorbic acid content during pineapple drying, in Drying 2004: Proc. 14th Int. Dry. Symp., M.A. Silva and M.C.S. Rocha, Eds., State University of Campinas, pp. 1984–1991, 2004. 19. Standley, L., Winterton, P, Marnewick, J.L., Gelderblom, C.A., Joubert, E., and Britz, T.J., Influence of processing staged on antimutagenic and antioxidants potentials of rooibos tea, J. Agric. Food Chem., 49(1), 114–117, 2001. 20. Shi, J. and Le Maguer, M., Lycopene in tomatoes: chemical and physical properties affected by food processing, Critic. Rev. Biotechnol., 20(4), 293–334, 2000. 21. Hansen, R.C., Keener, H.M, and Elsohly, H.N., Thin layer drying of cultivated taxus clippings, Trans. ASAE, 36(6), 1873–1877, 1993. 22. Mendez, L., Makhlouf, J., and Ratti, C., Garlic quality properties during constant convective drying, in Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan, and T. Kudra, Eds., McGill University, Montreal, Paper D-6, 2005. 23. Demir, V., Gunham, T., Yageioglu, A.K., and Degirmencioglu, A., Mathematical modelling and the determination of some quality parameters of air-dried bay leaves, Biosys. Eng., 88(3), 325–335, 2004. 24. Stuart, D.L. and Wills, R.B.H., Effect of drying temperature on alkylamide and cichoric acid concentrations of Echinacea purpurea, J. Agric. Food Chem., 51(6), 1608–1610, 2003. 25. Larrauri, J.A., Rupérez, P., and Saura-Calixto, F., Effect of drying temperature on the stability of polyphenols and antioxidant activity of red grape pomace peels, J. Agric. Food Chem., 45(4), 1390–1393, 1997. 26. Tang, J. and Yang, T., Dehydrated vegetables: principles and systems, in Handbook of Vegetable Preservation and Processing, Y.H. Hui, S. Ghazala, D.M. Graham, K.D. Murrell, and W.K. Nip, Eds., Marcel Dekker, New York, chap. 21, pp. 335–372, 2004. 27. Hsu, C.L., Chen, W.L., Weng, Y.M., and Tseng, C.Y., Chemical composition, physical properties and antioxidant activities of yam flours as affected by different drying methods, Food Chem., 83(1), 85–92, 2003. 28. Andrade, I. and Flores, H., Optimization of spray drying of roselle extract (Hibiscus Sabdariffa L.), in Drying 2004: Proc. 14th Int. Dry. Symp., M.A. Silva and M.C.S. Rocha, Eds., State University of Campinas, pp. 597–604, 2004. 29. Robert, P., Carlsson, R.M., Romero, N., and Masson, L., Stability of spray dried encapsulated carotenoid pigments from rosa mosqueta (Rosa rubiginosa) oleoresin. J. Am. Oil Chem. Soc., 80(11), 1115–1120, 2003. 30. Chua, K.J. and Chou, S.K., New hybrid drying technologies, in Emerging Technologies for Food Processing, Da-Wen Sun, Ed., Elsevier Academic Press, Amsterdam, chap. 20, pp. 535–551, 2005.

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31. Souza, C.R.F. and Oliveira, W.P., Spouted bed drying of bauhinia fortifica link extract: effect of the position of the feed atomizer and operating conditions on equipment performance and on product properties, in Drying 2004: Proc. 14th Int. Dry. Symp., M.A. Silva and M.C.S. Rocha, Eds., State University of Campinas, pp. 1150–1157, 2004. 32. Souza, C.R.F., Bott, R.F., and Oliveira, W.P., Dryer performance and physico-chemical properties in spray and spouted bed drying of medicinal plants, in Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan, and T. Kudra, Eds., McGill University, Montreal, Paper VI-2, 2005. 33. Runha, F.P., Cordeiro, D.S., Pereira, C.A.M., Vilegas, J., and Oliveira, W.P., Production of dry extracts of medicinal Brazilian plants by spouted bed process: development of the process and evaluation of thermal degradation during the drying operation, Trans. Inst. Chem. Eng., 79, Part C, 160–168, 2001. 34. Batungwanayo, A., Araya M., and Ratti, C., Freeze-Drying of Garlic (A. Sativum) and its Effect on Allicin Retention, in Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan, and T. Kudra, Eds., McGill University, Montreal, Paper D-5, 2005. 35. Tambunan, A.H., Yudistra, Kisdiyani, and Hernani, Freeze drying characteristics of medicinal herbs, Dry. Technol., 19(2), 325–331, 2001. 36. Kim, H.-O., Durance, T.D., Scaman, C.H., and Kitts, D.D., Retention of alkamides in dried Echinacea purpurea, J. Agric. Food Chem., 48(9), 4187–4192, 2000. 37. Kutovoy, V.A., Muminov, M.I., Karimov, M., and Sandalov, V.N., Intensification of vacuum drying of bioproducts, In: Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan, and T. Kudra, Eds., McGill University, Montreal, Paper II-1, 2005. 38. Abonyi, B.I., Feng, H., Tang, J., Edwards, C.G., Chew, B.P., Mattinson, D.S., and Fellman, J.K., Quality retention in strawberry and carrot purees dried with Refractance Window™ System, J. Food Sci., 67(3), 1051–1056, 2002. 39. Sharma, V.K., Colangelo, A., and Spagna, G., Experimental investigation of difference solar dryers suitable for fruit and vegetable drying, Renew. Energy, 6(4), 413–424, 1995. 40. Abdel-Rehim, Z.S. and Fahmy, F.H., Photovoltaic dryer with dual packed beds for drying medical herb, Dry. Technol., 16(3), 799–811, 1998. 41. Wisniewski, G., Drying of medicinal plants with solar energy utilization, Dry. Technol., 15(6–8), 2015–2024, 1997. 42. Tulasidas, T.N., Raghavan, G.S.V., and Mujumdar, A.S., Microwave drying of grapes in a single mode cavity at 2450 MHz, I: drying kinetics; II: quality and energy aspects, Dry. Technol., 13(8–9), 1949–1992, 1995. 43. Sunjka, P.S., Rennie, T.J., Beaudry, C., and Raghavan, G.S.V., Microwave/convective and microwave/vacuum drying of cranberries: a comparative study, Dry. Technol., 22(5), 1217–1231, 2004. 44. Venkatachalapathy, K. and Raghavan, G.S.V., Combined osmotic and microwave drying of strawberries, Dry. Technol., 17(4–5), 837–853, 1999. 45. Beaudry, C., Raghavan, G.S.V., and Rennie, T.J., Microwave finish drying of osmotically dehydrated cranberries, Dry. Technol., 21(9), 1797–1810, 2003. 46. Marcotte, M., Grabowski, S., Quan, D., Taherian, A.R., Zareifard, M.R., Poirier, M., and Kudra, T., Quality aspects of canadian blueberries and cranberries dried by osmoconvective method, in Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan, and T. Kudra, Eds., McGill University, Montreal, Paper IV-2, 2005. 47. Pan, Y.K., Zhao, L.J., Zhang, Y., Chen, G., and Mujumdar, A.S., Osmotic dehydration pretreatment in drying of fruits and vegetables, Dry. Technol., 21(6), 1101–1114, 2003.

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Membrane Separation Technology in Processing Bioactive Components Ashwani Kumar National Research Council of Canada

CONTENTS 8.1 8.2 8.3

Introduction ..................................................................................................193 Membrane Basics.........................................................................................194 Applications .................................................................................................195 8.3.1 Preconcentration...............................................................................196 8.3.2 Fractionation.....................................................................................197 8.3.3 Hybrid Processes..............................................................................199 8.3.4 New Membrane Processes ...............................................................201 8.3.5 Cleaning Chemical and Solvent Recycling .....................................203 8.4 Summary ......................................................................................................204 Acknowledgments..................................................................................................205 References..............................................................................................................205

8.1 INTRODUCTION In recent years there has been a lot of interest in functional foods and nutraceuticals, particularly their impact on human health and prevention of certain diseases. Manufacturers have responded to this sudden demand by releasing a large number of products. However, there is a lot of scope of research and development in making safe, reliable, and effective products from complex matrices of natural products. In addition to growing good-quality natural products, there are challenges in harvesting, postharvest treatment, extraction, refining, analysis of active ingredients, and safety before an effective and reliable product is made. Although all of the above activities

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play an important role in making a good product, special attention needs to be focused on separation and purification mainly due to the fact that 40% to 80% of the cost of production is assigned to separation and purification unit operations in making functional foods and nutraceuticals. Therefore, a lot of research effort has gone into developing efficient extraction and separation schemes. This chapter will mainly focus on refining and purification of bioactive compounds from natural products using membrane-based separation schemes. Membrane-based processes are usually more energy efficient than distillation, adsorption, and chromatography. Furthermore, membranes have the advantage of compatibility with a wide range of solvents and chemical products, ability to process thermally sensitive compounds, easy amenability to automation, smaller footprint, and seamless scale up. These advantages open up several possibilities of membrane application in the production of bioactive compounds in the areas of energy efficient preconcentration of dilute solutions, fractionation of diverse classes of compounds from a complex mixture, recovery of intermediates, and recycling of solvents. However, it is also important to be aware of the challenges posed by membranebased separation schemes, such as limited selectivity, fouling leading to performance decline, cleaning, and higher capital costs in certain installations. This chapter is divided in to three sections dealing with membrane basics, applications, and final remarks.

8.2 MEMBRANE BASICS Membranes could be described as barriers that provide selective and controlled passage of one component over the other from a multicomponent mixture. Membranes have been classified by pore sizes, materials, driving forces and devices, even specific applications. The basic definitions and principles of operation have been described in textbooks.1,2,3 The membranes are usually made of polymeric, ceramic, and metallic materials and are semipermeable to mixture components. These can be in flat sheet, spirals, hollow fibers, and tubular form and can be housed in specially designed housing or modules for applications. Membranes are often used in cross-flow filtration mode and permeation or rejection of a particular component mainly depends on the pore size, morphology, and interactions of feed components with membrane materials. A membrane’s ability to reject a solute is often expressed in molecular weight cutoff, which is determined by sieving experiments with standard solutes of varying molecular weights. A brief summary of the main types of membranes and their characteristics is given in Table 8.1 for those processes that are most relevant for functional foods and nutraceutical applications. In processing complex matrices of natural products, the extracts usually contain many dissolved and suspended species with varying physical and chemical properties. The separation and purification steps are complex. Considering that a variety of membrane-based processes that operate on different principles are available (Table 8.1), one can envision that these processes could have an effective role in separation schemes. Based on the knowledge of the characteristics of extracts and species of interest, one can select a suitable membrane process. The process development generally involves screening of commercially available membranes under different

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TABLE 8.1 Membrane Processes and Their Characteristics Membrane Process

Driving Force

Molecular Pore Sizes, Weight Cutoff, nm Dalton

Pervaporation

Vacuum

<1

—

Reverse Osmosis Nanofiltration

Pressure

<1

Low

Pressure

1–10

100–1000

Ultrafiltration

Pressure

10–100

1000–100,000

Microfiltration Pressure

Nonvolatile or affinity basis Ions, large molecules Multivalent ions, large molecules

Species Permeated Volatile or affinity basis Water, alcohol

— —

Non-ionic solutes Nonvolatile solute

Monovalent ions, water, alcohol Ions, water, alcohol, small molecules Some macromolecules, ions, water, alcohol Ionic solutes Water

—

Nonextractable species

Extractable species

100–10,000 —

Electrodialysis Electric current — Osmotic Partial pressure — distillation difference Membrane Concentration — solvent gradient extraction

Species Retained

Macromolecules, pathogens Large macromolecules, some pathogens

operating variables such as cross-flow velocities, permeate rate, operating pressure, and temperature. Longer-term process evaluation is also used to establish membrane cleaning schedules for restoring performance. This basic laboratory data is utilized to run pilot-scale experiments to ascertain the overall viability of the process for a particular application. These laboratory experiments and analysis of collected data are well described, particularly for food applications.1,3

8.3 APPLICATIONS It is obvious from the description given in Section 8.2 that separation characteristics of membranes are dependent not only on the physicochemical properties of the membrane but also on the feed characteristics and operating variables. This observation also provides a broad basis for developing applications of membranes in separation and purification to suit particular requirements of bioactive compounds from a complex mixture. The following sections present a summary of membranebased separation processes.

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8.3.1 PRECONCENTRATION Most of the process streams in the functional food and nutraceutical industries have very small amounts of dissolved solids of interest. Irrespective of the extracting solvent, a preconcentration step is essential prior to drying and making the final products. In traditional processes this step is usually thermal in nature, which is accomplished by thermal evaporation or distillation. Energy consumption for removing 1 kg of water from dilute solutions is listed in Table 8.2. It is obvious that a membrane-based preconcentration is very energy efficient. Furthermore, membranebased processing is suitable for thermally sensitive nutraceuticals and functional foods and helps in maintaining the natural taste of the final product by avoiding the heat-induced taste profile changes. In a U.S. Department of Energy study, it was clearly pointed out that current usage of membrane-based separation schemes in the food processing industry will expand and have a significant impact on reducing energy consumption.4 Concentration of several food products, such as fruit juices, maple sap, seafood cooker water, and corn light steep water, using reverse osmosis membranes have been reported.5,6,7 However, it should be noted that these applications are limited by the osmotic pressure of the dissolved species and fouling of membranes. Concentration applications are implemented more effectively using membrane-based separation as part of the overall process. Similar applications in nutraceuticals and functional foods are possible; however, published reports are scant. Ekanayake et al.8 patented a process describing the concentration of green tea extract with nanofiltration. These inventors claimed superior quality of the membrane-concentrated product in masking the taste of artificial sweeteners in beverages. Gugger and Grabiel9 utilized temperature-dependent solubilities of soy isoflavones for concentration by ultrafiltration membranes. The target compound in the feed stream was first adsorbed on a resin. The eluted isoflavone fractions were treated with either reverse osmosis or ultrafiltration to remove solvent and achieve a higher isoflavone concentration in the retentate, which was spray dried to make the final product. Recently, a similar approach for concentration of isoflavones from soybean process waste and extract of red clover flowers has been reported by Xu et al.10,11 In these studies concentration by membranes played an important role in making the final product.

TABLE 8.2 Energy Consumption in Preconcentration of Dilute Solutions by Different Unit Operations (Kumar et al. 1999) Unit Operation

Energy Consumption, kJ/kg Water Removed

Thermal evaporation Mechanical vapor recompression (MVR) Membranes

2100 350 90

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8.3.2 FRACTIONATION Considering that most nutraceuticals and functional foods are produced from complex matrices such as grains, botanicals, and fermentation broths, isolation and purification of the desired active components is very important for product efficacy. A membrane-based separation scheme has vast potential to make an impact in this area, particularly where compounds of interest are of comparable molecular weight, have similar chemical makeup, and impurities are significantly different from the target compounds. Several studies utilizing membranes in fractionation of food products from grains, meat, and poultry have been described.3,4 Recently the separation of individual whey proteins (alpha-lactalbumin and betalactoglobulin) for use as nutraceuticals was reported. This laboratory study utilized both cellulosic and polyethersulfone membranes and reported the optimal filtration conditions, showing that a diafiltration process yielded beta-lactoglobulin as the retentate product with a purification factor of 100 and recovery of 90%. Alphalactoalbumin was recovered in the filtrate with a purification factor of more than 10 and nearly 99% yield.12 A product made from oat extract in water/alcohol mixture and fractionated by membranes to get the active ingredient avenanthramides in reasonably high concentration for skin-care products was patented by Redmond and Fielder.13 The reported process claimed the use of ultrafiltration membranes for separating the active ingredients from crude extract and reverse osmosis membranes for concentration before making the final product. Singh14 has reported a process for making high soy protein concentrate (> 70%) with reasonably high concentration of isoflavones and saponins while the polysaccharide concentration was reduced. Fractionation of the protein was accomplished by adjusting the pH and using a 10 to 30 kDa ultrafiltration membrane. Yamaguchi et al.15 reported the use of ultrafiltration for isolating nicotianamine from water extracts of soybeans. Chou16 has patented a process to make antioxidant-rich compositions prepared from sugar cane and beet juices. A relatively high concentration of polyphenolics and flavonoids present in the juices were fractionated by various methods including ultrafiltration to get a retentate that showed very high oxygen radical absorbance capacity (ORAC) even at 50% recovery of thin juice. Mulligan and Gibbs17 reported using ultrafiltration for purifying glycolipids; however, only 77% of the product was retained. Buchholz et al.18 improved this process by adjusting the pH of the feed to retain 99% of the product. They have optimized the operation by using ultrafiltration in regular as well as diafiltration mode. Frangi et al.19 reported a procedure to extract procynidol from crude vegetable extract of Vitis vinifera using ultrafiltration membranes. Though the reported product was free from monomers, the procedure required longer processing time and chlorinated solvents, which are not desirable due to environmental and health concerns. NafisiMovaghar et al.20 patented a process that used only blends of polar solvent and water for extraction and employed ultrafiltration for separating proanthocynadins from the rest of the large molecular weight compounds while final extract was concentrated by reverse osmosis. Their flow sheet also included steps to recycle solvents. Nwuha21 screened a series of ultrafiltration membranes for separating green tea catechins and

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identified the alcohol stable membranes. However, rejection value for catechins and caffeine were not sufficiently different to make a caffeine-free catechin product. There has been concern among a section of consumer about the use of aggressive chemicals and solvents in processing nutraceuticals and functional foods. In keeping this in mind, process development for refining compounds of interest from a complex matrix has been attempted using only water extraction. Considering that membranebased processes largely involve inert material, these could be a natural choice for fractionation of such extracts. However, it is also important to note that the choice of extraction procedure plays an important role in downstream processing with membranes. One interesting example is development of a membrane-based process for refining natural sweeteners from stevia leaves extract.22,23 Extraction of glycoside sweeteners was accomplished in lightly packed column at an optimized pH, temperature, and flow rate to extract most of the sweetener and least amount of phenolics and lipids that might cause fouling problem during subsequent processing by membranes. Microfiltration of the extract removed suspended and colloidal impurities while sweeteners and dissolved compounds permeated through the membrane. Permeate from microfiltration was ultrafiltered with a membrane of 2 to 3 kDa molecular weight cutoff , followed by diafiltration in a batch mode until all sweeteners were washed out. Values of sweetener concentration in permeate at different diafiltration volumes of the original feed and permeate flux are listed in Table 8.3. It is obvious from the table that most of the sweetners are recovered at a diafiltration volume of 3 to 4. The permeate from ultrafiltration was processed at higher than ambient temperatures by a reverse osmosis membrane to concentrate the glycosides and remove lower molecular weight components that are perhaps responsible for bitter aftertaste of the product. It was also found that by raising the processing temperature from 50°C to 800°C, the amount of impurities removedwas increased from 55% to 89%. A conceptual process flow diagram is given in Figure 8.1. Though the final product from this process had a very good taste profile, the overall yield was lower than conventional processes based on solvent extraction. In a later study,24 a sequential extraction by solvents such as alcohols and hexane was followed with membrane processing of the extract. This process gave higher yields and good-quality product.

TABLE 8.3 Stevioside Concentration and Permeation Rates for Different Diafiltration Volumes in Ultrafiltration Step Diafiltration Volume

Sweetener Concentration, g/l

Permeation Rates, l.m–2h–1

0 2.0 3.0 4.0

1.5 0.75 0.25 ND

35 47 64 65

ND - not detected

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HEX DF

MF Perm DF

UF

DF NF

Sweetener concentrate

Water recycle

FIGURE 8.1 Conceptual process flow diagram for producing stevia sweetener concentrate; HEX is heat exchanger; DF, UF, and NF are diafiltration, ultrafiltration, and nanofiltration.

One very interesting finding of this study was that prior solvent treatment of stevia leaves was helpful in removing some of the natural products such as lipids, phenolics, and chlorophyll, which are known to foul membranes. Obviously, membrane processing of this extract was relatively easy and the overall yield of the sweeteners was higher than the previous water-based process.23

8.3.3 HYBRID PROCESSES Efficiency of a separation process to some extent depends on the type of material to be separated. In this regard membrane-based separations are no exception. Often, a membrane-based scheme could achieve good results; however, there are theoretical (e.g., osmotic pressure of the dissolved species) and material limitations (e.g., surface characteristics leading to fouling). Considering that functional food and nutraceuticals are present in complex matrices containing a large number of chemically different species, their separation and purification pose additional challenges. In order to develop these challenging applications, so-called hybrid processes are often very effective due to additional controls over the range of operating variables. Basically, a hybrid process combines more than one unit of operations to create overall synergy in the separation scheme. Hybrid processes have been reported1 utilizing pervaporation and distillation; reverse osmosis and multistage flash distillation; pressure swing adsorption and membrane permeation; condensation and vapor permeation; and reverse osmosis and freeze drying. Often the output stream from one process becomes the input stream for the next process. It appears that this hybrid process approach has not been often utilized in separation and purification of functional foods and nutraceuticals. Selected examples from literature are described below:

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In a process reported by Cheryan,25 extraction of two major proteins present in corn meal was done in aqueous alkaline medium and the proteins were separated by centrifugation. The individual proteins zein and glutelin were separated by using a combination of ultrafiltration and ethanol extraction. The permeate of ultrafiltration was also recycled using a reverse osmosis membrane. Seafood process cooker water usually contains suspended solids, protein, amino acids, and flavoring components. The suspended solids are removed by centrifugation or sieving, and proteins are concentrated by several folds with protein recoveries up to 90%.7,26 Nafisi-Movaghar et al.20 patented a process for extraction and refining of proanthocynadins from plant material. Their approach was different from earlier processes19 using a number of solvents, which allowed ultrafiltration only in the batch process. They used heat and pressure to extract proanthocynadins from plant matter, followed by ultrafiltration and chromatographic separation. Ethanol eluted proanthocynadins mixture was concentrated by reverse osmosis before drying the concentrate to powder. Borneman et al.27 reported a process for removing polyphenols from apple juice using a specially made ultrafiltration membrane that utilized adsorptive capabilities of polyvinyl pyrrolidine. A new process for making defatted soymilk with higher isoflavone concentration was reported by Xu et al.28 This process used wet grinding and centrifugation at a higher ratio of bean to water and separated fat by centrifugation. However, the protein concentration in the soymilk so produced was lower, which was brought up to 3% by ultrafiltration. The permeate from a 30 kDa ultrafiltration membrane was also rich in isoflavones. This stream was used to develop another process for recovering soy isoflavones (Xu et al.11) by ultrafiltering the permeate from milk production by a 1 kDa membrane. This lower molecular weight membrane was selected to reject phytates. The amount of isoflavones in ultrafiltration permeate at varying processing conditions is shown in Figure 8.2. It is obvious from this figure that permeate from the high-temperature ultrafiltration of cooked milk was richest in isoflavones. It is mainly due to the dissociation of isoflavones from proteins at higher processing temperatures. The permeate was concentrated with a commercial reverse osmosis membrane by 90%. The concentrate was further dried to make a powder that contained 11 mg/g isoflavones, which represented 8.4% mass yield of dried beans. Soy-based isoflavones are commonly used as nutraceuticals; however, these have been associated with estrogenic activity in a recent publication.29 Red clover flowerbased products have high amounts of isoflavones and do not show any estrogenic activity. Although there are compositional differences in isoflavones extracted from soy beans (aglicones and glycosides) and red clover flowers (bichanin A and formononetin), their absorption in humans is similar.30 A process for extraction and refining isoflavones from red clover flowers was developed31 that involved alkaline extraction, adsorption on polyvinylpolypyrrolidone powder, elution by ethanol, concentration, and finally freeze drying. The final product contained 20% isoflavones, which accounted for more tham 50% of the isoflavone content of dried red clover flowers. Although the recovery and the concentration of isoflavones in the final product were reasonably high, this process had numerous steps. A simpler process involving extraction by ethanol-water blends followed by processing with ultrafiltration and reverse osmosis membranes was recently developed.32 The process

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50 Permeate 45

Soymilk

Isoflavone yield in permeate (%)

40 35 30 25 20 15 10 5 0 No cooking, UF at 21°C

With cooking, UF at 21°C

With cooking, UF at 50°C

FIGURE 8.2 Yield of isoflavones in UF permeate as percentage of total amount in soybeans; results are presented as total amount of isoflavones in soybeans; error bars are based on duplicate runs.

utilized the solvation properties of ethanol-water blends and formation of micelles by partial removal of solvents. In this process the overall recovery of isoflavones from dried red clover flowers was 35% while the concentration of isoflavones in the final product was 9%, which is lower than the earlier process but was suitable for making nutraceutical supplements. Process flow diagrams of these two processes are shown in Figure 8.3. It is obvious that a membrane-based process has a reduced number of process steps with little penalty to overall recovery. Furthermore, this process offers the possibility of recycling the solvents. Obviously, in selecting a proper process, an overall assessment needs to be done.

8.3.4 NEW MEMBRANE PROCESSES In previous sections, membrane-based processes that are more frequently used in well-established food process industry are described with examples of applications in the nutraceutical and functional food industry. However, there are several new membrane-based processes that are at various stages of development and perhaps have potential for application in the industry. Selected examples of these developing membrane processes are given below. A review of the applications of pervaporation in food processing has been published by Karlsson and Trägårdh.33 Pervaporation of flavoring compounds from grape juice34,35 and ester-based flavoring compounds36 have been reported. It was observed that in the presence of increasing amounts of ethanol vapors the recovered

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Ground red clover flowers Ethanol solution Dried and powdered red clover blossom

Batch extraction in water at constant temperature and pH

Filter on a 50–200 mesh sieve

Dried isoflavone concentrate

Extraction

Freeze drying

Ethanol extract

Liquid isoflavone concentrate

Ultrafiltration

Washing

Alkali

Rejects Condensate

Vacuum evaporation

PVP adsorbent for reuse

Adsorbent PVP with isoflavones

Water washing of adsorbent

Ethanol-eluted isoflavones

Ethanol elution of isoflavones

Retentate

Permeate

Reverse osmosis Filtrate – + PVP and adjust pH

Centrifuge

Spent residue

Permeate

Retentate Ethanol evaporation

Aqueous solution Supernatant liquid

Wash water

Refrigeration

Supernatant

Micelles Drying

Isoflavone-enriched product

(a)

(b)

FIGURE 8.3 (a) Extraction and polymeric adsorption-based process flow diagram, (b) extraction and membrane-based process flow diagram.

aroma profile was changed significantly. A very comprehensive study on the screening of numerous commercial membranes for recovering peach flavors was also reported.37 A similar approach could be used to recover volatile components such as essential oils present in plant materials. A membrane-based solvent extraction process could overcome many of the problems encountered in a traditional solvent extraction process involving dispersion, coalescence, emulsification, and loading limits. Membrane hollow fibers made with hydrophobic material could be used to provide significantly higher contacting area that would provide very high extraction rates for selective solvent extraction of the desired species present in the aqueous phase across the membrane barrier. Prasad and Sirkar38,39 have reported the use of this techniques for purifying mevinolinic acid and thiazoles using different solvents. The purity of the products was very high and the membranes did not show any deterioration after long-term use. Dahuron and Cussler40 have reported extraction of proteins using polyethyleneglycol containing potassium phosphate. Recently Kertész et al.41 have reported a simultaneous solvent extraction and stripping process for recovering phenlanaline in n-alkanes

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containing a phosphoric acid derivative. These authors have presented a comprehensive study of mass transfer and kinetics of this system. Reviews of potential food application of electrofiltration have been published by Vradis and Floros42 and Bazinet et al.43 They have provided the theoretical basis as well as the potential of this technique to overcome some of the fouling problem in regular pressure driven membranes processes, particularly for the charged species in the feed. Pressure filtration of bioactive compounds is dependent on the ionic environment of the feed as it influences the hydrodynamic radius of species. Hofmann and Posten44 have given a sound basis for enhanced flux in separation of biopolymers and proteins under the influence of electric field. Recently, Labbé et al.45 have reported feasibility of separating green tea polyphenols by electrofiltration. These authors reported that up to 50% concentration of epagallocatechin and epagallocatechin gallate could be achieved without significant migration of caffeine. However, a similar study to recover polyphenols from tobacco extracts encountered serious membrane fouling.46 A feasibility study of separating bioactive peptide from casein isolates has been reported by Bargeman et al.47 These authors reported that active peptide was concentrated from 7.5% to 26%. Obviously, additional studies to develop these techniques for commercial applications are required. Osmotic distillation process uses the vapor pressure difference as the driving force while the feed stream and concentrated osmotic agent stream are separated by a hydrophobic porous membrane. This technique is particularly suitable for heat-sensitive compounds and has largely been used for concentration of fruit juices.48,49 Recently Babu et al.50 reported concentration of lime juice and phycocynin colorant by osmotic membrane distillation. They have studied the effects of various parameters on flux and developed models to predict flux using resistance models. Affinity membrane separation combines affinity chromatography and traditional pressure-driven membrane separation processes. This technique is suitable if the feed stream contains several compounds of comparable molecular weight and characteristics to the compound of interest. A ligand or binding agent that has affinity to the compound of interest is added to the feed solution and separated from other compounds by membranes. The retentate from the process is treated with a proper eluent to dissociate the ligand and the compound of interest is separated by a second membrane filtration step. Matthiasson and Ling51 successfully separated biologically active concavalin A from jack beans using S cerevisia cells as ligands and D-glucose as eluent. Considering that micelles are larger than the dissolved organic solutes, surface active agents also have been used to make micelles with the key component of the feed for easy separation.52 Ruckenstein and Zeng53 reported the development of chitin-silica membranes for separating lysozyme from ovalbumin. These membranes were an order of magnitude superior to the chitin beads for this separation and could be used for larger-scale separation.

8.3.5 CLEANING CHEMICAL

AND

SOLVENT RECYCLING

In a membrane-based separation process, various cleaning chemicals are periodically used for restoring membrane performance as well as cleaning and sterilizing the ancillary equipment. These procedures have added importance for the nutraceutical

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and functional food industry for maintaining a higher level of purity and safety of products for human consumption. The cleaning fluids are composed of caustic solution, surfactant solution, enzymes, and chemicals, or a combination of these ingredients. With repeated use, these cleaning fluids are contaminated with impurities and need to be replaced. This adds to the cost of operation in replacement chemicals as well as disposal fees. A membrane-based recycling of these cleaning fluids is attractive as properly selected membranes could remove suspended, colloidal, and dissolved species while water and active cleaning agent could be recycled. Caustic recycling in orange juice and dairy plants is in use and has replaced more energy-extensive evaporation systems.54 By using a multistage membrane separation scheme, other cleaning agents could also be recovered. As discussed in Section 8.3.3, organic solvents are used in extraction and elution of nutraceuticals extensively. A membrane-based approach can recover and concentrate the compound of interest and allow the recycling of solvent. For example, a typical reverse osmosis membrane will permeate water and ethanol while retaining compounds of interest. There are some commercial membranes that are suitable for alcohol and hexane recycling; however, there are very few membranes that are suitable for other solvents such as ketones and esters that are often used in the extraction of functional food and nutraceuticals. There have been several reports of new solvent-resistant nanofiltration membranes55,56,57,58 that might be suitable for recovery of compounds in the range of 300 to 900 Da molecular weights, which includes a large number of nutraceuticals.

8.4 SUMMARY In recent years there has been great interest in using plant-derived functional food and nutraceuticals. These compounds are usually present in low concentrations in a complex matrix; therefore, extraction, separation, and purification is generally very expensive. Opportunities offered by membrane-based processes in concentration, fractionation, and purification of these compounds have been identified in this work. Several examples of processes utilizing a membrane unit operation in combination with other unit operations such as extraction, adsorption, and thermal evaporation are presented. Multistage membrane processes for refining desired compounds are also discussed. Several new membrane processes are briefly described that need to be developed further for use in functional food and nutraceutical production. Membrane-based processes for recycling chemicals and solvents used in the functional foods are also highlighted as potential applications. Finally, the challenges for researchers in developing newer products and processes relevant for the functional food and nutraceutical industry are identified. Use of membranes in water purification and wastewater treatment alone or in combination with other unit operations is fairly well established. Membrane-based separation schemes are now making inroads into many other chemical processes, food, and dairy industries. Advantages of the membrane-based approach in nonthermal processing, energy efficiency, easy amenability to automation, modular nature, and generally smaller footprint have been clearly exploited. Potential of a

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hybrid-process approach needs to be exploited further even with the presently available technology. Many of the new membrane processes have potential applications in functional food processing; however, these need to be researched further with particular focus on material and process development. Membrane researchers have many challenges to overcome in the areas of membrane materials, morphologies, and surface characteristics that have an impact on compatibility of membrane with feed stream, separation performance, and resistance to fouling. Although many of the specifically targeted compounds are extracted from complex matrices using aggressive solvents, often at higher than ambient temperature and extremes of pH, there are very few membranes in the market to process such streams economically and effectively. Therefore, there is a need for developing membranes that are suitable for treating such feeds.

ACKNOWLEDGMENTS The author is thankful for the support of his colleagues in the Advanced Separation Technology Project.

REFERENCES 1. Ho, W.S.W. and Sirkar, K.K., Membrane Handbook, Chapman and Hall, New York, 1992. 2. Mulder, M., Basic Principles of Membrane Technology, 1st ed., Kluwer Academic Publishers, Dordrecht, 1996. 3. Cheryan, M., Ultrafiltration and Microfiltration Handbook, Technomic, Lancaster, PA, 1998. 4. Mohr, C.M., Engelgau, D.E., Leeper, S.A., and Charboneau, B.L., Membrane Applications and Research in Food Processing, Noyes Data Corporation, Park Ridge, NJ, 1989, 15. 5. Williams, M.E., Bhattacharya, Ray, and McCray, in Membrane Handbook, Ho, W.S.W. and Sirkar, K.K, Eds., Chapman and Hall, New York, 1992, 346. 6. Singh, N. and Cheryan, M., Membrane technology in corn refining and bioproductprocessing, Starch - Stärke, 50(1), 16, 1998. 7. Kumar, A., Croteau, S., and Kutowy, O., Use of membranes for energy efficient concentration of dilute streams, Appl. Energy, 64, 107, 1999. 8. Ekanayake, A., Bunger, J.R., and Mohlenkamp, M.J., Jr., Green Tea Extract Subjected to Cation Exchange Treatment and Nanofiltration to Improve Clarity and Color, U.S. Patent 6,268,009, July 31, 2001. 9. Gugger, E. and Grabiel, R., Production of Isoflavone Enriched Fractions from Soy Protein Extracts, U.S. Patent 6,171,638, January, 2001, and U.S. Patent 6,565,912, May 20, 2003. 10. Xu, L., Lamb, K., and Kumar, A., A membrane-based process for recovering isoflavones from a waste stream of soy processing, Food Res. Int., 37, 867, 2004. 11. Bhattacharjee, S., Bhattacharjee, C., and Datta, S., Studies on the fractionation of β lactoglobulin from casein whey using ultrafiltration and ion exchange membranes chromatography, J. Membrane Sci., 275, 141, 2006.

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12. Cheang, B. and Zydney A.L., Separation of alpha-lactalbumin and beta-lactoglobulin using membrane ultrafiltration, Biotechnol. Bioeng., 83(2), 201, 2003. 13. Redmond, M.J. and Fielder, D.A., Oat Extracts: Refining, Compositions and Methods of Use, U.S. Patent, 6,818,232, November 16, 2004. 14. Singh, N., Soy Protein Concentrate Having High Isoflavone Content and Process of Its Manufacture, U.S. Patent 6,818,246, November 16, 2004. 15. Yamaguchi, N. et al., Method for Producing Nicotianamine and Nicotanamine-Containing Matter, Japanese Patent 2003, 231,675A, August 19, 2003. 16. Chou, C. C., Preparation Antioxidants Enriched Functional Food Products from Sugar Cane and Beet, U.S. Patent Application 2003 0,198,694, published October 23, 2003. 17. Mulligan, J. and Gibbs, P., Recovery of biosurfactants by ultrafiltration, J. Chem. Tech. Biotechnol., 47, 23, 1990. 18. Buchholz, R., Ulrich, F., and Mixich, J., Process for Preparing Purified Glycolipids by Membrane Separation Process, U.S. Patent 5,440,028, August 8, 1995. 19. Frangi, E. et al., Process for Preparing Grape Seed Extracts Enriched in Procynidol Oligomers, U.S. Patent 5,4845,94, January 16, 1996. 20. Nafisi-Movaghar, K., Svanoe, T.T., and Seroy, W.A., Method for Extraction of Proanthocynadins from Plant Material, U.S. Patent 5,912,363, June 15, 1999. 21. Nwuha, V., Novel studies on membrane extraction of bioactive components of green tea in organic solvents: part I, J. Food Eng., 44, 4, 233. 22. Zhang, S.Q., Kumar, A., and Kutowy, O., Membrane-based separation scheme for processing sweeteners from stevia leaves, Food Res. Int., 33, 617, 2000. 23. Kutowy, O., Zhang, S.Q., and Kumar, A., Extraction of Sweet Compounds from Stevia Rebaudiana Bertoni, U.S. Patent 5,972,120, October 26, 1999. 24. Kutowy, O. and Kumar, A., Improved Stevia Extraction and Refining, Provisional U.S. Patent filed on February 18, 2004, Appl. No. 60/545,167. 25. Cheryan, M., Ultrafiltration and Microfiltration Handbook, Technomic, Lancaster, PA, 1998, 418. 26. Cheryan, M., Ultrafiltration and Microfiltration Handbook, Technomic, Lancaster, PA, 1998, 427. 27. Borenman, Z., Gokeman, V., and Nijhvis, H.H., Selective removal of polyphenols and brown color in apple juices using PES/PVP membranes in a single ultrafiltration process, Separ. Purific. Technol., 22, 53, 2001. 28. Xu, L., Kumar, A., and Lamb, K., A laboratory study for developing an aqueous process to make skimmed soymilk, J. Am. Oil Chem. Soc., 81, 91, 2004. 29. Atkinson, C. et al., Red clover-derived isoflavones and mammographic breast density: a double-blind, randomized, placebo-controlled trial [ISRCTN42940165], Breast Cancer Res., 6, R170, 2004. 30. Tsunoda, N., Pomeroy, S., and Nestel, P., Absorption in humans of isoflavones from soy and red clover is similar, J. Nutr., 132, 2199, 2002. 31. Xu, L. et al., Production of an isoflavone concentrate from red clover flowers, J. Food Sci., 70, 5558, 2005. 32. Xu, L. et al., Recovery of isoflavones from red clover flowers by a membrane-based process, Innov. Food Sci. Emerg. Technol., in press, 2006. 33. Karlsson, H.O.E. and Trägårdh, G., Applications of pervaporation in food processing, Trends Food Sci. Technol., 7, 78, 1996. 34. Rajgopalan, N. and Cheryan, M., Pervaporation of grape juice aroma, J.Membrane Sci., 104, 243, 1995. 35. Schafer, T. et al., Recovery of aroma compounds from a wine-must fermentation by organophilic pervaporation, Biotechnol. Bioeng., 62, 412, 2000.

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36. Baudot, A. and Marin, M., Improved recovery of an ester flavor compound by pervaporation coupled with flash condensation, Ind. Eng. Chem. Res., 38, 4458, 1999. 37. Song, B., Lee, J.-H., and Choi, Y.-H., A pervaporation technique for peach flavors using PDMS and DS-7 membranes, Food Eng. Progress, 6, 133, 2002. 38. Prasad, R. and Sirkar, K.K., Hollow fiber solvent extraction of pharmaceutical products, J. Membrane Sci., 47, 235, 1989. 39. Prasad, R. and Sirkar, K.K., Hollow fiber solvent extraction: performance and design, J. Membrane Sci., 50, 153, 1990. 40. Dahuron, L. and Cussler, E.L., Protein extraction with hollow fibers, Am. Inst. Chem. Eng. J., 34, 130, 1988. ˇ ˇ and Schlosser, S., ˇ Membrane-based solvent extraction and 41. Kertész, R., Simo, S., stripping of phenylalanine in HF contactors, J. Membrane Sci., 257, 37, 2005. 42. Vradis, I. and Floros, J., Electrically assisted membrane separation processes, in Food Process Design and Evaluation, R.K. Singh, Ed., 1995, CRC Press, Boca Raton, FL. 43. Bazinet, L., Lamrche, F., and Ippersiel, D., Bipolar-membrane electrodialysis: applications of electrodialysis in the food industry, Trends Food Sci. Technol., 9, 107, 1998. 44. Hofmann, R. and Posten C., Improvement of dead-end filtration of biopolymers with pressure electrofiltration, Chem. Eng. Sci., 58, 3847, 2003. 45. Labbé, D. et al., Electromigration feasibility of green tea catechins, J.Membrane Sci., 254, 101, 2005; Matthiasson, B. and Ling, T.G.I., Ultrafiltration affinity purification: a process for large scale bioseparation process, in Membrane Separations in Biotechnology, McGregor, W.C., Ed., 1986, Marcel-Dekker, New York. 46. Bazinet, L., DeGrandpré, Y., and Porter, A., Enhanced tobacco polyphenol electromigration and impact on membrane integrity, J. Membrane Sci., 254, 111, 2005. 47. Bargeman, G. et al., The development of electro-membrane filtration for the isolation of bioactive peptides: the effect of membrane selection and operating parameters on the transport rate, Desalination, 149, 369, 2002. 48. Cisse, M. et al., The quality of orange juice processed by coupling crossflow microfiltration and osmotic evaporation, Int. J. Food Sci. Technol., 40, 105, 2005. 49. Bui, A.V. and Nguyen, H.M., Scaling up of osmotic distillation from laboratory to pilot plant for concentration of fruit juices, Int. J. Food Eng., 2, 5, 2005. 50. Babu, B.R., Rastogi, N.K., and Raghavarao, K.S.M.S., Mass transfer in osmotic distillation of phycocyanin colorant and sweet-lime juice, J. Membrane Sci., 272, 58, 2006. 51. Matthiasson, B. and Ling, T.G.I., Ultrafiltration affinity purification: a process for large scale bioseparation process, in Membrane Separations in Biotechnology, McGregor, W.C., Ed., 1986, Marcel-Dekker, New York. 52. Scamehorn, J.F. and Harwell, J.H., An overview of surfactant-based separation processes, Surface Sci. Ser., 26, 341, 1987. 53. Ruckenstein, E. and Zeng, X., Macroporous chitin affinity membranes for lysozyme separation, Biotechnol. Bioeng., 56, 610, 2000. 54. Cheryan, M., Ultrafiltration and Microfiltration Handbook, Technomic, Lancaster, PA, 1998, 384. 55. Bhanusali, D. and Bhattacharyya, D., Advances in solvent resistant nanofiltration membranes: experimental observations and applications, Annals New York Acad. Sci., 984, 159, 2003. 56. Yang, X.J., Livingston, A.G., and Fretas dos santos, L., Experimental observations of nanofiltration with organic solvents, J. Membrane Sci., 190, 45, 2001.

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57. Musale, D.A. and Kumar, A., Solvent and pH resistance of surface crosslinked chitosan/poly(acrylonitrile) composite nanofiltration membranes, J. Appl. Polym. Sci., 1782, 77, 2000. 58. Musale, D.A. and Kumar, A., Effects of surface crosslinking on sieving characteristics of chitosan/poly(acrylonitrile) composite nanofiltration membranes, Separ. Purif. Technol., 21, 27, 2000.

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9

Bioprocessing Technology for Production of Nutraceutical Compounds Terry H. Walker, Caye M. Drapcho, and Feng Chen Clemson University

CONTENTS 9.1 9.2 9.3

Introduction ..................................................................................................212 Commodity Biochemicals and Nutraceutical Bioproduction......................214 Lipid-Based Nutraceuticals..........................................................................215 9.3.1 Polar Lipids ......................................................................................217 9.4 Long-Chain Polyunsaturated Fatty Acids....................................................219 9.5 Large-Molecule Nutraceuticals....................................................................221 9.5.1 Polysaccharide Compounds .............................................................221 9.5.2 Proteins and Nucleotides .................................................................224 9.5.3 Small Molecular Nutraceuticals.......................................................225 9.6 Bioprocess Design........................................................................................226 9.7 Process Analysis...........................................................................................227 9.8 Process Economics.......................................................................................227 9.9 Conclusions ..................................................................................................229 Acknowledgments..................................................................................................230 References..............................................................................................................230

211

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9.1 INTRODUCTION Nutraceuticals are natural compounds used to supplement the diet as a means to increase uptake of important nutrients in the diet of humans and other animals. Nutraceutical compounds are becoming an important commodity in the United States and globally as market demand increases for both purified and supplemented forms. In the 1990s, nutraceuticals from natural sources were most widely explored in “plants, marine organisms, and microorganisms, in particular actinomycetes, and fungi.”1 Development of biopharmaceuticals primarily from animal tissue culture in serum media has recently enabled obtaining these products in other eukaryotic systems such as plants, algae, and fungi. Many nutraceuticals are extracted and purified from plants (e.g., antioxidants) and animals (e.g., fish oil rich in essential fatty acids), which are an important component of the total market. However, there are vast sources of microorganisms capable of production of nutraceutical components and the database is rapidly increasing in recent years. Production of nutraceuticals in microorganisms is usually conducted in highly controlled closed systems capable of meeting U.S. Food and Drug Administration (FDA) approval criteria. Obvious expansion of this market requires innovation for new products, and the use of bioprocessing techniques commonly applied to the food and pharmaceutical industry may provide a means to enhance products through novel high-density cell culture techniques as well as new fractionation and bioseparation techniques. Researchers in biological and chemical engineering are finding ways to effectively utilize byproducts from the food, seafood, and agricultural industries for first fractionating valuable nutraceutical components and then further processing of coproduct to serve as feedstock for bioenenergy compounds like ethanol, biodiesel, methane, and hydrogen. The combined global market of nutraceuticals and functional foods surpassed $90 billon in 2004, up approximately 10% per year since 1993 where nearly half of the market comprised dietary supplements. The combined value of nutraceuticals and functional foods is expected to reach $130 billion by the year of 2007.2,3 One example is the Martek Biosciences process for production of nutraceutical oil from algal and fungal sources containing a high degree of omega-3 and omega-6 essential fatty acids primarily used as a supplement in baby formulas. Development of advanced biological conversion processes (enzymatic, microbial, and physicochemical processes) is an important part of the bioprocessing research agenda. Biological processes are the preferred paths for converting agriculture-based resources into industrial products. Bioprocesses tend to have higher reaction specificity, milder reaction conditions, and produce fewer toxic byproducts. These characteristics are very consistent with the goal of developing industrial processes and systems that are environmentally friendly. Innovative research is currently underway that links physicochemical processes, such as gasification, with microbial conversion processes. These novel reaction systems are evolving in response to the heterogeneity of most biomass resources and the realization that there are important autotropic microorganisms that are effective in converting common bioorganic compounds into more useful industrial or biomedical compounds.

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Raw materials, byproducts, agricultural “wastes” Bioprocessing, bioconversion, bioreactor design and control, “biorefineries”

Bulk chemicals, biofuels

Biomaterials

Specialty biochemicals

FIGURE 9.1 Biorefinery concept for conversion of by-products to bulk chemicals, biomaterials, and specialty biochemicals with emphasis on integrating the 3 Es (environment, education, and economics).

Land grant institutions across the United States represent a great repository of scientific and engineering knowledge to catalyze the transition of society from a fossil fuel to a biobased economy. With the biotechnology revolution currently in place, application of new technologies is sought to address the challenging arenas of specialty biochemical production (e.g., biopharmaceuticals and nutraceuticals), bioenergy compounds (e.g., ethanol), and biomaterials (e.g., polylactate, PLA). Figure 9.1 illustrates the basic flow of raw materials to end products proposed for a biorefinery concept4 in an initiative established by the U.S. Department of Agriculture (USDA).5 Integrated approaches to bioprocessing show the greatest promise to complete the cycle driven by the 3 Es including economics, environment, and educational aspects of the bioindustry. Bottom-line industry economics balanced with environmentally friendly technologies that contribute to carbon sequestration within the biological system require educating the increasing masses of people on the planet of this balance and creating a workforce to tackle the highly innovative challenges that lie ahead in a widely evolving biotechnological world. Environmental concerns encourage “green” technologies, economics requires feasibility of the proposed technologies, and education addresses the importance of all levels of learning including grade school, undergraduate studies, and graduate research opportunities as a mainstay to achieving the goals to establish a bio-based economy. Current breakthroughs are not limited to only molecular biology and genetics, but over the past 15 years, biological and chemical engineers have made notable advances in the fractionation and bioprocessing of biobased resources of raw materials required to obtain the principal building blocks for the synthesis of new bioproducts. For example, the critical first step in bioconversion of ligno-cellulosic biomass is the fractionation and modification of constituent biopolymers to facilitate either enzymatic or microbial conversion resulting in the efficient production of fermentable sugars. Several promising pretreatment technologies, such as ammonia fiber explosion (AFEX) and supercritical fluid explosion pretreatment have resulted

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in improved yields and reactivity.6 In addition, engineering advances in bioseparation technologies are improving capabilities to identify and separate secondary metabolites and other bioactive compounds from plant, animal, and microbial materials, such as simulated moving bed (SMB) chromatography and supercritical fluid fractionation (SFF). Advances in nanobiotechnology such as the fabrication of microfluidic channels in silicon wafers take advantage of differences in physical and chemical properties such as diffusion, electrophoretic mobility, or chemical affinity to produce rapid, efficient separations of proteins, secondary metabolites, and other organic compounds primarily for rapid identification and synthesis. In analytical biotechnology, chemical and biological techniques are combined and integrated into engineered devices or sensors for the detection and quantification of secondary metabolites and other organic compounds in a variety of bioprocesses.

9.2 COMMODITY BIOCHEMICALS AND NUTRACEUTICAL BIOPRODUCTION The biorefinery concept utilizes the bioconversion of natural raw materials or industry by-products to purified specialty biochemicals and bulk fuels (see Figure 9.1). Research to develop bioconversion processes to produce specialty chemicals and bioenergy consists of several phases: (1) screening for wild-type strains and metabolic engineering of cells to produce desired products; (2) characterization and pretreatment (hydrolysis) of feedstock prior to bioconversion; (3) determination of microbial growth, decay, and product formation kinetics of the target microorganism (filamentous fungi, bacteria, yeast, algae, etc.); (4) design/optimization of reactor systems to culture cells and produce desired products based on simulations using mass-balanced-based models and microbial kinetic parameters determined in phase 3; (5) downstream processing to separate, concentrate, and purify products and (6) recycle waste energy and materials to bio-refinery using sustainable technologies. The bioprocess involves chemical treatment of feedstocks to hydrolyze the cellulose and hemicellulose compounds to fermentable sugars, which consist of mostly glucose and xylose. The fermentable sugars are then fermented to industrial products (e.g., ethanol, lactic acid, or succinic acid) and nutraceuticals (e.g., amino acids, omega-3 fatty acids, or high-value biopharmaceuticals). Biopharma compounds range from smaller molecules such as traditional antibiotic products like penicillin or insulin from mammalian cell culture or modified E. coli (Eli Lilly process) to larger molecules that encompass bioactive proteins, lipoproteins and glycoproteins ranging from 1000 to 500,000 daltons with complex three-dimensional structures often dictated by their specific levels of glycosilation and chiral chemistry. Generally, the purity of substrate is required for increasing product value. For example the expense of serum media may exceed the value of most of the products shown in the table. Therefore, effort to utilize substrates from conventional agricultural byproducts for production of biopharmaceuticals with mammalian culture is of great interest to the biotechnology industry. Carotenoids including βcarotene astaxanthins, and more recently increasing markets for lutein and lycopene, make up a world market nearing $1 billion with a growth rate of about 3%/yr. Most

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carotenoids are produced synthetically by companies like BASF and DSM, but recent biotechnological advances for carotenoid metabolism have shown promise in fungi like Blakeslea trispora7 and have been extracted for the nutraceutical market (Nature Source®) from the algae Dunaliella salina.8–12 There is a great interest in biological chemicals, functional food ingredients and nutraceuticals regarding the rapid development in biochemistry, genetic engineering, genomics, biological engineering, food chemistry, and medical research fields. However, bioseparation and downstream processing equipment account for nearly half of the total cost of production of biological products and nutraceuticals.13,14 Therefore, the competitive nature of the biotechnology, pharmaceutical, and food industries for cost-effective manufacturing has provided much impetus for the development and use of new bioseparation techniques in large scale, but in lower cost. Except for the conventional bioseparation techniques, such as crystallization, precipitation, distillation, liquid-liquid extraction, and so forth, that have been well established and commercialized,15 a number of newer bioseparation techniques have been implemented at the commercial scale, including chromatography, supercritical fluid extraction, and membrane-based separation. These techniques have been implemented for purification of proteins and nucleotides, characterization of aromas, whey protein removal from dairy products, extraction of health-benefiting fish oil, and clarification of beverages including beer, fruit juices, and wine. The following section provides some examples of analytical- and process-scale bioseparations of biological products and nutraceuticals based on their polarity (lipid-based nutraceuticals) and molecular size (large and small molecular nutraceuticals). Table 9.1 lists several potential products from pretreated feedstocks to form high-value products.16

9.3 LIPID-BASED NUTRACEUTICALS Lipids include many broad classes of neutral and partially polar compounds with differing physiological function. Some contain important regulatory function such as sterols (e.g., cholesterol, phytosterol, ergosterol) produced by plants as well as fungi such as Saccharomyces cerevisae and Gibberella fujikuroi.17 Other antioxidants found in plant materials such as rice bran are the class of phytosterols,18 but also found in microorganisms like algae.19 These antioxidants include tocopherols and tocotrienols (see Figure 9.2) beneficial in lowering cholesterol as well as preventing cardiovascular diseases.20 Tocopherols are also believed to have anticancer effects.21,22 Many neutral lipids in mono-, di-, and triacylglycerol forms act as functional precursors to other metabolites or act as an energy storage source. Most lipids consumed by humans and other animals are derived from plants and animals due to their relative abundance in these sources. Bioproduction of lipids from microbial sources are also well known, but have only recently been established at the commercial level for production, primarily for lipids with considerable bioactivity such as very long-chain unsaturated compounds with high degrees of omega3 fatty acids or phospholipids and sulfolipids geared toward cancer research from algal or fungal sources.

