Separation, Extraction and Concentration Processes in the Food, Beverage and Nutraceutical Industries (Woodhead Publishing Series in Food Science, Technology and Nutrition)

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Separation, extraction and concentration processes in the food, beverage and nutraceutical industries

© Woodhead Publishing Limited, 2010

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Related titles: Separation processes in the food and biotechnology industries (ISBN 978-1-85573-287-2) This book reviews methods and techniques for separating food components and products of the biotechnology industry. The introduction focuses on food composition and some of the conventional separation techniques. Subsequent chapters deal with each specific type or area of application individually and include information on the basic principles, industrial equipment available, commercial applications and an overview of research and development. Novel enzyme technology for food applications (ISBN 978-1-84569-132-5) The food industry is constantly seeking advanced technologies to produce valueadded, nutritionally-balanced products for consumers in a sustainable fashion. Since enzymes are so specific in their action, they are a useful biotechnological processing tool and by controlling the action of enzymes, innovative food ingredients and higher quality food products can be produced. Part one of Novel enzyme technology for food applications covers the principles of industrial enzyme technology, including methods to develop and tailor enzymes for food bioprocessing. Part two introduces the reader to novel applications of enzymes for the production of improved ingredients and food products. Food processing technology (Third edition) (ISBN 978-1-84569-216-2) The first edition of Food processing technology was quickly adopted as the standard text by many food science and technology courses. The publication of a completely revised and updated third edition consolidates the position of this textbook as the best single-volume introduction to food manufacturing technologies available. The third edition has been updated and extended to include the many developments that have taken place since the second edition was published. In particular, advances in microprocessor control of equipment, ‘minimal’ processing technologies, functional foods, developments in ‘active’ or ‘intelligent’ packaging, and storage and distribution logistics are described. Technologies that relate to cost savings, environmental improvement or enhanced product quality are highlighted. Additionally, sections in each chapter on the impact of processing on food-borne micro-organisms are included for the first time. Details of these and other Woodhead Publishing books can be obtained by: ∑ visiting our web site at www.woodheadpublishing.com ∑ contacting Customer Services (e-mail: [email protected]; fax: +44 (0) 1223 893694; tel.: +44 (0) 1223 891358 ext. 130; address: Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK) If you would like to receive information on forthcoming titles, please send your address details to: Francis Dodds (address, tel. and fax as above; e-mail: francis. [email protected]). Please confirm which subject areas you are interested in.


iii Woodhead Publishing Series in Food Science, Technology and Nutrition: Number 202

Separation, extraction and concentration processes in the food, beverage and nutraceutical industries Edited by Syed S. H. Rizvi

Oxford

Cambridge

Philadelphia

New Delhi


iv Published by Woodhead Publishing Limited, Abington Hall, Granta Park, Great Abington, Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing, 525 South 4th Street #241, Philadelphia, PA 19147, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First published 2010, Woodhead Publishing Limited © Woodhead Publishing Limited, 2010 The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 978-1-84569-645-0 (print) ISBN 978-0-85709-075-1 (online) ISSN 2042-8049 Woodhead Publishing Series in Food Science, Technology and Nutrition (print) ISSN 2042-8057 Woodhead Publishing Series in Food Science, Technology and Nutrition (online)

The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elemental chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Replika Press Pvt Ltd, India Printed by TJI Digital, Padstow, Cornwall, UK


v

Contents

Contributor contact details..................................................................

xiii

Woodhead Publishing Series in Food Science, Technology and Nutrition............................................................................................... xvii Preface................................................................................................. xxvii Part I Developments in food and nutraceutical separation, extraction and concentration techniques 1 Principles of supercritical fluid extraction and applications in the food, beverage and nutraceutical industries................. Ž. Knez, M. Škerget and M. Knez Hrnčič, University of Maribor, Slovenia 1.1 Introduction....................................................................... 1.2 Thermodynamic fundamentals.......................................... 1.3 Cycle processes for extraction using supercritical fluids 1.4 Extraction of solids using SCF......................................... 1.5 Extraction of liquids using SCF........................................ 1.6 Conclusion......................................................................... 1.7 References......................................................................... 2 Principles of pressurized fluid extraction and environmental, food and agricultural applications................. C. Turner and M. Waldebäck, Uppsala University, Sweden 2.1 Introduction....................................................................... 2.2 Instrumentation and principles of pressurized fluid extraction . ........................................................................ 2.3 Applications of pressurized fluid extraction..................... 2.4 Future trends..................................................................... 2.5 Sources of further information and advice....................... 2.6 Conclusions ...................................................................... 2.7 References......................................................................... © Woodhead Publishing Limited, 2010

3 3 8 21 26 30 32 36 39 39 41 56 59 61 63 64

vi Contents 3 Principles of physically assisted extractions and applications in the food, beverage and nutraceutical industries..................................................................................... 71 E. Vorobiev, Compiègne University of Technology, France and F. Chemat, University of Avignon and Pays de Vaucluse, France 3.1 Introduction....................................................................... 71 3.2 Pulsed electric field-assisted extractions in the food industry.............................................................................. 72 3.3 Ohmic heating-assisted extractions in the food industry 83 3.4 Extraction assisted by high-voltage electrical discharges and applications in the food industry................................ 86 3.5 Ultrasound-assisted extraction (UAE) in the food industry.............................................................................. 90 3.6 Microwave-assisted extraction (MAE) in the food industry.............................................................................. 96 3.7 Combination of physical treatments for extraction in the food industry............................................................... 100 3.8 References......................................................................... 102 4 Advances in process chromatography and applications in the food, beverage and nutraceutical industries .................... M. Ottens and S. Chilamkurthi, Delft University of Technology, The Netherlands 4.1 Introduction....................................................................... 4.2 Basic principles of process chromatography.................... 4.3 Applications of process chromatography in the food, beverage and nutraceutical industries............................... 4.4 Recent developments in process chromatography............ 4.5 Process control in chromatography................................... 4.6 Future trends..................................................................... 4.7 Conclusions....................................................................... 4.8 Sources of further information and advice....................... 4.9 List of abbreviations......................................................... 4.10 References......................................................................... 5 Novel adsorbents and approaches for nutraceutical separation.................................................................................... B. W. Woonton, CSIRO Food and Nutritional Sciences, Australia and G. W. Smithers, Food Industry Consultant, Australia 5.1 Introduction....................................................................... 5.2 Molecular imprinted polymers and applications in the nutraceutical industry........................................................ 5.3 Organic monoliths and applications in the nutraceutical industry..............................................................................


109 109 113 118 128 135 135 137 137 137 138 148

148 149 153

Contents vii 5.4 5.5 5.6 5.7 5.8 5.9

Stimuli-responsive resins and applications in the nutraceutical industry........................................................ Mesoporous molecular sieves and applications in the nutraceutical industry........................................................ Peptide affinity ligands and phage display methodology and applications in the nutraceutical industry................... Membrane adsorbers, membrane chromatography and applications in the nutraceutical industry......................... Conclusions and sources of further information and advice................................................................................ References.........................................................................

6 Advances in the effective application of membrane technologies in the food industry.............................................. M. Pinelo, G. Jonsson and A. S. Meyer, Technical University of Denmark, Denmark 6.1 Introduction....................................................................... 6.2 Theoretical fundamentals of membrane separation.......... 6.3 Membrane technology in the dairy industry..................... 6.4 Membrane technology in the fruit juice industry............. 6.5 Membrane technology for treatment of wastewater in the food industry............................................................... 6.6 New applications of membrane technology for the food industry: concentration and fractionation of saccharides 6.7 Future trends..................................................................... 6.8 References......................................................................... 7 Electrodialytic phenomena, associated electromembrane technologies and applications in the food, beverage and nutraceutical industries............................................................. L. Bazinet, A. Doyen and C. Roblet, Laval University, Canada 7.1 Introduction....................................................................... 7.2 Principles of electrodialytic phenomena and associated membrane technologies..................................................... 7.3 Applications of electrodialytic phenomena and associated membrane technologies................................... 7.4 Future trends .................................................................... 7.5 References......................................................................... 8 Principles of pervaporation for the recovery of aroma compounds and applications in the food and beverage industries..................................................................................... S. Sahin, Middle East Technical University, Turkey 8.1 Introduction....................................................................... 8.2 Principles of pervaporation............................................... 8.3 Transport mechanism in pervaporation for the recovery of aroma compounds......................................................... © Woodhead Publishing Limited, 2010

159 163 166 169 172 173 180 180 181 182 185 190 191 195 197

202 202 203 204 213 214

219 219 220 221

viii Contents 8.4 8.5 8.6 8.7

Selection of membranes for pervaporation in the recovery of aroma compounds.......................................... Recovery of aroma compounds by pervaporation and applications in the food and beverage industries.............. Sources of further information and future trends............. References.........................................................................

9 Advances in membrane-based concentration in the food and beverage industries: direct osmosis and membrane contactors ................................................................................... E. Drioli and A. Cassano, Institute on Membrane Technology, ITM-CNR, Italy 9.1 Introduction....................................................................... 9.2 Conventional technologies in the food and beverage industries........................................................................... 9.3 Direct osmosis and applications in the food and beverage industries............................................................ 9.4 Membrane contactors and applications in the food and beverage industries............................................................ 9.5 Conclusions....................................................................... 9.6 Nomenclature.................................................................... 9.7 References......................................................................... 10 Separation of value-added bioproducts by colloidal gas aphrons (CGA) flotation and applications in the recovery of value-added food products.................................................... P. Jauregi and M. Dermiki, The University of Reading, UK 10.1 Introduction ...................................................................... 10.2 Colloidal gas aphrons (CGA) properties........................... 10.3 Applications of CGA in the recovery of value-added food products..................................................................... 10.4 Feasibility of industrial application of CGA..................... 10.5 Future trends..................................................................... 10.6 Sources of further information and advice....................... 10.7 References......................................................................... 11 Membrane bioreactors and the production of food ingredients................................................................................... M.-P. Belleville, D. Paolucci-Jeanjean and G. M. Rios, European Institute of Membranes, France 11.1 Introduction....................................................................... 11.2 Membrane bioreactors for the production of food ingredients......................................................................... 11.3 Applications of membrane bioreactors in food industries. 11.4 Future trends..................................................................... 11.5 References.........................................................................


227 230 239 240

244 244 245 248 250 275 275 278

284 284 285 293 307 308 309 310 314 314 315 322 331 331

Contents ix Part II Separation technologies in the processing of particular foods and nutraceuticals 12

Separation technologies in dairy and egg processing............. G. Gésan-Guiziou, INRA, France 12.1 Introduction....................................................................... 12.2 The dairy industry and composition of dairy products..... 12.3 Pretreatment of milk using separation techniques............ 12.4 Standardization and concentration of milk proteins in the dairy industry.............................................................. 12.5 Isolation of whole casein in the dairy industry................. 12.6 Separation techniques applied to whey and derivatives in the production of cheese............................................... 12.7 Fractionation of individual proteins and peptides in the dairy industry.................................................................... 12.8 Treatment of effluents and technical fluids in the dairy industry.............................................................................. 12.9 Conclusions and future trends in the dairy industry......... 12.10 The egg products industry and composition of egg products............................................................................. 12.11 Concentration and stabilization of egg white and whole egg..................................................................................... 12.12 Industrial extraction of egg-white proteins....................... 12.13 Industrial extraction of yolk components......................... 12.14 Conclusions and future trends in the egg-processing industry.............................................................................. 12.15 Sources of further information and advice....................... 12.16 References.........................................................................

13

341 341 343 347 351 354 357 360 366 368 369 371 371 374 375 376 377

Separation technologies in the processing of fruit juices....... G. Vatai, Corvinus University of Budapest, Hungary 13.1 Introduction....................................................................... 13.2 Characteristics of foods/fluids in the fruit juice product sector................................................................................. 13.3 Designing separation processes to optimize product quality in the fruit juice product sector............................. 13.4 Production of fruit juice concentrate ............................... 13.5 References.........................................................................

381

1 4

396

Separation technologies in oilseed processing ........................ M. A. Williams, Anderson International Corp., USA 14.1 Introduction....................................................................... 14.2 Preparation for oilseed processing.................................... 14.3 Extrusion preparation for oilseed processing.................... 14.4 Mechanical pressing of oilseeds....................................... 14.5 Percolation solvent extraction in oilseed processing........ 14.6 Solvent recovery in oilseed processing............................. © Woodhead Publishing Limited, 2010

381 382 383 386 394

396 397 399 403 415 422

x Contents

14.7 14.8 14.9 14.10

Obtaining oil from fruit pulps........................................... Future trends..................................................................... Sources of further information and advice....................... References.........................................................................

424 425 427 428

1 5

Separation technologies in brewing.......................................... G. J. Freeman, Campden BRI, UK 15.1 Introduction....................................................................... 15.2 Characteristics of brewery products.................................. 15.3 Selection of technology and raw materials appropriate to brewery products........................................................... 15.4 Wort production in the brewhouse.................................... 15.5 Whirlpools and applications in brewing........................... 15.6 Yeast flocculation and applications in brewing................ 15.7 Beer fining agents ............................................................ 15.8 Filter aid filtration and applications in brewing................ 15.9 Regenerable and reusable filter aids and applications in brewing......................................................................... 15.10 Bulk beer filtration by membranes.................................... 15.11 Recovery of cleaning detergents in brewing..................... 15.12 Dissolved gas control by membrane technology.............. 15.13 Future trends..................................................................... 15.14 References.........................................................................

430

1 6 Methods for purification of dairy nutraceuticals.................... C. J. Fee, J. M. Billakanti and S. M. Saufi, University of Canterbury, New Zealand 16.1 Introduction ...................................................................... 16.2 Components of acidic whey protein ................................ 16.3 Purification technologies for acidic whey proteins . ........ 16.4 Basic proteins in the dairy nutraceutical industry............. 16.5 Purification technologies for basic whey proteins in the dairy nutraceutical industry............................................... 16.6 Immunoglobulins in the dairy nutraceutical industry....... 16.7 Purification technologies for immunoglobulins in the dairy nutraceutical industry............................................... 16.8 Future trends..................................................................... 16.9 References ........................................................................ 17 Methods of concentration and purification of omega-3 fatty acids . ......................................................................................... S. P. J. Namal Senanayake, Danisco USA, Inc., USA 17.1 Introduction....................................................................... 17.2 Urea adduction in the concentration and purification of omega-3 fatty acids........................................................... 17.3 Chromatographic methods for the concentration and purification of omega-3 fatty acids................................... © Woodhead Publishing Limited, 2010

430 431 432 433 434 435 436 437 441 443 446 446 447 448 450 450 451 454 462 463 470 471 473 474 483 483 484 486

Contents xi 17.4 Low-temperature fractional crystallization for the concentration and purification of omega-3 fatty acids...... 17.5 Supercritical-fluid extraction for the concentration and purification of omega-3 fatty acids................................... 17.6 Distillation methods for the concentration and purification of omega-3 fatty acids................................... 17.7 Enzymatic methods for the concentration and purification of omega-3 fatty acids................................... 17.8 Integrated methods for the concentration and purification of omega-3 fatty acids................................... 17.9 Conclusions....................................................................... 17.10 References......................................................................... 18 Extraction of natural antioxidants from plant foods . ........... E. Conde, A. Moure, H. Domínguez and J. C. Parajó, University of Vigo, Spain 18.1 Introduction....................................................................... 18.2 Antioxidant activity in food systems................................ 18.3 Natural compounds with antioxidant activity and major sources............................................................................... 18.4 Biological activities of natural antioxidants..................... 18.5 Extraction of natural antioxidants from plant foods and residues.............................................................................. 18.6 Integration of extraction processes and purification......... 18.7 Future trends..................................................................... 18.8 Sources of further information and advice....................... 18.9 Acknowledgements........................................................... 18.10 References......................................................................... 19 Fractionation of egg proteins and peptides for nutraceutical applications.......................................................... B. P. Chay Pak Ting, Y. Pouliot and S. F. Gauthier, Laval University, Canada and Y. Mine, University of Guelph, Canada 19.1 Introduction....................................................................... 19.2 Composition and physicochemical characteristics of egg proteins and applications in the nutraceutical industry.............................................................................. 19.3 Biological activities of egg proteins and peptides and applications in the nutraceutical industry......................... 19.4 Available technologies for the fractionation of egg proteins and peptides, and applications in the nutraceutical industry........................................................ 19.5 Conclusion and perspectives............................................. 19.6 References.........................................................................


488 490 492 495 498 501 502 506 506 507 511 521 526 556 567 567 568 568 595

595 597 601 605 612 613

xii Contents 2 0 Supercritical-fluid extraction of lycopene from tomatoes...... J. Shi and S. Jun Xue, Agriculture and Agri-Food Canada, Canada, Y. Jiang, The Chinese Academy of Sciences, China and X. Ye, Zhejiang University, China 20.1 Introduction ...................................................................... 20.2 Supercritical-fluid extraction (SFE) of lycopene.............. 20.3 Factors affecting lycopene yield....................................... 20.4 Effects of pressure and temperature on the antioxidant activity of lycopene........................................................... 20.5 Effect of co-solvent and modifiers in lycopene extraction........................................................................... 20.6 Solubility of lycopene in supercritical fluids.................... 20.7 Conclusion and future trends ........................................... 20.8 References.........................................................................

619

619 622 623 628 631 634 639 640

Index..................................................................................................... 647


xiii

Contributor contact details

Chapter 2

(* = main contact) Editor S. S. H. Rizvi Department of Food Science Cornell University 114B Stocking Hall Ithaca, NY 14853-7201 USA E-mail: [email protected]

Chapter 1 Prof. Dr Ž. Knez*, Prof. Dr M. Škerget and M. Knez Hrnčič Faculty of Chemistry and Chemical Engineering University of Maribor Slomškov trg 15 2000 Maribor Slovenia

C. Turner* and M. Waldebäck Department of Physical and Analytical Chemistry Uppsala University P.O. Box 599 751 24 Uppsala Sweden E-mail: [email protected]

and C. Turner Department of Chemistry Lund University P.O. Box 124 221 00 Lund Sweden E-mail: [email protected]

E-mail: [email protected]


xiv Contributor contact details Chapter 3 E. Vorobiev* Groupe Technologies AgroIndustriels EA 4297 Département Génie des Procédés Université de Technologie de Compiègne B.P. 20529 60205 Compiègne France E-mail: [email protected]

F. Chemat UMR 408, Sécurité et Qualité des Produits d’Origine Végétale Université d’Avignon et des Pays de Vaucluse INRA 84000 Avignon France E-mail: [email protected]

Chapter 4 Marcel Ottens* and Sreekanth Chilamkurthi Delft University of Technology Department of Biotechnology Julianalaan 67 2628 BC Delft The Netherlands E-mail: [email protected]

Chapter 5 B. W. Woonton* CSIRO Food and Nutritional Sciences 671 Sneydes Road (Private Bag 16) Werribee Victoria 3030 Australia E-mail: [email protected]

G. W. Smithers Food Industry Consultant P.O. Box 158, Highett Melbourne Victoria 3190 Australia E-mail: [email protected]

Chapter 6 M. Pinelo and A. S. Meyer* Department of Chemical and Biochemical Engineering Center for BioProcess Engineering Building 229 Technical University of Denmark DK-2800 Kgs. Lyngby Denmark E-mail: [email protected]

G. Jonsson CAPEC Center for BioProcess Engineering Building 229 Technical University of Denmark DK-2800 Kgs. Lyngby Denmark Chapter 7 L. Bazinet*, A. Doyen and C. Roblet Institute of Nutraceuticals and Functional Foods (INAF) and Dairy Research Centre (STELA) Department of Food Sciences and Nutrition 2425 rue de l’agriculture, Pavillon Paul-Comtois Laval University Québec, QC, Canada G1V 0A6 E-mail: [email protected]


Contributor contact details xv Chapter 8

Chapter 12

S. Sahin Department of Food Engineering Middle East Technical University 06531, Ankara Turkey

Dr G. Gésan-Guiziou UMR1253 Science et Technologie du Lait et de l’œuf INRA – Agrocampus Ouest 65 rue de Saint Brieuc 35042 Rennes cedex France


E-mail: genevieve.gesan-guiziou@ rennes.inra.fr

Chapter 9 Enrico Drioli* and Alfredo Cassano Institute on Membrane Technology, ITM-CNR c/o University of Calabria via P. Bucci, 17/C I-87030 Rende (CS) Italy E-mail: [email protected];e.drioli@ unical.it; [email protected]

Chapter 10

Chapter 13 Prof. G. Vatai Corvinus University of Budapest Faculty of Food Science Department of Food Engineering 1114 Budapest Menesi út 44 Hungary E-mail: [email protected]

Dr Paula Jauregi* and Dr Maria Dermiki Department of Food and Nutritional Sciences The University of Reading Whiteknights, P.O. Box 226 Reading RG6 6AP UK E-mail: [email protected]

Chapter 14 M. A. Williams Anderson International Corp. 6200 Harvard Avenue Cleveland, OH 44105 USA E-mail: [email protected]

Chapter 11 Dr M.-P. Belleville*, Dr D. Paolucci-Jeanjean and Prof. G. M. Rios Institut Européen des Membranes UMR 5635 CC 047 – UM2 Place E. Bataillon F 34095 Montpellier cedex 05 France E-mail: marie-pierre.belleville@iemm. univ-montp2.fr

Chapter 15 G. J. Freeman Campden BRI Centenary Hall Coopers Hill Road Nutfield Redhill Surrey RH1 4HY UK E-mail: [email protected]


xvi Contributor contact details Chapter 16

Chapter 19

C. J. Fee*, J. M. Billakanti and S. M. Saufi Biomolecular Interaction Centre Department of Chemical and Process Engineering University of Canterbury Private Bag 4800 Christchurch 8020 New Zealand

Bertrand P. Chay Pak Ting, Yves Pouliot*, Sylvie F. Gauthier Département de Sciences des Aliments et de Nutrition and Institute of Nutraceuticals and Functional Foods (INAF) 2425 rue de l’agriculture, Pavillon Paul-Comtois Laval University Québec, QC Canada G1V 0A6



Chapter 17 S. P. J. Namal Senanayake Danisco USA, Inc. Four New Century Parkway New Century, KS 66031 USA E-mail: [email protected]

Chapter 18 E. Conde, A. Moure, H. Domínguez* and J. C. Parajó Department of Chemical Engineering Faculty of Sciences University of Vigo Campus Ourense Spain E-mail: [email protected]

Y. Mine Department of Food Science University of Guelph Guelph, ON Canada N1G 2W1 Chapter 20 J. Shi* and S. Jun Xue Guelph Food Research Center Agriculture and Agri-Food Canada Ontario Canada N1G 5C9 E-mail: [email protected]

Y. Jiang South China Botanical Garden The Chinese Academy of Sciences Guangzhou 510650 China X. Ye Department of Food Science and Nutrition School of Biosystems Engineering and Food Science Zhejiang University Zhejiang 310029 China


xvii

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xviii Woodhead Publishing Series in Food Science, Technology and Nutrition 16 Food emulsions and foams Edited by E. Dickinson 17 Maillard reactions in chemistry, food and health Edited by T. P. Labuza, V. Monnier, J. Baynes and J. O’Brien 18 The Maillard reaction in foods and medicine Edited by J. O’Brien, H. E. Nursten, M. J. Crabbe and J. M. Ames 19 Encapsulation and controlled release Edited by D. R. Karsa and R. A. Stephenson 20 Flavours and fragrances Edited by A. D. Swift 21 Feta and related cheeses Edited by A. Y. Tamime and R. K. Robinson 22 Biochemistry of milk products Edited by A. T. Andrews and J. R. Varley 23 Physical properties of foods and food processing systems M. J. Lewis 24 Food irradiation: a reference guide V. M. Wilkinson and G. Gould 25 Kent’s technology of cereals: an introduction for students of food science and agriculture Fourth edition N. L. Kent and A. D. Evers 26 Biosensors for food analysis Edited by A. O. Scott 27 Separation processes in the food and biotechnology industries: principles and applications Edited by A. S. Grandison and M. J. Lewis 28 Handbook of indices of food quality and authenticity R. S. Singhal, P. K. Kulkarni and D. V. Rege 29 Principles and practices for the safe processing of foods D. A. Shapton and N. F. Shapton 30 Biscuit, cookie and cracker manufacturing manuals Volume 1: ingredients D. Manley 31 Biscuit, cookie and cracker manufacturing manuals Volume 2: biscuit doughs D. Manley 32 Biscuit, cookie and cracker manufacturing manuals Volume 3: biscuit dough piece forming D. Manley 33 Biscuit, cookie and cracker manufacturing manuals Volume 4: baking and cooling of biscuits D. Manley 34 Biscuit, cookie and cracker manufacturing manuals Volume 5: secondary processing in biscuit manufacturing D. Manley 35 Biscuit, cookie and cracker manufacturing manuals Volume 6: biscuit packaging and storage D. Manley 36 Practical dehydration Second edition M. Greensmith 37 Lawrie’s meat science Sixth edition R. A. Lawrie 38 Yoghurt: science and technology Second edition A. Y Tamime and R. K. Robinson 39 New ingredients in food processing: biochemistry and agriculture G. Linden and D. Lorient 40 Benders’ dictionary of nutrition and food technology Seventh edition D A Bender and A. E. Bender


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xx Woodhead Publishing Series in Food Science, Technology and Nutrition 71 Safety and quality issues in fish processing Edited by H. A. Bremner 72 Minimal processing technologies in the food industries Edited by T. Ohlsson and N. Bengtsson 73 Fruit and vegetable processing: improving quality Edited by W. Jongen 74 The nutrition handbook for food processors Edited by C. J. K. Henry and C. Chapman 75 Colour in food: improving quality Edited by D MacDougall 76 Meat processing: improving quality Edited by J. P. Kerry, J. F. Kerry and D. A. Ledward 77 Microbiological risk assessment in food processing Edited by M. Brown and M. Stringer 78 Performance functional foods Edited by D. Watson 79 Functional dairy products Volume 1 Edited by T. Mattila-Sandholm and M. Saarela 80 Taints and off-flavours in foods Edited by B. Baigrie 81 Yeasts in food Edited by T. Boekhout and V. Robert 82 Phytochemical functional foods Edited by I. T. Johnson and G. Williamson 83 Novel food packaging techniques Edited by R. Ahvenainen 84 Detecting pathogens in food Edited by T. A. McMeekin 85 Natural antimicrobials for the minimal processing of foods Edited by S. Roller 86 Texture in food Volume 1: semi-solid foods Edited by B. M. McKenna 87 Dairy processing: improving quality Edited by G Smit 88 Hygiene in food processing: principles and practice Edited by H. L. M. Lelieveld, M. A. Mostert, B. White and J. Holah 89 Rapid and on-line instrumentation for food quality assurance Edited by I. Tothill 90 Sausage manufacture: principles and practice E. Essien 91 Environmentally-friendly food processing Edited by B. Mattsson and U. Sonesson 92 Bread making: improving quality Edited by S. P. Cauvain 93 Food preservation techniques Edited by P. Zeuthen and L. BøghSørensen 94 Food authenticity and traceability Edited by M. Lees 95 Analytical methods for food additives R. Wood, L. Foster, A. Damant and P. Key 96 Handbook of herbs and spices Volume 2 Edited by K. V. Peter 97 Texture in food Volume 2: solid foods Edited by D. Kilcast 98 Proteins in food processing Edited by R. Yada 99 Detecting foreign bodies in food Edited by M. Edwards 100 Understanding and measuring the shelf-life of food Edited by R. Steele © Woodhead Publishing Limited, 2010

Woodhead Publishing Series in Food Science, Technology and Nutrition xxi 101 Poultry meat processing and quality Edited by G. Mead 102 Functional foods, ageing and degenerative disease Edited by C. Remacle and B. Reusens 103 Mycotoxins in food: detection and control Edited by N. Magan and M. Olsen 104 Improving the thermal processing of foods Edited by P. Richardson 105 Pesticide, veterinary and other residues in food Edited by D. Watson 106 Starch in food: structure, functions and applications Edited by A-C Eliasson 107 Functional foods, cardiovascular disease and diabetes Edited by A. Arnoldi 108 Brewing: science and practice D. E. Briggs, P. A. Brookes, R. Stevens and C. A. Boulton 109 Using cereal science and technology for the benefit of consumers: proceedings of the 12th International ICC Cereal and Bread Congress, 24–26th May, 2004, Harrogate, UK Edited by S. P. Cauvain, L. S. Young and S. Salmon 110 Improving the safety of fresh meat Edited by J. Sofos 111 Understanding pathogen behaviour in food: virulence, stress response and resistance Edited by M. Griffiths 112 The microwave processing of foods Edited by H. Schubert and M. Regier 113 Food safety control in the poultry industry Edited by G. Mead 114 Improving the safety of fresh fruit and vegetables Edited by W. Jongen 115 Food, diet and obesity Edited by D. Mela 116 Handbook of hygiene control in the food industry Edited by H. L. M. Lelieveld, M. A. Mostert and J. Holah 117 Detecting allergens in food Edited by S. Koppelman and S. Hefle 118 Improving the fat content of foods Edited by C. Williams and J. Buttriss 119 Improving traceability in food processing and distribution Edited by I. Smith and A. Furness 120 Flavour in food Edited by A. Voilley and P. Etievant 121 The Chorleywood bread process S. P. Cauvain and L. S. Young 122 Food spoilage microorganisms Edited by C. de W. Blackburn 123 Emerging foodborne pathogens Edited by Y. Motarjemi and M. Adams 124 Benders’ dictionary of nutrition and food technology Eighth edition D. A. Bender 125 Optimising sweet taste in foods Edited by W. J. Spillane 126 Brewing: new technologies Edited by C. Bamforth 127 Handbook of herbs and spices Volume 3 Edited by K. V. Peter


xxii Woodhead Publishing Series in Food Science, Technology and Nutrition 128 Lawrie’s meat science Seventh edition R. A. Lawrie in collaboration with D. A. Ledward 129 Modifying lipids for use in food Edited by F. Gunstone 130 Meat products handbook: practical science and technology G. Feiner 131 Food consumption and disease risk: consumer-pathogen interactions Edited by M. Potter 132 Acrylamide and other hazardous compounds in heat-treated foods Edited by K. Skog and J. Alexander 133 Managing allergens in food Edited by C. Mills, H. Wichers and K. Hoffman-Sommergruber 134 Microbiological analysis of red meat, poultry and eggs Edited by G. Mead 135 Maximising the value of marine by-products Edited by F. Shahidi 136 Chemical migration and food contact materials Edited by K. Barnes, R. Sinclair and D. Watson 137 Understanding consumers of food products Edited by L. Frewer and H. van Trijp 138 Reducing salt in foods: practical strategies Edited by D. Kilcast and F. Angus 139 Modelling microorganisms in food Edited by S. Brul, S. Van Gerwen and M. Zwietering 140 Tamime and Robinson’s Yoghurt: science and technology Third edition A. Y. Tamime and R. K. Robinson 141 Handbook of waste management and co-product recovery in food processing Volume 1 Edited by K. W. Waldron 142 Improving the flavour of cheese Edited by B. Weimer 143 Novel food ingredients for weight control Edited by C. J. K. Henry 144 Consumer-led food product development Edited by H. MacFie 145 Functional dairy products Volume 2 Edited by M. Saarela 146 Modifying flavour in food Edited by A. J. Taylor and J. Hort 147 Cheese problems solved Edited by P. L. H. McSweeney 148 Handbook of organic food safety and quality Edited by J. Cooper, C. Leifert and U. Niggli 149 Understanding and controlling the microstructure of complex foods Edited by D. J. McClements 150 Novel enzyme technology for food applications Edited by R. Rastall 151 Food preservation by pulsed electric fields: from research to application Edited by H. L. M. Lelieveld and S. W. H. de Haan 152 Technology of functional cereal products Edited by B. R. Hamaker 153 Case studies in food product development Edited by M. Earle and R. Earle


Woodhead Publishing Series in Food Science, Technology and Nutrition xxiii 154 Delivery and controlled release of bioactives in foods and nutraceuticals Edited by N. Garti 155 Fruit and vegetable flavour: recent advances and future prospects Edited by B. Brückner and S. G. Wyllie 156 Food fortification and supplementation: technological, safety and regulatory aspects Edited by P. Berry Ottaway 157 Improving the health-promoting properties of fruit and vegetable products Edited by F. A. Tomás-Barberán and M. I. Gil 158 Improving seafood products for the consumer Edited by T. Børresen 159 In-pack processed foods: improving quality Edited by P. Richardson 160 Handbook of water and energy management in food processing Edited by J. Klemeš, R. Smith and J-K Kim 161 Environmentally compatible food packaging Edited by E. Chiellini 162 Improving farmed fish quality and safety Edited by Ø. Lie 163 Carbohydrate-active enzymes Edited by K-H Park 164 Chilled foods: a comprehensive guide Third edition Edited by M. Brown 165 Food for the ageing population Edited by M. M. Raats, C. P. G. M. de Groot and W. A Van Staveren 166 Improving the sensory and nutritional quality of fresh meat Edited by J. P. Kerry and D. A. Ledward 167 Shellfish safety and quality Edited by S. E. Shumway and G. E. Rodrick 168 Functional and speciality beverage technology Edited by P. Paquin 169 Functional foods: principles and technology M. Guo 170 Endocrine-disrupting chemicals in food Edited by I. Shaw 171 Meals in science and practice: interdisciplinary research and business applications Edited by H. L. Meiselman 172 Food constituents and oral health: current status and future prospects Edited by M. Wilson 173 Handbook of hydrocolloids Second edition Edited by G. O. Phillips and P. A. Williams 174 Food processing technology: principles and practice Third edition P. J. Fellows 175 Science and technology of enrobed and filled chocolate, confectionery and bakery products Edited by G. Talbot 176 Foodborne pathogens: hazards, risk analysis and control Second edition Edited by C. de W. Blackburn and P. J. McClure 177 Designing functional foods: measuring and controlling food structure breakdown and absorption Edited by D. J. McClements and E. A. Decker


xxiv Woodhead Publishing Series in Food Science, Technology and Nutrition 178 New technologies in aquaculture: improving production efficiency, quality and environmental management Edited by G. Burnell and G. Allan 179 More baking problems solved S. P. Cauvain and L. S. Young 180 Soft drink and fruit juice problems solved P. Ashurst and R. Hargitt 181 Biofilms in the food and beverage industries Edited by P. M. Fratamico, B. A. Annous and N. W. Gunther 182 Dairy-derived ingredients: food and neutraceutical uses Edited by M. Corredig 183 Handbook of waste management and co-product recovery in food processing Volume 2 Edited by K. W. Waldron 184 Innovations in food labelling Edited by J. Albert 185 Delivering performance in food supply chains Edited by C. Mena and G. Stevens 186 Chemical deterioration and physical instability of food and beverages Edited by L. H. Skibsted, J. Risbo and M. L. Andersen 187 Managing wine quality Volume 1: viticulture and wine quality Edited by A.G. Reynolds 188 Improving the safety and quality of milk Volume 1: milk production and processing Edited by M. Griffiths 189 Improving the safety and quality of milk Volume 2: improving quality in milk products Edited by M. Griffiths 190 Cereal grains: assessing and managing quality Edited by C. Wrigley and I. Batey 191 Sensory analysis for food and beverage quality control: a practical guide Edited by D. Kilcast 192 Managing wine quality Volume 2: oenology and wine quality Edited by A. G. Reynolds 193 Winemaking problems solved Edited by C. E. Butzke 194 Environmental assessment and management in the food industry Edited by U. Sonesson, J. Berlin and F. Ziegler 195 Consumer-driven innovation in food and personal care products Edited by S. R. Jaeger and H. MacFie 196 Tracing pathogens in the food chain Edited by S. Brul, P. M. Fratamico and T. A. McMeekin 197 Case studies in novel food processing technologies: innovations in processing, packaging, and predictive modelling Edited by C. Doona, K Kustin and F. Feeherry 198 Freeze-drying of pharmaceutical and food products T-C. Hua, B-L. Liu and H. Zhang 199 Oxidation in foods and beverages and antioxidant applications Volume 1: understanding mechanisms of oxidation and antioxidant activity Edited by E. A. Decker, R. J. Elias and D. J. McClements 200 Oxidation in foods and beverages and antioxidant applications


Woodhead Publishing Series in Food Science, Technology and Nutrition xxv

201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222

Volume 2: management in different industry sectors Edited by E. A. Decker, R. J. Elias and D. J. McClements Protective cultures, antimicrobial metabolites and bacteriophages for food and beverage biopreservation Edited by C. Lacroix Separation, extraction and concentration processes in the food, beverage and nutraceutical industries Edited by S. S. H. Rizvi Determining mycotoxins and mycotoxigenic fungi in food and feed Edited by S. De Saeger Developing children’s food products Edited by D. Kilcast and F. Angus Functional foods: concept to profit Second edition Edited by M. Saarela Postharvest biology and technology of tropical and subtropical fruits Volume 1 Edited by E. M. Yahia Postharvest biology and technology of tropical and subtropical fruits Volume 2 Edited by E. M. Yahia Postharvest biology and technology of tropical and subtropical fruits Volume 3 Edited by E. M. Yahia Postharvest biology and technology of tropical and subtropical fruits Volume 4 Edited by E. M. Yahia Food and beverage stability and shelf-life Edited by D. Kilcast and P. Subramaniam Processed Meats: improving safety, nutrition and quality Edited by J. P. Kerry and J. F. Kerry Food chain integrity: a holistic approach to food traceability, authenticity, safety and bioterrorism prevention Edited by J. Hoorfar, K. Jordan, F. Butler and R. Prugger Improving the safety and quality of eggs and egg products Volume 1 Edited by Y. Nys, M. Bain and F. Van Immerseel Improving the safety and quality of eggs and egg products Volume 2 Edited by Y. Nys, M. Bain and F. Van Immerseel Feed and fodder contamination: effects on livestock and food safety Edited by J. Fink-Gremmels Hygiene in the design, construction and renovation of food processing factories Edited by H. L. M. Lelieveld and J. Holah Technology of biscuits, crackers and cookies Fourth edition Edited by D. Manley Nanotechnology in the food, beverage and nutraceutical industries Edited by Q. Huang Rice quality K. R. Bhattacharya Meat, poultry and seafood packaging Edited by J. P. Kerry Reducing saturated fats in foods Edited by G. Talbot Handbook of food proteins Edited by G. O. Phillips and P. A. Williams


xxvi

xxvii

Preface

Separation, extraction and concentration of desirable components from their natural matrices are essential unit operations in the preparation of key ingredients for use in the food, pharmaceutical and chemical industries. These processes often account for somewhere between 50% and 70% of the product cost and are facing new challenges. Ever-tightening regulations on the use of organic solvents, environmental issues and process safety have accelerated the development of a variety of new technologies which are clean and efficient, and do not cause degradation of the products; these technologies therefore enhance the productivity and global competitiveness of the industries concerned. This book aims to provide a comprehensive overview of the most important technologies of interest for the production of high-value compounds. Based on the multidisciplinary expertise of 45 contributors from institutions with strong programs in separation, extraction and concentration processes, the book is organized in 20 peer-reviewed chapters, divided into two parts. Part I describes the latest advances in separation, extraction and concentration techniques, including supercritical fluid extraction, process chromatography and membrane technologies. It also reviews emerging techniques of particular interest, such as pervaporation and pressurized liquid extraction. Part II then focuses on advances in separation technologies and their applications in various sectors of the food, beverage and nutraceutical industries. Areas covered include dairy and egg processing, oilseed extraction and brewing. This part of the book discusses the characteristics of different foods and fluids, how food constituents are affected by separation processes and how separation processes can be designed and operated to optimize end product quality. This volume collectively provides valuable and timely information on the latest developments in the field. With its team of experienced international contributors, Separation, extraction and concentration processes in the food, beverage and nutraceutical industries is an important reference source for experienced professionals concerned with the development and optimization of these processes. It © Woodhead Publishing Limited, 2010

xxviii Preface is hoped that newcomers to this exciting and emerging field will also find valuable information in this book. I wish to thank the authors for their patience and support during the review and preparation of the manuscripts. I also wish to thank Sarah Whitworth of Woodhead Publishing who gave useful advice during the initial planning stages of the book. Syed S. H. Rizvi


Principles of supercritical fluid extraction and applications 3

1 Principles of supercritical fluid extraction and applications in the food, beverage and nutraceutical industries Ž. Knez, M. Škerget and M. Knez Hrn�i�, University of Maribor, Slovenia Abstract: The thermodynamic fundamentals of supercritical fluid extraction (SFE) are described and the environmental, health and safety benefits of using supercritical fluids are explored. Several hundred industrial-scale SFE plants are in operation worldwide for extraction of plant materials, such as hop constituents, decaffeination of tea and coffee, and separation of lecithin from oil, all high-pressure processes. Smaller industrial units are used for extraction of spices in the food industry and for natural substances used in cosmetics. The design of such an extraction plant is described. The unique thermodynamic and fluid dynamic properties of dense gases are also applied in integrated extractions and in in situ formulations, such as impregnation of solid particles, formation of solid powder emulsions, and particle coatings. Key words: extraction, supercritical fluids, dense gases, high pressure, thermodynamics, food industry.

1.1 Introduction The design of new products with special characteristics or of new processes that are environmentally friendly and have an impact on sustainability, present a great challenge to chemical engineers. Within the human environment, pressures range from 0.25 bar at the top of the highest mountain, up to 1000 bar at the bottom of the deepest ocean. Because human beings live on the surface of the globe, the first technologies for the production of various substances took place at atmospheric pressure. In the early 20th century, demand for new products like ammonia shifted the technological processes towards high pressure. Industrial high-pressure processes operate at ranges from about 50 bar (in particle formation processes) to over 200 © Woodhead Publishing Limited, 2010

4 Separation, extraction and concentration processes kbar (conversion of graphite to diamonds). High pressure is a relatively new tool and in several processes it has resulted in completely new products with special characteristics. Many of these new processes are environmentally friendly, low cost and sustainable. The advantages of using supercritical fluids (SCF) as solvents in chemical synthesis offer environmental benefits, health and safety benefits and chemical benefits (Jessop and Leitner, 1999). The environmental benefits of most SCFs in industrial processes result from their replacement of far more environmentally damaging conventional organic solvents. The low energy consumption of the process is a further environmental benefit. Health and safety benefits include the fact that the most important SCFs (SC CO2 and SC H2O) are non-carcinogenic, non-toxic, non-mutagenic, non-flammable and thermodynamically stable. One of the major process benefits is derived from the thermophysical properties of SCFs: high diffusivity, low viscosity and the density and dielectric constant of SCF, which can be fine tuned by changes in operating pressure and/or temperature. The motivation for using high pressure in a wide range of technologies and processes is based on chemical, physicochemical, physicobiochemical, physicohydrodynamic and physicohydraulic effects (Bertuco and Vetter, 2001). The extraction of hop constituents and the decaffeination of tea and coffee are the largest scale processes and are mostly performed on an industrial scale. Several industrial plants also extract spices for the food industry and natural substances for use in cosmetics. The advantages of using supercritical fluids for the isolation of natural products have been well described (Marr and Gamse, 2000) and include solvent-free products, low temperature, and no byproducts. One of the most important advantages of using supercritical fluids is the selective extraction of components or the fractionation of total extracts, which is made possible by use of different gases for isolation or fractionation of components and/or by changing the process parameters. A limitation on the further application of high-pressure technology for obtaining extracts is the relatively high price of the product compared with that of those produced conventionally. The legal restrictions on solvent residues and solvents (in products for human use) and the isolation or fractionation of special components from total extracts, in combination with different formulation (controlled release for example) and sterilisation processes, encourage the use of dense gases in extraction applications. There are fewer industrial units involved in using supercritical fluids for the separation of components from liquid mixtures. Some laboratoryscale studies involve extraction systems using liquid/supercritical fluid. Some data on binary systems liquid/SCF has been produced, but there are fewer data on liquid/liquid/supercritical fluid systems. As in all extraction processes, including the supercritical extraction of solid and liquid mixtures, the solubility of a single component or a mixture of components in SCF is the basic data for the design of separation processes. The components


Principles of supercritical fluid extraction and applications 5 or mixture of compounds to be extracted must be soluble in SCF/dense gas. As is known from thermodynamics, the solubility of compounds in SCF/ dense gases depends on the density of the SCF/dense gas, which depends on the pressure and temperature of the SCF. Another very important parameter influencing the solubility of compounds in SCF is its dielectric constant, which is influenced by the temperature and/or pressure of the SCF. The general flow sheet of the extraction process is presented in Fig. 1.1 and some industrial scale units are shown in Figs 1.2 to 1.4. In one extraction stage, the solubility of a compound or mixture of compounds has to be high whereas in another stage, the solubility of a compound in SCF has to be low. Therefore, the phase equilibrium data is the most important factor in the design of the operating pressure and temperature of SCF in an extraction plant. Thus, the theoretical amount of SCF necessary for separating compounds from a solid or a liquid mixture may be calculated. The design of process parameters has a very important influence on the investment costs of high pressure plants and subsequently, for the economics of the process. In addition to the solubility data of solute in SCF, mass transfer also has a large influence on the economics of the extraction process. Mass transfer models usually describe extraction yield versus extraction time, but a better presentation for the design of extraction apparatuses is yield versus mass of SC solvent/mass of solid material (S/F). Cascade operation is used in industrial-scale plants to increase the economy of the solid–SC solvent extraction process. To date, there has been some research on the continuous operation of plants for the extraction of solids with SC solvent (Eggers, 1996), but currently, no application for

Extractor

PEXT

PS

TEXT

TS

Solvent tank

Extract

Fig. 1.1 General flow sheet of SCF extraction plant. Subscripts EXT and S represent the extractor and separator respectively. © Woodhead Publishing Limited, 2010

6 Separation, extraction and concentration processes

Fig. 1.2 High-pressure extraction unit (700 bar). (Photo: courtesy of Uhde HPT, Hagen, Germany.)

Fig. 1.3 High-pressure extractor solids during manufacturing. (Photo: courtesy of Uhde HPT, Hagen, Germany.)

the continuous feed of solids has been applied on an industrial scale. In industrial scale operations, extractors are usually combined in series. By means of the cyclic operation of a battery of extractors, a quasi-continuous solid flow may be achieved. Such a mode of operation results in extremely high extraction yields because pure solvent is in contact with pre-extracted material, thus loading the solvent to maximum capacity.



Fig. 1.4 High-pressure extraction unit: closure of extractor. (Photo: courtesy of Uhde HPT, Hagen, Germany.)

1

2

3

Solvent tank

4

Extract

Fig. 1.5 Cascade of extractors for extraction of solid materials.

One of the major advantages of SFE processes is the fractionation of extracts. Multi-step separation may be performed by use of several separators by decreasing the solvent power. Decreased solvent power may be achieved by various methods, as described in sections 1.3 and 1.4.



Extractor

PEXT

PS1

PS2

PS3

TEXT

TS1

TS2

TS3

Fraction 1

Fraction 2

Solvent tank

Fraction 3

Fig. 1.6 Scheme of multi-step separation.

1.2 Thermodynamic fundamentals Extraction with dense gases is, as with solvent extraction and leaching, a separation process based on the solubility of compounds in phases present in the system. The driving force for mass and heat transfer is the difference from the equilibrium state under given conditions. High-pressure phase behaviour near critical points can be complex, even in simple binary mixtures, especially when the components differ in molecular size, shape, structure and polarity. Complex phase behaviour can be readily interpreted by P–T and P–x projections of P–T–x space diagrams. By using the phase rule (equation [1.1]), the geometrical limits for presentation of multiphase regions in the phase diagram are determined.

F = c + 2 – p

[1.1]

where F is the number of independent variables, c is the number of components and p is the number of phases. The phase equilibrium of compounds (1, 2, 3, ..., N) distributed in phases (a, b, g, ..., p) present in the system can be defined in terms of the fugacity (f) by the following equation:

fia = fib = fig = …… = fip

[1.2]

where i = 1, 2, 3, ..., N. Only binary systems will be discussed in detail in this chapter. The phase equilibria of ternary and multicomponent mixtures is discussed by Brunner (1994) and McHugh and Krukonis (1986), and Sadus (1992). 1.2.1 Solid–supercritical fluid equilibrium Phase diagrams In solid–SCF systems where the normal melting temperature of the solid is higher than the critical temperature (Tc) of the SCF, two possible types of © Woodhead Publishing Limited, 2010

Principles of supercritical fluid extraction and applications 9 phase behaviour exist. The simplest is presented in Fig. 1.7a and is typical of mixtures in which the components are chemically similar. The critical mixture curve runs continuously between the critical points of both components of the mixture. The solid–liquid–gas (SLG) line is continuous and begins at the normal melting point of the heavy component, moves toward lower temperatures as the pressure is increased, and ends at a temperature usually well below the critical temperature of the lighter component. The melting point of the pure solid normally increases with an increase in the hydrostatic pressure. However, in the presence of dense gas, the melting point of the solid decreases as the pressure increases owing to the increasing solubility of gas in the solid. The second type of solid–SCF phase behaviour (Fig. 1.7b) is typical for systems in which the solid and the SCF differ considerably in molecular size, shape and/or polarity and can be interpreted as type III fluid-phase behaviour (de Loos, 2006) according to the classification of van Konynenburg and Scott (1980). In this type of system, the light gas is not very soluble in the heavy liquid, even at high pressures. Therefore, the melting-point depression of the solid is relatively small. The SLG curve is no longer continuous; three phase SLG equilibria are presented by two branches of the SLG line in P–T diagram. The high-temperature branch of the SLG line starts at the normal melting point of the solid and intersects with the critical-mixture curve at the upper critical end point (UCEP). The low-temperature branch of the SLG line intersects with the critical-mixture curve at the lower critical end point (LCEP). At these two points, the liquid and gas phases merge into a single fluid phase in the presence of excess solid. Only solid–gas equilibria exist between these two branches of the SLG line. Possible phase behaviour for type III systems with interference of the solid phase is presented in detail by de Loos (2006). The course of the high-temperature branch of the three-phase SLG line of a binary system depends on the solubility of the gas in the liquid phase. SL

UCEP

1

C2

LG

C1

SLG

2

Pressure (P)

Pressure (P)

SL C1

LG

LG

C2

LCEP SLG

2

1 SLG

TP

TP

Temperature (T) (a)

Temperature (T) (b)

Fig. 1.7 Solid–SCF equilibrium: P–T projection of phase diagram for similar (a) and asymmetrical (b) binary systems. C, critical point; TP, triple point; L, liquid; G, gas; S, solid; UCEP, upper critical end point; LCEP, lower critical end point. Dashed curves are critical lines, lines denoted as 1 and 2 are the vapour pressure curves of the two components. © Woodhead Publishing Limited, 2010

10 Separation, extraction and concentration processes If a compressed gas is dissolved in the melting of a heavy component, two opposite temperature effects occur as given by equation [1.3] (Arons and Diepen, 1963):

fus Ê ∂T ˆ ˆ Ê dT = Á ˜ dP + Á ∂T ˜ dxA = TDVfus d + Ë ∂x A ¯ P Ë ∂P¯ x D A

2 A

fus

d

A

[1.3]

These two effects are that the increase of hydrostatic pressure increases the RT melting temperature of the heavy component, and Pthe dissolved gas x in the D H x H heavy component decreases the melting temperature. TheDhigher solubility of gas in the melted heavy component results in a larger melting-point depression. Four characteristic shapes of the SLG equilibrium lines in the P–T projection were observed experimentally for asymmetric binary systems of compressed gases and non-volatile components (Arons and Diepen, 1963; Loos, 2006; Tuminello et al., 1995; Weidner et al., 1997) (Fig. 1.8), with: ∑ negative dP/dT slope, where the effect of gas solubility predominates; ∑ positive dP/dT slope, where the effect of pressure predominates; ∑ temperature minimum, where both effects are competing; and ∑ temperature maximum and a temperature minimum. The last type of SLG line is a very rare phenomenon and can be explained by the higher solubility of the supercritical gas in the solid phase than in the liquid phase (de Loos, 2006). A SLG curve with a temperature maximum and a temperature minimum was reported for the systems CO2 + polyethylene glycol (Weidner et al., 1997), CO2 + tripalmitin (O’Connell et al., 2003), and

LG

UCEP

Pressure (P)

SLG 2

SLG 1 C2 SLG 4

SLG 3

1

TP

Temperature (T)

Fig. 1.8 Characteristic shapes of the SLG equilibrium lines.


Principles of supercritical fluid extraction and applications 11 CO2-tristearin (O’Connell et al., 2003). However, in most studies of these equilibria, it is assumed that the supercritical gas is insoluble in the solid phase of the non-volatile component (de Loos, 2006). The course of the SLG line is dependent on the gas and the chemical structure of the compound, i.e. the type and position of the functional groups. As an example, the melting point of vitamin K3 under the pressure of various gases is presented in Fig. 1.9 (Knez and Škerget, 2001) and the various paths of the SLG line can be observed. In the presence of CO2 and dimethyl ether, the negative slope dP/dT can be observed and the meltingpoint depression of vitamin K3 is highest under the pressure of dimethyl ether. The melting-point depression of vitamin K3 is less pronounced in the presence of propane, the SLG curve having a minimum at 94.9 °C and 39 bar. Under the pressure of inert gas (nitrogen and argon), the SLG curve has a positive dP/dT slope owing to the low solubility of gas in vitamin K3. Another example, which illustrates that isomers may have a different type of SLG line in the presence of a specific gas, is the vanillin–gas system. In Fig. 1.10, SLG phase lines for binary systems of vanillin (V) and o-vanillin (o-V) with fluorinated hydrocarbons (R23, R134a, R236fa) and CO2 are presented. For vanillin with –OH group in the para position, the meltingpoint depression in CO2 and fluorinated hydrocarbons is generally lower as for vanillin with –OH group in the ortho position. Thermodynamic modelling For a binary solid–SCF two-phase system at equilibrium, the fugacity of the solute in the solid phase is equal to that in the supercritical phase:

fiS (P, T , x ) = fiG (P, T , y)

[1.4]

350

Propane Dimethyl ether

300

CO2 250

N2

P (bar)

Ar 200 150 100 50 0 50

70

90

T (°C)

110

130

150

Fig. 1.9 SLG phase equilibria for binary system K3 – gas (CO2, propane, dimethyl ether, argon or nitrogen) (Knez and Škerget, 2001).


12 Separation, extraction and concentration processes 400 V - R23

CHO

350

V - R134a

300

V - R236fa OH

V - CO2 P (bar)

250

OCH3

o-V - R23 CHO

o-V - R134a

200

OH

o-V - R236fa 150

OCH3

o-V - CO2

100 50 0

0

10

20

30

40 50 T (°C)

60

70

80

90

Fig. 1.10 SLG phase lines for binary systems of vanillin (V) and o-vanillin (o-V) with dense gases.

By denoting the gas as component 1 and the non-volatile compound as component 2, and assuming that the solubility of the gas in the solid phase is negligible (x2 = 1), the fugacity of the solid in the solid phase is equal to the fugacity of pure solid (Prausnitz et al., 1986):

f2S (P, T , x ) = f2S,pure (P, T )

[1.5]

The fugacity of a pure compound is:

RT ln

f2S,pure (P,T ) = P

Ú0

RT ln

f2S,pure (P,T ) = P

Ú0

P

P2S

RT ˆ Ê ÁË v2 – P ˜¯ dP RT ˆ Ê ÁË v2 – P ˜¯ dP +

[1.6] P

ÚP

S 2

Ê S RT ˆ ÁË v2 – P ˜¯ dP [1.7]

where P2s is the sublimation pressure at the system temperature and v2S is the molar volume of the pure solid. The first term on the right side is the fugacity of the saturated vapour, which is equal to the fugacity of the saturated solid phase. The second term is the correction owing to the compression of the solid phase to pressure P.

RT ln

f2S,pure (P,T ) fS = RT ln 2S + P P2

P

ÚP

S 2

v2S dP S P – RT ln P2 P


[1.8]

Principles of supercritical fluid extraction and applications 13 By rearranging and inserting the expression for the fugacity coefficient fS j 2S = 2S : P2

Ê f2S,pure (P, T ) = P2Sj 2S exp Á 1 Ë RT

P

ÚP

S 2

ˆ v2S dP˜ ¯

[1.9]

where j2S is the fugacity coefficient at T and P2S. The fugacity of the gas phase is:

f2G (P, T , y) = j 2G y2 P

[1.10]

j2G

is the fugacity coefficient of solid component 2 in the mixture. where By inserting equations [1.9] and [1.10] into [1.5] and [1.4], the expression for solubility of solids in the gas phase is obtained:

P Ê ˆ P2Sj 2S expÁ 1 Ú S v2S dP˜ Ë RT P2 ¯ y2 = G Pj 2

[1.11]

Equation [1.11] can also be expressed in the form:

y2 =

P2S E P

[1.12]

where

P Ê ˆ j 2S expÁ 1 Ú S v2S dP˜ Ë RT P2 ¯ E∫ G j2

[1.13]

The enhancement factor E is the correction of the ideal-gas expression that is valid only at low pressures and contains three terms: j2S, which takes into account the non-ideality of the pure saturated vapour. For low sublimation pressure of the solid P2S, j2S almost equals unity. ∑ an exponential term called the Poynting correction, which gives the effect of pressure on the fugacity of the pure solid. It is small at low pressures but may become larger at high pressures or at low temperatures. ∑ j2G, the gas-phase fugacity coefficient in the high-pressure gas mixture. This term is the most important, because it is much lower than 1 and can produce very large enhancement factors (103 or higher). ∑

Assuming that the molar volume of the pure solid (v 2S) at the system temperature is pressure independent, the Poyning correction takes the simple form:



Ê vS (P – P2S )ˆ j 2S expÁ 2 ˜¯ RT Ë E= G j2

[1.14]

j2G can be calculated from an equation of state using following equation:

lnj i = 1 RT

•

ÚV

ÈÊ ∂P ˆ ˘ – RT ˙ dV – ln PV ÍÁ ˜ nT RT V ˙ ÍÎË ∂ni ¯ T ,V , n j ˚

[1.15]

where ni is the molar number of species i in the mixture, V is the total volume, v is the molar volume, and R is the gas constant. To calculate fugacity coefficients, the equations of state (EOS) are commonly applied in engineering practice because they can be used to predict the thermodynamic properties of fluids and describe the phase behaviour of mixtures over a wide range of temperature and pressure. Detailed reviews on the thermodynamic models applied for predicting phase behaviour and modelling aspects in supercritical fluid mixtures have been presented elsewhere (Anderko, 1990, 2000; Dohrn, 1994; Tuminello et al., 1995). Cubic EOS in combination with mixing rules are currently the most widely used models for the calculation of solubilities of components in SCF (Tables 1.1 and 1.2). Table 1.1 Cubic equations of state General form

Van der Waals (VDW: b = 0, c = 0) Redlich–Kwong (RK: c = 0)

Redlich–Kwong–Soave (RKS: c = 0) Peng–Robinson (PR: c = b)

a P = RT – v – b v(v + b ) + c(v – b )

[1.16]

a P = RT – 2c v–b v

[1.17]

acTc1/2 P = RT – v – b v(v + b )T 1/2

[1.18]

a P = RT – v – b v(v + b )

[1.19]

a P = RT – v – b v(v + b ) + b(v – b )

Where: a = aca

ac = Wa

[1.20]

[1.21]

RT Pc 2

2 c

[1.22]

a = [1 + c(1 – Tr1/2)]2

[1.23]

2

c = A 0 + B 0w + C 0w

[1.24]

RTc Pc

[1.25]

b = Wb


Principles of supercritical fluid extraction and applications 15 Table 1.2 Constants in cubic equations of state Konstanta

VDW

RK

RKS

PR

Wa Wb A0 B0 C0

0.42188 0.125 – – –

0.42478 0.08664 – – –

0.42747 0.08664 0.48 1.574 –0.176

0.45724 0.0778 0.37464 1.54226 –0.26992

In the equations in Tables 1.1 and 1.2, the parameters a and b reflect the contribution of attractive forces and molecular volume, respectively, and w, the acentric factor, is a measure of the acentric nature of intermolecular forces. In order to extend the use of a pure-fluid EOS to mixtures, it is assumed that the EOS for the mixture is the same as for a hypothetical pure fluid (Prausnitz et al., 1986) and that the characteristic constants a and b are dependent on composition. Usually, van der Waals one-fluid mixing rules with one or two adjustable parameters are used:

a = ∑∑ yi y j aij i j

b = ∑∑ yi y j bij i j

[1.26]

[1.27]

where a accounts for interactions between the species in the mixture and b accounts for the excluded volume of the species of the mixture. The cross coefficients aij and bij are related to the corresponding pure-component parameters by the following combining rules:

aij = aii a jj (1 – kij )

bij = 1 (bii + b jj )(1 – cij ) 2

[1.28] [1.29]

where kij and cij are the binary interaction and size parameters, respectively. When cij is set to zero, the co-volume b is expressed by a linear mixing rule, equation [1.29] reduces to:

b = ∑ yi bi i

[1.30]

In most practical applications, a linear mixing rule is used for the co-volume b (Anderko, 1990). However, many authors use a quadratic mixing rule for b in analogy with the mixing rule for a. In this way, a second binary parameter is introduced, which is usually found to be useful in correlating gas–liquid equilibria in mixtures with components of very different size (Anderko, 1990). Similarly, it usually improves results for solid–supercritical fluid equilibria © Woodhead Publishing Limited, 2010

16 Separation, extraction and concentration processes because of the large size differences in such systems (Anderko, 1990). Generally, kij and cij are of the same order of magnitude (Dohrn, 1994) and both are expected to have an absolute value of much less than 1 (McHugh et al., 1986). They can both be positive or negative. A negative value of kij usually indicates that specific chemical interactions, such as hydrogen bonding, are present in the mixture (McHugh et al., 1986). However, it is less apparent how to interpret a negative value for cij. By calculating partial derivatives of cubic EOS, inserting derivatives in equation [1.15] and after integrating the expression for the fugacity coefficient of the component, i is obtained. The expression for the fugacity coefficient of component i by using the Peng–Robinson (PR) EOS with the above defined van der Waals mixing rules is:

lnj i =

bN Ê Pv ˆ – 1˜ b ÁË RT ¯

Ê N ˆ 2∑y j aij Á b ˜ P(v – b ) j a – ln – – N˜ Á b˜ RT 2 2 RTb Á a ÁË ˜¯

¥l

[1.31]

where:

N

N

N –1 N

k

j

j =1i = j +1

bN = 2∑yk bik – ∑ y 2j b jj – 2 ∑ ∑ yi yi –j

ij

and for a binary system: v + (1 + 2)b y b ln bN = 2yi bij +v 2+y(1 yi2bbii – y 2j b jj – 2yi y j bi j b jj– – 2)

[1.32]

[1.33]

ij in the above equations The critical properties and acentric factors needed can usually be estimated by the use of group contribution methods, molar volume can be determined experimentally e.g. with a pycnometer, whereas the binary interaction and size parameters, kij and cij, respectively, are obtained by fitting the equation of state to measured phase-equilibrium data. The form of the mixing rules that extend the use of EOS developed for pure fluids to mixtures is, as reported (Anderko, 1990; Škerget et al., 2002), more important than the particular P–V–T relationship embodied in EOS. The shortcoming of the van der Waals one-fluid mixing rules is that they are only applicable to mixtures that exhibit relatively moderate solution nonidealities. Further problems, which persist when modelling phase equilibria in SCF mixtures with the use of cubic equations of state where the systems are highly non-ideal owing to the high pressures involved, are described by Prausnitz et al. (1986) and Škerget et al. (2002):

(a) the interaction parameters are often found to be temperature dependent; © Woodhead Publishing Limited, 2010

Principles of supercritical fluid extraction and applications 17 (b) reliable values for the necessary physical parameters are not always available; and (c) the equation does not fit the data equally well at all temperatures and pressures, and especially in the vicinity of the critical point, the deviation from the model is high. There have been a number of attempts to extend the range of EOS. Most equations retain the van der Waals separation of repulsive and attractive terms, but have introduced some modifications to either the attractive or repulsive term, or to both. In this way, a number of empirical and theoretical models have been developed (Sandler and Orbey, 2000, Sadus, 1992), which allow extrapolation and prediction over wide ranges of temperature and pressure and can describe greater degrees of non-ideality. Empirical approaches to eliminating the shortcomings of the van der Waals one-fluid model for a cubic EOS have been done to provide additional composition dependence or density dependence, by adding parameters to the combining rule for the parameter a, generally leaving the mixing rule for the parameter b unchanged (Sadus, 1992, Sandler and Orbey, 2000, Škerget et al., 2002). Some examples are the composition dependent combining rules of Adachi and Sugie (1986), Panagiotopoulos et al. (1986), Sandoval et al. (1989) and Schwartzentruber and Renon (1989 a,b). The density-dependent mixing rules have been revived by Anderko (1990) and Danner and Gupte (1986). A more empirical way was adopted by Luedecke and Prausnitz (1985). However, there are several problems associated with these multi-parameter combining rules which limit their use (Anderko, 1990; Škerget et al., 2002). The common difficulty is that they do not result in the correct treatment of ternary and multicomponent mixtures (de Loos, 2006). An alternative method is based on the combination of the equations of state with activitycoefficient models (Huron and Vidal (1979) and Wong and Sandler mixing rules (Wong and Sandler, 1992), which are usually fairly reliable for the prediction of multicomponent phase equilibria from binary data, except for the most strongly non-ideal systems. In binary three-phase SLG systems at equilibrium, the parameters needed in the PR EOS to model the SLG line are determined by solving the following equations:

f 2G(T, P, y2) = f 2L(T, P, x2)

[1.34]

f 2S (T,

[1.35]

f 1G(T, P, y1) = f 1L(T, P, x1)

P) =

f 2G(T,

P, y2)

[1.36]

Equation [1.35] transforms to equation [1.11] and equations [1.36] and [1.34] become:

y1j1G = x1j1L

[1.37]

y2j2G

[1.38]

=

x2j2L © Woodhead Publishing Limited, 2010

18 Separation, extraction and concentration processes Calculation of the pure solute fugacity The fugacity of a solute in a solid phase cannot be directly calculated by a conventional EOS, but it may be calculated by means of a sub-cooled liquid reference state. An equation developed by Prausnitz et al. (1986) is used to express the fugacity of the sub-cooled liquid at temperature T in terms of measurable thermodynamic properties: ln

f2L,pure DH 2fus Ê Tt,2 ˆ = ÁË T – 1¯ S,pure RT t,2 f2

Dcp,2 Tt,2 R T

Dcp,2 R

Tt,2 T [1.39]

where Tt,2 is the triple-point temperature, and DH 2fus the enthalpy of fusion Ê ˆ for component 2 at temperature T. ˜ – – 1˜ + ln ÁË ¯ ¯ Usually, it is sufficient to consider only the first term in equation [1.39], especially, if T and Tt are not far apart, and to neglect the contribution of cp. Furthermore, because there is little difference between the triple-point temperature and the normal melting temperature, it is common to substitute Tt for its normal melting point Tfus,i. The simplified equation thus obtained, valid at the triple point pressure Pt of the solute, is: f2L,pure DH 2fus Ê Tfus,2 = –1 [1.40] ˆ f2S,pure RTfus,2 Ê T ÁË ˜¯ To take into account the effect of pressure, the fugacities may be written as: ln

f2S,pure (P, T ) = f2S,pure (Pt , T ) exp

Ê 1 RT

ÊÊ 1 f2L,pure (P, T ) = f2L,pure (Pt , T ) expÁ Ë RT

P

ÚP v2S d P t

ˆ

P

ÚP v2L d P˜¯

[1.42]

t

P

ˆ ˜¯ (v – v ) dP˘ ˙˚

and the fugacity ratio is:

Ê ÁË f2S,pure (P, T ) f2S,pure (Pt , T ) = L,p exp È 1 L,pure ÍÎRT f2 (P, T ) f2 (P , T )

[1.41]

ÚP

t

By inserting equation [1.40] pureinto equation [1.43]:

S 2

L 2

[1.43]

t

f2S,pure (P, T )

and by integrating:

=

È f2L,pure (P, T )exp Í

1 RT Î

P

ÚP (v2S – v2L )dP + t

D

fus 2

Ê Á1 – fus,2 Ë

fus,2 ˆ ˘

˜¯ ˙ ˚

[1.44]

P

È(vS – v2L )(P – Pt f2S,pure (P, T ) = f2L,pure (P, T ) exp Í 2 RT Î –

)


+

T H RT T DH 2fus Ê Tfus,2 ˆ ˘ 1 RTfus,2 ÁË T ˜¯ ˙˚ –

[1.45]

Principles of supercritical fluid extraction and applications 19 1.2.2 Liquid–SCF equilibrium Phase diagrams According to the classification proposed by van Konynenburg and Scott (1980), six main types of fluid-phase behaviour in binary mixtures are distinguished by their critical properties. The corresponding P–T projections are shown in Fig. 1.11. Similar behaviour is observed for type I and type II, where a continuous LG critical line, which connects the critical points of the pure components, is observed. However, the difference between both types is observed because type II liquid mixtures are not miscible in all proportions and exhibit a LLG three phase line at low temperatures. The LLG line ends at the UCEP, where two liquid phases merge into one liquid phase. From this point, the LL critical line rises rapidly to higher pressures. In type III phase behaviour, the LG critical line is not a continuous line connecting critical points of the pure components, instead it has two branches; one going from the critical point of the component with the higher critical temperature, initially in the direction of the critical point of the other component and then rising sharply with pressure, whereas the other line goes from the critical point of the other component to the UCEP. Various sub-classes of type III behaviour have been observed, depending on the course of the main critical line. The critical line starting from C2 with a positive slope, indicates the existence of the so-called gas–gas equilibria Type I

Type II

1 Pressure (P)

C2

LG

C1

LL

Type III

1

2

Type V LG

C2 LL

C2

C1

LG

UCEP

LCEP

UCEP LLG

2

LCEP

1 LLG

UCEP

LL

2

1

C2

LG

C1

LG

LLG

2

LLG

Type VI LG

C1

1

LG

UCEP LLG

Type IV

C2

LG C1

1

2

C2

LG

C1

UCEP LLG

2

LCEP

Temperature (T)

Fig. 1.11 Classification of the phase behaviour of binary fluid systems. C, critical point; L, liquid; G, gas; UCEP, upper critical end point; LCEP, lower critical end point. Dashed curves are critical lines, lines denoted as 1 and 2 are the vapour pressure curves of the two components (Brunner, 1994; Prausnitz et al., 1986).


20 Separation, extraction and concentration processes (Sadus, 1992). The two phases are in equilibrium at a temperature higher than the critical temperature of either pure component. The three-phase LLG line is also observed near the vapour pressure line of the most volatile compound. In type IV phase behaviour, the critical line between the critical points of pure compounds is interrupted by a three-phase LLG line. One branch of the critical line goes from the critical point of the component with the higher critical temperature to the LCEP, whereas the other branch goes, as in type III, from C1 to the UCEP. Type V is similar to type IV except that there is also an LL critical line at low temperatures which ends at another UCEP. For this type of mixture, there are therefore two regions of limited liquid miscibility at lower and higher temperatures and pressures. In type VI there are two critical curves: a continuous LG critical line, which connects the critical points of the pure compounds, and an LL critical curve, which connects the UCEP and LCEP located at either end of threephase LLG line. Thermodynamic modelling of high-pressure vapour–liquid (V–L) equilibria Consideration of binary liquid in equilibrium with a gas phase: the equilibrium equation for a binary liquid–gas system at temperature T and pressure P is:

f 1G(T, P, y1) = f 1L(T, P, x1)

[1.45]

f 2G(T, P, y2) = f 2L(T, P, x2)

[1.46]

where xi is the mole fraction of component i in the liquid phase and yi is the mole fraction of component i in the gas phase. The fugacity in each phase can be written as:

f Li = xi jLi P

[1.47]

f Gi = yi jGi P

[1.48]

where jGi and jLi are the fugacity coefficients of i in the gas and liquid phases, respectively.

y1j1V = x1j1L

[1.49]

y2jV2 = x2 j2L

[1.50]

Fugacity coefficients for each component in the liquid and gas phase can be calculated by using equation [1.15].



1.3 Cycle processes for extraction using supercritical fluids 1.3.1 Solvent cycle A typical high-pressure extraction process basically comprises a separation stage for the feedstock (extractor and separator) and a regeneration stage for the solvent. In the separation stage, the components to be extracted become concentrated in the gas and are then precipitated in the separator by applying suitable methods (Fig. 1.12). The gas must be removed from the extract and the raffinate and cleaned for reuse in the extraction process. If a solvent mixture is applied in the extraction process, the composition of the mixture must be adjusted before reuse. The solvent recovery can be performed in various ways and the procedure chosen depends on the nature of substances, the scale of the process unit and the operating conditions (Brunner, 1994). The extract is separated from the solvent by changing the operating conditions in such a way as to reduce the solvent power of the SCF. This can be achieved by various means (Brunner, 1994): ∑

Isenthalpic throttling (expansion) to subcritical conditions (changing pressure and temperature), ∑ Changing the temperature and maintaining supercritical conditions, or cooling down to subcritical conditions, ∑ Employing an additional mass separating agent (absorbing medium, membrane adsorbents), whilst maintaining supercritical conditions for the solvent. Separation stage

Material to be extracted

Regeneration stage

Gas

Solvent–solute mixture

Extractor

Separator

Gas reservoir

Gas Raffinate

Gas

Solvent (compressed gas)

Extract (solute)

Fig. 1.12 Solvent cycle in extraction process using SCF. © Woodhead Publishing Limited, 2010

22 Separation, extraction and concentration processes The appropriate method and conditions in the separator are chosen according to the phase equilibrium data. In general, a change in temperature will not be effective in cleaning the solvent sufficiently for reuse in the extraction process, but a change in temperature can be applied in addition if total regeneration of the solvent is not necessary (Brunner, 1994). The advantage of using an absorbent or adsorbent for separation of the extract and the solvent is that the separation can be performed without significantly reducing the pressure. Operating at constant pressure reduces energy consumption. The disadvantage is that separation of the extract and the absorbent/adsorbent have to be performed subsequently, and this may be difficult and must be taken into account when designing the process and calculating its operating costs. The solvent in the subcritical (liquid) or supercritical state may be driven either by a compressor or by a pump. Solvent cycles with corresponding changes of conditions of state can be best presented in T–S diagrams and are used for calculating the heat balance of each step of the process. Pump process In Fig. 1.13 the temperature–entropy (T–S) diagram is schematically presented with areas of homogeneous liquid, gas, SCF and two-phase liquid–gas region (L–G). The dotted line separates the area of SCF from the gas and liquid state area and presents no phase border. The part of the line on the right of the critical point (CP) lies on the critical isobar (73 bar for CO 2). The extraction process is performed under constant conditions (3 in Fig. 1.13). The SCF phase following the extraction leaves the extraction unit and the dissolved substance is subsequently separated from the solvent by changing the pressure and temperature to reduce the solvent power of the SCF (isenthalpic H6

P5

P4

H5

Temperature (T)

P3 H4 H3 H2 2

H1 L

Pc

SCF

3

P2 P1

CP

G 5

1

6

L–G

4

Entropy (S)

Fig. 1.13 Gas extraction process in T–S diagram: solvent circuit in the pump mode.


Principles of supercritical fluid extraction and applications 23 throttling 3–4). The two-phase region of the solvent is obtained and the substance dissolved in the solvent precipitates to form a separate phase that can be removed. The gaseous part of the solvent is cooled and condensed to a liquid state (6) and then sub-cooled (1). To remove the substances that remain dissolved, the liquid solvent can be evaporated (6–5), subsequently liquefied (5–6) and sub-cooled (1). The sub-cooled liquid is then pumped to the operating pressure (1–2) and heated to the operating temperature (2–3). The area enclosed by the solvent circuit in the T–S diagram represents the thermodynamic work needed for the process of cycling the SCF. The lower the pressure drop needed for separating the extract and the solvent, the lower will be the work required. Compressor process The solvent circuit in the extraction process using a compressor is schematically presented in the T–S diagram in Fig. 1.14. After the extraction process, which is performed under constant conditions (3), the solute is separated from the SCF by isenthalpic throttling into the subcritical region (3–4) and this is followed by evaporation (4–5–1). The dissolved substances precipitate and the solvent, which is in a gaseous state, is then compressed to the pressure of the extraction, shown in Fig. 1.14 as an idealised, isentropic compression (1–2). During compression, the temperature of the SCF increases, therefore the solvent is cooled to the temperature of extraction (2–3). This completes the cycle, which may be repeated. The operating modes in Figs 1.13 and 1.14 are presented as examples. Solvent cycles may vary for different gases and depend upon the mode of H6

P5

H5

P4 P3

Temperature (T)

H4 H3

Pc

SCF

2

H2

H1

P2 P1

3

CP

G 1/5

L

4

L–G

Entropy (S)

Fig. 1.14 Gas extraction process in T–S diagram: solvent circuit in the compressor mode.


24 Separation, extraction and concentration processes operation, e.g. the stages involved in separating the extract from the solvent can be done by pressure decrease, temperature increase, temperature decrease or a combination of temperature and pressure change. The mass and heat losses should also be considered when calculating the balance for practical processes. 1.3.2 Separation of solute in extraction processes using SCF After the solubilisation of SCF soluble substances in the extractor, the dissolved substances must be separated from the solvent–SCF mixture. Several separation processes may be used, but the preferred option is to use processes in which no additional substances are introduced into the system, which have varying solvent powers for supercritical fluid, and where only small changes in the conditions of the extraction process are made. Adding substances to separate solute from the SCF increases the costs of separation, whereas re-compression and cooling conditioning of the SCF to operating extraction conditions, increases the energy costs of the process. One of main advantages of using SCF for extraction and fractionation of substances, is that by a stepwise reduction of solvent power, fractionation of extracts can be obtained. Separation of solute by reduced solvent power Reduction of pressure and temperature increase The mostly widely used process for the separation of solute from solvent, is reduction of the solvent power. A condensed phase is formed and the gaseous phase is separated. The solvent power of supercritical solvents depends upon pressure and temperature which influence the density of the SCF. Generally, the solubility of a solute increases with increasing density and decreases with decreasing density. A decrease in density can be obtained by decreasing pressure and/or increasing temperature. In many industrial operations, the concentration of solute in a solvent is reduced by decreasing the pressure and thus the density, whereas an increase in temperature increases the vapour pressure of the solute. In a low pressure range where the density decreases dramatically with a temperature increase, the concentration of solute in SCF decreases with increasing temperature when the vapour pressures of the substances are relatively low. The separation of a solute by temperature increase is not an efficient method (for example for separation of essential oils from SCF) when the vapour pressure of a solute is high and increases with a rise in temperature. High pressure, where density decreases slightly with an increase in temperature, will lead to a higher concentration of solute with increasing temperature (at constant pressure) in the SCF. Based on these observations, it is evident that the increase in temperature, which decreases the density and solvent power, should provide an efficient method for the separation of a solute with low or moderate vapour pressure within the low-pressure range. On the contrary,


Principles of supercritical fluid extraction and applications 25 for some substances, at certain process conditions, the solubility decreases with increasing pressure. For such systems, separation procedures of solutes by pressure reduction or reduction of the solvent power is not possible. It is evident that the design of efficient processes for the separation of solute from SCF by the reduction of solvent power is possible only when phase equilibrium data are available. After establishing thermodynamic conditions for reduced solubility in a separator, a condensed phase is formed and this is separated from the solvent with lower density in one, or several separators in series (in the last separator from the gaseous phase). Usually, such a series of separators will operate at different pressures and/or temperatures to fractionate the single components of the extract. The solute–solvent system must remain in the separator for a sufficient length of time to approximately achieve the phase equilibrium. Separation by expansion Under certain process conditions, the solute-loaded solvent phase is expanded in the separator where the solvent is a subcritical liquid. In this instance, both the gaseous solvent and the liquid solvent containing the solute are present in the separator, a three-phase mixture is present if the solute is insoluble or less soluble in the liquid and gaseous phases. Usually, the liquid solvent evaporates and the solute is removed from the separator, whereas the gaseous phase is reused. This set of separation sequences is effective for the separation of a solute from solute-loaded SCF, but it is extremely expensive owing to the high energy input required. For efficient separation of the condensed phase from low-density solvent, auxiliary devices are used to increase the separation of the solid or liquid phase from the low-density solvent. These include demisters consisting of wire mesh packing, deflectors and filters, and cyclones. Separation of solute and solvent by a mass separating agent The separation of solute from a solution by a mass separating agent is possible by: ∑ absorption, ∑ adsorption, ∑ use of membranes, or ∑ adding a substance of low solvent power. For separation of a solute from a solvent by absorption, the solvent circuit may operate at an almost constant pressure. The absorbing liquid must dissolve the solute and should not absorb the solvent. Separation of solute from the SC solvent by adsorption can be a very efficient process. This process, like that of absorption, can operate with practically no pressure and/or temperature drop. Therefore, both separation processes have a major influence on the economy of whole extraction process. Membrane separation processes may be efficiently used for the separation


26 Separation, extraction and concentration processes of solute from gas phase, owing to the difference in molecular mass. The pressure drop through the membrane is relatively low, therefore the solvent regeneration costs are low. The solvent power of a SC solvent may be reduced by adding a substance of low solvent power (e.g. adding nitrogen to SC CO2 for several substances). A similar effect may be obtained, when the entrainer is separated from the SC solution (if entrainer was applied in extraction process). For separating the solute, the entrainer has to be removed by adsorption or absorption. The advantage of such a separation process is its ability to operate at an almost constant pressure, thus keeping costs to a minimum.

1.4 Extraction of solids using SCF 1.4.1 Design of extraction plant The fundamentals of the design criteria for SCF extraction of solids in relation to process and equipment are reviewed in this section. For successful engineering and/or design of a SC extraction process, the following parameters should be defined: ∑ ∑

specific basic data, thermodynamic conditions for the operation of extraction and separation process, ∑ mass transfer data for the system, and ∑ energy consumption by means of T–S diagrams. The size of extractors for a certain capacity of plant are defined by specific basic data and thermodynamic conditions. Mass transfer data determines the time solids remain in contact with the SC solvent, so determining the solvent circulating system, including energy (heating and cooling) consumption, and therefore the capacity of the pump/compressor, the surface of heat exchangers and the piping system. Specific data and pretreatment of solids For the design of industrial plant, the following information is essential: ∑ raw material specification, ∑ final product specification, ∑ required plant size, ∑ plant location, sometimes taking into account the prevailing local conditions. The specification of the raw materials influences the quality of the extracts obtained and the overall economy of the process. If the raw material is contaminated, either by waste products or plant protection materials, a specified level of purity in the extract cannot usually be achieved. On the other hand, if the raw material contains a low concentration of substances to be extracted, the economy of the process will be questionable. © Woodhead Publishing Limited, 2010

Principles of supercritical fluid extraction and applications 27 The final product specification is usually based on customer needs, expectations and specifications and has a considerable influence on the costs of the extraction process. The process conditions in the extraction stage should be determined in order to obtain maximum yield and high selectivity for the required substance at minimal separation costs. However, a multistep separation is required for the fractionation of extracts. From a previous study (Gamse and Marr, 2001) it is known that the extractor volume influences the investment costs for an extraction unit (investment costs are a logarithmic function of extractor volume). Location will determine the mechanical construction of an extraction plant [e.g. location in Good Manufacturing Practice (GMP) area or in an earthquake area]. Climate conditions will also influence the design of heating and cooling devices and electrical drives. Pretreatment of solids Substances which should be extracted with SC fluids may be divided in two categories: ∑ ∑

raw materials where only unwanted substances should be removed and the geometry of the raw material is maintained during the process, and raw materials where pretreatment is allowed and extracted substances are the main product.

Examples of the first group of materials are coffee and tea decaffeination, the defatting of some seed plant materials, and the separation of pesticides from rice and ginseng. In the first process, high selectivity for the separation of unwanted compounds is necessary and therefore the process parameters or isolation of substances have to be very precisely selected. These processes are used for high-volume and relatively high-value market products. For the other group of materials, any pretreatment may typically be used to achieve a high yield at a low solvent to feed ratio with a low energy input. Therefore, the raw material should be ground before extraction to increase the bulk density. Some non-ground material could have a bulk density of 150–250 kg m–3, whereas the same material when ground would have a bulk density of 350–500 kg m–3. The ground feed material has an average particle size of 0.4 to 0.8 mm. Smaller particles have a higher specific area and therefore a higher mass flux could be expected. However, the linear solvent velocity through the ground raw material is decreased and consequently a greater drop in pressure is obtained. The linear solvent velocity through the raw material is 0.7 to 0.8 cm s–1. Finely ground material may also cause clogging of sintered plates on extraction vessels or filters and tends to cause channelling. If the bulk density of a ground substance is less than 250 kg m–3, the material should be formed into pellets. Bulk density has a strong influence on the economics of the process. If the bulk density in the extraction vessel is too low, less raw material is loaded and the yield per batch is reduced. Therefore, for a given product capacity, the volume of the extraction vessels should be greater. The moisture content


28 Separation, extraction and concentration processes of raw plant materials is between 8 to 15 wt%. The amount of water in plant material influences the economics of the process. In plant material having a higher water content, the relatively high solubility of water in CO2 means that polar substances are extracted at relatively low pressure. If the water concentration in the raw plant material is too low, the cells may shrink and hinder the mass transfer of substances which are extracted through the cell walls. The ideal moisture content for extraction of substances from plant material should be optimised by laboratory-scale tests. It is known that for the decaffeination of coffee and tea, the water content of green coffee beans should range from 35 to 45 wt% (Zosel, 1981), whereas in the extraction of astaxanthin from algae, it should be as low as possible (water–oil emulsions are formed and the yield of astaxanthin would therefore be very low). In the SC extraction process for the isolation of colorants and antioxidants from raw plant materials, the content of oils, essential oils and waxes has a particularly large influence on the extraction yield. These substances contained in raw plant material may act as entrainers for the extraction of valuable compounds (such phenomena could be observed for extraction of carotenoids from ground paprika, where, owing to the low solubility of carotenoids in pure CO2 even at ultra high pressure (1500 bar), the carotenoids could not be separated quantitatively from plant material. Therefore, in some processes, oils, essential oils and waxes are used as entrainers for extraction of valuable compounds. Thermodynamic data The solubility of an extracted substance in a supercritical solvent is critical to the economics of the extraction process. The highest possible loading of SC solvent should be achieved in the extraction stages of the process, whereas in the separation stage, the solubility of solute in solvent should be as low as possible. In some instances, the solvent power for solutes should be gradually reduced so the fractionated separation of solute from the solvent may be achieved. Details of the thermodynamic properties of the solute in SC solvents are described in section 1.2. Mass transfer Mass transfer data are essential for determining the extraction time and the capacities of pumps, compressors, and heat exchangers. The data on mass transfer should be optimised experimentally with raw plant material having defined humidity, particle size, and particle size distribution at defined optimal extraction pressure and optimal extraction temperature (data obtained from thermodynamic investigations) for a defined SC solvent. Based on mass transfer experiments, the mass of solvent per mass of feed should be determined for the highest possible yield of the substance to be extracted. Figure 1.15 presents the typical extraction curve for the isolation of a substance from solids. The extraction curve for the isolation of substances from solid raw plant material is generally divided into a period of constant



Yield (wt%)

Diffusion-controlled mass transfer

Solubility-controlled mass transfer

Mass of solvent/mass of feed (S/F)

Fig. 1.15 Typical extraction curve.

extraction rate and a period of falling extraction rate (Sovova, 2005). Solubility controlled mass transfer prevails in the initial period of the process, whereas at higher S/F (longer extraction time), the mass transfer is controlled by diffusion. The diffusion and hydrodynamics influence the mass transfer rates. Diffusion The substance to be extracted may be located in the cells of the raw material or adsorbed on the surface of a solid matrix. Therefore, the mass transfer depends on the location of the substance. If it is adsorbed on the surface, the mass transfer rates are high and, vice versa, when the solid particles of the substances diffuse through cell walls, the mass transfer rates will be low. In some instances, the substances to be extracted also form complexes which require release by a chemical reaction (usually hydrolysis with water). For substances that do not form a chemical complex, the diffusion may be influenced by: ∑ ∑

reduction of particle size (reduction of diffusion path), and destruction of cells (by swelling or cracking cells, by ultrasound, by milling procedure).