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TABLE 9.1 Bioconversions in Order of Increasing Value and Subsequent Substrate Purity. Pretreated Feedstocks (e.g., Mild Acid Hydrolysis) Are Converted to Product by Enzymes from Microbial “Factories” By-Product Source

Primary Substrate

Primary Enzyme (Microbial strain)

Product (Production)

Animal waste Corn syrup Potato/Sweet potato Wood fibers Dairy waste Oily waste Rice brokens Rice straw Switchgrass Sugarcane bagasse Corn stover Corn starch Molasses (sugarcane) Corn Syrup

Complex Glucose Glucose

(M. flagellate) (S. cerevisiae) Alcohol dehydrogenase (K. marxianus) Xylan (Clostridium sp. SAIV1) Lactose β-galactosidase Lipids Lipase (esterases) Glucose (C. acetobutylicum) Xylose Citrate synthase Xylose Lactose dehydrogenase (L. delbrueckii) Xylose Pyruvate Pyruvate decarboxylase (Acetobacter sp.) Acrylonitrile Nitrile hydratase D-xylulose Xylose reductase (C. tropicalis) Glucose Glucose isomerase Sucrose Fumarase (B. ammoniagenes) Glucose (Schizochytrium sp.)

Ethanol stillage3 Glucose Glucose Glucose Glucose Glucose Glucose

Specific Media

Glucose Glucose Glucose Glucose Serum Serum Serum

Methane Ethanol (26M tons/yr) Ethanol (thermophilic pathway) Ethanol Glucose and Galactose Fatty acids Acetone/butanol (30/60) Citric acid (1M tons/yr) Lactic acid (30 M kg/yr) Acetic acid Acrylamide (15,000 tons/yr) Xylitol High-fructose corn syrup (8 M tons/yr) L-malic acid

Oil rich in docosahexaenoic acid ($220 M/yr) (Phaffia rhodozyma mut.) astaxanthin ($2000/kg) AcetylCoA carboxylase L-glutamic acid (340,000 tons/yr) (C. glutamicum)-biotin MSG Aspartic amino L-phenylalanine (3000 tons/yr) transferase (E. coli) (Aspartame synthesis) Aspartase (E. coli) L-aspartic acid (4000 tons/yr) L-aminocaprolactam L-lysine (350,000 tons/yr) ($2/lb) hydrolase (C. laurentii) (P. fluorescens) L-histidine Penicillin amidase 6-aminopenicilloic acid (7500 tons/yr) (P. chrysogenum) (B. lichenformis) Proteases ($236 M/yr) (B. amyloliquefaciens) Amylases ($70 M/yr) (Rhizopus, Aspergillus) Other enzymes ($92 M/yr) (Anthrobacter simplex) Prednisone Hybridomas Monoclonal antibodies Human fibroblasts Interferon Monkey kidney cells Polio vaccine

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5

HO

H3C

H

H3C

H

2

7 8

O Tocopherol

5

HO

CH3

CH3

2

7 8

O Tocotrienol

FIGURE 9.2 Antioxidant sterol compounds tocopherol and tocotrienol.

9.3.1 POLAR LIPIDS Polar lipids are often associated with vital membrane structure and function depending on the lipid class (e.g., sulfolipid, SL; phosphatidyl choline, PC; phosphatidyl ethanolamine, PE), length and position of constituent fatty acids, and degree of saturation.23 Sphingolipids (e.g., sphingomyelin, a phospholipids, and ceramide) are another special class of bioactive compounds associated with cell membranes that act as lipid mediators important to intra- and extra cellular signaling, regulation of cell growth, differentiation, and apoptosis. Breakdown of the abundant membrane phospholipid, sphingomyelin, by sphinomyelinases produces sphinogosine and ceramide.24 Sphininogosine serves as a precursor to sphinosine-1-phosphate, which has cancer-promoting properties such as cell proliferation and angiogenesis, while ceramide primarily functions physiologically as a cancer preventive agent with properties of apoptosis and growth inhibition, thus together, placing the cell into a life and death balance.25 Cyanobacteria, like higher plants and green algae, have a glycerolipid composition that is made up of monogalactosyldiacylgylcerol (MGDG), sulfoquinovosyl diacylglycerol (SQDG) shown in Figure 9.3, digalactosyldiacylglycerol (DGDG), and phosphatidyl glycerol (PG). Sulfoquinovosyl diacylglycerols are structural components of chloroplast membranes in eukaryotic algae and lamellas in prokaryotic cyanobacteria, and appear to be the only natural lipid with sulfonic acid linkage.26 SQDG is characterized by its unique sulfonic acid head group, a 6-deoxy-6-sulfo-glucose.27 SQDG extracted fom cyanobacteria has been shown to inhibit HIV-1 in cultured human lymphoblastoid T-cell lines and thus was placed as high priority for further research by the National Cancer Institute.28 The cyanobacteria evaluated included species of lyngbya, phormidium, oscillatoria, scytonema, calothrix, and anabaeana. Sulfolipids comprised 10% by weight of the organic extractables from the cyanobacteria tested and all had similar activity, suggesting that acyl chain length and degree of unsaturation did not affect potency.28 Structurallyrelated acyl glycerols, lipids, and sulfonic acid derivatives did not inhibit HIV-1. SQDG was also found to reduce proliferation and

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−O SH C 3 2

HO

O

HO OH O

O SQDG

O O

O C R1

R2

FIGURE 9.3 Structure of SQDG where R1 and R2 represent acyl chains of different length and degree of unsaturation.27

viability of human gastric carcinoma cells SNU-1, by induction of apoptosis to direct necrosis at micromolar concentrations.29 These results indicate potential of whole algae or algal extracts containing sulfolipids as chemotherapeutic or preventive dietary supplements. For the cyanobacterium Anabaena 7120, SQDG content in algal basis at day 20 of batch incubation was unaffected (approximately 11 mg SQDG/g algal dry weight) by surface light intensities.30 SQDG content in the Anabaena biomass increased throughout the exponential and decreasing exponential growth phases, indicating SQDG may be characterized as a mixed primary and secondary product. For the green alga Scenedesmus, the combined SQDG and PG content of algal biomass did not differ as a function of light intensity (40 to 290 (ME m–2s–1) at day 5 of incubation, but decreased with increased light intensity by day 25 of incubation when cells had entered early stationary growth.31 Full characterization of the interaction between light intensity, growth rate, and SQDG production is needed for optimization of algal reactors. Unlike neutral galactolipids, which are easily separated from phospholipids by adsorption chromatography, SQDG is difficult to separate due to its polar nature.26 Quantification of SQDG in Anabaena extracts was accomplished by Archer et al.30 using total lipid extraction as per Folch et al.,32 followed by thin layer chromatography using silica gel plates and photodensitometric scanning after charring with copper sulfate and heat. Norman et al.33 developed a method for the extraction, separation, and analysis of SQDG from plant (spinach) biomass using the Bligh and Dyer34 extraction method for total lipids using sodium chloride in two-phase partitioning, separation of lipid classes using silica cartridges with SQDG and phospholipids (PL) eluted using methanol, and resolution of SQDG from the PL fraction using normal phase high-pressure liquid chromatography (HPLC) with silica column, heptane-isopropanol-0.001 M KCl 40:52:8 (v/v/v) as the mobile phase, and detector operated at 208 nm. The sulfolipid yield was found to be 0.18 mg SQDG/g fresh spinach. Cell lysis of algae and cyanobacteria for extraction of lipids is more difficult than for plant tissue, and techniques to shear cells include solvent addition with incubation, agitation, homogenization, and sonication.35 A cell-lysis method

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developed for the green alga Scenedesmus that included quartz sand and organic solvent addition, freeze drying, and extraction in ultrasound bath, was reported to provide excellent extraction efficiency of pigments and fatty acids.35 Supercritical fluid (CO2) extraction (SFE) provides an alternative for the extraction and recovery of algal nutraceuticals, and avoids use of toxic organic solvents. SFE of carotenoids and lipids from the green alga Chlorella has been found to compare favorably to organic solvent extractions.36 Shearing of algal cells using alternating mechanical and thermal shocking was required for efficient extraction. Yields of carotenoids and lipids were 5% of dry weight at 35 MPa and 55°C for whole algal cells, while yields were increased to 13.3% for disrupted cells. Similar results were achieved using SFE for extraction of carotenoids and lipids from Botryococcus braunii, Dunaliella salina, and Arthrospira maxima.37 SFE of sulfolipids may also prove comparable to organic solvent extraction. Recently, continuous countercurrent supercritical carbon dioxide fractionation has been successfully applied to fractionating low molecular fatty acids38 and antioxidants in plant oils such as corn and rice bran oil,39 and could be applied to fungal and algal systems.

9.4 LONG-CHAIN POLYUNSATURATED FATTY ACIDS Polyunsaturated fatty acids (PUFAs) are a class of compounds which has a variety of important functions in biological systems. Studies have shown that the longer-chain fatty acids, in particular EPA (eicosapentaenoic acid, C20:5n3; see Figure 9.4), ARA (arachidonic acid, C20:4n6), and DHA (docosahexaenoic acid C22:6n3), have important roles as biosynthetic precursors, as cellular membrane components, and as protection against oxidative stress.40,41 The relative levels of these compounds have been found to have profound effects on human health. Growing interest in health benefits of PUFAs has focused attention on providing suitable sources of these compounds. Isolation of highly efficient oleaginous microorganisms and development of related fermentation technologies may lead to fermentation as an alternative to agricultural and animal processes.42 Some microbial species produce high yields of certain PUFAs, which include Mortierella alpina,43–45 Mortierella elongata,46–48 Pythium irregulare,49–52 P. ultimum,53,54 and Entomophthora exitalis.55 Pythium irregulare is regarded as one of the most promising microbial species for possible commercial production of EPA due to its high EPA yield. This microorganism can also produce a significant amount of another important PUFA, arachidonic acid (AA).49–51 Research into fungal fermentation kinetics and modeling have been conducted with Pythium irregulare.56 Supercritical fluid extraction and 19

17

15

13

11

9

7

5

3

1 COOH

H3C cis-5, 8,1 1, 14, 47-eicosapentaenoic acid, 20:5 (n-3)

FIGURE 9.4 Chemical structure of EPA.

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modeling extraction behavior was implemented to develop a process for fractionation of important components (essential fatty acids and antioxidants) from both rice bran and fungi after fermentation.57 Beneficial health effects from consumption of certain fish oils have been attributed to the presence of the essential PUFA, EPA and docasapentaenoic acid (DHA). These omega-3 fatty acids have been linked to a reduced risk of coronary heart disease, arthritis, inflammation, hypertension, psoriasis, other autoimmune disorders, and cancer.58 PUFA are currently marketed as dietary supplements at health food stores in the form of concentrated fish oils. Supplementation into baby foods has lately received greater interest. PUFA are also prescribed medications for humans and pets. Declining marine resources and an increasing demand for PUFA have prompted the search for alternative sources of EPA and DHA. Filamentous fungi have the potential to produce large amounts of EPA within the mycelial walls when grown at optimal conditions.53,59 The filamentous fungus P. irregulare converts sweet whey permeate to significant amounts of EPA (24.9 mg EPA/g dry biomass), which could potentially add value to this food-processing by-product.50 Humans have a limited capacity to synthesize EPA, ARA, and DHA from shorterchain fatty acids. Additionally, typical American diets are extremely low in these compounds. The importance of EPA and ARA lies in that they are the biosynthetic precursors of the eicosanoid system that controls inflammatory and anti-inflammatory responses. There is increasing evidence that a variety of disorders, such as heart disease and hypertension, are related to malfunctions of the eicosanoid system caused by dietary imbalance of long-chain PUFA. DHA accumulates preferentially in the brain where it has been found to have rules both in neuronal impulse transmission and protecting the brain from oxidative stress. Dietary deficiencies of DHA have been linked to bipolar disorder and schizophrenia. Connor60 noted that diets rich in n-3 long-chain fatty acids tended to offset age-related degenerative diseases as a result of increased n-3/n-6 ratios in the blood and fatty acid profile in the brain. As a result, it has been found that dietary supplementation of PUFA has considerable efficacy in the treatment of these conditions and a considerable dietary supplement market has developed around these PUFA.61 The current commercial supplies of long-chain PUFAs primarily come from various oil seed plants, such as flax or borage, and from marine fish oil. Plant oils only contain the shorter-chain essential fatty acids, alpha-linolenic and linoleic acid, and do not contain the nutritionally important long-chain PUFA. Fish oil is a good source of EPA and DHA, which has major limitations including objectionable “fishy” smell and taste encapsulation, and growing concern of the presence of mercury and heavy metal accumulation in the extracted oils. While plants lack the enzymes necessary to create long-chain PUFA greater than 18 carbon units, microbes particularly in the algal and fungal classes possess oleaginous properties and an array of essential fatty acid desaturases required to synthesize short-chain PUFA, which are elongated to long-chain PUFA. Currently, microalgae are being used commercially to produce EPA and DHA. Microalgal production, however, has some drawbacks. Microalgal DHA production either requires the use of expensive photobioreactors or, in the case of tropically shifted algae, the use of expensive complex media.

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Common DHA-producing organisms include the algae Gonyaulax, Gyrodinium, and Cryptoconidium spp. and the bacteria Rhodopseudomonas and Shewanella spp. Mortierella alpina has been commercialized for production of long-chain PUFA primarily rich in ARA for use in infant formulas. Other ARA-producing fungi include Mortierella enongata, Entomorphthora exitalis, Pythium ultimum, and Pythium irregulare for use as an ingredient in infant formula to supply essential metabolic precursors for prenatal nervous system development. P. irregulare is an attractive candidate for PUFA production as it produces nearly equal amounts of both ARA and EPA.57 It is relatively easy to grow, can be adapted to both solid state and submerged culture techniques, and is capable of utilizing a wide variety of substrates. Specifically, P. irregulare has a comprehensive complement of metabolic enzymes and is able to be grown on substrates high in cellulose and hemicellulose. This allows the use of inexpensive waste materials as substrates for mass production EPA and ARA from P. irregulare. Research has indicated that culture conditions, such as medium composition, oxygen availability, pH, and temperature, could influence microbial lipid accumulation as well as PUFA composition.44,49,56 Due to the nature of the individual microorganism, the optimal conditions may differ from strain to strain. Some of the culture conditions for Pythium irregulare have been optimized.49–51,56 Glucose and yeast extract are considered as the preferred carbon and nitrogen sources for this microorganism.49 Nitrogen limitation is commonly believed to have significant effect on lipid accumulation in microbial fermentation.62 However, the optimal C/N ratio of Pythium irregulare for the target PUFA synthesis is not known, and few published studies have addressed this issue with other species. Therefore, optimizing medium composition and C/N ratio will be important for target PUFA production and process control. Zhu63 and Cheng et al.51 achieved the highest yields of the EPA and ARA (20 mg/g and 14.7 mg/g, respectively) with medium composed of 2% glucose and 0.25% yeast extract and C/N of approximately 32. Figure 9.5 shows the fungal culture of P. irregulare and oil production from P. irregulare grown on rice bran compared to commercial rice bran oil. Supercritical extraction of this oil compares favorably with the highly refined oil compared to conventional hexane extracted crude oil.64,65 Table 9.2 lists the primary fungal and algal sources considered for production of oils containing EPA and ARA, which serve as important precursors to an important class of metabolites including prostaglandins and leukotrienes.

9.5 LARGE-MOLECULE NUTRACEUTICALS Large-molecular nutraceuticals are generally made up of larger polymers typically in the 103 to 106 KDa range for proteins, and as high as 1010 KDa for polynucleotides.

9.5.1 POLYSACCHARIDE COMPOUNDS Many polysaccharides have been identified for therapeutic activity. Poly-branched β-D-glucans isolated from the cell walls of Saccharomyces cerevisiae76 and

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FIGURE 9.5 Photomicrograph of the mycelium of P. irregulare (1000). The mycelium contains about 20% oil rich in polyunsaturated fatty acids particularly high in EPA and ARA, which occur in about equal amounts.

Agrobacterium sp.77 has shown potent nonspecific immune-activation of macrophage immune cells, T-cells, NK-cells and B-cells and have shown high anti-AIDS activity with low side effects.77 A novel sulfated polysaccharide, calcium spirulan (Ca-SP) isolated from the cyanobacterium Spirulina platensis has been found to have both anti viral and anti cancer properties.78,79 Chitosan is the deacetylated from of chitin found in abundance in arthropod exoskeletal layers, but extractable chitosan is also in various fungi such as the industrial microoganism, Rhizopus oryzae.80–82 Chitosan is extensively marketed as a weight-loss nutritional supplement that binds to lipids, lowers serum cholesterol, and improves low-density lipoprotein to high-density lipoprotein (LDL/HDL) ratios. Chitosan has also shown anticarcinogenic properties83 as well as a tissue scaffold for implants and promotes healing of wounds.84 Purification of polysaccharides by different chromatographic processes that are differentiated from the lab scale from milligrams or even nanograms for chemical analysis up to the industrial scale in kilograms or tons of production for drugs and dietary supplements. Conventional gas chromatographic technique is commonly used for trace amounts of volatile chemical analysis, and preparative HPLC is suitable to collect biological chemicals up to milligrams for further chemical structural determination. However, both aforementioned methods are restricted by their loading capacities that are not suitable for industrial-scale processing of some functional carbohydrates. Recently, simulated moving bed (SMB) technique has drawn much attention because of its highly efficient productivity and suitability for sugar separation. The method has been found to be suitable for large-scale fractionation of xylitol, mannitol, and other specialty sugars.85,86 In addition, SMB technology has been used in pharmaceuticals, cosmetics, and fine chemicals. However, highly purified polysaccharides are seldom used except for their applications in biochemical research and in the bioprocessing industry. For instance, cellulose, agarose, and

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EPA Fungal Cultures

Carbon Source

P. irregulare P. irregulare P. irregulare P. irregulare P. irregulare M. alpina 1S-4 M. alpina M. alpina M. alpina M18 M. alpina 1S-4 M. alpina 1S-4 M. alpina 20-17 M. alpina M. elongata Saprolegnia Sp. C. minutissima P. cruentum

4% soy oil+ 2% glucose 5% rice bran 1% glucose^ SWP 1.8% glucose~ rice bran* 6.6% glucose 10% glucose" 3% linseed oil 2% linseed oil 1% linseed 5% glucose 2% linseed oil 1% olive oil 8000 lux

ARA

Lipid in Dry In Dry In Dry Biomass In Lipids Biomass In Lipids Biomass T °C (% w/w) (% w/w) (mg/g) (% w/w) (mg/g) 12 25 25 12 14 28 20 25 28 12 20 28 25 15 6 20 32

67 29 28 38 10 47

6 7 2 13 25

42 20 6.4 49 25

2 5 1 5 39 40 60 15

8 35 56 33 60

12 20 7

67 66 42

39 —

9 — 45 17

35 23 225

12 51

36

10 15 2 14 10 19 21 50

72

Reference Cheng et al. [51] Zhu et al. [56] Zhu et al. [56] Stintson et al. [49] O’Brien et al. [50] Higashiyama et al. [43] Jang et al. [66] Eroshin et al. [67] Yu et al. [68] Shimizu et al. [69] Jareonkitmongkol et al. [70] Shimizu et al. [71] Bajpai et al. [72] Bajpai et al. [72] Shirasaka et al. [73] Seto et al. [74] Nichols and Appleby [75]

+ supplemented with 1% soy meal; ^ spiked with 0.5% glucose 2 days before harvest; ~ supplemented with 0.1% soy oil; SWPsoy whey permeate. * solid-state culture (all others are submerged culture); "supplemented with 0.8 g/L glutamate.

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TABLE 9.2 Fungal and Algal Sources of EPA and ARA Grown on Various Carbon Sources Ranging from Pure Glucose to Complex Carbohydrates and Lipid Components

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dextran are commonly used as media of bioseparations such as electrophoresis and chromatography.

9.5.2 PROTEINS

AND

NUCLEOTIDES

In the past decades, the advance in biotechnology has enabled production of many desirable compounds from animals and plants. Future production of novel proteins such as scytovirin, isolated from the cyanobacterium Scytonema varium, which displays anticytopathic activity against laboratory strains of HIV-187 are expected to become increasingly important as new research developments are reported. Such controlled genetic activities involve the participation of many proteins and nucleotides. Therefore, recovery and purification of those biological macromolecules are essential for understanding and revealing the genetic activities. Harve and Bajpai88 have reviewed the steps of isolation and purification of those biological chemicals. In detail, common isolation steps often employ precipitation and crude adsorption followed by purification using ion exchange, affinity, and gel chromatography. Recently, capillary electrophoresis (CE), which is considered one of the most powerful separation technique for bioanalysts, has been commonly used regarding its rapid, high resolution of water-soluble components. Capillary electrophoresis has been accepted as a useful complementary tool for HPLC. However, CE and HPLC are limited by the prerequisite of several prepurification steps that often involve losses of useful targets. Therefore, expanded-bed adsorption has appeared to replace the conventional packed-bed operation to simplify purification of proteins and other biological chemicals. Purification of antibodies, in a large degree, is similar to the purification of other proteins. But its property in antigen specific recognition makes it also suitable for bioaffinity chromatography. To date, monoclonal antibodies and immunoglobulins represent by far the largest class of produced and purified proteins in numbers and mass. Recently, aqueous two-phase extraction (ATPE) has been successfully utilized to partition proteins into two partially immiscible phases in an aqueous environment. Hydrophobic interactions with proteins are factors in protein folding, transport across membranes, recognition, and enzyme catalysis. However, these hydrophobic entities are often buried within the folded lipoprotein structure and the introduction of hydrophobic tags (often aromatic rings) to the outer surface has been applied to increase solvent interactions as a technique to improve bioseparation.89 Another recent novel protein purification method particularly suitable to the class of large glycoproteins utilizes an affinity carbohydrate-based matrix called bioskin made of a coculture of Acetobacter xylinim, Saccharaomyces cerevisiae and Saccharomyces pombe90 with gradient elution with potassium phosphate.91 Table 9.3 shows the relationship between bioseparation cost relative to the general class of compound.92 Estimated global market demands for 2002 are also represented for these classes.2,3

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TABLE 9.3 Global Demand Value and Bioseparation Cost of Various Compound Classes

Product Functional foods, e.g., casein Nutraceuticals, e.g., scytovirin Industrial enzymes, e.g., cellulases Diagnostic enzymes, e.g., glucose oxidase Therapeutic enzymes, e.g., urokinase

Global Demand Market % of Total Value (2002) Expansion Relative Production (US$ billion) Rate (%) Cost Cost 33 47 2 8 27

10 10 6 9 14

1 2–10 5–10 50–100 50–500

10–30 30–50 30–50 50–70 60–80

9.5.3 SMALL MOLECULAR NUTRACEUTICALS Many natural nutraceuticals are small molecules with molecular weight less than 600 Da and typical radius of 0.5 nm. These often include monomer compounds such as sugars, fatty acids, amino acids, and organic acids. However, they are very diverse in chemical characterization in terms of their chemical polarity, natural existence in plants/animals, and biological activities. Many important small molecular nutraceuticals often found in plants, but also present in high concentrations within some microbial communities include various classes of antioxidants and alkaloids that stimulate the immune system by activation of T-cells, scavenge for free radicals, and inhibit tremors and analgesia.93 Compounds of commercial interest include carotenoids that function as pro-vitamin A, astaxanthin from red yeast Phaffia rhodozyma,94 canthaxanthin, and lycopene.95 A compound known to have anticonvulsant properties is the extracellular alkaloid compound pimprinine produced by Streptomyces sp.93 and processed by filtration, chromatography, and vacuum drying. Hydroxytyrosol is a powerful antioxidant naturally present in olive oil and is synthesized in strains of Pseudomonas aeruginosa.96 Cis -β-carotene is produced in Dunaliella salina algal culture and in fungi Blakeslea trispora, Neurospora crassa,97,98 and Phaffiea rhodozyma when stimulated by light.99 Due to the physicochemical and biological characteristics specific to these biomolecules, a variety of extraction and separation methods have been developed. Solvent extraction is a traditional method to extract phytochemicals or nutraceuticals, that is, extraction of anticancer polyphenolics from blueberry.100 Recently, supercritical fluid extraction (SFE) has been strongly recommended to extract nutraceuticals because of the extraction efficacy of SFE and concerns of the toxicity of solvents. But both extraction methods always bring a mixture of different classes of chemicals. Therefore, additional steps are often required for the removal of unwanted interfering substances. For example, solid phase extraction (SPE) and open column chromatography are now commonly used for further chemical fractionation. Successful methods of extraction of antioxidants from algae and fungi with use of supercritical CO294,95 and absolute ethanol101 have been identified.

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Naczk and Shahidi102 reviewed the extraction and analytical methods for many nutraceutical phytochemicals including phenolics, flavonoids, anthocynins, tannins, and so forth. It was concluded there is no uniform or completely satisfactory procedure for extraction of all phytochemicals or a specific class of phytochemicals in plant material. Considering the volatility of nutraceuticals, gas chromatography (GC) and high-pressure liquid chromatography (HPLC) have been frequently used for volatile and nonvolatile chemicals, respectively. Furthermore, different monitoring detectors such as UV and mass spectrometer are often combined with gas and liquid chromatography for quantitative and qualitative analysis of nutraceuticals.103,104 Enzyme-linked immunosorbent assay (ELISA) has also been frequently used in biological and clinical research.105 As a novel emerging technique, magnetic field combined with nanotechnology106 has been tried for drug delivery and analysis. Nevertheless, all of the methods mentioned are limited in lab-scale research, and only simulated moving bed (SMB) has been successfully and commercially used in large industrial scale. In general, bioseparations are essential in the good manufacturing practice (GMP) of production of pharmaceuticals and nutraceuticals, which generally require the products in an acceptable level of purity. Aside from basic separation issues of antioxidants from cell biomass in production systems, extraction procedures within clinical tissues such as the liver to measure the metabolic effect of antioxidants has hampered the analysis procedures due to poor sample preparation prior to HPLC analysis. Burri et al.107 demonstrated that the use of supercritical carbon dioxide without modifier proved to generate vitamin A and carotenoid extracts from calf liver tissue that did not require further pretreatment as typically needed for organic solvent techniques.

9.6 BIOPROCESS DESIGN Bioprocess design for nutraceutical production primarily involves the conceptual work to implement the technology for production and bioseparations, alterations, or scale up to the process plant design. A detailed overview of the procedures is covered in several texts.16,108 The process design of food, pharmaceutical, and nutraceutical production is a marriage of process analysis and cost estimation that include the life cycle of the product, use of engineering parameters and estimates to complete a series of mass and energy balances in a process flow simulation, and environmental impact assessment. The economic assessment begins with an order of magnitude estimation based on previous cost data for similar bioprocess resulting in large errors (up to 50%108) even with experienced teams of engineers, industrial biotechnologists, and economists. Further estimates include project planning based on major equipment needed, preliminary and detailed engineering design from process data, and contractor estimate with estimation error usually less than 10%.108 The life cycle analysis for products is considered to begin with preliminary research findings. Products first undergo a feasibility analysis for screening, then a project budget development is created, and finally, after product entry into the market, scale-up of commercial production is assessed based on market demand.108 Bioreactor design should consider microbial growth and product formation kinetics, impact of growth-limiting substrate or products, shear sensitivity of cells,

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temperature effects, immobilized vs. suspended culture, and characterization of products as primary, secondary, or mixed metabolite. Basic bioreactor design configurations include batch, fed-batch, and continuous-flow stirred tank reactors (CSTR) which are covered in detail in the literature.16,109–113

9.7 PROCESS ANALYSIS The first step of process analysis is to construct a generalized flow diagram or flow sheet where, during initial phases of process development, the unit operations may not be well defined. As research data containing vital biological information such as substrate (carbon, nitrogen, oxygen, etc.), biomass and product kinetics, pH range for the desired system (continuous, fed-batch, or batch mode), is made available, the appropriate material and energy balances may then be applied to determine reactor volume, product mass, temperature requirements, air flow rates, and so forth. Furthermore, molecular size, polarity, molecular charge, and orientation (degree of glycosylation, protein folding, etc.) of the bioproduct of interest generally determine the type of integrated bioprocess regime needed for production and separation of the product. Whether the biological function of the product is intracellular, extracellular, or membrane bound is another important factor to whether separation may occur during fermentation, as done in continuous or fed-batch situations, or additional bioseparation steps are required as in the case of intracellular and membrane bound products. Once reactor volume, mass of products, and product properties are known, subsequent bioseparation unit operations are placed in the flow diagram with corresponding material and energy balances conducted for each unit operation in a similar way conducted for the bioreactor step to determine equipment sizing and material requirements.114 Cost analysis is then integrated in the design process to narrow down the type of reactor and separation equipment to further optimize the process and enable some important initial order-of-magnitude cost estimates, including capital and operating costs discussed in further detail in the next section. Figure 9.6 is an example of a process flow chart constructed in SuperPro Designer® of a specific biorefinery concept proposed for the coproduction of fungal oil, fiber composites, ethanol, and nanocrystalline cellulose from conventional lignocellulosic biomass. Ethanol and carbon dioxide from the simultaneous saccharification and fermentation (SSF) step may be recycled back to the oil supercritical extraction unit as well as ethanol used to supply energy to the process.

9.8 PROCESS ECONOMICS Economics in preliminary bioprocess design typically includes research and development cost, estimation of capital and operating costs, and profitability analysis. Research and development cost may range in the millions of dollars depending on the ability to bring products to market, which is often less than 10% of many products tested. These efforts typically include screening of potential microorganisms, genetic modification for increased productivity, testing for contaminants and toxicity, process

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S-102

P-7 Oil

S-113 S-106

S-112

S-110

P-8 Fiber composite

P-Q/GBX-101 ethanol S-107

P-3/V-102 S-108 distillation S-109

S-105 S-103 P-6/V-101 Solid-state reactor P-2/DX-101 SFE

S-111 P-11/CY-1 Fiber separation

P-5/MSX-101 Hydrolysis (hot water/ mild acid)

P-4/V-103 SSF or SHF

S-114 P-10 Nanocrystalline cellulose

FIGURE 9.6 Biorefinery process flow schematic for production of nutraceutical and bioenergy products from lignocellusic by-products.

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S-104 S-101

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production and bioseparation technology, process scale-up studies, clinical trials (e.g., FDA-approved biopharma products), marketability and ethical sensitivity studies, and other research specific to handling and packaging of the product. Capital costs are often estimated using multipliers or unit costs that include total plant direct cost (TPDC) (i.e., installation, process piping, instrumentation and control mechanisms, electrical, building and facility cost), equipment cost (EC), total plant indirect cost (TPIC) (i.e., engineering and construction), and direct fixed capital (DFC) (TPDC + TPIC + contractors fees and contingency). Building and facility cost range from $2000/m2 for Class 100,000 process areas to as high as $8000/m2 for Class 100 areas (typical for biopharmaceutical processing subject to strict current GMP [cGMP], GLP standards). Individual equipment cost will increase with the key physical design scale-up parameters such as unit operation volume, membrane surface area, or flow throughput, but generally by a factor of about 52% referred to as the “economy of scale.”108 Operating cost include direct costs such as raw materials (10% to 80% total operating cost), consumables (membranes, process seals, etc.), labor (20% to 50% total operating cost), waste disposal (up to $0.5/m3), utilities (approximately $0.1/kWh, $8/1000 kg steam, up to $50/1000 kg purified water, $0.1/1000 kcal heat removed by refrigeration) and laboratory quality control (QC) and quality assurance (QA) costs (10% to 20% of the labor costs). Indirect operating costs include depreciation of fixed capital investment, maintenance (up to 10% of equipment cost per year), insurance (up to 1% of DFC), local property taxes (up to 5% of DFC), and other overhead expenses (accounting, payroll, security, fire protection, etc. which accounts for up to 10% DFC).108 Finally, profitability analysis includes various measures to assess profitability and marketability of the product. These factors include return on investment (ROI) or net profit per year per total investment value, net present value (NPV), internal rate of return (IRR), and payback time that considers cash flows (annual revenues, operating costs, etc.) and time value of money as a function of time of the projected life of the processing plant.115 Simulation software like Aspen Plus® and SuperPro Designer® have extensive capabilities to integrate both process analysis and economics for predicting cost necessary for key decision making toward conceptualizing a processing facility, determining scalability based on key processing properties and engineering parameters, and estimated profitability of a production plant.

9.9 CONCLUSIONS This chapter outlined the relevance of nutraceuticals in a newly developing biobased economy that is rapidly becoming an economic player in the food and agricultural industries. Bioprocessing techniques to produce high-value specialty biochemicals from food and agricultural by-products are emphasized, and greater need for bioenergy production to compensate for deficiencies in the present oil-based economy is described through the biorefinery concept. Specific examples of existing and proposed industrial bioprocesses that show substantial economic potential were outlined in terms of lipid-based, large and small molecular nutraceutical products.

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As the world population continues to increase posing an even greater health threat to third world countries, there is a continuing need for the integration of biology and engineering to address the issue for optimizing functional food, nutraceutical, and pharmaceutical production that incorporates important principles of environmentally friendly technologies and continuing education to support new advances for a bio-based economy.

ACKNOWLEDGMENTS The authors would like to acknowledge undergraduate students (particularly students from the 2005 Biochemical Engineering course) and graduate assistants (Jack Heaton, Hui Zhu, Kristy Conwel, Jared Powell, Keri Cantrell, Cheng-Yi Kuan, Meidui Dong, and Xiaohui Yu) in the Clemson University Biosystems Engineering Program who participated in bioprocessing projects for contributing to literature making this chapter possible.

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69. Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y., and Yamada, H., Microbial conversion of an oil containing alpha-linolenic acid to an oil containing eicosapentaenoic acid, J. Am. Oil Chem. Soc., 66, 342–347, 1989. 70. Jareonkitmongkol, S., Sakuradani, E., and Shimizu, S., A novel omega-5-desaturasedefective mutant of Mortierella alpina 1S-4 and its dihomo-gema-linolenic acid productivity, Appl. Environ. Microbiol., 59, 4300–4304, 1993. 71. Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y., and Yamada, H., Conversion of linseed oil to an eicosapentaenoic acid-containing oil by Mortierella alpina 1S-4 at low temperature, Appl. Microbiol. Biotechnol., 32, 1–4, 1989. 72. Bajpai, P.K., Bajpai, P., and Ward, O.P., Optimisation of culture conditions for production of EPA by Mortierella elongata NRRL 5513, J. Ind. Microbiol., 9, 11–18, 1992. 73. Shirasaka, N. and Shimisu, S., Production of eicosapentaenoic acid by Saprolegnia sp. 28YTF-1, J. Am. Oil Chem. Soc., 72, 1545–1549, 1995. 74. Seto, A., Wang, H.L., and Hesseltine, C.W., Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima, J. Am. Oil Chem. Soc., 61, 892–894, 1984. 75. Nichols, B.W. and Appleby, R.S., The distribution and biosynthesis of arachidonic acid in algae, Phytochem., 8, 1907–1915, 1969. 76. Miller, A., et al., The application of various protic acids in the extraction of 1→3-βD-glucan from Saccharomyces cerevisiae, Carbohydrate Res., 299, 203–208, 1997. 77. Kim, M., Ryu, K., Choi, W., Rhee, Y., and Lee, I., Enhanced production of 1→3-βD-glucan by a mutant strain of Agrobacterium species, Biochem. Eng. J., 16, 163–168, 2003. 78. Hiyashi, T. and Hayashi, K., Calcium spirulan, an inhibitor of enveloped virus replication, from Blue-Green Alga Spirulina platensis, J. Natural Products, 59, 83–87, 1996. 79. Mishima, T. and Murata, J., Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green algae, Spirulina platensis, Clin. Exp. Metastasis, 16(6), 541–550, 1998. 80. Hu, K.J., Hu J.L., Ho, K.P., and Yeung, K.W., Screening of fungi for chitosan producers and copper adsorption capacity of fungal chitosan and chitosanaceous materials, Carbohydrate Polymers, 58, 45–52, 2004. 81. Tan, S.C., Tan, T.K., Wong, S.M., and Khor, E., The chitosan yield of zygomycetes at their optimum harvesting time, Carbohydrate Polymers, 30, 239–242, 1996. 82. Nwe, N. and Stevens, W.F., Production of fungal chitosan by solid substrate fermentation followed by enzymatic extractions, Biotechnol. Letters, 24, 131–134, 2002. 83. Kiode, S.S., Chitin-chitosan: properties, benefits, and risks, Nutr. Res., 18, 1091–1101, 1998. 84. Madihally, S.V. and Matthew, H.W.T., Porous chitosan scaffolds for tissue engineering, Biomaterials, 20, 1133–1142, 1999. 85. Saska, M. and Chen, F., Process for the separation of sugars, U.S. Patent 6,451,123, 2002. 86. Saska, M., Chen, F., and Vatelot, A., Sugarcane bagasse-to-xylitol process: preliminary testing, J. Int. Sugarcane, 101(1207), 353–358, 1999. 87. Boekesch, H.R., O’Keefe, B.R., McKee, T.C., Pannell, L.K., Patterson, G.M.L., Gardella, R.S., and Sowder, R.C., A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium, Biochemistry, 42(9), 2578–2584, 2003.

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88. Harve, R. and Bajpai, R., Separation of nucleic acids and proteins, in Handbook of Bioseparations, Ahuja, S., Ed., Academic Press, San Diego, CA, 2000, chap. 8, pp. 365–378. 89. Bulow, L., Hydrophobic peptide tags as tools in bioseparation, Trends Biotechnol., 22(10), 511–516, 2004. 90. Vicente, C., Sebastian, B., Fontaniella, B., Marquesz, A., Filho, L.X., and Legaz, M.E., Bioskin as an affinity matrix for the separation of glycoproteins, J. Chromatogr. A, 917, 55–61, 2001. 91. Kishino, S., Nomura, A., Saitoh, M., Sugawara, M., Iseki, K., Kitabatake, A., and Miyazaki, K., Single-stop isolation method for six glycoforms of human 1-acid glycoprotein by hydroxylapatite chromatography and study of their binding capacities for disopyramide, J. Chromatogr. B, 703, 1–6, 1997. 92. Ghosh, R., Protein Bioseparation Using Ultrafiltration: Theory, Applications and New Development, Ghosh, R., Ed., Imperial College Press, 2003, chap. 1. 93. Nalik, S.R., Harindran, J., and Varde, A.B., Pimprinine, an extracellular alkaloid produced by Streptomyces CDRIL-312: fermentation, isolation and pharmacological activity, J. Biotechnol., 88, 1–10, 2001. 94. Lim, G., Lee, S., Lee, E., Haam, S., and Kim, W., Separation of axtaxanthin from red yeast Phaffia rhodozyma by supercritical carbon dioxide extraction, Biochem. Eng. J., 11, 181–187, 2002. 95. Sabio, E., Lozano, M., Montero de Espinosa, V., Mendes, R.L., Pereira, A. P, Palavra, A.F., and Coelho, J.A., Lycopene and B-carotene extraction from tomato processing waste using supercritical CO2, Ind. Eng. Chem. Res., 42(25), 6641–6646, 2003. 96. Allouche, N., Damak, M., Ellouz, R., and Sayadi, S., Use of whole cells of Pseudomonas aeruginosa for sythesis of the antoxidant hydroxytyrosol via conversion of tyrosol, Appl. Envir. Microbio., 70, 2105–2109, 2004. 97. Gessler, N.N., Sokolov, A.V., Bykhovshy, V.Y., and Belozerskaya, T.A., Superoxide dismutase and catalasse activites in carotenoid-synthesizing fungi Blakeslea trispora and Neurospora crassa fungi in oxidative stress, Appl. Biochem. Microbiol., 38(3), 205–209, 2002. 98. Rabbani, S., Beyer, P., Johannes, V.L., Hugueney, P., and Kleinig, H., Induced βcarotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil, Plant Physiol., 116, 1239–1248, 1998. 99. Ruddat, M. and Garber, E.D., Secondary Metabolism and Differentiation in Fungi, Bennett, J.W. and Ciegler, A., Eds., Marcel Dekker, New York, 1983, pp. 95–152. 100. Burns, K., et al., Chemopreventive potential of wild lowbush blueberry fruits in multiple stages in carcinogenesis, J. Food Sci., 7093, S159–166, 2005. 101. Roukas, T. and Mantzouridou, F., An improved method for extraction of b-carotene from Blakeslea trispora, Appl. Biochem. Biotechnol., 90, 37, 2001. 102. Naczk, M. and Shahidi, F., Extraction and analysis of phenolics in food, J. Chromatogr. A, 1054, 95–111, 2004. 103. Kim, H., Wu, C., Chen, F., Wang, H., and Jin, Z., Evaluation of antioxidant activity of tea tree (melaleuca alternifolia) oil and its components, J. Agric. Food Chem., 52, 2849–2854, 2004. 104. Kim, H.J., Chen, F., Wang, X., and Rajapaks., N.C., Effect of chitosan on biological properties of sweet basil (Ocimum basilicum L.), J. Agric. Food Chem., 53, 3696–3701, 2005. 105. Wang, X., Chen, F., Wan, P.J., and Huang, G., Development of monoclonal antibodybased enzyme linked immunosorbent assay for gossypol analysis in cottonseed meals, J. Agric. Food Chem., 52, 7793–7797, 2004.

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106. Son, S.J., Reichel, J., He, B., Schuchman, M., and Lee, S.B., Magnetic nanotubes for magnetic-field-assisted bioseparation, biointeraction, and drug delivery, J. Am. Chem. Soc., 127, 7316–7317, 2005. 107. Burri, B.J., Neidlinger, T.R., Lo, A.O., Kwan, C., and Wong, M.R., Supercritical fluid extraction and reversed-phase liquid chromatography methods for vitamin A and βcarotene heterogenous distribution of vitamin A in the liver, J. Chromatogr., 762, 201–206, 1997. 108. Harrison, R.G., Todd, P., Rudge, S.R., and Petrides, D.P., Bioseparations Science and Engineering, Oxford University Press, New York, 2003. 109. van Dam-Mieras, M.C.E., de Jeu, W.H., de Vries, J., Currell, B.R., James, J.W., Leach, C.K., and Patmore, R.A., Bioreactor Design and Product Yield, ButterworthHeinemann, Boston, 1992. 110. Grady, C.P.L., Daigger, G.T., and Lim, H.C., Biological Wastewater Treatment, 2nd ed., Marcel Dekker, New York, 1999. 111. Bailey, J. and Ollis, D., Biochemical Engineering Fundamentals, 2nd ed., McGrawHill, New York, 1986. 112. Shuler, M.L. and Kargi, F., Bioprocess Engineering: Basic Concepts, Prentice-Hall, New York, 1992. 113. Doran, P.M., Bioprocess Engineering Principles, Academic Press, San Diego, CA, 1995. 114. Ladisch, M.R., Bioseparations Engineering: Principles, Practice, and Economics, John Wiley and Sons, New York, 2001. 115. Peters, M.S. and Timmerhaus, K.D., Plant Design and Economics for Chemical Engineers, 4th ed., McGraw-Hill, New York, 1991.

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10

Microbial Modeling as Basis for Bioreactor Design for Nutraceutical Production Caye M. Drapcho Clemson University

CONTENTS 10.1 10.2 10.3 10.4 10.5

Introduction ...............................................................................................238 Xylitol........................................................................................................239 Summary of Unstructured Microbial Growth Models .............................240 Unstructured, Single Limiting Nutrient Models.......................................240 Inhibition Models ......................................................................................241 10.5.1 Substrate Inhibition ......................................................................241 10.5.2 Product Inhibition.........................................................................242 10.5.3 Inhibition by Xenobiotic Compounds .........................................243 10.6 Models for Multiple Limiting Substrates or Nutrients ............................243 10.6.1 Complementary Substrates...........................................................243 10.6.2 Substitutable Substrates ...............................................................244 10.7 Yield Parameters .......................................................................................246 10.8 Temperature Effects ..................................................................................247 10.9 Kinetic Rate Expressions ..........................................................................247 10.10 Bioreactor Design......................................................................................250 10.11 Batch Reactors ..........................................................................................250 10.12 Continuous Stirred Tank Reactors (CSTRs).............................................251 10.13 CSTR with Cell Recycle...........................................................................253 10.14 Fed-Batch Systems....................................................................................255 10.15 Bioreactor Design Strategies.....................................................................256 10.16 Modeling of Glucose/Xylose Utilization and Product Formation by Candida ..............................................................................258 10.17 Summary ...................................................................................................261 References..............................................................................................................261

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10.1 INTRODUCTION A wide variety of nutraceutical compounds, food additives, and foodstuffs may be produced through the bioconversion of substrates contained in agricultural crops and residues. Bioconverted products include traditional foods and beverages such as beer, wine, vinegar, yogurt, tofu, and baker’s yeast. Bioconverted nutraceutical and food products used in the food industry range from flavor compounds (roasted, nutty flavor of pyrazine produced through aerobic solid-state fermentation of soybeans by bacterium Bacillus subtilis,1 fruity flavor of citronellol, linalool, and geraniol produced by yeast Kluveryomyces lactis,2 vanillin flavoring produced by Pseudomonas and Corynebacterium,3 and bioflavors for beer produced by Saccharomyces cerevisiae4); essential fatty acids (omega-3 and omega-6 fatty acids eicosapentaenoic (EPA) and arachidonic (ARA) acids from aerobic submerged fungal cultures of Pythium irregulare, Mortierella elongata, and Mortierella alpina using soybean and rice bran substrates,5–9 and omega-3 fatty acid docosahexaenoic acid (DHA) and EPA from algal sources such as Gonyaulax, Gyrodinium, and Cryptoconidium10,11); organic acids (citric acid produced by aerobic fermentation of sugarcane or beet molasses by Aspergillus niger or Candida lipolytica,12–15 lactic acid produced by Lactobacillus helveticus from concentrated cheese whey,16 and pyruvic acid production by variety of yeasts and bacteria17); pigments (the carotenoid pigment and antioxidant astaxanthin produced by Xanthophyllomyces dendrorhous,18 red pigments produced by fungi Paecilomyces sinclairii during aerobic fermentation of sucrose and starch,19 and orange and red dyes produced by submerged culture of Monascus purpureus from hard wheat substrate20); emulsifiers (biosurfactants produced by filamentous fungi Curvularia lunata,21 and high molecular weight emulsifiers produced by yeast Candida utilis22); vitamins (vitamin B12 production by prokaryotic bacteria Propionibacterium shermanii and Pseudomonas denitrificans,23,24 or mixed cultures of methanogens,25,26 and vitamin K2-7 production by bacterium Bacillus subtilis using fermented soybeans27); and sugar substitutes (xylitol produced from microaerobic fermentation of D-xylose contained in hydrolysates of sugarcane bagasse, rice straw, and other lignocellulosic material and defined media by yeasts such as Candida and Pichia28–52). Bioprocess design involves consideration of all critical unit operations, including feedstock pretreatment and hydrolysis, bioreactor modeling and design, and extraction and separations technologies. Bioreactor modeling and design based on microbial growth and product formation kinetics may be used to optimize production of high-value compounds by a range of natural microorganisms such as fungi, bacteria, yeasts, and algae and recombinant organisms. Steps to the design based on microbial kinetics include expression of growth, decay, and product formation rates; development of mass-balance models of the bioreactor; simulation of substrate, biomass, and product concentrations and rates of biomass and product formation for various reactor configurations; selection of reactor configuration; and design optimization to maximize product formation rate or product concentration based on simulation. The focus of this chapter is on microbial modeling and bioreactor design for suspended growth, shear-tolerant, natural cells. Many bioprocessing examples could

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serve to illustrate the kinetic approach to modeling and design; we will highlight the bioconversion of xylose to the sugar substitute xylitol.

10.2 XYLITOL Xylitol, a 5-carbon sugar alcohol (C5H12O5), has a relative sweetness equal to sucrose, a negative heat of solution and is nonfermentable by cariogenic bacteria.53 Once ingested by humans, 80% to 85% of xylitol consumed is metabolized by the liver, and 20% to 80% of this portion is converted to glucose depending on metabolic need for glucose.53 The slow conversion of xylose to glucose reduces insulin stimulation. Due to these properties, it has found many uses in the food industry, especially in confectionaries, gums, oral hygiene products, and diabetic foods. Xylitol is found naturally at low levels in fruits and vegetables; for example, plums contain 1% xylitol by weight, while strawberries, raspberries, eggplant, spinach, and pumpkin contain 0.3% to 0.1% xylitiol, respectively.53 Xylitol can be synthesized chemically through the catalytic dehydrogenation of xylose, a 5-C sugar. However, extraction from natural foods and chemical production is costly, leading to investigations of bioconversion processes for xylitol production as an alternative. Bioconversion of xylose to xylitol can be accomplished by yeasts, primarily of the genus Candida and Pichia.29–52 These yeasts can utilize pentose and hexose sugars, with hexose sugars as the preferred substrate.34,50 The sequential use of glucose then xylose under batch conditions has been documented extensively.32,34,37,38,41,42,46 Under aerobic conditions, both glucose and xylitol are metabolized with biomass as the main product. Under conditions described variously as oxygen-limited or microaerobic, species of Candida can form xylitol from Dxylose29–52 and ethanol from glucose and xylose.33–35 Hemicellulosic hydrolysates from agricultural residues such as sugar cane bagasse, rice straw and wheat straw are plentiful and contain significant quantities of xylose and glucose in their hemicellulose and cellulose fractions. For example, 1 ton of milled sugar yields 180 to 280 kg of sugarcane bagasse, with the typical bagasse containing 19% to 24% hemicellulose, 32% to 48% cellulose, and 23% to 32% lignin.54 This composition represents typically 71% total reducing sugars, 25% xylitol, and 41% glucose on a dry weight basis.40 However, mild acid pretreatments with sulfuric or hydrochloric acids result in greater hemicellulose hydrolysis than cellulose hydrolysis.39 Varying conditions of temperature, time, and acid concentration result in greatly varied concentrations of sugars in sugarcane bagasse hydrolysate. A typical sugarcane bagasse hydrolysate may contain 17 to 105 g/L xylose and 7 to 30 g/L glucose, while a typical rice straw hydrolysate may contain 16 to 79 g/L xylose and 4 to 23 g/L glucose.39 Bioconversion of xylose can achieve xylitol yields as high as 0.7 to 0.8 g xylitol/g xylose in hemicullulosic hydrolysates30,37,43 and 0.8 to 0.9 in defined xylose media.35,38 The theoretical yield is 0.917 g/g. For example, batch fermentations of sugarcane bagasse hydrolysate using Candida guilliermondii FTI20037 achieved maximum xylitol production rates of 0.87 g/L-h with product yield of 0.67 g xylitol/g xylose at 0.45 v.v.m. aeration rate (kLa 27 h–1).49 Reported values of maximum

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specific growth rate for Candida range widely from 0.04 to 0.52 h-1 for xylose and glucose/xylose mixtures.38,40,50

10.3 SUMMARY OF UNSTRUCTURED MICROBIAL GROWTH MODELS Basic microbial growth models for suspended growth, shear-resistant cells should consider potential impact of multiple growth-limiting substrate and inhibitory substrates, products, and xenobiotic compounds. Structured models consider individual reactions occurring within the cell involving specific components, such as DNA or proteins. Unstructured models view the cell as an entity and model growth and death of the microorganism.55–63 Growth rates for unstructured models are expressed as v (cell number specific growth rate) or µ (biomass specific growth rate), both in units of time–1. Balanced growth occurs when v = µ and cell composition is constant with time. Unbalanced growth occurs as cell composition changes with time, resulting in a change in cell mass per cell; therefore v ≠ µ.56

10.4 UNSTRUCTURED, SINGLE LIMITING NUTRIENT MODELS The Monod model64 is a widely applied model used to describe microbial growth. The model was developed for the growth of a single microorganism (E. coli) growing on a media with a single limiting organic substrate (glucose) as shown below µ=

µˆ SS K s + SS

(10.1)

where µ = specific growth rate coefficient, h–1; µˆ = maximum specific growth rate, h–1; SS = soluble substrate concentration, mg/L; and KS = half-saturation constant, mg/L. The KS value is the substrate concentration at which the growth rate µ is equal to 1/2 of µˆ . The Monod model has been used successfully to model the growth of many pure cultures of heterotrophic and autotrophic organisms growing on single substrates and mixed microbial cultures using mixed substrates, such as wastewater treatment applications.59,60 Estimates of µˆ and KS parameter values may be obtained by collecting data of specific growth rate values as a function of soluble substrate concentration during exponential phase of batch growth, and applying a linearization technique, such as Lineweaver-Burk or Hanes (Hofstee) equations.56,57,59 Zero and first order approximations of the Monod model (Equation 10.2 and Equation 10.3) may be applied when the substrate concentration is high and low relative to the KS value, respectively. µ ≈ µˆ Zero order appropriation when SS>>KS

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µ≈

µˆ SS First order appropriation when SS<< KS Ks

241

(10.3)

Due to high substrate concentrations typically found in food/bioprocessing applications, many researchers report µˆ values and not KS .38 For Candida tropicalis utilizing xylose under batch microaerobic conditions, µˆ was determined to be 0.52 h–1 at initial xylose concentrations of 50 g/L.38 However, much lower µˆ values of 0.04 and 0.11 h–1 were reported for Candida guilliermondii41 cultures using xylose and glucose, respectively, and µˆ values of 0.057 to 0.137 h–1 were reported for Candida tropicalis cultures as a function of aeration rate.40 In these reports, observed specific growth rate was apparently assumed to be the maximum specific growth rate value. For example, Pessoa et al.40 reported that µˆ values were determined from the growth curve in the exponential phase. These values may represent the observed specific growth rate for culture conditions present rather than the maximum specific growth rate.

10.5 INHIBITION MODELS Expansions of the Monod model to include inhibition by substrate, product, or xenobiotic compounds have been developed. Many inhibition models for microbial growth are empirical in nature, while others apply enzyme kinetics concepts.56 In general, competitive inhibitors compete with substrate for binding site of the enzyme, and may be xenobiotic compounds or products that accumulate in reactor. Uncompetitive inhibition occurs when a compound binds to the enzyme-substrate (ES) complex. A special form of uncompetitive inhibition is substrate inhibition, where substrate binds to an alternative site on the enzyme leading to a nonreactive ES complex. Noncompetitive inhibitors can bind to either the free enzyme or the ES complex. Product inhibition may be modeled as noncompetitive inhibition.56

10.5.1 SUBSTRATE INHIBITION High substrate concentrations relative to the KS value may limit growth. Modifications of the Monod model, such as the Andrews’ Equation,65 may be used to describe substrate inhibition:59 µ=

µˆ SS 2

K S + SS + SS K I

(10.4)

where KI = inhibition coefficient, mg/L. As the value of KI increases, the equation above simplifies to the Monod model. In many cases of inhibitory substrate, the value of µˆ may never be observed and therefore KS cannot be calculated. In these cases, µ*, the maximum observed growth rate, and SS*, the substrate concentration at which µ* occurs, are determined and used to calculate µˆ and KS :59

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µ* =

µˆ and SS* = (KS /KI)0.5 2( K S / K I )0.5 + 1

(10.5), (10.6)

Bioprocessing studies have investigated the inhibition of cell growth rates due to elevated substrate levels. For example, Oh and Kim38 found that increasing the initial xylose concentration past 50 g/L for Candida batch fermentations on defined xylose media under microaerobic conditions resulted in reduced exponential-phase specific growth rates, from 0.52 h–1 at 50 g/L initial xylose concentration, to 0.49, 0.47, 0.35, and 0.23 h–1 at xylose concentrations of 100, 150, 200, and 250 g/L, respectively.38 However, Equation 10.5 does not model the response well, which may indicate that other factors, such as oxygen limitation with increasing substrate, lack of pH control, or product inhibition, may also have influenced the specific growth rate. For phototrophic microorganisms, Steele’s model66 is often used to describe algal growth considering potentially inhibitory light intensity66–68 

I 1 − µ = µˆ L e I opt

IL   I opt 

(10.7)

where IL and Iopt = light and optimal light intensity, µmol/m2-s.

10.5.2 PRODUCT INHIBITION High product concentrations may inhibit microbial growth. Product inhibition may be modeled as µ=

(

n P µˆ µˆ S  or µ = 1− 1− P P   m K Pm  KS + S  S    1 +  S

)

n

(10.8)

where P = product concentration, mg/L; Pm = product concentration where growth is completely inhibited; n = coefficient determined by data. For example, yeast growth under anaerobic conditions may be inhibited by ethanol concentrations above 5%.56,61 The growth of Candida shehatae was completely inhibited by ethanol concentrations of 25 g/L under microaerobic fermentations33 and 37.5 g/L under aerobic conditions.29 Another equation to describe noncompetitive inhibition by product is represented in several different forms as shown below56,58,61 µ=

µˆ SS  K P  µˆ  S  1 or µ = or µ = µˆ    P  K S + S  K S + SS  K P + P  KS   P   1+  1 + S   1 + K  Kp  S p (10.9)

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where Kp = product inhibition constant, mg/L.

10.5.3 INHIBITION

BY

XENOBIOTIC COMPOUNDS

A general form of uncompetitive inhibition of enzymes can be used to model inhibition by other compounds (rearranged from Blanch and Clark56) µ=

µˆ SS SS 1 + I K + K S I

(

)

(10.10)

where I = inhibitor concentration, mg/L, and KI = inhibition constant, mg/L. Another equation used to model general inhibition is given as  SS   K I  µ = µˆ   K S + SS   I + K I 

(10.11)

Sugarcane bagasse hydrolysate contains compounds such as furfural and 5-hydroxymethylfurfural (HMF) that may inhibit yeast growth at high concentrations. Inhibition of cell growth occurred at furfural and HMF concentrations of 1.0 and 1.5 g/L, respectively, for Candida guilliermondi.30 Use of KI value of 0.01 g/L in Equation 10.12 would result in a predicted 99% rate reduction of the specific growth rate at furfural concentration of 1.0 g/L.

10.6 MODELS FOR MULTIPLE LIMITING SUBSTRATES OR NUTRIENTS Substrate or nutrients can be thought of in a broad sense as complementary or substitutable. Complementary nutrients meet different needs for the microorganism; for example, oxygen may serve as electron acceptor for yeast growth while glucose serves as electron donor. Substitutable substrates meet the same need for the cell. Glucose and xylose used by yeasts such as Candida may be viewed as substitutable substrates.

10.6.1 COMPLEMENTARY SUBSTRATES To model multiple complementary nutrients, an interactive form of the Monod model may be used58–60 S2   S1   µ = µˆ  K + S  + K S 2   s1 1   s 2

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The interactive model is based on the assumption that both substrates influence the growth rate. This model predicts a lower specific growth rate than the noninteractive model, particularly when substrate concentrations are small compared to their KS values. This model is a continuous function and has been used in many applications, including describing the growth of hybridoma cells based on substrates glucose and glutamine with inhibition by products lactate and ammonia as follows58    Sglu Sglut K lact K NH   µ = µˆ        K glu + Sglu   K glut + Sglut   K lact + Plact   K NH + PNH 

(10.13)

with Sglu, Sglut, Plact, and PNH = soluble concentrations of glucose, glutamine, lactate, and ammonia, respectively, mg/L; and Kgl, Kglut, Klact, and KNH = K values for glucose, glutamine, lactate, and ammonia, respectively. An alternative approach to model complementary nutrients, called the noninteractive model, assumes that one nutrient limits growth59   S1   S2 µ = µˆ ∗ minimum of   ,    K s1 + S1   K s 2 + S2

    

(10.14)

The predicted growth rate will be the lowest value based on each substrate concentration. This model is a discontinuous function at the transition from one nutrient limitation to another. Both models have advantages and have been used successfully to describe microbial growth. Neither model was found in the bioprocessing literature investigated here to describe Candida or Pichia growth utilizing xylose and other substrates. However, these models could be applied for these fermentations where carbohydrate and oxygen or nutrients may be potentially growth-rate limiting. The KS value for oxygen, KS, O2, for most aerobic, heterotrophic bacterial cultures is quite low, and often is less than 1 mg/L.59

10.6.2 SUBSTITUTABLE SUBSTRATES When multiple substitutable substrates are present in the media, simultaneous use of the substrates may occur. In this case, one compound may be preferred by the microorganism over the other. If S1 is preferred, then its presence in the media will inhibit the use of S2. The growth rate of the microorganism using S2 is modeled as a function of S2 with inhibition by S1 as follows59,60 S1   µ1 = µˆ   K S1 + S1 

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  K  S2 S1 µ 2 = µˆ     K S2 + S2   K S1 + S1 

(10.16)

For example, both glucose and xylose may be used by yeasts such as Candida. Glucose is the preferred substrate, so its presence in media suppresses the use of xylose by reversibly inactivating the enzyme xylose reductase,41 while the presence of xylose in the media does not influence the use of glucose. The sequential use of glucose then xylose has been documented by many researchers.34,38,40–42 Thus, the growth rate of Candida based on glucose use may be modeled as follows  Sglu  µ Glu = µˆ   K glu + Sglu 

(10.15a)

where µGlu represents the specific growth rate using glucose as organic substrate, h–1. The growth rate of Candida based on use of xylose may be modeled as    K glu  SXyl µ Xyl = f * µˆ    K + S  Xyl Xyl   K glu + Sglu 

(10.16a)

where µXyl represents the specific growth rate using xylose as organic substrate, h–1, and f represents factor for decreased maximum specific growth rate for nonpreferred substrate. These equations represent the growth of Candida as exclusively using glucose when the glucose concentration is high. As the glucose concentration declines, the growth rate based on glucose declines, with concomitant increase in the growth rate based on use of xylose. Simulations of Candida specific growth rate values based on xylose use as a function of glucose and xylose concentrations (Equation 10.16a) using representative kinetic values are shown in Figure 10.1. 0.5

0 g/L glucose 2 g/L glucose 5 g/L glucose 50 g/L glucose

0.4 0.3 0.2 0.1 0 0

10

20 30 Xylose, g/L

40

50

FIGURE 10.1 Simulated specific growth rate (hr–1) of Candida based on xylose concentration using representative kinetic parameter values.

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If multiple complementary and substitutable substrates are present, the terms may be combined in multiplicative manner. The following expressions could be used to describe growth of Candida under aerobic conditions if oxygen, glucose, and xylose are growth-rate limiting substrates  Sglu   SO 2  µ Glu = µˆ     K glu + Sglu   KO 2 + SO 2 

(10.15b)

   K glu   SXyl SO 2  µ Xyl = f µˆ   K + S     K + S K + S  Xyl Xyl   glu glu  O2 O2

(10.16b)

Combining Equation 10.8 (product inhibition- ethanol), Equation 10.11 (xenobiotic inhibition-furfural), Equation 10.12 (complementary nutrients – O2 and glucose), and Equation 10.15 and Equation 10.16 (substitutable substrates – glucose and xylose) results in the following growth models for Candida   Sglu SO 2   Pm,ETOH − PETOH µ glu = µˆ     PETOH  K glu + Sglu   K O 2 + SO 2  

 K furfural    K  furfural + S furfural  (10.15c)

   K glu   Sxyl SO 2   Pm,ETOH − PETOH  µ xyl = f µˆ        PETOH  K xyl + Sxyl   K glu + Sglu   KO 2 + SO 2     K

 K furfural  S + furfural furfural  (10.16c)

10.7 YIELD PARAMETERS Biomass yield (YX/S) is defined as the mass of cells produced per mass of substrate utilized. Product yield (YP/S) is defined as the mass of product formed per mass of substrate utilized. Biomass and product yields may be determined from the stoichiometry of a balanced growth equation, or by measurement. For the balanced equation describing anaerobic fermentation of glucose by yeast61 given below, YX/S and YEtOH/S values are 0.078 mg biomass/mg glucose and 0.33 mg ethanol/mg glucose, respectively. 1000 C6H12O6 + 118 NH3 → 5.9 C100H174O45N20 + 1300 C2H5OH + 1540 CO2 + 430 C3H8O3 + 36 H2O (10.17)

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Biomass and product yields are generally assumed to not vary with respect to substrate concentration, but will vary for different electron acceptor environments. For example, xylitol yields from xylose for Candida tropicalis were reported by Kastner et al.35 as a function of redox potential of the culture environment as follows: YXylitol/xylose = 0.63, 0.87, 0.15, and 0.10 g/g for redox potentials of –150, –100, –50, and 0 mV, respectively.

10.8 TEMPERATURE EFFECTS Microbial growth models and design should consider impact of temperature on specific growth rate and decay constant values. Biomass yield, product yield, halfsaturation constant, and growth-associated product constant values are often considered independent of temperature. Maximum specific growth rate and decay constant values should be corrected for temperature differences using the Arrhenius or modified Arrhenius equation shown below59 k = Ae− EA / RT Arrhenius Equation

(10.18)

where k = temperature-dependent reaction rate constant; A = Arrhenius constant, EA = activation energy, kJ/mol; R = gas constant, kJ/mol K; T = absolute temperature, K. kT 1 = kT 2 θ(T1 −T2 ) Modified Arrhenius Equation

(10.19)

where kT1 and kT2 are the reaction rate constants at temperatures 1 and 2; θT = temperature correction factor, dimensionless, and T = temperature, °C or K. The Arrhenius expression is applicable over a wide range of temperatures. The modified Arrhenius expression should be applied only over a narrow range of temperatures, typically those encountered for mesophilic organisms.59

10.9 KINETIC RATE EXPRESSIONS Once the appropriate relationship to describe the specific growth rate is determined, the rates of utilization or formation can be developed for important reactor compounds. The rate of biomass formation is given as rXB = µ X B

(10.20)

where rXB = rate of biomass formation, mg/L-h. For growth rate expressions developed for cells using substitutable substrates, as in the case of xylose and glucose use by Candida, the rates of biomass formation using both substrates could be described as follows, following models proposed59,60 for wastewater treatment

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rXB = µ Glu X B + µ Xyl X B

(10.20a)

Products formed from bioconversion processes were classified by Gaden69 as one of three types: Type I, growth-associated products arising directly from metabolism of carbohydrate; Type II, products arising indirectly from carbohydrate metabolism that accumulate under abnormal metabolism; Type III, nongrowth-associated products formed as a result of processes other than growth/energy metabolism. Using this classification, ethanol produced by the anaerobic fermentation of glucose by yeasts is a Type I product and antibiotics such as streptomycin are Type III products.69 Citric acid produced as a result of energy metabolism by aerobic fermentation of molasses and accumulated under conditions of nutrient-limitation of trace metals such as manganese, iron, and zinc by Aspergillus niger14 would be characterized as a Type II product.69 This classification may be complex for some bioproducts. For example, polydroxyalkanoates (PHAs), which are internal energy/carbon storage products, are of interest for biodegradable polymers in food packaging and films.70 PHAs can be formed by two routes: microbes such as Alcaligenes eutrophus produce and accumulate PHAs under nutrient-limited conditions with excess carbon source, while microbes such as Alcaligenes latus do not require nutrient limitation for PHA production and accumulation but will accumulate greater quantities under nutrientlimited conditions.70 Therefore, PHA could be classified as a Type II or Type I product, depending on the microbial culture employed. Many have adopted a classification that combines the Type I and Type II products described by Gaden into one category, growth-associated (GA) products. The Type III products are nongrowth-associated (NGA) products. A middle category, called mixed products, are those that are formed from both growth and nongrowth metabolic processes. Using this convention, PHA would be considered a GA product.70 The rate of growth-associated product formation can be expressed as a function of the rate of biomass formation63 rpg= kpgrXB = kpgµXB

(10.21)

where rpg = rate of growth-associated product formation, mg/L-h; and kpg = growthassociated product formation ratio, mg product/mg biomass. For example, the kpg value for ethanol based on the anaerobic fermentation of glucose by yeast (Equation 10.17) 61 can be determined by the stoichiometry of the balanced equation as 4.26 g ethanol/g biomass. The value of kpg may also be calculated as the ratio of YP/S/YX/S or measured directly. The rate of nongrowth-associated (NGA) product formation is modeled solely as a function of the concentration of biomass in the reactor, and not the rate of biomass formation.63 rpn = kpnXB where rpn = rate of nongrowth-associated product formation, mg/L-h and

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(10.22)

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kpn = nongrowth-associated product formation constant, mg product/mg biomass-h. Products formed from a combination of growth and nongrowth-associated means may be modeled as 63 rP = k pg µX B + k pn X B

(10.23)

Examples of NGA products in food/agricultural bioprocessing include vitamins such as K2-7,27 pigments produced from Monascus fermentations,20 and emulsifier production by filamentous fungi Curvularia lunata.21 From graphs of xylose use and xylitol formation presented by several researchers, xylitol appears to be a mixed or NGA product.35,38,42 Other reactions that occur as a result of growth, such as substrate and oxygen utilization, are represented also as functions of the rate of biomass formation. The rate of organic substrate utilization is therefore rS =

µX B rX B = YX / S YX / S

(10.24)

The rate of substrate utilization that occurs as products are formed during nongrowth processes can be modeled as rS =

rpn YP /S

(10.25)

The rate of O2 utilization during aerobic growth can be expressed as a function of a stoichiometric factor based on the balanced growth equation or measured value. For example, in a representative equation describing organotrophic bacteria (C5H7O2N) grown on glucose, 0.578 mg O2 are required per mg XB formed59; therefore ro =ratio * rX B = 0.578* µX B

(10.26)

where rO = rate of oxygen utilization, mg/L-h O2 . If the stoichiometry is not known, then the rate of oxygen utilization can be expressed as follows if biomass and organic substrate are measured on a chemical oxygen demand (COD) basis59,60 ro = (1 − YX /S ) * rS =

1 − YX / S µX B YX / S

(10.27)

A final expression important to bioreactor modeling is the rate of microbial decay. This rate of natural death or decay may be modeled as first order with respect to biomass.

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rXB,d = –b XB

(10.28)

where rXB,d = rate of biomass decay, mg/L-h XB; b = decay constant, h-1.

10.10 BIOREACTOR DESIGN The kinetic rate expressions developed above may be used in mass balance equations with respect to substrate, biomass, and products for different bioreactor configurations. Basic bioreactor designs for suspended growth cultures are batch, continuous (flow) stirred tank reactor (CSTR), and CSTR with biomass recycle. To develop the mass balance equations with respect to products in the reactor, characterization of the product as a GA, NGA, or mixed metabolite is needed. Further characterization of the product as intracellular vs. extracellular, and “soluble” vs. “particulate” as compared to cell separation technique is needed for CSTR with recycle designs. If the means of cell separation (filtration, centrifugation, and settling) removes the compound with the biomass, then the product is considered particulate. Ethanol and xylitol are examples of soluble, extracellular products, while oils produced by filamentous fungi Pythium and PHAs are intracellular products. Products such as monoclonal antibodies are extracellular, “particulate” compounds, since they can be separated by ultrafiltration.

10.11 BATCH REACTORS The mass balance with respect to biomass for a batch reactor is given as follows, assuming growth and decay are the only reactions µX B − bX B =

dX B dt

(10.29)

Although there is a temporary balancing of the growth and decay terms during stationary phase, batch reactors never reach true steady-state conditions, where concentrations in the reactor do not change with time. During exponential growth in a batch reactor, decay is often assumed to be negligible. Integration of Equation 10.29 and substitution of the definition of biomass yield results in the logistic growth equation61 K S YX / S S K S YX / S   X  (t − t o )µˆ =  + 1 ln  Bt  + ln 0   X B 0 + S0YX /S   XB0 X B 0 + S0YX /S St

(10.30)

where t and tO = time and initial time, h; XBt and XBO = biomass concentration at time t and tO, and SSt and SSO = soluble substrate concentration at time t and tO. This equation describes how biomass increases while substrate decreases with time during exponential growth. The length of incubation in a batch reactor may vary depending on growth kinetics, accumulation of inhibitory products, and product type. Equation

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10.30 can also be used to determine the Monod kinetic parameters for batch growth data, a technique that is primarily utilized in the wastewater treatment literature71,72 but is being applied to bioprocessing applications.73

10.12 CONTINUOUS STIRRED TANK REACTORS (CSTRS) For a simple CSTR with a single influent and effluent flow, the mass balance with respect to biomass is

X Bi

Q Q dX B − XB + µX B − bX B = V V dt

(10.31)

where XBi = biomass concentration in influent flow, mg/L. For cases with no biomass in the influent flow, growth based on a single limiting nutrient and no substrate or product inhibition, the following equation results at steady state µ=

1 +b τ

(10.32)

where τ = hydraulic retention time in reactor, hr. Thus, the hydraulic retention time is the main design parameter for a CSTR, since it controls the microbial growth rate in the reactor. A simple CSTR can be operated at a short retention time to maintain a high specific growth rate to mimic a batch reactor during exponential growth, with resulting sustained formation of growth-associated products, or may be operated at a long retention time to maintain a low specific growth rate to mimic a batch reactor during declining exponential or stationary phases, resulting in continuous formation of nongrowth-associated products. Substitution of the single Monod model for µ results in SS =

K S (1 / τ + b ) µˆ − (1 / τ + b )

(10.33)

The effluent substrate concentration is a function of the hydraulic retention time and the kinetic parameters values, but not a function of the influent substrate concentration. The mass balance with respect to substrate for a simple CSTR is

SSi

Q µX B Q dSS − SS − = V V YX / S dt

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(10.34)

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After substitution of Equation 10.32, the resulting steady-state concentration of biomass is given as XB =

YX /S (SSi − SS ) 1 + τb

(10.35)

The mass balance with respect to a general product is

Pi

Q Q dP −P + k pg µX B + k pn X B = V V dt

(10.36)

The steady-state solutions for GA and NGA products are PGA = k pg µXb τ

(10.37)

where PGA is growth-associated product concentration, mg/L; and PNGA = k pn Xb τ

(10.38)

where PNGA is the nongrowth-associated product concentration, mg/L. At steady state, concentrations of substrate, biomass, and product will vary as a function of retention time. In general, the specific growth rate and effluent substrate concentration decline and biomass concentration increases with increase in hydraulic retention time. Thus reactor design may be based on achieving the desired effluent concentrations. Martinez et al.36 investigated a CSTR for xylitol formation using Candida guilliermondi cultures and sugar cane bagasse hydrolysate. In most comparisons, they found higher xylitol and biomass concentrations and lower substrate concentrations at a longer hydraulic retention time (100 vs. 20 h). To maximize rates of biomass or GA product formation or of substrate utilization, both a high specific growth rate and a high biomass value must be achieved. The hydraulic retention time that maximizes these rates can be estimated from the following63 0.5 1 ˆ   KS   = µ 1 −    τ   K S + SSi  

(10.39)

For an NGA product, there is no single hydraulic retention time that maximizes the rate of production. The equations above may be used for preliminary analysis and design of a CSTR. For more expanded analysis, and for systems where multiple substrates or inhibition occur, dynamic modeling may be used to determine the retention time that maximizes biomass or GA formation rates, or the product concentration as a function of retention time.

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10.13 CSTR WITH CELL RECYCLE A CSTR with biomass recycle includes some means to remove biomass from the reactor effluent flow, through external or internal filter, settling device, or centrifugation, and return biomass flow to the reactor. By separating biomass from the effluent flow, the length of time that cells remain in the reactor, the cell retention time, θ, can be controlled independently of the hydraulic retention time. To control θ, a portion of biomass is harvested or “wasted” from the reactor. When the harvest flow is pumped directly from the reactor, the biomass concentration in the reactor is equal to the concentration in the harvest flow, and θ is calculated as θ=

VX B V = QH X BH QH

(10.40)

where θ = cell retention time, h; V = reactor volume, L; XB = biomass concentration in reactor, mg/L; XBH = biomass concentration in the harvest flow, mg/L; and QH = harvest flow rate, L/h. The mass balance equation with respect to biomass for a CSTR with recycle is

X Bi

Q (Q − QH ) Q dX B − X B H − X Be + µX B − bX B = V V V dt

(10.41)

where XBe = biomass concentration in final effluent flow, mg/L. XBe ~ 0 if cell separation step is assumed 100% efficient. At steady state, the mass balance with respect to biomass simplifies to µ=

1 +b θ

(10.42)

Therefore, θ controls µ for a CSTR with recycle in the same manner as τ does for a simple CSTR. Substitution of the single Monod model for µ results in SS =

K S (1 / θ + b ) µˆ − (1 / θ + b )

(10.43)

The mass balance with respect to soluble substrate for a CSTR with recycle is

SSi

Q (Q − QH ) µX B dSS Q − SS H − SS − = V V V YX / S dt

(10.44)

Because soluble organic substrate will not be removed by the cell separation technique, the same concentration will be present in the harvest and main effluent flow. The mass balance at steady state simplifies to Equation 10.45, which describes the

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biomass concentration as a function of both the hydraulic and cell retention times. Influent organic substrate concentration and hydraulic retention time can be adjusted to achieve the desired cell concentration in the reactor. XB =

θ YX /S (SSi − SS ) × 1 + bθ τ

(10.45)

The mass balance with respect to a general product is given below.

Pi

Q (Q − Qw ) Q dP − P w − P + k pg µX B + k pn X B = V V V dt

(10.46)

Product concentrations at steady state will vary according to whether the product is GA or NGA, and particulate or soluble, as shown below

(

)

P = k pg 1 θ + b X B τ if product is GA, soluble

(

)

(10.47)

P = k pg 1 θ + b X B θ if product is GA, particulate

(10.48)

P = k pn X B τ if product is NGA, soluble

(10.49)

P = k pn X B θ if product is NGA, particulate

(10.50)

In general, the specific growth rate and effluent soluble substrate concentration will decline as cell retention time increases, and biomass and nongrowth-associated product concentrations, especially for particulate products, will increase with increase in cell retention time. In a CSTR with recycle, a single optimum cell retention time that maximizes the rate of product formation for all types of products does not exist. A model of the system based on mass balance equations may be used to run simulations to determine the product concentration and rate of product formation at different hydraulic and cell retention times. System optimization for a biological CSTR with recycle is a matter of determining the best combination of cell density and production rates for the system. A CSTR with biomass recycle inoculated with Candida guilliermondii FTI 20037 was shown to achieve xylitol volumetric productivity rates of 0.91 g/L-h at a hydraulic retention time of 16.7 h (dilution rate of 0.060/h) and essentially infinite cell retention time (i.e., all cell mass was filtered through 0.01-µm ultrafilter and continuously recirculated to reactor so the only loss of cell mass was through decay).48 This productivity was achieved with defined media containing 30 g/L xylose and no glucose. Recycling 100% of cell mass is not typically employed in

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CSTR with recycle designs, since control of the specific growth rate through wasting of cell mass is usually desired.

10.14 FED-BATCH SYSTEMS The important difference of fed-batch compared to batch and CSTR systems is that the specific growth rate decreases with time due to increasing volume at constant feed rates56 as shown by dµ d  Q  −Q 2 =  =  dt dt  V0 + Qt  (V0 + Qt )2

(10.51)

where VO = initial volume. However, steady-state operation may be achieved by exponentially adjusting the feed rate, Q as shown by dV = Q = λV dt

(10.52)

Q = λV0 eλt

(10.53)

which is integrated to obtain

where λ is the dilution rate or 1/τ. At steady-state operation, the expression for substrate concentration mimics that developed for the CSTR system (Equation 10.33) as shown below excluding the decay term µ=

KS τ 1 ˆ SS and SS ≅ =µ τ K s + SS µˆ − (1 τ )

(10.54)

Fed-batch operation may be represented at steady-state in terms of biomass and product formation excluding decay,61 respectively, as X t = X0t + QYB SSi t

(10.55)

where Xt is the total biomass produced (mg) and P = P0

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V0 t  V + k p XB  0 +  t  V 2τ  V

(10.56)

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where P is the product concentration (mg/L). A fed-batch bioreactor system has inherent advantages of both batch and continuous systems since the operation is semicontinuous with only substrate feed and no effluent stream. For this reason, these systems are often employed in many commercial applications within the food and pharmaceutical industry. The system works well for NGA production typically accomplished by introduction of secondary metabolite inducers, specific nutrient deprivation, or substrate inhibition at specific times during the process. The fed-batch system is also applied to form GA and mixed-growth-associated (MGA) products such as heterotrophic algal oils by adjusting substrate feed rate to obtain maximum conversion to oil stored in high cell density culture. Other common examples where fed-batch systems are employed in commercial settings include baker's yeast (GA) and penicillin (NGA) production. Fed-batch systems have been used to produce xylitol and ethanol from mixed sugars, glucose, and xylose, in Candida shehatae,34 Pichia stipitits, 45 and Candida boidinii50 cultures.

10.15 BIOREACTOR DESIGN STRATEGIES Bioreactor design may be based on maximizing the rate of product formation, biomass production, or substrate utilization, or on achieving target concentrations of product, biomass, or substrate. As revealed by the rate expressions above, rates of substrate, oxygen and nutrient utilization, and GA product formation will be high when the rate of biomass formation is high, while the rates of NGA product formation and decay will be high when the concentration of biomass is high. In general, rates of primary product formation in a batch reactor will be highest during mid to late exponential phase, while product concentration will be highest at end of exponential phase. Secondary product concentration in a batch reactor will peak at end of stationary phase. These responses are illustrated in Figure 10.2 and Figure 10.3 for representative GA and NGA products. For a GA, extracellular, soluble product such as ethanol, a batch reactor or simple CSTR would achieve high product formation rate. But for nongrowth-associated products or reactions that form inhibitory products, batch reactors may not be advantageous due to the low productivity rate or elevated product concentrations that may form. Selection of a simple CSTR allows for control of the specific growth rate through control of the hydraulic retention time to maximize the rate of a GA product. In a simple CSTR or CSTR with biomass recycle, the steady-state specific growth rate is inversely proportional to the hydraulic or cell retention time, respectively, while biomass concentration is, in general, directly proportional to the retention times. Therefore, these designs allow for efficient production of either primary or secondary products by achieving target specific growth rate and biomass or product concentrations through control of the retention time. Dynamic modeling and simulation of microbial growth and product formation are needed to optimize the design based on predicted production rates and concentrations. For NGA, intracellular products, such as certain lipids produced by fungi, or NGA extracellular, particulate products, such as monoclonal antibodies produced by hydridoma, the rates of formation and product concentrations will be maximized

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1: XB 60

2: GA product

257

3: NGA product

2

2

2

3

3

30 3 1 1 2

0 1 0.00

1

3 12.50

25.00 Time

1

37.50

50.00

FIGURE 10.2 Simulations of representative biomass (XB), growth-associated (GA), and nongrowth-associated (NGA) product concentrations (g/L) vs time (h) in a batch reactor. (Simulation conducted with STELLA® 8 software.)

1: XB form 20

2: GA product form

3: NGA product form

2

1

10

3

1

0 1 0.00

2

3

2 12.50

1 2 25.00 Time

3

1 2 37.50

3 50.00

FIGURE 10.3 Simulations of representative biomass (XB), GA, and NGA product formation rates (g/L-h) vs. time (h) in a batch reactor. (Simulation conducted with STELLA® 8 software.)

by a CSTR with biomass recycle design with fairly long cell retention time, which easily allows for high biomass concentration to be achieved without exposing cells to inhibitory high substrate concentrations. Alternating environments with respect to substrate level, electron acceptor, or nutrients can be used to maximize product formation or selecting desired biomass

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cultures.56,59 For example, high yields of the vitamin B12 by the bacterium Propionibacterium require microaerobic conditions; however, biosynthesis of a critical enzyme (5,6-dimethylbenzimidazole (DMBI)) requires oxygen. Therefore, biosynthesis of vitamin B12 requires two reactor environments. Martens et al.23 proposed the use of batch reactors with anaerobic conditions for the first three days of incubation for production of the vitamin B12 precursor cobamide, with subsequent aeration for three days for synthesis of DMBI and ultimately, vitamin B12. These bioprocesses would be a likely candidate for use of two-stage CSTR system with anerobic/aerobic environment for optimizing vitamin production. Xylitol fermentations are another example of a bioprocess where alternating environments can be beneficial. Microorganisms such as Candida will exhibit greater specific growth rate and greater biomass yield under aerobic conditions, while the desired products, ethanol or xylitol, are produced only under anaerobic or microaerobic conditions. Bioreactor designs with alternating environments allow for full utilization of glucose under aerobic conditions for biomass production, followed by anaerobic/microaerobic conditions to allow for ethanol or xylitol production. For example, use of a fed-batch, two-phase reactor with aerobic growth phase followed by anaerobic conditions to produce ethanol with Candida shehatae cultures produced yield of 0.4 g ethanol/g xylose and 75% to 100% conversion of xylose.34 Using this design scheme, the specific growth rate for Candida shehatae under aerobic utilization of D-xylose was µ = 0.32 h–1 producing 2 × 109 cells/mL, while in the second phase, ethanol yield was 0.23 g/g.34 Final ethanol concentrations (up to 50 g/L) showed greater success than the genetically modified strain of Zymomonas mobilis74 which resulted in 30 g/L ethanol. With a semicontinuous process, Rodrigues et al42 used Candida guilliermondi to ferment sugar cane bagasse hydrolysate. A maximum product yield of 0.79 g xylitol/g xylose was achieved, and a productivity of 0.66 g/L-h. The authors note that the critical first step is the rapid production of cell mass in the culture medium, which can be achieved by aerating throughout the first phase, then allowing oxygen levels to drop to stimulate xylitol production.42

10.16 MODELING OF GLUCOSE/XYLOSE UTILIZATION AND PRODUCT FORMATION BY CANDIDA Fermentation of xylose and glucose by Candida in sugarcane bagasse hydrolysate is an excellent example of a bioprocess with complex process kinetics that illustrates many of the concepts discussed here. First, the sequential use of glucose then xylose may be modeled as multiple substitutable substrates; second, the impact of xenobiotics, such as furfurals, and products such as ethanol, that have been identified as inhibitory may be modeled; third, the ability of species such as Candida to metabolize multiple sugars under aerobic, anaerobic, and microaerobic environments, with resulting products from each, demonstrates the advantage of using multiple phases of batch or multiple reactors of continuous flow reactors. To illustrate the xylitol formation by yeasts such as Candida, simulations of microaerobic batch, microaerobic CSTR, and two CSTRs in series design, with first CSTR under aerobic

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conditions and second CSTR under microaerobic conditions, were conducted. For simplicity, the modeling used here considered sequential use of glucose/xylose but assumed no inhibition by substrates, xenobiotics, or products. A typical concentrated sugarcane bagasse hydrolysate composition of 62 g/L xylose and 8 g/L glucose42 was used for these simulations. Representative kinetic and yield parameter values used were: µˆ = 0.5 h–1 using glucose for full aerobic conditions; f = 0.4 (assumed); YXylitol/xylose = 0.87 g/g for microaerobic conditions.32 Xylitol formation was modeled as a nongrowth-associated product, with kpn value of 0.3 g/g-h estimated from reported data.32 For microaerobic conditions, YX/Glu = 0.18 g/g and YX/Xyl = 0.05 g/g were calculated from data32 while biomass yield values for aerobic conditions (YX/Glu = 0.4 g/g and YX/Xyl = 0.2 g/g) were assumed. Mass balance equations were solved simultaneously using a program written with STELLA® software (High Performance Systems, version 8.0). As shown in Figure 10.4, the sequential use of glucose/xylose in a batch reactor can be modeled, with xylose consumption beginning at approximately 12 h, after glucose concentration has dropped considerably. Xylitol formation begins with use of xylose as substrate at approximately 12 h. The overall form of the concentration profiles match those presented for batch fermentations in mixed substrates.41,42 Results obtained by Pfeifer41 show that complete glucose utilization occurred by ~10 h of batch fermentation, and complete utilization of xylose by 30 to 35 h. Simulations of rates of substrate utilization and product formation (Figure 10.5) represent the sequential use of glucose/xylose and the lag in xylitol formation due to nongrowth-associated nature of product formation.

1: Glucose 62

2: Xylose

3: Xylitol

2

2 31

1

0 0.00

3

3

1 3

1 12.50

3 1 2 25.00 Time

1 37.50

2 50.00

FIGURE 10.4 Simulations of glucose, xylose, and xylitol concentrations (g/L) vs. time (h) in batch fermentation of sugarcane bagasse hydrolysate. (Simulation conducted with STELLA® 8 software.)

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1: rate of Glucose util 10

2: rate of Xylose util

3: rate Xylitol form

2 5

1 3 0 1 0.00

1

2

3

1 25.00 Time

12.50

2

3

1 37.50

2

3 50.00

FIGURE 10.5 Simulation of rates of glucose utilization, xylose utilization, and xylitol formation (g/L-h) vs. time (h) in batch reactor using sugarcane bagasse hydrolysate. (Simulation conducted with STELLA® 8 software.)

1: Glucose 62

2: Xylose

3: Xylitol

2

31

2 1

1

0 0.00

3

1 12.50

3

3

3 1 2 25.00 Time

1 2 37.50

50.00

FIGURE 10.6 Simulation of glucose, xylose, and xylitol concentrations (g/L) vs. time (h) in CSTR at hydraulic retention time of 20 h using sugarcane bagasse hydrolysate. (Simulation conducted with STELLA® 8 software.)

Simulation of substrate and product concentrations (Figure 10.6) for a single microaerobic CSTR with hydraulic retention time of 20 h also represents the sequential use of glucose followed by xylose. Simulated steady-state conditions are achieved by approximately 50 h. Simulation of substrate and product concentrations in a two-stage, aerobic/microaerobic CSTR system with a total system hydraulic retention time of 20 h is shown in Figure 10.7. Under aerobic conditions in the first

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2: Xylose1 2 4

3: Glucose2

261

4: Xylose2

5: Xylitol2

2

31 5

5 5

1

1

0 0.00

3

5

1 12.50

2 3 4

1 25.00 Time

3 4

1 2 37.50

3 4 50.00

FIGURE 10.7 Simulations of glucose, xylose, and xylitol concentrations (g/L) vs. time (h) in reactors 1 and 2 of a two-stage CSTR system with hydraulic retention time of 10 h per reactor (20 h system retention time). (Simulation conducted with STELLA® 8 software.)

reactor, no xylitol would be formed but high yields of yeast biomass would be achieved. The simulation suggests that the two-stage CSTR system would achieve greater xylitol concentration in the final effluent than the single-stage CSTR, through the use of alternating environments to achieve greater product formation. Optimization of this system would be accomplished by varying hydraulic retention times to achieve maximum concentration of xylitol.

10.17 SUMMARY The kinetics of microbial growth, substrate utilization, and product formation can greatly influence the effectiveness of a bioconversion process. Modeling involves characterization of growth and product formation kinetics and development of mass balance models of system. Dynamic modeling and simulation of the bioprocess can be used to select, design, and optimize a bioreactor for production of nutraceutical products.

REFERENCES 1. Besson, I. et al., Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans, Appl. Microbiol. Biotechnol., 47, 489, 1997. 2. Drawert F. and Barton, H., Bioysnthesis of flavor compounds by microorganisms.3: production of monterpenes by yeast Kluyveromyces lactis, J. Agric. Food Chem., 26, 765, 1978. 3. Priefert, H., Rabenhorst, J., and Steinbuchel, A., Biotechnological production of vanillin, Appl. Microbio.l Biotechnol., 56, 296, 2001. 4. Vanderhaegen, B. et al., Bioflavoring and beer refermentation, Appl. Microbiol. Biotechnol., 62, 140, 2003.

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5. Cheng, M.H. et al., Fungal production of eicosapentaenoic and arachidonic acids from industrial waste streams and crude soybean oil, Bioresource Technol., 67, 101, 1999. 6. Walker, T.H., Cochran, H.D., and Hulbert, G.J., Supercritical carbon dioxide extraction of lipids from Pythium irregulare, J. Am. Oil Chem. Soc., 76, 595, 1999. 7. Zhu, H., Drapcho, C.M., and Walker, T.H., Bioconversion of Rice Bran to Omega-3 Fatty Acids of Pythium irregulare Submerged Culture, ASAE paper, 01-7020, 2001. 8. Zhu, M., Zhou, P.P., and Yu, L.J., Extraction of lipids from Mortierella alpina and enrichment of arachidonic acid from fungal lipids, Bioresource Technol., 84, 93, 2002. 9. Yu, L.J., Qin, W.M., Lan, W.Z., Zhou, P.P., and Zhu, M., Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation, Bioresource Technol, 88, 265–268, 2003. 10. Sijtsma, L. and de Swaaf, M.E., Biotechnological production and applications of the omega-3 polyunsaturated fatty acid docosahexaenoic acid, Appl. Microbiol. Biotechnol., 64, 146, 2004. 11. Wen, Z.Y. and Chen, F., Heterotrophic production of eicosapentaenoic acid by microalgae, Biotechnol. Adv., 21, 273, 2003. 12. Mayilvahanan, D. et al., Citric acid productions: part 1, strategies for reduction in cycle time for targeted yields, Bioprocess. Eng., 15, 323, 1996. 13. Pazouki, M. et al., Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose, Bioprocess. Eng., 22, 353, 2000. 14. Zehentgruber, O., Kominek, J., and Salzbrun,W., Citric acid for beverages: a new economic submerged fermentation process, World Biotechnol. Report, 1, 687, 1985. 15. Grewal, H.S. and Kalra, K.L., Fungal production of citric acid, Biotechnology Advances, 13, 209, 1995. 16. Ghaly, A.E. et al., Batch propagation of Lactobacillus helviticus for the production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation, World J. Microbiol. Biotechnol., 20, 65, 2004. 17. Chen, Y.L. and Lun, S.-Y., Biotechnological production of pyruvic acid, Appl. Microbiol. Biotechnol., 57, 451, 2001. 18. Hu, Z.-C. et al., Effect of sugar-feeding strategies on astaxanthin production by Xanthophyllomyces dendrorhous, World J. Microbiol. Biotechnol., 21, 771, 2005. 19. Cho, Y.J. et al., Effect of carbon source and aeration rate on broth rheology and fungal morphology during red pigment production by Paecilomyces sinclairii in a batch reactor, J. Biotechnol., 95, 13, 2002. 20. Dominquez-Espinosa, R.M. and Webb, C., Submerged fermentation in wheat substrates for production of Monascus pigments, World J. Microbiol. Biotechnol., 19, 329, 2003. 21. Paraszkiewics, K., Kanwal, A., and Dlugonski, J., Emulsifier production by steroid transforming filamentous fungus Curvularia lunata: growth and product characterization, J. Biotechnol., 92, 287, 2002. 22. Shepherd, R. et al., Novel bioemulsifiers from microorganisms for use in foods, J. Biotechnol., 40, 207, 1995. 23. Martens, J.H. et al., Microbial production of vitamin B12, Appl. Microbiol. Biotechnol., 58, 275, 2002. 24. Haddadin, M.S.Y. et al., Utilisation of tomato pomace as a substrate for the production of vitamin B12: a preliminary appraisal, Bioresource Technol., 78, 225, 2001. 25. Yang, Y. et al., Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor packed with loofah, Bioresource Technol., 92, 285, 2004.

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26. Zhang, Z. et al., Study on the methane fermentation and production of vitamin B12 from alcohol waste slurry, Appl. Biochem. Biotechnol., 113, 1033, 2004. 27. Sato, T. et al., Production of menaquinone (vitamin K2)-7 by Bacillus subtilis, J. Biosci. Bioeng., 91, 16, 2001. 28. Canilha, L. et al., Use of response surface methodology to evaluate the optimum environmental conditions for the bioconversion of xylose into xylitol in eucalyptus hemicellulosic hydrolysate, J. Chem. Technol. Biotechnol., 78, 945, 2003. 29. Du Preez, J.C. et al., Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeast Candida shehatae and Pichia stipitis, Appl. Microbiol. Biotechnol., 25, 521, 1987. 30. Felipe, M.G.A. et al., Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolysate by Candida guilliermondii, J. Ind. Microbiol. Biotechnol., 18, 251, 1997. 31. Grootjen, D.R.J. et al., Reactors in series for the complete conversion of glucose/xylose mixtures by Pichia stipitis and Saccharaomyces cerevisiae, Enzyme Microbiol. Technol., 13, 828, 1991. 32. Kastner, J.R., Eiteman, M.A., and Lee, S. A., Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations, Biotechnol. Letters, 23, 1663, 2001. 33. Kastner, J.R. et al., Viability of Candida shehatae in D-xylose fermentations with added ethanol, Biotechnol, Bioeng., 40, 1282, 1992. 34. Kastner, J.R., Jones,W.J., and Roberts, R.S., Ethanol fermentation of mixed-sugars using two-phase, fed-batch process: method to minimize D-glucose repression of Candida shehatae D-xylose fermentations, J. Ind. Microbiol. Biotechnol., 22, 65, 1999. 35. Kastner, J.R., Eiteman, M.A., and Lee, S.A., Effect of redox potential on stationaryphase xylitol fermentations using Candida tropicalis, Appl. Microbial. Biotechnol., 63, 96, 2003. 36. Martinez, E.A., The influence of pH and dilution rate on continuous production of xylitol from sugar cane bagasse hemicellulose hydrolysate by C. guilliermondii, Process Biochem., 38, 1677, 2003. 37. Mussatto, S.I. and Roberto, I.C., Xylitol production from high xylose concentration: evaluation of the fermentation in bioreactor under different stirring rates, J. Appl. Microbiol., 95, 331, 2003. 38. Oh, D.-K. and Kim, S.-K., Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis, Appl. Microbiol. Biotechnol., 50, 419, 1998. 39. Parajo, J.C., Dominguez, H., and Dominguez, J.M., Biotechnological production of xylitol: part 3, operation in culture media made from lignocellulose hydrolsates, Bioresource Technol., 66, 25, 1998. 40. Pessoa, A., Mancilha, I.M., and Sato, S., Cultivation of Candida tropicalis in sugar cane hemicellulosic hydrolyzate for microbial protein production, J. Biotechnol., 51, 83, 1996. 41. Pfeifer, M.J. et al., Effect of culture conditions on xylitol production by Candida guilliermondii FTI 20037, Appl. Biochem. Biotechnol., 57, 423, 1996. 42. Rodrigues et al., Biotechnological production of xylitol from agroindustrial residues. Appl. Biochem. Biotechnol., 70, 869, 1998. 43. Roberto, I.C., et al., Influence of media composition of xylitol fermentation by Candida guiliiermondii using response surface methodology, Biotechnol. Letters, 17, 1223, 1995.

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44. Roberto, I.C., Mancilha, I.M., and Sato, S., Kinetics of xylitol fermentation by Candida guilliermondii grown on rice straw hemicellulosic hydrolysate, Appl. Biochem. Biotechnol., 77, 205, 1999. 45. Schneider, H., Conversion of pentoses to ethanol by yeasts and fungi, Crit. Rev. Biotechnol., 9, 2, 1989. 46. Sene, L. et al., Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugar cane bagasse hydrolysate for xylitol production, Appl. Biochem. Biotechnol., 91, 671, 2001. 47. Silva, S. et al., Batch fermentation of xylose for xylitol production in stirred tank bioreactor, Process Biochem., 31, 549, 1996. 48. Silva, S.S., The preliminary information about continuous fermentation using cell recycling for improving microbial xylitol production rates, Appl. Biochem. Biotechnol., 77, 571,1999. 49. Silva, A.A. et al., Maximizing the xylitol production from sugar cane bagasse hydrolysate by controlling the aeration rate, Appl. Biochem. Biotechnol., 63, 557, 1997. 50. Vandeska, E. et al., Fed-batch culture for xylitol production by Candida boidinii, Process Biochem., 31, 265, 1996. 51. Virginio Da Silva, D.D. et al., Evaluation of inoculum of Candida guilleriermondii grown in presences of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugar cane bagasse hydrolysate, Appl. Biochem. Biotechnol., 121, 427, 2005. 52. Yashashi, Y. et al., Production of xylitol from D-xylose by Candida tropicalis: the effect of D-glucose feeding, J. Ferment. Bioeng., 81, 148, 1996. 53. Makinen, K.K., Biochemical Principles of the Use of Xylitol in Medicines and Nutrition with Special Consideration of Dental Aspects, Birkhauser Verlag, Basel, 1978. 54. Scurlock, J., Bioenergy Feedstock Characteristics, Oak Ridge National Laboratory Publication, retrieved from http://bioenergy.ornl.gov/papers/misc/biochar_factsheet. html 55. Bailey, J. and Ollis, D, Biochemical Engineering Fundamentals, 2nd ed., McGrawHill, New York, 1986. 56. Blanch, H.W. and Clark, D.S., Biochemical Engineering, Marcel Dekker, New York, 1997. 57. Doran, P.M., Bioprocess Engineering Principles, Academic Press, San Diego, 1995. 58. Doyle, A., Griffiths, J.B., and Newell, D.G., Eds., Cell and Tissue Culture: Laboratory Procedures, John Wiley & Sons, New York, 1995. 59. Grady, C.P.L., Daigger, G.T., and Lim, H.C., Biological Wastewater Treatment, 2nd ed, Marcel Dekker, New York, 1999. 60. Henze, M., Grady, C.P.L., Jr., Gujer, W., Marais, G.V.R., and Matsuo, T. A., General model for single-sludge wastewater treatment systems, Water Res., 21, 505, 1987. 61. Shuler, M.L. and Kargi, F., Bioprocess Engineering, Prentice-Hall, Englewood Cliffs, NJ, 1992 62. van’t Riet, K. and Tramper, J., Basic Bioreactor Design, Marcel Dekker, New York, 1991. 63. van Dam-Mieras, M.C.E., de Jeu, W.H., de Vries, J., Currell, B.R., James, J.W., Leach, C.K., and Patmore, R.A., Bioreactor Design and Product Yield, ButterworthHeinemann, Boston, 1992. 64. Monod, J., The growth of bacterial cultures, Annual Rev. Microbiol., 3, 371, 1949. 65. Andrews, J.F., A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates, Biotechnol. Bioeng., 10, 707, 1968.

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66. Steele, J.H., Environmental control of photosynthesis in the sea, Limnol. Oceanogr., 7, 137, 1962. 67. Lehman, J.T., Botkin, D.B., and Likens, G.E., The assumptions and rationale of a computer model of phytoplankton population dynamics, Limnol. Oceanogr., 20, 343, 1975. 68. Welch, E.B., Ecological Effects of Waste Water, Cambridge University Press, Cambridge, 1980. 69. Gaden, E.L., Fermentation process kinetics, J. Biochem. Microbiol. Technol. Eng., 1, 413, 1959. 70. Wang, F. and Lee, S. Y., Poly(3-hydroxybutyrate) productions with high productivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation, Appl. Environ. Microbiol., 63, 3703, 1997. 71. Simkins, S. and Alexander, M., Nonlinear estimation of the parameters of Monod kinetics that best describe mineralization of several substrate concentrations by dissimilar bacterial densities, Appl. Environ. Microbiol., 50, 816, 1985. 72. Zhang C. et al., Aerobic biodegradation kinetics of four anionic and nonionic surfactants at sub- and supra-critical micelle concentrations (CMCs), Water Res., 33, 115, 1999. 73. Reeves, E.G.M., Kinetic Analysis of Kluyveromyces marxianus Yeast Strain, M.S. thesis, Louisiana State University, Baton Rouge, 2004. 74. Zhang, M., Eddy, C., Deanda, K., Finkelstein, M., and Picatoggio, S., Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science, 267, 240, 1995.