Hydrodynamics Particle properties such as particle size, particle shape and particle size distribution may cause channelling, which reduces the flow of the SC solvent through the material to be extracted. Some materials swell during the extraction process and may reduce the flow of the SC solvent through the extractor. For some substances, the direction of flow of the SC solvent is important. In industrial scale processes, the flow will usually be from bottom to top, whereas under some process conditions, the opposite direction of flow


30 Separation, extraction and concentration processes will give higher yields. Mechanical mixing in industrial scale plants is not feasible, but some flow restrictors that avoid channelling in the extraction bed are technically possible. An extended description on modelling mass transfer may be found in Brunner (1994). Energy consumption Energy consumption for an extraction process using SC solvents may be determined from the TS diagram as described in section 1.3. 1.4.2 Applications for extraction of solids using SCF There are numerous applications for extraction of solids using supercritical fluids. Several overviews are available (Brunner, 1994; Catchpole et al., 2009; Diaz-Reinoso et al., 2007; Eltringham and Catchpole, 2007; Fang et al., 2007; Gardner, 1993; King and Srinivas, 2009; Lack and Seidlitz, 1993; Lack and Simandi, 2001; Li, 2007; Meireles, 2007; Mendes, 2007; Moyler, 1993; Mukhopadhyay, 2007; Reverchon and Marco, 2007; Stahl et al. 1987; Teelli, 2009; Temelli et al., 2007. The web pages of equipment producers (Natex, Nova Swiss, Sitec, Uhde HPT) give references to their plants. From this information, it may be seen that the highest capacity equipment is installed for coffee and tea decaffeination. The second largest application is for the extraction of hop compounds. Extraction of spices for the production of oleoresins and the extraction of bioactive compounds from plants are also very widely used applications of SC fluids. One of the latest applications is the extraction of oil from the degumming residue to obtain highly concentrated and very pure lecithin (plant designed, manufactured and erected by Uhde HPT).

1.5 Extraction of liquids using SCF There are fewer industrial units using SCF for the separation of components from liquid mixtures. Extraction from liquid mixtures by SCF is similar to liquid–liquid extraction, where compressed gas is used instead of an organic solvent. In liquid–SCF extraction processes, pressure plays an important role. When pressure and/or temperature is changed, the physicochemical properties of the SCF, such as density, viscosity, surface tension, and dielectric constant are also changed. Selective extraction of components or the fractionation of total extracts is possible by the use of different gases for the different processes and/or by changing the process parameters. Another advantage is that depending on the feed material, the density difference between the two counter-current flowing phases can be adjusted. One of the most important advantages of using supercritical fluids is the simplicity of solvent regeneration in comparison with liquid–liquid extraction,


Principles of supercritical fluid extraction and applications 31 where in most cases, a re-extraction or distillation step is necessary; this step is energy consuming, thereby making the process more costly. Heat treatment of the extract or raffinate phase may cause the degradation of heat-sensitive substances. In extraction plants where SCFs are used, solvent regeneration is achieved by changing the pressure and/or the temperature after the extraction stage, thus changing the density and therefore the solvent power of the gas, which can be recycled after the separation of the solute. Compared with the extraction of solids with SCF, liquids may be continuously introduced and withdrawn from the high-pressure extraction unit. This offers the advantage of higher throughputs in continuous operating counter-current processes. There have been some laboratory-scale studies on extraction in systems using liquid/supercritical fluid. Data on binary systems liquid/SCF can be found, but there is less information on systems using liquid/liquid/supercritical fluid, which is necessary for the design of extraction processes of liquid mixtures with supercritical fluids. 1.5.1 Operation methods and apparatus As in conventional continuous liquid–liquid extraction in liquid/sub- or supercritical solvent, several operating modes of extraction are available. Single-stage extraction is the simplest and is used in systems where the separation factors for a solute are high. Multistage separation is necessary when the separation factor between the components is in the order of 1–10. Various modes of operation in multistage processes are used, such as multistage crossflow in which a relatively low loading of solvent with the extract is obtained in each stage. In multistage counter-current extraction, high loading of solvent with extracts is possible and a different configuration of the apparatus is possible. Counter-current liquid/sub- or supercritical fluid extraction (SFE) may be modelled by the use of typical commonly used basic equations: mass balance, energy balance, equilibrium distribution coefficients, and mass transfer rate equations. For extraction, the following data are necessary: ∑ ∑

determination of the number of theoretical stages/transfer units, size and type of a separation device in respect to separation performance, ∑ design of the solvent cycle. From the above facts and experimental data, the costs of separation using liquid/sub- or supercritical processes may be determined. The costs per tonne of the feed are influenced by the throughput and mode of operation (batch processes have higher operating costs, whereas in continuous processes the costs are lower) and are in the range from c. 607/kg feed at a throughput of c. 200 tonne per year in batch processes, down to approximately 0.067/ kg feed at a throughput of c. 60,000 tonne per year for continuous process. Brunner (2009) reported that the usual methods of calculation yield results


32 Separation, extraction and concentration processes with an error of ±30%. Even after completion of a project, it is unlikely that the margin of error in determining costs will be less than 5%. 1.5.2 Applications The applications of liquid/sub- or supercritical fluid extraction are numerous and are used in the separation of ethanol from water (Hsu and Tan, 1994; Knez et al., 1994), the separation of aromas in various alcoholic beverages (Gamse et al., 1999), the separation of components from citrus oils (Knez, 1989) and for the purification of tocopherols (Fleck et al., 2000). The separation of caffeine from CO2 is used widely in decaffeination processes. In Fig. 1.16, the high-pressure column of an industrial-scale decaffeination process is shown. In the future, further limitations on the use of organic solvents and the demands of new applications will be the deciding factors for sustainable processing.

1.6 Conclusion SCF-based technologies offer important advantages over organic solvent technology, such as ecological friendliness and ease of product fractionation. The extraction of hop components and the decaffeination of tea and coffee are the largest scale extraction processes using sub- or supercritical solvents to be used industrially. There are also several industrial plants in operation

Fig. 1.16 Column for liquid–SCF extraction under preparation for pressure test (length 12 m, diameter 0.8 m, operation pressure 500 bar, operation temperature 100 °C. (Photo: courtesy of Uhde HPT, Hagen, Germany.)


Principles of supercritical fluid extraction and applications 33 for the extraction of spices for the food industry and of natural substances for use in cosmetics. There are fewer industrial units engaged in the separation of components from liquid mixtures using sub- or supercritical fluids. The main advantages of using SCF for the isolation of natural products are: solvent-free products, no by-products and a low temperature in the separation process. In addition, the processes may be easily linked with direct micronisation and crystallisation from SC CO2 by fluid expansion. However, the most important advantage of the use of SCF, is the selective extraction of components or the fractionation of complete extracts. This is made possible by the use of various gases for the isolation/fractionation of components and/or by changing the process parameters. In addition to the wide use of gas for sub- or supercritical extraction, usually CO2, other sub- or supercritical solvents are also used. Sub- and supercritical CO2 and supercritical H2O are non-carcinogenic, non-toxic, non-mutagenic, nonflammable and thermodynamically stable. In addition, CO2 does not usually oxidise substrates and products, thus allowing the process to be operated at low temperatures. Water is currently the cheapest solvent and several substances are highly soluble in water. Further research on the use of subor supercritical water for the isolation and fractionation of substances is underway. One of the major process benefits is derived from the thermophysical properties of SCF: high diffusivity, low viscosity, density, and the dielectric constant of SCF, all of which may be fine tuned through changes of operating pressure and/or temperature. The limitation on further applications for obtaining extracts through highpressure technology is the price of the product; i.e. the price is relatively higher than for conventionally obtained extracts. The legal limitations on solvent residues and solvents (in products used for human applications) and the isolation/fractionation of special components from total extracts in combination with different formulations (controlled release for example) (Reverchon 2009; Weidner, 2009), chromatography (Taylor, 2009) and sterilisation processes, will lead to an increase in the use of dense gases for extraction applications. Figure 1.17 presents the number of compressed fluid extraction plants, by region (Lütge and Schuetz, 2007). It is evident that the number of extraction units will increase with time and the most ‘dense’ areas are in Europe and Asia, where a number of newly installed SCF extraction units may be observed. Figure 1.18 shows the pressures used for research and development. There is no clear trend towards higher or lower pressure. However, as part of research and development and in the filing of patents for SC extraction, both higher and lower pressures are used. Many substances have limited solubility at moderate pressure and the solubility of several substances increases by some orders of magnitude when using pressures over 500 bar. Because of this, the ultra high pressure range (up to 2500 bar) enables additional fractionation of substances in the extract


34 Separation, extraction and concentration processes 50 Europe

America

Asia

Australia

45 40 35

Number

30 25 20 15 10 5 0 1980

1985

1990

Year

1995

2000

2005

Fig. 1.17 Number of SFE plants having a total extraction volume >500 L by region. (Source: Schuetz Consulting/Uhde HPT.) 800 700

Pressure (bar)

600 500 400 300 200 100 0 1980

1985

1990

1995 Year

2000

2005

2010

Fig. 1.18 Maximum pressure of SFE processes. (Source: Schuetz Consulting/Uhde HPT.)

by pressure- and/or temperature-dependent precipitation. In Fig. 1.19 an ultra high pressure extraction unit operating up to 2500 bar is presented. The evaluation of costs for several plant materials processed by ultra high pressure extraction using SC CO2 is shown in Fig. 1.20. It is clear that with © Woodhead Publishing Limited, 2010


E2

S1

E1

S2

Fig. 1.19 Ultra high pressure extraction unit (operating pressure 2500 bar). (E1 and E2, extractors; S1 and S2, separators). (Photo: courtesy of Uhde HPT, Hagen, Germany.) 2.50

Specific processing costs (7/kg)

Operating costs (7/kg) Investment (7/kg) 2.00

1.50

1.00

0.50

0.00

300

500 1000 Pressure (bar)

1500

Fig. 1.20 Specific processing costs versus operating pressure (Lütge et al., 2007)

an increased operating pressure in a SC extraction plant (retaining the same capacity of equipment), the total processing costs decrease. In the future, further limitations on the use of organic solvents, new applications of several substances, changing customer requirements, sustainable © Woodhead Publishing Limited, 2010

36 Separation, extraction and concentration processes production and processing of substances, will all lead to new developments in high-pressure processing. We can be sure that advances in the field of high-pressure research into cheap and environmental friendly solvents, such as CO2 and some other gases and sub- or supercritical water, will open up new pathways for substances and products produced at high pressure.

1.7 References Y. Adachi and H. Sugie, Fluid Phase Equilib. 28(2) (1986) 103–118. A. Anderko, Fluid Phase Equilib. 61 (1990) 145–225. A. Anderko, in: J.V. Sengers, R.F. Kayser, C.J. Peters, H.J. White (Eds.), Equations of state for fluids and fluid mixtures, Elsevier, Amsterdam, 2000, pp. 75–126. J.D.S. Arons and G.A.M. Diepen, Rec. Trav. Chim. Pays-Bas, 82 (1963) 249–256. A. Bertuco and G. Vetter, High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2001. G. Brunner, Counter-current separations, J. Supercrit. Fluids 47 (2009) 574–582. G. Brunner, Gas extraction. An introduction to fundamentals of supercritical fluids and the application to separation processes, Darmstadt: Steinkopff; New York: Springer, 1994. O.J. Catchpole, S.J. Tallon, W.E. Eltringham, J.B. Grey, K.A. Fenton, E.M. Vagi, M.V. Vyssotski, A.N. MacKenzie, J. Ryan, Y. Zhu, The extraction and fractionation of specialty lipids using near critical fluids, J. Supercrit. Fluids 47 (2009) 591–597. R.P. Danner and P.A. Gupte, Fluid Phase Equilib. 29 (1986) 415–430. B. Diaz-Reinoso, A. Moure, H. Dominguez, and J.C. Parajó, Antioxidant extraction by supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 275–304. R. Dohrn, Berechnung von Phasengleichgewichten, Friedr. Vieweg & Sohn Verlagsgesellschaft mbH, Braunschweig, 1994. R. Eggers, Supercritical fluid technology in oil and lipid chemistry, AOCS Press, 1996, 35–62. W. Eltringham and O.J. Catchpole, Processing of fish oils by supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 141–188. T. Fang, M. Goto, M. Sasaki, and D. Yang, Extraction and purification of natural tocopherols by supercritical CO2, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 103–140. U. Fleck, G. Brunner and R. Karge, Purification of synthetic crude tocophenol acetate by means of supercritical fluid extraction, in: Proceedings of the 5th International Symposium on Supercritical Fluids, Atlanta, April 2000. T. Gamse, I. Rogler and R. Marr, 1999, Supercritical CO2 extraction for utilisation of excess wine of poor quality, J. Supercrit. Fluids 14 (1999) 123–128. T. Gamse and R. Marr in A. Bertucco, G. Vetter. High pressure process technology: fundamentals and applications, Industrial Chemistry Library, volume 9, 2001, 383. D.S. Gardner, Commercial scale extraction of alpha acids and hop oils with compressed CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 84–100. J.-H. Hsu and C.-S. Tan, Separation of ethanol/water solution with supercritical CO2 in the presence of a membrane, in S.S.H. Rizvi, Supercritical fluid processing of food and biomaterials, Chapman & Hall, 1994, 114–122. M.J. Huron and J. Vidal, Fluid Phase Equilib. 3 (1979) 255.


Principles of supercritical fluid extraction and applications 37 P.G. Jessop and W. Leitner, Chemical synthesis using supercritical fluids, Wiley-VCH, July 1999. J.W. King and K. Srinivas, Multiple unit processing using sub- and supercritical fluids, J. Supercrit. Fluids 47 (2009) 598–610. Ž. Knez, Separation of components of citrus oils – industrial project, unpublished data, 1989. Ž. Knez, F. Posel and I. Krmelj, High pressure extraction of organics from water, in S.S.H. Rizvi, Supercritical fluid processing of food and biomaterials, Chapman & Hall, 1994, 181–186. Ž. Knez and M. Škerget, Phase equilibria of the vitamins D2, D3 and K3 in binary systems with CO2 and propane, J. Supercrit. Fluids 20 (2001) 131–144. P.H. van Konynenburg and R.L. Scott, Critical lines and phase equilibria in binary van der Waals mixtures, Phil. Trans. Roy. Soc. London, Ser. A 298 (1980) 495. E. Lack and H. Seidlitz, Commercial scale decaffeination of coffee and tea using supercritical CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 101–139. E. Lack and B. Simandy in A. Bertucco and G. Vetter. High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2001, 537–575. S. Li, Application of supercritical fluids in traditional chinese medicines and natural products, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 215–242. T.W. de Loos, On the phase behaviour of asymmetric systems: The three-phase curve solid–liquid–gas. J. Supercrit. Fluids 39 (2006) 154–159. D. Luedecke and J.M. Prausnitz, Fluid Phase Equilib. 22(1) (1985) 1–19. C. Lütge and E. Schuetz, Market trends and technical developments in high pressure technology, in: Ž. Knez and M.J. Cocero, Proceedings of the 5th international symposium on high pressure process technology and chemical engineering, June 24–27, 2007, Segovia, Spain. C. Lütge, M. Bork, Ž. Knez, M. Knez Hrn�i�, M. Krainer, Ultra high pressure dense gas extraction and fractionation, in: Ž. Knez and M.J. Cocero, 5th International symposium on high pressure process technology and chemical engineering, June 24–27, 2007, Segovia, Spain. R. Marr and T. Gamse, High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2000, 396–402. M.A. McHugh and V.J. Krukonis, Supercritical fluid extraction: principles and practice, Butterworths, Stoneham, 1986. M.A. Meireles, Extraction of bioactive compounds from Latin American Plants, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 243–274. R. Mendes, Supercritical fluid extraction of active compounds from algae, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 189–214. D.A. Moyler, Extraction of flavours and fragrances with compressed CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 140–183. M. Mukhopadhyay, Processing of spices using supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 337–366. J.P. O’Connell, W. Weber and G. Brunner, Measurement and thermodynamics of triglyceride melting in near-critical fluids, in: Paper Presented at the AIChE Annual Meeting, Indianapolis, USA, 2003. A.Z. Panagiotopoulos, R.C. Reid, S. Watanasiri, Fluid Phase Equilib. 29 (1986) 525.


38 Separation, extraction and concentration processes J.M. Prausnitz, R.N. Lichtenthaler and E. Gomes de Azevedo, Molecular thermodynamics of fluid phase equilibria. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 1986. E. Reverchon and I. De Marco, Essential oils extraction and fractionation using supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 305–336. E. Reverchon, R. Adami, S. Cardea, G. D. Porta, Supercritical fluids processing of polymers for pharmaceutical and medical applications, J. Supercrit. Fluids 47 (2009) 484–492. R.J. Sadus, High pressure phase behavior of multicomponent fluid mixtures, Elsevier Amsterdam, 1992. S.I. Sandler and H. Orbey, Mixing and combining rules; in: J.V. Sengers, R.F. Kayser, C.J. Peters, H.J. White (Eds), Equations of state for fluids and fluid mixtures, Elsevier, Amsterdam, 2000, pp. 321–357. R. Sandoval, G. Wilczek-Vera, J.H. Vera, Fluid Phase Equilib. 52 (1989) 119. J. Schwartzentruber and H. Renon, Ind. Eng. Chem. Res. 28 (1989a) 1049. J. Schwartzentruber, H. Renon, Fluid Phase Equilib. 52 (1989b) 127. H. Sovova, Mathematical model for supercritical fluid extraction of natural products and extraction curve evaluation, J. Supercrit. Fluids 33 (2005) 35–52. E. Stahl, K.-W. Quirin, D. Gerard, Verdichtete Gase zur Extraktion und Raffination, Springer-Verlag, 1987, 82–225. M Škerget, Z. Novak-Pintari�, Ž Knez and Z. Kravanja, Estimation of solid solubilities in supercritical carbon dioxide, Peng–Robinson adjustable binary parameters in the near critical region. Fluid Phase Equilib. 5086 (2002) 1–22. L.T. Taylor, Supercritical fluid chromatography for the 21st century, J. Supercrit. Fluids 47 (2009) 566–573. F. Temelli, M.D.A Saldaña, P.H.L. Moquin, and M. Sun, Supercritical fluid extraction of specialty oils, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 51–102. F. Temelli, Perspectives on supercritical fluid processing of fats and oils, J. Supercrit. Fluids 47 (2009) 583–590. W.H. Tuminello, G.T. Dee and M.A. McHugh, Macromolecules 28 (1995) 1506. E. Weidner, V. Wiesmet, Z. Knez, M. Skerget, Phase equilibrium (solid–liquid–gas) in polyethylene glycol–carbon dioxide systems, J. Supercrit. Fluids 10 (1997) 139–147. E. Weidner, High pressure micronization for food applications, J. Supercrit. Fluids 47 (2009) 556–565. D.S.H. Wong and S.I. Sandler, AIChE J. 38 (1992) 671. K. Zosel, Process for the decaffeination of coffee, US Patent 4247570, January, 1981.


Principles of pressurized fluid extraction and applications 39

2 Principles of pressurized fluid extraction and environmental, food and agricultural applications C. Turner and M. Waldebäck, Uppsala University, Sweden

Abstract: The main principles of pressurized fluid extraction (PFE), including basic extraction theory and the effects of solvent selection and temperature variations are discussed. Methods for achieving selectivity during the extraction are described, and future trends explored. Examples of applications include extraction methods developed for environmental and food analysis. Some advice on building equipment in the laboratory is given. Key words: food analysis, pressurized fluid extraction, PFE, environmental analysis, solvent extraction.

2.1 Introduction When developing new products, processes and technologies, it is important to strive for sustainable development. This is especially important because chemists, chemicals, and the chemical industry as a whole are commonly regarded to be the cause of many of the current environmental problems. To ensure sustainability, new processes and new techniques should be studied from a life cycle point of view. Life cycle assessment (LCA) is a methodology in which the entire life cycle of a product or utility effect is analyzed, such as extraction and processing of raw materials, production, distribution, use, consumption and disposal as well as the potential ecological effects. In addition, energy conversions occurring in a life cycle and the resulting burden on the environment are assessed. The indoor environment and health perspective is as important as the outdoor environment. Chemicals should be used in smaller amounts and the ones used should be less hazardous.


40 Separation, extraction and concentration processes One process in which large volumes of organic solvents are used is the procedure of extraction. Traditional liquid–liquid extraction (LLE), also known as solvent extraction, and liquid–solid extraction (LSE) are techniques in which different compounds can be separated from each other based on their relative solubility. For a solid sample, the separation of a substance from the mixture occurs by dissolving that substance in an appropriate solvent. The extraction process usually requires several hours or even several days to perform, depending on the extraction temperature. These processes are slowly being replaced by more attractive alternatives. The most widely used extraction techniques today are still Soxhlet (developed in 1879) and sonication extraction (from 1960). These classical techniques are usually multi-step procedures based on exhaustive extractions from a sample matrix followed by successive clean-up steps before analysis. Such sample preparation procedures require large amounts of sample, sorbents and organic solvents, which are often hazardous and/or toxic, resulting in high costs of both purchase and disposal (Ramos et al., 2002). These methods also demand extensive manual handling, which often creates work-related health problems. During the past few decades new techniques have been developed and among the more successful ones are those employing high-diffusion liquids. Highdiffusion liquids are in this chapter defined as liquids of elevated temperature and pressure. By using high-diffusion liquids, the diffusion coefficient of the liquid is increased. This is the most effective way to increase the rate of the extraction process and decrease the required amount of organic solvent. Diffusion rates in liquids have been shown to increase about 2–20 fold upon increasing the temperature from 25 to 150 °C (Perry et al., 1984). Thus, the mass transfer rate can be increased and the whole extraction process becomes faster. Techniques that employ high-diffusion liquids with raised temperature and pressure are microwave-assisted extraction (MAE) and pressurized fluid extraction (PFE) (Majors, 1996). Another related extraction technique is supercritical fluid extraction (SFE), which usually employs carbon dioxide at pressures and temperatures above 74 atm and 31 °C, respectively, resulting in a liquid-like density in combination with high diffusion rates and low viscosity. In PFE, the solvent is kept in a liquid phase even at temperatures much above the atmospheric boiling point as a result of the applied pressure. This technique is also known as pressurized liquid extraction (PLE“), pressurized solvent extraction (PSE™), accelerated solvent extraction (ASE“) and enhanced solvent extraction (ESE). PFE was originally introduced at the Pittsburgh conference (Pittcon) in 1995 as ASE“ by the Dionex Corporation. The PFE technique utilizes the same basic principles as traditional liquid solvent extractions, but the extractions are carried out at higher temperatures and pressures. An increased temperature during the extraction gives more efficient extraction, and results in both time savings and lowered solvent consumption (Richter et al., 1996). The principle of PFE is similar to


Principles of pressurized fluid extraction and applications 41 MAE, where the solvent is heated by microwave energy, although in the PFE technique both higher temperatures and pressures can be obtained, independently. In this chapter, the basic instrumentation of dynamic (continuous flow) and static (batch mode) PFE as well as its principles of operation are described. Furthermore, fundamental extraction theory is explained including illustrative examples and classical extraction models from literature. A guide to solvent selection is included, which on an elementary level guides the reader to an appropriate choice of solvent in various applications, depending on the chemistry of the sample matrix and analytes to be extracted. Effects of changing the extraction temperature, time and pressure in PFE are described, in addition to some remarks on how to obtain selectivity as well as accuracy and precision. One section is devoted to describing common applications for this technique. Finally, future trends for PFE in terms of sustainability, ‘like-nature’ applications, hyphenated techniques, downscaling and transfer to industry are depicted. The final paragraph also lists commercial vendors of PFE equipment and some advice for building equipment in the laboratory. The next subsection is largely based on a doctoral thesis written by one of the authors of this chapter (Waldeback, 2005).

2.2 Instrumentation and principles of pressurized fluid extraction 2.2.1 Basic instrumentation Extractions using a pressurized liquid above its atmospheric boiling point require open/close valves and/or pressure restrictors, in order to maintain pressure during the extraction. Two main types of instrumentation can be used: static PFE, which is a batch process with one or several extraction cycles with replacement of solvent in between; and dynamic PFE, in which the extraction solvent is continuously pumped through the extraction vessel containing the sample. A schematic of a static PFE instrument is shown in Fig. 2.1. Temperatures applied usually range from room temperature to 200 °C and pressures are generally between 35 and 200 bar. Filter paper is inserted into a stainlesssteel extraction cell followed by the sample, if necessary mixed with a drying agent. The cell is either loaded on a carousel and automatically placed in the oven, or, for simpler equipment, manually placed into the oven. Most equipment has a pump that fills the extraction cell with solvent. Commonly, a static valve in combination with a pressure relief valve control the pressure in the sample vessel during the static extraction; either by adding more solvent to the cell or by opening the static valve, whichever is needed to maintain the desired pressure. The first part of the extraction is a pre-heating step to reach thermal equilibrium. During this heating, thermal expansion of the


42 Separation, extraction and concentration processes Purge valve

Pressure relief valve Extraction cell Oven

Pump

Solvent A Solvent B Static valve

Vent Solvent C Nitrogen

Waste vial

Solvent D Collection vial

Fig. 2.1 Schematic diagram of a PFE system (courtesy of Dionex Corporation).

solvent occurs and causes an increase in pressure within the cell. When the set values are achieved static extraction is performed during a selected time, typically 5–10 min. After the static time, part of the solvent in the extraction cell is replaced with fresh solvent, to start the next extraction cycle. After the last cycle, the sample cell is purged with an inert gas such as nitrogen to remove the remaining solvent from the cell and the lines to a vial that contains the extract. In some equipment, sequential extractions can easily be performed by repeating the procedure with a new solvent, and purging in the same or a different collection vial. Dynamic PFE is quite similar to static PFE, but requires a more sophisticated high-pressure or HPLC pump as well as a pressure restrictor rather than a static open/close valve. The equipment is similar to that used for ‘superheated water chromatography’ (Smith et al., 1999), but the column is replaced with an extraction cell and the tubing has slightly wider inner-diameter. Currently, there is no dynamic PFE equipment available on the market. In fact, there are only a few different types of commercial instruments available (Section 2.5.1), but many examples of home-made instrumentation have been described. Some of them perform both dynamic and static extractions at temperatures above 200 °C (Bautz et al., 1998; Hawthorne et al., 2000; Lou et al., 1997; Vandenburg et al., 1998). A short paragraph about building


Principles of pressurized fluid extraction and applications 43 your own equipment can be found in Section 2.5.2, including Fig. 2.8 that shows a schematic of what a dynamic PFE system may look like. 2.2.2 Extraction strategy In order to perform efficient and smart extractions, an understanding of the matrix characteristics and the different steps of the extraction is of great importance. The nature of the sample matrix (water and organic/inorganic content) and its physical characteristics (homogeneity, porosity, particle size) should also be considered. To better understand the extraction process, two models showing the distribution of analytes in different types of sample matrices are illustrated below. Figure 2.2 is a conceptualization of an aggregate of matrix particles from a source-separated household waste, and the possible sites where analytes in this instance chlorinated paraffins, are expected to be found (Nilsson et al., 2001). Figure 2.2 works as a model showing the variety of possible (and likely) positions and status of analytes in many different types of sample matrices, i.e. the analyte can be: 1. 2. 3. 4. 5.

adsorbed at the surface of the matrix, dissolved in the pore solvent and/or adsorbed at the pore surface, dissolved/adsorbed in a micro/nano pore, chemically bonded to the matrix, or dissolved in the bulk solution.

A simpler extraction model can be described for extracting small organic compounds from polymer particles, for instance the antioxidant Irganox

5 1 2

4 3

Household waste particle Stagnant solvent layer

Fig. 2.2 Schematic of a household waste particle and some possible sites (for explanation of numbers see text) where the analyte (chlorinated paraffin) might be adsorbed or chemically bonded (Waldeback, 2005).


44 Separation, extraction and concentration processes 1076 from the polymer LLDPE (Waldeback et al., 1998), Fig. 2.3. The extraction process of compounds from polymer particles, or other similar sample matrices, generally follows the following steps: 1. diffusion of the solvent into the matrix, 2a. desorption of the analytes from the matrix (including breaking of chemical bonds), 2b. solvation of the analyte into the extraction solvent, 3. diffusion of the analyte out from the matrix, and 4. diffusion of the analyte through the stagnant solvent layer and into the bulk solvent. Knowledge about distribution coefficients and distribution ratios are useful tools to provide guidance to the selection of solvent for the extractive separation process. Solubility of the analytes, their diffusivity in the solvent and matrix characteristics are the main factors to consider, when choosing a solvent for a successful extraction process. It is also important to understand the mass transfer mechanism across chemical/physical interfaces in order to design liquid/liquid and liquid/solid extraction processes. Toxicity and sustainability aspects of the solvent should also be considered. Additionally, in most of the analytical-scale applications, the concentration of the target molecule is very low, and thus the rate of the extraction is not limited by the analyte concentration in the extraction solvent, but rather determined by the rate of mass transfer out of the matrix. In order to perform a fast and complete extraction, a solvent has to be chosen that has the right chemical properties to dissolve and release the analyte, but should preferably not dissolve other solutes in the sample, i.e. the solvent power should not be higher than needed. Solubility theory has been discussed and proposed in classic works by J. Hildebrand, who combined the correlation between vaporization and intermolecular forces, van der Waal forces and hydrogen bonding, to the correlation between vaporization and solubility behaviour (Hildebrand and Scott, 1962; 1964). This model assumes that the same intermolecular attractive forces have to be overcome to vaporize a liquid as to dissolve an analyte. The term ‘solubility parameter’ d was described by Hildebrand as the square root of the cohesive energy density c, giving a numerical value indicating the analyte behaviour in a specific solvent, equation [2.1], where DH is the heat of vaporization (J mol–1) of the solvent, R the gas constant (J K–1 mol–1), T the temperature (K) and V is the molar volume of the analyte.

d = c = DH – RT [2.1] V Hansen (1967) took Hildebrand’s work further and assumed that the total cohesive energy is a linear addition of three components; dh (hydrogen bonding ability contribution), dd (dispersion coefficient contribution), and


Principles of pressurized fluid extraction and applications 45 dp (polarity contribution). They are linked by equation [2.2], where dt is the total solubility parameter (Fitzpatrick and Dean, 2002).

d t2 = d h2 + d d2 + d p2

[2.2]

Fitzpatrick and Dean (2002) predicted a suitable solvent to extract persistent organic pollutants (POPs) from contaminated soil and certified reference material using the Hildebrand solubility parameter and confirmed the results by experiments. The ideal extraction solvent from the calculations was a mixture of acetonitrile and dichloromethane (1:1 v/v), perhaps the best solvent from the view of solvent power, but not from a health and environmental view. When choosing a solvent it is a good start to have the Hildebrand and Hansen’s theories in mind. However, the choice of solvent in a particular situation involves other factors apart from the solvent power. As described in Fig. 2.2 and 2.3, the solvent has to penetrate the matrix thoroughly, break the bonds between the matrix and the analytes of interest, help the dissolved analytes to diffuse out from the matrix and finally be dissolved in the extraction solvent. The process of solute transfer across an interface between two liquid phases may be rate-controlled by molecular diffusion, by motion of eddies, by irregular surface disturbances or even by chemical reactions in the bulk of a phase or in the interface region. Local velocities in the interface region could be of importance as well as other factors affecting the local conditions, not least the presence of surface-active agents. The dependence of matrix geometry has been reported for SFE of mineral oils applied to metal devices (Bjorklund et al., 1996). Irganox 1076

1

2 3

Polymer particle Stagnant solvent layer

4

Fig. 2.3 Schematic showing the extraction of Irganox 1076 from an LLDPE polymer particle (Waldeback, 2005).


46 Separation, extraction and concentration processes Partitioning processes have a central role of concern in the extraction procedure. These involve partitioning of the analytes between the surface of the matrix and the solvent, as well as chemisorption of the analytes on active surface sites and within the solvent. Different matrices behave somewhat differently, e.g. polymeric samples usually build up a layer of stagnant liquid around the polymeric particles, as seen in Fig. 2.3, through which the analyte has to transfer into the extracting solvent. In this case, the partitioning of the analyte between the stagnant liquid and the extraction solvent has to be considered. Soils differ strongly in surface physicochemical properties and grain-size characteristics. Sediments, on the other hand, contain water having various types of bonding, from free water available for the plants, to water strongly bound to the particles, therefore a variety of equilibria take place. In milk powder, hard shells of lactose are formed during the short crystallization time when the milk is spraydried (Walstra and Jenness, 1984), and this may lead to difficult extraction of lipids and fat soluble vitamins. If the extraction solvent is nonpolar, it may be necessary to add a more polar alcohol such as methanol or ethanol to the sample before extraction (Turner and Mathiasson, 2000). There are only a few theoretical models suggested for the PFE technique. For instance, Vandenburg et al. applied the ‘hot ball’ model, originally described for SFE, for the extraction of additives from polymeric samples using PFE (Vandenburg et al., 1998). By plotting ln(m1/m0), where m1 is the mass of analyte remaining in the particle of radius r at time t, m0 is the initial amount of analyte and D the diffusion coefficient of the analyte in the solvent, a linear portion is given in equation [2.3] (Cotton et al., 1993). In this instance, when ln(m1/m0) is plotted against time, the line falls steeply initially and shortly after becomes linear where it follows equation [2.3] (Vandenburg et al., 1998):

ln(m1/m0) = – 0.4977 – (p2Dt/r2)

[2.3]

The physical explanation of the shape of the curve is that the analyte near the surface is rapidly extracted until a smooth falling concentration gradient is established across the particle. The extraction rate is then completely controlled by the rate at which the analyte diffuses to the surface. By plotting the amount of extracted analyte versus the extraction time for different solvents at different temperatures, the resulting curves showed a good fit to the ‘hot ball’ model. With a good kinetic model of the extraction process it would be possible to predict experimental parameters, and find out where the extraction is expected to be only diffusion dependent. However, the models mentioned here can at most be considered to give useful hints when developing new extraction methods based on PFE. There is no theoretical model that includes strong solute-matrix interactions caused by chemical bonding between the solute molecules and active sites on the matrix. In addition, sample matrices are seldom homogeneous, thus the penetration of the solvent is difficult to foresee. As a result, accurate descriptions are by


Principles of pressurized fluid extraction and applications 47 no means easy to provide because of experimental difficulties, as well as the chemical and mathematical complexity of the total extraction process. Empirical approaches, in combination with multivariate chemometric methods, are the most widely used. 2.2.3 Solvent selection As mentioned in Section 2.2.2, a solvent has to have the right chemical properties to desorb and dissolve the analyte preferably without dissolving other solutes in the sample, i.e. the solvent power should not be higher than needed. Richter et al. (1996) have suggested the conventional LLE solvent to be used when developing a new PFE method. Generally, when choosing a solvent the rule-of-thumb is ‘like dissolves like’, i.e. polar solvents dissolve polar analytes, and nonpolar solvents dissolve nonpolar analytes. In addition, the dipole moment and/or dielectric constants of solvents are useful for selecting an appropriate extraction solvent, see Table 2.1. Other important aspects to consider when facing a new extraction problem/challenge is in how the analyte of interest is dissolved or attached Table 2.1 Chemical properties of common extraction solvents (from: Handbook of chemistry and physics, CRC press, 52nd Edition, 1971–1972; and Handbook of chemistry and physics, CRC Press, 60th Edition, 1979–1980) Solvent Boiling Density point (°C) (g mL–1)

Vapour Dipole pressure moment at 20 °C (debye) (mbar)

Dielectric constant at 20 °C (mbar)

Polar, protic solvents Water 100.0 Methanol 64.9 Ethanol 78.5 n-Propanol 97.1 2-Propanol 82.4 n-Butanol 117.7

23 128 44 – 43 –

1.85 1.70 1.69 1.68 1.66 1.66

80.2 32.2 24.3 20.1 18.3 17.8

3.96

47.2

1.000 0.791 0.789 0.803 0.785 0.810

Polar, aprotic solvents Dimethyl sulfoxide 189 (DMSO) Acetonitrile 81.6 Acetone 56.2 Dichloromethane 40.0 Tetrahydrofuran 66 Ethyl acetate 77.1

0.786 0.790 1.326 0.886 0.894

93 233 453 200 97

3.92 2.88 1.60 1.63 1.78

37.5 20.7 9.1 7.4 6.0

Nonpolar solvents Chloroform Diethyl ether Toluene Benzene n-Hexane

1.498 0.714 0.867 0.879 0.660

211 507 29 108 160

1.0 1.15 0.4 0 0

4.8 4.3 2.4 2.3 1.9

61.7 34.5 110.6 80.1 68.9

1.092

0.56


48 Separation, extraction and concentration processes to the matrix, see Fig. 2.2 and 2.3, and also the type and strength of the intermolecular forces between the solvent molecules, which partly can be elucidated by the ΔHvap value, alternatively by the vapour pressure of the solvent (Table 2.1). When dissolving a solute, the intermolecular forces, i.e. hydrogen bonding, dipole–dipole and/or van der Waals interactions between the solvent molecules have to break and form new bindings/interactions with the solute. In instances when the solute is chemically bonded to the matrix, as in Fig. 2.2 (site 4), the extraction solvent has to overcome such bindings and then form new solute–solvent interactions, which altogether will lead to a more stable system of lower energy. Methanol and acetonitrile, two organic solvents commonly used in extraction and separation processes, have somewhat similar dielectric constants, 32 and 37, respectively, but differ strongly with regard to intermolecular forces as methanol forms hydrogen bonds, whereas acetonitrile relies on dipole–dipole interactions. Hence, acetonitrile, which is a less environmentally friendly solvent than methanol, should only be used in cases where such dipole–dipole interactions are more suitable. What are the partitioning processes or equilibrium reactions taking place during the extraction? For example, when extracting samples of high water content, such as wet sediments, vegetables or animal muscles, using organic solvents, there is always a risk that a nonpolar solvent will not be able to penetrate water-sealed pores that contain the analyte, and thus result in a lower extraction yield and unrepeatable results. The more knowledge about the chemistry of the target solutes, pertinent coextractable (unwanted) compounds and chemical and physical properties of the sample matrix and solute–matrix interactions, the easier it is to make a good selection of extraction solvent for instance from the ones listed in Table 2.1. One interesting aspect with PFE is the possibility of choosing a solvent, apart from very low or high pH solvents that may not be applicable in all kinds of equipment owing to possible corrosion of the tubings. In a collaborative study with an oil company, the objective was to predict the amount of process chemicals that could migrate into the sea if there was an oil discharge. By using a water solution of the same salinity as seawater, oil samples containing added process chemicals were extracted at different temperatures and times. The results could be used to predict what would probably happen in a real world situation (data not presented – only used internally at the company). This type of investigation is of course more environmentally relevant than a study performed with traditional extraction techniques based on organic solvents. How does the PFE technique and the solvent selection fit to a more sustainable chemistry? One of the advantages with PFE is that owing to the higher temperature and higher mass transfer during the extraction, more environmentally friendly solvents or solvent mixtures can be used according to Nilsson et al., 2001). As described above, the solvation power of a liquid increases with higher temperature and higher pressure. Furthermore, the


Principles of pressurized fluid extraction and applications 49 dielectric constant decreases with increasing temperature, which implies that the solvent polarity can be tuned by changing the temperature. This is especially true for water, the dielectric constant of which decreases from e = 78 at 25 °C to e = 56 at 100 °C; e = 36 at 200 °C; and e = 8 at 400 °C, since hydrogen bonding between the water molecules become less and less substantial at higher temperatures (Hawthorne et al., 1994). This can be compared to other polar, protic solvents and nonpolar solvents in Table 2.1. Hence, water is a very interesting alternative as an extraction solvent, and can potentially replace many of the organic solvents conventionally used in solvent extraction. The first work describing the use of water at elevated temperature and pressure for extraction of nonpolar solutes is one by Hawthorne et al. (1994). In this work, polar organics (e.g., chlorinated phenols), low-polarity organics (e.g., PAH), and nonpolar organics (alkanes) were extracted from soils and sediments using water at temperatures ranging from 50 to 400 °C. In our own work, water at 120 °C and 50 bar was used as a solvent for the extraction of polyphenolic glycosides from onions, as compared with using a conventional water/methanol (1:1) mixture at 80 °C (Turner et al., 2006). The developed method was also faster, 15 min compared with 2 h for the conventional. In a continuous study, a life cycle assessment was conducted to assess environmental impacts of the two methodologies, showing that the production and use of methanol compared with water has a large impact in terms of carbon dioxide emissions and energy usage, whereas the difference in energy usage for heating the solvents in the two extraction methods was much smaller (Lindahl et al., 2010). If more nonpolar solutes are to be extracted, and these solutes are slightly thermolabile, then ethanol is an interesting alternative to water since not as high a temperature is needed to obtain the same solvent strength. For instance, betulin was extracted from birch bark using ethanol at 120 °C and 50 bar in only 10 min (Co et al., 2009). Initially, the study tested to see if water at elevated temperature close to 200 °C could be an appropriate solvent for betulin, because pressurized liquid water at this temperature has a dielectric constant of around 36, and this is similar to that of methanol at room temperature (e = 32, see Table 2.1), which is indeed a good solvent for betulin. However, it turned out that it is not sufficient to use the dielectric constant as a measure of solubility of a solute in a solvent, as other chemical properties are also important. For this reason, the total (sometimes called ‘relative’) solubility parameter taking dh, dd and dp into account (equation [2.2], Hansen, 2007) was plotted as a function of temperature for betulin, water and ethanol, showing that even at 250 °C, the solubility of betulin in water is estimated to be very low (Fig. 2.4), whereas ethanol at temperatures between 50 and 150 °C should be appropriate, which was confirmed by experiment (Co et al., 2009).