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Food Pasteurization and Sterilization with High Pressure Alberto Bertucco University of Padova, Italy

Sara Spilimbergo University of Trento, Italy

CONTENTS 11.1 Introduction ..................................................................................................270 11.2 Ultrahigh Hydrostatic Pressure Treatment (UHHP)....................................271 11.2.1 Process and Equipment Fundamentals ............................................271 11.2.2 State of the Art of UHHP ................................................................272 11.2.2.1 Microorganisms ................................................................272 11.2.2.2 Enzymes............................................................................274 11.2.2.3 Microbial Inactivation Mechanism...................................274 11.2.3 Commercial Application of UHHP .................................................278 11.3 Dense CO2 Treatment (DCO2).....................................................................280 11.3.1 Process and Equipment Fundamentals ............................................281 11.3.2 State of the Art of DCO2 .................................................................281 11.3.2.1 Microorganisms ................................................................281 11.3.2.2 Enzymes............................................................................284 11.3.2.3 Microbial Inactivation Mechanisms .................................285 11.3.3 Commercial Application of DCO2 ...................................................287 11.4 Potentials of High-Pressure Technologies and Conclusions .......................288 References..............................................................................................................289

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11.1 INTRODUCTION Manufacturers of food products are currently under increasing and stringent demands to control their production process. The entire course of manufacturing starting from raw materials to the final packing is closely monitored and wherever possible inspected as well. This is happening essentially because of the growing demand for high-quality products and for energy-saving and safer production processes. Consumers and the business class today prefer fresh or mildly processed and user-friendly food products. Typically, such products are low in sugar, salt, and fat, and contain as few preservatives as possible. However, these factors offer an excellent breeding ground for microorganisms. Thus, finding the right method for extending the shelf-life for these highly perishable products without affecting their quality is indeed an open challenge. Microbiological stabilization of a product is not concerned with the removal of all existing microorganisms but to reduce the number of undesirable ones below a specific critical value during the shelflife of the product. On the other hand, the use of food preservatives and additives is viewed with more and more concern by consumers, as shown by the increase in restrictive measures appearing in the food regulations of industrialized countries. The apprehension about these substances is associated with the concerns for heat treatments that are still the most widely used procedure in food industry to microbiologically stabilize foodstuff. Thermal pasteurization (up to 80°C) and sterilization (up to 120°C) are successful treatments in eliminating the degradation effects of enzymes and microorganisms, but they may also decrease food quality by causing alterations in the taste and sensory attributes of food products. As a consequence, in the future, the food industry is expected to turn toward new and alternative technologies for extending shelf life while retaining all original nutritional properties in the end products. In this regard, systems based on different bactericidal agents, such as microwave, electrical conductivity, pressure gradient, and so forth, seem to offer new possibilities. They include high pressure (both hydrostatic and with CO2), pulsed electric field,1 ohmic heating,2,3 pulsed x-ray,4 ultraviolet light,5 ultrasound,6 filtration,7 microwave and radio frequency processing,8 and oscillating magnetic fields.9 Although these technologies have been studied for more than one century, at the present time none of them is ready to market. In particular, as far as high-pressure treatments are concerned, for many years researchers have investigated the effect of high hydrostatic pressure both on microorganism viability and on foodstuff properties.10,11 The main result has been that such a technique can be successfully applied at room temperature, however, the high plant costs will restrict its development for large-scale production. On the other hand, the use of supercritical CO2 seems to be a more feasible approach, since it allows operations at milder temperature (close to ambient) and pressure (70–300 bars).12 However, this new technique has been proposed only recently, thus no industrial application is ready to enter the large-scale market yet. This chapter will describe and discuss the current knowledge in the pasteurization and sterilization of foodstuff by means of UltraHigh Hydrostatic Pressure (UHHP) method and Dense CO2 (DCO2) method. The most significant achievements

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will be summarized, including fundamental concepts, main applications in both simple and complex solutions, and results on inactivation of different microorganisms and enzymes. At the end of the chapter the current knowledge on the different hypotheses of bacterial inactivation mechanisms involved will be discussed and evaluated and some conclusions about the two high-pressure techniques will be drawn.

11.2 ULTRAHIGH HYDROSTATIC PRESSURE TREATMENT (UHHP) Pressure is one of nature’s fundamental forces that is known to have unique effects on the thermodynamics of substances. Likewise, the preservation action of high pressure on food substances has been acknowledged in the food science community for over 100 years.13 The isostatic nature of pressure results in no shear stress within the food and thus the food’s shape is not destroyed. However, the thermodynamic conditions achieved under UHHP treatment are sufficiently hostile to living bacteria that they are inactivated or irreparably damaged. In this way potential foodborne pathogens can be destroyed and food spoilage organisms can also be greatly reduced. Since this process is typically carried out at temperatures not far from ambient, the food quality can be retained.

11.2.1 PROCESS

AND

EQUIPMENT FUNDAMENTALS

The use of UHHP treatment of food is carried out in batch mode. A process schematic is reported in Figure 11.1. The UHHP equipment essentially consists of a relatively large pressure-resistant vessel and a high-pressure generating system. A booster pump and an intensifier pump are used to reach the required pressure. Food packaged in waterproof sealed elastic containers ranging from bottles to bags are placed into the vessel, which is then filled with water (or water-glycol solution) and pressurized to typically 500–600 MPa for a time period usually between 2 and 3 minutes. The pressure is applied by a direct or indirect compression technique but the more widespread method is the indirect one, with a pump compressing the liquid from the medium-pressure tank to the cell (see Figure 11.1), until the desired pressure value is reached. The temperature control is ensured by a simple electrical resistance if only heating is required. Otherwise a heating/cooling jacket, or a heat exchanger inside the cell, can be applied. The form of the container is designed to minimize the dead space inside the autoclave. It is important for reaching a high volumetric efficiency in order to reduce the cost per unit. Under routine operation, foods are now processed at pressures up to 600 MPa. Single plant production rates of 40 million lb/yr are already in operation. Production costs typically range from over 6 to about 4 cents/lb.14

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High-pressure cell Pressure medium tank

Pressure booster

Pump

P

FIGURE 11.1 Schematic of typical UHHP apparatus.

11.2.2 STATE

OF THE

ART

OF

UHHP

11.2.2.1 Microorganisms Relevant studies about the effect of high pressure on different kind of microbes and food substrates will be briefly discussed in this section. Early experiments, dated in the late 19th and early 20th centuries, showed that short treatments with an operating pressure of a few thousand bars were able to reduce the microbial activity by many orders of magnitude.13 In 1899 it was observed that pressurized milk would remain fresh and unspoiled for a longer time than untreated milk; also microbes contained in vegetables and fruits could be inactivated if they underwent a high-pressure treatment for a few minutes.15,16 Many later studies also showed that short treatments with an operating pressure of a few thousand bars were able to reduce the vegetative forms of microbial activity by various orders of magnitude.17 For instance, Timson and Short in 1965 started a systematic investigation to test the resistance of bacterial spores, and to try to inactivate them completely, that is, to get sterilization. These authors studied the behavior of spores under UHHP treatment with longer processing times at constant pressure in a range of temperatures between –25°C and 95°C.18 With the works of Gould and coworkers,19,20 the existence of optimal hydrostatic pressure to inactivate spores was demonstrated. After much research on various bacterial forms, these

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authors confirmed their hypothesis about the induction of spore germination by the action of pressure. More recently, Ludwig’s group investigated the behavior of spores under different conditions of UHHP21 and introduced the cycle-type treatment that proved to be more efficient than the double-level treatment.22,23 In recent years the effect of high hydrostatic pressure on the survival of different kinds of microorganism in various food substrates under different conditions was addressed24–33 and reviewed.26,27 Generally, the most recent results confirm the findings of early studies that food can be pasteurized under high pressure (400–600 MPa) and low to moderate temperatures (up to 60°C) but these products require refrigeration during storage and distribution to ensure microbiological stability. The specific operating conditions depend on the history of the bacterial cultures, that is, the type and growth phase of the test microorganisms considered.25 Bacterial spores were demonstrated to require more severe pressure treatments, in combination with other preservation techniques, principally heat treatment, to achieve inactivation.31,34 Recently, high hydrostatic pressure inactivation of vegetative microorganism, aerobic, anaerobic spores in pork Marengo (a low acidic particulate food product) was studied by Moerman,35 who showed that Saccharomyces cerevisiae and the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli are more pressure sensitive than cocci Enterococcus faecalis and Staphylococcus aureus (Gram-positive): their inactivation at room temperature was successful only with pressures as high as 600 MPa, whereas Gram-negative bacteria were more easily killed at pressure of about 400 MPa. The UHHP method was also applied to solid substrates, in particular air-dried alfalfa seeds inoculated with E. coli and L. monocytogenes. These samples were subjected to different pressure conditions (from 275–575 MPa for 2 min and 475 MPa for 2–8 min, at 40°C).36 It was shown that a maximum reduction of 2 log can be obtained, and that treated seeds took a longer time to germinate compared to the untreated seed. The effect of high-pressure processing on the safety, quality, and shelf life of ready-to-eat meats (low-fat pastrami, Strasburg beef, export sausage, and Cajun beef) was investigated at 600 MPa and 20°C for 3 min.10 After processing, samples were stored at 4°C for 98 days. After storage their counts of aerobic and anaerobic mesophilies, lactic acid bacteria, Listeria spp., staphylococci, Brochothrix thermosphacta, coliforms, yeasts, and molds were undetectable or at low levels. Furthermore, sensory analyses revealed no difference in consumer acceptability and no sensory quality degradation. An interesting paper concerning the effect of high-pressure-induced inactivation of Listeria innocua in buffer frozen suspension was published by Luscher et al.;37 a cycle pressure treatment above 200 MPa resulted in inactivation of about 3 log, probably due to the mechanical stress associated with phase transition of ice into its different polymorphs. The effect of various pressure levels (50–600 MPa) and holding times on color and microbiological quality of bovine muscle was also investigated.38 The reported experiments, carried out at 10°C and pressure higher than 300 MPa, induced modifications of meat color parameters. Pandey et al.39 studied the effect of high pressure treatment (250–400 MPa) for various holding times (0–80 min) at 3°C and 2°C on raw milk with high count of indigenous microflora. It was found that higher pressures, longer holding times, and lower temperatures resulted in larger destruction of microorganisms.

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Finally, we quote an interesting paper by Linton et al.40 who found that UHHP treatment (at 20°C and 300–600 MPa) readily inactivated psychotropic bacteria, coliforms, and pseudomonas in different types of shellfish such as mussels, prawns, scallops, and oyster. Table 11.1 is a compilation of interesting UHHP applications to food that can be found in the literature from the year 2000 on, with indication of the substrate, the type of microorganism, the pressure conditions, and the maximum inactivation ratio achieved. 11.2.2.2 Enzymes Food quality deterioration is caused by a wide range of phenomena, including physical conditions and both chemical and biochemical reactions. As far as enzymatic reactions are concerned, the effect of pressure on protein structure and functionality can vary dramatically depending on the magnitude of the pressure, the reaction mechanism, and the overall balance of forces responsible for maintaining the protein structure.41 To date, it is difficult to establish precisely the general effect of hydrostatic pressure on different enzymes in various food substrates and environment. Pressure has always been recognized as a potential denaturant of proteins, but examples of pressure-induced stabilization have also been reported. For instance, both polyphenol oxidases (PPO) and peroxidases (POD) which play an important role in food quality, as they influence the visual appearance, flavor and health-promoting properties, are known to be pressure-stable enzymes.42 This stability depends on plant source and type of product (e.g., whole fruit, rather than puree or juice). The global effect of pressure on enzymes is quite complex and can be explained in terms of individual molecular interactions within proteins, including hydrophobic, electrostatic, and van der Waals interactions.41 The information available on this topic is scarce and often contradictory; thus it is still impossible to discuss exhaustively the effect of UHHP on most enzymes catalyzing chemical reactions which influence quality and degree of deterioration of foodstuff. Table 11.2 provide a summary of recent studies on the effect of pressure on enzymatic reactions related to food, with indication of enzyme type, treatment conditions, and substrate utilized. 11.2.2.3 Microbial Inactivation Mechanism Microorganisms are inactivated when they are exposed to factors that substantially alter their cellular structure or physiological functions. Structural damage includes DNA strand breakage, cell membrane rupture, and mechanical damage to cell envelope. The reason for the effect of high pressure on microorganisms is not completely clear. It is well known that pressures in the range of 20–180 MPa delay microbial growth and tend to inhibit protein synthesis, while at pressures higher than 180 MPa inactivation causes the loss of viability. Lethal UHHP treatment disrupts membrane integrity and denatures many proteins. Another fundamental requirement for the survival and viability of microorganisms is the regulation of the cytoplasmatic pH

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TABLE 11.1 Applications of UHHP on Microbial Cells in Simple and Complex Solutions Reference 35

Treatment Regime 400 MPa/30 min

Temperature Maximum (°C) Reduction 20–50

Bacillus subtilis

0.14 log

Bacillus stearothermophilus Clostridium sporogenes Clostridium tyrobutyricum Clostridium saccharolyticum Enterococcus faecalis Staphylococcus aureus Escherichia coli Pseudomonas fluorescens Saccharomyces cerevisiae Alfalfa seeds Escherichia coli Listeria monocytogenes

0.69 log 1.63 log 1.79 log 3.35 log 6.49* log 3.51* log 575 MPa/2 min 40 475 MPa/2–8 min 300 MPa/5 min 20 400 MPa/1 min 700–800 MPa/5 min 900 MPa/1 min

1.4 log 2.0 log

10

600 MPa/180 s 20

4* log

37

200 MPa

83

>75 MPa/30 min 200 MPa/8 min 150–250 MPa

82

84 85

4* log

Subzero 3 log temperature 37

* means total inactivation

© 2007 by Taylor & Francis Group, LLC

Solution/ Substrate

1.31 log

0.74 log 0.21 log

36

Inoculated Microorganism

Pork marengo

Salmonella typhimurium Escherichia col Yersinia enterocolitica Vibrio parahaemolyticus Bacillus cereus Staphylococcus aureus Listeria monocytogenes Anaerobic mesophiles Lactic acid bacteria Listeria spp. Staphylococci Brochothrix thermosphacta Coliforms yeast and molds Listeria innocua

0.1% Buered peptone water (pH 7.4)

Escherichia coli

Desoxycholate Agar (DESO)

Escherichia coli Not Listeria monocytogenes inactivated at 250 MPa

Low-fat pastrami Strasburg beef Export sausage Cajun beef

Buffer solution

Chilled coldsmoked salmon

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TABLE 11.1 (CONTINUED) Applications of UHHP on Microbial Cells in Simple and Complex Solutions Reference

Treatment Regime

Temperature Maximum (°C) Reduction

86

350–550 MPa

30–45

7* log

87

350 MPa/20 min

50

5 log

38

50–600 MPa/20–300 s 250–400 MPa/0–80 min 300–600 MPa/2 min

10

2.5

20

81

50–400 MPa/15 min

47

39 40

88 89

90 91 92

Inoculated Microorganism Listeria monocytogenes Staphylococcus aureus; Gram-negative: Escherichia coli Salmonella enteritidis Alicyclobacillus acidoterrestris

Solution/ Substrate UHT 1% low fat milk

Total flora

Model system (BAM broth) and orange, apple, and tomato juices Bovine muscle

Escherichia coli

Raw milk

96%

Psychrotrophic bacteria Coliforms Pseudomonas

25

4 log

Total aerobic mesophilic and psychrotrophic bacteria

400 MPa/10 min 450 MPa/15 min 400 MPa/20 min

20

9* log

Listeria monocytogenes

40

99.97%

Room 2 log temperature

Listeria monocytogenes

500 MPa/60 s 300–600 MPa

Room 4–5 log temperature

Natural flora

Shellfish, mussels, prawns, scallops, oysters Tomato puree + natural additives (citric acid and sodium chloride) pH 5,6 citrate buffer Porcine blood plasma ACES buffer (N-(2-acetamido)-2aminoethanesulfonic acid) Green beans

400–500 MPa/5–30 min

10–40

2–31

* means total inactivation

© 2007 by Taylor & Francis Group, LLC

Milk and diary products Red blood cells fraction from porcine blood

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TABLE 11.1 (CONTINUED) Applications of UHHP on Microbial Cells in Simple and Complex Solutions Reference

Treatment Regime

Temperature Maximum (°C) Reduction

75

300, 800 MPa

93

300 MPa/15 min

94

200–600 MPa

95

50–400 MPa/1–60 min

20–80

6* log 3.67 log 2.27 log

96

1000 MPa/15 min

20

34

400 MPa/25 min

8–30

97

98 99

400 MPa/10 min 600 MPa/10 min 200 MPa/12h 545 MPa

Not Fruit and inactivated vegetable at 1000 products MPa 0.4 log Listeria monocytogenes Ultrahigh Bacillus cereus temperature Pseudomonas fluorescens milk Listeria monocytogenes Citrate buffer and phosphate buffer

100

600 MPa/5min

101

400 MPa/three 5-min cycle

102

400 MPa/5 min 20

103

100–500 MPa/15 min

Room temperature 5–50

25

7

4-25-50

* means total inactivation

© 2007 by Taylor & Francis Group, LLC

4,7 log

2 log

7 log

Inoculated Microorganism L. plantarum Escherichia coli Lactic acid bacteria Baird Parker flora Pseudomonas sp. Enterobacteria Lactobacillii harmful to beer Bacillus subtilis Bacillus stearothermophilus Streptococcus faecalis B. cereus

Psychrophilic bacteria Escherichia coli Listeria monocytogenes enteroxigenic Staphylococcus aures Yeast and lactic acid bacteria Microbial load

Solution/ Substrate

Sliced cooked ham and ground pork patties Beer Meat batters

Tilapia fillets Tomato-based salsa

Beer

Chilled hake (Merluccius capensis) Escherichia coli Milk cheese Escherichia coli Ringer solution Pseudomonas fluorescens and ovine Listeria innocua milk Staphylococcus aureus Lactobacillus helveticus

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TABLE 11.1 (CONTINUED) Applications of UHHP on Microbial Cells in Simple and Complex Solutions Reference 104

Treatment Regime

Temperature Maximum (°C) Reduction

400 MPa/10 7 min continuous pressure and pulsed pressure in two 5-min steps

5 log

Inoculated Microorganism H2S-producing microorganisms Lactic acid bacteria Brochothrix thermosphacta Coliforms

Solution/ Substrate Oysters

which plays an important role in secondary transport of several compounds; an irreversible internal pH decrease during UHHP treatment (200–300 MPa) probably promotes denaturation of proteins required for pH homeostasis.43 Another effect to be taken into account is a phase transition of the cytoplasmatic membrane from the physiological liquid-crystalline to the gel phase, which can induce leakage of sodium and calcium ions and increase membrane permeability.44 The main structural and functional changes in microorganisms at different pressures are summarized in Figure 11.2. It is worth noting that these microbial inactivation hypotheses were established recently, thanks to the possibility of exploiting new analytical methods which can determine structural and physiological cell modifications and loss of cytoplasmatic content by quantitatively and qualitatively estimating viable cells. This field of research was started because the traditional methods of analysis were not able to assess the physiological state of damaged cells after UHHP treatment. Furthermore, classic culture techniques were widely recognized to underestimate the number of truly viable bacteria, especially when cells had been damaged by physical treatment. Fluorescent staining, detection by microscopy, flow cytometry, and differential scanning calorimetry are presently the most widely used techniques.37,45–47

11.2.3 COMMERCIAL APPLICATION

OF

UHHP

In the last 15 years the food industry has been successful in marketing a number of UHHP treated products, which meet evolving regulatory concerns for greater food safety and a growing consumer demand for higher-quality and convenient (i.e., ready-to-eat) foods. Commercialization is being achieved thanks to collaboration between food scientists, microbiologists, and high-pressure equipment providers. Products and processes using UHHP are now available. In 1990 the Meidi−ya Food Company of Japan, introduced the first UHHP-pasteurized products, namely strawberry, kiwi, and apple jams. From 1993 the range of this kind of products has become larger and larger, and a variety of jams, juices, sauces, milk-desserts, fruit jellies, raw beef, and fish have been commercially produced. These products are

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TABLE 11.2 Applications of UHHP on Enzymes in Food Substrates Reference

Treatment Regime

Temperature (°C)

105

>300 MPa/ 20 min

9

106

400, 600, 800 MPa/5, 10, 15 min

18÷22

107 108

110 111 112 74 113

114 115 116 117

Proteolytic enzymes: Cathepsin B-like Cathepsin B+L-like Calpains Beta-glucosidase Peroxidase Polyphenoloxidase Pectin methylesterase PME Oxidase enzymes

109

300 450 MPa/ 15 min 400 MPa/10 min 300 MPa 100–400 MPa

0–450 MPa/15–30 min 207–310 MPa/0–2 min 395–445 MPa/8–11 min 0–800 MPa

118 119

50 MPa/72 h

120

Up to 700 MPa

121

600–900 MPa

122

300–400 MPa, 30–120 min

Proteolytic enzymes Beta-lactoglobulin 7

7-40-75-100

Red raspberry and strawberry Fruits and vegetables Carrots

Muscadine grape juice Octopus arm muscle

Oysters

Proteins Alphalactalbumin Beta-lactoglobulin Microbial load and autolytic activity Lipase

Milk

Octopus muscles Pacific oysters Carrot juice

20÷65

Tomato pectin methylesterase polygalacturonase Protein Rhizomucor miehei lipase Lactoperoxidase

4.4 log 3.09 log

Nonflavonoid phenolics Pectin methyl esterase

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Cold-smoked salmon

Polyphenoloxidase

70

÷ in the range – separates single set points

Solution/ Substrate

Enzymes

Tomato

Cheese

Bovine milk and acid whey Grape juice Orange juice

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Pressure (MPa)

300

Irreversible protein denaturation leakage of cell content

Intracellular pH decrease

200

Membrane damage signs of cell content leakage

Threshold of lethality

Reversible protein denaturation compression of gas vacuoles

100

50

Atmospheric pressure

Inhibition of protein synthesis reduction in the number of ribosomes

0.1

FIGURE 11.2 Main structural and functional changes in microorganisms as a function of pressure (adapted from Ref. 29).

packed in plastic bags and can be stored at 4°C for two months if sealed, or for one week after opening. At the present time, fruit, vegetables, shellfish, meat, and other products are in commercial UHHP production.48 In Europe, at least two wellestablished UHHP pasteurized products are worth mentioning, fruit juice in France and ham in Spain, while in Mexico companies are treating avocado puree for the U.S. market at a processing pressure of 700 MPa.17 For the sake of completeness, it must be mentioned that UHHP can be applied at the industrial scale to reduce the microbial activity, as well as to modify the consistency of foodstuffs; active research has been developed to obtain UHHP precooked foods such as meat and rice-based products.49

11.3 DENSE CO2 TREATMENT (DCO2) The antimicrobial effect of CO2 under pressure (or dense CO2) was discovered in the second half of last century, but specific research began only about 20 years ago. For instance, in the book on dense gas extraction by Quirin et al.,50 part of a chapter is dedicated to “sterilization” by dense gases, showing considerable reduction (i.e., 5 to 8 log) of microbial counts after appropriate CO2 pressure application at room temperature. Soon afterward, a systematic investigation was started to exploit this effect in order to develop a new nonthermal pasteurization technology, suitable for applications where processing temperatures close to ambient have to be used. As detailed in the section “state of the art” below, DCO2 was found to be lethal to basically all forms of microorganisms to which it had been applied. In addition,

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it has been shown51 that the antimicrobial action can be exerted at pressures as low as 7 MPa, that is, much lower than the ones required by UHHP treatment. Thus, DCO2 qualifies as the best candidate to replace traditional thermal treatment, especially when heat-labile components need to be preserved, as often happens with products related to food, pharmaceuticals, and cosmetics. A major limitation of the DCO2 technology is that direct contact between CO2 and the microorganism to be killed must be ensured. Therefore, at present, pasteurization can be achieved successfully only when liquid substrates or slurries are processed; treating solid materials by this technique remains problematic.

11.3.1 PROCESS

AND

EQUIPMENT FUNDAMENTALS

Treatment of liquid solutions and suspensions (i.e., pumpable substrates) by DCO2 is quite a simple task, which can be accomplished in many ways. The most efficient type of equipment is a continuous contactor, where the feed to be pasteurized and the CO2 stream can flow either cocurrently or countercurrently. A flow sheet of a pilot unit is sketched in Figure 11.3. The plant basically consists of a CO2 surge tank, two high-pressure pumps (one for CO2, the other for the substrate to be pasteurized), a mixer where the two streams are suitably contacted, a thermostatically controlled holding tube to ensure the proper retention time at the desired temperature, a suitable depressurization system, and eventually a degasser section. Energy requirements of this process are generally low, as liquids are easier to pump than gases. The consumption of CO2 is quite low as well: in the case of aqueous-based substrates the ratio between the CO2 and feed flow rates is never more than 1:20 on a weight basis, because the solubility of CO2 in these liquids is usually less than 0.05 (weight fraction). If desired, after depressurization, CO2 may be recompressed and recycled back to the contactor, but this option increases substantially the energy requirement. From our experience, the retention (holding) time to get a 6-log count reduction depends on the type of microorganism, but is usually in the range of 5 to 30 min, and can be further reduced by a suitable design of the holding tube.52 So far, we have processed a number of fresh fruit juices (such as orange, apple, grape, and pear) in a unit like the one depicted in Figure 11.3, always obtaining good results in terms of inactivation, provided that a temperature not less than 30°C–32°C was used.

11.3.2 STATE

OF THE

ART

OF

DCO2

11.3.2.1 Microorganisms The possibility of using CO2 under pressure to inactivate microbes was initially addressed by Fraser in the 1950s,53 who reported the disruption of bacteria cells by rapid release of CO2 gas from about 35 bars to ambient pressure. The first patent in the field was obtained by Swift & Co. in 1969, who claimed that food products could be sterilized by CO2 without degradation of their flavor at “superatmospheric” conditions and by exposing them to relatively low radiation dosages.54 Since 1985 many papers have reported on the bacteriostatic action of CO2 and on the growth and metabolism of different microorganisms, but it is from the paper

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TI1 VP1 A1

A2

TI2 VP 2 TI3 V-S

V1

V2

VP3 VP4 TI4 VP5 TI5 VP6 TI6

V4 TI8 P1

TI7

VP 6

PI3 VNR

TCI P3

BT 1

PC VL V-3

BT2

D

PI2

PI1

PI4 V5

V3

B1 CO2

B2

V6

P2 F

C

PV R

FIGURE 11.3 Schematic of DCO2 continuous equipment (adapted from Ref. 52); P1, P2, P3: pumps; PV: vacuum pump; A1, A2, B1, B2, D: tanks; F: filter; V1, V2, V3, V4, V5, V6: on-off valves; VNR: no-return valve; V-S: safety valve; VL: control valve; VP: sampling valves; TI: temperature indicators; PI: pressure indicators; BT1, BT2: thermostatic baths; C: chiller; R: flow meter.

by Kamihira et al. in 198755 that the inhibitory effect of CO2 under pressure toward microbes started to be addressed systematically and quantitatively. The number of publications on this topic has increased over the years since 1990. Many authors have reported experimental evidence on the effect of dense CO2 on different substrates and different kinds of microbes commonly present in foodstuff, both in their vegetative and latent forms. A review on inactivation of bacteria by the DCO2 method

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has been published recently,12 and it discusses research published in 83 articles and patents, half of which were published from 1999 to 2003. In the last two years several new articles have been published. They include an article by Liu et al.56 which describes a study on the influence of compression and decompression rate, and the concerted effects of temperature, pressure, exposure time, water content, and initial pH on the physiology of Absidia coerula and Saccaromices cerevisiae. The paper by Erkmen57 deals with the mathematical analysis of high-pressure CO2 regarding the inactivating effect on S. cerevisiae at different temperatures and pressures. His study allowed the prediction of yeast inactivation when exposed to different CO2 operating conditions. Watanabe et al.58 compared DCO2 treatment with other methods, in particular UHHP and thermal processing, for the inactivation of spores at different temperatures and exposure times. In the work by Furukawa et al.,59 the effect of CO2 on the germination of spores was studied at 65 bars, 35°C, and 120 min. In the past two years, several patents have also been issued, including the one claimed by Balaban,60 which deals with a continuous method to reduce microorganism and enzyme activities in liquid beer and wine products, and the one claimed by Praxair Inc. (Burr Ridge, IL, USA), which validates a continuous DCO2 process as a nonthermal pasteurization technology of fruit juice from lab-scale to commercialization for a feed flow rate up to 120 L/min (Better than Fresh®). In this last patent the presence of a CIP (Cleaning In Place) equipment is evaluated as a key design feature, to assure frequent cleaning and sanitization of the apparatus.61 Very recently, more studies have been discussed at international symposiums on high pressure and supercritical CO2, which demonstrate the increasing interest in this new mild pasteurization technology. Daiminger et al.62 presented a study on the efficiency of a continuous apparatus able to ensure 8-log count reduction of different kinds of microbes, both inoculated in orange juice and naturally present in an active sludge. The effect of the main operating parameters (flow rate and pressure) was discussed and an inactivation mechanism was proposed. Inactivation of Staphylococcus in liquid whole egg products by means of DCO2 in a batch stirred pilot device was presented by Van Ginneken et al.63 and patent application on this promising process has been filed.64 Zhang and coworkers65 investigated the synergistic effect of DCO2 in conjunction with low levels of H2O2 to deactivate spores and found that at least 4-log reduction can be reached at 40°C. On the inactivation mechanisms, disruption of the exospores, morphological changes, and release of dipicolinic acid (DPA) have been observed by means of TEM, SEM, and DPA fluorescence assay. From these reports, it is apparent that DCO2 has been shown to be effective as a bactericidal agent on vegetative forms of microorganisms at a near-ambient temperature and a relatively low pressure. The experiments, performed using both simple suspensions and complex substrates, resulted in inactivation levels sufficient to assure the pasteurization of foodstuffs. However, it should be noted that the texture of some solid products was disrupted and the color was changed after the DCO2 treatment,66 thus further work is needed in this respect. Mainly batch devices have been used as experimental apparatus at the laboratory scale and different operating conditions of pressure (55–300 bars) and temperature (10°C–50°C) have been tested. The presence of a mixing system has been shown to be beneficial in terms of inactivation efficiency.

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As far as latent forms are concerned, DCO2 alone is not suitable for practical application in the food industry, unless higher temperatures (at least more than 60°C) are applied.67 In this case, some hurdle approach can be beneficial.25,68 11.3.2.2 Enzymes As mentioned above, an irreversible pressure-induced denaturation of proteins generally requires values of pressure greater than 300 MPa, much larger than the ones used in DCO2 treatment. A different hypothesis must therefore be proposed to justify the inactivation of enzymes caused by DCO2. Apart from pressure, a number of factors can be taken into account when dealing with the influence of high-pressure CO2 on enzymes: pH of the medium, temperature, processing time, surface tension at the gas-water interface, type of microbes, and nature of the substrate. Current research results show that DCO2 treatment can either activate or inactivate enzymes.52 The interaction between dense CO2 and enzyme molecules is expected to cause conformational changes that will result in either loss or increase of activity. Recent reports dealing with the influence of high-pressure CO2 on enzymes are listed in Table 11.3 with indication of enzyme type, treatment conditions, and substrate utilized. It can be noticed that, despite the number of experimental data so far collected on enzyme activity changes after DCO2 treatment, no correlation nor definite enzymatic inactivation hypotheses are available in the literature. For instance, the decrease of activity has been related to the loss of secondary structures in enzyme molecules.69 Studies using gel electrophoresis showed that there are differences in the isoelectric profiles and protein patterns between the untreated and CO2-treated polyphenol oxidase (PPO), an enzyme causing undesirable browning on the surface of fruits and vegetables. Spectropolarimetric analysis revealed that CO2 treatment also changes the secondary structure of this enzyme.70 An enzyme H+-ATPase, located in the cell membrane, which is also important for the regulation of bacterial internal pH, was shown to maintain its initial specific activity in L. plantarum cell membrane even though viability of the cells was reduced by several log counts after high-pressure treatment.71 In contrast, Wouters et al.72 observed stimulation of ATPase activity in the membrane vesicles when L. plantarum was exposed to a hydrostatic treatment at 250 MPa for 80 min. The activity assessment of several enzymes by using the APIZYM system from B. subtilis cells before and after DCO2 treatment was exploited to demonstrate selective enzymatic inactivation as one of the probable cause of microbial inactivation.71 Recently, Habulin et al.73 observed that proteinase from Carica papaya latex showed improved stability at 300 bars of CO2 pressure, when compared to ambient pressure, and that addition of water also increased the activity of this enzyme (optimum amount was between 0.5 and 0.7 g/L). On the so-called hurdle approach, two papers have recently been published. Both of them deal with the combined effect of UHHP and DCO2 treatments on the activity of selective enzymes: Park and Lee74 observed that the residual activity of polyphenoloxidases, lipoxygenase, and pectinmethylesterase were less than 11.3%, 8.8%, and 5.1%, respectively, after a combined treatment of DCO2 at 4.9 MPa and UHHP at 600 MPa. Corwin et al. studied the UHHP processing of CO2-added solutions (at

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TABLE 11.3 Applications of DCO Solutions Reference 123 69 124

2

Treatment Regime

Treatment on Enzymes in Simple and Complex Temperature (°C)

31 MPa/10 min 25 MPa/30 min Supercritical and gaseous liquid phase, pH 3 ÷ 6 30MPa/30 min 35, 2 MPa/15min; 62, 1 MPa/15 min 30 MPa

35–50

71

7 MPa/10 min

30

75

300–800 MPa (0.2% CO2)

25–30

128

Micro-bubble methods Batch, 300 bar

125 126 127

73

285

30–60 35-45-55 25–50

35 40–55

50

Enzymes

Solution/Substrate

Pectinesterase Pectinesterase Acid protease, alk. protease, papain, and glucoamylase Myoglobin Lipoxygenase, peroxidase

Orange juice Valencia orange juice McIlvaine buffer

Aqueous solution 30% sucrose solution

Alpha-amilase Buffer solution acid protease H+-ATPase, costitutive Saline solution enzyme in L. plantarum Pectin methylesterase (PME), polyphenol oxidase (PPO) Alpha-amilase Buffer solution acid protease Proteinase from C. papaya

0.2% CO2 mole fraction). After treatment at 300÷800 MPa a synergistic effect on enzyme inactivation of pectin methyl-esterase and polyphenol-oxidase in L. plantarum and E.coli was found.75 11.3.2.3 Microbial Inactivation Mechanisms If inactivation of microorganisms in a pumpable substrate can be achieved in a simple apparatus such as the one outlined in Figure 11.3, the reason for the ability of DCO2 to inactivate microorganisms is not clear yet. Over the years, a number of hypotheses have been proposed to explain the inactivation mechanisms, but to date none appears completely satisfactory. For instance, it was demonstrated that, under standard operating conditions, cell death is not caused by explosive decompression of CO2 upon pressure release.76 Also, the inactivation of key enzymes as a consequence of direct interaction with CO2 has not been sufficiently proven, and remains a possible hypothesis. On the other hand, extraction of cell wall lipids by DCO2 based on the extraction capacity of CO2 as a supercritical fluid cannot be invoked at all as a reason for deactivation. Studies have shown that when the operating pressure is as low as 7 MPa and the temperature is around 30°C, the solubility of phospholipids in DCO2 is quite negligible. Thus, both intracellular acidification and modification of the cell membrane properties remain the two main reasons for

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explaining cell deactivation. It is interesting that both reasons are associated with the CO2 dissolution in the substrate. To better explore this hypothesis, the data presented in Figure 11.4 may be helpful. Let us consider the acidification aspect first. When a suitable CO2 pressure is applied over a physiological aqueous solution where model microorganisms are suspended, the pH of the solution is a strong function of pressure. It can be either measured, for example, by online UV spectroscopy77 (results are shown in Figure 11.4a), or calculated according to a suitable equation of state (SAFT)78 (Figure 11.5), so that curves such as those displayed can be obtained, depending on the system temperature. Clearly, when a pressure between 60 and 80 bars is reached, the pH value is lowered to about 3 by CO2 dissolution, but at higher CO2 pressures it cannot be further reduced; this behavior is compatible and can be correlated with the trend of CO2 solubility in water.52,78 On the other hand, a saline solution at neutral pH can be completely pasteurized by the DCO2 treatment carried out at 80 bars of pressure, so that it might be concluded that such a pH decrease (down to about 3) is responsible for the death of microorganisms. However, when a different aqueous solution such as orange juice was considered for the same experiment, no pH effect could be detected upon application of CO2 pressure (as shown in Figure 11.4b), even though total deactivation was achieved. This is an indication that solution (i.e., external) acidification itself is not sufficient to explain the inactivation of microorganisms. As far as interaction with the cell membrane is concerned, it was suggested that CO2 can dissolve into phospholipids, even at pressures of 60–80 bars.79 If the CO2 concentration in the phospholipids becomes high enough, the physical properties of the cell membrane can be modified enough to allow the enzymes linked to the membrane double layer to be released. On the other hand, the higher CO2 concentration in the membrane helps penetration of CO2 into the cytoplasm, with subsequent decrease of intracellular pH value. As a consequence of internal acidification, other phenomena such as enzyme deactivation and salt precipitation are likely to occur. It is noteworthy that both external acidification and modification of cell membrane properties are driven by the pressure-increased chemical potential of CO2 in the gas phase contacting the aqueous solution. If sufficient time is allowed for equilibration after pressure is applied, thermodynamics dictates that the chemical potential has to be the same in all the phases involved, that is the liquid bulk, the cell membrane, and the cytoplasm, leading to the possible consequences discussed above. Experimental evidence of the proposed hypothesis is under current investigation. With respect to intracellular acidification, we have recently shown that it can be measured and correlated to the microbial inactivation level.79 As far as the property changes of cell membrane are concerned, preliminary results suggest that when centrifuging an E.coli suspension after DCO2 treatment, significant and unexpected key enzyme activity can be measured in the liquid phase (supernatant), a clear indication of the enzyme release from the cell membrane itself.52

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35°C 3.40 3.30

Water

pH

3.20 3.10 3.00 2.90 2.80 0

20

40

60

80 100 120 Pressure (bar)

140

160

180

(a) 35°C 3.60 3.50 3.40

pH

3.30 3.20 3.10 3.00 Juice

2.90 2.80 0

20

40

60

80 100 120 Pressure (bar)

140

160

180

(b)

FIGURE 11.4 Experimental extracellular pH profiles as a function of operating CO2 pressure at 35°C in water, pH = 7 at atmospheric condition (4a) and in orange juice, pH = 3.47 at atmospheric condition (4b).

11.3.3 COMMERCIAL APPLICATION

OF

DCO2

Despite the huge research and development efforts performed in the last 20 years, at present no industrial applications of DCO2 to food pasteurization are known, even though several companies are actively working in this field. It appears that commercialization could be a matter of time. In this respect it is interesting to quote the opinion of Praxair, a company which is particularly active in developing a commercially sustainable DCO2 process,61 as reported in the proceedings of the recent Fifth

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3.35

3.30 313.15 K

pH

3.25

3.20 328.15 K

333.15 K

3.15

3.10

3.05 0

50

100

150

200 300 250 Pressure (bar)

350

400

450

500

FIGURE 11.5 Calculated pH profiles as a function of CO2 pressure at different temperatures (taken from Ref. 78).

International Symposium on Supercritical Fluids (Orlando, FL, May 2005):80 “… The validation of the dense phase carbon dioxide process is two-fold. First, it demonstrated a greater than 5-log reduction of the pertinent pathogens on juice using this process, to meet the juice HACCP guidelines set by FDA. As a result, this technology is now considered by the FDA as an alternative to thermal pasteurization. Second, product quality was validated by demonstrating the retention of physical, nutritional quality and sensory profile. …” Under these circumstances, it could be argued that the use of DCO2 as a nonthermal pasteurization treatment is going to be reality within a short time.

11.4 POTENTIALS OF HIGH-PRESSURE TECHNOLOGIES AND CONCLUSIONS From the results and discussion presented in this chapter, it can be concluded that high pressure provides efficient alternative processes for the pasteurization of foodstuffs when avoidance of thermal effects becomes an issue. It has been shown that UHHP treatment is already a commercial technology, at least for niche products, whereas the DCO2 process needs more scale-up development before achieving industrialization. On the other hand, it is clear that neither UHHP nor DCO2 alone can be exploited as sterilization techniques, and that high-pressure processing has to be performed in combination with other physical or chemical treatments81 in order to kill all microorganisms, including latent forms. Of course, coupling different techniques could also be beneficial to achieve pasteurization, for instance by allowing

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the use of lower pressures and processing time, which would reduce the investments in pressure equipment25,27 and improve the process economics. It is also interesting that in recent works the coupled effect of DCO2 and UHHP has been given special attention.74,75

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122. Basak, S., Ramaswamy, H.S., and Simpson, B.K., High pressure inactivation of pectin methyl esterase in orange juice using combination treatments, J. Food Biochem., 25, 509, 2001. 123. Balaban, M.O. et al., Inactivation of pectinesterase in orange juice by supercritical carbon dioxide, J. Food Sci., 56, 743, 1991. 124. Ishikawa, H. et al., pH-dependent inactivation of enzymes by microbubbling of supercritical carbon dioxide, Food Sci. Technol. Res., 6, 212, 2000. 125. Ishikawa, H. et al., Irreversible unfolding of myoglobin in an aqueous solution by supercritical carbon dioxide, J. Agric. Food Chem., 48, 4535, 2000. 126. Tedjo, W., Eshtiaghi, M.N., and Knorr, D., Impact of supercritical carbon dioxide and high pressure on lipoxygenase and peroxidase activity, J. Food Sci., 65, 1284, 2000. 127. Yoshimura, T. et al., Inactivation kinetics of enzymes by using continuous treatment with microbubbles of supercritical carbon dioxide, J. Food Sci., 66, 694, 2001. 128. Yoshimura, T. et al., Effect of CO2 flow rate on enzyme inactivation by continuous method with microbubbles of supercritical carbon dioxide, J. Faculty Agric., Kyushu University, 46, 345, 2002.

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Microencapsulation and Delivery of Omega-3 Fatty Acids Luz Sanguansri and Mary Ann Augustin Food Science Australia

CONTENTS 12.1 12.2 12.3 12.4

Introduction ..................................................................................................298 Drivers for Delivery of Omega-3 Fatty Acids into Foods ..........................299 Incorporation of Omega-3 Oils into Foods .................................................300 Microencapsulation ......................................................................................301 12.4.1 Microencapsulation in the Food Industry........................................301 12.4.2 Microencapsulation of Omega-3 Oils..............................................301 12.5 Manufacture of Microencapsulated Omega-3 Ingredients ..........................302 12.5.1 Elements of Microcapsule Production.............................................302 12.5.1.1 Omega-3 Oil Core Selection ............................................302 12.5.1.2 Encapsulant Selection.......................................................304 12.5.1.3 Formulation Design ..........................................................304 12.5.1.4 Choosing the Process........................................................304 12.5.1.5 Format for Delivery ..........................................................305 12.5.2 Encapsulating Materials...................................................................305 12.5.2.1 Challenges for Selection of Materials for Omega-3 Oil Encapsulation .............................................306 12.5.2.2 Classes of Encapsulant Materials Used for Omega-3 Oil Encapsulation .............................................306 12.5.3 Mechanical Processes ......................................................................308 12.5.3.1 Emulsion Preparation and Stabilization ...........................309 12.5.3.2 Spray Drying.....................................................................310 12.5.3.3 Extrusion ...........................................................................311 12.5.3.4 Gelation and Particle Formation ......................................312 12.5.3.5 Secondary Coating Application........................................312 12.5.4 Chemical-Based Processes...............................................................313 12.5.4.1 Coacervation .....................................................................313 12.5.4.2 Inclusion Complexation....................................................314 12.5.4.3 Liposome Encapsulation...................................................314

297

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12.5.5 Emerging Technologies....................................................................315 12.5.5.1 Supercritical Fluid Spraying.............................................315 12.5.5.2 Nanoencapsulation............................................................315 12.6 Properties of Microencapsulated Omega-3 Ingredients ..............................315 12.7 Food as Delivery Systems for Omega-3......................................................316 12.7.1 Infant Formula..................................................................................316 12.7.2 Bakery and Cereal Products ............................................................317 12.7.3 Dairy Products..................................................................................317 12.7.4 Meat and Fish Products ...................................................................318 12.7.5 Drinks and Beverages ......................................................................318 12.7.6 Confectionery ...................................................................................318 12.8 Considerations for Successful Delivery of Omega-3 Oils into Commercial Food Products .........................................................................318 12.8.1 Compliance with Regulatory Standards ..........................................318 12.8.2 Definition of Final Product Application to Ensure the Correct Delivery Format ..................................................................319 12.8.3 Understanding of the Protection and Release Requirements during Processing .....................................................319 12.8.4 Understanding of How and Where to Add the Microencapsulated Ingredient..........................................................319 12.8.5 Understanding Possible Interactions with Other Ingredients..........319 12.8.6 Understanding the Shelf-Life, Sensory, and Physical Properties of the Final Product........................................................319 12.8.7 Working in Partnership through Collaboration ...............................320 References..............................................................................................................320

12.1 INTRODUCTION The functional food revolution and increasing consumer awareness of the link between health and food have resulted in a growing demand for foods that offer benefits for healthy living.1,2 Significant among the growing class of functional foods are foods that are fortified with omega-3 fatty acids. The high degree of unsaturation of omega-3 oils makes them very susceptible to oxidation. Oxidation of omega-3 oils can occur at all stages in the supply chain, from the handling of raw materials through to processing, storage, and handling of the oil during food manufacturing process and subsequently in the storage and handling of the final omega-3 enriched food products. This makes the incorporation of omega-3 fatty acids into foods a significant challenge as their susceptibility to oxidation and development of off-flavors affects the sensory properties of the omega3 fortified foods.3–6 To overcome some of the issues relating to stability of omega-3 oils, the use of microencapsulation technology has been explored. Microencapsulation, which involves the packaging of a core within a secondary material in small particles, is proving to be a successful strategy for protecting sensitive ingredients and enabling

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their delivery into foods.7 Microencapsulation technology has proven to be a powerful technique for tailoring the properties and functionality of sensitive ingredients for use in specific systems. It has the potential to offer novel solutions for stabilization and improved delivery of omega-3 fatty acids in food. This chapter reviews the issues relating to incorporation of omega-3 fatty acids in food. It includes a discussion of microencapsulation technology and its application for delivery of omega-3 fatty acids in the food industry, the reasons for its use, the materials used as encapsulants, the techniques used, and examples of its use in product applications. Trends and innovations in new materials, emerging processes and technical challenges associated with the selection of encapsulants, and appropriate techniques for the production of microencapsulated products are discussed. Important considerations for successful delivery of omega-3 oils into the functional foods market are highlighted.

12.2 DRIVERS FOR DELIVERY OF OMEGA-3 FATTY ACIDS INTO FOODS Omega-3 fatty acids have been associated with many health benefits relating to heart health, brain function, eye health, joint health, mood, behavior, cancer, diabetes, skin disorders, pregnancy, and lactation.3,8–14 Interest in delivery of omega-3 fatty acids into foods has primarily been driven by consumer awareness of the health benefits of omega-3 fatty acids and an increased consumer demand for foods fortified with omega-3 fatty acids that have desirable nutritional, sensory, and functional attributes. There is also a growing recognition that modern diets are deficient in omega-3 fatty acids. The average intake of omega-3 fatty acids by consumers has been found to be grossly inadequate due to low consumption levels of fish, one of the major sources of omega-3 fatty acids in the diet. Major health and nutrition organizations and scientific authorities recommend that intakes of long-chain polyunsaturated fatty acids (PUFA) should be increased by eating two to three fatty fish meal per week, or by supplementation. The International Society of the Study of Fatty Acids and Lipids (ISSFAL), American Heart Association (AHA), Institute of Medicine (IOM), and British Nutrition Foundation (BNF) all make recommendations on the daily intake of omega-3 fatty acids. As a general guideline, the minimum recommended daily intake for adults is approximately 500 mg of combined docosahexaenoic acid, C22:6n-3 (DHA) and eicosapentaenoic acid, C20:5n-3 (EPA) per day for cardiovascular health, or 900 mg of combined DHA and EPA if you have coronary heart disease.15–18 In 2000, the Food and Drug Administration (FDA) announced a qualified health claim for dietary supplements containing EPA and DHA and the reduced risk of coronary heart disease. The FDA recommends that consumers do not exceed more than a total of 3 grams per day of EPA and DHA with no more than 2 grams per day from a dietary supplement. In 2004, the FDA allowed a qualified health claim for omega-3 fatty acids (DHA and EPA) and cardiovascular health. To qualify for the health claim in the U.S., foods must contain a minimum of 125 mg long-chain

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omega-3 fatty acids per serving, and be part of a food that is low in fat, saturated fat, trans fat, and cholesterol.19 In Australia and New Zealand, a nutritional claim has been allowed for omega-3 fatty acids if the food contains a minimum 30 mg total EPA and DHA, or 200 mg alpha-linolenic acid, C18:3n-3 (ALA) per serving, when part of a food that is low in saturated fat and trans fatty acids.20 The accumulating evidence about the health benefits of omega-3 fatty acids, the increasing awareness of consumers about the benefits of omega-3 fatty acids, and advances in processing of PUFA ingredients are making omega-3 enriched foods a reality in the market place.5,21–24 The advent of the nutritional and health claims has opened up new opportunities for foods rich in omega-3 fatty acids. More products enriched with omega-3 fatty acids are becoming available in the market and starting to gain the acceptance of consumers.

12.3 INCORPORATION OF OMEGA-3 OILS INTO FOODS Omega-3 oils, such as fish oil, are prone to oxidative deterioration and to the development of objectionable off-flavors and odors even when precautions are taken such as protection from light and oxygen and low-temperature storage.25 Oxidation also affects the nutritional quality and safety of the oil to consumers. The fishy taste and odor associated with omega-3 oils from marine sources is another hurdle for incorporation of omega-3 fish oils into food products. The development of oxidative rancidity of omega-3 oils is a factor that has limited its use in foods. The biggest challenge to the omega-3 oil manufacturers and suppliers is overcoming the development of undesirable taste and odors in the end products and the difficulties in using the oil as an ingredient in food and beverage applications.26 As the oxidation of unsaturated fatty acids is an autocatalytic process, unsaturated oils used for incorporation into foods must be of the highest quality practically achievable in order to minimize the development of off-flavors. This means that the oils should be extracted from raw materials of good quality and the bulk oils should be refined, stabilized, packaged, and stored under appropriate conditions, which minimize exposure to factors that promote oxidation such as air, oxygen, and light. Antioxidants may be added to protect the oil from oxidation. Synthetic antioxidants, such as tertiary butyl hydroquinone, butylated hyroxylanisole, and butylated hyroxytoluene, singly or as mixtures, have been used in fish oils. The push toward natural ingredients has resulted in natural antioxidants such as tocopherols, ascorbic acid, carotenoids, and rosemary extracts becoming a more popular option.25,27 Mixtures of tocopherol, lecithin, and ascorbyl palmitate were found to be effective for retarding oxidation in fish oil.28 Even with the addition of antioxidants, unprotected oils can still oxidize very rapidly once they are exposed to a food environment. Omega-3 oils may be added directly to foods but this approach has its limitations. The oil is not protected from the food matrix during processing of the food product, thereby exposing the oil to environments (e.g., air, oxygen, heat) that promote oxidation of unprotected fats. This has undesirable consequences on the shelf

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stability of the omega-3 fortified food product. This has in part been addressed by addition of antioxidants to the bulk oil and to the food systems. An alternative approach is to use microencapsulated omega-3 oils.29 With the use of microencapsulation, the oil is protected from the harsh processing environments during processing. A good microencapsulation system also protects the oil from interactions with the food matrix and environment during storage. This allows the omega-3 fortification of food products without affecting the taste and shelf life of the final food. The principles of designing microcapsules and the use of microencapsulation and delivery technologies for incorporation of omega-3 fatty acids into foods is discussed in more detail below.

12.4 MICROENCAPSULATION Microencapsulation is the science of packaging components (referred to as the core or active) within a secondary material (referred to as the encapsulant or coat) and delivering them in small particles (i.e., microcapsules). Microencapsulation is used as a means of isolating an ingredient from the reactions of the surrounding materials or environment.30 It may be used for stabilizing a sensitive ingredient, masking flavors, and for controlled delivery of active components. An associated benefit is the improved ease of handling when microencapsulated ingredients are delivered in a powder format.

12.4.1

MICROENCAPSULATION

IN THE

FOOD INDUSTRY

Microencapsulation technology has been used for flavor encapsulation since 1930.31 In recent years the functional food and ingredient industries have been identified as potentially significant growth areas for the technology, especially for the encapsulation of unstable new functional ingredients. There are greater demands being made on the integrity of the microcapsules to provide controlled delivery of the core material at the desired time and site.7,32–36 Microencapsulation is an enabling technology that provides novel solutions for the fortification of food, while ensuring that the taste, aroma, or texture of food is not compromised.7,34,37

12.4.2 MICROENCAPSULATION

OF

OMEGA-3 OILS

The main advantage of omega-3 oil encapsulation is the protection afforded to the oil against oxidation during storage and processing. This is because the microencapsulated oils have a built-in barrier (i.e., the encapsulant) that isolates the oil from the surrounding environment. Hence, the oxidation of polyunsaturated oils is inhibited, and the shelf life of these products during storage and processing may be improved.29,38 The level of protection afforded against oxidation depends on the encapsulant used and the integrity of the microcapsule. The oil may be protected from direct exposure to light and oxygen during storage and from the other components in the food matrix during food processing and storage, thus reducing potentially undesirable reactions with other food ingredients in a complex food system.