50 Separation, extraction and concentration processes 50

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Fig. 2.4 Solubility parameters as a function of temperature for betulin, ethanol and water (Co et al., 2009).

2.2.4 Temperature effects An increase in temperature usually tends to promote solubility, as the thermal kinetic energy rises. Increasing the temperature also facilitates analyte diffusion and/or reduces interactions between analytes and the matrix by disrupting intermolecular forces such as van der Waal’s forces, hydrogen bonding and dipole attractions. Higher temperatures also decrease the viscosity of a liquid solvent, thus enabling better penetration of matrix particles. An increased temperature will also decrease the surface tension of the solvent, allowing the solvent to better ‘wet’ the sample matrix (Richter et al., 1996). Both lower viscosity and lower surface tension facilitate better contact of the solvent with the solutes and thereby enhance the extraction. Besides the selection of an extraction solvent, temperature can be considered as the second most important parameter in PFE. The advantages of using higher temperatures are described in every study published on PFE. As a general rule of thumb, a higher temperature gives a better extraction yield. It needs to be remembered that the range of solvents applicable in PFE is much wider than that of Soxhlet extraction. A poor solvent for Soxhlet extraction can, however, be a good solvent in PFE owing to the higher extraction temperatures used (Lou et al., 1997). Hence, as described below, by using a higher temperature, a more environmentally friendly and/or less toxic solvent can be used, for instance water or ethanol. However, other factors might hinder the use of the highest temperature even if it should give the best yield, e.g. as predicted in an experimental design. For example, Waldeback et al. (1998) found that the sample matrix (granules of LLDPE) started to dissolve/melt and block the tubings of the instrument depending on solvent and temperature, e.g. at temperatures above 75 °C © Woodhead Publishing Limited, 2010

Principles of pressurized fluid extraction and applications 51 using tetrahydrofuran as a solvent. Pihlstrom et al. (2002) found that, where pesticides from canola seed were extracted, the results from the screening study showed that the interpretation of the chromatograms was difficult owing to the large number of co-extracted compounds. It was obvious that higher temperatures gave more matrix peaks (Nemoto and Lehotay, 1998), which were most probably derived from co-extracted lipids. In another study (Waldeback et al., 2004), where squalene was extracted from olive biomass, the thermostability of the analyte was a limiting factor in the choice of extraction temperature. To determine the concentration of squalene in the olive biomass, an optimization study using experimental design was performed for the variables extraction temperature, extraction time and concentration of acetone in a mixture of acetone and 2-propanol. Significant factors were determined to be temperature, extraction time, the interaction between temperature and extraction time, and the square of the extraction time. This study showed that, at temperatures above 100 °C, the yield of squalene decreased with extraction times longer than 12–15 min, indicating that squalene either decomposed or reacted with other sample components or with the solvent. An example of extremely thermolabile compounds are anthocyanins, which are antioxidants found in vegetables and fruits, such as grapes, red cabbage, red onions, blueberries and red beets. PFE has been used to extract anthocyanins from red cabbage and red onions using water/ethanol/formic acid (94:5:1, vol%) at 99 °C and 50 bar (Arapitsas and Turner, 2008; Petersson et al., 2008, 2010). However, preliminary results show that anthocyanins extracted from red onion start to degrade after only a few minutes at a set temperature. Hence, extraction and degradation naturally occurs simultaneously in PFE. The kinetics of extraction and degradation depend on the nature of the sample matrix, on the chemical properties of the solutes to be extracted, and on temperature. Theoretical extraction curves were calculated using experimental extraction curves (with simultaneous degradation) combined with experimental degradation curves, as schematically described in Fig. 2.5 below. The conclusion was that, theoretically, a 20 to 30% higher yield of anthocyanins could be obtained from red onions if there were no degradation problems. This would require an extraction time of around 30 min (plus 8 min pre-heating time). In another study (Co et al., 2009), an increasing extraction temperature resulted in higher antioxidant activity in both water and ethanol extracts of birch bark. Furthermore, the antioxidant activities did not level out at high extraction temperature; hence, it appeared that some types of reactions occurred, and that these resulted in higher antioxidant activity. For instance, hydrolysis of polyphenolic glycosides leads to aglycons, which results in compounds of higher antioxidant activity because of the higher number of ‘reactive’ hydroxyl groups. Hence, an important conclusion is that a higher temperature may result in higher extraction yield of the target molecules, but also degradative reactions as well as extraction of more unwanted compounds, i.e. a less selective extraction.



25

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Fig. 2.5 Calculation of an ideal extraction curve (continuous lines on top) from experimentally obtained data on degradation (dashed line) and extraction with degradation (continuous line with circles), respectively, for cyanidin-3-(6≤ malonoylglucoside) in red onion (Petersson et al., 2010).

Another type of degradative reaction that may occur in food and agricultural samples is caramelization of sugars. When water is used as a solvent at temperatures of around 160 °C and above, sugars such as glucose caramelize (Montilla et al., 2006). Depending on the length of the extraction time, this obstacle may cause erratic results as well as plugging of tubings and filters. If such high temperatures are to be used, it is advisable to carry out the extraction as fast as possible, to minimize the occurance of caramelization. Maillard reactions may also occur during PFE, involving reducing sugars and amino acids or proteins, but also flavonoids, ascorbic acid and other carbonyl compounds (Manzocco et al., 2000). Maillard polymers can be a major contributor to antioxidant capacity at higher temperature (>170 °C), when using for instance water or water/ethanol mixtures as extraction solvents in food applications (Howard and Pandjaitan, 2008). Hence, in food applications, to be on the safe side, it is advisable to use an extraction temperature of less than 160 °C. 2.2.5 Extraction time The third most important parameter after extraction solvent and temperature, and maybe the absolutely most interesting one, is extraction time. Several studies have shown that PFE offers faster extraction methods relative to those based on Soxhlet, sonication and SFE, and similar to MAE (Dean, 1996;


Principles of pressurized fluid extraction and applications 53 Focant et al., 2004; Vandenburg et al., 1999). For instance, a comparison study was made for the extraction of PAHs from contaminated soil using Soxhlet, two different MAE methods, SFE and PFE (Saim et al., 1997). The Soxhlet method needed 24 h, the SFE method 35 min, the two MAE methods 20 min and the PFE method only 10 min, and Soxhlet extraction gave the highest recovery and the worst precision. In another study, Heemken et al. (1997) made a comparison of PFE and SFE with Soxhlet extraction of PAHs, aliphatic hydrocarbons and chlorinated hydrocarbons from marine samples. Statistical evaluations of accuracy and precision showed that equivalent results were achieved. The extraction time needed for the PFE method was 15 min, whereas SFE needed 90 min and Soxhlet 24 h. In general, during method development, extraction time and temperature are the most important parameters that need to be optimized once the solvent or solvent mixture has been chosen, as well as the mode of sample pre-treatment. Typically, extraction times are in the range of 10–15 min, plus a preheating step of 5–10 min depending on the temperature used and instrumental capabilities. However, if a dynamic extraction system is used (Section 2.5.2), the solvent can be preheated and continuously pumped through the sample without needing to preheat the cell. In commercially available PFE systems, dynamic extraction is mimicked using several short cycles of static extraction, replacing a certain volume of extraction solvent in between each cycle. The most recent Dionex PFE system, ASE-350®, can accomplish semi-dynamic extractions by step-wise replacement of small volumes of solvent continuously during the extraction. 2.2.6 Extraction pressure An increased pressure in the PFE technique is mainly applied to keep the solvents as liquids, at temperatures above their atmospheric boiling point. Most PFE studies observe no difference in extraction yield when the pressure is varied in the range of 34–204 bar (Lundstedt et al., 2000), although it has been reported (Richter et al., 1996) that a higher pressure rendered a higher extraction yield, when a standard mixture of PAH and polychlorinated hydrocarbons from a reference material was spiked onto wet silica of different pore sizes, using methylene chloride/acetone (1:1 v/v) at 100 °C as a solvent. Higher pressure gave higher extraction yield, when the pore size of the wet silica was 300 Å, but no difference in recovery was observed with the dry silica. It was suggested that higher pressure could probably facilitate the extraction of analytes trapped inside matrix pores because the pressure would force the solvent into the pores of the matrix that normally would not be contacted by solvents at atmospheric pressure. Pressurized flow would also aid in the solubilization of air bubbles so that the solvent could more easily penetrate the sample matrix. Kremer et al. (2004) also obtained higher yield of acidic herbicides in soil when pressure was increased from 69 to 138 bar, at 100 °C with dichloromethane as solvent.


54 Separation, extraction and concentration processes 2.2.7 Selectivity during the extraction Selective extractions are in general obtained by changing the solvent or the solvent power. Under ambient conditions (room temperature and atmospheric pressure) the solvent power of a liquid is essentially constant regardless of extraction conditions. In PFE, selectivity can mainly be obtained by varying the nature of the solvent or the solvent mixture (Majors, 1999; Ramos et al., 2002). It has been pointed out that a potential disadvantage with PFE is that the extraction tends to be exhaustive, therefore leading to nonselective extractions (Reighard and Olesik, 1996). Hawthorne et al. (2000) compared the difference in color of the extracts, when soils contaminated with PAH were extracted by Soxhlet, SFE, PFE and pressurized hot water extraction (PHWE). The Soxhlet and the PFE extracts were much darker mainly owing to the greater amount of extracted compounds, i.e. the extractions were less selective. Hence, to avoid coextracting compounds with maintained high yield of the target analytes, it is essential to choose the most selective solvent for the analyte combined with an appropriate temperature, i.e. high enough for efficient extraction of the target compounds, but not too high to avoid coextraction of unwanted molecules. Extraction time is also an important factor: the minimum time necessary should be used to achieve targeted extraction rather than exhaustive extraction of other matrix solutes. However, by using adsorbents in the extraction cell as an in-line clean-up step, more selective PFE extractions can be obtained. For instance, activated acidic alumina has been employed to adsorb fat when PCBs were extracted from spiked freeze-dried fish tissue, and in this way better chromatogram separations were achieved (Ezzell et al., 1996). Sulfuric acid impregnated silica has similarly been used to adsorb the lipids from fat-containing food and feed when extracting PCBs to obtain lipid-free extracts (Sporring and Björklund, 2004). In our own study (Nilsson et al., 2001), when extracting chlorinated paraffins from household waste, clean extracts and chromatograms were obtained when the drying agents Hydromatrix and sodium sulfate were used, a result which is in agreement with other studies (Hawthorne et al., 2000; Ramos et al., 2002). On the other hand, the use of adsorbents such as XAD-2, XAD-4, and XAD-16 did not provide the removal of unidentified interfering compounds. In another work of ours, squalene and a-tocopherol have been selectively extracted from olive oil without coextraction of triglycerides when utilizing Amberlite XAD-16 as adsorbent (data not published). A liquid chromatogram of the olive oil before and after PFE is illustrated in Fig. 2.6. 2.2.8 Accuracy and precision Accuracy and precision obtained with the various PFE methods are similar to those accomplished with other extraction techniques such as Soxhlet, sonication, SFE and MAE (Helaleh et al., 2005; Saim et al., 1997). As with any extraction technique, accuracy and precision of the chemical analysis



2.5¥105

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Fig. 2.6 Liquid chromatograms of (a) olive oil diluted with ethanol/acetone and (b) a PFE extract of olive oil, obtained using XAD-16 as adsorbent and methanol as extraction solvent.

method heavily depend on how well the sample is homogenized, and if the collected sub-sample is an accurate representation of the entire sample under study. These problems with sample preparation can of course be minimized by taking out a larger number of sub-samples, i.e. replicates, for analysis. Another important variable is the particle size, i.e. too large a particle size leads to inefficient extraction and inaccurate results. This was demonstrated by Björklund et al. (1999) for sediment samples of different particle size distributions. Similarly, Isaac et al. (2005) in a study of the extraction of lipids from homogenized cod tissue obtained a higher yield compared with extracting intact wet cod muscle. The conclusion was that the proteins in the sample denaturated to a hard pellet during the extraction making the diffusion of the solvent into the matrix not efficient enough to extract all the lipids. On the other hand, too small a particle size led to a relatively compact sample, which in turn may result in channeling inside the sample and again inefficient extraction.



2.3 Applications of pressurized fluid extraction Most of the studies on PFE deal with either environmental analysis or food and agricultural samples, targeting either pollutants or health-beneficial compounds such as antioxidants. Figure 2.7 shows the publication record from 1995 to 2008, taken from Web of Science. There is not enough space in this chapter to discuss in detail the wide range of different applications for PFE, hence, the reader is referred to a number of reviews of the field: on applications for environmental analysis (Giergielewicz-Mozajska et al., 2001; Nieto et al., 2008); food and drugs (Beyer and Biziuk, 2008; Carabias-Martinez et al., 2005; Herrero et al., 2006; Mendiola et al., 2007); and medicinal plants, herbs and agricultural (Huie, 2002; Wang and Weller, 2006). 2.3.1 Environmental applications Pesticides were extracted from fruits and vegetables (Adou et al., 2001; Barriada-Pereira et al., 2007; Blasco et al., 2005; Herrera et al., 2002; Pihlstrom et al., 2002; Tanaka et al., 2007; Wennrich et al., 2001) and from agricultural crops and soils (Hildebrandt et al., 2007; Otake et al., 2008; Popp et al., 1997; Schreck et al., 2008) using solvents and solvent mixtures such as acetone, acetone/dichloromethane, acetone/hexane, methanol, hexane saturated with acetonitrile, and acetonitrile and water, at temperatures between 60 and 130 °C. For polyhalogenated persistant organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), it is more common to use hexane, toluene/acetone and hexane/ acetone, at temperatures between 100 and 150 °C (Antunes et al., 2008; Bjorklund et al., 1999; Brandl et al., 2006), but water has also been used at temperatures between 250 and 300 °C (Yang et al., 1995). Number of publications

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Fig. 2.7 Publication record (Web of Science 2009-08-02, search term: ‘pressurized liquid extraction’ OR ‘accelerated solvent extraction’ OR ‘pressurized solvent extraction’ OR ‘subcritical water extraction’ OR ‘pressurized fluid extraction’). In total 1469 publications between 1995 and 2008.


Principles of pressurized fluid extraction and applications 57 Organometallic compounds containing tin, arsenic and mercury have been extracted from sediments and soils (Beichert et al., 2000; Chiron et al., 2000), vegetables (Marcic et al., 2005) and seafood (Mato-Fernandez et al., 2007; Wahlen and Catterick, 2004) using methanol, water, methanol/water and methanol/ethyl acetate mixtures, at temperatures between ambient and 160 °C. There are also a few studies on the extraction of explosives from soil samples (Ragnvaldsson et al., 2007) and biological samples (Pan et al., 2005); and antibiotics from soil (Jacobsen et al., 2004; Schlusener et al., 2003) and meat samples (Berrada et al., 2008). Pharmaceutical applications are less common than environmental ones, and most of them consider the extraction of pharmaceutical drugs and their metabolites in environmental samples such as sediments, sludge and fish/ shell fish. For instance, pharmaceutical residues have been extracted from sludge using methanol/water (1:1) at 60 °C as solvent (Barron et al., 2008). There is also an increasing number of studies on endocrine disruptors in the environment, for instance, 17-b-estradiol has been extracted from soil using acetone/hexane (1:1) at 100 °C (Chun et al., 2005); and alkylphenolic compounds have been extracted from river sediment using methanol/acetone (1:1) at 50 °C as a solvent (Petrovic et al., 2002). 2.3.2 Food and agricultural applications Food and agricultural applications considering endogeneous compounds commonly involve the use of more environmentally friendly solvents such as water and ethanol, because these applications often aim at isolating healthbeneficial compounds for later use as food additives, or alternatively, the compounds that are extracted are fairly polar, hence stronger organic solvents are not needed. Exceptions are in applications concerning the extraction of lipids such as acylglycerols, sterols, terpenoids and essential oils, for which stronger organic solvents are generally employed, e.g. isopropanol, ethyl acetate and hexane. Flavonoids such as quercetin, kaempferol, catechin and anthocyanidins have been extracted from vegetables and herbal plants using pressurized hot water, ethanol or water/ethanol mixtures as solvents at temperatures between 50 and 160 °C (Arapitsas and Turner, 2008; Howard and Pandjaitan, 2008; Ibanez et al., 2003; Ollanketo et al., 2002; Turner et al., 2006). For instance, water and a 70:30 mixture of ethanol and water at temperatures of 50 to 190 °C were used to extract flavonoids from dried spinach (Howard and Pandjaitan, 2008). The results showed that the total phenolic content as well as the antioxidant capacity were the highest in extracts obtained with the highest extraction temperatures (170–190 °C) for both water and the water/ethanol mixture, although the solvent mixture was somewhat more efficient than pure water in extracting flavonoids. However, when separating the extracts into low (1000 Da) molecular weight fractions, it turned out that at temperatures above 130 °C for water and


58 Separation, extraction and concentration processes above 150 °C for ethanolic solvents, polymeric Maillard reaction products were responsible for the increase in antioxidant capacity. Howard and Pandjaitan (2008) concluded that, in order to extract flavonoids effectively, water should be used at temperatures between 50–130 °C and for ethanolic solvents, 50–150 °C. In one of our own studies, quercetin and its glycosides as well as isorhamnetin and kaempferol were extracted from yellow and red onion using water at 120 °C and 50 bar, employing three consecutive 5-min extraction cycles (Turner et al., 2006). Thermostable b-glucosidase was used to convert the polyphenolic glucosides into their respective aglycons within only 10 min of reaction, using water at 90 °C and pH 5 as a reaction media. Results obtained compared well with a conventional extraction method employing a 1:1 mixture of methanol and 2.4 M HCl at 80 °C and 2 h combined extraction/ reaction, followed by filtration. Phenolic compounds such as rosmarinic and carnosic acids, carnosol and methyl carnosate have been extracted by PHWE, ultrasonication-assisted methanol extraction, hydrodistillation, and maceration with 70% ethanol from sage (Ollanketo et al., 2002). It turned out that PHWE, performed at 100 °C, gave the highest antioxidant activity of the extracts. Furthermore, PHWE was faster than the conventional techniques used in this work. In another study (Ibanez et al., 2003), antioxidative compounds such as carnosol, rosmanol, carnosic acid, methyl carnosate, cirsimaritin and genkwanin have been extracted from rosemary leaves using subcritical water at temperatures between 25 and 200 °C, showing that the selectivity of the extraction can be tuned by varying the temperature. At 25 °C a high concentration of rosmanol was obtained in the extract, while at 200 °C, the dominant compound was carnosic acid. The antioxidant activity was high in all of the extracts obtained at 100, 150 and 200 °C, respectively (Ibanez et al., 2003). Phenolic acids have been extracted from fruits and vegetables using water, methanol and water/methanol mixtures, at temperatures ranging between 20 and 100 °C (Alonso-Salces et al., 2001; Chen et al., 2007a; Mukhopadhyay et al., 2006; Waksmundzka-Hainos et al., 2007). In general, the main concern for the extraction of phenolic acids as well as many of the flavanoids is the thermal stability, or rather the tendency to degrade during the extraction, as discussed in Section 2.2.4. Terpenoids including limonene, pinene, artemisinin, retinol, taxol, squalene, lycopene and carotene, have only limited water solubility even at elevated temperatures, hence these are extracted with, for instance, 2-propanol or methanol/ethyl acetate/light petroleum (1:1:1) at temperatures ranging from 40 to 190 °C (Breithaupt, 2004; Fojtova et al., 2008; Schaneberg and Khan, 2002; Waldeback et al., 2004). There are, however, also studies in which pressurized hot water is used as a solvent; Yang et al. (2007) used water at temperatures between 100 and 250 °C to extract a-pinene, limonene, camphor, citronellol, and carvacrol terpenoids from oregano and basil leaves. However, it was shown that thermal degradation occurred at all temperatures tested,


Principles of pressurized fluid extraction and applications 59 although least severe at 100 °C – between 10 and 25% for a 30-min treatment in static mode. In a work by Breithaupt (2004), carotenoids were extracted by PFE and compared with a conventional solvent extraction, in both cases using a solvent mixture of methanol/ethyl acetate/light petroleum (1:1:1). The PFE extraction conditions were optimized by varying the temperature (25–80 °C) and the pressure (70–140 bar), giving the highest yield at 40 °C and 70 bar. Both methods were used for the extraction of a large number of different carotenoids from beverages, pudding mixes, cereals, cookies and sausages (Breithaupt, 2004). Acylglycerols and sterols have been extracted from eggs and egg-containing food products, using chloroform/methanol 2:1 (v/v) and hexane/isopropanol 3:2 (v/v), at various extraction temperatures and pressures (60 °C and 150 bar, 100 °C at 150 bar, and 120 °C and 200 bar (Boselli et al., 2001). Results showed that the hexane/isopropanol mixture employed at 60 °C and 150 bar could successfully be used to extract lipids from egg products. In another study, lipids were extracted from freshly ground corn kernels and ground rolled oats, using four different organic solvents: hexane, dichloromethane, isopropanol, and ethanol, at two temperatures, 40 and 100 °C (Moreau et al., 2003). The results showed that oat oil with the highest levels of digalactosyldiacylglycerol (DGDG) was obtained using ethanol at 100 °C. Comparison of the extraction effects with seeds of two different species of grains indicated that the extractability is greatly dependent on the polarity and temperature of the solvent. Tocopherols and tocotrienols are fat-soluble compounds extractable using solvents such as methanol and acetone. For instance, tocopherols and tocotrienols have been extracted from cereals using methanol as extraction solvent at 50 °C and 110 bar, using only one 5-min extraction cycle (Bustamante-Rangel et al., 2007). Other food and agricultural applications include for instance alkaloids (Mroczek and Mazurek, 2009); saponins (Chen et al., 2007b); coumarins (Waksmundzka-Hajnos et al., 2004); oligosaccharides (Bansleben et al., 2008); lignans (Smeds et al., 2007); and various essential oils (JimenezCarmona et al., 1999; Ozel et al., 2003; Schaneberg and Khan, 2002).

2.4 Future trends The authors believe that some of the most important future trends for PFE are: (i) switching to more environmentally friendly solvents; (ii) hyphenation and automation; (iii) miniaturization; (iv) mimic nature; and (v) transfer to industry.


60 Separation, extraction and concentration processes All these trends are somewhat connected with the growing push for a more sustainable development. In order to minimize the use of organic solvents that need to be produced and then disposed of, both switching to more environmentally friendly solvents and miniaturizing the extraction equipment is vital. The use of solvents at elevated temperatures implies that more efficient extractions can be obtained, even when using more environmentally friendly solvents such as water and ethanol rather than the classical ones such as hexane and ethyl acetate. Hence, future trends will most probably involve switching from conventional organic solvent based extraction methods to PFE using water, ethanol and other ‘green’ alternatives. This has already been demonstrated (Burkhardt et al., 2005; Co et al., 2009; Crescenzi et al., 1999; Curren and King, 2001; Fernandez-Perez and de Castro, 2000; Hawthorne et al., 1998; Hyotylainen et al., 2000; Morales-Munoz et al., 2002; Smith, 2002; Turner et al., 2006; Yang and Li, 1999), but more applications are expected in the near future. Regarding miniaturization of PFE, we found only one example (Ramos et al., 2000), in which PAHs were extracted from soil and sediment using an extraction vessel constructed by a 10 mm ¥ 3 mm inner diameter stainlesssteel tubing. The only extraction solvent used was 100 mL of toluene at 200 °C and 150 bar, in an extraction process taking 10 min, with subsequent direct injection into large-volume injection gas chromatography–mass spectrometry (GC–MS). Future trends will probably include more examples of miniaturized PFE systems, probably also combined with analytical separation and detection systems. It has been recognized that in order to minimize the production of waste and emissions of carbon dioxide, it is advantageous to use as few steps in a process as possible, i.e. combined (hyphenated) methods and automation are relevant. There are several good examples showing that PFE can be coupled on-line with separation by HPLC (Hyotylainen et al., 2000; Li et al., 2000), but also with GC using a miniaturized PFE system (Ramos et al., 2000). Smith showed that subcritical water extraction can be coupled on-line with chromatography using subcritical water as the only mobile phase (Smith, 2002; Tajuddin and Smith, 2002). In the future, technology development enabling combination with several other separation and detection techniques is expected, as well as in situ analysis instrumentation. In order to better understand processes occurring in real life or in the environment, laboratory-scale methods that mimic nature in an efficient way are important. Thus, by using PFE, an interesting possibility is to simulate what could happen in a real life situation (in the human body, in the environment or within an industrial process). This can be accomplished using a suitable solvent, similar to the surroundings in question, for example water with appropriate pH and ion concentrations (such as seawater). Anaerobic conditions can be simulated using a nitrogen purge between sequential extractions. Hence, it is possible to predict what would happen if a specific chemical is used by a specific company in a specific environment


Principles of pressurized fluid extraction and applications 61 and, in this way, obtain detailed information for an environmental regulatory impact analysis. Another challenge would be to follow the migration of any hazardous compound and their degradation products in soil, sediment, sludge or biota. Ongoing research in this area is expected to address important environmental issues. A final note on future trends of PFE is that several of the smaller-scale processes will be transferred to industry to replace conventional solvent extraction methods in chemical analysis applications, as well as scaled up for industrial processes in applications regarding isolation of interesting (valuable) compounds from the plant kingdom. For instance, a standard method (US EPA SW-846 Method 3545) for the extraction of PCBs from sediments using hexane/acetone (1:1) as a solvent at 150 °C has replaced a traditional Soxhlet method. Both new applications and replacement of technology in old applications are expected to be developed in the near future.

2.5 Sources of further information and advice 2.5.1 Commercially available equipment There is equipment available on the market that performs static PFE, with full or semi-automation. Currently, the following vendors have been identified: Dionex (www.dionex.com) products include ASE® 150 (one extraction vessel at the time, of sizes 1, 5, 10, 22, 34, 66 or 100 mL) and ASE® 350 (carousel with up to 24 extraction vessels of the same sizes as for the 150-system). A solvent controller allows mixing and delivery of up to three solvents. Temperature range applicable is 40–200 °C and the pressure is fixed to 1500 psi (103 bar). pH hardened pathway with Dionium™ components and extraction vessels make the instrument compatible with acid or alkaline pretreated sample matrices. ∑ Applied Separations (www.appliedseparations.com) has two extraction systems, one PSE™ and fast-PSE™, which operate with one and six in-parallel extraction vessels, respectively. Available vessel sizes are 11, 22 and 33 mL. An automated solvent dispenser is optional, and up to four different solvents can be handled. Temperature range is 50– 200 °C and maximum pressure is 150 bar. ∑ Fluid Management Systems (www.fmsenvironmental.com) market PFE™ systems that handle one to six samples (six modules) at the time in parallel, with the option of in-line clean up by column chromatography. Temperature and pressure ranges obtainable are 70–200 °C and 1500– 3000 psi (103–207 bars), respectively. Extraction vessels come in sizes ranging between 5–250 mL. ∑


62 Separation, extraction and concentration processes 2.5.2 Designing your own equipment In our laboratory, we usually build our own equipment, since the ones commercially available are all based on static PFE and, in addition, operate within a strictly limited temperature range. Hence, in order to run dynamic PFE, even at temperatures above 200 °C, it is necessary to custom build PFE instrumentation. What we recommend is to use the following parts: (a) an optional heater plate set to a temperature of around 20–30 °C below the boiling point of the solvent; (b) a bottle containing the extraction solvent or solvent mixture; (c) an ordinary HPLC pump; (d) a long stainless-steel tubing that enables the solvent to reach desired temperature before passing through the extraction vessel; (e) a type K thermocouple temperature probe for measuring the temperature of the solvent just before entering the extraction vessel; (f) a stainless-steel extraction vessel (for instance a preparative HPLC column) in which the sample is placed before extraction; (g) a simple GC oven (only temperature control is needed, hence no need for injector or detector); (h) a cooling bath to bring down the temperature of the extractant to below the boiling point of the solvent; (i) a needle valve or a back pressure regulator; (j) collection vial(s); and (k) optional nitrogen gas to flush out the entire system after extraction. It is advisable to install an in-line filter to protect the needle valve/back pressure regulator. It is also recommended to install a pressure gauge for control of the pressure as well as a burst disc in case the pressure rises too high in the system. There are burst discs available for different pressures and solvents. Figure 2.8 shows a schematic of a home built dynamic PFE system. (c)

(b)

VALCO (a)

(k)

(e)

(g) (d)

(i)

(f) (h) (j)

Fig. 2.8 Schematic of a home-built dynamic PFE system. (a) Heater plate; (b) extraction solvent; (c) HPLC pump; (d) stainless steel tubing; (e) temperature probe; (f) extraction vessel; (g) oven; (h) cooling bath; (i) needle valve; (j) collection vial; (k) nitrogen gas.


Principles of pressurized fluid extraction and applications 63 A factor to consider is which solvents and temperatures are allowable by the different parts of the extraction system. For instance, an HPLC pump cannot take too high a temperature, and especially not with some of the organic solvents. If water is used as a solvent, caution must be taken if temperatures above ca. 200 °C are used, since water becomes more and more corrosive at higher temperature. Hence, higher-quality nickel steel alloy, such as Hastelloy® that exhibits high resistance to corrosion, should be used. However, owing to obstacles such as caramelization and Maillard reactions occurring in food and agricultural samples, temperatures applied are rarely above 160 °C. Finally, if an organic solvent is used, safety arrangements around your homebuilt device, such as ventilation and burst discs connected to stainless-steel tubings leading to a secure waste container, should be provided.

2.6 Conclusions In industrial and governmental analytical and research laboratories, PFE is a promising extraction technique that easily replaces conventional techniques such as Soxhlet extraction – especially since the same solvents can be used. Compared with conventional extraction techniques, PFE is faster; more automated; and enables a ‘friendlier’ work environment. Furthermore, new PFE methods are fairly easy to develop by optimizing temperature and extraction time. Since PFE operates at elevated temperatures, it is possible to use less harmful solvents than in Soxhlet with maintained extraction efficiency. However, caution has to be taken regarding degradation of thermolabile target compounds during the extraction, even though the problems are usually minor if the extraction time and temperature are carefully optimized. Finally, selectivity can be obtained in PFE by firstly selecting an appropriate extraction solvent, secondly by varying the temperature of the solvent, which is especially effective for water, or thirdly by using adsorbents in the extraction cell. How does PFE fit into a sustainable development? PFE uses smaller amounts of organic solvent, thereby preventing unnecessary waste, because less organic solvent needs to be manufactured and less waste is thereby produced by the manufacturer. In addition, less organic solvent waste is produced at the laboratory and, in total, there will be a decrease in volatile organic carbon emissions and a reduced risk for photochemical smog formation. Accidental emissions to municipal wastewater plants will do less harm. By being able to substitute hazardous solvents with more environmentally friendly solvents such as ethanol and water, the toxicity is reduced with positive effects on both the environment and health in general. Creating high diffusion liquids requires a significant amount of energy, which is a disadvantage. However, because the extraction times are shorter, minutes compared with hours in Soxhlet and modified Soxhlet techniques, and, as these traditional solvent extraction techniques are usually performed © Woodhead Publishing Limited, 2010

64 Separation, extraction and concentration processes just below the boiling point, a significant amount of energy is saved. Lifecycle assessment should be conducted to calculate the overall energy usage in different methodologies.

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Principles of physically assisted extractions and applications 71

3 Principles of physically assisted extractions and applications in the food, beverage and nutraceutical industries E. Vorobiev, Compiègne University of Technology, France and F. Chemat, University of Avignon and Pays de Vaucluse, France Abstract: The physical methods used to enhance pressure and solute extraction methods in the food industry are reviewed. The quality of extracted products (e.g. purity, colour, texture, flavour and nutrients) may be degraded by conventional mechanical, thermal or chemical pretreatments. Several physical treatments (pulsed electric fields, power ultrasound, microwaves, ohmic heating, arc discharges) have been shown to be important and of great interest for the food industry in terms of enhancing solute and pressure extraction, and dehydration processes. Emerging extraction technologies that show promise for commercial food processing are discussed. Key words: plants, food, extraction, pulsed electric field, ultrasonic extraction, microwave extraction.

3.1 Introduction Pressure and solvent extractions are widely used in the food industry for the production of juices, wine, sugar and vegetable oil; these methods are also frequently applied in the extraction of various target compounds, such as colorants, antioxidants, essential oils, and aromas, from raw plant materials. Pressing, hot water and organic solvent extractions are long established processes that have excellent efficacy when applied in optimal conditions. These extractions can be done concurrently but more often they compliment each other and are technologically combined (e.g. the hot water extraction of sugar from sugar beets is combined with the subsequent pressing of pulps; the pressing of oilseeds is combined with the subsequent solvent extraction of oil from press-cake; the pressing of apples or grapes can be combined © Woodhead Publishing Limited, 2010

72 Separation, extraction and concentration processes with the subsequent solvent extraction of bioactive compounds from mash). The yield of extracted compounds can be very high in optimal conditions. Unfortunately, the quality of solutions and extracted products (e.g. purity, turbidity, colour, texture flavour and nutrients) may be degraded in the course of the raw material treatments that are necessary to increase the yield (grinding, heating, chemicals/enzyme addition). Moreover, a significant quantity of waste is often produced from the purification of impure extraction solutions, when undesirable loss of solvents and other additives occurs. These problems need to be resolved or minimised for the future development of environmentally sustainable food technologies. In recent decades there has been a growing interest in alternative food technologies that allow for nonthermal or mild thermal food preservation. Several emerging technologies are prominent and of great interest to the food industry, in particular pulsed electric fields (PEF), power ultrasound (PU), microwave (MW), pulsed light (PL), ohmic heating (OH), irradiation (IR), radio-frequency (RF) heating, high pressure processing (HP), high voltage electric discharges (HVED), amongst others (Barbosa-Cánovas et al., 1998; Zeuthen and Bogh-Sorensen, 2000; Povey and Mason, 1998). Such success in the development of novel preservation technologies has encouraged further research and has renewed the industrial interest in extractions assisted by nonthermal or mild thermal physical treatments (PU, PEF, HVED, OH, MW). Recently, these treatments were found to be effective for the enhancement of solute and pressure extractions, and dehydration processes (Knorr et al., 2001; Kulshrestha and Sastry, 2003; Li et al., 2004; Virot et al., 2007; Vorobiev and Lebovka, 2006, 2008). The potential benefits of physically assisted extractions are important. For instance, they can lead to the future substitution of hot water or organic solvent extractions with cold, or mild thermal, aqueous or pressure extractions. Moreover, the alternative physical treatments are found to be less invasive methods for the processing of plant foods, making it possible to avoid many undesirable changes in products, pigments, vitamins, and flavouring agents, which are typical of other extraction techniques, including thermal, chemical and enzymatic techniques. Some of these novel technologies remain very much in the research arena, whereas some others are on the brink of commercialisation. This chapter provides an overview of the emerging extraction technologies that are promising in terms of their potential applications in commercial food processing.

3.2 Pulsed electric field-assisted extractions in the food industry 3.2.1 Principles of pulsed electric field (PEF) treatment Pulsed electric field processing is a technique in which a food is placed between two electrodes in a batch or continuous treatment chamber and © Woodhead Publishing Limited, 2010

Principles of physically assisted extractions and applications 73 exposed to a pulsed voltage (typically 15–80 kV cm–1 with several pulses of 1–5 ms for the killing of micro-organisms; and 0.1–5 kV cm–1 with pulses of 50–1000 ms for the electroporation of plant cells and nonthermal extraction from solid foods). To generate such short pulses, various pulse-forming networks are used, with main components that include power supply at the selected voltage, one or several capacitor banks, inductors and/or resistors (Barbosa-Cánovas et al., 1998; Bluhm, 2006). Various different pulse shapes can be generated, including the simplest exponential decay pulses and squarewave pulses (mono-and bipolar). The duration and/or number of pulses are limited in order to prevent any significant temperature elevation, which is usually less than 3–5 °C. Mechanism of cell membrane electroporation When the intensity of an applied electric field increases, the potential difference across a cell membrane (transmembrane potential um) also increases. If um exceeds a stated threshold value (typically 0.2–1 V), a temporary loss of membrane semipermeability occurs. This phenomenon of membrane damage is named electroporation (electropermeabilisation) (Fig. 3.1). A number of models have been proposed in the biophysical literature to describe the electroporation mechanism (Konduser and Miklavcic, 2008; Zimmermann,

E < Ecr

Compression

Compressed membrane

E > Ecr

(a) Repulsion

Pore

Membrane

2r

(b)

Fig. 3.1 Membrane electroporation. With application of electric field E, the cell membrane is compressed owing to the electric charges attraction (a). (b) When the electric field exceeds some critical value (E > Ecr), the transmembrane potential increases to a threshold value and electroporation occurs (from Vorobiev et al., 2005).


74 Separation, extraction and concentration processes 1986). The transmembrane potential um of a spherical cell depends on the angle q between the external field E direction and the normal vector on the membrane surface. The transmembrane potential may be given by the following equation (Zimmermann, 1986):

um = 0.75fdcEcosq [1 – exp (– t/tc)]

[3.1]

where dc is the cell diameter, tc is the time of membrane charging, and f is a parameter depending on the electrophysical and dimensional properties of the cell, membrane, and surrounding media. The values of f and tc vary for different cell sizes and ratios of extracellular/intracellular conductivities se/ si (Lebovka et al., 2000; Vorobiev and Lebovka, 2008). At complete cell damage, it follows that se/si = 1 and f ≈ 1. The critical value of transmembrane potential required for biological membrane electroporation at ambient temperature is estimated as um ≈ 1 V (Zimmerman, 1986) and can vary in plant and animal cells from 0.7 to 2.2 V (Knorr et al., 2001). The typical size of a cell in plant tissue is dc ≈ 50–100 mm. Therefore, the required threshold PEF intensity for the electroporation of food plants tissues (for f = 1 and tc 7.5 kV cm–1) (Schrive et al., 2006;


Principles of physically assisted extractions and applications 83 Zhang et al., 1994), though smaller fields can also affect the structure of cells. For instance, the early stages of damage in the yeast cells were observed at E < 7.5 kV cm–1, in a PEF treatment of long duration (El Zakhem et al., 2006a,b). The electroextraction of proteins from suspensions of S. cerevisiae was also observed at 3.2 kV cm–1 (Ganeva et al., 2003), but high efficiency (yield of 85%) required long extraction following PEF treatment (>4 h at 30 °C). Recently, Shynkaryk et al. (2009) demonstrated that a rather long application of PEF treatment (1000 trains, 200 pulses of 100 ms in each train) was needed to result in a high level of membrane disintegration (Z > 0.8), even at E = 10 kV cm–1. Moreover, the quantity of released high molecular weight intracellular components extracted with PEF was lower than that extracted with other methods (HVED and high-pressure homogenisation) (Shynkaryk et al., 2009).

3.3 Ohmic heating-assisted extractions in the food industry 3.3.1 Principles of ohmic heating (OH) treatment Ohmic heating (OH) processing is a technique in which a food is placed between two electrodes in a batch or continuous treatment chamber and exposed to a dc, ac or pulsed voltage (typically 20–80 V cm–1) to heat the food and kill any micro-organisms. OH generators, which use ac and highfrequency electric fields permit reduced electrolysis and product contamination, compared with dc designs (Lima et al., 1999; Sastry, 2005). The basic principle of OH is the dissipation of electrical energy in the form of heat, resulting in the generation of internal energy (Sastry, 2005):

q = sE2

[3.3]

where q is the heat power per unit volume, s is the electrical conductivity of food and E is the electric field intensity. OH processing is currently commercialised for the pasteurisation and sterilisation of particulate foods (Biss et al., 1989; Sastry, 2005). However, the effects of OH are not purely thermal. Several studies have noted that electropermeabilisation is also induced by OH (Kemp and Fryer, 2007; Kulshrestha and Sastry, 2003; Lima et al., 2001; Praporscic et al., 2006; Schreier et al., 1993). Lebovka et al. (2005) have studied the membrane damage of ohmically heated potato and apple tissues. The OH experiments conducted under ac treatment were started from room temperature T = 22 °C at different electric field strengths E, and the treatment was stopped when the temperature reached T = Tf = 50 °C. The total treatment time te was higher with lower E (Fig. 3.5), but the samples were not kept in the warm juice (at 40–50 °C) for more than 15 min. The thermal damage and softening of potato and apple tissues at such mild thermal treatment conditions can be neglected (Lebovka et al., 2004a; 2004b). But the conductivity disintegration index Z © Woodhead Publishing Limited, 2010

84 Separation, extraction and concentration processes increased considerably at the electric fields E > 20 V cm–1, both for apples and potatoes (Fig. 3.5), and these data provide evidence for the significance of electrically induced tissue damage. More recently, Lebovka et al. (2007) estimated the variation of Z values of sugar beet tissue during OH. They obtained electrical conductivity graphs for intact, maximally damaged and ohmically treated tissues at various temperatures and calculated Z(T) from equation [3.2] for various electric fields E. The results provide evidence for the significance of the electroporation effect in OH. This effect becomes more significant as E is increased from 40 to 100 V cm–1. Imai et al. (1995) have found that the OH behaviour and impedance values of Japanese white radish depended on the frequency of the electric field, which varied from 50 Hz to 10 kHz. At a constant voltage of 40 V cm–1, the time required to heat the sample to the desired temperature was longer for higher frequencies of electric field. It was concluded that the initial rapid heating of white radish at low frequency (50 Hz) is caused by the electroporation effect. 3.3.2 Juice expression and solutes extraction enhanced by OH The first studies of extractions enhanced by OH were completed in the former Soviet Union, and summarised by Rogov and Gorbatov (1974) and by Lazarenko et al. (1977). In Soviet publications, electroporation was known as ‘electroplasmolysis’, and the combined thermal and electric effects caused by OH were called ‘thermo-electroplasmolysis’ or ‘low-gradient ac, Tf = 50 °C

Potato Apple 103

0.6

te (s)

Conductivity disintegration index, Z

0.8

0.4 102

0.2

0

0

20

40 60 80 Electric field strength, E (V cm–1)

100

101

Fig. 3.5 The conductivity disintegration index Z and time of treatment te versus electric field strength E for potato and apple tissues ohmically heated to 50 °C by ac treatment.


Principles of physically assisted extractions and applications 85 plasmolysis’ to underline the relatively low electric fields (typically 10–100 V cm–1) used for OH. Research interest in extractions assisted by OH processing was renewed in Europe and the USA during the 1990s and 2000s (Sastry, 2005; Schreier et al., 1993; Wang and Sastry, 2002) probably due to the successful commercialisation of OH systems for food sterilisation (Biss et al., 1989). This interest in OH extractions focused on the synergy of electrical and thermal effects on cell tissue, and the lower temperatures needed for effective membrane damage compared with conventional heating. On the other hand, the electrical field applied in OH-assisted extraction is lower than that for nonthermal PEF treatment. Therefore, electrical equipment can be simplified to deliver a lower output voltage than typical high voltage PEF generators. Various studies have recently demonstrated the effectiveness of OH pretreatment for the intensification of pressing and solute extraction (Kemp and Fryer, 2007; Kulshrestha and Sastry, 2003; Lima et al., 1999; Praporscic et al., 2005, 2006; Sensoy and Sastry, 2004; Wang and Sastry, 2002). For instance, Lima et al. (1999) ohmically heated apple cuts up to 40 °C with a voltage of E = 40 V cm–1 and wave frequencies of 4 and 60 Hz. Subsequent pressing at a constant rate (3 cm min–1) resulted in a higher juice yield than that obtained for untreated cuts, and the 4 Hz pretreatment was more effective than the 60 Hz pretreatment. More recently, Wang and Sastry (2002) have demonstrated that OH pretreatments of 40 or 50 °C by ac with 40 V cm–1 and 60 Hz enhanced the juice yield from apple samples. Compared with untreated samples, or those preheated by microwaves, the juices extracted from ohmically preheated apple tissue were visually similar in quality, and the yields were higher. The lower frequency of OH compared with MW heating may be the important factor that caused the greater increase in juice yield (Wang and Sastry, 2002). There may be other mechanisms involved in intra- and extracellular moisture flow during OH. In addition to its thermal effects and electrical breakdown, electro-osmosis may also be the reason for moisture flowing inside of cell tissue. Bazhal and Vorobiev (2000) have demonstrated that electro-osmotic flow was induced in apple tissue by a dc electric field of low voltage, which resulted in a significant increase in juice yield, even at ambient temperature. Solute extraction can also be enhanced by OH pretreatment. Kemp and Fryer (2007), Kulshrestha and Sastry (2003), and Schreier et al. (1993), showed that there is an increased diffusion of betanine dye from red beets treated by OH. In their interesting study, Kulshrestha and Sastry (2003) treated red beet cuts at 45 °C over a range of voltages from 0 to 23.9 V cm–1, and at frequencies of 0 (dc), 10, 50, 250 and 5000 Hz. As the electric field frequency decreased, the betanine content in the solution increased. This was explained by the increased time for which the cell membrane was charged at lower field frequencies. An exception to this tendency was detected for those tissues treated by dc, which was less effective than OH treatment at 10 Hz. All the mechanisms involved in tissue modification by


86 Separation, extraction and concentration processes OH treatment have not yet been elucidated. Electrolysis phenomena might also be important in terms of process optimisation. Other fresh materials, for example, mint leaf (Sensoy and Sastry, 2004), potato (Praporscic et al., 2006), and sugar beets (Praporscic et al., 2005) can also be processed by OH-assisted extraction.

3.4 Extraction assisted by high-voltage electrical discharges and applications in the food industry 3.4.1 Principles of underwater high-voltage electrical discharges (HVED) Underwater (electrohydraulic) HVED is a technique in which solid particles are placed in a dielectric liquid (typically tap water) inside a chamber, containing a high-voltage (HV) needle electrode and a plated grounded electrode, and exposed to pulsed shockwaves (typically 40–60 kV cm–1, 2–5 ms) to produce a liquid breakdown and particle fragmentation. This technique has various applications (defragmentation of solid materials, such as concrete, treatment of municipal solid waste, and drilling) (Bluhm, 2006). Some publications propose the use of the HVED application for the deactivation of micro-organisms and for the enhancement of extractions (Grémy-Gros et al., 2008). Figure 3.6 shows a schematic diagram of a HVED generator, as well as typical voltage and current curves measured during a HVED (Boussetta et al., 2009a). Effects of HVED The mechanisms of HVED are very complex and not yet well understood. The phenomenon is based on the electrical breakdown of water (Bluhm, 2006). Air bubbles that are already present in the water, or formed as a result of local heating, participate in and accelerate this phenomenon. If the electrical field is intense enough, the avalanche of electrons becomes a starting point for streamer propagation from the HV needle electrode to the grounded one (Fig. 3.7). A crack can be attracted from the discharge channel into the solid inclusion if their dielectric properties are different. The electrical breakdown is accompanied by a number of secondary phenomena (high-amplitude pressure shock waves, bubbles cavitation, and creation of liquid turbulence). For instance, the pressure exerted by the expanding channel almost always exceeds the tensile strength of the solid inclusions, and leads to the formation of cracks in the solids. Several hundred to thousands of discharges are sufficient to fragment the stones into small pieces of less than 1 mm (Bluhm, 2006). For biological particles, the cell structure can be damaged by HVED, which accelerates solute extraction (Grémy-Gros et al., 2008). If electrical breakdown does not occur, water carries electricity and behaves


Principles of physically assisted extractions and applications 87 HVED generator

Vent hole

Insulation

Air Water + sample

Needle electrode Plate electrode

Metal

HVED treatment cell (a) 15

40

10

20 5

0

0

Current (kA)

Voltage (kV)

tf ª 0.5 ms

–5 0

2

4 6 Time (ms) (b)

8

10

Fig. 3.6 HVED treatment (a) experimental setup and (b) typical voltage and current graphs.

like a good electrical conductor; then electrical signals have exponential form. When HVED are produced, electrical signals look like damped oscillations, as presented in Fig. 3.6 (Boussetta et al., 2009a; Grémy-Gros et al., 2008). 3.4.2 Solute extraction enhanced by HVED HVED have been used to accelerate the extraction of solutes from soybeans, potato, tea leaves, peat, and fennel (Barskaya et al., 2000; El-Belghiti, 2005; El-Belghiti et al., 2007) as well as from other food materials.



(a)

(b)

(c)

HV electrode

Shock wave

Charging unit

Ground Solid (d)

Fig. 3.7 Type of electric discharge in aqueous solution: (a) streamer, E = 4.4 kV cm–1; (b) streamer and spark, E = 13.3 kV cm–1; (c) spark, E = 33.3 kV cm–1 (Sugiarto et al., 2001). (d) Schematic diagram of electro-hydraulic disintegration process.

Oil The application of HVED can enhance the aqueous extraction of oil from oilseeds (Grémy-Gros et al., 2008; Gros et al., 2003). Linseeds were crushed and pressed with a hydraulic press, and then the press-cake obtained was reduced to powder and dissolved in demineralised water at ambient temperature. The mixture was treated with HVED (1–1640 discharges) and centrifuged (Gros et al., 2003). Approximately 26% of oil remained in the residue after 1640 pulses. The process was then optimised by varying the number of pulses, pH, water/press-cake ratio and temperature (Grémy-Gros et al., 2008). The HVED treatment also made it possible to enhance mucilage extraction from whole linseed (Gros et al., 2003). Seeds were dissolved in demineralised water and treated for 10 min (i.e. 300 pulses of 0.5 Hz frequency). A centrifuge separation was then performed to obtain solution and residue. The residue was then treated a second and a third time with fresh water under the same conditions. During the HVED treatment, linseeds were defragmented under the action of shock waves. Three successive 10-min treatments were sufficient to extract the mucilage almost entirely. The extraction of proteins began during the third treatment. However, the oil–protein–water emulsions obtained by HVED treatment then had to be separated. The following research study


Principles of physically assisted extractions and applications 89 resulted in the development of membrane separation of emulsions created by HVED (Li et al., 2009). Grape pomace Grape pomace (composed of stems, seeds and skins) has a high polyphenol content. These polyphenols have attracted great interest as they exhibit antibacterial, antiviral, and antioxidant properties, and can help prevent cardiovascular diseases (Dugand, 1980). Boussetta et al. (2009b) studied the application of HVED in the extraction of polyphenols from the pomace obtained as a residue from pressed white grapes (Vitis vinifera L., cultivar ‘Chardonnay’, vintage 2007). The treatment chamber (Fig. 3.6) was initially filled with pomace mixed with distilled water (the liquid-to-pomace ratio was 3:1, the water temperature was 20, 40 or 60 °C), and 80 successive discharges were applied (40 kV, pulse repetition rate 0.5 Hz). Afterwards, the total solute and polyphenol diffusion from the untreated and treated pomace mixtures were studied at the desired temperatures. Figure 3.8 shows that both temperature and HVED treatment improved the extraction of polyphenols. The difference between the yield of solutes for experiments with HVED treatment and without HVED treatment decreased with temperature elevation (Boussetta et al., 2009b). On the contrary, the opposite tendency was observed for the yield of polyphenols. The application of HVED enhanced the polyphenol yield, not just for fresh grape pomace, but also for sulfured

1

20

T (°C) 40

60 With HVED treatment

0.8

Ypolyphenols (%)

0.6

0.4

Without HVED treatment 0.2

0.0034

0.0032 1/T (K)

0.003

Fig. 3.8 Effect of treatment temperature on final yield of polyphenols after 1 h of extraction of fresh grape pomace with and without HVED treatment.


90 Separation, extraction and concentration processes and frozen pomaces. The final yields of solutes, reached after HVED and subsequent diffusion for 40 min were more than twice those reached after 240 min without HVED. Yeast cells Shynkaryk et al. (2008) studied the efficiency of HVED-assisted extraction from wine yeast cells (S. cerevisiae) in aqueous suspension (1% w/w). HVED induced damage in yeast membranes, leakage in cytoplasmic ions, and the release of intracellular bioproducts; it also resulted in an increase in the disintegration index Z. For HVED treatment at 40 kV, the high level of membrane disintegration (Z > 0.8) required more than 100 discharges, which corresponded to the effective time of HVED treatment between 100 and 200 ms. HVED treatment decreased the average cell size somewhat, owing to cell disruption. However, the quantity of high molecular weight intracellular components released, controlled by the absorbance analysis, was lower than that obtained by high-pressure homogenisation (Shynkaryk et al., 2008).

3.5 Ultrasound-assisted extraction (UAE) in the food industry 3.5.1 Principles of UAE Power ultrasound, which has frequencies between 20 kHz and 1 MHz, is now well known to have significant effects on the rate of various physical and chemical processes. Cleaning and solubilisation are its more developed applications, and a large variety of ultrasound baths exist for chemical laboratory use. The effect of ultrasonic waves on solid samples is widely used for the extraction of aromas from plant materials, or metal impurities from soils. Degassing and stripping are widely used for flavour analysis, and in environmental and polymer research. Other interesting ultrasound applications include homogenisation, emulsification, sieving, filtration, and crystallisation. The most interesting effect of ultrasound-based operational units is the reduction of processing time and the increase in product quality. All these effects are attributed to acoustic cavitation: when a liquid is irradiated by ultrasound, micro-bubbles form, grow and oscillate extremely fast, and eventually collapse powerfully (if the acoustic pressure is high enough). When the size of these bubbles reaches a critical point, they collapse during a compression cycle and release large amounts of energy. The temperature and pressure at the moment of collapse have been estimated to be up to 5000 K and 5000 atmospheres. This creates hotspots that are able to dramatically accelerate chemical reactivity in the medium. When these bubbles collapse onto the surface of a solid material, the high pressure and


Principles of physically assisted extractions and applications 91 temperature released generate microjets directed towards the solid surface. These microjets are responsible for the degreasing effect of ultrasounds on metallic surfaces, which is widely used for cleaning materials. Another application of microjets in the food industry is the extraction of vegetal compounds (Mason, 1990; Suslick, 1988). As shown in Fig. 3.9, a cavitation bubble can be generated close to the surface of the plant material. Then, during a compression cycle, this bubble collapses and creates a microjet (400 km h–1) directed toward the plant matrix. The high pressure and temperature involved in this process will destroy the cell walls of the plant matrix, and its content can be released into the medium. Power ultrasound involves the mechanical and chemical effects of cavitation. The mechanism can be explained by two competing theories. The hot spot theory assumes that the high pressures and temperatures generated in the bubbles during the last nearly adiabatic compression, just before collapse, are responsible for the breakage of molecular bonds and the formation of radicals. On the other hand, the electrical theory describes micro-discharges, produced by the high electrical fields generated by the deformation and fragmentation of the bubbles. The most commonly used frequencies in sono-extraction are between 20 and 40 kHz. With higher frequencies, cavitation would be more difficult to induce because the cavitation bubbles need a slight delay to be initiated during the rarefaction cycle. The higher the frequency, the shorter the rarefaction cycle, so the less time available for the bubble to be created (Povey and Mason, 1998).

Cavitation bubble with negative pressure

Maximum and critical bubble size 2

1

A new cavitation bubble appears and cycle is repeated

3

4

Implosion of bubble by compression

Fig. 3.9 Ultrasonic cavitation phenomenon and its action on extraction of natural products.


92 Separation, extraction and concentration processes 3.5.2 UAE reactors The two most common types of ultrasound equipment that are used for extraction are the ultrasonic cleaning bath and the more powerful probe system. For small extraction volumes, an ultrasound horn with its tip submerged in the fluid can be sufficient. Large volumes of fluids have to be sonicated in an ultrasound bath or in continuous or recycled-flow sono-reactors. There are also ultrasonic reactors conceived especially for solvent extraction on a laboratory scale (3 l), in a pilot plant (30 l) and on an industrial scale (150–1000 l). Pump systems are coupled to the ultrasonic reactor to stir the mixture and to empty the system at the end of the experiment. The intensity of the ultrasound is about 1 W cm–2, with a frequency of 25 kHz. In order to maintain constant temperature, the reactor is made of a double mantle into which cooling water can circulate. The main advantage of this type of apparatus is that the natural products and extraction solvent are mixed into a container, and the ultrasound is directly applied to the mixture (Fig. 3.10). Recently, the new methodology of continuous-flow systems has been used in analytical chemistry. Most UAE applications have been developed in discrete systems, using a bath or an ultrasonic probe, particularly in the extraction of food samples. Less frequent has been the design of on-line UAE systems in the same field. However, it is noted that the latter approach is considerably faster. It consists of an open system, in which fresh solvent flows continuously through the sample. This induces the displacement of mass transfer equilibria toward the solubilisation of analytes into the liquid phase (Priego-Capote and Luque de Castro, 2007). 3.5.3 Factors affecting UAE All the research carried out over the last 50 years strongly supports the importance of each identified factor in the ultrasound process, where frequency, intensity, treatment time, temperature, pressure, and treatment

(a)

(b)

Fig. 3.10 (a) Laboratory (3 l) and (b) industrial (150 l) ultrasound extraction reactors (with permission, www.etsreus.com).


Principles of physically assisted extractions and applications 93 media define the extraction and the separation kinetics of ingredients from natural products (De Gennaro et al., 1999). Generally, the highest efficiency of UAE, in terms of the yield and composition of the extracts, can be achieved by increasing the ultrasound power, reducing the moisture of food matrices to enhance solvent–solid contact, and optimising the temperature to allow a shorter extraction time. Solvent choice is dictated by the solubility of the ingredients of interest, the interactions between the solvent and matrix, and the intensity of ultrasound cavitation phenomena in the solvent. The impact of ultrasound is attributed to intracellular cavitation, that is, micro-mechanical shocks that disrupt cellular structural and functional components up to the point of cell lysis (Butz and Tauscher, 2002). Figure 3.11 shows a proposed mechanism for ultrasound-induced cell damage. Cavitation is the formation, growth and, sometimes, the implosion of microbubbles created in a liquid when ultrasound waves propagate through it. The collapse of the bubbles leads to energy accumulation in hot spots, where temperatures of above 5000 °C and pressures of approximately 500 MPa have been measured. This phenomenon can cause enzyme inactivation through three mechanisms, which can act alone or combined. The first one is purely thermal, owing to the enormous temperatures achieved during cavitation. The second is caused by free radicals, generated by water sonolysis; and the third is the result of the mechanical forces (shear forces) created by microstreaming and shock waves (Raso et al., 1999). 3.5.4 UAE: main applications Essential oils and aromas UAE has also been developed for the extraction of essential oils from aromatic plants, such as peppermint leaves (Shotipruk et al., 2001), artemisia (Asfaw et al., 2005) and lavender (Da Porto et al., 2009), or from other vegetal matrices, such as garlic (Kimbaris et al., 2006) and citrus flowers (Alissandrakis et al., 2003). Increased yields of essential oil were found for peppermint leaves (up to 12%) and for artemisia when using UAE; and yields of the main compounds of lavandula essential oil were found to increase by Ultrasound (US)

US

H 2O

Cell

US

Cell

Pore initiation

US

H2O influx and swelling

Cell

Membrane rupture and cell lysis

Fig. 3.11 Mechanism of ultrasound-induced cell damage.


94 Separation, extraction and concentration processes 2- to 3-fold when comparing UAE with conventional distillation. Moreover, UAE not only improved yields, but also showed less thermal degradation in the final extracts. Several studies have been conducted into the extraction of the main aroma compounds from spices. For example, vanillin was extracted from vanilla pods (Jadhav et al., 2009) carvone from caraway seeds (Chemat et al., 2004) and safranal from Greek saffron (Kanakis et al., 2004). Yields of vanillin obtained after 1 h using UAE were similar to those obtained after 8 h using conventional extraction methods. Wines and other alcoholic beverages contain many volatile compounds, which play an important role in organoleptic characteristics. The ultrasound-assisted maceration of fruits and herbs in alcoholic beverages to extract volatile compounds is an important process in the food industry (Fig. 3.12). Antioxidants A wide variety of fruits and vegetables have been studied by UAE because antioxidants are present in different amounts in different varieties of plants, and these antioxidants come from different families. One of the most common antioxidants is the lycopene extracted from tomatoes (Lianfu and Zelong, 2008). In this particular work, authors not only worked on UAE, but also on coupling ultrasounds with microwaves, which produces high lycopene extraction in only 6 min. Herrera and Luque de Castro (2005) showed that the amount of phenolic compounds that could be extracted from strawberries in 2 min, was similar to the amount extracted over 20 h using the conventional method, and 3 h using supercritical fluid extraction. Another example is the extraction of anthocyanins from raspberries, developed by Chen et al. (2007). In this work, the same yields of anthocyanins were achieved in 3 min when using UAE, compared with 53 min when using the conventional extraction system. The difference between ultrasound and conventional methods will be more or less significant, depending on the part of the plant studied. This has been highlighted by studies of grapes and grape seeds (Palma and Barroso, Sono-extraction

0.100

Conventional extraction

Yield (%)

0.075 0.050

0.025 0.000

0

30

60

90

120 150 180 210 240 270 Time (min)

Fig. 3.12 Rapid sono-extraction of orange peels in alcoholic beverages.


Principles of physically assisted extractions and applications 95 2002). In this work, authors found that the use of ultrasound instead of maceration was more interesting when working on seeds than on the whole fruit (Table 3.1). Oil and fat Many papers have reported on the UAE of oil and fat from various food samples. According to Luque-Garcia and Luque de Castro (2004), extraction from oleaginous seeds is difficult. Indeed, only 75–85% of the oil is solubilised in the solvent. The rest of the oil content is strongly bound to the matrix and cannot be extracted without additional treatments. For instance, Li et al. (2004) described the UAE of soybean oil in an optimised yield and reduced operating time, compared with conventional maceration. Luque-Garcia and Luque de Castro (2004) have reported a device consisting of an ultrasoundassisted Soxhlet apparatus, which is an attractive alternative to traditional Soxhlet extraction. Using this device, it takes only 90 minutes to obtain the same yield (99% of fat recovery) as can be obtained over 12 h with the traditional Soxhlet extraction method (Table 3.1). 3.5.5 Hazard analysis critical control point (HACCP) for UAE processing operation The hazard analysis critical control point (HACCP) system is a process that identifies and assesses the hazards and risks associated with the manufacture, Table 3.1 Ultrasound-assisted extraction of food ingredients Matrix (reference)

Extracts

Essential oils and aromas Citrus flowers (Alissandrakis et al., 2003) Caraway seeds (Chemat et al., 2004) Garlic (Kimbaris et al., 2006) Lavender flowers (Da Porto et al., 2009) Greek saffron (Kanakis et al., 2004) Antioxidant extracts Strawberries (Herrera and Luque de Castro, 2005) Raspberries (Chen et al., 2007) Tomatoes (Liangfu and Zelong, 2008) Grape (Palma and Barroso, 2002) Fat and oil extraction Olive seeds (Luque-Garcia and Luque de Castro, 2004) Soybean germ and seaweed (Li et al., 2004) Cocoa powder and nibs (Luque- Garcia and Luque de Castro, 2004) Bakery products (Luque-Garcia and Luque de Castro, 2004)

Linalool Carvone and limonene Essential oil (organosulfur compounds) Essential oil (1,8-cineol, camphor and linalyl acetate) Safranal Polyphenolic compounds Anthocyanins Lycopene Phenolic Oil Oil Fat content Fat content


96 Separation, extraction and concentration processes storage and distribution of foods and implement the appropriate controls, aiming at the elimination or reduction of these hazards at specific points of the production line. In UAE processing, parameters such as the temperature and physical properties of the product, the timescale of treatment in the ultrasound chamber, the frequency and power of the ultrasound, and the nature of the probe are points which need to be monitored. For each part of the food process, including ultrasound processing, the evaluation and classification of hazards are completed. In this way, a grade is given to each hazard that has been identified according to its gravity, its risk of occurrence and the ability to detect it. For each hazard, preventive measures are set up, with procedures that indicate who will be responsible for them, as well as how, when and where. Preventive measures are aimed primarily at avoiding the occurrence of the hazard. For example, a good maintenance plan decreases the risk of metal contamination from the ultrasound horn (Table 3.2).

3.6 Microwave-assisted extraction (MAE) in the food industry 3.6.1 Principles of MAE Microwaves are electromagnetic waves with a frequency range of between 100 MHz and 3 GHz that comprise electric and magnetic field components and thus constitute propagating electromagnetic energy. This energy acts as a non-ionising radiation, which causes molecular motion of ions and rotation of the dipoles, but does not affect molecular structure. Table 3.2 Possible critical control point limits and associated corrective actions in an ultrasound-assisted extraction processing Critical control point

Danger

Target

Deviation

Processing Temperature Adequate Does not conform operation to the requirements Ultrasound Adequate Does not conform frequency to the requirements Ultrasound Adequate Does not conform power to the requirements Flow rate Below Does not conform specifications to the requirements Probe Absence Detected spoilage

Corrective actions

Adjust temperature reject or reprocess the product Adjust frequency, laboratory controls, or reprocess the product Adjust the power, laboratory controls, or reprocess the product Adjust flow, laboratory controls, or reprocess the product Reject


Principles of physically assisted extractions and applications 97 When dielectric materials containing either permanent or induced dipoles are placed in a MW field, the rotation of the dipoles in the alternating field produces heat. More precisely, the applied MW field causes the molecules to spend slightly more time, on average, orienting themselves in the direction of the electric field rather than in other directions. When the electric field is removed, thermal agitation returns the molecules to a disordered state in the relaxation time and thermal energy is released. Thus, MW heating results from the dissipation of the electromagnetic waves in the irradiated medium (Fig. 3.13). The amount of dissipated power in the medium depends on the complex permittivity of the material and the local time-averaged electric field strength (Metaxas and Meredith, 1993). In conventional heating, heat is transferred from the heating medium to the interior of the sample, whereas in MW heating; heat is dissipated volumetrically inside the irradiated medium, and thus heat transfers occur from the sample to the colder environment. This causes a significant difference Moisture (%)

Classical heating

Microwave heating

Microwave heating Heat transfer

1 2

1. Solid friction desorption 2. Internal diffusion 3. External diffusion

Mass transfer

3

Conventional heating Heat transfer Mass transfer

Fig. 3.13 Microwave versus conventional extraction.


98 Separation, extraction and concentration processes between conventional and MW heating. In conventional heating, heat transfer depends on thermal conductivity, on the temperature difference across the sample, and, for fluids, on convection currents. As a result, the temperature increase is often rather slow. In contrast, the volumetric heating effect in MW heating enables much faster temperature increases to be obtained, depending on the MW power and the dielectric loss factor of the material being irradiated. The influence of MW energy on chemical or biochemical reactions is strictly thermal. The MW energy quantum is given by the standard equation W = hn. Within the frequency domain of microwaves and hyper-frequencies (300 MHz–300 GHz), the corresponding energies are respectively 1.24 ¥ 10–6 eV to 1.24 ¥ 10–3 eV. These energies are much lower than the usual ionisation energies of biological compounds (13.6 eV), of covalent bond energies like OH (5 eV), hydrogen bonds (2 eV), and van der Waals intermolecular interactions (lower than 2 eV) and even lower than the energy associated with Brownian motion at 37 °C (2.7 ¥ 10–3 eV). From a scientific point of view, the direct molecular activation of microwaves should be excluded. 3.6.2 MAE reactors It is now possible to use a wide range of vessels and instrumentation when working with microwaves, depending on the intended purpose and the analytes to be extracted. Two types of MW ovens can be found in laboratories and they differ in terms of their cavity type: single-mode (also called monomode or focused mode) and multi-mode. A single-mode cavity permits the generation of one type of frequency, allowing only one mode of resonance to be excited. Thus, the sample is placed in such a way as to receive the maximum amount of MW energy, ensuring a direct and efficient application of a high density of MW energy to the sample matrix, and inducing a rapid heating of the medium with a fast extraction of the analyte. However, monomode systems are quite limited in terms of the volume and quantity of the sample that is ultimately useable. On the other hand, multi-mode ovens are larger and allow several modes of resonance. Thus, a large number of incident waves can be applied to the sample being studied. This kind of oven allows for a large quantity of matrix, and can be used with several types and forms of vessels. There are also MW reactors conceived especially for solvent extraction in the laboratory (0.1 to 1 l) and on an industrial scale (100 l h–1) (Fig. 3.14). 3.6.3 MAE: main applications Microwaves are very useful for rapid extraction and obtaining high quality aromatic compounds from garlic, lavender flowers, orange peel and rosemary leaves. The extraction of essential oils from orange peels with solvent-free MW extraction is better in terms of energy saving, extraction time (30 min



(a)

(b) –1

Fig. 3.14 (a) Laboratory (1 l) and (b) industrial (100 l h ) MAE reactors (with permission, www.milestonesrl.com and www.rchimex.com).

versus 3 h), product yield (0.42 versus 0.39%) and product quality (AbertVian et al., 2008). Hemwimon et al. (2007) made a comparison between the MAE of antioxidative anthraquinones from the roots of Morinda citrifolia, and extraction with other conventional techniques (maceration and Soxhlet). The efficiency of extraction using MAE (15 min) was much higher than that using maceration (3 days) and was also similar to that of Soxhlet (4 h). Similarly, the best recoveries of antioxidants in short time periods, and with a lower consumption of polar solvent were also observed from sweet grass by Grigonis et al. (2005). As well as obtaining a higher yield, Pan et al. (2008) extracted phenolic compounds with scavenging ability, compared with synthetic antioxidants from longan peel with MAE. Elkhori et al. (2007) described the MAE of cocoa powder with hexane/ isopropanol, which resulted in the rapid determination of fat contents, with recoveries similar to, or better than, those of the conventional method, and with the added advantages of low solvent consumption, short extraction time, low energy consumption and excellent reproducibility. Focused MW-assisted Soxhlet extraction was used by Pérez-Serradilla et al. (2007) for acorn oil determination, which resulted in trans fatty acid-free oil, probably because of the reduced exposure to drastic conditions in just 30 min, which is much less than the time required by the Soxhlet (8 h) and stirring (56 h) reference methods. Lianfu and Zelong (2008) described the extraction of lycopene from tomatoes with the combined innovative techniques of ultrasound and MW-assisted extraction (UMAE) in comparison with ultrasound-assisted


100 Separation, extraction and concentration processes extraction (UAE). Similarly, a higher yield of anthocyanins in red raspberries was also observed by Sun et al. (2007) with optimal conditions of MAE. MAE proved to be more rapid and efficient as it extracts various types of anthocyanins, without any destruction of their chemical structure, in a very short time, owing to the intensive disruption of tissue structure under MW irradiation. Table 3.3 clearly shows some applications of MAE for various useful metabolites, carried out using several vegetal matrices. 3.6.4 Safety considerations The MAE process is simple and can be readily understood in terms of the operating steps to be performed. However, in inexperienced hands, the application of MW energy can pose serious hazards. All persons dealing with microwaves must exercise a high level of safety and attention to detail when planning and performing such experiments. They have to ensure that they seek proper information from knowledgeable sources and that they do not attempt to implement this technique unless proper guidance is provided. Only approved equipment and scientifically sound procedures should be used.

3.7 Combination of physical treatments for extraction in the food industry Various physical treatments can induce various effects on cell and tissue structure, which may be more or less beneficial for the extraction of intracellular components. Unfortunately, the studies comparing or combining two or more Table 3.3 Microwave-assisted extraction of food ingredients Matrix (reference)

Extracts

Antioxidant extracts Green tea leaves (Pan et al., 2003) Noni plant roots (Hemwimon et al., 2007) Sea buckthorn (Sharma et al., 2008) Longan peel (Pan et al., 2008)

Polyphenols Anthraquinones Phenolic constituents Flavonoids

Fat and oil extraction Olive seeds (Virot et al., 2007) Oleaginous seeds (Pérez-Serradilla et al., 2007) Soybean germ and seaweed (Cravotto et al., 2008) Cocoa powder and nibs (Elkhori et al., 2007)

Oil Oil Oil Fat content

Natural food colour extraction Tomato paste (Lianfu and Zelong, 2008) Curcuma (Mandal et al., 2008) Red raspberries (Sun et al., 2007) Paprika (Csiktusnádi Kiss et al., 2000)

Lycopene Curcumin Anthocyanins Carotenoids


Principles of physically assisted extractions and applications 101 physically assisted extractions are rather rare. El-Belghiti (2005) compared the application of PEF (680 V cm–1, 1000 pulses of 100 ms) and HVED (40 kV, 100 discharges) for the cold (20 °C) aqueous extraction of solutes from two dried products: tea leaves and Datura innoxia (common name: moonflower) roots. PEF treatment did not have any influence on the process of extracting from dried and rehydrated products, probably because of the absence of the cell membrane element, which had been disrupted by previous drying. The non-efficacy of moderate electric treatment for the dried cell tissue was reported earlier in Sensoy and Sastry (2004). In contrast to PEF, the HVED smashed products to small fragments and the extraction kinetics were significantly enhanced for both tea leaves and Datura innoxia. El-Belghiti et al. (2007) compared PEF, HVED and PU as treatments to enhance cold (20 °C) aqueous extraction from fennel gratings. The objective was to obtain extracts used as natural food preservatives (antioxidants). The three assisted extractions led to the same final yield of solutes, and all preserved the antioxidant substances. However, their kinetics were different. The most rapid and the slowest kinetics were offered by HVED and PU, respectively. The final yield of 98% was reached in 20 min with HVED, in 40 min with PEF, and in more than 180 min with PU. The amounts of energy needed for these treatments were also different: the PEF treatment appeared to be the most economic energetically. Recently, Shynkaryk et al. (2008) compared PEF, HVED, and high-pressure homogenisation (HPH) for disruption of wine yeast cells (S. cerevisiae bayanus). The PEF and HVED were applied at electric field strengths of 10 kV cm–1 and 40 kV cm–1, respectively. The HPH was applied within a pressure range of 30–200 MPa for a different number of passes (1–20). It was shown that the releasing efficiency of ionic components, enzymes, proteins and other bioproducts dramatically depended on the applied method of disruption. Extraction efficiency was rather high for ionic components and small for high molecular weight components in PEF or HVED pretreated suspensions. The PEF treatment removes membrane barriers and accelerates the release of ionic contents, but has practically no influence on the cell walls. The efficiency of releasing high molecular weight content is questionable and may depend on the cell strain, the age of culture, the time of extraction, temperature and many other factors. For example, high levels of PEF extraction of proteins for baker’s yeast was previously reported (Ganeva et al., 2003), but the efficiency of the PEF extraction of proteins was found to be rather low for wine yeast in the study by Shynkaryk et al. (2008). On the contrary, the application of the combined HVED–HPH technique resulted in a material loss of integrity in the yeast cell walls. Even a small disintegration (Zi = 0.15±0.05), initially induced by HVED pretreatment, resulted in noticeable acceleration of HPH disruption kinetics. Although most of the research effort in UAE has concentrated on ultrasound itself, some studies have also examined the coupling between ultrasound and other techniques. For instance, UAE is currently being employed in


102 Separation, extraction and concentration processes combination with MW energy (Lagha et al., 1999), supercritical fluid extraction (Hu, 2007) or simply with conventional methods such as Soxhlet extraction (Luque-García and Luque de Castro, 2004). When combined with supercritical fluid extraction, UAE enhances the mass transfer of the species of interest, from the solid phase to the solvent used for extraction. Soxhlet extraction can also be improved by ultrasound when applied at the cartridge zone before siphoning, thus permitting the removal of lipid fractions from very compact matrices. The efficiency of combining MW and ultrasound has been clearly shown in applications such as the extraction of food ingredients. Studies combining assisted extractions present undeniable interest as synergistic effects of different treatments may be discovered.

3.8 References Abert-Vian M, Fernandez X, Chemat F (2008), ‘Microwave hydrodiffusion and gravity, a new technique for extraction of essential oils’, Journal of Chromatography A, 1190, 14–18. Alissandrakis E, Daferera D, Tarantilis P A, Polissiou M and Harizanis P C (2003), ‘Ultrasound-assisted extraction of volatile compounds from citrus flowers and citrus honey’, Food Chemistry, 82, 575–579. Angersbach A, Heinz V and Knorr D (2002), ‘Evaluation of process-induced dimensional changes in the membrane structure of biological cells using impedance measurement’, Biotechnology Progress, 18(3), 597–603. Asfaw N, Licence P, Novitskii AA, Poliakoff M (2005), ‘Green chemistry in Ethiopia: the cleaner extraction of essential oils from Artemisia afra: a comparison of clean technology with conventional methodology’, Green Chemistry, 7, 352–356. Barbosa-Cánovas G V, Pothakamury U R, Palou E and Swanson B G (1998), Nonthermal preservation of foods, Marcel Dekker, New York. Barskaya A V, Kuretz B I and Lobanova G L (2000), ‘Extraction of water soluble matters from vegetative raw material by electrical pulsed discharges’, 1st International Congress on Radiation Physics, High Current Electronics, and Modification of Materials, Tomsk, Russia, 533–535. Bazhal M and Vorobiev E (2000), ‘Electrical treatment of apple cossettes for intensifying juice pressing’, Journal of the Science of Food and Agriculture, 80, 1668–1674. Bazhal M I, Lebovka NI and Vorobiev E I (2001), ‘Pulsed electric field treatment of apple tissue during compression for juice extraction’, Journal of Food Engineering 50, 129–139. Biss C H, Combes S A and Skudder P J (1989), ‘The development and application of ohmic heating for the continuous processing of particulate food stuffs’, In Processing engineering in the food industry (Field RW, Howell JA, eds), Elsevier, London, 17–27. Bluhm H (2006), Pulsed power systems, Springer, Berlin, Heidelberg, New York. Bluhm H and Sack M (2008), ‘Industrial-scale treatment of biological tissues with pulsed electric field’, In: E. Vorobiev and N. Lebovka (Eds), Electrotechnologies for extraction from food plants and biomaterials, Springer, 237–269. Boussetta N, Lebovka N, Vorobiev E, Adenier H, Bedel-Cloutour C, and Lanoisellé J-L (2009a), ‘Electrically assisted extraction of soluble matter from Chardonnay grape skins for polyphenols recovery’, Journal of Agricultural and Food Chemistry, 57, 1491–1497.


Principles of physically assisted extractions and applications 103 Boussetta N, Lanoisellé J-L, Bedel-Cloutour C and Vorobiev E (2009b), ‘Extraction of polyphenols from grape pomace by high voltage electrical discharges: effect of sulphur dioxide, freezing process and temperature’ Journal of Food Engineering, 95, 192–198. Bouzrara H (2001), Amélioration du pressage de produits végétaux par Champ Electrique Pulsé. Cas de la betterave à sucre, PhD thesis, UTC, Compiègne, France. Bouzrara H and Vorobiev E (2000), ‘Beet juice extraction by pressing and pulsed electric fields’, International Sugar Journal, CII(1216), 194–200. Bouzrara H and Vorobiev E (2003), ‘Solid/liquid expression of cellular materials enhanced by pulsed electric field’, Chemical Engineering and Processing, 42, 249–257. Butz P and Tauscher B (2002), Emerging technologies: chemical aspects, Food Research International, 35, 279–284. Chemat S, Lagha A, AitAmar H, Bartels P, Chemat F (2004), ‘Comparison of conventional and ultrasound-assisted extraction of carvone and limonene from caraway seeds’, Flavour and fragrance journal, 19, 188–193. Chen F, Sun Y, Zhao G, Liao X, Hu X, Wu J, Wang Z (2007), ‘Optimization of ultrasoundassisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography-mass spectrometry’, Ultrasonics Sonochemistry, 14, 767–772. Cravotto G, Boffa L, Mantegna S, Perego P, Avogadro M, Cintas P (2008), ‘Improved extraction of vegetable oils under high-intensity ultrasound and/or microwaves’, Ultrasonics Sonochemistry, 15, 898–902. Csiktusnádi Kiss G, Forgács E, Cserháti T, Mota T, Morais H, Ramos A (2000), ‘Optimisation of microwave-assisted extraction of pigments from paprika powders’, Journal of Chromatography A, 889, 41–49. Da Porto C, Decorti D and Kikic I (2009), ‘Flavour compounds of Lavandula angustifolia L., to use in food manufacturing: comparison of three different extraction methods’, Food Chemistry, 112, 1072–1076. De Gennaro L, Cavella S, Romano R and Masi P (1999), ‘The use of ultrasound in food technology I: inactivation of peroxidase by thermosonication’, Journal of Food Engineering 39, 401–407. Dugand L R (1980), ‘Natural antioxidants’, In M.G. Simic, M. Karel (Eds), Autooxidation in food and biological systems, Plenum Press, New York, 261–295. El-Belghiti K and Vorobiev E (2004), ‘Mass transfer of sugar from beets enhanced by pulsed electric field’, Transactions of the Institution of Chemical Engineers, 82, 226–230. El-Belghiti K (2005), Effets d’un champ électrique pulsé sur le transfert de matière et sur les caractéristiques végétales, PhD thesis, UTC, Compiègne, France. El-Belghiti K, Moubarik R and Vorobiev E (2007), Use of moderate pulsed electric field, electrical discharges and ultrasonic irradiations to improve aqueous extraction of solutes from fennel (Foeniculum vulgare), unpublished data. El-Belghiti K and Vorobiev E (2005a), ‘Kinetic model of sugar diffusion from sugar beet tissue treated by pulsed electric field’, Journal of the Science of Food and Agriculture, 85, 213–218. El-Belghiti K and Vorobiev E (2005b), ‘Modelling of solute aqueous extraction from carrots subjected to a pulsed electric field pre-treatment’, Biosystems Engineering, 90(3), 289–294. Elkhori S, Jocelyn Paré J, Bélanger J, Pérez E (2007), ‘The microwave-assisted process: extraction and determination of fat from cocoa powder and cocoa nibs’, Journal of Food Engineering, 79, 1110–1114. El Zakhem H, Lanoisellé J-L, Lebovka N I, Nonus M and Vorobiev E (2006a), ‘Behavior of yeast cells in aqueous suspension affected by pulsed electric field’, Journal of Colloid and Interface Science, 300(2), 553–563. El Zakhem H, Lanoisellé J-L, Lebovka N I, Nonus M and Vorobiev E (2006b), ‘The early


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Advances in process chromatography and applications 109

4 Advances in process chromatography and applications in the food, beverage and nutraceutical industries M. Ottens and S. Chilamkurthi, Delft University of Technology, The Netherlands Abstract: The basics of process chromatography and its application to separation and purification in the food, beverage and nutraceutical industry are discussed. Basic principles and modes of chromatography are described together with guidelines for appropriate modeling; all illustrated with examples. New developments are described in the area of novel ligands, new materials, and modes of operation and control. Finally, future trends with respect to the production of high-value nutraceuticals, regulation and process control are given. Key words: process chromatography, nutraceuticals, proteins, model, simulated moving bed.

4.1 Introduction 4.1.1 Chromatography as a unit operation in food, beverage and nutraceutical processing The global food, beverage, and agriculture industry caters to the population of the entire world. It is a complex, global collective of diverse businesses that together supply much of the food energy consumed by the world population. Global market forces are driving the continuous evolution of the food and beverage industry. Consolidation, changing consumer preferences and increasing government regulations are significantly impacting manufacturing and business strategy. Producing quality goods at the lowest possible cost is a major aim. Consumers are increasingly interested in the health benefits of foods and have begun to look beyond the basic nutritional benefits of food to the


110 Separation, extraction and concentration processes disease-prevention and health-enhancing compounds contained in many foods. This, combined with a more widespread understanding of how diet affects disease, health-care costs and aging populations, has created a market for functional foods and natural health products. There is a growing interest in functional foods, which are dietary supplements that increase a person’s well being. A functional food contains one or more ingredients that are bioactive, that is: they promote desired biological activity in the human body. Process-scale chromatography is an indispensable unit operation in biotechnology and finds wide application in the food and beverage industry (Fig. 4.1). Even though the food industry does not rely on process scale chromatography to the same extent as perhaps other industries such as the pharmaceutical sector, the sheer size of the industry results in a considerable application for chromatography. Other than process-scale applications of chromatography, many applications are related to safety, quality control, and research of new food ingredients and products. Many product streams in the food industry contain components that may have applications for, for example, functional foods. These product streams are typically large in volume and contain only a small amount of the component (or mixture of components) of interest. For economical application of large-scale preparative protein, peptide or oligosaccharide chromatography from agricultural/biotechnology streams, there is a need for ‘cheap’ resins (the costs for resins may be as high as 70% of total processing costs). The standard technology for fractionation or enrichment of food streams is using a packed bed. The close contact between interstitial liquid and carrier beads ensures effective mass transfer. For the treatment of large process streams

Fermentation Cell disruption Separation of cell debris

Removal of ‘like’ molecules such as mis-folds, multimers, degradation products via: Crystallization (extraction) Chromatography

Concentration Purification

Physical adsorption Ion exchange Affinity, size (sorption processes)

Formulation

(membrane separations) (electrokinetic separations)

Market

Fig. 4.1 Positioning of chromatography in a general layout of a bioprocess. For food processes, the fermentation step may not be present.