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Microencapsulation can also be used to mask the undesirable taste and odor that may be imparted by the oil, thus enabling the production of omega-3 fortified foods with desirable sensory properties.39

12.5 MANUFACTURE OF MICROENCAPSULATED OMEGA-3 INGREDIENTS The production of a microencapsulated omega-3 product involves the selection of the omega-3 oil (i.e., the core), choosing the ingredients to be used as encapsulants, developing the formulation comprising the required amount of omega-3 oil to encapsulant ratio, and applying an appropriate process for production. To ensure that the microencapsulated product is used effectively for fortification of foods, the properties of the microcapsule need to be tailored to suit the food application. It is important to ensure that the omega-3 oil core is protected from the harsh processing environments and that the release of the core is not triggered prematurely. Many different encapsulant materials combined with different processing technologies have been used for the encapsulation of omega-3 oils resulting in microcapsules with different properties.25,40–46 However, there are still limitations to the use of microencapsulated ingredients in some applications. An integrated approach to microcapsule production and its final product application is required for the success of a microencapsulated ingredient (Table 12.1).

12.5.1 ELEMENTS

OF

MICROCAPSULE PRODUCTION

12.5.1.1 Omega-3 Oil Core Selection The first step is to choose a source of omega-3 oil. It may be chosen from a range of polyunsaturated oils that are rich in omega-3 fatty acids. The long-chain omega3 fatty acids, DHA and EPA, are found mainly in fish, such as tuna, salmon, sardines, and mackerel, and in marine algae and seaweeds. The shorter-chain omega-3 fatty acid, ALA, can be found in many plant sources such as linseed, canola, hemp, perilla, and some vegetables such as spinach, green peas, and beans.3,4,47 The major commercial source of DHA and EPA is presently from fish. However, there is increasing interest in algal oils48,49 and transgenic oil seeds50 as alternative sources of longchain omega-3 fatty acids. It is essential to use high-quality oil with a low degree of oxidation. The specific refining and deodorizing process applied in the processing of commercial omega-3 oil, the source of the raw material (e.g., fish oil, algal oil, plant oils, etc.), the fatty acid composition, and addition of antioxidant mixtures to the oil will influence the stability and the sensory properties of the microencapsulated oil product. The oxidative stability of microencapsulated omega-3 oils is dependent also on the type of antioxidant used. Tocopherol proved to be more effective than ascorbyl palmitate for protection of microencapsulated fish oil powder against oxidation.51

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TABLE 12.1 Elements of Microencapsulation: An Integrated Approach to the Development of Microencapsulated Ingredients Technological Activities Identify core Characterize core Physical and chemical properties Stability Choose material Use without modification Modify to alter functionality Characterize material Physical and chemical properties Stability

Production of Microencapsules Core

+ Encapsulant Material

↓ Design formulation Formulation of Consider interactions between components Microcapsule Stability of formulation System Triggers for release of core

Choose appropriate process Conventional New/Emerging Define unit process variables and apply process Assess process consistency efficiency Determine composition Gross composition Proportion of encapsulated and free core Characterize microcapsule Physical and chemical properties Stability Triggers for release

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↓ Processing of Microcapsule

Market-Related Activities Work with a supplier of the core and user of the ingredient Obtain specifications Consider cost Establish reliable source of material Obtain specifications Consider cost

Consider what is available in the market place Range of formulations available Limitations of existing formulations Market need for microcapsules with differentiated functionality Consider need for capital investment Processing cost Process efficiency and reliability Alternative processes

↓ Microencapsulated Develop product specifications and Product applications Align with end users Assess suitability Provide technical service

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12.5.1.2 Encapsulant Selection The encapsulant can be selected from a wide variety of natural or synthetic polymers depending on the characteristics desired in the final microcapsule. The composition as well as the physical and chemical properties of the encapsulant material can influence the functional properties of the final microcapsule and the choice of processing technologies to be used for microencapsulation. For encapsulation of omega-3 fatty acids, it is desirable to choose materials from a natural source, which have good oxygen barrier properties in order to afford protection of the core from oxidation. 12.5.1.3 Formulation Design The design of formulations for encapsulation of omega-3 fatty acids requires an understanding of the properties of the oil and the required function of the encapsulant materials and any other ingredients or additives chosen for the formulation. The primary aim is to design a formulation that results in high encapsulation efficiency and a microcapsule that gives high oxidative stability to the omega-3 oil core. A high oil load in the microcapsule is also desirable provided this does not compromise the quality of the microcapsule. The development of a successful formulation for a target application requires knowledge about the stability of the chosen oil; the encapsulating properties of the ingredients; the likely interactions between the omega-3 oil core, the encapsulant and other components in the formulation; and the reactivity of the microcapsule formulation in the final product application. It has been shown that factors such as the type of encapsulant material, addition of antioxidant, chelating agent, emulsifier, stabilizers, and salts affect the stability of microencapsulated oil products.52. 12.5.1.4 Choosing the Process The selection of unit processes and process conditions for omega-3 oil encapsulation requires some consideration. A number of standard unit processes suitable for production of microencapsulated omega-3 oils are widely available in the food industry. Standard unit processes (e.g., mixing, homogenization, and drying) have traditionally been used for the production of microencapsulated ingredients such as omega-3 oil powders as it usually involves the preparation of an emulsion and drying. There are possibilities for application of emerging food-processing operations (e.g., microfluidization, ultrasound, high-pressure processing) in the manufacture of microencapsulated ingredients. The use of these technologies at various stages of the microencapsulation process, in place of standard unit processes, may be used to produce microcapsules with unique properties. The choice of the processes used for microencapsulation will depend on the properties required and the cost for production of microcapsules. Even with using standard unit processes, the cost of production of microcapsules can vary significantly depending on which microencapsulation technology is being used. If nonstandard unit processes are used, there may be a need for capital investment. It is

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important to consider the essential requirements of the microencapsulated ingredient in the final product, and whether the product can carry the additional cost. 12.5.1.5 Format for Delivery For addition into a dry or powdered food product formulation, it is necessary to convert the liquid microcapsules containing omega-3 oils into powder to make it commercially feasible for dry blending. This means that a drying step is needed as an integral part of the microencapsulation process. It is important to match the physical properties (e.g., particle size and bulk density) of the microcapsule to that of the powder with which it is to be dry blended in order to minimize or avoid physical separation or segregation of the ingredients during transport and storage. For liquid food applications, a liquid format (e.g., emulsion) is sometimes preferred, but a powder format that can easily be rehydrated into a stable emulsion or dispersion is also suitable.53

12.5.2 ENCAPSULATING MATERIALS Proteins, carbohydrates, lipids, gums, polysaccharides, and cellulose materials are commonly used for microencapsulation of omega-3 fatty acids and oils (Table 12.2). They can be used alone or in combination to achieve certain functions either during production or in the final product. Some of the new developments in materials for microencapsulation of omega-3 fatty acids involve the application of clever chemistry and processing to modify the properties of the native food ingredient to achieve new functional properties not present in their standard form.

TABLE 12.2 Materials Used as Encapsulants of Omega-3 Oil Encapsulation Material Class Proteins Sugars and sugar products Starch and starch products Gums Cellulose material

Other carbohydrates Lipids

Types of Materials Albumin, caseinate, gelatin, gluten, peptides, soy protein, whey proteins, zein, other vegetable proteins Fructose, galactose, glucose, maltose, sucrose, oligosaccharide, corn syrup solids, dried glucose syrup Maltodextrins, dextrins, starches, modified starches Agar, alginates, carrageenan, gum acacia, gum arabic, pectins Acetylcellulose, carboxymethyl cellulose, cellulose acetate butylate phthalate, cellulose acetate phthalate, ethyl cellulose, methyl cellulose, nitrocellulose Chitosan, cyclodextrins Acetoglycerides, beeswax, diglycerides, natural fats and oils, fractionated fats, hardened fats, lecithin, liposomes, monoglycerides, paraffin, tristearic acid, waxes

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A good encapsulant for omega-3 fatty acids protects the oil in the microcapsule from degradation during processing and storage and also masks the undesirable taste and odor associated with the microencapsulated omega-3 oil ingredient when added into a food product. The encapsulant must be approved for food use if the formulation is to be incorporated into food products. The encapsulant materials may be used in combination with other components in the formulation such as oxygen scavengers, antioxidants, chelating agents, and surfactants. Cost is also a consideration for the selection of the encapsulant. 12.5.2.1 Challenges for Selection of Materials for Omega-3 Oil Encapsulation It is important to consider the encapsulant that will be used in the formulation and process for the production of the microcapsule. The ability of the encapsulant to stabilize and protect the oil core during processing and storage of the microencapsulated omega-3 oil ingredients, and the mechanisms required for release of omega3 fatty acids at the appropriate time and site needs to be taken into account. Other criteria that need to be considered during encapsulant selection include the mechanical strength requirement of the microcapsule, the compatibility of the encapsulant material with the target food product, its nutritional value, sensory properties, and aesthetic properties.34,35 The availability of a very limited range of suitable encapsulant materials allowed for food use still remains the biggest challenge in encapsulant material selection, especially when more sophisticated microcapsule properties are required by food manufacturers and consumers. The requirement for good powder flow properties, dispersibility, and solubility are now becoming standard prerequisites for omega-3 oil powder microcapsules, with the requirements for oxygen, moisture, heat, shear, and pH barrier properties becoming important characteristics of the encapsulant surrounding the core. Food-grade materials commercially available meet some of these requirements. Modification of existing food-grade materials to achieve differentiated encapsulant functionality may be required to achieve new properties in microcapsules and improved performance of microencapsulated ingredients. 12.5.2.2 Classes of Encapsulant Materials Used for Omega-3 Oil Encapsulation 12.5.2.2.1 Proteins Proteins possess many excellent functional properties. They have been used as encapsulants for omega-3 fatty acids and oils because of their good film-forming and emulsifying properties.54-58 The most commonly used proteins are gelatin, whey protein, casein, soy protein, and albumin. The good water solubility of proteins is desirable during preparation of the encapsulant in order to achieve the required encapsulant functionality when using an all-aqueous-based process. However, the good water solubility characteristics of the encapsulant generally result in rapid dissolution of the encapsulant when

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the microcapsule is in contact with water in the final application. This may be accompanied by the release of the core to the environment, which presents a disadvantage in some applications. In order to improve the resistance of protein encapsulants to moist environments, the solubility of protein wall materials can be modified using a number of techniques. This may be achieved by coagulating the protein by heat or by the action of pH.59 Lee and Rosenberg60 used a double emulsification and gelation process to prepare whey protein microcapsules (containing a milkfat core), which had limited water solubility for controlled core release in food applications. Others have improved stability of protein-based fish oil capsules by using transglutaminase to cross-link soy proteins in double emulsions.61 The good oxygen barrier properties of some proteins41,62,63 makes them desirable as a choice of encapsulant for protecting oxygen sensitive cores like omega-3 fatty acids and oils against oxidation. Proteins have been used in combination with carbohydrates in the formulation of microencapsulated oil ingredients.64,65 Lin et al.44 reported that wall materials containing gelatin, caseinate, and maltodextrin provided optimal protection against oxidation of microencapsulated squid oil. Further, the oil stability was improved with the addition of emulsifier (lecithin) and a stabilizer (Avicel). 12.5.2.2.2 Carbohydrates Maltodextrins, corn syrup solids, and sugars have been used in formulations for omega-3 oil encapsulation, but not on their own because they have poor emulsifying and film-forming properties. Starches have also been used for encapsulation of oil and oil-soluble cores. They have been made more suitable for encapsulating oils by chemical modification to increase their lipophilicity and improve their emulsifying properties. Starches, such as high amylose cornstarch, may also be used for sitespecific delivery as demonstrated by the application of high amylose starch as a food-grade enteric encapsulant.66 Gums have traditionally been used in the flavor industry but also have applications as encapsulating materials for omega-3 oils. The gum most commonly used is gum acacia, although it is the protein fraction in acacia gum preparations that is responsible for its emulsification properties. Modified celluloses such as methylcellulose and hydroxypropyl methylcellulose in combination with maltodextrin have been demonstrated to be suitable encapsulants for stabilization of fish oil in high oil load powders with 40%–50% oil.67 Cyclodextrins, which are cyclic molecules derived enzymatically from starch, have some unique encapsulation properties. They have the ability to form inclusion complexes with flavors and oil-soluble nutrients and can provide stabilization, during manufacturing and storage of some core materials that cannot be stabilized by other methods. The use of cyclodextrin is currently limited because of its high cost. Nevertheless, β-cyclodextrin offers advantages as a microencapsulant because it has good controlled release properties, with binding occurring up to 200°C. The release of the complexed material is influenced by moisture. β-cyclodextrin has been approved by the FDA and granted GRAS status.

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12.5.2.2.3 Lipid Materials Fats have been used mainly as a secondary coating to primary microcapsules to improve their water barrier properties.68 Liposomes are artificially made microscopic membrane vesicles consisting of one or more concentric layers of lipids.35, 69 These versatile materials can be used for delivery of water- or oil-soluble core materials and are therefore suitable for microencapsulating a range of food ingredients, including vitamins, omega-3 oils, minerals, and flavors. Stable microcapsules, ranging from a few nanometers to microns in diameter, may be made which makes them suitable for a range of applications. The microcapsules produced by liposome entrapment are more versatile and less fragile than fat-encapsulated ones. One promising area of application where liposomes could have the advantage over other methods is the prevention of oxidation of unsaturated fats in food emulsions such as spreads, margarines, or mayonnaise. However this needs further development to optimize loading, protection, and delivery. 12.5.2.2.4 Protein—Carbohydrate Conjugates An alternative to the use of physical blends of protein and carbohydrates is to form protein-carbohydrate conjugates. Kato and Kobayashi70 demonstrated that proteindextran conjugates, developed by coupling proteins to cyanobromide-activated dextran or by linking proteins with dextran through the naturally occurring Maillard reaction, have excellent emulsifying properties. These conjugates were superior to the native protein, commercial emulsifiers from sucrose-fatty acid esters and polyglycerine esters, especially at high salt concentration and at acidic pH. The emulsifying properties of protein-dextran conjugates were greatly enhanced by preheating at 100°C. Protein-carbohydrate conjugates, formed as a result of the Maillard reaction, have been shown to be superior to physical blends of protein and carbohydrate for encapsulation of omega-3 oils. The conjugates have excellent film-forming, emulsifying, and oxygen scavenging properties. Omega-3 oils encapsulated within a Maillard reaction product of film-forming protein and carbohydrate have longer shelf life compared to those encapsulated with proteins and carbohydrate blends.71

12.5.3 MECHANICAL PROCESSES The selection of appropriate processing technologies for microencapsulation of omega-3 fatty acids depends on the desired properties of the final microcapsules such as oil loading, shelf life, release properties, microcapsule format, and the final product application.72 Microencapsulated omega-3 oil products are generally produced in powder form for better stability and ease of handling. Liquid emulsions are also available for more specialized liquid food applications. A generalized process used for production of microencapsulated omega-3 oil ingredients is shown in Figure 12.1. Stable omega3 oil-in-water emulsions may be heat treated in liquid state or converted by spray drying into free-flowing microencapsulated powders in order to improve their shelf stability.

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Omega-3 oil + encapsulant material

Emulsification

Stable oil emulsion (Liquid emulsion microcapsule)

Spray drying

Omega-3 oil powder (Primary powder microcapsule)

Secondary coating in fluidized bed (Optional process)

Omega-3 oil powder with secondary coating

FIGURE 12.1 Generalized process for production of spray-dried microencapsulated omega3 oil powder with optional secondary coating.

12.5.3.1 Emulsion Preparation and Stabilization Preparation of an emulsion involves the dispersion of the oil phase into very fine droplets in which the action of an emulsifying agent at the surface of the oil droplets provides protection or functionality to form stable oil-in-water emulsions. High shear processes such as homogenization or microfluidization may be used for dispersing the oil into droplets and forming the emulsion. Emulsifiers, antioxidants, and other additives may be incorporated into both the oil phase and the aqueous phase prior to homogenization.73 The emulsion formulation and other variables such as temperature, total solids, pH, and storage conditions influence the emulsion properties and stability.74–76 Stable emulsion microcapsules can be dried into a powder for greater shelf stability. The selection of appropriate materials that can form a strong film around the oil droplets, which will remain intact during processing and storage, is essential for providing protection and stability to the omega-3 oil droplets. Both the physical stability of the emulsion during processing and the storage and oxidative stability need to be considered in the development of omega-3 oil emulsions. A range of proteins, emulsifiers, and stabilizers have been used for the stabilization of omega-3 oil in water emulsions. The oxidative stability of omega-3 oil-inwater emulsions is dependent on the type of protein and emulsifiers used in the formulation.77–82 This is because the oxidation of an oil-in-water emulsion droplet is influenced by the properties of the interfacial membrane surrounding the lipid

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core as well as the properties of the bulk phase. Important determinants of oxidative stability of emulsions include the thickness of the interfacial membrane of an emulsion droplet,83 the size of the surfactant hydrophobic tail group size,84 the type of antioxidants, the partitioning of the added antioxidants in the emulsions,85,86 and the level of hydroperoxides, which may be present in both lipids and surfactants.80 Advances in emulsion technology provided the possibility of designing multiple emulsions for the controlled delivery of bioactives. An example is a method of producing an oil-in-water-in-oil (o/w/o) emulsion where the oil or oil-soluble core (flavors) is emulsified in a protein to form the o/w emulsion and then this emulsion is further emulsified in a liquefied fat that is solid at room temperature. The resulting o/w/o emulsion is then atomized into a food product as a delivery vehicle for the core.87 Benichou et al.88 have also developed hybrids of natural polymers for entrapment and slow release of active matters in double emulsion systems. 12.5.3.2 Spray Drying Spray drying has been generally referred to as an encapsulation technology because it is one of the oldest encapsulation methods and has been in use since the 1930s.31 It is still the most commonly used microencapsulation method in the food industry. The process of spray drying is efficient and cost effective, uses equipment that is readily available, and produces particles of reasonably good quality. Food ingredients microencapsulated by this method include fats, oils, flavors and oil-soluble materials. Encapsulant materials are usually proteins, carbohydrates, or combinations of both. The general process involves the dispersion of the core material into a polymer solution, forming an emulsion or dispersion, followed by homogenization of the liquid and atomization of the mixture into the drying chamber.89 This leads to evaporation of the solvent (water) and the formation of matrix-type microcapsules. The advantage of the spray drying process is that it can be operated on a continuous basis. The microcapsules produced are soluble in water. Proper adjustment and control of the processing conditions enables the production of a desired bulk density and particle size distribution of the powder. Advances in spray-dried microencapsulated ingredients have been made possible by new dryer and atomizer designs. New dryer designs allow drying at lower outlet temperatures (multistage dryers), longer residence time (tall-form dryer), and therefore provide milder drying conditions for minimizing deterioration of heat-sensitive microcapsules. New atomizer designs can improve the properties of microcapsules produced in spray dryers. New atomizer designs have been developed by Particle Coating Technology, USA, and Niro A/S, Denmark. Both these atomizer designs can be fitted into a spray dryer setup for the production of particles with a narrower size distribution than conventional atomizers. Many spray-dried formulations have involved the dispersion of liquid cores in a carrier material prior to spray drying.90–93 For the production of microencapsulated omega-3 oils, it is essential to design the formulation and apply the appropriate process to obtain stable liquid microcapsules before conversion of these into powder by drying. The formulation and processing steps that take place prior to spray drying are important steps in the production of stable omega-3 oil powder microcapsules.

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Prior to the spray-drying step, the omega-3 oils are essentially encapsulated oil droplets in an aqueous system (e.g., emulsion, dispersion, and coacervate systems) and the formation of a stable liquid microcapsule is essential for the manufacture of acceptable microencapsulated oil powders. Omega-3 fish oils have been encapsulated using a variety of encapsulant formulations which are subsequently spray dried into powder microcapsules. These formulations include proteins (sodium caseinate and whey protein isolate),94 proteins in combination with carbohydrates as wall materials95–97 and protein-carbohydrate conjugates,71 which improved the stability of the omega-3 oil core against oxidation. Spray drying can easily be substituted by any other drying method for production of powdered omega-3 oils such as freeze drying,98-100 fluid bed drying,64 refractive windows drying, and filtermat drying, depending on final powder properties desired and the cost that the microencapsulated product can bear in the market. Although drying is only one of the steps involved in the production of microcapsules, the control of the processing conditions during drying has a significant influence on the physical properties of the final powder microcapsule. Processing conditions such as temperature, shear, pH of the emulsion, total solids, drying rate, and order of addition of each of the components used during manufacture of omega-3 oil powders influence the microcapsule properties and stability. Depending on the formulation, the drying method (e.g., spray drying, freeze drying) used to convert emulsions and dispersions into powder can also influence the physical structure and stability of microcapsules during storage. 12.5.3.3 Extrusion Extrusion is used to produce high-density encapsulated products via mixing of molten carriers with active ingredients followed by solidification. It involves projecting a dispersion or an emulsion of the core and the encapsulant through a die or nozzle at high pressure. It has been mainly used in flavor encapsulation, but the process has also been used for omega-3 oil encapsulation. The cost of extrusion is believed to be twice that of spray drying;101 however, it still ranks second behind spray drying as the most frequently used process for encapsulating flavors.102 The use of food-grade hydrating liquid or solvent to solidify the extruded product into pellets or powder is an important factor in considering this method for food ingredient production. The formulation and the processing conditions during extrusion affect the properties of the microcapsule. Particle size was reduced with increased screw speed and melt temperature during processing and increasing the hydrophilic-lipophilic balance (HLB value) of the emulsifier in the formulation when sunflower oil was encapsulated in starch matrices via extrusion.103 There are a number of modifications to the extrusion process aimed at improving the process and the microcapsule release properties. These include the following: High pressure method: This method, developed by Ottens Flavors and Delta Food Group, USA, was designed to microencapsulate key volatile compounds, flavors, and fragrances by using high pressure during the coating

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process. This process is said to keep flavors from evaporating during processing. The core is protected in matrices of carbohydrates and fats. Release temperatures are selected such that flavors added to the food before processing are retained unaltered after cooking. The new capsule forms are said to provide controlled release, which enables multistage release or break-on-demand targeted delivery. Modified extrusion process: This method, developed by Fuisz Technologies Ltd., utilizes low-level heating to entrap a particulate dispersion within a thin matrix. The final products are said to have enhanced flavors, or sustained-release flavor delivery, such as is required in chewing gums. Extrusion has been used to encapsulate both particulate and liquid cores in a protein carrier that has been heated to create a melt and then denatured by pH adjustment and treated with an enzyme to form the microcapsule.104 Heat sensitive and readily oxidizable cores were encapsulated by extrusion using a range of proteins and carbohydrate materials to achieve discrete solid microcapsules with controlled release properties.105 Cores, including lipids, have been encapsulated in a carbohydrate or protein matrix that is gelatinized or polymerized during extrusion process. The microcapsule is claimed to release in the digestive tract.106 A recent patent describes the use of extrusion process for protection of omega-3 oils.107 12.5.3.4 Gelation and Particle Formation A process often used for encapsulation of oil and oil-soluble cores involves the use of biopolymer gels as entrapment matrices. Examples include oil and oil-soluble cores encapsulated in alginate beads by extrusion to form the particles, followed by drying,108 and oils that are sensitive to oxygen (e.g., wheat germ oil, evening primrose oil) encapsulated in alginate by emulsification.109 A number of methods have been developed for more efficient particle formation. These include the following: Centrifugal extrusion: This is one of the more established methods of microcapsule particle formation. It is generally a liquid coextrusion process utilizing nozzles consisting of concentric orifices located on the outer circumference of a rotating cylinder.110 Microject method: This has been developed by Ganan-Calvo111 for the production of a core- and shell-type microcapsule particle. Ripoll et al.112 have also developed an electrified coaxial jet method for production of micro- and nano-metric-size microcapsule particles. All these methods which result in the formation of wet gelled particles require a method for segregation and collection of the microcapsules prior to drying to produce dry free-flowing microcapsules. 12.5.3.5 Secondary Coating Application Secondary coating application has been widely used in the flavor industry to produce agglomerated powders or granules subsequently coated with another material,113 to

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improve shelf life and sensory properties of oil based flavors.114,115 This method has also been used to apply a thin polymeric layer of secondary coating to spray-dried fish oil microcapsules to increase the degree of protection and controlled release of the primary fish oil microcapsules.116 Fluidized bed coating is the most common method used for coating applications. It is accomplished by suspending or fluidizing particles of a core material in an upward stream of air and applying an atomized coating material to the fluidized particles. The coating to be applied may be a molten fat (e.g., hydrogenated fats, stearines, fatty acids, emulsifiers, waxes) or materials from a range of film-forming materials (such as proteins, starches, gums, or maltodextrin) dissolved in a solvent (e.g., water). When molten fat is used, cool air is used to harden the coating. When a soluble coating is used, hot air is used to evaporate the solvent and harden the coating. Fat-coated core ingredients are released by an increase in temperature or by physical fracture, whereas core ingredients coated with water-soluble materials are released when water is added to redissolve the coating. This process is limited to the microencapsulation of powder particles with diameters of greater than 100 microns, as particles of less than 100 microns tend to either clump to form agglomerates or get carried away in the exhaust air.117,118 This method is not suitable for primary encapsulation of an oil or fat-based product such as omega-3 fatty acids; however, it can be used to apply a secondary coating to the primary powder microcapsule (Figure 12.1). Fluidized bed coatings can be used to enhance, time, or tune the release properties of the primary omega-3 oil microcapsules. Another method used for coating application is the spinning disk system. It is a new fast coating method for controlled release applications, developed by Particle Coating Technologies.119 The coating materials used in fluid bed coating processes may be applied to this system.

12.5.4 CHEMICAL-BASED PROCESSES 12.5.4.1 Coacervation Coacervation has been defined as the separation of two liquid phases in colloidal systems. The more concentrated phase is termed the coacervate phase and the other phase is called the equilibrium solution. Complex coacervation occurs when two oppositely charged colloids interact to form a complex.120 The underlying cause of coacervation is the phase separation of one or more hydrocolloids from the initial biopolymer solution. Coacervation may be induced by a change in temperature or by the addition of a second substance such as a concentrated aqueous ionic salt solution or a nonsolvent. The coacervate coats the emulsified oil droplets by phasing polymers out of solution. The coat can then be hardened by using a chemical or enzymatic cross-linker. It is the hardening of the shell that remains the greatest challenge, especially for food applications.121,122 Complex coacervates between proteins and polysaccharides that interact to form an electrostatically bound complex are most commonly used in the food industry. Protein-polysaccharide complexes have been shown to exhibit better functional properties than that of the proteins and polysaccharide combination, in a number of

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applications.123 The complex generally has superior interfacial properties, an effect that is attributable to the simultaneous presence of the two biopolymers, and hence it functions well in an encapsulant capacity. In the food industry, coacervation has typically been used for encapsulation of flavor oils but it has potential for application to a range of other microencapsulated food ingredients.124 Microcapsules have been produced using a plant protein (e.g., soy protein) and a polyelectrolyte with an opposite charge to the protein subjected to complex coacervation and followed by hardening with cross-linking agent (e.g., dialdehydes and tannins). Microcapsules produced are suitable for a range of applications including food.125 A complex coacervate with gelatin-caboxymethylcellulose cross-linked with glutaraldehyde has been used for flavor delivery.126 Chitosan coacervated microcapsules have been shown to have good diffusion release characteristics.127 This technique may be adapted for encapsulation of omega-3 fatty acids.128 Soeda et al.129 describes a simple coacervation technique for encapsulation of flavors and oil-soluble cores including omega-3 fatty acids, using transglutaminase as the crosslinking agent for hardening the capsule wall formed by salting out. The resulting product has been shown to be suitable for a wide range of food applications. Oilsoluble cores (e.g., vitamins and polyunsaturated fatty acids) have been encapsulated by cross-linking a protein, sugar, and a water-soluble salt, by heating the mixture under specific conditions, to produce a microcapsule that is substantially insoluble in boiling water for at least 3 minutes.130 A double emulsification and an enzymatic gelation method using transglutaminase cross-linked protein encapsulant has been suggested by Cho et al.61 to improve the storage stability of fish oil and achieve controlled release. 12.5.4.2 Inclusion Complexation Inclusion complexation is the term used when cyclodextrins are used for microencapsulating core materials. Unlike other techniques, inclusion complexation takes place at a molecular level and utilizes cyclodextrin molecules as the encapsulating medium. A -cyclodextrin complex has been shown to enhance oxidation stability of onion and garlic oils as well as mask undesirable odor and flavor.131 Inclusion complexes using cyclodextrins have been used for the encapsulation of omega-3 fatty acids to enhance their stability to temperature, light, oxidation, polymerization, or doublebond migration and mask undesirable taste and odor.132–134 Yoshii et al.135 demonstrated that a complex between DHA oil and cyclodextrin prepared by a twin-screw kneader is able to produce a microencapsulated omega-3 powder with very high resistance to oxidation in fish meal paste application even without an antioxidant in the formulation. 12.5.4.3 Liposome Encapsulation Liposome encapsulation means to microencapsulate or trap core materials using liposomes. Liposomes have been used to encapsulate omega-3 fatty acids by

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dissolving the oil in phospholipid before water is added and sonicated to form the encapsulated material. The entrapped oil is protected from oxidation and the preparation can be added directly to food products as a dispersion or it can be dried into a free-flowing powder.136 Food approved proliposomes are now available, which can be used for the microencapsulation of food ingredients, and are much simpler to use.

12.5.5 EMERGING TECHNOLOGIES 12.5.5.1 Supercritical Fluid Spraying This is a relatively new technique, primarily developed for the pharmaceutical industry, in which the core material and the microencapsulating material (typically a polymer) are dispersed and dissolved in a supercritical fluid, such as carbon dioxide. The supercritical fluid is ejected from a nozzle in the form of a spray. The carbon dioxide flashes off very rapidly, leaving residual particulate material. By careful control of the operational parameters and choice of materials, successful microencapsulation has been achieved and the production of very uniform powder particles is possible.137,138 12.5.5.2 Nanoencapsulation Nanoencapsulation uses vesicular systems in which the active ingredient is confined to a cavity surrounded by a unique polymeric membrane. The preparation of nanocapsules containing a membrane-forming molecule, a coemulsifier, and a lipophilic component has been used to encapsulate a range of food ingredients, including omega-3 fatty acids. The microcapsules are 40–80 nm in size and are suitable for delivery of components into clear liquid drinks, as well as other beverages and foods.139 Hydrophobic nanospheres encapsulated in pH- or moisture-sensitive microspheres have been shown to improve shelf life of foods and beverages and prolong the sensation of flavor.140

12.6 PROPERTIES OF MICROENCAPSULATED OMEGA-3 INGREDIENTS Omega-3 powder microcapsules are small particles, typically containing 25%–60% oil surrounded by a matrix or encapsulant. The physical properties of the microencapsulated omega-3 oil ingredients and their stability need to be defined as these are important considerations for suppliers and users of these ingredients. The final powder properties need to be tailored to meet the requirements in the final food product. Most microencapsulated omega-3 ingredients are supplied as powders that are reconstituted and hence the ability of the powder to form a stable dispersion or emulsion upon reconstitution is a desirable attribute. Powder particle size, encapsulation efficiency, solubility, and dispersibility can vary depending on the properties of the emulsion or dispersion before drying, process conditions used during drying,

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and the method of drying. Particle size is an important consideration in the design of the microcapsule for targeted delivery and application.119 Storage conditions such as temperature, humidity, oxygen availability, light, and packaging can greatly influence the stability of the microcapsules, depending on the core and encapsulant properties as well as the structure and morphology of the powder microcapsule. Most of the microencapsulated omega-3 products developed have had significant limitations in terms of storage stability and shelf life. Only very few encapsulated products has been successfully commercialized without requiring low temperature storage (e.g., 4°C–10°C). Understanding lipid oxidation of microencapsulated oil ingredients is important because oxidation results in loss of nutritional value and development of undesirable flavors and odors during storage of the ingredient and when it is used in a wide range of commercial food applications. The stability of microencapsulated omega3 oil powders to oxidation can be improved by appropriate selection of encapsulant materials, processing, packaging, and storage conditions.

12.7 FOOD AS DELIVERY SYSTEMS FOR OMEGA-3 Omega-3 fatty acids and oils are key ingredients for the development of functional food products. Higgins et al.141 have highlighted the potential of fortification with omega-3 fatty acids in food as a vehicle for increasing omega-3 intake by consumers. With the use of microencapsulation technology, new and innovative ways for the protection and delivery of omega-3 fatty acids into a range of food products is made possible.9 However, there are significant challenges involved during production and storage of the final food product containing omega-3 fatty acids, because of the susceptibility of the these components to oxidation and development of undesirable flavors and odors. Developments in microencapsulation technologies and delivery strategies have resulted in increasing numbers of successful omega-3 fortified products in the marketplace. Omega-3 enriched products launched worldwide include pet foods, dietary supplements, dairy products, processed fish, meat and egg products, snacks, meals, infant formula and baby foods, bakery products, and beverages. At the forefront of omega-3 enriched foods are infant formula and baby follow-on foods, bread, dairy products, nutrition bars, and margarines. Additionally, niche products fortified with omega-3 oils such as soups, ice tea drinks, cakes, biscuits, and seafood products where there has been restoration of omega-3 fatty acids are commercially available.21

12.7.1 INFANT FORMULA Infant formula has been a major segment of focus for delivery of omega-3 fatty acids to food because of its benefits to early childhood development.29 Fortification of powder infant formula with omega-3 fatty acids is quite challenging, due to the long shelf life of the product (usually 3 years from manufacture) and the limited number of ingredients allowed in infant formula. The major challenge is to be able to add the health benefits of omega-3 oil to infant formula without affecting its odor, flavor, and shelf life during storage. The first successful commercial entry

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into this market was seen in Australia and New Zealand in 1998, using microencapsulated omega-3 oils that had been specially formulated using ingredients already in use for infant formula manufacture. Omega-3 encapsulated powder was incorporated without affecting the sensory properties, shelf life, and the ingredient listing of the original product.142 There are now a number of commercial infant formula products fortified with omega-3 fatty acids using a whole range of incorporation technologies.

12.7.2 BAKERY

AND

CEREAL PRODUCTS

Bread is a very popular food delivery vehicle for a number of functional ingredients including omega-3 fatty acids. Fortification of bread with omega-3 fatty acids requires that the omega-3 oil ingredient be protected from the liquid environment, shear, and heat during the bread making process. As fresh bread is a short-shelf-life product (< 1 week), long-term oxidative stability of the omega-3 oil is not required but it is essential that the oil be not released from the microcapsule during the bread manufacture. The incorporation of encapsulated omega-3 oils into bread has been made possible by microencapsulation without affecting sensory properties and shelf life.143 A number of other cereal-based products such as breakfast bars and muesli bars fortified with microencapsulated omega-3 oils are available worldwide. Omega-3 oils can also be delivered as a coating onto a dry food particle (soy nuggets and cereals) where a PUFA coated particle is further coated with a sugar solution and dried to a moisture content of the same level as the original dry food particle.144 Encapsulated sardine oil in egg-white protein has been incorporated into cookies without affecting the taste and quality of the final product.45

12.7.3 DAIRY PRODUCTS There are opportunities for microencapsulated ingredients for fortification of dairy foods.145 Dairy products have been targeted by a number of food companies as a delivery vehicle for omega-3 fatty acids. Delivery into dairy products has been attempted in two ways; by incorporation of encapsulated omega-3 oil into the cow diet146 or by fortification of the dairy product during its manufacture. Omega-3 fish oil (refined, deodorized, and hydrogenated) was incorporated with an all-vegetable fat blend or a dairy blend mixture of butter and vegetable oils with a view to the manufacture a longer-shelf-life, low-calorie spread (40% fat).147 The use of yogurt as a delivery vehicle for omega-3 oils is less challenging. The sensory properties of omega-3 oil fortified yogurt were found to be superior with the use of microencapsulated tuna oil compared to tuna oil in the free form.148 A yogurt drink (Omega-3 laban drink) has been successfully commercialized in the Middle East under the Live Well brand. Cheese and cheese-based products have also been successfully fortified with omega-3 fatty acids without affecting their flavor and shelf life. The fortification of fresh milk with omega-3 oils is difficult because of the delicate flavor of milk. Milk products fortified with microencapsulated nutrients including omega-3 fatty acids have been developed, where the nutrients, which

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comprise both water-soluble and oil-soluble components, were dispersed in a hydrogenated oil containing lecithin and encapsulated in gelatin and sorbitol forming the outer layer.149

12.7.4 MEAT

AND

FISH PRODUCTS

Microencapsulated omega-3 fatty acids have been successfully incorporated into meat and fish products in Australia. Examples of these are the Hans low-fat range of meat products such as Strassburg, roast chicken, and Vienna sausage, and Birds Eye crumbed fish fillets under the “Smart Choice – Omega 3” brand with a range of different flavors.

12.7.5 DRINKS

AND

BEVERAGES

New functional orange juice has been successfully commercialized in Australia by Golden Circle and in the United Kingdom by a company under the Supa Jus brand.

12.7.6 CONFECTIONERY Even with sweet and strong-flavored confectionery products the addition of omega3 fatty acids can be difficult. New orange- and mint-flavored pastille-like products have been developed as a vehicle for omega-3 fatty acids delivery to increase intake of omega-3 fatty acids by consumers.150 Their most promising formulation had EPA and DHA concentration of only 0.8%–1.0% even with strong flavorings added to mask the fishy flavor in the final product.

12.8 CONSIDERATIONS FOR SUCCESSFUL DELIVERY OF OMEGA-3 OILS INTO COMMERCIAL FOOD PRODUCTS Ideally it is desirable to design omega-3 oil microcapsules that are stable in many different applications, but in reality this is not a practical option, as the microencapsulated ingredient has to be matched to the target food application. Food ingredient manufacturers will normally have microcapsule formulations designed for different food category applications; for example, infant formula, bakery, dairy, and liquid beverage applications, each of which has different requirements. There are different challenges in the final food formulation with regards to food standard regulations, shelf life, and release properties of the omega-3 oil microcapsules. Some of the key issues for successful delivery of omega-3 fatty acids into commercial food products include the following:

12.8.1 COMPLIANCE

WITH

REGULATORY STANDARDS

Prior to deciding on the microcapsule formulation, it is mandatory to check the regulatory standards to ensure that ingredients chosen as encapsulants are approved for food use in each country where the product will be marketed or sold. This step will minimize the time for regulatory approval when the final product is launched

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into the market, and also avoids possible reformulation and testing as in the case where a nonfood-grade material has been used during the initial trials.

12.8.2 DEFINITION OF FINAL PRODUCT APPLICATION THE CORRECT DELIVERY FORMAT

TO

ENSURE

For dry blending with other food powders, it is necessary for the microencapsulated powder to be matched in terms of water activity, bulk density, and particle size so as to achieve a homogeneous product and avoid possible physical separation of the different powders during transport and storage. For liquid product applications, the rehydration and redispersion behavior of the powder is important if a powder format is chosen. Alternatively, a liquid format for microencapsulated omega-3 ingredients could be used.

12.8.3 UNDERSTANDING OF THE PROTECTION REQUIREMENTS DURING PROCESSING

AND

RELEASE

Knowledge and understanding of the processing steps and unit operations involved in the manufacture of the final food product is important to be able to design the microcapsule properties in such a way that that the core will be protected during the process and released at the desired time.

12.8.4 UNDERSTANDING OF HOW AND WHERE MICROENCAPSULATED INGREDIENT

TO

ADD

THE

Familiarity with the food manufacturing plant setup and layout is also required in order to identify the appropriate stage of addition of the microcapsule (e.g., how and where the microcapsule can be added) without the need for any process modification.

12.8.5 UNDERSTANDING POSSIBLE INTERACTIONS INGREDIENTS

WITH

OTHER

When the new microencapsulated omega-3 food ingredient is added to a food product, the final food product properties and its shelf life may be affected due to possible ingredient interactions. Sometimes these interactions may be minimized by minor modification to the process or the formulation. However, sometimes even small changes are not possible due to limitations in the plant layout/setup or further modifications could add significant capital investment. Therefore formulating the new omega-3 food ingredient has to be approached in such a way that possible negative ingredient interactions are minimized.

12.8.6 UNDERSTANDING THE SHELF-LIFE, SENSORY, AND PHYSICAL PROPERTIES OF THE FINAL PRODUCT Addition of omega-3 food ingredients to short-shelf-life products with very strong flavor is much less of a problem than addition to food products with delicate flavors

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and longer shelf life. Therefore the requirements of the final food product have to be considered when designing the microcapsule formulation to suit the final application.

12.8.7 WORKING

IN

PARTNERSHIP

THROUGH

COLLABORATION

There is great advantage when the food ingredient supplier/company who develops the microencapsulation technology and the food company manufacturing the final food product can work in partnership during the development of the new food product with added omega-3 oils.143,151 The food ingredient supplier or the company who develops the microencapsulated omega-3 ingredient will understand the performance and limitations of the microcapsule when added to the food product. In addition, the food manufacturer will have a better understanding of the market and consumer requirements of the final food product. By choosing an appropriate encapsulant and microencapsulation process, controlled, sustained, or delayed release of omega-3 oil in the final product application or for targeted release at specific sites in the gastrointestinal tract after consumption may be achieved. With carefully fine-tuned controlled release properties, microencapsulation is no longer just an added-value technique, but the source of totally new ingredients with desirable properties.124 The food industry expects increasingly complex properties from food ingredients and such complex properties can often only be provided by microencapsulation. With the use of appropriate microencapsulation technologies, the food industry has the ability to develop functional omega-3 ingredients and products and improve the nutritional content of food without affecting the taste, aroma, and texture of food, and without reducing their bioavailability and functionality.65

REFERENCES 1. Klont, R., Healthy ingredients driving innovation, World Food Ingred., March/April, 34, 1999. 2. Augustin, M.A., Functional foods: an adventure in food formulation, Food Aust., 53, 428, 3. Michelsen, B., Madsen, S.L., and Gotfredsen, P., Polyunsaturated fatty acids, in Guide to Functional Foods, Young, J., Ed., Leatherhead Publishing, Surrey, England, 2001, p. 141. 4. Trautwein, E.A., N-3 Fatty acids: physiological and technical aspects for their use in food, Eur. J. Lipid Sci. Technol., 103, 45, 2001. 5. Ennen, S. The rising tide of omega-3, Food Process, Wellness Foods, Sept/Oct, 19, 2002. 6. Augustin, M.A. and Sanguansri, L., Polyunsaturated fatty acids: delivery, innovation and incorporation into foods, Food Aust., 55, 294, 2003. 7. Pszczola, D.E., Encapsulated ingredients: providing the right fit, Food Technol., 52, 12, 70, 1998. 8. Muggli, R., Health benefits of long-chain polyunsaturated fatty acids, Food Technol. Int., 22, 24, 2001.

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77. Miyashita, K. et al., Oxidative stability of triglycerides from orbital fat of tuna and soybean oil in an emulsion, Fish. Sci., 61, 273, 1995. 78. Donnelly, J. L, Decker, E.A., and McClements, D.J., Iron-catalyzed oxidation of menhaden oil as affected by emulsifiers, J. Food Sci., 63, 997, 1998. 79. Pin Der Duh, Wen Jye Yen, and Gow Chin Yen, Oxidative stability of polyunsaturated fatty acids and soybean oil in an aqueous solution with emulsifiers, J. Am. Oil Chem. Soc., 76, 201, 1999. 80. Nuchi, C.D., McClements, D.J., and Decker, E.A., Impact of Tween 20 hydroperoxides and iron on the oxidation of methyl linoleate and salmon oil dispersions, J. Agric. Food Chem., 49, 4912, 2001. 81. Formuso, L.B., Corredig, M., and Akoh, C.C., Effect of emulsifier on oxidation properties of fish oil-based structured lipid emulsions, J. Agric. Food Chem., 50, 2957, 2002 82. Hu, M., McClements, D.J., and Decker, E.A., Lipid oxidation in corn oil-in-water emulsions stabilized by casein, whey protein isolate and soy protein isolate, J. Agric. Food Chem., 51, 1696, 2003. 83. Silvestre, M.P.C. et al., Ability of surfactant headgroup size to alter lipid and antioxidant oxidation in oil-in-water emulsions, J. Agric. Food Chem., 48, 2057, 2000. 84. Chaiyasit, W. et al., Ability of surfactant hydrophobic tail group size to alter lipid oxidation in oil-in-water emulsions, J. Agric. Food Chem., 48, 3077, 2000. 85. Frankel, E.N. et al., Evaluation of antioxidant activity of rosemary extracts, carnosol and carnosic acid in bulk vegetable oils and fish oil and their emulsions, J. Sci. Food Agric., 72, 201, 1996. 86. Hu, M., McClements, D.J., and Decker, E.A., Emulsion droplets engineered to improve the oxidative stability of n-3 fatty acids in functional foods, Lipid Technol., 16, 79, 2004. 87. Merchant, Z.M. and Nicholson, V.J., Microcapsule flavour delivery system, U.S. Patent 6,136,364, 2000. 88. Benichou, A., Aserin, A., and Garti, N., Double emulsions stabilized with hybrids of natural polymers for entrapment and slow release of active matters, Adv. Colloid Interface Sci., 108–109, 29, 2004. 89. Re, M.I., Microencapsulation by spray drying, Drying Technol., 16, 1195, 1998. 90. Witteveen, F. et al., Stable spray-dried composition in a carbohydrate substrate and process for obtaining said composition, Int. Patent Application WO01/35764-A1, 2001. 91. Subramaniam, A. et al., Spray-dried compositions and method for their preparation, U.S. Patent 6,723,359-B2, 2004. 92. Minemoto, Y. et al., Oxidation of linoleic acid encapsulated with soluble soybean polysaccharide by spray drying, Biosci. Biotechnol. Biochem., 66, 1829, 2002. 93. Partanen, R. et al., Encapsulation of sea buckthorn kernel oil in modified starches, J. Am. Oil Chem. Soc., 79, 219, 2002. 94. Fair-Flow Europe, Fighting heart disease doesn’t have to be a fishy business, FairFlow Reports, FFE 411/01/CG9, p. 1, 2001. 95. Kagami, Y. et al., Oxidative stability, structure, and physical characteristics of microcapsules formed by spray drying of fish oil with protein and dextrin wall materials, J. Food Sci., 68, 2248, 2003. 96. Keogh, M.K. et al., Stability to oxidation of spray dried fish oil powder microencapsulated using milk ingredients, J. Food Sci., 66, 217, 2001. 97. Barrier, P. and Rousseau, J.Y., Powder comprising microcapsules based on fish oils rich in polyunsaturated fatty acids, French Patent Application FR2758055-A1, 1998

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98. Heinzelmann, K. et al., Microencapsulation of fish oil by freeze drying techniques and influence of process parameters on oxidative stability during storage, Eur. Food Res. Technol., 211, 234, 2000. 99. Heinzelmann, K. et al., Protection of fish oil from oxidation by microencapsulation using freeze drying techniques, Eur. J. Lipid Sci. Technol., 102, 114, 2000. 100. Grattard, N. et al., Influence of physical state and molecular mobility of freeze dried maltodextrin matrices on the oxidation rate of encapsulated lipids, J. Food Sci., 67, 3002, 2002. 101. Tuley, L., Breaking free: update on controlled flavour release, Int. Food Ingred., 3, 14, 1996. 102. Reineccius, G.A., Carbohydrates for flavor encapsulation, Food Technol., 45, 3, 144, 1991. 103. Yilmaz, G. et al., Encapsulation of sunflower oil in starch matrices via extrusion: effect of the interfacial properties and processing conditions on the formation of dispersed phase morphologies, Carbohydr. Polym., 45, 403, 2001. 104. Janda, J., Bernacchi, D., and Frieders, S., Microencapsulation process, U.S. Patent 5,418,010, 1995. 105. Van Lengerich, B.H., Embedding and encapsulation of controlled release particles and encapsulated products, European Patent Application EP1342548-A1, 2003. 106. Katzen, S. Preserved nutrients and products, U.S. Patent 3,962,416, 1976. 107. Saleeb, F.Z. and Arola, V.K., Method of preparing glass stabilized material, U.S. Patent 5,972,395, 1999. 108. Truter, P.A., Xulu, P.M., and Igwe, N., Microparticles and process for making microparticles, Int. Patent Application WO00/33673, 2000. 109. Chan, L.W., Lim, L.T. and Heng, P.W.S. Microencapsulation of oils using sodium alginate, J. Microencapsul., 17, 757, 2000. 110. Schlameus, W., Centrifugal extrusion encapsulation, in Encapsulation and Controlled Release of Food Ingredients, Risch, S.J. and Reineccius, G.A., Eds., ACS, Washington, 96, 1995. 111. Ganan-Calvo, A.M., Enhanced food products, U.S. Patent 6,589,579-B2, 2003. 112. Ripoll, A.B. et al., Production of capsules and particles for improvement of food products, U.S. Patent Application 2004/0161498-A1, 2004. 113. Schleifenbaum, B. et al., Encapsulated flavour and/or fragrance preparations, U.S. Patent Application 2004/0081735-A1, 2004. 114. So, R.S., Encapsulated liquid product, U.S. Patent 6,174,554-B1, 2001. 115. Pacifico, C.J., Wu, W.-H., and Fraley, M., Sensitive substance encapsulation, U.S. Patent 6,251,478-B1, 2001. 116. Souza-Neves-Cardoso, F. de., Grosso, C., and Almeida-Vitali, A. de, Starch microcapsules obtained by spray drying and coated using fluid bed, Brazilian J. Food Technol., 4, 131, 2001. 117. Jackson, L.S. and Lee, K., Microencapsulation and the food industry, Lebensm. Wiss. Technol., 24, 289, 1991. 118. Balassa, L.L. and Fanger, G.O., Microencapsulation in the food industry, Crit. Rev. Food Technol., 2, 245, 1971. 119. Labell, F., New, precise methods for encapsulation, Prepared Foods, 171, 1, 55, 2002. 120. IUPAC, Compendium of Chemical Terminology, 2nd ed., 1997, 1972, 31, 611. 121. Courts, A., Gelatin emulsions in the formation of microcapsules, Chem. Ind., 19, 943, 1973. 122. Versic, R.J., Coacervation for flavour encapsulation, in Flavour Encapsulation, Risch, S.J and Reineccius, G.A., Eds., ACS, Washington, 370, 1988.

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Packaging Technologies of Functional Foods Louise Deschênes Agriculture and Agri-Food Canada

CONTENTS 13.1 Introduction ..................................................................................................329 13.2 Packaging Practices versus the Requirements of Functional Foods...........330 13.2.1 Fruits and Vegetables .......................................................................330 13.2.1.1 Fresh Plant Products .........................................................330 13.2.1.2 Processed Plant Products..................................................331 13.2.2 Probiotics..........................................................................................331 13.2.2.1 Yogurts ..............................................................................331 13.2.2.2 Dried Cultures...................................................................332 13.2.3 Intermediate Moisture Products.......................................................332 13.2.4 Oils and Fats ....................................................................................332 13.3 Choice of Packaging Materials....................................................................333 13.4 Active Packaging..........................................................................................334 13.5 Summary ......................................................................................................335 References..............................................................................................................335

13.1 INTRODUCTION The emerging functional foods market presents a considerable challenge to all manufacturers to keep abreast of the use of adequate packaging materials for their products. The interest in functional foods is intrinsically linked to the potential health benefits of their bioactive components. Preserving these bioactive components is therefore a crucial factor to providing the anticipated health benefits of functional foods. However, recurring failure in maintaining the quality of functional foods has been reported.1−3 Functional foods include a wide variety of products such as prebiotics and probiotics, dried foods (fibers, probiotics, tea, herbs, etc.), fermented products (yogurts, kefirs, vegetables), and fresh fruits and vegetables. Their bioactive

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components (minerals, vitamins, fibers, peptides, proteins, n-3 polyunsaturated fats, antioxidants, enzymes, and symbiotics) range from very simple molecules to complex living organisms such as bacteria. The chemical nature of the bioactive components and the way functional foods are offered to consumers (in the form of dried extracts, fresh foods, or processed foods) dramatically influence the shelf life of these functional foods and their packaging requirements. Unfortunately, only a limited number of documents addressing this critical issue are available. Furthermore, the data are fragmented and dispersed within the literature on studies from other fields of research to which functional foods were incidental. A comprehensive review of the current literature would be useful to summarize the existing knowledge on what is required to preserve functional foods and to develop appropriate packaging guidelines.

13.2 PACKAGING PRACTICES VERSUS THE REQUIREMENTS OF FUNCTIONAL FOODS The effect of packaging on food composition during storage depends on the technology used and the storage conditions. Generally, functional foods packaging and preservation practices are based on the conditions applied to products of similar origin and format. This approach can give satisfactory results for several products but should not be taken as a universal rule regarding all functional foods. In some cases, the usual technologies and packaging materials could be detrimental to the shelf life of certain functional ingredients. In other cases, minor adaptations of the packaging technologies could likely improve the shelf life of functional foods greatly. Some examples are given below.