Advances in process chromatography and applications 111 and/or process streams where suspended solids or fouling components are present, problems may arise: the pressure drop over the column might be too high. The workhorse of preparative protein purification is ion exchange chromatography (IEC). For dilute feeds, IEC can be used in a capture step. Another way of pre-concentration is precipitation followed by resolubilization and IEC fractionation. Large-scale preparative chromatography can be done more efficiently using counter-current chromatography, which has larger driving forces for mass transfer and more efficient use of desorbent and (expensive) resin (see Section 4.4). As an example of the fractionation of protein hydrolyzates, simulated moving bed (SMB) chromatography has been recently applied (Ottens et al. 2006b). An example of large-scale purification of biomolecules being applied in industry is, for example, the purification of lysine (an amino acid) in SMB carousels (Van Walsum and Thomson, 1997). However, using a larger number of protein fractions, more side streams are needed, or a different approach will be needed (similar to distillation with different side streams). Resin screening is an important aspect of the development of an economical process (with respect to resin capacity, regeneration cycles, resin lifetime, costs of resins, food grade resins: some resin is always lost in the feed and the need for ‘water-soluble elution liquids’, as many food companies are not used to processing organic solvents). Last but not least, fouling characteristics are important as well as a proper cleaning program. The current challenges that are associated with the use of chromatography on a large scale in the food industry are the following (see also section 4.4): Large diluted stream with minor component (mg m–3) from large product streams (m3 h–1). – For high throughputs, expanded bed adsorption (EBA) or SMB chromatography might be considered, as indicated above. Considerable effort in the application and development of EBA was made by Kula and co-workers (e.g. Hubbuch et al., 2006). ∑ Pressure drop. Possible solutions include: – use of micro-structured materials, multimodal pores, perfusion resins; monoliths, structured woven packing, all allowing for a large convective flow and creating an interfacial area in the (functionalized) micropores, – (partly) fluidization, as in EBA, – radial flow chromatography. ∑ Fouling: – removal of debris, proteins and fats from the feed by membrane filtrations preferably in combination with a pretreatment. ∑ An understanding of the required purity of the targeted molecule. Mixtures with enriched fractions or pure targeted component. ∑

These considerations are addressed in this chapter.


112 Separation, extraction and concentration processes Several examples of functional food products that can be enriched or purified by chromatography are briefly introduced next. Peptides are known to have a versatility of bioactivity, e.g. ranging from a metal binding function, through influencing mood functions, to a reduction of blood pressure. The peptides can be obtained from various (food) proteins; dairy liquids such as milk and whey have often been investigated (e.g. Meisel and Schlimme, 1996). Typically, the production of a bioactive ingredient for functional food is obtained by enzymatic hydrolysis of the native protein. From the resulting complex mixture, the peptide(s) of interest need to be isolated. Currently, this is often done by membrane filtration or chromatography. Membrane filtration is regarded as a cheap technology, but the selectivity is rather low. Electromembrane filtration has been suggested as an alternative isolation route for charged components. In this membrane-based separation, the selectivity is increased by applying an electrical field as the (selective) driving force for migration over the membrane (Bargeman et al., 2002a; 2002b), which contributes to separation costs and complexity in comparison to regular membrane filtration. On the other hand, chromatography is highly selective, but is often regarded as expensive, as a result of the relatively high costs of the column media and eluents. The use of SMB technology is investigated as an alternative separation route, as this counter-current chromatographic separation method has been shown to be able to efficiently reduce sorbent inventory and the use of buffers by one order of magnitude in comparison to regular (fixed bed) column chromatography (e.g. Ballanec and Hottier, 1993; Cavoy et al., 1997). Furthermore, the use of chromatography for glucose–fructose separation (Azevedo and Rodrigues, 2005; Luz et al., 2008) sugar recovery from molasses (Hannu and Jarmo, 2000), stabilization of beers (Rehmanji et al., 2002), separation of proteins from various sources, such as milk (Kim et al., 2003a) and rapeseed, are only a few of the process scale applications of chromatography in the food and beverage industry. In addition to this several other functional components such as amino acids (Kostova and Bart, 2007a,b), flavors and fragrances (Gunther and Armin, 1993), nutraceuticals (Genovese et al., 2007), organic acids (Yoshikawa, et al., 2007), and oligosaccharides (Berensmeier and Buchholz, 2004) have also been separated on process and analytical scales. All these separations involve various interaction mechanisms, including ligand-exchange chromatography, LEC (Azevedo and Rodrigues, 2005, Luz et al., 2008, Steffenson and Westerlund, 1996), size-exclusion chromatography, SEC (Thurl et al., 1993), IEC (Kim et al., 2003a), and reversed-phase highperformance liquid chromatography, RP-HPLC (Stah et al., 1994; Thurl et al., 1991). The various interaction modes are described in the next section. Sanitation and hygienic design are key characteristics of food, beverage and nutraceutical processing. Major chromatographic companies that operate in the field are amongst others Universal Oil Products (UOP) and Novasep, and they can readily assist in designing large-scale chromatographic separations.


Advances in process chromatography and applications 113 4.1.2 Main chapter themes In the remaining part of this chapter first the basic principles of chromatography are described and the various types and basic modeling outlined. Then some applications are presented to indicate how chromatography is currently employed and its future potential in the food field. Subsequently, a section on recent developments in the chromatographic field is given with implications and applications for use in the food industry. This section is followed by an overview of future trends in the food chromatography field and finally some conclusions and suggestions for further reading are given.

4.2 Basic principles of process chromatography 4.2.1 Types of chromatography used The different modes or types of chromatography that can be used are categorized and depicted in Fig. 4.2 (see also GE website for further information; GE Healthcare is one of the major suppliers of chromatography equipment and resins: http:\\www.gehealthcare.com). The various methods are ranked according to their interaction strength, with the weakest on top. Size-exclusion chromatography is based on differences in the size of the solute molecules, by using a porous resin with distinct pore sizes (Horneman et al., 2004, 2006). Hydrophobic interaction chromatography and reversed-phase chromatography involve separation based on the differences in (surface) hydrophobicity of the solutes. The most powerful and most often used in food processing is ion-exchange chromatography, which separates solutes based on the differences in charge, which can be modulated by the pH and ionic strength of the solution. The strongest and most selective method is Sorption mechanisms

Weak

∑ Size

∑ Gel filtration (GF)

∑ Van der Waals

∑ Hydrophobic interaction (HIC) and reversed phase (RPC)

∑ Polar

d+

d–

d+ d+ +

∑ Ionic Strong ∑ Affinity

+

+

∑ Ion exchange (IEC)

+ ∑ Affinity chromatography (AC)

Fig. 4.2 Illustration of the basic interaction modes of chromatographic separation.


114 Separation, extraction and concentration processes affinity chromatography, and it is most often used in the pharmaceutical field, because of its selectivity and because its high cost can be met here. The purpose of process chromatography is to separate a target component from a mixture. Liquid chromatographic separations are based upon the different degrees of interaction of solutes dissolved in a mobile phase with a chromatographic medium or resin (the stationary phase), resulting in differential migration of the solutes through the column as illustrated in Fig. 4.3. The feed to the chromatography column is a binary mixture of solutes A and B, moving with concentration (wave) velocities wA and wB, respectively, through the column, with wB > wA. That is, solute B is more strongly retained or retarded by the resin than solute A. Each solute band becomes more dispersed as the chromatographic process progresses (depicted with fainter shading in Fig. 4.3b). Therefore, a chromatographic purification that is mainly based on adsorption and desorption of the target compound consists of several steps as follows. Equilibration of the resin For equilibration of the resin, the adsorbent material is prepared for the chromatographic separation. Depending on the mechanism of adsorption the equilibration step may include equilibration with the desired ionic strength, pH, and solvent concentration. Usually the equilibration solvent is chosen such that upon adsorption of the target compound and its impurities no change in composition (pH, ionic strength, and solvent composition) of the mobile phase occurs. In some instances it may be necessary to change the composition of the mobile phase to enhance the adsorption process. Loading In the loading process step, the target compound (and some impurities) is adsorbed onto the media. The capacity of the resin and/or the difference in affinity between target compound and its impurities determine the loading volume and loading conditions. It may be necessary to change or alter the feed stream properties to optimize the adsorption process. Washing In the wash phase, a washing buffer or solvent is used to desorb impurities from the resin. The washing buffer may be similar to the equilibration buffer, but this is not necessarily the case. Elution of the target compound needs to be avoided in this step. Elution In the elution phase the target compound is desorbed into an elution buffer solution. This can be done both in isocratic mode (constant mobile phase composition) and in gradient mode in which a gradient in the eluting buffer is applied to achieve optimal purity and/or minimal elution volume.


Advances in process chromatography and applications 115 Feed Mobile liquid phase

WB

Mobile phase

WA

WB

Mobile phase

WA

WA

WB

More strongly retained Less strongly retained

Stationary solid phase

t = 0 min

t = 10 min

t = 20 min Concentration

tR,B-tR,A

A

(a)

B A

B A

tR,A

tR,B

wA

wB

Time

Regenerant (eluent)

Feed B A

B

Flush

A

A B

B

B

A

A

B

A

A

A

A

Time

(b)

Fig. 4.3 (a) Illustration of the basic principle of chromatographic separation. Chromatogram showing the exit concentrations of A and B as a function of time (arbitrary times are used to show the progress of the chromatographic separation). (b) Adsorption, flush and regeneration.

Sanitization After elution a sanitization step may be applied in which compounds that show a higher affinity to the resin than the target compound are desorbed. For polymeric media, often a solvent is used to remove very apolar compounds from the resin. For hydrophilic media, a caustic solution is often used to © Woodhead Publishing Limited, 2010

116 Separation, extraction and concentration processes hydrolyze any irreversibly bound proteins. Acidic washes are applied to remove salts with low solubility (often calcium salts). Varying the salt concentration, pH or solvent composition can also be used to deliberately shrink and swell the resin which may enhance sanitization. After sanitization, the process starts again from equilibration. For processes in which there is very little affinity difference between the target compound and a limited number of impurities, it is often decided to use a continuous mobile phase with constant composition. The compounds are collected sequentially at the exit of the column and no intermediate equilibration, washing and sanitization is applied. A typical example of such a process is the separation of glucose and fructose on strong cationexchange resins in the Ca form. The requirements for sorbents to be used in chromatography and the different basic types are outlined in Fig. 4.4. 4.2.2 Modes of operation There are three different modes of operating a chromatographic process: frontal, elution and displacement. These stem from the three different ways of desorbing bound solutes from the chromatographic medium. Requirements ∑ Insoluble

∑ Hydrophilic Compromise

∑ Macroporous ∑ Mechanically stable

∑ Proper shape ∑ (Bio-)chemical stability

Main types

Composite

Organic ‘polymers’

Inorganic ‘pellets’

(a) ∑ Inorganic

∑ Organic polymers

Activated carbon

Polystyrene

Silica

Poly(meth)acryl

Alumina

Polysaccharides (dextran, agarose, etc.)

Pellet

Dense gel

Apolar

Macroreticular

Polar

Open gel

(b)

Fig. 4.4 (a) Requirements for sorbents and the main types available. (b) Examples of inorganic and organic polymers used as sorbents.


Advances in process chromatography and applications 117 In frontal chromatography, the sample is also used for elution. In this case, the sample feed is continuously applied to the column and the sample components displace each other in order of decreasing affinity for the chromatographic medium. The least retained solute is obtained in a pure form (i.e. depleted of the more strongly retained components) until the other solutes break through. Eventually, the column is saturated with the strongest retained component and the effluent has the same composition as the feed. In elution chromatography, the solutes are desorbed from the chromatographic medium by the action of a competing or modifying agent in the mobile phase, such as a salt. The concentration of the modifying agent is a key parameter. Three situations can be distinguished: isocratic, gradient or step change in the mobile phase composition. By keeping the composition (e.g. ionic strength) of the eluting buffer constant, the capacity factor is also kept constant; this is called isocratic elution and it is primarily used for separation of small molecules for which the variation of retention factor with mobile phase composition is not as large as for larger molecules. In gradient elution, the composition of the mobile phase is continuously changed (e.g. increasing ionic strength as in ion exchange chromatography), resulting in a continuous change in the retention factor. Instead of using a continuous gradient, the mobile phase composition may be changed in a stepwise fashion, resulting in discontinuous changes or ‘shifts’ in the retention factor. Gradient or step elution may be necessary for separation of large biomolecules for which the binding strengths may be too strong for isocratic elution to be feasible. In displacement chromatography, the affinity of the competing agent for the chromatography media is much higher than that of the solute, such that the agent effectively displaces the solute. This takes place irrespective of the concentration of the displacing agent (or displacer), in contrast to elution chromatography. Displacement chromatography has the advantage of being able to concentrate samples. However, selecting an appropriate displacer may not be easy. Several important aspects of using chromatography in food separations are outlined in Fig. 4.5. 4.2.3 Basic modeling The use of mathematical modeling is helpful in the design of chromatographic separators to size the chromatographic piece of equipment and to determine its optimal operation conditions. There is a wealth of information available on modeling of chromatography in different levels of detail. Guiochon gives a good basic overview (see section 4.8), and some recent modeling efforts using the general rate model and the linear driving force model for mass transfer are given by Nilsson (2005) and Horneman et al. (2006), respectively. Although the general rate model is the more accurate one, a less computationally involved but still accurate one is the linear driving force


118 Separation, extraction and concentration processes Eluent recycle ∑ Operating conditions and short-cut design Raffinate product Feed

∑ Complex phase behavior (azeotropic separations) ∑ Non-equilibrium fractionation ∑ Reactive separations

Extract product

Eluent make-up

∑ Multiple side/product streams ∑ Column dynamics

Sorbent recycle

Fig. 4.5 Schematic diagram of the operation of a large-scale chromatographic fractionator, such as a simulated moving bed, and the main factors for successful operation.

model, as shown in Fig. 4.6. It contains two partial differential equations describing the concentration of solutes in the bulk liquid and solid phase as function of time and axial position in the column. The equations are connected by a mass transfer term containing the isotherm, which gives the ratio of partitioning over the solid and liquid phase of a solute under certain environmental conditions. Several isotherms can be used, the most common being the Langmuir one, which gives a linear relation between the concentration in the solid and liquid phase at low concentrations and levels off at a constant saturated value at higher concentration at the maximum loading capacity of the specific resin used. Any appropriate PDE solver can be used (e.g. PDESol, Comsol) to solve the set of obtained partial differential equations. Commercial packages that are available on the market are for example ASPEN Chrom, gPROMS, and for flowsheeting SuperProDesign (Intelligen).

4.3 Applications of process chromatography in the food, beverage and nutraceutical industries Process scale chromatography has been effectively used in the food, beverage and nutraceutical industry (Fig. 4.7 and Table 4.1). Examples of these applications are presented here and by doing so a brief overview is provided of the industrial areas where process chromatography is applied. The various sources and products that can be concentrated, fractionated and purified by chromatography are given in Fig. 4.7 and Table 4.1, as well as scales of some chromatographic separations actually in industrial practice with its operation mode (SMB, and fixed bed chromatography, FB).


Advances in process chromatography and applications 119 4.3.1 Glucose–fructose separation The technology used previously (up to the middle 1970s) in the preparation of fructose was based on the application of a scarce and expensive raw material, inulin, which is a fructose polymer. This prevented the wide-scale use of fructose in the food and medical industry. Only in the 1980s did the application of chromatographic methods permit the large-scale manufacture of fructose by its isolation from glucose–fructose syrups obtained either as ∑ ∑ ∑ ∑

Which phases? Which convective streams? Is there dispersion? Mass transfer between phases?

Void fraction f

(Interstitial) velocity v

Feed flow Q MT

Cross-section A

Q = v fA

Mass transfer

Dispersion

(a) Mass balance over a slice Convection

(fA)vc

Dispersion

Ê ˆ (fA)Á – E ∂c ˜ Ë ∂z ¯

Mass transfer Accumulation

z + Dz

– [(1 – f)A] MTDz (fADz ) ∂c ∂t

Change = in – out ∂c = – ∂t Equation 1

z

Per volume solid

Dz (vc ) |z+ z

Dz

+E

Ê ∂c ˆ ÁË ∂z ˜¯

∂c = – v ∂c + E ∂2c – 1 – f MT ∂t ∂z f ∂z 2

z+Dz z

Dz

–

1–f MT f

E = 2vdp

(b)

Fig. 4.6 Mathematical modeling for chromatography (a). Different (partial differential) equations to be used (b) and (c), with ‘closing relations’ for mass transfer (d). Symbols: z axial length scale, D difference, c concentration, t time, k mass transfer coefficient, a interfacial area, K partition coefficient, Sh Sherwood number, Re Reynolds number, Sc Schmidt number, D diffusion coefficient, d particle diameter, n kinematic viscosity, v velocity, A cross- sectional area, Q flow rate, f void fraction, MT mass transfer, E dispersion coefficient. Subscripts: f film, p particle. Superscripts: * at equilibrium, vinculum solid phase.


120 Separation, extraction and concentration processes z + Dz

z

Mass balance over a slice of solid Mass transfer MTDz = ka (c* – c) Dz Overall coefficient Area per volume of solids Equilibrium concentration Accumulation

∂c Dz ∂t

Equation 2

∂c = ka (c* – c) = MT ∂t

Overall MT coefficient k–1 = (Kkf)–1 + k–1 p

Solve two coupled PDEs (c) dp = 10–3 m

In the ‘film’ Shf = 4 + 1.1Re0.6 Scf0.33 kf dp

Shf ∫ Re ∫

Df

; Scf ∫

nf ; Df

vdp nf

Df = 10–9 m2 s–1 v = 10–3 m s–1 Re = 0.5 Scf = 1000 Shf = 10 kf = 10

Df dp

= 10–5 m s–1

In the ‘particle’ Dp = 10–10 m2 s–1

Shp = 10 Shp ∫

k pd p Dp

; Dp π Df

kp = 10

Dp dp

= 10–6 m s–1

(d)

Fig. 4.6 Continued

a result of the inversion of sucrose or by converting glucose with the aid of glucose isomerase. The most common type of fructose syrup, usually called high-fructose syrup, contains 42% fructose, 52% glucose, and 6% oligosaccharides on a dry basis, and is used in place of sucrose in some foods and beverages (Kishihara et al., 1992). For some purposes, because fructose is sweeter and more soluble in water at low temperatures than glucose, syrup with 55 to 90% fructose, called higher-fructose syrup, is desirable. Therefore, to produce syrup containing more than 50% fructose a process to separate fructose from the equilibrium mixture is desirable. This mixture can be separated by chemical and physical means. The most widely used method for this kind of separation is chromatographic separation using cation exchange resins in Ca2+ form (Barker et al., 1984). In this instance, SMB is a widely used technology (see Section 4.4.3) (Bubnik et al., 2002). © Woodhead Publishing Limited, 2010


Dairy industry

Marine

Milk Eggs

Fish Algae Peptides Proteins

Omega-3, -6 fatty acids

Agriculture Sugars Flavors

Fig. 4.7 Source and product areas where process chromatography is being used or has potential. Table 4.1 Industrial scale chromatographic separations. Taken from Lecture notes from the Advanced Course on Downstream Processing, Delft, 2009, and Ganetsos and Barker (1992) (see Section 4.8) System

Process*

Scale (tonne a–1)

Xylene isomers Glucose–fructose Lactic acid, citric acid Amino acids Chiral compounds Peptides Milk proteins Flavors

SMB SMB FB/SMB FB/SMB SMB SMB FB FB

400 000 100 000 10 000 1500 1–10 0.1–1 0.1 0.01–01

*FB, fixed bed; SMB, simulated moving bed.

When large-scale separations are required, the SMB process is often the technology of choice in order to reduce separation costs. Compared with batch chromatography, SMB technology uses less eluent; in addition the separation productivity is higher thus using less adsorbent (Broughton, 1984; McCulloch et al., 1994). In recent years, fructose and glucose were separated from complex mixtures such as cashew apple juice using SMB technology (Azevedo and Rodrigues, 2005; Luz et al., 2008). 4.3.2 Recovery of sucrose from molasses Chromatographic separations are used to separate valuable components from beet molasses in beverage industries. Characteristic products that are © Woodhead Publishing Limited, 2010

122 Separation, extraction and concentration processes currently produced in addition to sugar include betaine, inositol, peptides, amino acid mixtures and several individual amino acids. The recovery of the other components can significantly improve the economy of sucrose recovery, but still sucrose remains as the most important product controlling the overall economy of the process. Chromatographic separation can also be applied to treat intermediate juices in the beet sugar industry. Thick juice separation has also been suggested. The resolution of the peaks may change owing to the changing composition of the feed material. Industrial batch systems were first built in the 1960s and the 1970s. In the mid-1980s the continuous simulated moving bed process was applied in molasses separation. The sequential SMB process, which can simultaneously recover multiple product fractions, was introduced in the late 1990s (Hyöky, 1999; Lancrenon, 1997; Paananen, 1996; Rousset, 1997). Hyöky et al. (1998) described the multi-profile FAST separation, which effectively doubled the efficiency. Further development includes a patented two-stage process (Hyöky et al., 1998). It is a combination of two chromatographic fractionators to improve the recovery and the purity of overlapping components, such as sucrose and betaine. It is uniquely suitable for recovering multiple value-added products from the process streams of the sugar industry (Paananen and Kuisma, 1999). Chromatographic systems are used in production of sweeteners based on beet, cane and starch as described by Paillat (1999). Sugar beet produces sucrose, betaine, raffinose, invert and many other water-soluble components. These components are extracted from the beets as juice. Part of these watersoluble components are removed or destroyed in the juice purification, but a large part of them will end up in the by-product: molasses. Molasses is the most common raw material in chromatographic desugarization plants. The main product is sucrose, but in addition betaine, inositol, amino acid mixtures and individual amino acids have been commercially separated from molasses (Hyöky et al., 1998; Paananen, 1996; Paananen and Kuisma, 1999). Low green or B molasses separation is already in industrial use. Kearney has suggested thick juice separation (Kearney, 1997; Kearney and Rearick, 1995). The positioning of the chromatographic step in the beet process has distinct effects on the recovery of sucrose and especially on the recovery of the other components. 4.3.3 Stabilization of beers Brewery production of some corns and hops for beer is a universal process in the beer industry. The quality of beer depends on its taste, clearness, color and foam retention. One of the key processes is the procedure of removing non-micro-organisms impurities. Fixed-bed chromatography with several resins like silica gel (SiO2nH2O) are in use to remove proteins that make beer muddy. Beers are known to contain a wide variety of phenolic compounds, most of which originate from the raw materials of brewing, i.e., barley and hops


Advances in process chromatography and applications 123 (Gardner and McGuinness, 1977). Of these phenolic compounds, the flavanoids are of particular interest to brewers, as they have long been thought to be precursors of non-biological haze in unstabilized beers (McMurrough et al., 1983). The flavanols have been classified into three groups on the basis of their chromatographic behavior. The first group, the simple flavanols, comprise flavanol monomers [e.g. (+)-catechin and (–)-epicatechin)], dimers (e.g. prodelphinidin B3 and procyanidin B3), and trimers. The second groups, the polymeric flavanols, are formed by oxidation and polymerization of simple flavanols. The complexed flavanols, the third group, result from the interaction of polyphenols with proteins to form complex structures (McMurrough, 1979). Stabilization of beer against haze formation may be achieved by decreasing the simple flavanol content, thereby limiting further flavanol polymerization and complexation. Increasingly, this stabilization is being achieved in breweries by treatment of beer with polyvinylpolypyrrolidone (PVPP) before packaging (McMurrough, 1993; McMurrough et al., 1992). Stabilization is achieved by sorption of phenolics on PVPP, which is subsequently removed by sheet filtration. This procedure can be carried out in conjunction with silica hydrogel treatment to remove haze-forming proteins (McMurrough and Madigan, 1996). These amorphous, non-additive stabilizers offer selective removal of haze forming proteins, improving colloidal stability of beer with no adverse effects on head or taste. Typically these silicas have been used with body feed diatomaceous earth filter aid (D.E.). However, by changing the conditions in the manufacturing process, it is possible to vary permeability (filterability) of silica hydrogel products. An innovation in the stabilization of beer is the ‘combined stabilization system’ (CSS) which is capable of a combined removal of turbidity forming protein and polyphenols in a single step (Janey and Katzke, 2002). As the economic alternative to PVPP and silica stabilization, the CSS is a compact rig mounted fully automated stabilization system, which can be integrated into any existing filter line. The adsorbent is permanently retained between an inlet and an outlet screen, making the dosing of precoat and feed suspension before each filtration unnecessary. A CSS adsorber has a particle size of 100– 300 mm. The solid phase adsorber resin is based on a high-grade, cross-linked, insoluble agarose (polysaccharide). Protein and polyphenols are adsorbed and then removed from the agarose adsorbent during the regeneration. Neither substance is dissolved in the beer nor is the beer quality affected in a negative way. The beer’s organoleptic properties, its foam stability, color and bitterness units remain unaltered with this kind of approach. 4.3.4 Separation of lysozyme from egg white Lysozyme has a number of possible applications; for example it can be used as an additive to baby milk or ophthalmic preparations; for treatment of ulcers, wounds and infections, as a potentiator of some antibiotics; as an


124 Separation, extraction and concentration processes antioxidant; and for its antimicrobial properties (Owen and Chase, 1997). The expanding potential for application of lysozyme in many fields of science and technology dictates the development of efficient and simple methods for lysozyme purification. Several chromatographic techniques that have been used for the separation of hen egg white lysozyme include ion exchange chromatography (Banka et al., 1993; Li et al., 1999; McCreath et al., 1997), affinity chromatography (Chiang et al., 1993; Yamada et al., 1985; Yamasaki and Eto, 1981), dye-binding chromatography (Tejeda-Mansir et al., 2003), affinity membrane separation (Arica and Bayramoglu, 2005; Bayramoglu et al., 2003; Ruckenstein and Zeng, 1997), ultrafiltration (Ghosh and Cui, 2000; Ghosh et al., 2000), PEG/salt aqueous two-phase system (Su and Chiang, 2006), reverse micelles (Noh and Imm, 2005), metal-affinity precipitation (Roy et al., 2003) or adsorption to plant residues (Hou and Lin, 1997). Commercial cation-exchange resins such as Duolite C-464, Amberlite, CM Sephadex have been used for the separation of lysozyme on process scales with recoveries of 90–95% (Li-Chan et al., 2006). Nonetheless, the lengthy steps and dilution of product at the end of the process have hindered their application at process scales. More recently this problem has been overcome by the use of magnetic separation techniques for the separation of egg white lysozyme and several other proteins (Safarik and Safarikova, 1993; 2004). These techniques usually enable simple, onestep separation of target proteins, and in most instances magnetic affinity adsorbents can be used. However, large-scale application may be difficult to achieve. 4.3.5 Separation of whey protein from milk Milk proteins are the most important source of bioactive peptides. Typical bovine milk contains 13% solids, with 4% fat present as an emulsion of globules with diameters up to 10 mm and caseins present as a colloidal suspension of particles with diameters up to 0.1 mm (Bylund, 2003). Fat globules normally cause problems for chromatographic separations, as they block packed columns as soon as the feed is introduced. Raw whole milk contains larger suspended particles than whole (full fat) processed milk, as the latter is homogenized to produce a uniform consistency. The composition of bovine milk includes water, fat, lactose and minerals, and up to 6% of the mass is made up by proteins and peptides, among them a number of high-value substances (Lourdes et al., 2000). In particular, milk contains two major protein groups, caseins and whey proteins, which differ greatly with regard to their physicochemical and biological properties. Normal milk contains 30–35 g L–1 proteins, approximately 80% of which are caseins with the remainder being the whey proteins (Korhonen et al., 1998). Whey proteins can be acquired as a by-product in cheese manufacturing process. The required long-term stability in functional performance of these proteins is often lacking. In general, whey is dilute liquid composed of lactose, a variety


Advances in process chromatography and applications 125 of proteins, minerals, vitamins and fat. Whey contains about 6% solids of which 70% or more is lactose and about 0.7% is proteins (Gerberding and Byers, 1998). Whey protein components are a-lactalbumin, b-lactoglobulin, immunoglobulins A, M and G, bovine serum albumin (BSA), lactoferrin and lactoperoxidase. b-Lactoglobulin is the major whey protein in bovine milk; it has a molecular weight of 18.4 kDa, possesses 162 amino acid residues and its concentration is 2–4 g L–1. a-Lactalbumin is an albumin which has 123 amino acid residues. It possesses a molecular weight of 14.2 kDa and its concentration in milk is 0.6–1.7 g L–1 (Ye et al., 2000). Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media. Immunoglobulin G (IgG) and immunoglobulin A (IgA), present in bovine whey, have high pharmaceutical value (Hahn et al., 1998). a-Lactalbumin can be used in infant formula and as a nutraceutical because of its high tryptophan content. b-Lactoglobulin is used in the production of confections (Zydney, 1998). Oral administration of bovine IgG is known to be an effective treatment of various infections of newborn infants (Hutchens et al., 1990). Typical concentrations, molecular weights and isoelectric points of whey proteins are given in Table 4.2 (Al-Mashikhi et al., 1988). Several techniques are used for the partitioning of whey proteins by e.g. aqueous two-phase systems (Jose et al., 2000; da Silva and Meirelles, 2000; Rito-Palomares and Miguel, 1998). Also, some attempts have been made to isolate whey proteins by using membrane filtration (Lucas et al., 1998; Gerd et al., 2000). The separation of whey proteins by using ion exchange chromatography on a process scale has been investigated by many researchers and several methods have been reported (Gerberding and Byers, 1998); Ionexchange separations take advantage of electrostatic interaction between surface charges on biomolecules, such as amino acids or proteins, and clusters of charged groups on the resin phase. An adsorbing biomolecule displaces counterions associated with the surface, discharging a complementary buffer salt in the process. Adequate buffering is required to shield native protein structures from changes in pH adjacent to exchange surfaces (Donnan effect) and pH effects were induced by sorption. Selection of an appropriate buffer Table 4.2 Typical concentration of whey proteins and their isoelectric points (pI) Protein

Approximate MW (kD) concentration (w/w%)

Isoelectric point (–)

b-Lactoglobulin a-Lactalbumin Immunoglobulins (A, M, G) BSA Protease-peptones Lactoferrin (LF) Lactoperoxidase (LP)

0.3 0.07 0.06 0.03 0.14 0.003 0.002

5.35–5.49 4.2–4.5 5.5–8.3 5.13 3.3–3.7 7.8–8.0 9.2–9.9

18.4 14.2 150–900 69 37–55 78 78


126 Separation, extraction and concentration processes is critical to the success of ion-exchange separations (Keith and Edwin, 1995). 4.3.6 Separation of lactoferrin and lactoperoxidase In addition to the caseins and whey proteins, which are generally isolated from milk, some specialty proteins are also identified as byproducts in these separations. Two very important such proteins of commercial interest, which are part of the whey protein fraction, are lactoferrin (LF) and lactoperoxidase (LP). Although production of such high-value whey proteins is a commercial reality, two aspects of dairy processing may not be optimal for their production. First, the proteins are subjected to a series of processing steps before being extracted. It is a generally accepted principle of bioseparation process design that proteins should be separated from a source material as fast and in as few steps as possible to avoid loss of activity and yield (Ladisch, 2001; Harrison et al., 2003; Ottens et al., 2006a). Lactoferrin (LF) and lactoperoxidase (LP) being basic proteins are usually captured from whey or skim milk by cationexchange chromatography and sold as specialty ingredients (Lonnerdal and Carlsson, 1977). The costs associated with the separation of these types of proteins is high, but the value and wide range applications in various functional food and nutraceutical products outweigh the cost. Lactoferrin, a 70–80 kDa single subunit glycoprotein, consist of two domains, each with one iron-binding site that requires synergistic binding of a bicarbonate anion (Al-Mashikhi and Li-Chan, 1988). A conformational change in the binding site, between open or closed forms, accompanies iron binding and release. The affinity of lactoferrin for iron is much greater than of transferrin which functions as an iron carrier in serum. It is present in human milk, and many exocrine fluids (saliva, mucous secretion) as well as in mammalian. Lactoferrin is a complex molecule with a number of interesting functional properties. Based upon these, lactoferrin may enter the composition in a wide range of products. Lactoperoxidase belongs to the category of peroxidase and acts as a biopreservative. Both these products find several applications based on their antimicrobial and antiviral activities such as in baby foods, food supplements, cosmetics, oral care, and preservation of meat. The production of high-value dairy proteins such as lactoferrin and lactoperoxidase normally requires extensive pre-treatments of milk to remove fat and caseins by centrifugation, precipitation, Ca2+ chelation and/ or filtration. Similarly, fat and caseins are normally removed before capture of recombinant proteins from the milk of transgenic animals (Morison and Joyce, 2005). Such pre-treatments can result in significant loss of protein yield and/or activity. Currently, high-value dairy proteins are viewed as by-products, with the major income of the industry coming from commodity dairy foods such as milk powder, cheese and butter. Economies of scale for production of commodity


Advances in process chromatography and applications 127 dairy products mean that centralized processing is the industry norm. Milk is typically cooled and held at 4 °C in vessels on the farm for up to 2 days before being transported to a dairy factory. There, it is pumped to holding tanks and then undergoes a series of unit operations such as cream (fat) separation, pasteurization, homogenization and blending for standardization before further processing into individual products (Bylund, 2003). After a number of such operations (which varies from factory to factory), LF and LP may be extracted from skim (low fat) milk or, more commonly, from whey, which is produced as permeate during membrane concentration of milk or after precipitation of caseins much further down the processing chain as a by-product of casein production or cheese making (Tomita et al., 2002). Extensive pre-treatments of milk and whey before ion-exchange capture of proteins are by no means restricted to industry but are also used in the laboratory. Many authors have examined the capture and analysis of whey proteins by chromatography (Al-Mashikhi and Li-Chan, 1988; Al-Mashikhi and Nakai, 1987; Andrews et al., 1985; Chaplin, 1986; de Frutos et al., 1992; Donnelly, 1991; Doultani et al., 2004; Elgar et al., 2000; Etzel et al., 2000; Felipe and Law, 1997; Francis and Regester, 1995; Geberding and Byers, 1998; Hahn et al., 1998; Humphrey and Newsome, 1984; Konecny et al., 1994; Lonnerdal and Carlsson, 1997; Morr and Ha, 1993; Noppe et al., 1999; Torre and Cohen, 1996; Visser et al., 1991; Xu et al., 2000; Ye et al., 2000; Yoshida, 1988). Hahn et al. (1998) examined the performance of a range of commercially available pharmaceutical grade cation exchangers for protein capture from acid whey. Doultani et al. (2004) used cationexchange chromatography to produce a number of protein products from mozzarella cheese whey. Ye et al. (2000) used both anion and cation exchange chromatography to isolate a-lactalbumin, b-lactoglobulin, lactoferrin and lactoperoxidase from rennet whey. Cation exchange membrane chromatographic systems have been successfully used for the separation of lactoferrin and lactoperoxidase on an industrial scale from sweet whey concentrate with yields of more than 90% for lactoferrin. It has also been demonstrated that these system can be scaled up to handle 1 ¥ 105 tonne/year of whey (Plate et al., 2006). 4.3.7 Separation of napin from rapeseed meal Preparative chromatography of napin and other proteins from rapeseeds was successfully carried out with a combination of ion exchange followed by hydrophobic interaction and size-exclusion chromatography. The yield of this combined process was 18% for napin and 40% for cruciferin (Berot et al., 2005). Rapeseed proteins were underexploited as it was being used only as animal feed until its non-food applications were demonstrated in a European project (Green chemicals and biopolymers from rapeseed meal with enhanced end-user performance. European contract Enhance QLK5 CT 199901442). Hence large quantities were needed to demonstrate its potential © Woodhead Publishing Limited, 2010

128 Separation, extraction and concentration processes in functional additives. Preparative purification processes of seed storage proteins were demonstrated for 11S and 7S globulin-type proteins in pea and soy (Crevieu et al., 1996; Gueguen et al., 1984; Howard et al., 1983; Lehnhardt et al., 1983; Larre and Gueguen, 1986; Nielsen, 1985; Thanh and Shibasaki, 1976). But with rapeseed storage proteins, which are composed of 11S globulin and 2S albumin-type proteins, only analytical purifications were developed, except procedures based on ammonium sulfate selective precipitation of proteins (Raab and Schwenke, 1984). SEC was successfully used to achieve the separation of 11S and 2S proteins (Dalgalarrondo et al., 1986), and cation-exchange chromatography (CEC) to separate 2S isoforms (Monsalve and Rodriguez, 1990). More recently a new procedure for fractional enrichment and purification of plant (e.g. soy beans) and milk-based proteins was demonstrated by the use of volatile electrolytes for isoelectric precipitations (Hofland et al., 2003; Golubovic et al., 2005). Rapeseed protein meal has two major classes of seed storage proteins: 12S globulin (cruciferin), which represents 25–65% of its protein content (Raab et al., 1992) and 2S albumin (napin). In addition to these two proteins it also contains some minor proteins of interest, such as thionins, trypsin inhibitors and a lipid transfer protein (LTP). From the structure and physicochemical properties of these proteins it is evident that cruciferin shows very different characteristics from other proteins including: high molecular weight, neutral isoelectric point pI; but aggregation could occur during the purification process. On the contrary, napin and LTP show rather close characteristics in their molecular weights and pIs, which could complicate the purification process. Separation of napin and cruciferin can be obtained by a combination of nanofiltration in combination with several chromatographic steps of cationexchange, size-exclusion and hydrophobic interaction chromatography on process scales.

4.4 Recent developments in process chromatography Figure 4.8 shows some of the areas where there is potential for development in process scale chromatography. The innovations can be categorized in several fields, materials, modes of operation, and equipment. In the subsequent subsections some of the innovation in these areas will be highlighted. 4.4.1 Structured matrices and monoliths Monoliths for enhancing reactions have been around for several decades. For example, PolyHipe is a highly porous macrocellular cross-linked styrene–divinylbenzene (DVB) copolymer, prepared by polymerization of a high internal phase emulsion (HIPE) of water droplets dispersed in a styrene/DVB continuous phase (Ottens et al., 2000). This type of monolith © Woodhead Publishing Limited, 2010

Advances in process chromatography and applications 129 ∑ Materials – optimization of selectivity, MT, and hydrodynamics, magnetic beads, monoliths, structured matrices ∑ Mode of operation – exploiting non-linear effects of coupled multi-component equilibria ∑

Equipment – CPC: centrifugal partitioning chromatography – Radial flow: annular chromatography – SMB (counter-current chromatography) – EBA (expanded bed adsorption)

∑ Chromatographic reactors – coupled reaction – separation

I Feed A,B

Waste

II III IV

Solids B Raffinate A Extract

Desorbent

Fig. 4.8 Areas of innovation in chromatography and a schematic diagram of a simulated moving bed.

was used to enhance a multiphase reaction, but had structural properties that also made it suitable for high capacity, low-pressure drop chromatographic separations. Structured monoliths from Corning that have been used in chemical conversions in the work of Kreuzer et al. (2005) would also suit chromatographic separations purposes. Pictures of monoliths are given in Fig. 4.9. For more than a century, chromatography practitioners have been separating the components of chemical mixtures by using columns packed with various types of particulate matter. Monoliths are a relatively new class of stationary phases, completely different from conventional stationary phases. A good overview of this relatively new stationary chromatographic phase in the biotechnology area is given by Jungbauer and Hahn (2004). The material is cast into a chromatography column as a continuous block of one piece interlaced with channels (Jungbauer et al., 2002a). The ramified channels do not have dead ends. Owing to this structure, the transport of the solute to the surface is solely by convection instead of diffusion as observed in conventional media (Hahn and Jungbauer, 2000; Hahn et al., 2002;). These nontraditional column materials have recently been commercialized and are being recommended by manufacturers and users for the enhanced speed and thoroughness with which they can separate complex mixtures of biological molecules. The most important characteristics of monolithic media are the excellent mass transfer properties and the low pressure drops. The large channel diameter makes monoliths excellent stationary phase materials for chromatographic separations. The first monoliths were developed by Hjerten et al. (Hjerten and Liao, 1988; Hjerten et al., 1989, 1992) and Tennikova et al. (1990). Hjerten and coworkers compressed polyacrylamide gels and observed excellent resolution. Scale up was initially difficult but recently was successfully performed (Podgornik et al., 2000). Another important development is in the use of monoliths as a support for solid-phase synthesis (Jungbauer et al., 2002c). An interesting application is the direct synthesis of peptides on © Woodhead Publishing Limited, 2010


(a)

(b)

Fig. 4.9 Photographs of monoliths: (a) SEM photograph (15 kV; scale, 1 cm = 32.7 mm) of PolyHipe-type X20PV90 on an ISI-DS-130 SEM apparatus (Ottens et al., 2000); (b) Corning monoliths, as used in catalytic multiphase conversions by Kreutzer et al. (2005), with potential for use in large-scale food, beverage and nutraceutical purification.

monolithic columns. Because the synthesis is performed on polymethacrylate monoliths, the directly grown peptide can be used as an affinity ligand without any further treatment (Pflegerl et al., 2002a; 2002b; 2002c). This strategy provides an excellent screening platform for affinity ligands. The screening of the ligand can be performed by microtitration and the same resin can be used for screening and large-scale separation. Monoliths are also interesting supports for enzyme reactors. In packed-bed reactors, the efficiency is often limited by pore diffusion. The high porosity of monoliths ensures that the enzymatic process is not mass transfer limited (Jungbauer et al., 2002b). © Woodhead Publishing Limited, 2010

Advances in process chromatography and applications 131 A relatively new stationary phase is the combination of chromatographic resin beads and a membrane. Mixed matrix membranes (MMMs), which incorporate adsorptive particles during membrane casting, can be prepared simply and have performances that are competitive with other membrane chromatography materials. The application of MMM chromatography for fractionation of b-lactoglobulin from bovine whey was recently shown (Saufi and Fee, 2009). The dynamic binding capacity of b-lactoglobulin in whey solution was about 80 mg g–1 membrane (24 mg mL–1 of MMM), which is promising for whey fractionation using this technology. 4.4.2 Novel ligands Affinity separations are normally very expensive; therefore, application in the food industry requires either very high value products or a lower ligand cost. Affinity ligands are the basic moieties on which the various affinity chromatographic separations run. Affinity chromatography has been used widely in biomedical research and biotechnology (Wilchek and Chaiken, 2000). It is based on molecular recognition where one recognition partner is immobilized on a base matrix, and soluble target molecules can be retained from a crude mixture. The target molecule can then be released and recovered in a functional form. The basis of elution is to reduce the affinity between immobilized ligand and analyte. This is most often accomplished by use of a bond-breaking buffer (i.e., by changing pH, ionic strength, and solvent) or by use of competitive elution (Firer, 2001). In either instance, the diffusion of immobilized ligand is always strictly limited, and therefore, the local concentration of immobilized ligand is constant. The environment surrounding the immobilized ligand changes to modulate the ligand–analyte affinity. Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. Although affinity chromatography is used extensively on a laboratory scale, its widespread acceptance has been limited on the preparative scale because of the high cost of the affinity ligands and their biological and chemical instability (Lowe et al., 2001). Only recently, the development of new methods for screening, selection and design of stable synthetic ligands, has opened the opportunity of exploitation of such materials on a large-scale (Ladner and Ley, 2001). The rapid growth of bioinformatics and molecular docking techniques and the introduction of combinatorial methods for systematic generation and screening of large numbers of novel compounds, has made feasible the rapid and efficient generation of ligands for affinity chromatography (Clonis, 1990; Labrou, 2002; 2003; Labrou et al., 2004; Spalding, 1991). Immobilized lectin affinity chromatography (Endo, 1996; Kobata, 1994) using Ricinus communis agglutinin (RCA) has been used for separation of several glycopeptides and oligosaccharides (Merkle and Cummings, 1987;


132 Separation, extraction and concentration processes Shinohara et al., 1987). RCA binds specifically to nonreducing-end galactose residues (Wei and Koh, 1978). Competitive elution using lactose, a hapten sugar, is generally used to elute the target molecule from the RCA immobilized column. Separation of bioactive peptides from defatted sunflower meal using immobilized lung extracts or immobilized angiotensin converting enzyme (ACE) (Megıas et al., 2004; 2006a; 2006b; 2007a; 2007b) has been used. Peptides that have angiotensin converting enzyme (ACE)-inhibitory activity are of great interest because of their potential antihypertensive effect, and have been found in hydrolysates of plant and animal origin (Murray and FitzGerald, 2007; Ottens et al., 2006a; 2006b). These are a few examples of using novel ligands in the separation of several valuable products from food sources. Enrichment by affinity chromatography generally allows further purification using classical chromatographic techniques. 4.4.3 Mode of operation Counter-current chromatography: simulated moving bed In conventional chromatography, the resin is fixed in a column, and the liquid flows through the column. As in extraction and distillation processes, the counter-current movement of the two phases improves the efficiency of the separation process, by establishing a higher driving force for mass transfer. A typical moving bed system is shown in Fig. 4.10b. It is equivalent to a distillation column. The liquid flows from section I to section IV, the sorbent moves from section IV to section I. A feed mixture (F) is introduced in the middle of the system. It is fractionated in an extract stream (E) containing the more retained component(s), and a raffinate stream (R), containing the less retained component(s). Similar to distillation, the movement of the components depends on the flow rates of the liquid and solid phases and the equilibrium distribution coefficient over both phases. Commonly, a four section SMB is used, the middle sections II and III serving to achieve the actual separation and section I cleaning the sorbent for reuse while section IV cleans the liquid for (partial) reuse. A real continuous movement of the solid phase is difficult to achieve in chromatography without losing resolution, owing to attrition of the solid phase. In practice, the movement of the solid phase is ‘simulated’ by periodic switching of a carousel of fixed chromatographic columns (Fig. 4.10a). The efficient use of the resin and liquid (desorbent D) is an important contribution to the efficiency of the SMB compared with that of fixed bed (FB) chromatography. Normally a reduction by a factor of 2–10 in resin and buffer use per amount of product produced can be achieved in an SMB compared with a FB. Chiral separation was the main driving force for the establishment of SMB separations in the fine chemicals and pharmaceutical industry over the past two decades (e.g. Cavoy et al., 1997; Pais et al., 1997; Rodriques and Pais, 2004). It is now a well-known and regularly applied chiral binary separation


Advances in process chromatography and applications 133 Feed Liquid flow cycle

Liquid flow direction

Raffinate

Extract Liquid flow direction

Desorbent

(a) Sorbent recycle q4

q3

q2

IV c4

c3

II c2 c¢2

R

q0

q1

III

cF F

I c1

c0

D

E

N species: 11 N unknowns (c, q)0, …, 4 + c¢2 11 N independent equations (all linear)

4N concentration ratio 4N overall mass balances 3N balances: feed, desorbent, sorbent [A] x = b (b)

Fig. 4.10 (a) Schematic of the concept and operation of continuous multi-column simulated moving bed chromatography. Different tints indicate the extent of concentration of the two solutes. (b) A simple steady-state model for mathematical modeling of four-zone linear SMB chromatography: q solute solid-phase concentration, c solute liquid-phase concentration, R the raffinate stream, F the feed stream, E the extract stream, D the desorbent stream. The different zones are indicated by I–IV and 1–4.

technique. SMB is also a well-studied operation. However, some areas of application need to be developed. Separations of biologicals (Houwing, 2003; Houwing et al., 2002, 2003a; 2003b; Paredes et al., 2005) as well as multi-component (MC) separations in SMBs (e.g. Abel et al., 2004; Wang and Ching, 2005) are important research fields with a large industrial relevance. Multi-component (MC) separations can be performed in SMBs and some studies are devoted to it, mainly by serial SMB systems. Multiplezone (or section) SMBs have been reported (up to nine zones, Wooley © Woodhead Publishing Limited, 2010

134 Separation, extraction and concentration processes et al., 1998). Five-zone SMBs are being investigated and design criteria have been presented, for example, by Kim et al. (2003b). Wang and Ching (2005) presented a straightforward extension of a four-section SMB to a five-section SMB. An extra raffinate or extract port was added to the standard four-section SMB to perform a ternary separation. The pros and cons of two raffinate or two extract streams were discussed. However, in all these cases, a single component was purified from a three-component mixture. An equal or larger volume of industrial separations, particularly in the food industry, concern enrichment of a (MC) product in a particular functionality, for instance from a health or nutritional point of view. When using chromatography, the functionality can be associated to a particular MC fraction of an (also) MC feedstock. Developing and optimizing an efficient separation has to deal with the inherent MC nature of the feedstock and product(s). Also in chromatographic separations in food and biotechnology, consumption of costly resin and salty buffers is high, which are reasons to use SMB technology for these applications as well. As yet, few biotechnology applications of SMB technology have been described in open literature. The few examples found mainly concern small biomolecules, such as amino acids (e.g. Maki et al., 1987; Van Walsum and Thompson, 1997; Wu et al., 1998) disaccharides (Geisser et al., 2005) polypeptides (e.g. Mun et al., 2003) but also protein separations and purifications (e.g. Adachi, 1994; Gottschlich et al., 1996; Gottschlich and Kasche, 1997; Hashimoto et al., 1988; Horneman et al., 2006, 2007a; Houwing, 2003; Houwing et al., 2002, 2003a; Huang et al., 1988), plasmid purifications (Paredes et al., 2005) and viral clearance (Horneman et al., 2007b). In biotechnology, a whole range of chromatographic techniques is available for separation of proteins. SMB is applicable to any of these systems. Considering size exclusion; several forms have been investigated: separation of dilute mixtures (i.e., the linear isotherm case), surfactant-aided gel filtration (Horneman et al., 2004, 2006, 2007a; 2007b), separation of concentrated mixtures (the non-linear isotherm case) (Houwing, 2003), and separation of multicomponent mixtures (Ottens et al., 2006a). In the latter work, the technological and economic feasibility of SMB technology for multi-component product (and feedstock) separations is described, using the fractionation of peptides from protein hydrolysates as functional food ingredients as an example. The impact on the environment was also assessed. The model system consisted of an enzymatic hydrolysate of casein, which was fractionated using gel filtration (size-exclusion chromatography). The technical feasibility of isolating peptides, capable of inhibiting the ‘angiotensinconverting enzyme’ (ACE), from a casein hydrolysate by means of size-exclusion chromatography was investigated experimentally. In another study, the successful separation of the disaccharide lactose from a complex mixture of human milk oligosaccharides (HMOS) with the continuous chromatography of SMB technique is described (Geisser et al., 2005).


Advances in process chromatography and applications 135 Expanded bed adsorption A major concern when operating a chromatographic column is fouling and, clogging of the column causing high pressure drop and preferential flow, or channeling, and thereby loss of capacity. To overcome this problem a technique was developed over recent decades that overcomes this problem, by operating the adsorption and washing step in the fluidized mode. Liquid enters the column from below at sufficient velocity to drag the sedimented solid stationary material with it, thus creating a fluidized or expanded bed. This mode of operation allows processing of crude feed streams with a high load of particulates, and can be used as a combined solid–liquid separation step and an initial capture step (e.g. Hubbuch et al., 2006). A recent application of expanded bed adsorption (EBA) in the food industry is the recovery fractionation and purification of valuable proteins from potato juice (Løkra et al., 2009). Horneman et al. (2008) used EBA in SMB mode to optimally recover and purify potato proteins to obtain an economically viable process.

4.5 Process control in chromatography In order to have an optimal separation at high productivity, yield and purity, it is necessary to operate under the appropriate conditions. The conditions may change owing to fouling or loss of capacity, a fluctuating feed composition or the inherent unsteady state of the operational procedure (i.e. with SMB chromatography). To operate at optimal conditions, necessitates control of the operation of the industrial-scale chromatography column(s). For SMB a safe regime is usually 10% below the optimal boundary. Proper control strategies may allow higher productivity, while maintaining the production constraints of purity and yield (Grossmann et al., 2008). With the availability of massive, cheap computer power and detailed mathematical models (as outlined in section 4.2.3), process scale chromatography will benefit more and more in the near future from proper control of the operation of the unit.

4.6 Future trends 4.6.1 High-value nutraceuticals Functional foods and nutraceuticals provide an opportunity to improve health while reducing health care costs. Functional foods involve the broad class of prebiotics and probiotics. Probiotics are viable microbial dietary supplements that influence the host and have a beneficial effect in the gastro-intestinal tract (Jardine, 2009; Salminen et al., 1998), whereas prebiotics are nondigestible food ingredients that benefit the host organism by stimulating the growth or activity of one or limited number of bacteria in the colon (Gibson and Roberfroid, 1995; Jardine, 2009). © Woodhead Publishing Limited, 2010

136 Separation, extraction and concentration processes Nutraceuticals refers to extracts of foods claimed to have a medicinal effect on human health. An important trend in food technology is the production of these ingredients from sources such as aromatic plants or spices. Among the nutraceuticals, antioxidants receive much attention in the food industry (Madhavi et al., 1996), not only as preservatives in food products to prevent or retard oxidation of fats and oils, but also because of their beneficial effects on human health. Supercritical-fluid chromatography (SFC) is one of the widely used process options in nutraceutical separations. Preparative-scale SFC is an environmentally clean technology whose main advantage, compared with preparative LC, is the easy recovery of the isolated compounds by a simple decompression of the supercritical fluid (Coleman et al., 1999). Fish oil preparation and separation of polyunsaturated fatty acid esters such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Alkio et al., 2000) is a well-documented example of preparative SFC and has been used in nutraceutical production since the early 1990s. Novel methods such as continuous membrane chromatography reactor system (CMCRS) are used for the synthesis and separation of galacto oligosaccharides (GOS) (Engela et al., 2008). Membrane separations using ion-exchange chromatography is another widely used technique for functional components from milk sources (Goodall et al., 2008). 4.6.2 Good manufacturing practice (GMP), quality assurance (QA) and quality control (QC) and Food and Drug Administration (FDA) regulation Good manufacturing practice (GMP) is a term that is recognized worldwide for the control and management of manufacturing and quality control of foods, pharmaceutical products, and medical devices. GMPs are guidelines that outline the aspects of production that affect the quality of a product. The guiding principle of GMP is that quality is built into a product, and not just tested into a product. Therefore, the assurance is that the product not only meets the final specifications, but that it has been made by the same procedures, called standard operating procedures (SOP), under the same conditions each and every time it is made. In the food industry GMPs address all factors involved in a manufacturing process: personnel, building, premises, machinery and apparatus, documentation, and quality control, that generally affect product quality or impact quality monitoring. Besides these factors, an important theme that is part of GMP is validation. It is that part of GMP that ensures that facility systems, equipment, processes, and test procedures are under control and therefore consistently produce quality product. This is a special quality assurance method, which is explicitly required by GMP codes and mandated by law. GMPs are enforced in the United States by the FDA (http://www.fda.gov); within the European Union, GMP inspections are performed by National


Advances in process chromatography and applications 137 Regulatory Agencies, e.g. GMP inspections are performed in the United Kingdom by the Medicines and Healthcare products Regulatory Agency (MHRA)(http://www.mhra.gov.uk), in the Republic of Korea (South Korea) by the Korea Food and Drug Administration (KFDA), and by similar national organizations worldwide. 4.6.3 Process analytical techniques (PAT) and control Process analytical techniques (PAT) are now being implemented in the pharmaceutical industry to monitor and control proper operation of these processes. Also in chromatographic fractionating and purification in the food industry such an application is seen. It allows for GMP, QC, better operation, better control, higher productivity and on spec production/higher number of on spec batches.

4.7 Conclusions Process chromatography is being applied in food, beverage and nutraceutical processing. Chromatography has a high resolving power which can give highpurity food products. With the advent of the nutraceutical field, also more expensive forms of chromatography, based on affinity, may find application, as the potential higher sales revenues balance the higher operating costs. Chromatography is a flexible purification method that is likely to play a role in food, beverage and nutraceutical processing for years to come.

4.8 Sources of further information and advice ∑

Ganetsos, G., Barker, P.E. Preparative and production scale chromatography. Marcel Dekker, Inc., 1992. ∑ Guiochon, G., Shirazi, S.G., Katti, A.M. Fundamentals of preparative and nonlinear chromatography. Academic Press, Inc., 2nd Edition, 1994. ∑ Sofer, G., Hagel, L. Handbook of process chromatography – a guide to optimization, scale-up and validation; Academic Press, London, 3rd Edition, 2001.

4.9 List of abbreviations ACE CEC CMCRS

angiotensin converting enzyme cation-exchange chromatography continuous-membrane chromatography reactor © Woodhead Publishing Limited, 2010

138 Separation, extraction and concentration processes CSS DHA DVB EBA EPA FB FDA GMP GOS HIPE HMOS IEC IgG LEC LF LP LTP MC MHRA MMM MT PAT PDE QA QC RCA RP-HPLC SEC SFC SMB SOP

combined stabilization system docosahexaenoic acid divinylbenzene expanded bed adsorption eicosapentaenoic acid fixed bed Food and Drug Administration good manufacturing practice galacto oligosaccharides high internal phase emulsion human milk oligosaccharides ion-exchange chromatography immunoglobulin G ligand-exchange chromatography lactoferrin lactoperoxidase lipid transfer protein multi-component Medicines and Healthcare products Regulatory Agency mixed matrix membranes mass transfer process analytical techniques partial differential equation quality assurance quality control ricinus communis agglutin reversed-phase high-performance liquid chromatography size-exclusion chromatography supercritical-fluid chromatography simulated moving bed standard operating procedure

4.10 References Abel, S., Babler, M.U., Arpagaus, C., Mazzotti, M., Stadler, J., (2004) Two-fraction and three-fraction continuous simulated moving bed separation of nucleosides, J Chromatogr A, 1043(2): 201. Adachi, S., (1994) Simulated moving bed chromatography for continuous separation of two components and its application to bioreactors, J Chromatogr, 658: 271. Alkio, M., Gonzales, C., Jäntti, M., Aaltonen, O., (2000) Purification of polyunsaturated fatty acid esters from tuna oil with supercritical fluid chromatography, J Am Oil Chem Soc, 77: 315–321. Al-Mashikhi, S.A., Li-Chan, E., Nakai, S., (1988) Separation of immunoglobulins and lactoferrin from cheese whey by chelating chromatography, J Dairy Sci, 71: 1747–1755.


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5 Novel adsorbents and approaches for nutraceutical separation B. W. Woonton, CSIRO Food and Nutritional Sciences, Australia and G. W. Smithers, Food Industry Consultant, Australia

Abstract: In this chapter, an overview is provided of novel chromatographic adsorbents that are perhaps on or just over the horizon but hold promise as the basis for future industrial technologies in the cost-effective extraction of nutraceuticals from agri-food streams. Such adsorbents include molecular imprinted polymers, organic monoliths, stimuli-responsive resins, mesoporous molecular sieves, peptide affinity ligands, and membrane adsorbers. These adsorbents have the potential to improve specificity, selectivity, simplicity, robustness, and productivity, and to reduce the environmental impact of nutraceutical manufacture. Key words: nutraceuticals, separation, molecular imprinted polymers, monoliths, stimuli-responsive resins, molecular sieves, peptide affinity ligands, membrane adsorbers.

5.1 Introduction Nutraceuticals have entered the mainstream of foods and beverages. By definition, these components derived from agricultural and food raw materials have a positive influence on human health through a specific prophylactic or therapeutic physiological effect against chronic disease when consumed (Hardy, 2000). Nutraceuticals have usually been delivered to the consumer in a medicinal vehicle such as a tablet, capsule or in powdered form. More recently, the functional foods market, driven by consumer demands for healthpromoting foods and drinks, has seen nutraceuticals enter the food industry by way of the active component(s) in ‘functional’ varieties of everyday food and beverage products like yoghurt, bread, margarine, juice, and even water. This market is becoming a lucrative outlet for new and novel nutraceuticals, and © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 149 food companies are keen to capture a slice of this market. These companies are thus exploring new sources of nutraceuticals, and the means to isolate, characterize and substantiate the bioactivity of such components to support the development of successful functional foods and beverages. In an earlier chapter (Chapter 4), more traditional approaches (e.g. fixed and moving bed chromatography) to the extraction, separation and concentration of nutraceuticals have been addressed. Chromatography is a powerful technique for the cost-effective separation and isolation of nutraceuticals from agrifood streams, including food processing co-product streams. Developments in continuous chromatography have enabled the commercial separation and isolation of biologically active proteins from milk and whey. For example, continuous separation (CSEP) technology is being used to isolate valuable dairy-derived molecules, including nutraceuticals (De Silva et al., 2003). Central to the chromatographic process is the relevant adsorbent that is responsible for effecting the separation. In this chapter, we introduce novel chromatographic adsorbents and approaches for the isolation of nutraceuticals, those that are perhaps on/over the horizon but hold promise as future industrial technologies for the cost-effective isolation of nutraceuticals, and the potential to deliver on consumer demands for effective functional foods and beverages. This overview is not intended to be exhaustive or inclusive of all novel adsorbents and technologies, but rather to provide a summary of the most promising adsorbents and approaches, that with further research and development can be employed for large-scale nutraceutical manufacture. The chapter provides an account of developments in: (i) (ii) (iii) (iv) (v) (vi)

molecular imprinted polymers; organic monoliths; stimuli-responsive resins; mesoporous molecular sieves; peptide affinity ligands; and membrane adsorbers.

5.2 Molecular imprinted polymers and applications in the nutraceutical industry Molecular recognition is an important process in many biological systems, for example the specific recognition of an antigen by an antibody. Such recognition mechanisms have been utilized in the development of biological affinity-based separation media (e.g. mono- or poly-clonal antibodies and affinity peptide resins) (Tharakan et al., 1992). Molecular imprinted polymers (MIPs) differ from these biological affinity systems in that they are synthetic organic materials designed with cavities specific to a template molecule employed during synthesis. In effect, they are tailored synthetic affinity materials that can be used in a range of applications including the separation © Woodhead Publishing Limited, 2010

150 Separation, extraction and concentration processes and analysis of molecules, the sensing of molecules, as artificial enzymes, and as antibody mimetics. MIPs are worthy of discussion as they have the potential to reduce the complexity of nutraceutical separation processes, thus reducing processing costs and the environmental impact associated with such processes. The concept of molecular imprinting was first reported by Kikuchi et al. (1972) and Wulff & Sarhan (1972), and since these early reports, there has been a significant amount of research undertaken to improve MIPs using alternative polymer chemistries and synthesis methodologies. There are a large number of publications reporting the development and application of MIPs for a range of target molecules including small organic molecules such as pharmaceuticals, pesticides, amino acids, peptides, high molecular weight proteins, polypeptides, steroids, sugars and cells (Andersson et al., 1995; Bolisay et al., 2006; Bossi et al., 2007; Nicholls et al., 1995; Rachkov & Minoura, 2001; Sellergren, 1994; Shi et al., 1999; Steinke et al., 1995). 5.2.1 Synthesis The synthesis of MIPs involves the organization of a cross-linked polymer matrix around a template molecule, followed by removal of the template, leaving recognition sites complementary to the template or similar molecules (Fig. 5.1). There are three main steps involved in the synthesis of a MIP, and these include: (i) pre-arrangement of selected monomers (such as vinyl or acrylic) around the target molecule; (ii) polymerization of the monomers (e.g. radical polymerization) with the addition of a cross-linker; and (iii) extraction of the target molecule leaving binding sites that recognize the target molecule (Gupta & Kumar, 2008). Two different approaches may be employed to manufacture MIPs, these are covalent and non-covalent imprinting. Non-covalent imprinting is the most common and involves the interaction of the template molecule with the monomers via hydrogen bonding, ionic bonding, hydrophobic interactions and van der Waals forces (Mosbach, 1994). After polymerization and removal of the template, the functional groups of the polymer are positioned to rebind the target molecule using the same non-covalent interactions. The non-covalent approach can only be employed where template molecules have functional groups capable of strong non-covalent interactions with the monomers. However, this can be overcome by using the covalent imprinting method, which involves covalently attaching the template molecule to a monomer followed by polymerization, cleavage of the covalent bond to liberate the template, and removal of the template. The reactive groups are positioned within the polymer cavities to allow covalent bonds to be reformed with the target molecule and provide selectivity. As there is a relatively small number


(i) Complexation Template

(ii) Polymerization

(iii) Washing

+ cross-linker

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Template

Fig. 5.1 Schematic illustration of the method for preparing molecular imprinted polymers (Takeuchi & Haginaka, 1999). Reproduced with permission. Copyright Elsevier (1999).

Novel adsorbents and approaches for nutraceutical separation 151


Functional monomer

152 Separation, extraction and concentration processes of suitable reversible covalent bonding reactions that can be employed, the covalent imprinting method has limited applications (Gupta & Kumar, 2008). MIPs are often manufactured using bulk polymerization methods to produce monolithic materials which can be used for in situ separations, or ground and sieved to produce particles suitable for column packing and separation applications (Haginaka, 2009). MIPs can also be manufactured using suspension, seed and dispersion/precipitation polymerization methods to produce micro- or nano-spheres (Haginaka, 2009; Kempe & Kempe, 2006; Ye & Mosbach, 2001). Using such suspension polymerization methods, MIPs can be generated in aqueous environments; however, hydrogen bonding between the template molecule and the functional monomer, and therefore MIP selectivity, can be reduced when using aqueous solvents (Haginaka, 2009). Suspension polymerization techniques in liquid perfluorocarbon or mineral oil do not reduce hydrogen bonding and electrostatic interactions and the resultant MIP microspheres have been shown to be selective for biologically active molecules (Perez-Moral & Mayes, 2006). MIPs are usually packed into columns of various dimensions to allow (a) sample preparation before analysis; (b) removal of valuable molecules; or (c) removal of undesirable molecules from various raw material streams. 5.2.2 Advantages and disadvantages The benefit of MIPs is their stability in various chemical and physical environments for an extended period of time without any loss in template recognition (Ciardelli et al., 2006; Kriz & Mosbach, 1995). In addition, they are physically robust, resistant to elevated temperatures and pressures, and inert towards acids, bases, metals ions and organic solvents. The reported limitations of MIPs include binding site heterogeneity and slow mass transfer, which subsequently leads to peak broadening and tailing. In addition, MIPs prepared by bulk polymerization often have irregular sized and shaped particles which can lead to poor chromatographic performance and large pressure drops (Turiel et al., 2005). These limitations may be overcome by: (i) optimizing the MIP loading solvent; (ii) optimizing the MIP washing regimen to remove contaminant molecules; and (iii) finer control over the polymerization reaction leading to more regularly shaped particles. 5.2.3 Applications The characteristics of MIPs make them ideal for use in separation applications including chromatography, capillary electrophoresis, solid phase extraction and membrane separations. They also have potential to be used as microreactors, in immunoassays, as antibody mimics and artificial enzymes. Owing to their © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 153 selectivity and robustness, MIPs have been applied in the selective isolation, concentration and analysis of food-derived molecules such as theophylline, caffeine, catechin, cholesterol, glycyrrhetinic, theanine, a-tocopherol and flavonoids (Haginaka, 2009). For example, Puoci et al. (2007) developed a MIP and optimized conditions for the selective extraction and analysis of a-tocopherol from bay leaf (Fig. 5.2). This method recovered approximately 60% of the a-tocopherol with high purity as measured by HPLC. Many other MIPs have been reported for the separation and analysis of agricultural herbicides, pesticides and fungicides, toxins, antibiotics and other chemicals in food (Haginaka, 2009). The ability to produce MIPs for the selective isolation of proteins and peptides is improving (Bossi et al., 2007; Rachkov & Minoura, 2001; Shi et al., 1999), and being employed to selectively target bacteria through their surface expressed proteins (Xue et al., 2009). 5.2.4 Commercialization MIPs can be employed in analytical applications as solid-phase extraction media to selectively increase the concentration of analyte, decrease sample preparation time and increase method sensitivity. MIPs for the solid phase extraction and analysis of food contaminants such as antibacterials (chloramphenicol, nitroimidazoles, fluoroquinolones) and herbicides (triazines) have been manufactured by MIP Technologies AB (Lund, Sweden) and distributed by Sigma-Alridch (St Louis, USA). MIPs for solid phase extraction and analysis of vitamins in food such as riboflavin have recently become available (MIP Technologies AB, Sweden). There are few reports in the public domain verifying large-scale applications of MIPs in the food, pharmaceutical and chemical industries. However, given the reported benefits of MIPs including stability and specificity, with further research and development into large-scale manufacture of these adsorbents together with scaled applications, it is likely that they will find targeted use in the isolation of nutraceuticals.

5.3 Organic monoliths and applications in the nutraceutical industry In contrast to columns packed with small particles to facilitate separation (e.g. silica or agarose beads), monoliths are continuous solid phases which can be synthesized in situ in confined spaces such as columns, capillaries, and microfluidic and microchip devices. Monoliths are unique as they have an interconnected skeletal structure with flow-through paths that provide very high permeability to a mobile phase, high flow rates, and low back pressures during chromatography. These characteristics make monoliths promising for the large-scale cost-effective purification of nutraceuticals


154 Separation, extraction and concentration processes 0.9 0.8

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Fig. 5.2 HPLC chromatograms of (a) crude bay leaf extract and (b) bay leaf extract after isolation and concentration of the a-tocopherol (a-TP) (Puoci et al., 2007). Reproduced with permission. Copyright Elsevier (2007). © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 155 in the food industry. Monoliths can be synthesized using either inorganic materials such as silica or organic materials such as methacrylate polymers. As silica has poor stability and operational robustness during the caustic cleaning procedures employed in the food industry, this chapter focuses on the development and application of organic polymeric monoliths. 5.3.1 Synthesis Polymeric monoliths were first introduced in the late 1980s (Hjerten et al., 1989; Svec & Fréchet, 1992). Since then there have been several publications detailing developments in their manufacture and use in liquid chromatography, capillary electrochromatography and microscale separations (Nordborg & Hilder, 2009). Polymeric monoliths are synthesized by mixing an initiator, organic monomers (including a cross-linking monomer) such as styrene, methacrylate, acrylamide-based materials and pore forming solvents (porogens). The reagents are placed within a suitable mould, such as a column, capillary or channel of a microfluidic device and polymerized using radical polymerization initiated by either thermal treatment, ultraviolet, gamma or microwave radiation (Nordborg & Hilder, 2009). As polymerization proceeds, the polymer chains formed become insoluble in the polymerization mixture, phase separation takes place and the polymer chains precipitate out of solution, forming the monolith. The properties of the monolith synthesized, including the size and distribution of macro-, meso- and micropores are determined by the composition of the polymer mixture and the polymerization conditions. For example, the mode of initiation, type and amount of cross-linker, porogen, and the polymerization temperature. The large number of monomers that can be chosen to impart charged groups, other monomers, cross-linking agents and porogens results in a very large number of variations that can be examined and optimized to produce the desired monolith (Nordborg & Hilder, 2009). Organic monoliths can also be synthesized using sub-zero temperatures, where ice-crystals act as the pore-forming elements. After formation of the monolith, the ice crystals are allowed to thaw, the resulting water removed, leaving behind macropores between 10 and 100 mm in diameter. Such monoliths are known as cryogels (Arvidsson et al., 2002, 2003). Solid granules such as sodium sulfate can also be employed during the synthesis to improve bi-model pore formation and the hydrolytic characteristics of the resulting polymeric monoliths (Fig. 5.3) (Du et al., 2007). 5.3.2 Functionalization To improve the applicability of polymeric monoliths for separation applications, functionalization either during or after polymerization is required. In the first approach, the choice of monomers with charged functional groups results in material having charged groups throughout the monolith, including its surface.


156 Separation, extraction and concentration processes (a)

Acc. V Spot Magn Det WD 20.0 3.0 2000¥ SE 9.3

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10 mm

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Fig. 5.3 SEM image of a glycidyl methacrylate-co-ethylene glycol dimethacrylate monolith prepared in the presence of the organic porogens dodecanol and cyclohexanol, (a) without and (b) with sodium sulfate granules (Du et al., 2007). Reproduced with permission. Copyright Elsevier (2007).

Examples of charged monomers that can be employed include methacrylic acid and ethylene glycol dimethacrylate. Optimization of monolith chemistry must take into account that the charged monomers participate in the polymerization process and will not all be available for separation interactions on the surface © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 157 of the monolith. The approach of post-polymerization modification has been examined and reported to be advantageous as the number of variables is reduced and the monolith chemistry can be optimized followed by modification of the surface with the desired functionality. Monoliths can be modified to achieve separation based on ion-exchange, affinity recognition, reversedphase, and hydrophobic interaction. 5.3.3 Advantages and disadvantages Polymer monoliths have similar benefits to their polymeric particles. Their pH and pressure stability are two advantages that allow them to be employed in a range of environments where high flow rates may lead to large pressure drops across the bed. However, as monoliths are made from one piece of material with large interconnected pores (Fig. 5.3), diffusion mass transfer is minimized and convective mass transfer is maximized. As convective mass transfer is faster than diffusive transport, higher mobile phase flow rates can be employed and faster separation of molecules can be achieved. Breakthrough curves of lysozyme and immunoglobulin G using a polymethacrylate monolithic column (Connective Interaction Media, BIA Separations; Ljubljana, Slovenia) and a column packed with Source 30S particles (GE Healthcare Life Sciences Uppsala, Sweden) have been studied (Hahn et al., 2002). As expected, mass transfer in the particle packed column was very dependent on the linear velocity of the mobile phase, molecule size, and feed concentration, whereas mass transfer in the monolith was not significantly affected by velocity, molecule size or feed concentration (Hahn et al., 2002). A disadvantage of monoliths is their low surface area as a result of their high porosity. Thus, monoliths have been reported to be more suitable for the isolation of large biomolecules (e.g. proteins) rather than small molecules. Monoliths also have a tendency to be hydrophobic and thus adsorb proteins via both ion-exchange and hydrophobic interaction. This characteristic makes proteins difficult to elute from the monolith without solvent which can lead to protein denaturation (Nordborg & Hilder, 2009). Research to develop polymeric monoliths with less hydrophobic characteristics or modification of monolith surfaces with hydrophilic coatings is being undertaken to improve their effectiveness in the isolation of biological molecules. 5.3.4 Applications Monoliths have been employed to separate a range of food-derived biomolecules and nutraceuticals. These include enzymes such as pectin methylesterase, endo-polygalacturonase (Vovk et al., 2005; Vovk & Simonovska, 2007), lysozyme (Svec & Fréchet, 1995), and proteins such as trypsin inhibitor (Svec & Fréchet, 1995), bovine serum albumin (Du et al., 2007), lactoferrin (Adam et al., 2008), casein peptides (Gu et al., 2006), and virus particles


158 Separation, extraction and concentration processes (Kramberger et al., 2004). Hilder et al. (2004) has also demonstrated that monoliths coated with quaternary amine-functionalized latex particles can effectively separate sugars (Fig. 5.4), and separate glucose and maltose from amylase-hydrolyzed corn starch. There are a number of studies indicating that monolithic adsorbents have potential for the preparative-scale separation and purification of biomolecules, such as immunoglobulins (Brne et al., 2007), a-lactalbumin, enzymes, plasmid DNA, viruses and cells (Jungbauer & Hahn, 2008). In addition, monolithic columns have been evaluated for their potential to isolate immunoglobulins using simulated moving bed chromatography (Pennings et al., 2008). 5.3.5 Commercialization Currently, a number of companies are manufacturing and distributing polymeric monolithic columns for analytical applications. These include BIA Separations (Villach, Austria), Dionex (Sunnyvale, USA), and BioRad (Hercules, USA). BIA Separations are one of only a few companies designing and supplying organic monoliths for both small- and large-scale purification. Their disk format monoliths are manufactured for small-scale separations using axial flow design characteristics, and their tube format monoliths are prepared for 3 1

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Fig. 5.4 Separation of carbohydrates within 10 min using an optimized anionexchange latex-coated polymeric monolithic capillary column. Peaks: (1) d(+) galactose, (2) d(+)glucose, (3) d(+)xylose, (4) d(+)mannose, (5) maltose, (6) d(–) fructose, (7) sucrose (Hilder et al., 2004). Reproduced with permission. Copyright Elsevier (2004).


Novel adsorbents and approaches for nutraceutical separation 159 larger scale separation using radial flow design characteristics. The latter design is employed for large columns (e.g. up to 8 L) and is reported to allow for a significant increase in the linear velocity through the monolithic bed, without adversely affecting performance or pressure characteristics. Optimization of surface chemistry and adsorption capacity is a prerequisite for the application of organic monoliths in large-scale isolation of nutraceuticals, together with a determination of the economic benefit of using such adsorbents over traditional particle-packed columns. As manufacturing processes are optimized, and costs reduced, larger monolithic columns will deliver cost-efficient nutraceutical separation and isolation processes in the food industry.

5.4 Stimuli-responsive resins and applications in the nutraceutical industry Polymers that undergo a significant change in their structure and behavior (e.g. solubility) in response to an external physical, chemical or electrical stimulus are termed ‘stimuli-responsive’, ‘smart’ or ‘intelligent’ polymers. A number of polymers have this property and various stimuli, including temperature, pH and light can be applied to induce a change in polymer conformation and subsequent properties. Stimuli-responsive polymers with temperature responsiveness have been extensively studied and characterized. These polymers have been grafted onto chromatographic supports to produce stimuli-responsive resins. These novel resins have the potential to reduce solvent usage, processing costs and the environmental impact of nutraceutical isolation in the food industry. The best characterized temperature-responsive polymer is poly(Nisopropylacrylamide (poly-NIPAAm). This polymer differs from traditional polymers as its solubility decreases as the temperature of the poly-NIPAAm solution increases. When a critical temperature is reached (32 °C for polyNIPAAm), hydrogen bonding of water to the amide group is reduced and the poly-NIPAAm becomes unstable, contracts and enters a globular state (Fig. 5.5). The temperature at which this occurs is called the lower critical solution temperature (LCST) (Ayano & Kanazawa, 2006). The LCST of temperature-responsive polymers including poly-NIPAAm can be modified by incorporating hydrophilic or hydrophobic monomers into the polymer (Ayano & Kanazawa, 2006). Hydrophilic monomers generally increase the LCST of the polymer whereas incorporation of hydrophobic monomers generally decrease the LCST of the polymer. 5.4.1 Synthesis Temperature-responsive resins are synthesized by grafting temperatureresponsive polymers to the surface of solid chromatographic supports. Silica © Woodhead Publishing Limited, 2010


H 2O PNIPAAm

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Above LCST

Fig. 5.5 Coil to globule transition and subsequent solution turbidity change when poly-NIPAAm (PNIPAAm) is heated above or cooled below the lower critical solution temperature (LCST) (Ayano & Kanazawa, 2006). Reproduced with permission. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

has been the predominant support employed using radical co-polymerization of initiator immobilized silica or activated ester amine coupling to aminopropylmodified silica (Maharjan et al., 2008). Temperature-responsive co-polymers that have been grafted onto silica beads for chromatography include polyNIPAAm, poly-NIPAAm-co-butyl methacrylate, poly-NIPAAm-co-acrylic acid, and poly-NIPAAm-co-acrylic acid-co-N-tert-butylacrylamide (Maharjan et al., 2008). The use of silica matrices for the separation of nutraceuticals in the food industry is limited owing to their lack of operational robustness, including their instability at the high pH employed during cleaning processes (Maharjan et al., 2008). A temperature-responsive resin employing a porous crosslinked agarose support was recently reported. This resin was synthesized by functionalizing agarose beads with the initiator 4,4-azobis(4-cyanovaleric acid), followed by polymerization of N-isopropylacrylamide, tert-butylacrylamide, acrylic acid and the cross-linking agent, N,N-methylenebisacrylamide onto the surface using free radical polymerization (Maharjan et al., 2009). 5.4.2 Applications Ion-exchange chromatography There have been a number of studies examining the effect of pH and temperature-responsive resins on retention and separation of biomolecules by ion-exchange chromatography. Temperature-responsive poly-NIPAAmco-acrylic acid-co-N-tert-butylacrylamide silica beads with cation-exchange functionality have been synthesized and employed to separate bioactive peptides and amino acids. This temperature-responsive silica resin showed greater retention and separation of basic bioactive peptides at higher temperatures


Novel adsorbents and approaches for nutraceutical separation 161 than at lower temperatures when using aqueous conditions (Kobayashi et al., 2002; 2003). Similarly, silica beads grafted with poly-NIPAAm-cobutylmethacrylate-co-dimethylaminopropyl acrylamide show better separation of small bioactive molecules (anti-inflammatory compounds) at 50 °C than at 10 °C when using an isocratic aqueous mobile phase (Ayano et al., 2006). Although changes in the retention and separation behavior of molecules have been demonstrated with temperature responsive silica resins, there have been a minimal number of reports showing the effective capture and release of biological molecules. Research recently published using porous cross-linked agarose resins has demonstrated the potential of temperature-responsive cation-exchange agarose-based resins to selectively capture and isolate charged cationic proteins such as lactoferrin from mixed protein systems (Maharjan et al., 2009). The temperature-responsive agarose resin had a 3-fold higher lactoferrin adsorption capacity at 50 °C than at 20 °C (Fig. 5.6), and was able to selectively adsorb lactoferrin from a mixture of lactoferrin, a-lactalbumin and b-lactoglobulin. In addition, dynamic studies indicated that approximately 50% of the adsorbed lactoferrin could be eluted from the agarose by simply reducing the temperature. The remaining lactoferrin was eluted using low concentrations of NaCl (Maharjan et al., 2009). Hydrophobic-interaction chromatography A significant amount of research has been undertaken to develop and understand temperature-responsive resins for hydrophobic-interaction chromatography. For example, temperature-responsive poly-NIPAAm-co-butyl methacrylate functionalized silica beads have been employed to separate a mixture of insulin chains A and B, and b-endorphin fragment 1–27 (Fig. 5.7). Temperature Adsorbed lactoferrin (mg mL–1)

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Fig. 5.6 Equilibrium adsorption isotherms of lactoferrin onto poly-NIPAAm-coacrylic acid-co-N-tert-butylacrylamide agarose resin at 20 °C (continuous line) and 50 °C (dashed line) (error bars represent the standard deviation from the mean) (Maharjan et al., 2009). Reproduced with permission. Copyright Elsevier (2009).


162 Separation, extraction and concentration processes 1, 2, 3 2

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Fig. 5.7 Separation of a mixture of insulin chains A and B, and endorphin fragment 1–27 on a poly-NIPAAm-co-butyl methacrylate modified silica resin. Column temperature: (a) 0 °C, (b) 40 °C. Mobile phase: aqueous 0.17M NaCl. Flow-rate: 0.5 ml min–1. Detection: 215 nm (Kanazawa et al., 1997). Reproduced with permission. Copyright Elsevier (1997).

gradients were able to alter the hydrophobicity of the resin, resulting in temperature-modulated peptide elution from the column (Kanazawa et al., 1997). In addition, temperature-responsive poly-NIPAAm silica beads have been employed to separate steroids with an aqueous mobile phase (Kanazawa et al., 1996). The steroids were unresolved at temperatures below the LCST and well resolved above the LCST. The improved resolution at temperatures above the LCST was most probably the result of increased hydrophobic interaction of the steroids with the poly-NIPAAm stationary phase at the higher temperatures. Size-exclusion chromatography Temperature-responsive polymers have also been applied to synthesize temperature-responsive size-exclusion resins. For example, poly-NIPAAm was grafted onto porous glass beads, the poly-NIPAAm end functionalized with mercaptopropionic acid, and the resulting resin employed for gel permeation size-exclusion chromatography (Gewehr et al., 1992). The elution time of the dextrans was substantially altered between 25 °C and 32 °C, possibly owing to a change in the effective pore size of the resin via transition of the poly-NIPAAm chains from coils to globules on the surface of the pores of the glass beads. Porous polystyrene beads grafted with poly-NIPAAm have also shown temperature-responsive size-exclusion characteristics (Hosoya et al., 1994). In this example, an increase in temperature prolonged the elution time of higher molecular weight dextrans, possibly owing to collapse of the poly-NIPAAm causing the pore size to expand, and thus permitting the dextrans to penetrate the pores.


Novel adsorbents and approaches for nutraceutical separation 163 Affinity separations Temperature-responsive polymers can also be conjugated to proteins and employed as affinity separation materials. Cycling below and above the LCST of the temperature-responsive polymer induces reversible precipitation/ solubilization of the bioconjugates in the aqueous solution, thereby allowing separation of the bioconjugate and the target molecule. Proteins such as a-chymotrypsin (Kim & Park, 1998) and a-glucosidase (Hoshino et al., 1998) were purified using this method. In addition, temperature-dependent isolation of lactate dehydrogenase from porcine muscle was achieved using a dye-affinity agarose modified by physically adsorbing the smart polymer poly-N-vinylcaprolactam onto the surface. Using this method, the lactate dehydrogenase was adsorbed to the column at 40 °C and eluted by reducing the temperature to 23 °C and adding KCl (Galaev et al., 1994). 5.4.3 Commercialization Temperature-responsive polymers can be employed to separate a wide spectrum of molecules using a range of separation mechanisms (e.g. charge, size, affinity). However, temperature-responsive resins are not currently available commercially for large-scale separation applications. With further research and development into specific applications and scale-up, temperature-responsive resins with silica or other matrices could be employed commercially to isolate hydrophobic and charged nutraceutical molecules. Such processes should require less solvent (e.g. salt, ethanol), which would reduce the cost and environmental impact of such separations, and potentially improve retention of the biological activity of such isolated molecules.

5.5 Mesoporous molecular sieves and applications in the nutraceutical industry Mesoporous molecular sieves, also termed mobile crystalline materials (MCMs), are a family of inorganic solids with regular nanostructure, large internal surface area (>1000 m2 g–1) and void volume, and narrow pore size distribution. These remarkable materials, exhibiting defined and tailored pore diameters from approximately 2 nm through to 50 nm, offer new possibilities for designing highly effective adsorption media based upon their pore size and large internal surface area. Mesoporous molecular sieves, notably the MCM-4X family, were first reported in the early 1990s (Beck, 1991; Beck et al., 1992; Kresge et al., 1992), and have attracted considerable research attention since then as novel catalysts, guest supports in chemical reactions, and potential adsorbents for the isolation of biological components (Beck & Vartuli, 1996; Brady et al., 2008; Kisler et al., 2001a; 2001b; Selvam et al., 2001).


164 Separation, extraction and concentration processes 5.5.1 Synthesis Mesoporous molecular sieves are prepared using a technique called liquidcrystal ‘templating’, illustrated schematically for MCM-41 in Fig. 5.8. In this process, mesoporous solids are synthesized by thermal treatment and ultimate calcination of aluminosilicate gels in the presence of suitable surfactants. Unlike other mesoporous inorganic materials, such as silica, the MCM family of mesoporous molecular sieves comprise regular arrays of non-intersecting uniform hexagonal channels (Fig. 5.8 and 5.9), the dimensions of which can be tailored within 1.5–50 nm through the selection of different surfactants (‘template molecules’) and reaction conditions (Gusev et al., 1996; Beck, 1991; Kresge et al., 1992). The pore size can be tailored during synthesis to accommodate biological molecules of almost any size (Fig. 5.9). Hexagonal array

Silicate

Calcination

MCM-41

Fig. 5.8 Schematic illustration of the liquid-crystal ‘templating’ technique for manufacture of mesoporous molecular sieves such as MCM-41 (Kresge et al., 1992). Reproduced with permission of Macmillan Publishers Ltd: Nature. Copyright Macmillan Publishers Ltd (1992).