13.2.1 FRUITS

AND

VEGETABLES

The effects of processing and preservation technologies on minerals and vitamins have been extensively documented for decades.4,5 In addition to vitamins and minerals, several other important functional phytochemicals such as flavanoids, prostaglandins, and sulfur compounds are present in fresh and processed plant products. Unfortunately, their stability under processing and storage conditions is not well known.6 13.2.1.1 Fresh Plant Products Minimal processing is often a way to preserve the bioactive components of functional foods. Packaging, therefore, is a significant factor in extending the shelf life of minimally processed food products. However, the effect of minimal processing and of packaging technologies on the nutritional quality of functional foods is not well established.7 Modified atmosphere packaging (MAP) is a process well known for its capacity to enhance the shelf life of fresh fruits and vegetables.8 The improvement in shelf life obtained using MAP is usually associated with the preservation of normal appearance and firmness. The MAP process slows down or matches the metabolism of packaged fresh products and microflora to avoid fermentation, thus minimizing

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microbial growth. In fresh fruits and vegetables, the level of antioxidants (vitamins, phenolics, and carotenoids) depends on storage conditions. Gil et al. observed constant levels of flavanoids over a seven-day period for spinach maintained at 10°C.9 However, the total antioxidant capacity of the product decreased over the same storage period. The spinach subjected to MAP was more affected than the unpackaged spinach. These results indicate that spinach packaged in the conventional manner — but with a shorter shelf life — could provide more functional benefits than MAP spinach. However, the lack of data in the literature prevents the extension of such a conclusion to the entire class of fresh fruits and vegetables. 13.2.1.2 Processed Plant Products Plant products contain phenolic compounds, which are closely associated with the nutritional quality of foods. Their properties are based on their antioxidant capability.10 As with other antioxidant compounds, these molecules easily oxidize in the presence of oxygen. Phenolic compounds are present in cereals, legumes, oilseeds, fruits, and beverages of plant origin (tea, cocoa, beer, wine, fruit juices, etc.). Processed plant products with medium to high water activity will be the most susceptible to browning and phenolic degradation. Conditioning and packaging these products require controlling the oxygen level to which they are exposed.

13.2.2 PROBIOTICS The viability of microorganisms may be affected by several factors, including packaging. The viability of both Lactobacillus and Bifidobacterium species may diminish drastically during storage, resulting in low numbers of living cells at the time of consumption of the food product.11 Oxygen is often reported to be a concern for the survival of these cell cultures. Lactobacillus acidophilus is microaerophilic, and surface growth on solid media is generally enhanced by anaerobiosis or reduced oxygen with 5%–10% CO2. The other popular probiotics, Bifidobacteria, are usually considered strictly anaerobic but some strains of Bifidobacterium lactis have demonstrated a relative tolerance to oxygen.11 13.2.2.1 Yogurts Yogurts, whether they contain probiotics or not, are usually packaged in regular high-impact polystyrene (HIPS) cups. This type of packaging is not ideal for probiotics because of their high oxygen transmission rate. Dave and Shah have demonstrated that for living cells contained in yogurts, oxygen concentration in the product and container significantly influences the survival of probiotics.12,13 Lactobacillus and Bifidobacterium spp. are particularly sensitive to oxygen toxicity.14 Strains of these bacteria grown in defined oxygen concentrations (0%, 5%, 10%, 15%, and 21% oxygen) showed a drastic decrease in lactate production with increased oxygen uptake.15 More recently, the Champagne research group study on probiotics in nonfermented milks indicated that the oxygen level in these types of products must also be controlled to ensure the survival of probiotics.16 Comparative packaging tests showed that the use of glass hermetic containers can significantly

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increase the survival rate of probiotics in yogurts.12 However, given the cost of this form of packaging, it is not a viable commercial alternative. The use of active packaging, conversely, could be an option worthy of consideration. Polystyrenebased cups coated with a Nupak (gas barrier layer combined with an oxygen scavenger (Zero2™)) significantly improved the survival of Lactobacillus and Bifidobacterium spp. in yogurts.17,18 13.2.2.2 Dried Cultures Dried probiotic cultures are also affected by storage conditions such as temperature, moisture, light, and air.19 Alur and Grecz20 showed that problems with freeze-dried bacteria could be attributed to surface phenomena. Other authors have also demonstrated that oxygen and moisture affect survival kinetics.21,22 Encapsulation could be used as a primary packaging system able to contribute to the preservation of dried probiotic cultures.23,24 As a general guideline, oxygen and water vapor should be carefully controlled to ensure the optimal packaging conditions of dried cultures.

13.2.3 INTERMEDIATE MOISTURE PRODUCTS Numerous food products can be classified as intermediate moisture foods (IMFs). Typical examples of IMFs are cakes, syrups, toaster pastries, fruit rolls, and softbars. The intermediate moisture levels in foods are generally related to a water content of 10%–40% and a water activity of 0.60–0.9.25 Intermediate moisture nutraceutical foods, such as semisoft food bars, belong to this category. Despite relative stability at ambient temperatures, the rates of some deterioration processes are very high at intermediate moisture levels.26 Browning reactions are particularly significant in IMFs27 and lipid oxidation is also a problem, provoking rancidity and unacceptable sensory defects.28 The rate of lipid oxidation increases as the water activity (aw) decreases below the monolayer moisture content. IMFs would greatly benefit from high barrier materials integrating oxygen scavengers.

13.2.4 OILS

AND

FATS

Extracted natural oils and fats contain molecules with unsaturated molecules and natural antioxidants such as tocopherols. These molecules are easily oxidized when exposed to oxygen and light. Consequently, light and gas barrier packaging materials should be mandatory to preserve the functionality of these products. Conjugated linoleic acid (CLA) is a natural fatty acid recognized for its anticancer properties.29,30 When CLA is present in its original matrix, it remains quite well preserved within the usual shelf life of fresh products. The CLA can be found in dairy products and meat. Lavillonnière et al. showed that the CLA level in dairy products (e.g., yogurt, cheese, and ice cream) stored and packaged in usual conditions does not vary significantly over a six-week period.31

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13.3 CHOICE OF PACKAGING MATERIALS The degradation mechanisms of the bioactive components in packaged functional foods are mainly related to ultraviolet (UV) sensitivity, water activity, microbial load, and oxidation processes. Given these considerations, packaging technologies should be selected based on the likelihood that they will contribute to controlling light, humidity level, temperature, and atmosphere conditions. Table 13.1 summarizes some factors to be taken into consideration in the choice of appropriate packaging conditions for various types of functional foods. This list is not exhaustive and specific tests are recommended to ensure the preservation of minimal levels of bioactive components. Common packaging technologies could be a simple solution to extending the shelf life of functional foods. Such technologies include gas flushing, barrier, controlled atmosphere, modified atmosphere, and vacuum packaging. Further, new advanced packaging technologies could provide additional tools to increase the shelf life of bioactive functional food components.

TABLE 13.1 Functional Foods and Packaging Requirements Deterioration Factor Oxygen

Products Fermented milks

Oils and fats Processed fruits and vegetables

Moisture

Fresh fruits and vegetables Dried probiotics

Light

Oils and fats Fermented milks Protein-based products

Microorganisms

Broccoli sprouts

Temperature

Dried herbs

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Deterioration Effect

Packaging Requirements

Vitamin oxidation Decrease in survival probiotic bacteria Lipid oxidation Rancidity General nutritional quality loss Antioxidant destruction Vitamin destruction

Controlled oxygen barrier

Decrease in the active load Oxidation, rancidity Oxidation of vitamins Modification of proteins and amino acids Flavor problems Pathogen development

Moisture barrier

High barrier High barrier

Controlled atmosphere

Light barrier

Hermeticity moisture control High barrier

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13.4 ACTIVE PACKAGING Active packaging is a class of technologies in which dynamic physical and chemical interactions between the content and the containers induce modifications of the internal conditions resulting in shelf-life improvement. Active packaging contributes to the preservation of functional foods by integrating sophisticated systems of oxygen scavenging, enzyme and antioxidant release, moisture control, and antimicrobial surface properties. Table 13.2 presents a list of various active packaging systems. A list of commercially available active packaging systems for food applications were published by Brody et al.32 Antioxidant release is one of the first active packaging systems to be implemented in the industry. Traditionally, hindered phenolic compounds are added to the food contact layer and act as oxygen scavengers and chelators. The most popular additives are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butyl hydroquinone (TBQH).33 The packaging material of cereals, nuts, and oils often contains such additives to increase their storage stability.34,35 For functional foods, complex chains of chemical interactions involving vitamins, polyphenols, and enzymes are critical.36 The release of antioxidants naturally involved in the biochemical processes would present a better preservation strategy than the use of BHA and BHT. New active packaging systems are using natural molecules as antioxidants. Deschênes et al.37 observed that the replacement of Irgafos 168 with vitamin E in the contact packaging layer increased the preservation of vitamin C and polyphenols in sauerkraut and fermented vegetable juices by 50%. There was a synergistic effect between the vitamin in the packaging and vitamins C and E and polyphenols in the product, a phenomenon that was not observed when the synthetic antioxidant was used. The advantages of using natural compounds in packaging materials are particularly beneficial to the preservation of functional foods. TABLE 13.2 Active Packaging Systems Parameter to Control Moisture Gas atmosphere

Mechanism of Action Physical dehydrators Oxygen scavengers Ethylene absorbers

Odors absorbers CO2 absorbers CO2 generators Microbial load

Bacteriocins, proteins, vegetable extracts, metals, organic acids, chelators, fungicides, antibiotics

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Packaging Active Constituent Anhydrous salts, starch, clay Ascorbates, ferrous compounds, oxidizible polymers, enzymes Zeolites, cristobalites, clays, alumino-silicates, active carbon, permanganates Active carbon, zeolites, catalyzers Calcium hydroxide Ascorbic acid/sodium bicarbonate, iron carbonate Nisin, lactoferrin, silver, acetic acid, imazalil, benomyl, EDTA

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With the use of active packaging, new materials can be tailored to ensure the efficacy and safety of functional foods through the incorporation of agents to control the internal environment.38 In addition, active packaging can enhance the stability, bioavailability, and sensory quality of these products.39

13.5 SUMMARY Guaranteeing significant levels of beneficial bioactive components in functional foods is a great challenge to manufacturers. The shelf life of functional foods is highly dependent on the storage and packaging conditions that are applied throughout the entire distribution chain. These conditions must be precisely adapted to the specific needs of each and every functional food. Unfortunately, research on the effect of packaging conditions on functional foods is lacking. To optimize the preservation of these sensitive products, there is an urgent need for appropriate data. Further, the establishment of specific labeling regulations and standards to functional foods will, it is hoped, stimulate further research in this area. These studies should be undertaken to supply data supporting the determination of shelf life and expiration dates ensuring health benefit claims. Active packaging systems constitute a promising approach to improving the preservation of functional foods.

REFERENCES 1. Labuza, T., The search for shelf life: an update on continued efforts in understanding practical strategies for determining and testing the shelf life of food products, Food Test. Anal., 6(2), 26–36, 2000. 2. Schmidl, M. and T. Labuza, Essentials of Functional Foods, Aspen Publishers, Gaithersburg, MD, 2000. 3. Labuza, T., Functional Foods, retrieved from http://courses.che.umn.edu/01fscn43461s/4346PDFLecturefiles/ShelfLife-Color3.pdf, 2005. 4. Schroeder, H.A., Losses of vitamins and trace minerals resulting from processing and preservation of foods, Am. J. Clin. Nutr., 24(5), 562–573, 1971; retrieved from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&term=22 Schroeder+HA%22%5BAuthor%5D”\o 5. Jongen, W., Ed., Fruit and Vegetable Processing: Improving Quality, Woodhead Publishing, Cambridge, UK, 2002. 6. Delaquis, P. and G. Mazza, Functional vegetable products, in Functional Foods: Biochemical and Processing Aspects, Mazza, G., Ed., Technomic, Lancaster, PA, 1998. 7. Anonymous, retrieved from http://www.agro.nl/innovatienetwerk/doc/Functional_ Foods2.pdf, 2005. 8. Ooraikul, B. and Stiles, M.E., Eds., Modified Atmosphere Packaging of Foods, Ellis Horwood, London, 1991. 9. Gil, M.I., F. Ferreres, and F. A. Tomás-Barberán, Effect of post-harvest storage and processing on the antioxidant constituents (flavanoids and vitamin C) of fresh-cut spinach, J. Agri. Food Chem., 46(6), 2213–2217, 1999. 10. Shahidi, F. and M. Naczk, Food Phenolics: Sources, Chemistry, Effects, Applications, Technomic, Lancaster, PA, 1995.

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11. De Vuyst, L., Technology aspects related to the application of functional starter cultures: application of functional starter cultures, Food Technol. Biotechnol., 3(2), 105–112, 2000. 12. Dave, R.I. and N.P. Shah, Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures, Int. Dairy J., 7(1), 31–41, 1997. 13. Dave, R.I. and N.P. Shah, Effectiveness of ascorbic acid as an oxygen scavenger in improving viability of probiotic bacteria in yoghurts made from commercial starter cultures, Int. Dairy J., 7(6/7), 435–443, 1997. 14. Talwalkar, A. and K.A. Kailasapathy, A review of oxygen toxicity in probiotic yogurts: influence on the survival of probiotic bacteria and protective techniques, comprehensive, Rev. Food Sci. Food Safety, 3, 117–124, 2004. 15. Talwalkar, A. and K. Kailasapathy, Metabolic and biochemical responses of probiotic bacteria to oxygen, J. Dairy Sci., 86(8), 2537–2546, 2003. 16. Bolduc, M.P., C. Champagne, P. Fustier, Y. Raymond, and J. C. Vuillemard, Sensitivity of Bifidobacteria to Oxygen and Redox State of Non-Fermented Pasteurized Milk, 4th NIZO Dairy Conference, 15–17 June, Papendal, The Netherlands, 2005. 17. Miller, C.W., M.H. Nguyen, M. Rooney and K. Kailasapathy, The influence of packaging materials on the dissolved oxygen content in probiotic yoghurt, Packag. Technol. Sci., 15, 133–138, 2002. 18. Miller, C.W., M.H. Nguyen, M. Rooney, and K. Kailasapathy, The control of dissolved oxygen content in probiotic yoghurt by alternative packaging materials, Packag. Technol. Sci., 16, 61–67, 2003. 19. Percival, M., Choosing a probiotic supplement, Clin. Nutr. Insights, 6(1), 1–4, 1997. 20. Alur, M.D. and N. Grecz, Mechanism of injury of escherichia coli by freezing and thawing, Biochem. Biophys. Res. Commun., 62(2), 308–312, 1975. ˇ T.F., M. Özilgen, and U. Bakir, Survival kinetics of lactic acid starter 21. Bozoglu, cultures during and after freeze drying, Enzyme Microb. Technol., 9, 531–537, 1987. 22. Greiff, D., Protein structure and freeze-drying: the effects of residual moisture and gas, Cryobiology, 8, 145–152, 1971. 23. Champagne, C., The potential of microencapsulation for the protection of probiotics destined to foods, in Pre- and Probiotics – Ingredients for Nutraceuticals and Functional Food, presented at Binzen, 11–13 May, 2004; presentation available at http://www.ncapsolutions.com/IMG/pdf/Probiotics.pdf. 24. Chan, E.S. and Z. Zhang, Encapsulation of probiotic bacteria lactobacillus acidophilus by direct compression, Food Bioprod. Process., 80(C2), 78–82, 2002. 25. Erickson, L.E. Recent developments in intermediate moisture foods, J. Food Prot., 45, 484–491, 1982. 26. Schmidl, M. and T. Labuza, Essentials of Functional Foods, Aspen Publishers, Gaithersburg, MD, 2000. 27. Fontana, A.J., Water Activity: Why It Is Important for Food Safety, in Proceedings of the First NSF International Conference on Food Safety, November 16–18, 1998, Albuquerque, NM, 177–185. 28. Labuza, T.O., Oxidative changes in foods at low and intermediate moisture levels, in Food Science and Technology, a series of monographs on water relations of foods, Duckworth, R.B., Ed., Academic Press, New York, 1975. 29. Corpet, D.E., Aliments fonctionnels et réduction du risque de développer un cancer, in Aliments fonctionnels, Roberfroid, M., Ed., Tec&Doc, Paris, chap. 5, 2002.

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30. Thompson, H.J., Z. Zhu, S. Banni, K. Darcy, T. Loftus, and C. Ip, Morphological and biochemical status of the mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk, Cancer Res., 57, 5067–5072, 1998. 31. Lavillonnière, F., J.C. Martin, P. Bougnoux, and J.L. Sébédio, Analysis of conjugated linoleic acid isomers and content in French cheeses, J. Am. Oil Chem. Soc., 75(3), 343–353, 1998. 32. Brody, A.L., E.R. Strupinsky, and L.R. Kline, Active Packaging for Food Applications, Technomic, Lancaster, PA, 2001. 33. van Aardt, M., S.E. Duncan, S. O’Keefe, J.E. Marcy, T.E. Long, and S. Nielsen, Controlled Release of Antioxidants from Polymeric Films to Control Lipid Oxidation in Milk, in International Animal Agriculture and Food Science Conference, Indianapolis, 2001; http://www.fass.org/fass01/pdfs/Duncan.pdf. 34. Miltz, J., P. Hoojjat, J.K. Han, J.R. Giacin, B.R. Harte, and I.J. Gray, Loss of antioxidants from high-density polyethylene: its effect on oatmeal cereal oxidation, in Food and Packaging Interactions, Hotchkiss, J.H., Ed., American Chemical Society, Washington, 83–93, 1988. 35. Sharma, G.K., C.V. Madhura, and S.S. Arya, Interaction of plastic films with foods, II, effect of polyethylene films on the stability of vegetable oils, J. Food Sci. Technol., 27, 328–331, 1990. 36. Kikugawa, K., A. Kunugi, and T. Kurechi, Chemistry and implications of degradation of phenolic antioxidants, in Food Antioxidants, Hudson, B.J.F., Ed., Elsevier, New York, 1990, pp. 85–94. 37. Deschênes, L., F. St-Germain, and R. Bélanger, Conservation de Légumes LactoFermentés, internal report, Agriculture and Agri-Food Canada, 1998. 38. Grower, J.L., K. Cooksey, and K.J. Getty, Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes, J. Food Prot., 67, 475–479, 2004. 39. Lutter, C.K. and K.G. Dewey, Proposed nutrient composition for fortified complementary foods, J. Nutr. 133, 3011S–3020S, 2003.

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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite Aditya Kulkarni and Hideo Etoh Shizuoka University, Japan

CONTENTS 14.1 Introduction ..................................................................................................341 14.2 Peroxynitrite Formation In Vivo ..................................................................342 14.3 Carotenoids...................................................................................................343 14.3.1 Carotenoids as Antioxidants ............................................................344 14.3.2 Reactions of Peroxynitrite with Some Important Carotenoids .......345 14.3.2.1 Reaction of Peroxynitrite with Zeaxanthin ......................345 14.3.2.2 Reaction of Peroxynitrite with Astaxanthin (Astaxanthin Structure-Activity Relationship).................346 14.3.2.3 Reaction of Retinol and Retinyl Acetate with Peroxynitrite......................................................................347 14.4 Conclusions ..................................................................................................349 14.5 Summary ......................................................................................................350 References..............................................................................................................350

14.1 INTRODUCTION Peroxynitrite is a potent and versatile oxidant that can attack many types of biological molecules and produce a rich variety of products. Because of its potential to injure or destroy biological molecules or molecular systems, leading to the pathogenic process of human diseases, peroxynitrite has received considerable attention worldwide. The anion of peroxynitrite (O = NOO–), which forms in vivo from nitrogen monoxide and superoxide anion, is a powerful oxidant and known to induce breakage of DNA strands, inactivation of enzymes, protein modification by nitration and hydroxylation, and lipid peroxidation in LDL. In recent years, it was reported that biological antioxidants have a tendency to readily react with peroxynitrite. However, chemical processes and mechanism involved in these phenomena remain to be

341

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elucidated in many respects due to lack of relevant information on the nature of peroxynitrite. Our past research has reported some new modes of reactions exhibited by peroxynitrite, which included quenching of peroxynitrite by lycopene in vitro, reactivity of retinol and astaxanthin with peroxynitrite, and its consecutive reaction products and mechanisms involved. In this chapter, we have elaborated these results further and have extended our study to include biochemical studies on peroxynitrite and peroxynitrite quenching mechanisms by biological antioxidant systems.

14.2 PEROXYNITRITE FORMATION IN VIVO A number of studies have indicated that peroxynitrite is formed in vivo from the combination of nitric oxide and superoxide with a rate constant near the diffusion controlled limit and that peroxynitrite is in equilibrium with peroxynitrous acid. ×10 M S Oi2– + i NO 7 → – O–O–N=O 9

–

–1 –1

O–O–N=O + H + ← → H–O–O–N=O

Superoxide and nitric oxide can react together to produce peroxynitrite and each can modulate the effects of the other. For example, superoxide can diminish the effects of nitric oxide by diverting it to form peroxynitrite. Similarly, .NO can capture superoxide and divert its effects, even to the extent of acting as an antioxidant. A number of systems are now known in which the presence of nitric oxide can alter, or even protect against, the effects of superoxide and superoxide-derived reactive oxygen species. A very high concentration of nitric oxide is found in cigarette smoke. In fresh smoke, .NO concentration can be as high as 500 ppm, and as smoke ages, the nitric oxide is oxidized to nitrogen dioxide. Cigarette smoke can also initiate lipid peroxidation, producing lipid peroxyl radicals. In smoke systems, the lipid peroxyl radicals can react with either nitric oxide or nitrogen dioxide, thus inhibiting lipid peroxidation. These reactions produce either peroxynitrite esters or peroxynitrate esters. Other studies have also revealed that nitric oxide can scavenge superoxide in vivo. In the DNA of macrophages, nitric oxide and superoxide induce oxidative damage in addition to deamination. Beckman et al.1,2 showed independent existence of peroxynitrite in vivo using immunohistochemical techniques. They revealed the presence of nitrotyrosine residues in atherosclerotic human coronary arteries. This was major evidence for peroxynitrite formation in arterial plaques, since tyrosine is nitrated by peroxynitrite but not by nitric oxide itself. Figure 14.1 represents potential sources and reactions of nitric oxide (Figure 1a) and superoxide (Figure 1b). The peroxynitrite anion is relatively unreactive, and its conjugate acid known as peroxynitrous acid (HO-O-N=O), which is an inorganic peroxyacid, is more reactive. Peroxyacids, while having a peroxide O-O bond, do not usually dissociate to form radicals. The reasons for this include the fact that the O–O bond in these

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1 Arg

NOS

1

NADPH oxidase

O2 Cigarette smoke •NO

2 •NO

+e−

Nitrosation (e.g., RS–NO)

8 O2

NO−

−OONO

11 H+ 9 O2•− −

14 Hb

OONO

O2•− QH2

3 H2O NO2− O2 6 H+ 4 −e− 5 +NO 7

343

10 H+

HOONO 12 13 S NO2− NO3− +SO

2O2•−

2

O2 Xenobiotics

3 2H+

H2O2

4 Fe

HO•

O2 5 Oxidation (e.g., catechols) & Reduction (e.g., ferretin)

NO 6

−OONO

Met Hb (a)

(b)

FIGURE 14.1 Representations of sources and reactions of nitric oxide and superoxide. (Reprinted with permission from The American Psychological Society, (701, 1995.)

compounds is relatively strong for peroxides, and peroxyacids are polar with a tendency for undergoing even-electron, nonradical reactions. One of the striking features of peroxynitrite chemistry is that a scavenger has not yet been found that can capture 100% of the reactive intermediate purported to be formed: HO. or HOONO*. Because HOONO* would be less reactive than the hydroxyl radical, a mechanism involving HOONO* as an intermediate can also explain the inability of hydroxyl-radical scavengers to completely block peroxynitrite reactions.

14.3 CAROTENOIDS The carotenoids are yellow, orange, or red pigments that are widely distributed in the plant and animal kingdom. They are the major pigments of certain yellow, orange, or red flowers, vegetables, berries, mushrooms, insects, bacteria, feathers, and egg yolk. In plants, they are found in leaves together with chlorophyll, and in animals they are dissolved in fats or combined with protein in the aqueous phase; ordinarily, they are fat soluble. Color and antioxidant activities of carotenoids are a consequence of their unique structure, an extended system of conjugated double bonds. Carotenoids are tetraterpenes formed by tail-to-tail linkage of two C-20 units, and in many carotenoids the end-groups are modified into five- or six-membered rings giving monocyclic or dicyclic compounds. There are two main groups of carotenoids, the hydrocarbons (carotenes) and the xanthophylls (a range of which are shown in Figure 14.2). The xanthophylls have oxygenated functional groups most often as either hydroxy-, methoxy-, epoxy-, carboxy-, or carbonyl groups. Most of the substituents are usually on the ring positions 1−6 and 1’−6’, although the 5,8 epoxides are the main exceptions.

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β-carotene (carrot, mango)

Lycopene (tomato)

α-carotene (orange) OH

HO Lutein (orange, egg yolk) OH

HO Zeaxanthin (mango, egg yolk) O OH

HO O Astaxanthin (salmon, lobster)

FIGURE 14.2 Structures of selected carotenoids.

14.3.1 CAROTENOIDS

AS

ANTIOXIDANTS

Carotenoids have the capacity to quench or inactivate excited states of molecules. It was originally thought that carotenes and xanthophylls served as light filters for chlorophyll or functioned as protectors of enzymes in cells. Some of the well-established functions of carotenoids are their pro-vitamin A and antioxidant activity, and their abilities to quench singlet oxygen and inhibit the growth of certain tumors. Research on the antioxidant activity of carotenoids was sparked by the description of their singlet-oxygen-(1O2)-quenching properties and their ability to trap

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peroxyl radicals. Singlet oxygen quenching by carotenoids occurs via physical or chemical quenching. The efficacy of physical quenching greatly exceeds that of chemical quenching and involves the transfer of excitation energy from 1O2 to the carotenoid, resulting in ground-state oxygen and excited triplet-state carotenoid. The energy is dissipated through rotational and vibrational interactions between the excited carotenoid and the surrounding solvent to yield the ground-state carotenoid and the thermal energy. In the process of physical quenching, the carotenoid remains intact and can undergo further cycles of singlet oxygen quenching, thus acting like a catalyst. The quenching activity of carotenoids mainly depends on the number of conjugated double bonds and is influenced to a lesser extent by carotenoid end groups (cyclic or acyclic) or the nature of substituents in carotenoid containing cyclic end groups.

14.3.2 REACTIONS OF PEROXYNITRITE CAROTENOIDS

WITH

SOME IMPORTANT

14.3.2.1 Reaction of Peroxynitrite with Zeaxanthin Two carotenoids have been identified in the macular area of the human retina: the xanthophylls zeaxanthin and lutein. Humans accumulate both β-carotene and xanthophylls, oxygenated carotenoids, in plasma and tissues, but zeaxanthin and lutein appear to be specifically concentrated in the macula. The biological function of xanthophylls has not yet been clearly established; for example, compared to βcarotene, xanthophylls have little pro-vitamin A activity.3 The oxygenated carotenoids zeaxanthin and lutein may offer protection against or repair of oxidative damage associated with the degenerative diseases of aging. Macular pigments like zeaxanthin, could protect the highly unsaturated lipids of retina cell membranes from oxidation by peroxynitrite. β-carotene, cantaxanthin, and zeaxanthin have been reported to inhibit macrophage-mediated low-density-lipoprotein oxidation,4 in which peroxynitrite presumably plays a role. Scheidegger et al.5 studied the kinetics of the reaction of peroxynitrite with zeaxanthin in liposomes constructed from the fully saturated lipid L-α-dimyristoyl-phosphatidylcholine (C14: O) and from the unsaturated egg lecithin and its absorbance spectrum in liposomes strongly resembles in shape and amplitude that of zeaxanthin dissolved in methanol. They found that zeaxanthin reacts with peroxynitrous acid slowly in a second order reaction. The formation of hydrogen bonds between peroxynitrous acid and the distal hydroxyl groups of zeaxanthin may limit the rate of reaction. The research revealed that the carotenoid, zeaxanthin, could be easily incorporated into liposomal vesicles prepared from egg lecithin or L-α-dimyristoylphosphatidylcholine, dimyristoyl (C14: O). In electron micrographs, liposomes containing zeaxanthin have had a rougher appearance than liposomes prepared in the absence of zeaxanthin. Gabrielska and Gruszecki6 have shown by proton NMR that incorporation of zeaxanthin in the lipid bilayer is associated with rigidity of the membrane which is not observed in liposomes prepared with β-carotene; they suggest that zeaxanthin molecules adopt a regular orientation in the membrane, with the polar ends contacting the aqueous milieu and the hydrophobic chain in the

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membranes the hydrophobic core, and that β-carotene, by contrast, which has no polar moiety, becomes randomly organized in the membrane. In the reaction between zeaxanthin and peroxynitrite, the change in the membrane spectrum resembled spectral changes found in the trans- to cis-isomerization of β-carotene.7 However, after longer reaction times, that is, 5 s, the cis-like absorbance is lost, and at very long reaction times (over days), spectral changes in zeaxanthin resulting from reaction with peroxynitrite are similar to those found with photo-oxidation reactions, although the peroxynitrite itself is isomerized to nitrate within seconds of initiating the reaction. Kinetic fits of the spectral data indicate an A to B to C type reaction, consistent with initial production of one or more cisisomers of zeaxanthin followed by further reaction, possibly including cleavage of the carbon chain. The subsequent reactions cannot be attributed to further reaction with peroxynitrite, but most likely result from the initial formation of a reactive intermediate. The overall reaction of zeaxanthin with peroxynitrous acid is second order. The order of the reaction with respect to zeaxanthin could not be determined directly from a linear plot of the apparent first order rate constant as a function of zeaxanthin concentration since that dependence was negative. The negative dependence could be attributable to hydrogen bonding interactions between peroxynitrous acid and the distal hydroxyl groups of zeaxanthin. The pH profile indicates that the reaction with zeaxanthin involves peroxynitrous acid and not the conjugate anion. Solubilization of zeaxanthin in liposomes allowed the observation of the reaction between the water insoluble carotenoid and water-soluble peroxynitrite, which is one of the first demonstrations of a reaction between a carotenoid and peroxynitrite. Liposomes represent a good model system for biological membranes, and are very suitable for in vitro studies of membrane-associated biological antioxidants. It can be estimated that zeaxanthin plays a major role in protection of macular tissue from oxidative damage. Other studies have also shown that zeaxanthin is more active than other carotenoids in preventing lipid oxidation, and that peroxynitrite plays a pivotal role in mediating oxidative damage in vivo is becoming increasingly clear. It can be guessed that the direct reaction of peroxynitrite with zeaxanthin and other carotenoids is less important in vivo than reactions of peroxynitrite with other targets in the lipid bilayer. However, carotenoids may play a role in repair of other target molecules damaged through reaction with peroxynitrite or other reactive oxygencontaining species. 14.3.2.2 Reaction of Peroxynitrite with Astaxanthin (Astaxanthin Structure-Activity Relationship) Many carotenoids such as zeaxanthin, canthaxanthin, and astaxanthin are known to inhibit lipid peroxidation significantly. Of these, astaxanthin, which is found as a common pigment in algae, fish, and birds, is reported to be more effective than βcarotene in preventing lipid peroxidation in solution and various biomembrane systems, such as egg yolk phosphatidylcholine liposomes and rat liver microsomes. However, the reason why the antiperoxidation effect of astaxanthin is more than that of β-carotene is not well understood. Many attempts were made in order to solve

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MDA (nmol/ml)

0.6

0.4

0.2

0

0

1 2 Carotenoids (mol%)

3

FIGURE 14.3 Effects of β-carotene (●) and astaxanthin () on the formation of lipid peroxides in terms of MDA in liposomes.

this question. In a recent study done by Goto et al.8 the effects of astaxanthin and β-carotene on lipid peroxidation of liposomes induced by ADP/ Fe2+ under various conditions were examined. For this, a certain amount of carotenoid was added to an etherial solution of EyPC (egg yolk phosphatidylcholine) and BhCL ( cardiolipin from bovine heart) in a molar ratio of 9:1 (EyPC/BhCL liposomes), and then the liposomes were prepared by reverse-phase evaporation method at pH 7.4. Lipid peroxidation was initiated with 1mM ADP and 0.1 mM Fe2+. Progress of lipid peroxidation was determined as the amount TBARS (thiobarbituric acid-reactive substances) measured in terms of MDA concentration 45 min after addition of ADP/ Fe2+. At this time, peroxidation without carotenoids had completely stopped. As shown in Figure 14.3, both β-carotene and astaxanthin inhibited liposomal lipid peroxidation. The production of lipid peroxides was inhibited progressively by these carotenoids with increases in their amounts in the liposomal membrane up to 1 mol%, and their effects almost leveled off at 1 mol%. At 3 mol%, astaxanthin was almost two times more effective than β-carotene, being consistent with its relative radical trapping effects in organic solvents and antiperoxidative effects in membrane systems, such as EyPC liposomes and rat liver microsomes. 14.3.2.3 Reaction of Retinol and Retinyl Acetate with Peroxynitrite Retinol is a fat-soluble vitamin that plays an important role in the visual cycle and is essential for the normal growth of bones, reproduction, embryonic development, and in differentiation of epithelial tissues. Retinol is a circulating form of vitamin A and is generated from either β-carotene or retinyl esters. Retinol is stored as retinyl esters in the liver. Retinols are sensitive to oxidation.9−13 The extended polyene structure of retinols is reactive toward peroxyl radical addition reactions, and is thought to trap lipid-peroxyl radicals, since they inhibit lipid peroxydation. However, the peroxynitrite reaction mechanism of retinol is not yet understood. Some studies have been done in this direction to understand this mechanism. In this regard, retinyl

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OCH3

OCOCH3

OH 14-hydroxy-13-methoxyretinyl acetate

O

OCOCH3

O 5, 6, 11, 14-diepoxyretinyl acetate

OCOCH3

O 5, 6-epoxyretinyl acetate

O

OCOCH3

11, 14-epoxyretinyl acetate

FIGURE 14.4 Structures of retinyl acetate and reaction products.

acetate, an acetate derivative of retinol, has been subjected to reactions with an alkylperoxyl radical.13 Retinyl acetate was reacted with an alkylperoxyl radical in two solvent systems, methanol and benzene. The alkylperoxyl radical was generated by the thermal decomposition of a free radical initiator, 2, 2’-azobis (2, 4-dimethylvaleronitrile) (AMVN), at 37°C. The main product of the reaction was 14-hydroxy-13-methoxyretinyl acetate in addition to some minor products. The reaction with benzene gave some products with low yield. They were 5,6-epoxyretinyl acetate, 11,14-epoxyretinyl acetate, and 5,6,11,14-diepoxyretinyl acetate (Figure 14.4). These results also show that the lipid peroxidation initiated by AMVN is inhibited by retinyl acetate. The inhibition of the peroxidation by conjugated polyene compounds has been attributed to their lipid peroxyl radical-trapping ability. These compounds seem to exert antioxidant activities by a mechanism in which the chain-propagating peroxyl radical is trapped by addition to the conjugated polyene system rather than the mechanism of hydrogen donation. On the other hand, conventional chain-breaking antioxidants such as tocopherols trap peroxyl radicals by donating hydrogen atoms. Samokyszyn et al.14 have demonstrated that peroxyl radicals preferentially reacted with 13-cis-retinoic acid via addition and not by hydrogen-atom abstraction reactions. The lack of a distinct induction period during the inhibition of methyl linoleateperoxidation by retinyl acetate, in contrast to α-tocopherols, can be explained by

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the idea that the reaction rate of retinyl acetate with peroxyl radicals is similar to the rate of chain propagation of methyl linoleate peroxidation. The reaction in benzene gave only epoxy compounds, demonstrating that the peroxyl can attack every double bond of the polyene structure. The reaction of conjugated polyenes with an alkylperoxyl radical can be accomplished by several processes. The alkylperoxyl radical (AOO.) adds to the double bond of polyenes in Reaction 14.1. (1)

AOO. +

C

C

AOO

C.

C

Reaction 14.1

Addition can be followed by the unimolecular decomposition of the β-alkylperoxylalkyl radical, affording epoxide and an alkoxyl radical (AO. , Reaction 14.2), or by the reaction with oxygen, giving polyperoxides (Reaction 14.3). (2)

O AOO

C

C.

AO. +

C

C

C

C

Reaction 14.2

(3)

AOO

C

C. + O2

AOO

OO. Reaction 14.3

The results indicate that the alkylperoxyl radical reacts with retinyl acetate to form epoxides by Reaction 14.1 and Reaction 14.2. Low yields of the epoxy compounds in benzene indicate these compounds are unstable and decompose into some other products. The oxygenated products obtained in this study suggest that retinyl acetate is capable of scavenging lipidperoxyl radicals by its conjugated polyene system. Keeping in mind the reaction of peroxyl radicals with retinyl acetate, further studies on reaction of retinol with peroxynitrite are under way in our laboratory.

14.4 CONCLUSIONS The versatility of the reaction mode is suggestive of the involvement of several different active species in the reaction of peroxynitrite. There are many yet unidentified products, the identification of which may provide additional new reaction modes of reaction of peroxynitrite with various biological antioxidation systems. These reactions would probably be involved in vivo and contribute to the degradation of biological systems, eventually leading to the pathogenic process of diseases. Better understanding about the behavior of peroxynitrite toward a wide variety of biological

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antioxidation systems should enable us to predict the role of peroxynitrite in vivo and should provide valuable information on its physiological significance.

14.5 SUMMARY Peroxynitrite, the reaction product of superoxide and nitric oxide, is a powerful oxidant produced by macrophages and neutrophils. Peroxynitrite and its decomposition products are known to induce DNA strand scission, protein modification by nitration and hydroxylation, and lipid peroxidation in LDL. Recently, it was reported that biological antioxidants readily react with peroxynitrite. In this review we present results of biochemical studies on peroxynitrite and peroxynitrite quenching mechanism by biological antioxidant systems. We hope to clarify peroxynitrite quenching mechanisms by considering interactions with antioxidants, intracellular localization, and biological concentrations.

REFERENCES 1. Beckman, J.S., Ye, Y.Z., Anderson, P., Chen, J., Accavitti, M.A., Tarpey, M.M., and White, C.R., Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry, Biol. Chem. Hoppe-Syler, 375, 81, 1994. 2. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D., and Beckman, J.S., Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase, Arch. Biochem. Biophys., 298, 431, 1992. 3. Weiser, H. and Kormann, A.W., Provitamin A activities and physiological functions of carotenoids in animals: relevance to human health, in Carotenoids in Human Health, New York Academy of Sciences, New York, 1993, p. 213. 4. Carpenter, K.L.H., van der Veen, C., Hird, R., Dennis, I.F., Ding, T., and Mitchinson, M.J., The carotenoids β-carotene, canthaxanthin and zeaxanthin inhibit macrophage mediated LDL oxidation, FEBS Lett., 401, 262, 1997. 5. Scheidegger, R., Pande, A.K., Bounds, P.L., and Koppenol, W.H., The reaction of peroxynitrite with Zeaxanthin, Nitric Oxide, Biol. Chem., 2, 8, 1998. 6. Gabrielska, J. and Gruszecki, W.I., Zeaxanthin (dihydroxy-β-carotene) but not βcarotene rigidifies lipid membranes: a 1H-NMR study of carotenoid-egg phosphatidylcholine liposomes, Biochem. Biophys. Acta Bio-Membr., 1285, 167, 1996. 7. Frank, H.A., Physical and chemical properties of carotenoids, in Carotenoids in Human Health, New York Academy of Sciences, New York, 1993, p. 1. 8. Goto, S., Kogure K., Abe, K., Kimata, Y., Kitahama, K., Yamashita, E., and Terada, H., Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin, Biochim. Biophys. Acta, 1512, 251, 2001. 9. Jungalwala, F.B., and Cama, H.R., Preparation and properties of 5,6-monoepoxyvitamin A acetate, 5,6-monoepoxyvitamin A alcohol, 5,6-monoepoxyvitamin A aldehyde and their corresponding 5,8-monoepoxy (furanoid) compounds, Biochem. J., 95, 17, 1965.

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10. Takashima, Y., Nakajima, T., Tanaka, S., Washitake, M., Anmo, T., and Matsumaru, H., Stability of retinal analogs, IX, stability of vitamin A acetate in aqueous ethanolic solutions and quantitative analysis of its decomposition products by high performance liquid chromatography, Chem. Pharm. Bull., 27, 1553, 1979. 11. Ikeda, A., Iwahashi, H., Negoro, Y., and Kido, R., Detection of radical species in mixtures of some retinoids with hematin by using the ESR spin-trapping technique, J. Biochem., 101, 743, 1987. 12. Oyler, A.R., Motto, M.G., Naldi, R.E., Facchine, K.L., Hamburg, P.F., Burinsky, D.J. Dunphy, R., and Cotter, M.L., Characterization of autoxidation products of retinoic acid, Tetrahedron, 45, 7679, 1989. 13. Yamauchi, R., Miyake, N., Kato, K., and Ueno, Y., Peroxyl-radical reaction of retinyl acetate in solution, Biosci. Biotech. Biochem., 56, 1529, 1992. 14. Samokyszyn, V.M. and Marnett, L.J., Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid, Free Radic. Bio. Med., 8, 491, 1990.

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15

Stability of Lycopene During Food Processing John Shi1 , Yukio Kakuda2, Yueming Jiang3, Gauri S. Mittal4 1

Agriculture and Agri-Food Canada University of Guelph 3 Chinese Academy of Sciences 4 University of Guelph 2

CONTENTS 15.1 Introduction ..................................................................................................353 15.2 Physical and Chemical Properties of Lycopene ..........................................354 15.3 Lycopene Degradation during Processing ...................................................356 15.3.1 Effect of Temperature on Lycopene Degradation ...........................356 15.3.2 Effect of Light Irradiation on Lycopene Degradation.....................360 15.3.3 Effect of Oxygen on Lycopene Degradation...................................361 15.4 Lycopene Isomerization in Food Processing...............................................364 15.5 Summary ......................................................................................................366 References..............................................................................................................368

15.1 INTRODUCTION Lycopene is a natural pigment that imparts red color to tomato, guava, rosehip, watermelon, and pink grapefruit.1 Tomatoes (especially deep-red fresh tomato fruits) and tomato products are considered to be the most important sources of lycopene in the human diet.2,3 Recent interest in the consumption of lycopene-rich foods as a means of reducing the risk of cancer has prompted researchers to investigate the level of lycopene in foods frequently consumed by people. The ability of lycopene to function as an antioxidant may contribute to a reduction in disease risk.4 Increasing clinical evidence supports the role of lycopene as an important micronutrient, since it appears to provide protection against prostate cancer, lung cancer, and a broad range of epithelial cancers.5–7 With increasing interest and awareness of the health benefits of lycopene, lycopene concentrates and lycopene-rich food products have

353

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been used in functional foods, and its stability during food processing and storage has drawn more and more attention. In order to ensure lycopene stability, there is a need to understand the influence of processing parameters and conditions on the oxidation and isomerization of lycopene. Lycopene belongs to the carotenoid family and exists in nature in the all-trans form. Heat, light, oxygen, and different food matrices are factors that have a marked effect on its isomerization and autoxidation. Lycopene may isomerize to mono- or poly-cis forms and reisomerization can take place during storage. The oxidation of lycopene splits the molecule, which causes a loss in color and the production of offflavors. It is generally accepted that the all-trans isomer has the highest bioactive stability, and the cis-isomers have the lowest bioactive stability. Bioactive potency is dependent on the extent of isomerization and oxidation. A true assessment of the nutritional quality of tomato products depends not only on the total lycopene content but also on the distribution of the lycopene isomers. The reports concerning the effects of heat, oxygen, and light on the stability of lycopene are generally in agreement in much of the literature; however, in some areas controversy still exists such as the exact conditions required for the isomerization reaction.

15.2 PHYSICAL AND CHEMICAL PROPERTIES OF LYCOPENE Lycopene is the major carotenoid pigment found in tomatoes The chemical structure of lycopene is shown in Figure 15.1. Lycopene is a polyene hydrocarbon characterized by a symmetrical and acyclic structure containing 13 double bonds of which 11 are conjugated double bonds arranged in a linear array and having a molecular formula of C40H56. Color and antioxidant activities of lycopene are a consequence of its unique structure, an extended system of conjugated double bonds. In nature, lycopene exists in the all-trans form, the most thermodynamically stable form. However, under certain conditions, seven of these bonds can isomerize from the trans form to the less stable mono- or poly-cis form. Lycopene is soluble in oils and apolar organic solvents. In aqueous systems they tend to aggregate and precipitate as crystals; such reactions are thought to hinder the bioavailability of lycopene in humans. In ripe tomato fruits, lycopene takes the form of elongated, needle-like crystals, which are responsible for the typical bright red color of ripe tomato fruits. Lycopene is more soluble in chloroform, benzene, and other organic solvents than in water. Lycopene is also very sensitive to light, heat, oxygen, acids, and some metallic ions such as Cu 2+, Fe 3+ which catalyzes its oxidation.

FIGURE 15.1 Molecular structure of lycopene.

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TABLE 15.1 Comparison of Antioxidant Activities of Carotenoids: Singlet Oxygen Quenching Lycopene (a) Singlet oxygen quenching, Kq × 109 (m–1s–1) (b) Radical scavenging (Trolox equivalents) (c) Reaction of carotenoid radical anions with O2; k × 108 (m–1s–1)

31 2.9 2

Other carotenoids’ singlet oxygen quenching, 109 × Kq (m–1s–1) γ-carotene α-carotene β-carotene Lutein Astaxanthin Bixin Canthaxanthin Zeaxanthin

25 19 14 8 24 14 21 10

Source: Data from Di Mascio et al.8,11; Conn et al.9,12; Miller et al.13

Lycopene exhibits many unique and distinct biological properties. One of its main activities is to function as an antioxidant. Through its conjugated double-bound system, lycopene is able to quench efficiently the energy of deleterious forms of oxygen (singlet oxygen) and to scavenge a large spectrum of free radicals. Lycopene is among the most efficient singlet oxygen quenchers of the natural carotenoids.8,9 The quenching activity of the different carotenoids depends essentially on the number of conjugated double bonds. It is modulated by the end groups or the nature of the substituents in the carotenoids that contain cyclic end groups.10 There are considerable differences in the quenching rate constants (Kq) for various carotenoid species (Table 15.1). The antioxidant activities of lycopene and other carotenoids are highlighted by their singlet oxygen quenching properties and their ability to trap peroxyl radicals.14 Lycopene is known to exist in a variety of geometric forms, including all-trans, mono-cis, and poly-cis forms. The all-trans isomer of lycopene is the predominant geometrical isomer in fresh tomatoes, and is the most thermodynamically stable form. However, lycopene can undergo trans-to-cis isomerization during tomato processing and storage. In various tomato-based foods, the all-trans isomer is composed of 35% to 96% of total lycopene.15 In general, cis isomers are more polar than their all-trans counterparts, and are less prone to crystallization due to their kinked forms. The cis isomers are also more soluble in oil and hydrocarbon solvents than the all-trans isomers. The bioactive potency of the cis isomers is not the same as the all-trans isomers, because of the differences in their structural shapes. Most stability studies of lycopene in food systems focused on its degradation. Lycopene is a conjugated polyene and is susceptible to at least two reactions during tomato processing, that is, isomerization and oxidation. Lycopene may be partially

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destroyed in food products by heat, light, oxygen, or the presence of metallic ions (Cu2+, Fe3+, etc.). The isomerization of lycopene occurs when pure lycopene is treated and when the lycopene in food products is subjected to processing conditions. On the other hand, the conversion of the cis isomer to the trans isomer, which is the more favorable reaction, occurs during the storage of the product. The cis isomers are the less stable form, while the trans isomers are in the more stable ground state.

15.3 LYCOPENE DEGRADATION DURING PROCESSING More than 80% of the tomatoes produced are consumed in the form of processed products such as tomato juice, paste, puree, ketchup, sauce, and salsa. The lycopene content in concentrated tomato products is generally lower than expected, because of losses during tomato processing.16 The main causes of lycopene degradation in food processing are isomerization and oxidation. It is widely assumed that lycopene in general undergoes isomerization during thermal processing. These changes in lycopene content and in the distribution of trans-cis isomers result in a change in biological activity. Physical and chemical factors known to degrade lycopene include elevated temperature, exposure to light, oxygen, extremes in pH, and active surfaces.17

15.3.1 EFFECT

OF

TEMPERATURE

ON

LYCOPENE DEGRADATION

Lycopene content, mg/100 g

The native lycopene is located within the tomato cell matrix which gives it some protection from degradation during heat processing. The effect of heating on total lycopene and cis-isomer content in tomato puree are shown in Figure 15.2. Lycopene retention in tomato juice is shown in Table 15.2. The length of cooking time has less effect on the degradation of lycopene if the heating temperature is less than 8 7 6 5 4 3 2 1 0

0

1

2

3

4

5

6

Time (hr) 90°C

100°C

120°C

150°C

FIGURE 15.2 Effect of heat treatment on total lycopene degradation.18 (a) HPLC chromatogram of lycopene in oil solution after being treated at 25°C for 90 min. (b) HPLC chromatogram of lycopene in oil solution after being treated at 100°C for 90 min. (c) HPLC chromatogram of lycopene in oil solution after being treated at 180°C for 90 min.

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TABLE 15.2 Lycopene Loss Rate in Tomato Juice during Heating Lycopene Loss (%)

Heating Temperature (°C)

Heating time 1 min

Heating time 3 min

Heating time 7 min

90 100 110 115 118 121 124 127 130

0.6 0.9 2.2 2.7 3.7 4.6 5.5 6.5 7.4

0.9 1.4 3.2 4.5 6.0 7.3 8.5 9.9 11.5

1.1 1.7 4.4 7.0 9.1 10.6 12.5 14.6 17.1

Source: Data from Miki and Akatsu.19

100°C. However, temperatures greater than 120°C will result in more lycopene loss. It has also been reported that serious losses of lycopene can occur when the holding times at high temperature are long (Table 15.2). The changes in trans- and cis-isomer concentrate during heat treatment are shown in Figure 15.3. Increasing the temperature from 100°C to 180°C increased the degradation of both the trans and cis isomers of lycopene. It was observed that the cis isomers increased with thermal treatment at 100°C, but dropped significantly with treatment at 180°C. An increase in temperature from 100°C to 180°C caused a 76% decrease in total lycopene content (Figure 15.3). A 90-min treatment at 180°C resulted in lycopene degradation and a greater loss in total lycopene, compared to cis-isomer formation. Total lycopene concentration decreased at all treatment times, but the cis isomers appeared only during the first hour of heating. After 1 h of heating, the rate of cis-isomer accumulation decreased. It was observed that increasing the temperature from 100°C to 180°C or increasing thermal treatment time would increase the degradation of trans isomer and cis isomer of lycopene (Figure 15.3). It was suggested that degradation of lycopene was the main mechanism of lycopene loss when heated above 100°C, and that lycopene in general underwent isomerization with thermal processing. An increase in temperature from 90°C to 150°C caused a 35% decrease in total lycopene content. The high temperature and large amount of air incorporated in the tomato juice during the breaking and straining operations can quickly destroy substantial amounts of lycopene. The results suggest that length of heating is a critical factor controlling the degradation of lycopene. It appears that deaeration and “high temperature-short duration” heat treatment of tomato juice can have beneficial effects on tomato juice quality. Research by Agarwal et al.21 showed that subjecting tomato juice to cooking temperatures in the presence of corn oil resulted in the formation of cis isomeric forms. According to Ax et al.,22 total lycopene content in oil-in-water emulsions decreased during thermal treatment with and without exposure to oxygen. Higher

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40 cis-isomers

mAU

30

all-trans

20 10

5-cis

0 0

5

10

15

20

Min (a). HPLC chromatogram of lycopene in oil solution after being treated at 25°C for 90 min

40

mAU

30 20

cis-isomers all-trans

10

5-cis 0 0

5

10

15 20 Min (b). HPLC chromatogram of lycopene in oil solution after being treated at 100°C for 90 min

FIGURE 15.3 Lycopene oxidation and isomerization during thermal treatment.20

temperatures were directly correlated with increasing lycopene losses, and thermal treatment led to a significant decrease in the concentrations of all-trans and 13-cis isomer, while the concentration of the 9-cis isomer increased. Zanoni et al.23 performed a study on the loss of lycopene during air drying. Tomato halves were dried in a cabinet air dryer and the drying was carried out at 80°C and 110°C, with an airflow rate of 1.5 m/s. Lycopene losses of 12% occurred at 110°C. According to Sharma and LeMaguer,24 heating tomato pulp at 100°C for 120 min decreased lycopene content from 185.5 to 141.5 mg/100g total solid, and the freeze drying and oven drying (25°C to 75°C) of tomato pulp solids did not cause any loss in lycopene content. Anguelova and Warthesen25 found that 70% of all-trans lycopene were retained in tomato products at 6°C, but storage at 45°C affected lycopene stability to a greater extent as only about 40% of all-trans lycopene were retained under the same conditions of six weeks of light irradiation.

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all-trans 40

mAU

30 20 cis-isomers

10

5-cis

0 0

5

10

15

20

Min ( c). HPLC chromatogram of lycopene in oil solution after being treated at 180°C for 90 min

FIGURE 15.3 (Continued).

Lycopene in the pure form, without plant matrix protection, will undergo degradation more quickly than in tomato tissue. Lee et al.26 studied the stability of lycopene by heating lycopene (lycopene dissolved in hexane) under 50°C, 100°C, and 150°C, respectively. For the 50°C treatment, there was no significant change in the all-trans lycopene content within the first 12 h; however, the content began to decline thereafter. The levels of the mono-cis forms of lycopene were found to decrease with increasing heating time, which indicates that the degradation rate of mono-cis lycopene may be greater than the formation rate. Unlike the mono-cis forms of lycopene, the percentage change of two di-cis isomers showed increasing trends, which were probably due to the conversion of mono-cis lycopene. There was a significant decrease in total lycopene after heating at 150°C for 9 h. The result revealed that isomerization was the main reaction during heating in the first 9 h, after which the degradation reaction dominated. Similar results were observed for the concentration change of all-trans lycopene and its cis isomers during heating at 100°C, the level of all-trans lycopene decreased by 78% after 120 min heating. The mono-cis forms of lycopene showed a decreasing trend. The levels of two di-cis isomers were found to rise in the first 60 min and then decreased, implying that dicis lycopene might be converted to the mono-cis lycopene or undergo degradation after prolonged heating. The result indicated that the isomerization reaction was favored at the beginning and then the degradation dominated after prolonged heating at 150°C. When comparing the results, it appears that with increasing temperature and heating time, degradation dominated over isomerization. Shi et al.20 dissolved extracted lycopene into canola oil, and heated the sample at 25°C, 100°C, and 180°C, respectively. It was observed that increasing the temperature from 100°C to 180°C or increasing thermal treatment time increased the degradation of trans isomer and cis isomer of lycopene (Figure 15.4). Compared with the treatment at 25°C, the cis isomers increased with thermal treatment at 1005°C, but dropped significantly with treatment at 180°C.

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Lycopene

CHO

O 2-methy-2-hepten –6-one

O Pseudo-ionone

Geranial

O 6-methyl-3.5-heptadien-2-one

CHO Neral

FIGURE 15.4 Molecular structural changes of lycopene during heating. (Reprinted with permission from J. Agric. Food Chem., 38, 1238–1242, 1990. Copyright 1990 American Chemical Society.) 27

It is also possible to form peroxyl radical capable of acting as a prooxidant and propagate autoxidation. The proposed degradation pathway of lycopene is shown in Figure 15.3. It is widely assumed that lycopene in general undergoes isomerization with thermal processing. This isomerization results in the conversion of the all-trans isomer into cis isomers. The cis isomers are present in processed food samples and their concentration increased with increasing temperature and time of thermal treatment. A large loss of lycopene during processing would indicate a longer and more drastic thermal procedure. The changes in lycopene content and the formation of cis isomers may result in a reduction in bioactive potency.28,29,30

15.3.2 EFFECT OF LIGHT IRRADIATION ON LYCOPENE DEGRADATION Increasing illumination increases the loss of lycopene. Lee et al.26 studied lycopene stability by dissolving standard lycopene in hexane and illuminating (illumination intensity ranged 2000 to 3000 lux) the samples for 6 days at 25°C. The content of all-trans lycopene was found to decrease with increasing illumination time. After 144 h of exposure to light the losses amounted to 94% (Figure 15.5). All the monocis isomers of lycopene showed an inconsistent change. For instance, the level of 5-cis-lycopene was found to increase in the beginning and then decrease after the illumination time reached 2 h. Likewise, a similar trend was observed for 9-cis-, 13cis-, and 15-cis lycopene. These results appear to indicate that isomerization and degradation of lycopene and its cis isomers, during illumination, were proceeding simultaneously. The increased level of mono-cis lycopene was probably due to the initial conversion of all-trans lycopene. With continued illumination, a decrease in the mono-cis lycopene could occur due to a further conversion to another cis form

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0.50 0.00

In (CA/CAD)

−0.50

y = −0.0176x + 0.1435 R2 = 0.9609

−1.00 −1.50 −2.00 −2.50 −3.00

0

20

40

60

80 Time (h)

100

120

140

160

FIGURE 15.5 First-order plot for the degradation of total amount of lycopene during illumination at 25°C for 144 h.26

through intermediate all-trans lycopene or undergoing degradation. The percentage changes of all-trans lycopene and its cis isomers showed a different trend when compared to the changes in the total amount of lycopene. The all-trans lycopene may have been isomerized to form mono-cis or di-cis lycopene. These results may account for the percentage increase of all the mono-cis and di-cis forms of lycopene during illumination. The effects of light irradiation on the content of total lycopene, trans isomers and cis isomers in pure lycopene samples under different light irradiation intensities of 2010 (outdoor), 900, 650, and 140 (indoor) µmol m–2 s–1 for 24 h are shown in Figure 15.6. The results indicate that isomerization and degradation of lycopene and its cis isomers proceed simultaneously during illumination. The loss in total lycopene, trans-I somers and cis isomers increased significantly as the intensity of the light irradiation increased. The small amount of cis isomer formed during light irradiation indicates that either less cis isomers were formed, or that the oxidation reactions were predominating. It is possible that any cis isomer formed was quickly degraded into oxidative by-products, indicating that the rate of cis-isomer oxidation was much greater than formation of cis isomers by light irradiation. This would suggest that cis-isomer oxidation is the main reaction pathway. The content of total lycopene, trans isomers, and cis isomers in lycopene concentrates decreased under all light irradiation conditions, which indicates that light irradiation induces lycopene oxidation.

15.3.3 EFFECT

OF

OXYGEN

ON

LYCOPENE DEGRADATION

Henry et al.31 studied the effect of oxygen on the degradation of lycopene in an aqueous model system. The standard lycopene was exposed to continuous flow of water saturated with oxygen at 30°C, and the result showed that 90% of the lycopene was lost after 2 h. Ax et al.22 dissolved lycopene in the oil phase of an oil-in-water

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17.5 15

mAU

12.5 10 7.5

cis-isomers

5

all-trans

2.5

5-cis

0 0

5

10

15

20

Min (a). HPLC chromatogram of lycopene in oil solution after irradiation by light at 2010 μmol m−2 s−1 intensity (outdoor) for 24 hrs

17.5 15

mAU

12.5 10 7.5

cis-isomers

5

all-trans 5-cis

2.5 0 0

5

10

15 20 Min (b). HPLC chromatogram of lycopene in oil solution after irradiation by light at 900 μmol m−2 s−1 intensity (outdoor) for 24 hrs

FIGURE 15.6 The changes of all-trans-and cis-isomers of lycopene of oxidation and isomerization during sun-light irradiation.20 (a) HPLC chromatogram of lycopene in oil solution after irradiation by light at 2010 µmol m–2 s–1 intensity (outdoors) for 24 h. (b) HPLC chromatogram of lycopene in oil solution after irradiation by light at 900 µmol m–2 s–1 intensity (outdoors) for 24 h.

emulsion, and the emulsions were poured inside a glass flask with a sintered-glass frit allowing continuous flushing with either synthetic air or nitrogen gas, thus providing oxygen saturation or oxygen-free conditions. At 25°C, about 25% of the lycopene was degraded within 30 h in the oxygen-removed emulsions, whereas 80% was lost in the oxygen-saturated emulsion. In the presence of oxygen, lycopene destabilization was about three times higher than in the absence of oxygen. A nitrogen or argon headspace can be employed to keep the exposure to atmospheric oxygen to a minimum.17 However, according to Ribeiro et al.,32 removing oxygen

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17.5 15

mAU

12.5 10

all-trans

7.5

cis-isomers

5 5-cis

2.5 0 0

5

10

15

20

Min (c ). HPLC chromatogram of lycopene in oil solution after irradiation by light at 650 μmol m−2 s−1 intensity (indoor) for 24 hrs

17.5

all-trans

15 mAU

12.5 10 7.5

cis-isomers

5

5-cis

2.5 0 0

5

10

15 20 Min (d). HPLC chromatogram of lycopene in oil solution after irradiation by light at 140 μmol m−2 s−1 intensity (indoor) for 24 hrs

FIGURE 15.6 (c) HPLC chromatogram of lycopene in oil solution after irradiation by light at 650 µmol m–2 s–1 intensity (indoors) for 24 h. (d) HPLC chromatogram of lycopene in oil solution after irradiation by light at 140 µmol m–2 s–1 intensity (indoors) for 24 h.

in water by flushing the system with nitrogen achieved no improvement of stability. Instead, lycopene degradation was accelerated. On the other hand, complete exclusion of oxygen using the enzyme glucose oxidase was shown to produce far better stability. Anguelova et al.33 subjected tomato powder to three treatments: light exposure at room temperature, 6°C, and 45°C in the dark. Differences among the storage treatments are not obvious from the data, but the amount of cis isomers as a percentage of the total lycopene increased to the 14% to 18% range, regardless of the storage conditions (Table 15.3). The treatments at 6°C did not have less 5,5′ dicis lycopene than the other two treatments; this was explained as the degradation of

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TABLE 15.3 Contents of Presumptive 5, 5’ di- cis Lycopene in Tomato Powders (Percentage of Total Lycopene in Sample after Given Storage Period under Fluorescent Light 38,500 lux) 33 Tomato Powder T1 Weeks 0 1 2 3 4 5 6

Light Exposure 4.3 5.6 5.8 7 8.6 8.1 14.2

6°C

45°C

4.3 4.8 5.4 5.5 9.4 11 18.4

4.3 6.5 5.3 5.4 9.1 12.8 14.6

Tomato Powder T2 Light Exposure 6.2 6.7 5.2 5.5 7.7 11.8 14.1

6°C

45°C

6.2 6.1 7.6 6.4 6.8 11.7 18.2

6.2 5.9 6.3 7 7.8 12.1 14.1

cis isomer due to autoxidation, since cis isomers were more susceptible to autoxidation than the all-trans isomer.34

15.4 LYCOPENE ISOMERIZATION IN FOOD PROCESSING Nguyen and Schwartz17 assessed the effect of several different heat treatments on lycopene’s isomeric distribution in a variety of tomato products (Table 15.4). Schierle et al.18 showed that heating tomato-based food in oil caused increased lycopene isomerization than heating in water as shown in Table 15.5. Lycopene isomerization

TABLE 15.4 Lycopene Isomers in Various Thermally Processed Tomato Products Sample

Total Lycopene (mg/100g, dry basis)

Cis Isomers (%)

Peeled tomato Tomato juice (hot-break) Tomato juice (retorted) Tomato (whole, retorted) Tomato paste (concentrated) Tomato paste (retorted) Tomato soup (retorted) Tomato sauce (retorted)

149.89 161.23 180.10 183.49 174.79 189.26 136.76 73.33

5.37 5.98 3.56 3.67 5.07 4.07 4.34 5.13

Source: Data from Nguyen and Schwartz.35

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TABLE 15.5 Effect of Heating Treatment on Lycopene Trans to Cis Isomerizationin Aqueous and Oily Dispersions of Tomato Paste (70°C) Heating Time (min)

All-trans (%)

5-cis (%)

9-cis (%)

15-cis (%)

Other cis (%)

In Water 0 15 30 60 120 180

92.6 92.3 88.1 87.1 86.2 83.4

4.5 4.4 5.1 5.2 5.5 6.1

0.9 0.9 2.1 2.2 2.7 3.6

1.6 1.6 2.3 2.7 2.6 3.2

0.5 0.5 2.5 3.0 3.1 3.8

In Olive Oil 0 30 90 120 180

87.4 85.2 83.5 80.3 76.7

4.8 5.8 6.2 7.0 8.1

4.3 5.5 5.9 6.9 8.8

3.0 2.9 3.3 3.2 3.1

0.5 0.5 1.2 2.6 3.3

* Reprinted from Food Chem., 59(3), Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and Schuep, W., “Content and isomeric ratio of lycopene in food and human blood plasma,” 459–465, 1996, with permission from Elsevier. Source: Data from Schierle et al.15

and the amount of cis isomers increased as a function of processing time during heating of tomatoes. Heat treatment clearly increased the percentage of the cis isomers. It is obvious from these results that food processing can enhance cis isomerization in tomato-based foods. This indicates that not only the duration and temperature of heat treatment, but also the food matrix components such as oil or fat, influence lycopene isomerization. It was observed that lycopene loss and the rate of thermal isomerization was less while heating tomato pulp than when heating pure lycopene in an organic solution . The tomato tissue matrix may offer protection for lycopene. It is generally accepted that the all-trans form of lycopene has the highest stability and the cis isomers have the lowest stability. Bioactive potency depends on the extent of isomerization and oxidation as well as the stability.4,28,29 Thus isomerization would lead to degradation of lycopene. Although the typical processes such as cooking, freezing, or canning do not usually cause significant changes in total lycopene content, it is widely assumed that lycopene undergoes isomerization upon thermal processing. Heat, light, acids, and other factors have been reported to cause isomerization of lycopene.15,20,35 The changes in lycopene content and the distribution of trans-cis isomerization will result in a reduction in biological potency when lycopene concentrates are subjected to processing.28,29,30 The key question now is how to maintain high bioactive properties during food processing and storage. The isomerization and oxidation of lycopene greatly depends on the treatments used since each treatment produces a different form of energy

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All-trans-isomers

Cis-isomers

Oxidized products

FIGURE 15.7 Pathway of lycopene degradation.

(such as heat and light, respectively). It was found that the light irradiation caused more losses in total lycopene than the heating treatment. In all treatments, the rate of trans-isomer loss was greater than the cis-isomer formation, which suggests that degradation through oxidation of lycopene was the predominant mechanism, rather than the isomerization of trans isomer to cis isomer. The losses could be coupled to the conversion of trans- to cis-isomers, followed by the direct degradation into smaller oxidized by-products which are not isomers of lycopene.36 Because lycopene is a highly unsaturated molecule, comprising many conjugated double bonds, it is very susceptible to oxidation. The pathway of lycopene degradation is described in Figure 15.7. Bioactive potency depends on the content of total lycopene and the extent of isomerization.4,28,29 Thus isomerization can lead to a change in the bioactivity of lycopene. Characterization and quantification of lycopene isomers would provide a better understanding of the potential bioactive properties and health benefits of the lycopene-based products. Controlling isomerization and oxidation of lycopene during the preparation of a lycopene-based food would be of benefit in improving product quality. A true assessment of the relationship between nutritional quality and health benefits of dietary lycopene depends not only on the total lycopene content, but also on the distribution of lycopene isomers. Characterization and quantification of lycopene isomers would provide better insight into the potential nutritional quality and healthy benefit of the processed tomato products, and more accurately predict the lycopene bioactivity than just a total lycopene content with no knowledge of its isomeric composition. The control of lycopene oxidation and isomerization during production and storage can be of benefit in improving the retention of biological activity and health-promoting effect. Lycopene isomers in various thermally processed tomato products are listed in Table 15.6.

15.5 SUMMARY Consumers, researchers and the leaders in the food industry have become dramatically more interested and aware of the health benefits of lycopene from tomatoes. Lycopene in the human diet has significant protective and beneficial physiological effects, because lycopene has many unique and distinct biological properties. Among these is its strong antioxidant activity which plays an important role in improving human health and providing protection against a broad range of epithelial cancers. The antioxidant activities of lycopene and other carotenoids are highlighted by their singlet oxygen quenching properties and their ability to trap peroxyl radicals.

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TABLE 15.6 Lycopene Isomers in Various Commercial Tomato Products

Sample

Total Lycopene (mg/100g All-trans 5-cis 9-cis 13-cis Other cis wet basis) (%) (%) (%) (%) (%)

Tomato paste (“Tomatenmark,” Panocchia, Italy) Tomato paste (“'Maracoli,” Kraft, Germany) Tomato Ketchup (“Hot Ketchup,” Del Monte, Italy) Tomato Ketchup (“Hot Ketchup,” Heinz, USA) Instant Meal (“Eier-Ravioli,” Hero, Switzerland) Sauce (“Hamburger Relish,” Heinz, The Netherlands) Sauce (“Sauce Bolognaise,” Barilla, Italy) Canned tomatoes (“Chris,” Roger Sud, Italy)

52

96

4

<1

<1

<1

3.7

91

5

1

2

<1

9.5

88

7

2

3

1

3.0

77

11

5

7

1

0.6

76

8

5

6

5

3.0

93

5

<1

3

<1

9.2

67

14

6

5

8

7.1

84

5

3

5

3

* Reprinted from Food Chem., 59(3), Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and Schuep, W., “Content and isomeric ratio of lycopene in food and human blood plasma,” 459–465, 1996, with permission from Elsevier. Source: Data from Schierle et al.15

Lycopene is known to exist in a variety of geometric forms, such as the all-trans, mono-cis, and poly-cis isomers. The all-trans isomer of lycopene is the most predominant geometrical isomer in fresh tomatoes. Lycopene can undergo trans-to-cis isomerization during tomato processing and storage. Lycopene can undergo at least two changes during tomato processing, that is, isomerization and oxidation. Lycopene may be partially destroyed in processed tomato products by heat, light, oxygen, and by the presence of metallic ions. The opposite reaction, the conversion of the cis isomer to the trans- form can occur during the storage of the product. Cis isomers are less stable than the all-trans isomer. Lycopene is a very efficient antioxidant that quenches highly reactive singlet oxygen (O2.) and traps peroxyl radicals (ROO.). In processed tomato products, oxidation is a complex process that depends upon many factors, such as processing conditions, moisture, temperature, and the presence of pro- or antioxidants.Certain steps can be taken to minimize lycopene degradation during food processing and storage. Products containing lycopene need to be protected from excessive heat, extreme pH conditions, exposure to oxygen, and light. Processing technology should be optimized to prevent lycopene oxidation. The reduction of lycopene content and trans-cis isomerization result in a reduction in biological activity. It is generally accepted that the all-trans form of lycopene has the highest stability and the cis isomers have the lowest stability. Bioactive potency depends on the extent of isomerization and oxidation. A true assessment of the

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nutritional quality and healthy benefit of processed tomato-based food depends not only on the total lycopene content, but also on the distribution of lycopene isomers. Lycopene can undergo degradation via isomerization and oxidation under different processing conditions, which impacts on its bioactivity and reduces its health-promoting functionalities. How to maintain the high bioactive properties of lycopene during food processing and storage is a key question. Controlling isomerization and oxidation of lycopene during tomato processing or other lycopene-based food products can improve product quality. The mechanism of lycopene destruction depends on many parameters during food processing and storage. The main cause of damage to lycopene during food processing and storage is oxidation. Low storage temperature, low oxygen content, low light, low water activity, and low moisture content will help to limit the oxidation of lycopene.

REFERENCES 1. Holden, J.M., Eldridge, A.L., Beecher, G.R., Buzzard, I., Marilyn, B., Seema, C.S., Douglass, L.W., Gebhardt, Susan H.D., and Schakel, S., Carotenoid content of U.S. foods: an update of the database, J. Food Composit. Analy., 12, 169, 1999. 2. Rao, A.V., Waseem, Z., and Agarwal, S., Lycopene content of tomatoes and tomato products and their contribution to dietary lycopene, Food Res. Int., 31, 737, 1998. 3. Rao, A.V. and Agarwal, S., Role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: a review, Nutr. Res., 19, 305, 1999. 4. Sies, H., Stahl, W., and Sundquist, A.R., Antioxidant function of vitamins; vitamins E and C, -carotene, and other carotenoids, Ann. N. Y. Acad. Sci., 669, 7, 1992. 5. Micozzi, M.S., Beecher, G.R., Taylor, P.R., and Khachik, F., Carotenoid analyses of selected raw and cooked foods associated with a lower risk for cancer, J. Natl. Cancer Inst., 82, 282, 1990. 6. Olson, J., Carotenoid, vitamin A and cancer, J. Nutr., 116, 1127, 1986. 7. Levy, J., Bisin, E., Feldman, B., Giat, Y., Minster, A., Danilenko, M., and Sharoni, Y., Lycopene is a more potent inhibitor of human cancer cell proliferation than either β-carotene or -carotene, Nutr. Cancer, 24, 257, 1995. 8. Di Mascio, P., Kaiser, S., and Sies, H., Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Arch. Biochem. Biophys., 274, 532, 1989. 9. Conn, P.F., Schalch, W., and Truscott, T.G., The singlet oxygen and carotenoid interaction. J. Photochem. Photobiol. B. Biol., 11, 41, 1991. 10. Stahl, W. and Sies, H., Uptake of lycopene and its geometrical isomers is greater from heat-processed than from unprocessed tomato juice in humans, J. Nutr., 122, 2161, 1992. 11. Di Mascio, P., Murphy, M.C., and Sies, H., Antioxidant defense systems, the role of carotenoids, tocopherols and thiols, Am. J. Clin. Nutr. Suppl., 53, 194, 1991. 12. Conn, P.F., Lambert, C., Land, E.J., Schalch, W., and Truscott, T.G., Free Radicals, Res. Commun., 16, 401, 1992. 13. Miller, N.J., Sampson, J., Candeias, L.P., Bramley, P.M., and Rice-Evans, C.A., Antioxidant activities of carotenes and xanthophylls, FEBS Lett., 384, 240, 1996. 14. Burton, G.W. and Ingold, K.U., -carotene: an unusual type of lipid antioxidant, Science, 224, 569, 1984.

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15. Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and Schuep, W., Content and isomeric ratio of lycopene in food and human blood plasma, Food Chem., 59, 459, 1996. 16. Tavares, C.A. and Rodriguez-Amaya, D.B., Carotenoid composition of Brazilian tomatoes and tomato products, Lebensm.-Wiss. U.–Technol., 27, 219, 1994. 17. Nguyen, M.L. and Schwartz, S.J., Lycopene: chemical and biological properties, Food Technol., 53,38, 1999. 18. Shi, J., LeMaguer, M., Bryan, M., Kakuda,Y., Kinetics of lycopene degradation in tomato puree by heat and light irradiation, J. Food Proc. Eng., 25, 485, 2003. 19. Miki, N. and Akatsu, K., Effect of heating sterilization on color of tomato juice, J. Japan. Food Ind., 17, 175, 1970. 20. Shi, J., Wu, Y., Bryan, M., and LeMaguer, M., Oxidation and isomerization of lycopene under thermal treatment and light irradiation in food processing, Nutraceut. Food, 7, 179, 2002. 21. Agarwal, A., Shen, H., Agarwal, S., and Rao, A.V., Lycopene content of tomato products: its stability, bioavailability and in vivo antioxidant properties, J. Med. Food, 4, 9, 2001. 22. Ax, K., Mayer-miebach, E., Link, B., Schuchmann, H., and Schubert, H., Stability of lycopene in oil-in-water emulsions, Eng. Life Sci., 4, 199, 2003. 23. Zanoni, B., Peri C., Nani, R., and Lavelli, V., Oxidative heat damage of tomato halves as affected by drying, Food Res. Int., 31, 395, 1999. 24. Sharma, S.K. and LeMaguer, M., Kinetics of lycopene degradation in tomato pulp solids under different processing and storage conditions, Food Res. Int., 29, 309, 1996. 25. Anguelova, T. and Warthesen, J., Lycopene stability in tomato powders, J. Food Sci., 65, 67, 2000. 26. Lee, M.T. and Chen, B.H., Stability of lycopene during heating and illumination in a model system, Food Chem., 78, 425, 2002. 27. Kanasawud, P. and Crouzet, J.C., Mechanism of formation of volatile compounds by thermal degradation of carotenoids in aqueous medium, 2, Lycopene degradation, J. Agric. Food Chem., 38, 1238, 1990. 28. Emenhiser, C., Sander, L.C., and Schwartz, S.J., Capability of a polymeric C30 stationary phase to resolve cis-trans carotenoids in reversed phase liquid chromatograph, J. Chromatogr. A., 707, 205, 1995. 29. Wilberg, V.C. and Rodriguez-Amaya, B.D., HPLC quantitation of major carotenoids of fresh and processed guava, mango and papaya, Lebensmittel-Wissenschaft und Technol., 28, 474, 1995. 30. Stahl, W. and Sies, H., Perspectives in biochemistry and biophysics, Arch. Biochem. Biophys., 336, 1, 1996. 31. Henry, L.K., Catignani, G.L., and Schwartz, S.J., Oxidative degradation kinetics of lycopene, lutein, 9-cis and all-trans beta-carotene. J. Am. Oil Chem. Soc., 75, 823, 1998. 32. Ribeiro, H.S., Ax, K., and Schubert, H., Stability of lycopene emulsions in food systems, J. Food Sci., 68, 2730, 2003. 33. Anguelova, T. and Warthesen, J., Lycopene stability in tomato powders, J. Food Sci., 65, 67, 2000. 34. Abushita, A.A., Daood, H.G., and Biacs, P.A., Change in carotenoids and antioxidant vitamins in tomato as a function of varietal and technological factors, J. Agric. Food Chem., 48, 2075, 2000. 35. Nguyen, M.L. and Schwartz, S.J., Lycopene stability during food processing, Exp. Biol. Med., 218, 101, 1998.

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36. Boskovic, M.A., Fate of lycopene in dehydrated tomato products: carotenoid isomerization in food system, J. Food Sci., 44, 84, 1979.

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Fruits with High Antioxidant Activity as Functional Foods Shiow Y. Wang U.S. Department of Agriculture

CONTENTS 16.1 Introduction ..................................................................................................372 16.2 Antioxidants in Fruits ..................................................................................373 16.3 Effect of Preharvest Factors.........................................................................374 16.3.1 Genotypes and Maturity...................................................................374 16.3.2 Geographical Variation and Year-to-Year Differences.....................375 16.3.3 Climate, Temperature, and Light .....................................................376 16.3.4 Cultural Practices .............................................................................376 16.4 Effect of Postharvest Handling....................................................................378 16.4.1 Storage Duration and Temperature..................................................378 16.4.2 Controlled Atmospheres...................................................................380 16.4.3 Heat Treatment and Irradiation........................................................383 16.4.4 Treatment with Natural Compounds or Chemicals.........................385 16.5 Effect of Processing .....................................................................................386 16.5.1 Heat ..................................................................................................387 16.5.2 Freezing ............................................................................................389 16.5.3 Sugar, Ascorbic Acid, and Other Chemicals ...................................389 16.5.4 Drying and Dehydration ..................................................................391 16.5.5 Extraction Techniques ......................................................................392 16.5.6 High Pressure ...................................................................................393 16.5.7 Microwave and pH...........................................................................394 16.5.8 Oxygen and Light ............................................................................395 16.5.9 Enzymes ...........................................................................................395 16.6 Genetic Engineering Techniques .................................................................396 16.7 Conclusions ..................................................................................................397 References..............................................................................................................398

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16.1 INTRODUCTION Foods that we eat supply our bodies with nutrients essential for good health. Foods that are classified as functional foods contain components that impart health benefits beyond basic nutrition. An example of a functional food is fruits, which are good sources of antioxidants. In addition to the usual nutrients such as vitamins and minerals, fruits are also rich in anthocyanins, flavonoids, and phenolic acids. Today’s consumers are more educated and increasingly demand functional foods. Though the mechanisms through which they work are not always clear, when eaten on a regular basis as part of a varied diet, functional foods may lower the risk of developing certain diseases. It is estimated that more than 5000 phytochemicals have been identified. However, a large percentage remains unknown and needs to be identified before their health benefits are fully understood.1 Some of these beneficial chemicals appear to work alone. Others may work in conjunction with vitamins and other nutrients to stimulate protective enzymes or to block various hormonal actions and metabolic pathways that are associated with the development of cancer and heart disease. Active oxygen species are generated as by-products of normal metabolism. Exposure to free radicals from external sources such as cigarette smoke, pollutants, chemicals, and environmental toxins may enhance the production of active oxygen species. Increased levels of these active oxygen species or free radicals can create oxidative stress, which leads to impairment of metabolism and DNA damage. The different antioxidant components found in fruits provide protection against harmful free radicals and have been associated with lower incidence and mortality rates of various diseases, in addition to a number of other health benefits.2,3 Fruits are considered excellent functional foods because they contain high levels of natural antioxidants such as carotenoids, vitamins, phenols, flavonoids, dietary glutathionine, and endogenous metabolites. These antioxidants are capable of performing a number of functions including free radical scavengers, peroxide decomposers, singlet and triplet oxygen quenchers, enzyme inhibitors, and synergists.4 Antioxidants can also delay or prevent the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidizing chain reactions. Growing evidence suggests that eating more fruits and vegetables can aid in preventing stomach, lung, mouth, esophagus, colon and rectal cancers. The incidence of other chronic diseases, such as coronary heart disease, atherosclerosis, and stroke may also be reduced through increased fruit and vegetable consumption. Fruits and vegetables may also play a preventive role in birth defects, cataract formation, hypertension, asthma, diverticulosis, obesity, and diabetes.5−9 Excellent reviews on the health benefits of fruits and vegetables are contained in recent articles by Birt et al.,10 Heber,11 Le Marchand,12 Wise,13 and IARC handbooks of cancer prevention.14 Research is in full force to find the best genotypes, optimum cultural practices, and most desirable postharvest handling and processing techniques to enhance and preserve the natural antioxidants in fruits and vegetables for their use as functional foods. This chapter summarizes the antioxidants in various fruits and describes the factors affecting these antioxidant content and activities. The possible use of genetic engineering and biotechnology techniques to enhance phytonutrients in fruits is also discussed.

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16.2 ANTIOXIDANTS IN FRUITS Antioxidants are compounds that can delay or inhibit the oxidation of lipids, nucleic acids, or other molecules by inhibiting the initiation or propagation of oxidizing chain reactions.15 In general, there are two basic categories of antioxidants, natural and synthetic. Recently, interest has been increasing considerably in finding naturally occurring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their potential carcinogenicity.16 Fruits are good sources of natural antioxidants and contain thousands of phytonutrients.7,9,17 The phytonutrients in fruits responsible for antioxidant activity can largely be attributed to phenolic compounds such as anthocyanins, phenolics, and to other flavonoid compounds. These compounds may act independently or in combination as anticancer or cardio-protective agents by a variety of mechanisms. The antioxidant activities of phenolic compounds are mainly due to their redox properties which can play an important role in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxide.4 Fruits showed a remarkably high scavenging activity of chemically generated active oxygen species. The range of hydrophilic antioxidant capacities (H-ORACFL) was large among the different fruits, such as prunes, raisins, blackberries, black currants, blueberries, raspberries, strawberries, grapes, pomegranates, and some varieties of apples.18−22 H-ORACFL for all of the melons was relatively low. Among the small fruits, black raspberries and blackberries have higher antioxidant activities than red raspberries, while strawberries generally have lower values of total antioxidants.21 Häkkinen et al.23 also found that extracts of crowberry, cloudberry, whortleberry, cowberry, aronia, rowanberry, and cranberry had high antioxidant activities, while red currant, black currant, and strawberry had relatively low activities. These results correlate with the findings that black raspberries and blackberries contain high amounts of cyanidin glycosides, a strong antioxidant, while strawberries contain pelargonidin 3-glucoside and ascorbic acid, which are weak antioxidants.24−26 Among the anthocyanins, the relative antioxidant strength in preventing oxidation of human low-density lipoprotein is as follows: delphinidin > cyanidin > malvidin > pelargonidin.27 Among the dried fruits, such as figs, raisins, and dates, the H-ORACFL value was about 30 µmol TE/g, whereas those of prunes were nearly three times higher.19,20 Major antioxidant compounds in prunes are caffeoylquinic acid isomers, which have been shown to have a high antioxidant capacity.28,29 There is also a large range for lipophilic antioxidant capacities (L-ORACFL) for different fruits. Compared to H-ORACFL in fruits, L-ORACFL values were generally low.19,20 L-ORACFL values for the dried fruits were usually ranged from 0.30 to 1.8 µmol of TE/g. L-ORACFL values for fresh fruits could be as low as 0.07 µmol of TE/g fresh weight for tangerines and as high as 5.52 µmol of TE/g fresh weight in avocados. Avocado contains linoleic acid and vitamin E, but which lipophilic constituents contribute to the high lipophilic antioxidant capacity is largely unknown.19,20 Among all berry fruits, cranberries, raspberries, and blackberries have the highest L-ORACFL values with >1µmol of TE/g fresh weight.19,20 The peroxyl radical is the most common free radical in human biology, but radicals of superoxide radicals (O2• –), hydrogen peroxide (H2O2), hydroxyl radicals

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(•OH), and singlet oxygen (1O2) are all present in biological systems. However, relatively little information is available on the scavenging of these radicals. Fruits such as blackberries, blueberries, cranberries, raspberries, and strawberries not only possess peroxyl radical (ROO•) absorbance capacity, but also have antioxidant activities against superoxide radicals (O2• –), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2).30 Different species or different cultivars showed varying degrees of scavenging capacity on different active oxygen species (O2•-, H2O2, •OH, and 1O2). Among all the species and cultivars, blackberries had the highest inhibition of active oxygen species (O2• –, H2O2, •OH, and 1O2) production.30 Antioxidant enzyme activities are usually positively correlated to antioxidant activities. The production of antioxidant enzymes in fruits is a complex process that is not yet totally understood. The antioxidant enzyme defense system consists of hundreds of different substances and mechanisms. The main antioxidant enzymes in fruits are catalase, glutathione-peroxidase (GSH-POD), superoxide dismutase (SOD), guaiacol peroxidase (G-POD), ascorbate peroxidase (AsA-POD), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR).31 Antioxidant enzymes have the capacity to lower the free radical burden and neutralize excess free radicals created by stress conditions. Antioxidant enzymes serve as catalysts that can act at one or more of the three stages of free radical formation: initiation, propagation, and termination. Therefore, it is possible that antioxidant enzymes can prevent cellular and tissue damage in the human body.

16.3 EFFECT OF PREHARVEST FACTORS 16.3.1 GENOTYPES

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Genetic factors play an important role in determining antioxidant capacity in crops. For many crops, a large variety of genotypes exist and thus there is potential for genetic variability relating to antioxidant activity. The influence of variety on the content of phenolic compounds and antioxidant activity in the berries of the genus Vaccinium, Ribes, Rubus, and Aronia have been studied by several researchers.15,18,21,32−47 In blackberry, ‘Hull Thornless’ cultivar yields higher antioxidant values (ORAC) compared to ‘Chester Thornless,’ and ‘Triple Crown’ cultivars. For raspberry fruit, ‘Jewel’ (a black raspberry) has higher values compared to red raspberry cultivars (‘Autumn Bliss,’ ‘Canby,’ ‘Sentry,’ and ‘Summit’). ORAC, anthocyanin, and total phenolic contents are also different among the different cultivars of strawberry fruits. ‘Earliglow’ has the highest ORAC value. There is a linear relationship between both ORAC and total phenolics and between ORAC and anthocyanins of ripe berry fruits.18,21 Various species and cultivars of blueberry and bilberry also have different antioxidant capacities.18 The effect of genotype variation on antioxidant activities has also received much attention. in grapes,48−50 prunes, and plums,51−55 sea buckthorn,56,57 apples,58–61 sweet cherries,62 citrus,63 guavas,64 and nectarines and peaches.54 The cultivated varieties of apricot possess significantly higher vitamin C, vitamin E, β-carotene, and selenium contents than the wild type.65 The antioxidant potential is significantly

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higher in the grapefruit hybrids than in white grapefruit.66 Different cultivars of clingstone peaches also have different antioxidant activities.67 Antioxidant capacity also varies considerably with different stages of maturity. Many phytonutrients are synthesized in parallel with the overall development and maturation of fruits. Fully ripened fruits offer not only the ultimate in taste and flavor but also maximum color intensity and high antioxidant activities. Blackberry, raspberry, and strawberry fruits harvested during their ripe stage consistently yield higher antioxidant values than those harvested during the pink stage.21 Unripe strawberries contain lower concentrations of chlorogenic acid, p-coumaric acid, quercetin, and kaempferol compared with ripe strawberries.68 Immature blueberries harvested at an early stage (immediately after turning blue) have lower ORAC values and total anthocyanin content when compared to more mature blueberries harvested 49 days later.18 Kalt et al.69 found anthocyanin content was substantially higher in highbush blueberries of more advanced stages of ripeness than in berries of less ripeness. In grapes, the content of all polyphenolics generally increase as fruit ripen, the highest concentrations being found in the skins.48 Free ellagic acid, ellagic acid glycosides, and total ellagic acid increase during maturation in the skin of grapes.48 Antioxidant activities also increase with ripening in Rubus L. hybrids.43 Ripening also results in an increase of total antioxidant activity and vitamin C content in both peel and flesh of ‘yuzu’ citrus.70 However, while the amounts of all total phenolics, hesperidin, and naringin increased with ripening in peel, they decreased slightly in flesh.70 Both ascorbic acid and total carotenoids increase with maturation and ripening in ‘Clingstone’ peaches and ‘Solo’ papayas.71,72

16.3.2 GEOGRAPHICAL VARIATION DIFFERENCES

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The effect of geographical variation and year-to-year difference on the antioxidant capacity of various fruits is well described and is likely due to environmental conditions. Anthocyanin production can be stimulated by cold growing conditions.73 Abiotic conditions (temperature, moisture, irradiation, soil fertility) can vary markedly from year to year and affect the content of phenolic components.74 Kalt and McDonald75 found that seasonal variation in anthocyanin content among lowbush blueberry cultivars over seven seasons was quite marked in fruit harvested from the same site. Anthocyanin content varied by up to 2.4-fold for ‘Blomidon,’ 1.8-fold for ‘Cumberland,’ and 2.0-fold for ‘Fundy.’75 Connor et al.76 found that antioxidant content varied significantly in blueberry fruit harvested in Minnesota, Michigan, and Oregon among the cultivars across location and years. ‘Bluegold’ and ‘Northland’ had the highest overall antioxidant activity in Minnesota and Oregon. The antioxidant activities of blueberries were substantially lower in Michigan than in Minnesota and Oregon. Prior et al.18 found no substantial difference in antioxidant activities in blueberry ‘Jersey’ harvested at sites in Oregon, Michigan, and New Jersey in a single year, but Connor et al.76 showed differences between these locations and also showed variability between years. This may reflect differences in climate and cultural practices among locations, including differences in ultraviolet radiation, temperature, or water stress, or mineral nutrient availability.76

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Different origins and harvesting dates affect vitamin C, tocopherols, tocotrienols, and phytosterols content in sea buckthorn.56,57 Apricot fruit from different geographical regions had different vitamin C content. Cultivars of apricot grown in highaltitude regions had significantly higher vitamin C content than the same cultivars grown in other regions.65 Goncalves et al.77 found that flavonol and flavan-3-ol contents in cherry fruit remained quite constant, but the level of phenolic acids were higher in 2001 and anthocyanin levels were higher in 2002. Ascorbic acid content in strawberries is also highly affected by climate conditions and growing area.78 Olsson et al.68 showed some variation in antioxidant activity, ascorbic acid content, and ellagic acid content between 1999 and 2000 within each strawberry cultivar. The total folate content in 13 strawberry cultivars harvested in 1999 and 2001 were higher than in the harvest from 2000. These data suggest a significant influence of climatic conditions on these compounds and antioxidant activity. The effect of geographical variation and year-to-year difference on ORAC values will be described more in the following sections.

16.3.3 CLIMATE, TEMPERATURE,

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Growing temperature and light intensity have been shown to influence the nutrient content of fruits, especially carotenoids, thiamine, ascorbic acid, and riboflavin. Increased light intensity resulted in higher ascorbic acid content in strawberry fruit than of the same varieties produced under lower light intensity conditions.71 Strawberries grown under high temperature conditions showed significantly higher content of flavonoids and antioxidant capacities. Plants grown in cool day and night temperatures generally had the lowest antioxidant capacity in fruit.79 Month-to-month variability in vitamin C content dependent on growing conditions has been documented in processed Florida citrus products such as orange and grapefruit juices.80 One explanation for this difference could be related to different flavonoid concentrations.79 The composition of flavonols in red raspberry juice was also influenced by cultivar, processing, and environmental factors.81

16.3.4 CULTURAL PRACTICES Soil types, composts, mulching, and fertilization influence the water and nutrient supply to the plant and can affect the nutritional composition and antioxidant activity of the harvested fruit. Increasing the nitrogen and/or phosphorus supply to citrus trees results in somewhat lower acidity and ascorbic acid content in citrus fruits, while increasing potassium fertilization increases their acidity and ascorbic acid content.82 Strawberry plants grown with different soil nutrients also show differences in ascorbic acid content. Plants grown in low-organic-matter and low-cationexchange-capacity sandy soil amended with calcium, magnesium, and nitrogen produce more ascorbic acid in their fruit than plants without supplemental fertilizer.83 In green-flesh honeydew muskmelons, the total ascorbic acid and folic acid are higher when grown on clay loam vs. sandy loam soil.84 Sandy loam soil produces less β-carotene as compared with sandy loam soils in orange-fleshed muskmelon fruit.85

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Cultural practices, such as conventional, organic cultivations or use of compost as a soil supplement can all affect antioxidant capacity in fruits. Compost as a soil supplement increases organic matter in the soil, which also enhances antioxidant content in strawberries. Compost significantly enhances content of ascorbic acid (AsA), glutathione (GSH), and the ratios of AsA/dehydroascorbic acid (DHAsA) and the GSH/oxidized glutathione (GSSG) in fruit of strawberry.86 The oxygen absorbance capacity for peroxyl radical, as well as the superoxide radical, hydrogen peroxide, the hydroxyl radical, and singlet oxygen in strawberries increases significantly with increasing compost use.86 Plants grown with compost yield fruits with high levels of phenolics, flavonol, and anthocyanin content.86 It is possible that compost causes changes in soil chemical and physical characteristics, increases beneficial microorganisms, and increases nutrient availability and uptake, thus favoring plant and fruit growth. Woese et al.87 reported that the optimal conditions for plant growth generally result in the highest levels of antioxidants. Using different mulches for growing strawberries also affects strawberry fruit quality. Different mulches probably lead to differences in canopy temperature, soil temperature, and moisture content, and the quantity and quality of light transmitted, reflected, or absorbed. These differences in turn affect plant growth, development, fruit quality, and carbohydrate metabolism in strawberry plants. Fruit from a hill plasticulture (HC) production system have higher flavonoid content and antioxidant capacities compared to fruit from plants grown in a matted row (MR) system.88 In general, phenolic acid and flavonol content, as well as cyanidin- and pelargonidinbased anthocyanins and total flavonoids are greatest in the HC system. Fruits from plants grown in the MR system generally have the lowest content of phenolic acids, flavonols, and anthocyanins. Fruit grown under HC conditions have the highest peroxyl radical absorbance capacity.88 Although food products from organic farming are believed to be healthier than the corresponding conventional foods, clear experimental evidence supporting this assumption is still lacking and assessment of the nutritional potential of these products requires further research.87 Carbonaro et al.89 found a parallel increase in polyphenol content and polyphenoloxidase activity of organic peaches and pears compared with the corresponding conventional samples. Ascorbic and citric acids were higher in organic than conventional peaches and α-tocopherol also increased in organic pears. Lombardi-Boccia et al.90 grew plums in conventional (tilled soil) and organic cultivations (soil covered with trifolium, and soil covered with natural meadow) and found that differences in macronutrients were marginal, whereas antioxidant vitamins and phenolic compounds concentration markedly differed among cultivations. Ascorbic acid, α-, γ-tocopherols, and β-carotene were higher in organic plums grown on soil covered with natural meadow. The highest phenolic acid content was detected in plums grown on soil covered with trifolium. Total polyphenols and quercetin were higher in conventional plums, but myrecitin and kaempferol were higher in organic plums. Asami et al.91 showed that higher levels of total phenolics were consistently found in organically and sustainably grown cultivations of marionberry and strawberry as compared to those produced by conventional agricultural practices. These data provide evidence that an improvement in the antioxidant defense system of the plant occurred as a consequence of the organic cultivation practice. Therefore, the type of soil

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management turned out to be of primary importance in influencing the concentration in these health-promoting compounds. Carbon dioxide concentrations may also have an effect on antioxidant capacity. Higher CO2 (300 and 600 µmol mol–1 above ambient) concentrations in the field result in increases in anthocyanins, phenolics, AsA, GSH, and ratios of AsA to DHAsA and GSH to GSSG in strawberry fruit.92 These increases are associated with increased antioxidant capacity and increased removal of free radicals. Growing strawberry plants under CO2 enrichment conditions significantly enhances fruit flavonoid content as well.92 Fruit of strawberry plants grown in CO2 enrichment conditions also have higher oxygen radical absorbance activity against ROO•, O2• –, H2O2, •OH, and 1O2 radicals.92 This indicates that strawberry fruit grown with CO2 enrichment have high scavenging activity for chemically generated active oxygen species. Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MJ) are a class of oxylipins derived from the lipoxygenase-dependent oxidation of fatty acid. Both compounds have been found to occur naturally in a wide range of higher plants. JA/MJ play key roles in plant growth and affect a great diversity of physiological and biochemical processes.93 Preharvest spray of MJ significantly enhances anthocyanin, total phenolic, flavonoid content, and antioxidant capacity in raspberries.94 MJ results in stimulation of anthocyanin biosynthesis in strawberry ripening.95 MJ vapor also promotes β-carotene synthesis and chlorophyll degradation in Golden Delicious apple peel.96

16.4 EFFECT OF POSTHARVEST HANDLING 16.4.1 STORAGE DURATION

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Concentrations of nutrients in fruits tend to decrease during postharvest handling and storage. Losses of vitamins and other nutrients in fruits vary by nutrient, type of fruit, physical damage, temperature, and storage environment. Fresh fruits are perishable because they are composed of living tissues. These tissues must be kept alive and healthy throughout marketing. Respiration is one of the essential processes of living cells. High respiration rates reflect fast consumption of energy and rapid depletion of reserves such as carbohydrates, organic acids, amino acids, proteins, fatty acids, and lipids. Refrigeration is the best method of slowing down respiration and retarding the deterioration. Strawberries respire about 10 times as fast at 20°C as at 0°C.71 High temperature also increases moisture loss and promotes growth of decay-causing microorganisms. Therefore, rapid removal of field heat and prompt cooling of the harvested fruits by applying precooling procedures immediately following harvest are essential for quality maintenance. Postharvest handling can also affect phytonutrient levels in fruits. Most fruits reach their maximum vitamin and nutrient content when fully mature. However, many of them are usually harvested at a slightly immature stage with firmer texture in order to facilitate handling and transportation and to minimize mechanical damage of the commodities. Therefore, those fruits could continue to mature and increase in antioxidant and nutrient values in storage.

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In cranberries, postharvest storage temperatures between 0°C and 15°C increase antioxidant capacity, anthocyanins, and total phenolic content.97 Strawberries stored at 4°C retain vitamin C well but less chlorgenic acid and quercetin.68,98,99 Kalt et al.99 found that anthocyanin concentration and surface color increases in strawberries during storage. Temperature, and to a lesser extent light, affects the rate of strawberry color development. At 20°C anthocyanins accumulate rapidly and at 30°C the pigments form more slowly than at 20°C and there is a significant increase in the surface color rating of white-harvest berries after storage in the light.99 Gil et al.26 reported that the content of ellagic acid in strawberries increase slightly during storage for five days in 5°C. Strawberries and raspberries stored at temperatures greater than 0°C also result in an increase in antioxidant capacity, anthocyanin, and total phenolic content, and the magnitude of this increase is related to storage temperature. There are no ascorbate losses in strawberries or highbush blueberries during eight days of storage at the various temperatures.100 Increases in anthocyanin content during storage have also been reported for lowbush blueberries (V. angustifolium Aiton),75 rabbiteye blueberries (V. asheii Reade),101 pomegranates,102 highbush blueberries,100 and other cultivar of strawberries.103−−105 Connor et al.106 demonstrate that increases in antioxidant activity, total phenolic, and anthocyanins content in blueberry during cold storage are cultivar dependent. Tomas-Barberan et al.105 found an increase in anthocyanin concentration in nectarine, cherry, grape, and strawberry fruits and an increase in ellagic acid in strawberries during air storage whereas flavonoids and hydroxycinnamic acid derivatives remained constant. Goncalves et al.77 studied the changing of cherry phenolic profiles during storage at 15 ± 5°C and 1°C to 2°C and found phenolic contents generally decreased with storage at 1°C to 2°C and increased with storage at 15 ± 5°C. Anthocyanin levels increased at both temperatures. In ‘Van’ cherry, anthocyanin levels increased up 5-fold during storage at 15 ± 5°C (from 47 to 230 mg/100g of fresh weight). Kalt et al.69 found ORAC was positively correlated with total phenolic content, but not with anthocyanin in highbush blueberry cultivars Bergitta, Bluegold, and Nelson during ripening and storage. Kiwifruit slices stored at 5°C and 10°C exhibited a gradual decrease in ascorbic acid and an increase in dehydroascorbic acid content. The total vitamin C was 8%, 13%, or 21% lower than initial values in slices kept for six days at 0°C, 5°C, or 10°C, respectively.107 Piretti et al.108 found that the most important phenolics in ‘Granny Smith’ apple skin, epicatechin, quercetin glycosides, and procyanidins decreased from day 100 to the end of storage at day 205, both in air and controlled atmosphere (CA) storage (1.0% O2 + 2.0% CO2). Further decreases were found during one week of shelf life at 20°C. However, Curry109 found the level of antioxidants in ‘Delicious’ apple increased 2 to 10 times during the first two months of storage at −1°C and in ‘Granny Smith’ apples, whereas antioxidant content significantly decreased during the following four months of storage. Leja et al.110 found that increase of soluble peroxidase (POD) activity was much stronger in apple kept in air than in CA, while after subsequent seven-day storage at high temperature, a further increase in enzyme activity was observed in all treatments. Ju et al.111 found ‘Delicious’ and ‘Ralls’ apple, held for four or five months in cold storage, showed no changes in the concentrations of simple phenols, flavonoid, and anthocyanin. However, during seven days at 20°C following storage, simple phenols and flavonoids decreased rapidly.

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Coseteng and Lee112 have shown the total phenol concentration in apples is relatively stable during storage. Burda et al.113 reported that the concentration of epicatechin, procyanindin B2, and phloridzin in ‘Golden Delicious,’ ‘Empire,’ and ‘RI Greening’ in both the flesh and the skin remained relatively constant during six months of cold storage. Lin et al.114 found that cyaniding 3-galactoside in ‘Starkrimson’ apple stored at 2°C and 73% relative humidity for about seven months still remained constant. Reay115 also reported that both anthocyanin and quercetin glycoside concentrations did not change significantly in ‘Gala’ apple during storage at 1°C for about five months. Perez-Ilzarbe et al.116 found that in ‘Granny Smith’ apple stored at 4°C for 10 days, the concentration of phenolic compounds increased in the skin but not in the flesh. During rewarming the fruit at 22°C for 21 days and during cold storage, their concentration remained stable in the skin while it decreased in the flesh. Golding et al.117 found that individual phenolics in the peel of ‘Granny Smith,’ ‘Lady Williams,’ and ‘Crofton’ apples remained relatively stable after storage in air at 0°C up to nine months. However, Zhang et al.118 reported that flavan-3-ols monomers and dimers and cyanidin 3-glucoside decline in litchi fruit following storage at ambient temperature (20°C to 25°C) for seven days and at 4°C for 35 days. Postharvest treatment of strawberries with a high electric field has been reported to increase their shelf life.119 High electric field exposure for short periods was reported to suppress the respiration rate in some fruits and vegetables.120 Antioxidant capacity of ‘Irwin’ mango fruit stored at low temperature after high electric field pretreatment remained unchanged for 20 days of storage at 5°C, but decreased thereafter. Total phenol and carotenes increased during storage, and antioxidant capacity of fruits was significantly correlated to ascorbic acids in fruit.121

16.4.2 CONTROLLED ATMOSPHERES Controlled atmospheres (CA) means removal or addition of gases resulting in an atmosphere different from that of air. The potential benefits from this method are retardation of senescence, suppression of biochemical and physiological changes, reduction of physiological disorders (e.g., chilling injury) in some fruits, and retardation of microbial growth, maintenance of quality, and extension of storage life of fresh produce. This storage technique is considered a supplement to refrigeration. The storage life of certain fruits can be greatly extended by this storage technique. The potential benefit of using controlled atmosphere is dependent upon commodity, variety, physiological age, atmosphere composition, and temperature and duration of storage. Increasing the carbon dioxide concentration around fruits inhibits the postharvest increase in anthocyanin, by affecting its biosynthesis, degradation, or both.26,102,103 Carbon dioxide-enriched atmospheres (10% to 20%) are especially effective in retarding decay and softening of strawberries. However, exposure to high concentrations of carbon dioxide can adversely affect the color change in strawberry fruit.26 In strawberries, high carbon dioxide storage does not affect anthocyanin content in external tissues, but causes a reduction in red color intensity and a decrease in anthocyanin content of internal tissue of strawberry fruit. It is possible that high carbon dioxide causes an increase in pH which in turn affects the stability of

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anthocyanins. As carbon dioxide levels increase, the concentration of pelargonidin glycosides in the internal tissue decreases.26 In red raspberries, the ascorbic acid increases slightly while red pigment shows no significant changes after CA (10% O2 + 15% CO2, 10% O2 + 31% CO2) storage for seven days at 2°C.122 Agar et al.123 stored berry fruits in high CO2 (10% to 30% CO2), with or without a parallel reduction in O2, and found a decrease in vitamin C content, particularly in strawberries. This reduction in vitamin C was moderate in black currants and blackberries and almost absent in raspberries and red currants when compared with strawberries. Ascorbic acid was more diminished at high CO2 than dehydroascorbic acid. This suggests a stimulating effect of high CO2 concentrations on the oxidation of ascorbic acid and/or an inhibition of mono- or dehydroascorbic acid reduction to ascorbic acid. Agar and Streif 124 also reported a decrease in ascorbic acid content in red raspberries in CA storage. ‘Stevens’ cranberries had a higher phenolics content and total antioxidant activity than ‘Pilgrim’ fruits. The storage atmosphere did not affect the content of total phenolics or flavonoids. The total antioxidant activity of the fruits increased overall by about 45% in fruits stored in air. This increase was prevented by storage in 30% CO2 plus 21% O2.125 Holcroft and Kader103,104 and Holcroft et al.102 reported that concentrations of ellagic acid, catechin, quercetin, and kaempferol derivatives in strawberries increased during air storage, but remained relatively stable in high CO2 storage. Vitamin C content of fresh-cut kiwifruit slices decreased by 14%, 22%, or 34% when kept in 5, 10, or 20 kPa CO2, respectively, and decreased by 7%, 12%, or 18% when kept in 0.5, 2, or 4 kPa O2, respectively, for 12 days at 0°C.107 Veltman et al.126 found that “Conference” pears stored in CA (2 kPa O2 + 10 kPa CO2) resulted in 60% loss in ascorbic acid, but the concentration of total phenolics was not affected by storage conditions.127 Larrigaudiere et al.128 also showed that total ascorbate and glutathione in “Conference” pears decreased rapidly during storage, especially when the fruit was stored in CA (2% O2 and 5% CO2). Fruit exposed to CA showed a sharp burst in ascorbate peroxidase and glutathione reductase activity immediately after storage. A significant increase in superoxide dismutase activity and a late decrease in catalase were also found in the CA-stored fruits. Increased lipoxygenase activity and higher amounts of hydrogen peroxide were found in storage especially when the fruit were exposed to CA.128 Enhanced losses of vitamin C in response to CA may be due to their stimulating effects on oxidation of ascorbic acid and inhibition of dehydroascorbic acid reduction to ascorbic acid.107 In ‘Selva’ strawberries, Holcroft and Kader103,104 reported the activities of phenylalanine ammonia lyase (PAL) and UDP glucose: flavonoid glucosyltransferase (GT) decreased in both external and internal tissues of strawberries stored in air + 20% KPa CO2 and the effects were more obvious in the internal tissues. Bangerth 129 found that losses of ascorbic acid in apples and red currants were reduced by storage in reduced O2 atmospheres, but these losses were accelerated by storage in elevated CO2 atmospheres. Mareczek et al.130 reported that the level of total phenolic in the peel of ‘Golden Reinders’ and ‘Gala Must’ apples were similar in fruit stored for about seven months at 0°C in air and in CA (3% O2 + 5% CO2), but an additional eight days at 16°C reduced phenolics concentration only in the peel of ‘Gala Must’ fruit previously stored in air. Leja et al.110 found that the increase

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of soluble peroxidase (POD) activity was much stronger in apple kept in air than in CA (2% O2 and 2% CO2) at 1°C, but after a subsequent seven days’ storage at high temperature, a further increase in enzyme activity was observed in all treatments. ‘Jonagold’ and ‘Sampion’ apple showed a slight decrease in anthocyanins stored in air, while the CA (2% CO2 and 2% O2) treatment did not cause any significant changes, and antioxidant capacity of fruit peel increased significantly after cold storage and in CA.110 Additional storage at 16°C caused a slight further increase in total phenols, antioxidant activity and radical scavenging activity of ‘Jonagold’ fruit stored previously in a regular cold chamber. This was probably due to synthesis of phenols in the peel.131,132 However, Mazza and Miniati133 observed that anthocyanins in apples were relatively stable when stored at 2°C, but decreased in low O2 and high CO2. Awad and de Jager134 reported that during storage of ‘Jonagold’ apple for three, six, and eight months and ‘Elstar’ apple for two, four, and six months plus one or two weeks shelf life, the concentrations of cyanidin 3-galactoside and quercetin glycosides were relatively constant, while the concentrations of catechins, phloridzin, and chlorogenic acid showed only minor decreases. Moreover, there were no significant differences in the concentration of flavonoids and chlorogenic acid between fruits stored at 1°C under ultralow oxygen storage (1.2% O2 + 5% CO2) compared to air conditions. It was concluded that flavonoids present in apples are stable, following harvest. Zheng et al.135 studied the effects of superatmospheric O2 treatments (40%, 60%, 80%, or 100% O2 at 5°C) on ‘Duke’ highbush blueberries and showed that the antioxidant levels were markedly increased by 60% to 100% O2 treatments as compared with 40% O2 treatment or air control during 35 days of storage. Elevated O2 between 60% and 100% also promoted increases of total phenolics and total anthocyanins, in particular, malvidin-based anthocyanins, as well as the individual phenolic compounds. Stewart et al.136 also reported that soft fruit stored under elevated O2 levels exhibited good antioxidant capacity over the first four days of storage, but then declined with prolonged storage, possibly due to O2-promoted oxidation of the main antioxidants including anthocyanins and other phenolic compounds.136 This was confirmed by Pérez and Sanz137 who found that, in comparison with fruits stored in air, strawberries held in 80% O2 + 20% CO2 had significantly higher levels of total anthocyanins during the first four days of storage, but significantly lower levels of total anthocyanins at the end of storage. It seems that the effect of high O2 on total phenolics, total anthocyanins, and ORAC value may vary depending on the commodity, O2 concentration, and storage time and temperature. Data obtained in these studies suggest that high-oxygen treatments may improve the antioxidant capacity of fruits. Taira et al.138 treated ‘Hiratanenashi’ persimmom fruit after harvest with 15% ethanol vapors at 20°C or with 80% CO2 at 20°C and showed that the soluble tannin decreased during treatment and fruits were not astringent after seven days of ethanol treatment or three days of the CO2 treatment. Zavrtanik et al.139 treated ‘Hachiya’ and ‘Fuyu’ persimmons with CO2 or N2 at 99.9% for 24 h at 20°C , then stored the fruits at 1°C. The total phenol concentration decreased very quickly and fruits were edible after one day of the CO2 treatment; N2 treatment reduced polyphenol content less quickly and fruit were edible after 5 to 7 days while control fruit lost polyphenols

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slowly and were edible after 10 days of storage. Wright and Kader140 showed CA (2% O2, air + 12% CO2 or 2% O2 + 12% CO2) had no significant effect on changes in total ascorbate content for persimmons and strawberries after seven to eight days at 5°C. Al-Redhaiman 141 also found that total tannin content in ‘Barhi’ date fruit did not change significantly after 26 weeks at 0°C under 20% CO2 storage. Ozone, the triatomic form of oxygen (O3), is an unstable compound that decomposes either spontaneously, producing hydroxyl radicals and other free radical species, or upon contact with oxidizable surfaces. Ozone has been used in different applications in the food industry, and has been recommended to be used as a GRAS (generally recognized as safe) disinfectant or sanitizer for foods in the United States.142 Ozone, applied as a gas or as ozonated water, has been tested for postharvest treatment of fruits and vegetables. Pérez et al.143 reported that at the end of cold storage, the vitamin C content of ozonated strawberries was three times that of control fruits. A decrease in the anthocyanin content of treated and nontreated samples was observed after three days at 2°C, with a significantly lower value in ozonated fruits (639. 08 ± 11.01 nmol/g fresh weight) than in nontreated fruits (811.34 ± 6.81 nmol/g fresh weight). When strawberries were placed at 20°C, a slight increase in anthocyanin accumulation was detected in both treated and nontreated fruits. No significant differences were found after four days at 20°C.143 Blackberry fruit stored over 12 days at 2°C in a 0.3 ppm ozone atmosphere showed a sharp decrease in anthocyanin levels.144 Artés-Hernández et al.145 showed an increase of up to twofold in total stilbenoid content in ‘Napoleon’ grapes after shelf life for CA (5 kPa of O2 + 15 kPa of CO2 + 80 kPa of N2) and O3 (8 ppm) treatments up to 38 days at 0°C. Piceid concentration remained unaltered or slightly changed, whereas large increases were observed after shelf life for resveratrol, even up to three- and four-fold for O3-treated grapes and two-fold for CA-treated ones. Therefore, improved techniques for the keeping quality of ‘Napoleon’ table grapes during long-term storage seem to maintain or enhance their antioxidant compound content.

16.4.3 HEAT TREATMENT

AND IRRADIATION

One of the postharvest techniques that has been demonstrated to effectively maintain quality of fresh fruits is prestorage heat treatment. In addition to controlling diseases and insects, adequate heat treatment can also retard senescence or degradative processes in fresh produce. Application of thermal treatments reduced the softening rate of apples, avocado, peach, papaya, pear, and banana.146−149 Civello et al.150 also found that heat treatments delay ripening and postharvest decay of strawberry fruit. Strawberries were treated for 1 to 5 h at temperatures ranging from 39°C to 50°C . After treatments, fruits were placed at 0°C overnight and then held at 20°C for three days. Most of the heat treatments improved strawberry shelf life, with best results obtained for fruit heated at 42°C and 48°C for 3 h. However, both treatments reduced anthocyanin accumulation and PAL activity relative to the controls. Civello et al. also showed that heat treatment (48°C air, 3 h) reduced anthocyanin accumulation in strawberries. According to these results, the lower fungal development and the slower ripening rate shown by heat-treated fruits suggest that this physical method could be useful to extend the postharvest shelf life of strawberries. In contrast,

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Yoshikawa et al.151 treated ‘Chandler’ strawberries with humid air at 43°C and 46°C for 80 min and found severe damage in the fruits. These contradictory results could be due to cultivar-dependent responses of strawberries to heat treatments. This phenomenon has been described for other fruits as well.148,152 Red pigmentation of apples can be improved after harvest using artificial light. A range of wavelengths from UV-B (280 to 320 nm) to red light (680 to 780 nm) is effective.153,154 Red light alone is only slightly effective in stimulating anthocyanin production, UV-B has considerable effect, and both together have synergistic effects.153 Marais et al.155 showed that postharvest irradiation with high-pressure sodium light-enhanced anthocyanin synthesis in apples, but not in pears. Postharvest irradiation with UV rays at 2.35 W m−2 alone markedly promoted red color development and increased the anthocyanin content in the skin of ‘Satohnishiki’ sweet cherries within 48 h.156 In grapes, accumulation of anthocyanin occurred in the skins when berry sections were exposed to UV irradiation.157 It was proposed that UV irradiation induced and activated decay-resistance mechanisms, for example, by an increase in antifungal compounds in the fruit peel. An additional positive effect of UV treatment is the enhancement of levels of anthocyanins in strawberries and red apples.154,158 Accumulation of anthocyanins in apple skins occurred from de novo synthesis of PAL and chalcone isomerase following UV-B treatment, and it was suggested that there could be a similar effect of short exposure to high-energy UVC irradiation. By lowering the temperature during irradiation from 23°C to 11°C , the effectiveness of UV irradiation was suppressed. The decrease in temperature may cause a suppression of the enzymatic activities related to phenylpropanoid synthesis which was stimulated by UV radiation.156 Irradition (2 to 3 kGy) combined with refrigeration has been shown to extend the shelf life of strawberries.159 During storage, ascorbic acid significantly increased while dehydroascorbic acid decreased in irradiated strawberries.159 Stilbenes are nonflavonoid phenolics that are mainly present in grape, and they have been reported to have a number of health-beneficial properties.160,161 The induction of stilbenes in response to stress, such as preharvest and postharvest UV-C irradiation has been described.162−−165 Cantos et al.160 demonstrated that postharvest UV-C irradiation (510 W, 40 cm, 60 s) enhances stilbene content in red grape varieties ‘Flame,’ ‘Red Globe,’ ‘Crimson,’ and ‘Napoleon,’ as well as the white varieties ‘Superior,’ ‘Dominga,’ and ‘Moscatel Italica.’ The most inducible stilbenes in grapes were trans-resveratrol, trans-piceatannol, and viniferins. The net resveratrol induction ranged from 3.4-fold (‘Flame’) to 2315-fold (‘Red Globe’). The highest viniferins content was observed in the variety ‘Flame’ (0.73 mg/100 g fresh weight), although the variety ‘Red Globe’ presented the highest viniferins induction (175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fresh weight and 173-fold, respectively) was observed in the variety ‘Flame.’ Cantos et al.162 found that the content of most phenolics in ‘Napoleon’ grapes remained quite constant during postharvest refrigerated storage (10 days at 0°C) while the resveratrol derivatives increased twofold. Postharvest treatment of grapes with UV-C and UVB light induced a large increase in resveratrol derivatives (three- and twofold, respectively).163−−165 The biological activity of resveratrol as an anticarcinogenic and an antioxidant has been reported.166,167 These results showed that UV irradiation of

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table grapes can be beneficial in terms of increasing the content of potentially healthpromoting resveratrol.163−−165 Patil et al.168 demonstrated that the response of ‘Rio Red’ grapefruit to irradiation depended on harvest time. The early season grapefruit exposed to low doses of irradiation (70 and 200 Gy) followed by storage (35 days) at 10°C had significantly higher naringin, narirutin, and total flavanone concentrations compared to the initial flavanone concentrations. This increase may be attributed to an increase in PAL activity during low-temperature storage and low dose irradiation exposure.169,170 The early-season grapefruit also had higher levels of β-carotene after 35 days of storage than at their initial (0 day) level, irrespective of the irradiation. On the contrary, in late-season fruit, irradiation had no effect on β-carotene in grapefruit before or after 45 days of storage conditions. Higher doses of irradiation (400 and 700 Gy) and 35 days of storage had detrimental effects on quality of early-season grapefruit; however, no significant effect was observed on the quality of late-season fruit.168 Oufedjikh et al.171 also reported that the irradiated fruit (300 Gy) had lower concentration of flavanone glucoside and polymethoxylated flavones. The decrease in flavanone content was ascribed to their role in counteracting the oxidative stress induced by the gamma irradiation. Variation in the flavanone content at different doses of irradiation treatment may be due to equilibrium between gamma irradiation-induced stress and de novo synthesis of flavonoids by increased PAL activity.171 The UV-C (9.2 kJ m−2) and heat treatments (45°C) retained fruit quality and antioxidant activity better than in control fruit of boysenberries.172 The effect of UV rays on promoting the accumulation of anthocyanin has also been reported in apples, grapes, sweet cherry, and several kinds of fruits.24,25,105,153−−158,162

16.4.4 TREATMENT

WITH

NATURAL COMPOUNDS

OR

CHEMICALS

A naturally occurring compound, methyl jasmonate (MJ), was found to reduce chilling injury, retard decay, and improve storage quality of several fruits.173,174 The MJ treatment maintained higher levels of ORAC in blueberries than the untreated control fruit, especially during the latter part of storage.175 The high antioxidant activities were associated with better overall quality of MJ-treated raspberries, which included high levels of sugars, organic acids, and low incidence of decay.176 Chanjirakul et al.177 studied the effect of various natural volatiles such as methyl jasmonate (MJ), allyl isothiocyanate (AITC), essential oil of Melaleuca alternifoli (tea tree oil or TTO), and ethanol (EtOH) on antioxidant capacities and antioxidant enzymes in berry fruits and found that strawberries and blackberries treated with MJ had the highest antioxidant capacity expressed as oxygen radical absorbance capacity (ORAC) values after seven days of storage (40.77 and 67.70 µmol TE/g fresh weight, respectively).177 Moreover, MJ treatment enhanced antioxidant capacity in strawberries and blackberries as measured by the radical DPPH• and ABTS•+ scavenging activity in both 7 and 14 days after storage. The ED50 of free radical DPPH• scavenging capacity ranged from 31.79 to 36.94 mg after 7 days of storage and from 32.57 to 42.72 mg after 14 days of storage in strawberries, but only from 4.29 to 6.65 mg and from 5.10 to 7.73 mg in blackberries after 7 and 14 days of storage, respectively. The MJ-treated fruits showed the highest percent inhibition for DPPH

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radicals among all the treatments in both strawberries and blackberries. The MJ treatment also increased scavenging capacities on (ROO•) superoxide radicals (O2•–), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2) in strawberries and blackberries, except for O2•– scavenging capacity in blackberries stored for 14 days. Treatment with TTO or EtOH enhanced most of these free radical scavenging capacities, except for H2O2 in strawberries, and for O2•– and 1O2 in blackberries. It is possible that the elevated capacity in scavenging various free radicals by these natural volatile compounds increased the resistance of tissues to decay. In raspberries, treatment with MJ enhanced the activity of several antioxidant enzymes, including SOD, G-POD, AsA-POD, GSH-POD, GR, MDAR, and DHAR. Moreover, raspberries treated with MJ showed the highest amount of AsA, DHAsA, GSH, and GSSG compared to other treatments. Treatments with TTO or EtOH also enhanced the antioxidant system. Although AITC treatment promoted the O2•– scavenging capacity in blackberries after 14 days of storage, it had little effect on scavenging capacities of other radicals or the antioxidant enzyme activities. These results indicate that MJ may increase the resistance of tissues to decay through enhancing their antioxidant system and their free radical scavenging capability, while AITC may retard the decay directly by its antimicrobial properties. Therefore, it is possible to enhance the antioxidant system, reduce decay, and extend storage life of berry fruits by treatment with natural volatiles.177 Calcium dips may be used to reduce physiological disorders and maintain firmness in apples, cherries, and other fruits. Bangerth178 observed an increase in vitamin C content of apples treated with calcium chloride. When dipped in 1% CaCl2 after cutting, and kept in an ethylene-free atmosphere, kiwifruit slices had a slightly higher ascorbic acid content than those treated with 1% CaCl2 only.107 Diphenylamine (DPA) has been used in commerical postharvest treatment to suppress the storage disorder superficial scald during long-term storage of apples. Superfical scald is a serious physiological disorder that affects some susceptible apple cultivars, such as ‘Red Delicious’ and ‘Granny Smith.’ Golding et al.117 treated apple (‘Granny Smith,’ ‘Lady Williams,’ and ‘Crofton’) with DPA at harvest then stored in air at 0°C for nine months and found that the concentrations of benzoic acid derivatives, cinnamic acid derivatives, flavan-3-ols, flavonols, and procyanidins of stored fruit were higher in the treated peel than in the control peel. However, Duvenage and DeSward 179 showed that DPA inhibited both the synthesis and oxidation of flavonols during storage.

16.5 EFFECT OF PROCESSING Many changes in physical and chemical properties occur in fruits during processing. Several types of oxidative reactions may take place in which electrons are removed from atoms/molecules leading to the formation of an oxidized form. These reactions cause browning reactions, loss or changes to flavor or odor, changes in texture, and loss of nutritional value from destruction of vitamins and essential fatty acids.180 The health-promoting capacity of processed foods in the diet is still not completely known. The concentration of nutrients may be changed, influencing the overall

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antioxidant potential of fruits by the processing methods.181,182 In general, fruit processing may be responsible for a decrease, increase, or no change in content and functionality of phytochemicals. Additional information on the influence of processing on antioxidants can be found in Klein and Kurilich,183 Nicoli et al.,184 and Pokorny´ and Schmidt.185

16.5.1 HEAT It is generally recognized that processing can negatively affect the original antioxidant properties of raw material. Generally, thermal processing results in a decrease in ascorbate, anthocyanins, and tocopherols. But recent research has shown that food processing also has some positive effects on the quality and health properties of the processed food. The most notable positive effect of processing on the overall quality or health capacity of fruit is increased bioavilability of β-carotene, resulting in an increased antioxidant status. Carotenoid terpenes are plant pigments that are present in fruits such as oranges, pink grapefruit, mango, autumn olive, and papaya. Different carotenoids have different degrees of sensitivity to heat treatment. For example, epoxy carotenoids were sensitive to heat treatment, while lutein, neurosporene, αand β-carotene, lycopene, phytofluene, and phytoene survived heat treatment.186 Thermal processing has been shown to convert all-trans forms of β-carotene to cis forms.187 These compounds enhance immune response and protect skin cells against the sun's ultraviolet radiation. Loss of vitamin C after heat treatment has been widely reported.188 Freshsqueezed orange juices are higher in ascorbic acid and anthocyanins than processed juices.189 Fruits are high in vitamin C content and processing tends to cause loss from as little as 10% to almost 100% during cooking or canning.190 Vitamin A loss in canning totals about 10%, whereas the individual nutrients in 13 fruits and vegetables were 35% to 96% less for fat-soluble nutrients (96% tocopherols, 42% total carotenoids, 35% vitamin) as compared with frozen fruits.191 The canned fruits also showed about twice as much vitamin C, niacin, and riboflavin loss and over three times as much thiamin loss compared with frozen fruit. Blueberry puree was relatively high in its antioxidant capacity, with values close to those in intact fresh fruit and frozen fruit samples. The antioxidant capacity (ORAC) of blueberry pie filling prepared from canned fruit had only about one-third of the ORAC of fresh fruit, suggesting that substantial damage and/or losses of antioxidants occurred during canning processing.192 Lo Scalzo et al.193 showed that thermal treatments reduced ascorbic acid in blood orange juice. However, an increase in anthocyanins, hydroxycinnamic acids, and antioxidant capacity in thermally treated juice was found.193 Kalt et al.192 showed that extraction of lowbush blueberry at 60°C resulted in higher anthocyanins content and antioxidant capacity, compared to extracts obtained at 25°C. Subsequent room temperature storage resulted in losses in anthocyanin and antioxidant capacity only in those extracts obtained at 60°C. On the other hand, Jackman et al.194 indicated that heat is one of the most destructive factors of anthocyanins in berry juices. Increasing storage temperatures from 20°C to 100°C decreased half-life time of strawberry preserves. In dry powder of elderberry extracts, the stability of anthocyanins increased six to nine times when

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temperature was reduced from 20°C to 4°C.195 Anthocyanin degradation in aqueous solution increased from 30% to 60% after 60 days when storage temperatures were increased from 10°C to 23°C.196 Therefore, high-temperature, short-time processing is recommended for maximum anthocyanin retention of foods containing anthocyanins.197 Asami et al.198 demonstrated that both storage and thermal processing conditions had a profound impact on the levels of polyphenolics in clingstone peaches and showed a dramatic decrease in procyanidins in thermally processed peaches. Cold storage at 4°C for 14 days or freezing and storing at −12°C for three months produced no loss in total phenolics and procyanidins. Peaches stored at 30°C for 24 h resulted in a 1.7-fold increase in total phenolics. Peaches processed at temperatures of 100°C for 40 min, 104°C for 10 min or 110 °C for 2.4 min showed that processing above 100°C decreased levels of both total phenolics (up to 21%) and procyanidins (up to 100%). Processing at 100°C for 40 min produced no significant loss in total phenolics, but a 30% to 43% loss in phenolic levels was found during the first three months in storage after canning. This result indicated that freezing has relatively little impact on total phenolics and procyanidins, whereas peeling and canning using high temperatures typically employed in commercial sterilization have detrimental impacts on both total phenolics and procyanidins levels.198 The processing temperatures at 120°C also showed reduction of the free radical scavenging capacity in red grape pomace peels (28.3%) and white grape pomace peels (22.9%) sample extracts as compared to those at 20°C.199 Skorikova and Lyashenko200 obtained a negative correlation between the heating period and the polyphenol content of apple and pear juices, with the leucoanthocyanins being the least stable polyphenol fraction. Similarly, Sato et al.201 found a correlation between wine color and the ability of the wine constituents to scavenge superoxide radicals. Specifically in red grape pomace peels, losses in the total polyphenols, color, and antioxidant capacity were reported when they were dried at 100°C and 140°C, with the anthocyanins being more affected than other compounds.202 Bioactive compounds (such as polyphenols, flavonoids, etc.) that are associated with the antioxidant capacity of the samples are usually oxidized at high temperatures.203 Therefore, a lower reduction in the free radical scavenging capacity of white grape pomace peel extracts with processing temperature as compared to that from red grape pomace peels, suggests that the bioactive constituents of white grape pomace peel extracts are more heat resistant than those from red grape pomace peels. Food processing, especially if heat-treatment based, can result in undesired reactions, mostly of a chemical nature. For instance, oxidation can result in antioxidant and vitamin depletion. On the other hand, heating can also induce the formation of compounds with new antioxidant properties, as occurs during the development of the Maillard reaction in which a reducing sugar reacts with amino groups. Antioxidant depletion in thermally treated fruits may also be attributed to consumption of ascorbic acid, polyphenols, sugar and amino acids as reactants in the Maillard’s reaction.184,204 Maillard reaction products have an effect on the nutritive value (loss of the essential amino acid lysine), as well as the development of new compounds, which improve flavor and color in many food products during baking, cooking, roasting, or frying.205 Although they have been extensively studied, it is not still clear whether the Maillard reaction produces mutagenic or antimutagenic

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compounds or substances, which favor or block microbial growth or enzymatic activity.206,207 Jeong et al.208 showed that the antioxidant activities of citrus peel extracts increased as heating temperature increased. Heat treatment of citrus peels at 150°C for 60 min increased the total phenolic content, radical scavenging activity, and reducing power. Several low-molecular-weight phenolic compounds such as 2, 3diacetyl-1-phenylnaphthalene, ferulic acid, p-hydroxybenzaldoxime, 5-hydroxyvaleric acid, 2,3-diacetyl-1-phenylnaphthalene, and vanillic acid were newly formed in the citrus peels heated at 150°C for 30 min. These results indicated that the antioxidant activity of citrus peel extracts was significantly affected by heating temperature and duration of treatment on citrus peels, and that the heating process can be used as a tool for increasing the antioxidant activity of citrus peels.208 The formation of new phenolic compounds with antioxidant properties in the citrus peels heated at 150°C for 30 min may be attributed to the development of Maillard reaction products.

16.5.2 FREEZING Overall, fruits retain more nutrients if they are frozen rapidly, stored in airtight packaging, and thawed rapidly.209 Freezer temperature should be −18°C (or 0°F) or lower. If a constant temperature of −22°C is maintained, many foods will only lose about 25% vitamin C in one year.210 Mullen et al.211 studied the effects of storage and freezing on raspberries and showed that the antioxidant capacity was not significantly different in fresh and frozen berries. It was also demonstrated that the freezing process had little effect on the ellagic acid released by acid hydrolysis, total phenol, vitamin C content, and antioxidant capacity.212 However, during long-term frozen storage (12 months), the level of ellagic acid declined 14% to 21% and vitamin levels fell 33% to 55%, although the antioxidant capacity of the berries was unchanged.212 Ancos et al.212 also analyzed the anthocyanins of raw, just frozen, and long-term frozen storage at −20oC of four raspberry cultivars (cvs. ‘Heritage,’ Autumn Bliss, Zeva, and Rubi) and found that the stability of anthocyanins after freezing and frozen storage was dependent on the seasonal period of harvest. Heritage and Autumn Bliss (early cultivars) were less affected by processing and longterm frozen storage (one year), and the total pigment extracted increased 17% and 5%, respectively. ‘Rubi’ and ‘Zeva’ (late cultivars) exhibited a decrease in total anthocyanin content of 4% for “Rubi” and 17.5% for “Zeva.” Cyanidin 3-glucoside degraded the most during processing and storage. Bushway et al.213 evaluated five raspberry cultivars harvested at the red-ripe stage and during long-term frozen storage at −20°C. They rated color by instrument and by a sensory panel and found that the effect of freezing on instrumental hue and sensory color was not significant for any of the five varieties investigated.

16.5.3 SUGAR, ASCORBIC ACID,

AND

OTHER CHEMICALS

High sugar concentrations (> 20%) are protective of anthocyanins.214 Pasteurized ascorbic-acid-enriched chokeberry extracts with 65% sucrose had three times longer

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anthocyanin half-life compared with sucrose-free extracts. Moderate sucrose levels (13%) also showed slightly improved anthocyanin stability.215 Glucose, sucrose, and maltose are more effective for this protection than fructose, arabinose, lactose, and sorbose.197 Strawberries frozen with added sugar retain more vitamin C than unsweetened strawberries.209 Heat processing did not result in a loss of total anthocyanins, total phenolics, and antioxidant activity when the values in syrup and cherries were combined. There was little loss of total anthocyanins with canning of cherries, but about 50% of anthocyanins and polyphenolics were redistributed to the syrup. About half of anthocyanins and polyphenolics were leached from fruits into the brine solution.216 This may be due to increased extraction efficiency in the softened fruits. High temperature may increase membrane permeability in the macerated peel tissue facilitating phenolic extraction.217 Moreover, with the breakdown of the cellular constituents, bound phenolic compounds may be released.218 The phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries (bayberry, black currant, blackberry, blueberry, cowberry, cranberry, raspberry, red currant, and strawberry) remained stable during jam processing.219 This indicated that flavonoids and phenolic acid retained their conjugate forms in the samples during jam processing. Zafrilla et al.220 found the flavonol content of raspberry jams decreased slightly with processing and more markedly during storage of the jams. The ellagic acid derivatives remained quite stable with processing and during six months of jam storage. The content of free ellagic acid increased 2-fold with processing, and it continued increasing up to 3.5fold after one month of storage of the jam. The increase observed in ellagic acid could be explained by a release from ellagitannins with the thermal treatment. The industrial processing of pomegranates juices showed an increase of their antioxidant capacity and phenolics content,22 whereas strawberry processing to produce jams decreased the total ellagic acid content by 20%221 and the flavonols by 15% to 20%.222 Cooking also affects flavonoid content. Cooking strawberries with sugar to make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking of bilberries with water and sugar, 40% of quercetin was lost.222 Strålsjö et al.223 found almost no losses of folate when strawberries were cooked to jam or stewed to strawberry desserts from frozen berries. This finding is contradictory to data reported in four European food tables, which indicate that only 3% to 30% of folate was retained in various strawberry products such as jam and stewed desserts.224 Fruits commonly lose nutrients when they are used to make juices.191 Important variables affecting nutrient content are temperature, pH, exposure to air or light, type of storage container, and presence of antioxidants or preservatives.225 It was reported that vitamin C is most stable in pineapple juice and less stable in apple juices blended with other fruits, and carbonated fruit beverages. Vitamin A is quite stable when added to juices and other beverages, but least stable in pineapple juice and pineapple juice drinks. Carotenoids are very stable in juices and the B-vitamins are stable in frozen beverages.191,225 Common food additives (such as sodium nitrate) cause folate destruction.226 Sulfur dioxide and sulfite have been widely used in fruit processing. Sulfur dioxide enhances the extraction of anthocyanins from fruit tissues.227 At low concentrations (30 ppm), it inhibits the enzymatic degradation of anthocyanins; at moderate concentrations (500 to 2000 ppm), it forms a reversible,

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colorless SO2-anthocyanin complex; at high concentrations (0.8% to 1.5%), it forms an irreversible complex, as in the bleaching of red cherries during brining.228 Use of sulfite in food processing to inhibit browning reactions can lead to extensive losses of thiamine.226 The presence of vitamin C in food can improve folate retention during heating whereas the presence of metals can increase loss.226 Antioxidant activity of whole apple juices which were fortified with ascorbic acid declined after 10 days of storage at room temperature or at 4°C and unfortified whole apple juice remained relative stable.229

16.5.4 DRYING

AND

DEHYDRATION

Dry anthocyanin powders are stable for several years when stored in hermetically sealed containers.197,230 Zajac et al.195 reported that anthocyanin content of dry black currant powder decreased 14% after 15 months of storage at 20°C, but when starch syrup or maltodextrins were included as carriers during the drying of extracts, the anthocyanin losses were reduced to 2% and 3%, respectively. Larrauri et al.202 found that polyphenolic content, color, and antioxidant activity of red grape pomace peels were not significantly affected when dried at 60°C. However, when drying temperature was either100°C or 140°C, a significant reduction in both total extractable polyphenols (18.6% and 32.6%) and condensed tannins (11.1% and 16.6%) were observed, as well as a decrease of 28% and 50%, respectively, in the antioxidant activity of the samples. Hue angle and total color difference in the samples dried at 140°C were significantly higher than in the freeze-dried reference material. This may be due to the thermal degradation of the phenolic compounds at high temperatures.202 Dried blueberries were varied in their ORAC levels, and appeared to be lower in fruit that had been dried more thoroughly. The ORAC value of juice concentrate was only about 65% that of the fresh.192 Vacuum and osmotic concentration of red raspberry juice decreased total quercetin-forms compared to unconcentrated juice.81 Freeze drying, vacuum microwave drying, air drying, and a combination of air drying and vacuum microwave drying all reduced total phenolics and anthocyanin contents in addition to reduced antioxidant activities, as compared with fresh frozen Saskatoon berries.231 The air-dried and freeze-dried apple peels had the highest total phenolic, flavonoid, and anthocyanin content as compared to the oven dried at 40°C, 60°C, or 80°C.232 Piga et al.233,234 dehydrated fresh plums to prunes and found that drying destroyed anthocyanins and flavonols. High temperatures and high oxygen concentrations involved in the air-drying process lead to the rapid degradation of the anthocyanins.133 During drying, the balance between the various types of anthocyanins shifts toward either the quinoid base or the colorless chalcones, which are destroyed by different oxidation mechanisms to produce different types of compounds.235 A marked decrease in ascorbic acid content was found after the dehydration process. The fruits dried at 85°C showed significantly higher losses, ranging from a minimum of 61% to a maximum of 80%. Total polyphenol values decreased in the samples dried at 60°C, but were two and a half times higher in ‘President’ plums dried at 85°C. No significant changes were found in the chlorogenic and neochlorogenic acid when processing was conducted at the higher temperature (85°C), whereas a decrease was found in the prunes obtained at

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60°C. It has been reported that during the dehydration process, the polyphenoloxidase (PPO) activity remains high for long periods when the drying temperature is around 55°C, whereas only moderate activity is observed at temperatures higher than 75°C.236 Raynal et al.236 also found a decrease of 25% and 30% in chlorogenic and neochlorogenic acids, respectively, for plums dried at 85°C. The antioxidant capacity tended to increase significantly with the higher drying temperature in the ‘Sugar’ variety, while in the ‘President’ plum sample, dried at 60°C, antioxidant capacity was significantly lower than in the fresh fruits. Processed plums, dried at 85°C, exhibited doubled antioxidant activity in both cultivars (‘Sugar’ and ‘President’).233,234 This may be due to high temperature stabilization procedures that may lead to the formation of new compounds with higher antioxidant activity. This is essentially the case in the Maillard reaction, in which Maillard reaction products (MRPs) are created with markedly higher antioxidant power, often by a chainbreaking type mechanism.237−239 Osmotic dehydration has been used as a prestep to further processing.240 These processes use a sequence of technological steps to achieve controlled changes of the original properties of the raw material. Torreggiani and Bertolo241 found that the pigment retention of strawberry halves was significantly higher in osmotic dehydration than that observed in the fruits frozen without a concentration pretreatment. An osmotic step could also improve the stability of vitamin C and color during air drying and frozen storage of osmodehydrofrozen apricot cubes by the modification of sugar composition.241,242 Therefore, the quality characteristics of processed fruit could be improved through the application of an osmotic step.

16.5.5 EXTRACTION TECHNIQUES Processing apples into juice had resulted in large flavonoid losses. Conventional apple juice production (straight pressing of apple pulp or pressing after pulp enzyming) resulted in a juice poor in flavonoids with only 3% to 10% of the antioxidant activity of the original fruit.243 However, by applying an alcoholic extraction step, either on the pulp or on the pomace, the levels of flavonoids and chlorogenic acid in enriched juice are between 1.4 (chlorogenic acid) and 9 (quercetin glycosides) times higher than in conventional apple juice. In enriched juice the antioxidant activity was five times higher than in conventional apple juice, with 52% of the antioxidant activity of the originating fruits.244 Häkkinen et al.35 showed that traditional preservation of crushed lingonberries in their own juice caused a considerable (40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in unprocessed berries were retained in juices made by common domestic methods (steam-extracted black currant juice, unpasteurized lingonberry juice). Cold pressing was superior to steam extraction in extracting flavonols from black currants. During nine months of storage at −20°C, quercetin content decreased markedly (40%) in bilberries and lingonberries, but not in black currants or red raspberries. Myricetin and kaempferol were more susceptible than quercetin to losses during storage. When juices were made by common domestic processing methods, considerable losses of flavonols were observed. Cold pressing was superior to steam extraction in extracting the flavonols. Also, crushing the berries resulted in a considerable loss of quercetin.

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Rommel and Wrolstad 81 reported that the red raspberry juice produced by highspeed centrifugation contained the most total quercetin forms and much more than juices made by other techniques. Juice following high-speed centrifugation, combined with addition of pectinases, contained less total quercetin forms (ca.190 ppm) than the juice prepared without pectinases (280 ppm). Diffusion-extracted juice contained even less total quercetin forms. This was probably due to deglycosylation of flavonol glycosides to less-stable aglycones in the presence of pectinases. In the crowberry juices, such an effect was not observed. Rommel and Wrolstad41 also found that juices of red raspberry made by diffusion extraction and a standard process had the highest concentration of ellagic acid and its forms. High-speed centrifugation reduced total ellagic forms by half compared to diffusion extraction and depectinization and concentration decreased total forms even further. Spanos et al.217 reported that a high temperature during initial diffusion extraction produced up to a fivefold increase in the recovery of phloretin glucosides as compared to that obtained in a conventionally pressed juice without temperature elevation. Gil-Izquierdo et al.245 evaluated phenolic compounds, vitamin C, and antioxidant capacity in orange juices by different extraction processing techniques on commercial (squeezing, mild pasteurization, standard pasteurization, concentration, and freezing) and domestic squeezing (a hand processing technique) and found that commercial squeezing extracted 22% more phenolics than hand squeezing. The freezing process caused a dramatic decrease in phenolics, whereas the concentration process caused a mild precipitation of these compounds into the juice cloud. In pulp, pasteurization led to degradation of several phenolic compounds, that is, caffeic acid derivatives, vicenin 2 (apigenin 6,8-di-C-glucoside), and narirutin (5,7,4′-trihydroxyflavanone-7-rutinoside) with losses of 34.5%, 30.7%, and 28%, respectively. Orange juice produced by commercial squeezing contained 25% more vitamin C than by domestic squeezing. Mild and standard pasteurization slightly increased the total vitamin C content as the contribution from the orange solids parts, whereas concentration and freezing did not show significant changes. The content of Lascorbic acid provided 77% to 96% of the total antioxidant capacity of orange juice. Mild pasteurization, standard pasteurization, concentration, and freezing did not affect the total antioxidant capacity of juice, but they caused a 47% reduction in pulp.

16.5.6 HIGH PRESSURE Food processing under high pressure and low to moderate temperature (below 70°C) conditions has been introduced as an alternative to high temperature preservation.246,247 High pressure has been used to inactivate pectinesterase and pectin methylesterase for bypassing the use of extreme heat during processing which results in a loss of fresh fruit flavor in the juice.248,249 There are a few studies that have been published on the effect of high hydrostatic pressure on postprocessing total antioxidant activity of orange juice or other food systems. Polydera et al.250−252 studied the total antioxidant activity of high pressure processed (500 MPa, 35°C, 5 min or 600 MPa, 40°C, 4 min) compared to thermally pasteurized (80°C, 30 s or 80°C, 60 s) fresh orange juice as a function of storage at different isothermal conditions (0°C to 30°C) and found higher antioxidant activities for high-pressure pressurized

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compared to thermally pasteurized orange juice during their storage. This may be due to high-pressure treatment leading to better ascorbic acid retention compared to conventional pasteurization. Nienaber and Shellhammer253 found that stabilization of fresh orange juice at pressures of 800 MPa and 25°C for 1 min and use of thermally pasteurized pulp yielded the lowest level of residual pectinmethylesterase activity (3.9%) and good cloud stability at 4°C and 37°C over a period of more than two months. Ascorbic acid loss was less than 20% after storage for three months at 4°C or two months at 15°C. Color values were stable during storage at 4°C, 15°C, and 26°C. DeAncos et al.254,255 reported stability or even improvement of radical scavenging activity of persimmon fruit purees after high hydrostatic pressure treatment. This may be due to the stability of carotenoids which are related to the increase of vitamin A. Sancho et al.256 surveyed the retention of ascorbate on ultra-high-pressurized strawberry coulis over 30 days, and found that vitamins B1 and B6 showed no significant losses but vitamin C degraded slightly after ultra-high-hydrostatic pressure treatment. Therefore, high-pressure processing of fruit products offers the chance of producing food of high quality, greater safety, and increased shelf life.

16.5.7 MICROWAVE

AND PH

When foods are microwave cooked at low power, nutrient retention is equal to or better than that of the same food prepared by conventional methods or held hot in food service operations.257−259 Vitamin retention improves if small amounts of water are used.259 Low-power warming techniques show equal or better retention of thiamine, riboflavin, vitamin B6, folate, and ascorbic acid when compared with foods that are conventionally reheated.257 To date, no information on the effects of microwave cooking on flavonoid content in fruits has been reported. The red color of anthocyanins is present only at low pH in aqueous solution. By raising the pH above 4, yellow, colorless, and blue compounds are produced. Anthocyanins exhibit the highest stability as the red flavylium cation around pH 1 to 2, whereas the other forms, especially the chalcone, are unstable and eventually lead to the degradation of the anthocyanins. In juices and wines, where the pH typically is between 2.5 and 4, anthocyanins are present as their most stable red flavylium cation, due to copigmentation with various other compounds.260 Nielsen et al.261 found that the stability of anthocyanins (cyanidin 3-ο-β-glucoside, cyanidin 3-ο-β-rutinoside, delphinidin 3-ο-β-glucoside, and delphinidin 3-ο-β-rutinoside) in black currant juices are pH dependent. More than 90% of each anthocyanin remained intact up to pH 3.3. At pH 3.8 a minimum instability was detected, suggesting the presence of an unstable intermediate, and at pH > 4.5 the stability rapidly decreased. In acidic foods, vitamin C and thiamine are more stable, while flavonoids are also less stable if the pH is alkaline.226 Cabrita et al.196 also studied the effect of pH on anthocyanin stability. Anthocyanin 3- glucoside solutions in the range of pH 1 to 12 were stored in dark over 60 days at 10°C and 23ºC. At pH 1 to 3, more than 70% of initial anthocyanin concentrations remained after 60 days at 10ºC, while considerable losses (> 90%) occurred at pH 5 to 6 after 8 days. Similar stability patterns occurred at the higher temperature of 23ºC, although the rate of anthocyanin degradation was higher. Only

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40% of the initial anthocyanins were detectable after 60 days. Kalt et al.192 showed that anthocyanin content and antioxidant capacity in lowbush blueberry fruit were greatest in pH 1 extracts, compared to extracts at pH 4 and 7.

16.5.8 OXYGEN

AND

LIGHT

Atmospheric oxygen is responsible for most losses of vitamin C during storage.194,225 Oxygenation is also detrimental to both anthocyanins and antioxidant capacity in fruit products.192 It is known that anthocyanins stored under vacuum or nitrogen are more stable than when exposed to oxygen.194 Kalt et al.192 found that in vials completely filled with blueberry juice that had little oxygen-containing head space, there was no loss in anthocyanins after 6 h, while in the half-full vessels, 76% of monomeric anthocyanins were lost during the same period. Total phenolic content and antioxidant capacity did not decrease in sealed vials, but decreased 30% and 46%, respectively, in those with air, over the same period. In strawberry juice at 45°C, degradation of pelargonidin 3-glucoside is very weak from pH 2.0 to 4.5 in the absence of O2, whereas degradation increased considerably at pH 4.0 in the presence of O2.228 It is generally advisable that oxygen should be eliminated during processing and storage of juice or when anthocyanins are used as a food colorant. In cranberry juice, elimination of oxygen tripled the half-life of color.262 Addition of ascorbic acid can protect anthocyanins from oxidation and degradation of juice color.263 Flavonoids are highly sensitive to degradation in the presence of light, visible and UV.183,197Light has been reported to be a major cause of anthocyanin degradation in elderberry extract.264 The short wavelength had been proven to have a more deteriorating effect. Packing material with proper light barriers in visible and the ultraviolet range of spectrum showed protective effects. Therefore, fruit juices and fruit-based beverages should be kept in the dark during storage. Glycosidation, acylation, and copigmentation have been reported to improve light stability for anthocyanins.265,266 Light has also been shown to reduce carotene and to convert all trans form of β-carotene to cis forms of β-carotene, which are then oxidized. The rate of cis-isomer oxidation was much greater than the rate of formation under heat and light treatment, resulting in loss of carotenoid activity.187 Light also destroys vitamin C. Therefore, thawing fruits in the dark can protect vitamin C.

16.5.9 ENZYMES In beverages, as in other food products, enzymes may occur naturally or may be added. Enzymes perform many functions in beverages. They can help increase yields, provide nutrients for the fermentation process, facilitate processing, and affect the color, flavor, and clarity of the finished product. Adding pectinase in making grape wine can accelerate the increase of pigments from the grape skin. In juice processing, pectin methyl esterase, polygalacturonase and pectinase are mainly used to increase yield and clarify the juice.267 Nariginases (a combination of enzymes containing αrhamnosidase and β- naturally) have been used to reduce naringenin by total hydrolysis or decrease naring 7-β-D-glucoside, a compound with low bitterness in citrus

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juice, particularly grapefruit juice.268 Limonin, another bitter citrus compound, may also be present in citrus. This too can be enzymatically broken down.269 Enzymes can affect the anthocyanin content in fruits and juices. Polyphenoloxidase is the major enzyme causing anthocyanin destruction during blueberry juice processing.270,271 Skrede et al.271 used frozen blueberries to process into juice and concentrate, and to monitor the changes of anthocyanin and polyphenolics (cinnamates, procyanidins, flavonol glycosides). While juice yield was 83%, only 32% of the anthocyanins were recovered in juice. Flavonol, procyanidin, and chlorogenic acid recoveries in juice were 35%, 43%, and 53%, respectively. The proportion of polyphenolics remaining in the press-cake residue ranged from 1% (chlorogenic acid) to 18% (anthocyanins). Pronounced losses of anthocyanins and polyphenolics during milling and depectinization are believed to be due to polyphenol oxidase. Losses during concentration ranged from 1.5% (anthocyanins) to 20% (procyanidins). Malvidin glycosides were the most stable and delphinidin glycosides the least.271 Peroxidase activity has been shown to be responsible for undesirable browning reactions, which appear in processed canned syrup strawberries after three months. This undesirable browning reaction has been associated with turnover and degradation of anthocyanin pigments.272,273 Glycoside enzymes can also be very destructive of anthocyanins in berry fruits.214,274

16.6 GENETIC ENGINEERING TECHNIQUES Genetic engineering techniques are considered to be among the most powerful and economically promising techniques for use in many areas. Using traditional genetic modification methods, such as cross-fertilization, scientists can produce desired traits. But in doing so, they mix thousands of genes, requiring many attempts over many years to remove the unwanted traits that occur. Genetic engineering techniques are more precise, predictable, and sometimes faster. By controlling the insertion of one or two genes into a plant, scientists can give it a specific new characteristic without transferring undesirable traits. Examples of genetically engineered foods that have been established so far include: rice, corn, and soybeans engineered to contain altered levels of nutrients; corn, soybeans, tomato, and canola plants that withstand herbicide application; corn, tomatoes, and potatoes that have their own “built-in” pesticides; corn, tomatoes, potatoes, soybeans, grapes, cantaloupe, canola, and other plants that have been manipulated to resist plant viruses; tomatoes, peas, peppers, and fruits engineered to improve processing and extend shelf life; and various enzymes used to make wine, fruit juice, sugar, beer, and oil.275 While little information is available on modifying phytonutrients in fruits, successful genomic-based approaches to metabolically engineer the vitamins in other crops has been reported. A good example is the engineering of high levels of βcarotene in the endosperm of “golden rice.”276−278 In the “golden rice,” daffodil phytoene synthase and lycopene β-cyclase were placed under the control of the endosperm-specific glutelin promoter, while phytoene desaturase was placed under the control of the CaMV 35S promoter (35S::tp::crtI). As a result, phytoene synthase and phytoene desaturase were sufficient not only for the synthesis of lycopene, but also for that of β-carotene and zeaxanthin. This led to “golden rice” which contained

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up to 200 µg β-carotene per 100 g (i.e., a tenth of the RDA in a daily intake of 300 g of rice).276−278 Although this is far from the RDA, it will have a high dietary impact on populations of Asia, Africa, and Latin America where avitaminosis A is widespread and rice consumption is high. Tomatoes contain lycopene and a variable amount of β-carotene. “Good” tomato cultivars contain >10 mg per 100 g fresh weight lycopene, which is a dietary antioxidant reported to lower the risk of certain cancers.279 Another aim of genetic engineering has been to increase the content of lycopene in tomatoes. Mehta et al.280 demonstrated that the lycopene levels in engineered tomatoes increased 2 to 3.5 times compared to the nonengineered tomatoes. Canola is another example. Canola seeds and oil contain negligible amounts of β-carotene. In this case, the simple overexpression of a bacterial phytoene synthase, crtB, fused to a plastidic transit peptide and under the control of a seed-specific napin promoter, was sufficient to boost β-carotene levels 300-fold, to a level similar to that found in oil palm.281 Therefore, it seems to be possible to enhance the flavonoid content in fruits by genetic modification. These research findings show great promise for increasing the flavonoids and vitamins of agricultural crops and are a good example of the potential that exists for manipulating phytonutrients in fruits. Thus, the genetics of the fruits can sometimes be altered to create plants that offer higher levels of phytonutrients, so-called “designer functional foods” for enhancing human health.282,283 Furthermore, the advent of the human genome project has led to the concept of “nutrigenomics,” where a person’s nutritional intake would be custom-tailored to their DNA profile. Therefore, the foods of the future are emerging. Rather than taking out certain ingredients, we can begin to engineer new nutrients that have been clinically proven to help protect against diseases and can be individualized according to a person’s needs. In the future, fruits and other functional foods will be seen as a powerful new tool that consumers can use to safeguard against diseases.283−285

16.7 CONCLUSIONS Of the 10.3 million new cases of cancer diagnosed around the world each year, as many as two-thirds may be caused by dietary and lifestyle factors. Diets rich in fruits and vegetables may decrease the risk of many types of cancer and other degenerative diseases. The reason fruits and vegetables have such health benefits is because they contain abundant phytonutrients. Many of these phytonutrients have antioxidant capacity. Recent advances in structural and functional genomics, as well as technical advances in plant breeding, bioengineering, and biotechnology make it possible now more than ever to create designer foods for consumers. Carrots have been bred to contain elevated levels of β-carotene, and tomatoes have been selected for increased concentrations of lycopene. New hybrids of various fruits with high phytonutrient content and high antioxidant capacity can be achieved for providing the protective effects they may confer in cellular detoxification processes. Therefore, enhanced antioxidant capacity of fruit crops can be achieved through plant breeding and genetic engineering and biotechnology techniques.

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Selection and breeding of fruit crops from a diverse genetic base with known concentrations of phytonutrients is a key step in developing an improved food supply. In addition, preharvest factors may influence the concentration and stability of phytonutrients with nutritional value. For example, climate, temperature, and light all affect antioxidant activity in various fruit crops. The stage of crop maturity also significantly influences the antioxidant capacity of various fruits. One of the most nutritionally significant applications of modern technologies may well be to retard the softening process so that fruits can be harvested and marketed at a more mature stage, when more of the phytocompounds have already been biosynthesized. Postharvest evaluation of the phytochemical composition of fruit crops is an important component in assessing the impact of handling procedures on the nutritional content of a fruit. New research is being initiated to develop a better understanding of how transport and storage influence their chemical composition. Research is needed to develop the best processing techniques to reduce the degradation of nutritive factors such as flavonoids, carotenoids, catechins, and bioactive peptides. Despite increasing media attention in the past few years, the science of phytonutrients is still an embryonic discipline. A number of priority research areas on phytonutrients need to be emphasized and evaluated. Future research should include: (a) improved selection criteria among different fruit crops including variety selection, molecular genetic breeding, and maturity determination at time of harvest; (b) investigation of the influence of production practices on the formation of selected phytonutrient compounds; and (c) evaluation of postharvest conditions of transport, storage, and processing techniques on the stability of phytonutrient compounds in fruits. Knowledge gained from these research studies will be helpful in improving human health by optimizing the nutritional content and quality of fruits in the diet.

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Part I Supercritical Fluid Extraction Technology

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Part II Pressurized Low Polarity Water Extraction, Membrane Separation, Distillation, and Dehydration Technologies

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Part III Bioprocessing Technology

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Part IV Preservation and Packaging Technologies

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Part V Antioxidant Properties and Material

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