Fig. 5.9 Model of the molecular sieve MCM-41 illustrating methane and ethane inside one of the hexagonal pores (pore diameter ~3 nm).


Novel adsorbents and approaches for nutraceutical separation 165 5.5.2 Advantages and disadvantages The high internal surface area, tailored pore size, and very narrow pore size distribution of these mesoporous molecular sieves are characteristics that make them ideal candidates as ‘designed media’ for size-exclusion and adsorptionbased separation of biological molecules, notably proteins, peptides and other functional ingredients (Brady et al., 2008; Kisler et al., 2001a,b). For these types of applications, the best studied mesoporous molecular sieves include MCM-41 and MCM-48 (Melo et al., 1999; Zhao et al., 1996). Although the uniform mesoporous pore size makes these materials attractive candidates for adsorption and separation of biological macromolecules, their stability in aqueous systems is limited (Kisler et al., 2001b). Such instability has been a major limitation in the widespread use of these materials for waterbased extractions, and in the separation and isolation of targeted bioactive components for use as nutraceuticals. In order to overcome this shortcoming, many researchers have modified the mesoporous materials with a hydrophobic coating to assist in stabilizing the material in water (Brady et al., 2008; Kisler et al., 2001b). Such modification of MCM-41, for example, has enhanced the utility of this mesoporous molecular sieve in biochemical applications (Brady et al., 2008; Kisler et al., 2001a; 2001b). These materials have also been studied as processing aids in water treatment and in the generation of potable water (Cooper & Burch, 1999). 5.5.3 Applications Size-based separations The utility of mesoporous molecular sieves as size-exclusion materials in the isolation and separation of proteins was first examined by Diaz and Balkus (1996) and subsequently by Washmon-Kriel et al. (2000) in studies of cytochrome C permeation and adsorption. This research showed a correlation between protein adsorption and the pore size of the molecular sieve being examined, as would be expected, and retention of biological activity, an important consideration when designing a separation protocol for isolation of bioactive components like nutraceuticals. Takahashi et al. (2000; 2001) and Vinu et al. (2004) also showed strong correlation between enzyme immobilization and pore size in a study of several macroporous molecular sieves including MCM-41. The isolation of both large and small biological molecules using MCM-41 and MCM-48 has been explored (Brady et al., 2008; Kisler et al., 2001a; 2001b; Xue et al., 2004). Trypsin, lysozyme and riboflavin have been studied as model biological solutes in order to examine the size-exclusion characteristics of these mesoporous materials and their potential as bioseparation media (Kisler et al., 2001a). Similarly, Brady et al. (2008) explored the potential of hierarchical mesoporous silica sieves in the isolation of valuable dairy bioactive components as functional food ingredients, and the feasibility of such materials as industrial-scale adsorbents.


166 Separation, extraction and concentration processes Protein stabilization Stabilization of labile enzyme activity, both in aqueous and organic environments, may well be an important application for mesoporous molecular sieves. MCM-48 was used to immobilize penicillin G acrylase and to stabilize the activity of this enzyme (Xue et al., 2004), and was shown to be superior to MCM-41 for this application. Kim et al. (2006) provided a comprehensive review of the probable mechanisms involved in stabilization of enzymes by various nanostructures, including mesoporous molecular sieves, which include confinement, charge and hydrophobic interaction, and multipoint attachment to the protein. Wang & Caruso (2005) reported the usefulness of mesoporous silica and modified forms thereof as supports for immobilization and encapsulation of enzymes, and stabilization of biological activity, demonstrating success of the technology with catalase. Mesoporous sieves may also provide utility in expanding the application scope of enzymes for processing in organic solvents by stabilizing their catalytic activity in such environments (Takahashi et al., 2001; Yan et al., 2002). 5.5.4 Commercialization The highly regular structure, tailored and tuneable properties, uniform pore size distribution and high internal surface area of selected mesoporous sieves represent attractive properties for the design of modern separation materials and protocols based upon size-exclusion and surface-interaction chemistry. However, before their industrial potential can be realized and these characteristics exploited, the long-term stability and safety, effectiveness in processing agrifood streams, and scaleability and process economics must be established.

5.6 Peptide affinity ligands and phage display methodology and applications in the nutraceutical industry Affinity chromatography is a method for separating biochemical components, often macromolecules (proteins, peptides, nucleic acids), from complex mixtures based on a highly specific biological interaction such as that between an antigen and antibody, substrate and enzyme, or ligand and receptor. The rationale for this process was first reported in the early part of the 20th century (Starkenstein, 1910). However, it was not until the late 1960s, associated with developments in modern chromatographic resins and support materials, and chemistry/linking reactions for immobilization of functional ligands, that affinity chromatography emerged as a viable industrial technology for the isolation of valuable biological components, including nutraceuticals (Cuatrecasas & Anfinsen, 1971; Wilchek & Miron, 1999). Although affinity chromatography is a very powerful technique and has revolutionized the fields of modern biochemistry, biotechnology and medical science over the past 40 years, it is not without its challenges. These relate © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 167 to the preparation of the affinity adsorbent (ligand and resin support) and the use of the adsorbent in processing, and include considerations of selectivity, functionality, stability, efficiency, capacity, repeatability, and productivity. The choice of functional ligand to be immobilized and to effect the affinity separation is vast, and is dictated by the characteristics of the molecule targeted for isolation. Such functional ligands include proteins, enzymes, peptides, nucleic acids, sugars, vitamins, lipids, synthetic organic molecules (e.g. dyes), and metal ions. Once the ligand is selected, immobilization chemistry, maintenance of biological functionality, stability, avoidance of steric effects associated with the resin support, and effectiveness of the adsorbent under processing conditions, among others, must be considered (Wilchek & Miron, 1999). In the design of modern affinity adsorbents, the selection of ligands with broad applicability that meet most of these requirements is important in addressing the cost-effectiveness consideration critical for industrial processes. In this regard, peptides offer an attractive option (Amatschek et al., 2000; Pflegerl et al., 2002). 5.6.1 Advantages and disadvantages The main advantage of small peptides is their high affinity and selectivity. In addition, they are usually stable to mild chemical, physical and biological conditions, and not prone to degradation by protease enzymes found in most agri-food streams. Peptides are also amenable to a variety of linkage chemistries that provide for a very stable covalent bond between peptide and resin support. Tailored peptides of almost any composition and sequence can be readily synthesized through modern solid-phase techniques. Parallel and combinatorial approaches to directed peptide synthesis can readily result in large peptide libraries which can be rapidly screened for their affinity to a target compound (Pflegerl et al., 2002). Although peptide affinity resins have high affinity and selectivity, they can have relatively low binding capacity, increasing the overall cost of nutraceutical manufacture and the cost of the final product. In addition, manufacturing specific peptides on a large scale can be time consuming and expensive. Research groups are currently working on methodologies to improve the binding capacity of peptide adsorbents and significant competition in the field of peptide production is rapidly improving manufacturing technologies and reducing costs. There are also concerns regarding the toxicity and biocompatibility of novel affinity peptides when employed to produce products for human consumption. Before peptide affinity ligands can be employed in the food industry, detailed studies to ensure that they are stable during processing and non-toxic will be required. 5.6.2 Peptide identification A technique developed recently that will perhaps revolutionize the development and selection of peptide ligands for affinity adsorbtion and other applications


168 Separation, extraction and concentration processes is called phage display, based on a concept reported in the mid-1980s (Smith, 1985). Phage display is a powerful technique for the study of protein–protein, protein–peptide, and protein–nucleic acid interactions. This approach uses bacteriophage vectors to express a protein or peptide on the surface of the virus particle coded by genetic material residing on the inside of the particle. Phage display thus creates a connection between genotype and phenotype, and facilitates the creation of a library of variants of a peptide (or protein) expressed on the outside of the virus, and allows for rapid in vitro screening of these variants based upon their binding affinity to a target molecule (e.g. enzyme, antibody, receptor, cell structural protein). This in vitro selection process is called panning, and allows for the high-throughput screening of protein interactions (Kay et al., 2001; Koivunen et al., 1999). In its simplest form, panning is carried out by incubating a library of phage-expressed peptides with the target molecule, usually in a 96-well plate, washing away any unbound phage, and then eluting the specifically bound phage. The eluted phage is then taken through additional binding and amplification cycles to enrich the pool in favor of the specific binding peptide sequence (Kay et al., 2001). In this way, phage display/panning allows for the rapid discovery of useful peptide ligands with specific affinity for the target protein or other molecules (nutraceuticals), and thus the development of effective peptide affinity adsorbents for research and industrial applications. The phage display/panning technique has been widely used in drug discovery, notably for the identification of promising new ligands (e.g. enzyme inhibitors, and receptor agonists and antagonists) to target proteins. Perhaps the most successful commercial outcome from phage display/panning technology has been the therapeutic antibody drug adalimumab (Bain & Brazil, 2003; Scheinfeld, 2003). This antibody, developed by Cambridge Antibody Technology (Cambridge, UK), targets the inflammatory cytokine TNF-a and is marketed commercially by Abbott Laboratories as HUMIRA (‘human monoclonal antibody in rheumatoid arthritis’) (Scheinfeld, 2003). 5.6.3 Commercialization The use of phage display/panning in the discovery of specific peptide– biomolecule interactions that may lead to effective affinity adsorbents was simplified through the development and sale of commercial ‘kits’ containing pre-made random peptide libraries, as well as a cloning vector for construction of custom libraries. Such ‘kits’ and related reagents are available from a number of commercial biotechnology/analytical companies, including for example New England BioLabs (Ipswich, USA), V-Biolabs (Sawston, UK), Mimotopes (Melbourne, Australia), and Cambridge Antibody Technology (Cambridge, UK) (now part of AstraZeneca). Development and refinement of phage display technology by the pharmaceutical industry provides an example of a biotechnology that can be applied in food processing applications and nutraceutical isolation. Peptide


Novel adsorbents and approaches for nutraceutical separation 169 affinity technologies are not yet commercially available for the isolation of nutraceuticals at commercial scale. However, the phage display technology represents a robust tool for research into novel peptide affinity adsorbents. With further research and development to optimize capacity and manufacturing procedures, peptide affinity resins aimed at cost-effective isolation and separation of nutraceuticals should become available in the near future.

5.7 Membrane adsorbers, membrane chromatography and applications in the nutraceutical industry A recent development in the arena of chromatographic adsorbents is that of membrane adsorbers aimed at improving the efficiency and simplicity of the chromatographic process by allowing the targeted separation to occur on and within a macroporous membrane support (see Charcosset, 1998; Klein, 2000; Przybycien et al., 2004 for reviews of alternative bioseparation processes including the use of membrane adsorbers). This process has also been termed membrane chromatography (Charcosset, 1998; Thömmes and Kula, 1995). Membrane adsorbers are porous membranes with functional ligands (such as ion-exchange or affinity groups) covalently attached to the membrane (Demmer et al., 1989) or immobilized within the membrane structure. 5.7.1 Advantages and disadvantages Membrane adsorbers have several potential advantages over traditional particle matrices and adsorbents (Charcosset, 1998). Ion-exchange and other functionalized membranes avoid problems with swelling or packing and can easily be scaled up. Membrane adsorbers have low compressibility characteristics and also tend to demonstrate lower back pressures than traditional chromatographic resin materials thus allowing for the rapid processing of large volumes of fluid. Large targeted molecules, such as proteins and large peptides, are convected through the membrane, rather than having to diffuse into a resin bead before adsorbing to the functional ligand, and thus diffusion limitations are minimized. Overall mass transfer is improved, leading to shorter cycle times and higher overall throughput (Brandt et al., 1988). These properties facilitate the use of processing, maintenance and cleaning protocols more diverse and adaptable to existing procedures in the food processing industry. Based on the advantages of membrane adsorbers over traditional chromatographic resins (Charcosset, 1998), these adsorbents, once scaled to industrial size, would appear to be best suited for the separation and isolation of targeted components, notably proteins and peptides, found in low concentrations in large volumes of liquid (Gebauer et al., 1997). Membrane adsorbers have similar disadvantages to traditional membrane technologies. For example, when processing protein


170 Separation, extraction and concentration processes containing fluids, a secondary boundary layer can form, reducing flux rates and increasing membrane cleaning requirements. In addition, as membrane adsorbers are made from organic materials, they are sensitive to the elevated temperatures and cleaning regimes often used in the food industry. These membranes must therefore be cleaned using mild conditions and replaced when their adsorption capacity or flux rates fall outside critical process specification. As with traditional membranes, large pressure drops across the membrane should also be avoided as they can cause deformation and irreversible changes in pore-size characteristics. 5.7.2 Functionalization The functional ligand chemistry available with membrane adsorbers is almost endless and is similar to that for traditional chromatographic resins, including strong and weak anion and cation exchange, affinity interaction, reversed phase, and hydrophobic interaction. Selection of the appropriate ligand chemistry, and membrane support material and configuration, will depend upon the specific targeted component of interest and separation strategy, source and ultimate end-use application of the isolate. 5.7.3 Configurations Improvements in membrane materials, equipment for conducting membrane chromatography, and the means by which the targeted fluid is presented to the membrane give the user choice in designing the best combination of membrane adsorber, device, and geometric configuration to achieve the most cost-effective separation of the targeted compound. Macroporous functionalized membranes are available as single or stacked flat sheets, hollow fibers, spiral-wound sheets, dead-end/single-use filters, and cassettes from several established commercial manufacturers (e.g. Pall, Sartorius) and some new entrants in the field (e.g. Mosaic Systems, Natrix Separations). These configurations allow for simple dead-end filtration applications, but also the more commercially relevant cross-flow processing for the treatment of large volumes of liquid in a short timeframe. The benefits of membrane adsorbers are most apparent in flow-through applications because capacity constraints in retention mode, particularly at high loading rates, make resin chromatography a more attractive option for capture steps. 5.7.4 Applications Ion-exchange separations Ion-exchange (anion and cation) functionalized membrane adsorbers have been the most extensively researched as cost-effective alternatives to traditional resin chromatography for the isolation of valuable minor protein and peptide constituents from agri-food streams, notably dairy whey (Adisaputro et al.,


Novel adsorbents and approaches for nutraceutical separation 171 1996; Mitchell et al., 1994; Yang et al., 1999; Zietlow & Etzel, 1995). Attention has been devoted to the use of strongly acidic membrane adsorbers in the isolation of lactoferrin and lactoperoxidase from cheese whey (Chiu & Etzel, 1997; Mitchell et al., 1994), although some attention has also been paid to the growth factor constituents (Mitchell et al., 1994). Glycosylated and non-glycosylated forms of the glycomacropeptide have been isolated from whey using anion-exchange membrane chromatography (Kreub & Kulozik, 2009; Kreub et al., 2008). In the former study, the authors describe a direct-capture method for the isolation of glycosylated glycomacropeptide at pilot scale using a membrane adsorption process. Membrane ion-exchange chromatography has also been described for the isolation of bovine serum albumin (He & Ulbricht, 2008; Sarfert & Etzel, 1997), soybean trypsin inhibitor (He & Ulbricht, 2008; Josic & Strancar, 1999), patatin from potato (Alt et al., 2004), a-lactalbumin (Yang et al., 2002), a large protein, thyroglobulin (Yang et al., 2002), and in the large-scale production of antibodies (Zhou & Tressel, 2005; 2006; Zhou et al., 2006). Affinity separations Integration of membrane processing and affinity chromatography by way of affinity membrane adsorbers provides a number of advantages over traditional affinity chromatography with resin packed columns, notably in respect to time and recovery of bioactivity (Brandt et al., 1988; Klein, 2000; Zou et al., 2001). Such membrane adsorbents have been applied to the isolation of pepsin and chymosin using an immobilized pepstatin A membrane (Suen & Etzel, 1994), and to the separation of specific antibodies using membraneimmobilized bovine serum albumin, soybean trypsin inhibitor, Protein G, and immunoglobulin G (Kochan et al., 1996; Ostryanina et al., 2002). Affinity membranes can be dominated by slow sorption kinetics thus potentially impacting the performance of the separation (Suen & Etzel, 1994). Many of the protein and peptide examples shown have the potential to serve as valuable nutraceuticals in functional food applications (Etzel, 2004; McIntosh et al., 1998), and as natural antibacterial and bioactive agents and thus enter areas of both human and veterinary medicine, and biotechnology (Dionysius et al., 1993; Perraudin, 1991; Pouliot & Gauthier, 2006; Regester & Belford, 1999; Smithers, 2004; 2008). Micro-organism removal Membrane adsorbers provide an attractive alternative to traditional resinbased chromatography columns used to remove trace impurities and microbial contaminants (e.g. virus particles) in downstream processing applications, notably those requiring good manufacturing practice (GMP) compliance (Etzel & Riordan, 2006; Zhou et al., 2008). These membranes have been reported to reproducibly achieve greater than 4-log removal of mammalian viruses and greater than 3-log removal of endotoxin and contaminant DNA (Zhou et al., 2008).


172 Separation, extraction and concentration processes 5.7.5 Commercialization Pall Filtration (Port Washington, USA) and Sartorius (Goettingen, Germany) are perhaps the best known manufacturers of membrane adsorbers for research and small industrial use. These manufacturers have targeted agrifood, biotechnology and biopharmaceutical processing applications for their membrane adsorber products, primarily the ion-exchange offerings. There are a number of citations in the scientific literature on the effective use of membrane ion exchangers in the isolation of biological molecules (He & Ulbricht, 2008; Mitchell et al., 1994; Przybycien et al., 2004; Yang et al., 1999). Several new manufacturers have introduced further innovations into the field that improve the inherent efficiency and simplicity of membrane adsorbers. For example, Mosaic Systems (Ba Breda, The Netherlands) is a biotechnology enterprise with a specialization in commercial-scale separation and isolation of components for the food ingredient and biopharmaceutical industries. This company has developed a novel mixed-matrix membrane adsorber that comprises functionalized resin beads (5–10 mm) immobilized in a macroporous membrane. The company claims that the embedded resin beads retain full chromatographic functionality resulting in the products’ selectivity and binding capacity, but that the macroporous membrane decouples pressure drop from the resin particle size leading to enhanced hydrodynamic performance. This mixed-matrix system leads to improved throughput and productivity compared with an equivalent traditional chromatographic system, minimal fouling, reduced water and buffer use, and retention of efficiency and specificity through multiple adsorption cycles. Mosaic Systems claims their mixed-matrix adsorbers have been proven in trials with customers for the manufacture of food ingredients, biopharmaceuticals, and the isolation of valuable components from dairy streams. Natrix Separations (Burlington, Canada) (formerly Nysa Technologies) has developed a patented polymeric hydrogel technology that combines the high binding capacity, selectivity and specificity associated with traditional chromatographic resins with the high throughput and ease of use of macroporous membranes (Childs et al., 2008). The membrane adsorber consists of a polymeric hydrogel formed within a flexible porous support matrix. The matrix provides mechanical strength, whereas the hydrogel characteristics determine the separation chemistry of the product. The hydrogel polymer provides high binding site density, a large surface area for binding, and rapid mass transfer supporting high flow rates while providing highly efficient capture of the target molecule (Childs et al., 2008).

5.8 Conclusions and sources of further information and advice There appear to be many advantages to the application of ‘next-generation’ separation materials, such as those covered in this chapter, in the isolation © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 173 of nutraceuticals. These advantages include improved specificity, selectivity, simplicity, robustness, productivity and reduced environmental impact. However, several of these materials do remain on or over the horizon from an industrial-scale perspective. Further research is needed to establish: (i) large-scale manufacturing procedures for a number of these novel adsorbents; (ii) specific scaled applications in nutraceutical manufacture; (iii) their processing advantages over traditional chromatographic materials and devices; and (iv) perhaps of most importance from an industrial perspective, process economics and commercial feasibility. The reader is directed to the following websites for further information about the novel adsorbents and approaches covered in this chapter. ∑ ∑ ∑ ∑ ∑ ∑

Molecular imprinted polymers: www.MIPtechnologies.com www.molecular-imprinting.com http://www.sigmaaldrich.com/analytical-chromatography/samplepreparation/spe/supelmip.html Organic monoliths: www.bio-labs.com www.BIAtechnologies.com www.dionex.com Stimuli-responsive polymers: http://macromolecules.case.edu/research_stimuli-responsive.htm Mesoporous molecular sieves: www.mrs.org Peptide affinity ligands and phage display methodology: www.neb.com www.vbiolabs.com www.mimotopes.com www.cambridgeantibody.com Membrane adsorbers and membrane chromatography: www.pall.com www.sartorius.com www.mosaicsystems.nl www.natrixseparations.com

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6 Advances in the effective application of membrane technologies in the food industry M. Pinelo, G. Jonsson and A. S. Meyer, Technical University of Denmark, Denmark Abstract: This chapter focuses on the recent advances in the use of membrane technology for efficient separation and concentration of solutes in the dairy and fruit juice industry, as well as advances in the purification of bioactive compounds to be used as food additives. The importance of fouling reduction is emphasized because this is necessary for membrane processes to become economically feasible. Key words: membranes, dairy industry, fruit juice, fouling, bioactive compounds, membrane bioreactors.

6.1 Introduction Membrane separation technologies have attracted much attention in the food processing industries over recent decades. The main reason for the interest is that membranes allow efficient separation and concentration of solutes without changing phase, maintaining worthy chemical and physical properties of food components and systems with particular economical relevance. This chapter primarily focuses on the use of membrane technology in the dairy and fruit juice industries. The incorporation of new, advanced membrane filtration techniques generally occurred first in the dairy industry, and was only later incorporated into other food manufacturing processes. Fouling is still the main challenge to overcome before use of membrane technologies becomes economically feasible in industrial processes. Various techniques to reduce fouling, related to the dairy industry, are discussed in the first section of the chapter. In the fruit juice industry, membrane technology is mainly used for concentration and clarification purposes. The main challenge is to


Advances in the effective application of membrane technologies 181 reduce the volume of juice maintaining the aroma compounds, which are otherwise lost when concentration methods such as evaporation are employed. The most recent advances in reverse osmosis and membrane distillation are reviewed, as these techniques are the most promising ones to maintain aroma compounds and juice quality. Wastewater treatment in food processing is also discussed in the chapter, as regulation regarding purified water quality and landfill management are becoming more and more restrictive. We pay special attention to wastewater treatment by using membrane bioreactors (MBR), particularly anaerobic MBR, on which research has focused on the recent years. Membrane separation is also a promising down-stream technique for purification of food ingredients and additives. We present the current challenges and limitations of applying membrane separation, coupled or not with enzyme technology, to purification and fractionation of oligosaccharides, as an example of an integrated process where membrane technology can act in synergy with other unit operations and processes.

6.2 Theoretical fundamentals of membrane separation Implementation of membrane technology in the dairy and fruit juice industry is determined by the economical feasibility of the process, which is directly associated with the separation efficiency and impact of fouling. Several theories and models have been developed in order to relate separation efficiency to the separation set-up, molecular features of solute, solvent and membrane, and operational conditions during filtration. One of these models (Jonsson and Boesen, 1975; Jonsson et al., 2008) is based on the transport of solvent and solute through the membrane pores and the friction of the solute particles and the pore wall. The model relates separation efficiency to several physical parameters: bˆ cm Ê t l Jv ˆ Ê [6.1] = 1 = b + 1 – ˜ exp Á – cp 1 – R K ÁË K¯ Ë e Di ˜¯ where cm and cp are the solute concentrations on the membrane surface and at the permeate, respectively. R is the rejection factor, b is the friction coefficient (dependent on solute and pore diameter), K is the distribution factor between pore fluid and bulk solution, t is a tortuosity factor, l is the real thickness of the fouling layer, e is the fractional pore area, Jv is the volumetric flow per unit membrane area and Di is the diffusion coefficient for a component of the solute. In certain conditions, e.g. high fluxes, concentration polarization can have a particular relevance on membrane performance. There are equations to quantify the influence of concentration polarization (e.g. Jonsson, 1985):

cm – cp ˆ Ê = exp Á J v d ˜ Ë Di ¯ cb – cp


[6.2]

182 Separation, extraction and concentration processes and

Di =

kT 3 ph di

[6.3]

where c b represents the solute concentration in the bulk solution, d the polarization layer thickness, k the Boltzmann’s constant, and h the viscosity. These models and equations can be used as a tool to determine preliminarily, within a certain error interval, the efficiency of a particular process that could eventually be implemented at a large scale.

6.3 Membrane technology in the dairy industry Use of membrane technology in the dairy industry represents ~40% of the total applications of membranes in food processes (Daufin et al., 2001). Both polymeric and ceramic membranes are used for concentration purposes in the dairy industry. Among the polymeric ones, spiral cartridges of polysulfone or polyethersulfone are usually chosen, and tubular ceramic membranes of zirconium or aluminum oxide are also widely used. Ultrafiltration is the most widely used membrane separation process in the dairy industry (D’Souza et al., 2005). Ultrafiltration is used, for example, for concentration of milk before the cheese-making process or to reduce shipping volumes as well as for whey concentration, resulting in a protein-enriched stream which is being examined for novel applications, and which may have potential antiinflammatory and anti-cancer properties. 6.3.1 Fouling as a major concern in the dairy industry Fouling is the major concern for the dairy industry nowadays. In recent years, research efforts have aimed at the development of ultrafiltration systems with enhanced life time that can efficiently reduce the concentration polarization and fouling (Nigam et al., 2008; Rice et al., 2009a). Concentration polarization is provoked by an increasing solute concentration on the membrane surface owing to the transport of solute during convective solvent flux, which is rejected and accumulated on the membrane surface. The accumulation results in a back-diffusion of solute into the solution in the retentate side, hence creating a concentration gradient. As a consequence, an initial drop of permeate flux is observed, followed by a further progressive decrease caused by fouling. During microfiltration of skim milk for removal of bacterial spores, Guerra et al. (1997) observed a peak of permeate flux at 0.25 bar of transmembrane pressure, followed by a continuous decrease of permeate flux at increasing pressures (Fig. 6.1). After reaching 1 bar, the pressure was diminished, resulting in lower permeate fluxes than the ones obtained during


Advances in the effective application of membrane technologies 183 the increasing pressure phase. This confirmed the influence of fouling on the permeate flux. Although theoretically the flux stabilizes when the steady-state is reached, in practice, the flux continues to decrease slowly with continued filtration. Also, fouling reduces dramatically the useful life of membranes (Rice et al., 2009b). The cleaning treatment is a key factor influencing the whole plant productivity. 6.3.2 Cleaning of membranes in the dairy industry D’Souza et al. (2005) made a complete study about the causes of membrane fouling as well as the main cleaning processes used in the dairy industry. They classified the cleaning agents into six different categories: (1) alkalis, which hydrolyze proteins and carbohydrates; (2) acids, for dissolving salts and oxides; (3) enzymes, particularly proteases and lipases; (4) surfactants, which decrease surface tension and charge; (5) sequestrants, for removal of minerals; and (6) disinfectants, since one of the main targets of the cleaning process is also the reduction of microbial load. A cleaning process generally involves three steps: product removal from the membrane and water rinsing; use of a single or a combination of appropriate cleaning agents followed by water rising; and disinfection (Trägårdh, 1989). In most cases, an acid–alkali treatment is used, the concentration of each cleaning agent being a critical parameter for each cleaning situation (Tohammadi et al., 275 250 225 Flux (L h–1 m–2)

200 175 150 125 100 75 50 25 0 0.0

0.1

0.2

0.3

0.4 0.5 0.6 0.7 0.8 Transmembrane pressure (bar)

0.9

1.0

1.1

Fig. 6.1 Permeate flux versus transmembrane pressure during microfiltration of skim milk for removal of bacterial spores using a ceramic membrane.The arrows show the direction in which the transmembrane pressure was changed. Adapted from Guerra et al. (1997).


184 Separation, extraction and concentration processes 2002). Moreover, temperature and mechanical aspects i.e. flow rate of the cleaning solution and applied pressure during the cleaning process have to be carefully selected for each particular situation (Bartlett et al., 1995; Bird et al., 1995). The election of a proper cleaning method is also an important factor to consider in nanofiltration and reverse osmosis processes, which also have a major presence in the dairy industry. Nanofiltration is currently used, for example, to remove salt from whey as well as for the recovery of lactose from the permeate of the ultrafiltration step. Reverse osmosis has proved to be useful for whey concentration and for obtaining concentrated lactose by removing salt and minerals. 6.3.3 Recent advances for fouling reduction in the dairy industry In recent years, patents and publications on membrane technology applied to the dairy industry have focused, almost exclusively, on fouling reduction by (a) proposing new cleaning processes, (b) by optimizing the existing ones or (c) by coupling devices to the membrane set-up particularly designed to reduce the fouling, e.g. rotation/vibration and electric fields (see Section 6.7). Some have reported the advantage of using rotating or vibrating systems to enhance permeate flux and membrane selectivity (Frappart et al., 2006; Genkin et al., 2006; Jaffrin, 2008), providing promising results ready to be extrapolated to large-scale processes. Al-Akoum (2006) found a strong correlation between the permeate flux and the vibration speed in ultrafiltration of soy milk at 25 °C. The permeate flux increased ~10 times when using disks with 6-mm vanes at 11,000 rpm compared with smooth disks at 2500 rpm. Genkin et al. (2006) doubled the critical flux when using transverse vibration on submerged hollow fiber membranes, and with combined axial and transverse vibration, a five-fold increase of critical flux was observed. Frappart et al. (2006) increased the permeate flux from 130 to 230 L h–1 m–2 during recovery of lactose from diluted milk when using a rotating disk with vanes at 2000 rpm compared with a smooth disk at 1000 rpm. The same authors compared the performance of a vibrating system to the rotating disk, concluding that the rotating system is more efficient in reducing the fouling, owing to its higher shear rate. A recent patent (Makardij-Tossonian, 2009) proposes the use of an enzyme solution propelled by injecting gas to synergistically combine the effect of the enzyme solution on the breakdown of the particles causing fouling with the mechanical de-fouling effect promoted by the gas. The patent was especially developed for dairy food and beverage processing. Other inventions are more focused on the use of cleaning solutions than on the use of mechanical systems to prevent fouling. Flemming and Skou (2006) patented a system to reuse the cleaning solution by filtering it through the membrane, providing good results for ultrafiltration of dairy products. Omprakash et al. (2008) found that a 0.2% caustic solution provided the best performance for cleaning of fouled membranes used for concentration of whey protein. Measurement of fouling is another critical factor which


Advances in the effective application of membrane technologies 185 has also been the subject of some patents. Mickols et al. (2008) conceived a system for on-line measurement of fouling in spiral wound membranes with particular potential for the dairy industry. It is based on the difference between acoustic impedances on the outer surface for a cylindrical wall of a permeate collection tube. Other systems applied other physical principles to measure the degree and nature of fouling, but the high costs associated with their implementation on a large scale is still hampering utilization at an industrial level. It can be concluded that the implementation and degree of profusion of membrane technology into the dairy industry over the coming years will be dependent on the economic impact that the several presented strategies for fouling reduction can have on the overall economy of the process. The most promising strategies are: (1) mechanical removal of fouling by using, for example, vibrating systems; (2) optimization of the conditions of use of specific chemical agents; and (3) use of enzymes able to breakdown the structure of the particular compounds causing the fouling, e.g. proteases for whey solutions (Nigam et al., 2008).

6.4 Membrane technology in the fruit juice industry Membrane technology is mainly used for clarification and for concentration of fruit juices, replacing the traditional evaporation (Table 6.1). There are several advantages of membrane processes over evaporation in fruit juices manufacture: (1) a higher quality of the final juice because of the reduction in the loss of aroma and other compounds with nutritional value; (2) lower energy consumption; and (3) versatility of the equipment which can be used to treat different products (Jiao et al., 2004). Use of traditional vacuum evaporation for concentration of fruit juices usually results in generation of off-flavours, the loss of aroma compounds and, in some cases, color degradation. Despite the drawbacks mentioned, vacuum evaporation is still the most widely used technique for concentration of juices in industry (GEA, 2009). This is probably ascribable to the fact that evaporation permits higher concentration efficiency than membrane treatments, which also in general, have, higher operational costs. 6.4.1 Reverse osmosis Amongst the membrane separation techniques used for juice concentration, reverse osmosis is the one of major interest for industry. The main advantage


186 Separation, extraction and concentration processes Table 6.1 Some examples of utilization of membrane technology for concentration and clarification of fruit juices Application

Type of filtration

Conditions

Reference

Concentration of apple juice

Reverse osmosis

Spiral wound cellulose acetate and polyamide membranes 21.1–26.7 °C

Chua et al., 2007


Reverse osmosis and ultrafiltration

Plate and frame cellulose acetate membrane 20 °C

Sheu and Wiley, 1983

Clarification of cactus pear juice

Ultrafiltration

Hollow fiber polysulfone membrane 25 °C

Cassano et al., 2007a

Concentration of cactus pear juice

Osmotic distillation

Microporous polypropylene Cassano et al., hollow fiber 28 °C 2007a

Clarification of kiwifruit juice

Ultrafiltration

Hollow fiber polysulfone membrane 20–30 °C

Cassano et al., 2007b

Concentration of pineapple juice

Osmotic evaporation

Polytetrafluoroethylene membrane 25 °C

Hongvaleerat et al., 2008

Concentration of Reverse osmosis blackcurrant juice

Tubular polyethersulfone membrane 25 °C

Banvolgyi et al., 2009

Concentration of noni juice


Hollow fiber polypropylene Valdes et al., 30 °C 2009

Concentration of Membrane blackcurrant juice distillation

Hollow fiber polypropylene Kozak et al., membrane 15 and 19 °C 2009

Recovery of the Vacuum main pear aroma membrane compound (ethyl distillation 2,4-decadienoate)

Hollow fiber module of polypropylene 25 °C

Diban et al., 2009

is that the process is carried out at low temperatures, which results in higher retention of juice components and reduction in the consumption of energy. The efficiency of the process is highly determined by the operational variables and type of membrane. Polyamide and cellulose acetate are the most common materials used for reverse osmosis of fruit juices. Polyamide membranes used for concentration of apple juice have been found to be more resistant, and have provided higher retention of flavors and higher flux than the cellulose acetate ones, and similar results were obtained for other fruit juices (Chua et al., 1987; Sheu et al., 1983; Palmieri et al., 1990). A different configuration also seems to play a main role in the performance of reverse osmosis. The plate and frame configuration have, in general, a higher retention capacity for flavor aromas than the spiral wound, owing to the lower membrane package density and area (Chou et al., 1991). Increases in transmembrane pressure and decreasing temperatures favored the retention of flavor compounds such as ethyl-2-methyl butanoate, hexanol and hexanal in concentration of apple juice (Alvarez et al., 1998; Chou et al., 1991). There are no results


Advances in the effective application of membrane technologies 187 of advances or a significant research focus on reverse osmosis applied to concentration of fruit juices in recent years. This is probably ascribable to the limited capacity of reverse osmosis to achieve high concentration levels. Jiao et al. (2004) set 25–30 °Brix as the maximum achievable concentration of fruit juice when reverse osmosis is used, the low efficiency ascribable to the osmotic pressure, versus ~80 °Brix that can be reached by multistep vacuum evaporation. This suggests the use of reverse osmosis as a pre-step in the concentration process, which is coupled, for instance, to a subsequent evaporation. This is already being implemented in commercial plants (Gadea, 1987), being energy-saving and resulting in a higher production capacity. 6.4.2 Membrane distillation Membrane distillation can be defined as the transfer of a target solute between two solutions subjected to different temperatures and separated by a hydrophobic membrane. The temperature gradient creates a vapor pressure difference between the two interfaces, resulting in a water flux from the high-temperature to the low-temperature side. The volatile compounds evaporate and diffuse and/or convect across the membrane from the feed side, and are condensed in the other side (Khayet et al., 2002; Lawson and Lloyd, 1997). The maximum achievable concentration when using membrane distillation was reported to be 60–70 °Brix, which is very close to the one reachable by traditional evaporation (Jiao et al., 2004). The selectivity of the membrane for each of the components in the feed solution is determined by the vapor–liquid equilibrium. Therefore, the highest permeation rate will correspond to the component with the highest partial pressure (Mulder, 1996; Bandini and Sarti, 1999). The pore size of the membrane used for membrane distillation are usually between 0.2 and 1.0 mm and hydrophobic materials such as polyvinyldifluoride and polytetrafluoroethylene are the most used polymers (Mulder, 1996). The material of the membrane must be carefully selected. If the affinity between the material of the membrane and the properties of the feed solution is too high, an undesired wetting of the membrane can occur (Mengual et al., 2004; Mulder, 1996). As expected, the flux rate across the membrane will increase with higher porosities and thinner membranes. A higher temperature difference between juice and water results in a flux increase, however, temperature cannot be increased indefinitely, as some of the solute components of fruit juice are thermosensitive. As expected, permeate flux only slightly decreases with increasing juice feed concentration compared with reverse osmosis and, in general, flux is higher than for reverse osmosis at high concentration ratios (Calabro et al., 1994). Depending on the method by which the vapor is recovered from the membrane pores, the membrane distillation process can be performed by using several configurations (Fig. 6.2): (a) direct-contact membrane distillation (DCMD) uses an aqueous solution for vapor recovery; © Woodhead Publishing Limited, 2010

188 Separation, extraction and concentration processes Membrane

Aqueous solution

Flow Aqueous solution

Sweep gas Vapor

Vacuum or Air gap

(a) Vacuum Aqueous solution

Aqueous solution

Sweeping gas

(b)

(c) Boundary layers

Cfeed

Cfm

Cpermeate Cpm

Tfeed

Tfm Tpm Tpermeate

Membrane (d)

Fig. 6.2 Membrane distillation processes employed for concentration of juices: (a) cross-sectional view of a hydrophobic membrane in contact with an aqueous solution illustrating the vapor–liquid interfaces in MD; (b) configuration of SGMD; (c) configuration of VMD; (d) schematic illustration of temperature (T) and concentration (C) profiles in VMD and SGMD during the concentration process; membrane–feed boundary layer (fm), membrane–permeate boundary layer (pm).

(b) osmotic membrane distillation (OMD), an osmotic medium; (c) vacuum membrane distillation (VMD) couples to a vacuum system; (d) sweeping gas membrane distillation (SGMD) uses a sweeping gas; and


Advances in the effective application of membrane technologies 189 (e) air-gap membrane distillation (AGMD), a stagnant air gap plus a cold plate. Of these, DCMD has probably been the most studied configuration. About 5 years ago, a very limited number of studies on application of membrane distillation for juice concentration or aroma recovery were available (Calabro et al., 1994; Khayet et al., 2002; Lagana et al., 2000). However, the number of studies on this topic has dramatically increased recently. Calabro et al. (1994) succeed in concentrating orange juice by membrane distillation with total retention of sugars and organic acids, but a considerable degradation of vitamin C caused by oxidation and high temperature. Temperature seems to be the most important factor for process improvement in membrane distillation. Bui et al. (2004) reported a flux increase of 3–4 times when feed temperature was 15 degrees higher in glucose solutions of 30 to 60%. A reduction of the flux is normally caused by viscosity increase when the concentration is higher than 50%. In all the studies, however, membrane distillation is still coupled to a prior evaporation or membrane filtration step. Kozak et al. (2006) used OMD as the final step for concentration of must and apple juice in laboratory and in a large-scale installation. After reverse osmosis, grape and apple juice reached a concentration of 23 °Brix, which increased up to ~62 °Brix by using OMD at 20 °C. No advances in the use of new membrane materials or setups are being investigated for improving membrane distillation performance. Instead, most of the studies are focused on the optimization of the operating variables for each particular fruit, in an attempt to enhance the total soluble solids concentration in the particular juice. Temperature was the major factor in the concentration of ethyl 2,4decadienoate, a pear aroma compound, over feed flow rate, feed concentration and pressure when a commercial hollow-fiber polypropylene membrane was used for VMD (Diban et al., 2009). Changes in temperature were also reported to have a higher effect than feed flux in the concentration of volatile aroma compounds from blackcurrant juice (Bagger-Jørgensen et al., 2004). The low temperature (10 °C) favored the concentration of blackcurrant aroma esters compared with 45 °C by VMD. Kozak et al. (2009) reported an increase of 80% of the permeate flux by increasing the temperature by only four degrees (from 15 to 19 °C) between both sides of the membrane, confirming the dramatic effect of this variable in blackcurrant juice concentration. In this case, a microfiltration treatment was used for pre-concentration, and the membrane distillation contribute to concentrate the juice from 22 to 58.2 °Brix. Many recent works have also measured antioxidant capacity and content of phenolic compounds, which are reported to exert beneficial health effects (Del Rio et al., 2010), in juices subjected to membrane distillation to prove the advantages of this technique over the traditional methods. The antioxidant capacity and the content of phenols remained constant after the membrane distillation treatment in cactus pear juice, noni juice and other red fruit juices (Cassano et al., 2007a; Valdés et al., 2009; Koroknai et al., 2008). The practically non-existent patents on the application of membrane © Woodhead Publishing Limited, 2010

190 Separation, extraction and concentration processes distillation for concentration of fruit juices suggest that interest in this topic is still confined to academic circles. However, the higher concentration degree attainable by using membrane distillation compared with reverse osmosis suggests a major use of this technique for the future. Integrated processes As stated above, reverse osmosis and membrane distillation processes are commonly coupled to pre-evaporation or, to a micro/ultrafiltration step at industrial level. Micro- or ultrafiltration has been proved to be useful not only for clarification purposes, but also to reduce the viscosity of the juice by removing solids and pectin, easing the posterior membrane processes by increasing the permeate flux (Jiao et al., 2004). Johnson et al. (1996) developed a method for concentrating orange juice, combining ultrafiltration for clarification with traditional evaporation, reaching a concentration higher than 80 °Brix. A more complex method has been proposed by Álvarez et al. (2002) for concentrating apple juice, including a membrane bioreactor for clarification, reverse osmosis for pre-concentration, pervaporation to recover aromas and traditional evaporation as a final step. Enzyme treatment and ultrafiltration has become the most usual clarification treatment. It has been used for concentration of blackcurrant juice coupled with reverse osmosis in an integrated process (Banvolgyi et al., 2009). In turn, membrane filtration techniques themselves are sometimes coupled to other techniques with the aim, in most instances of reducing fouling. Fernandes et al. (2007) patented a hybrid method that combines nanofiltration and electrodialysis for concentration and fouling removal of grape must, respectively.

6.5 Membrane technology for treatment of wastewater in the food industry Again, the dairy industry is the first food processing industry incorporating the new advances in membrane technology applied to wastewater treatment. It has been estimated that the dairy industry generates 0.2–11 l of waste water per liter of milk (Daufin et al., 2001). Dairy industry effluents have been reported as one of the highest volume industrial effluents (Wheatley, 1990). Carbohydrates, lipids and proteins are the most abundant organic compounds in the effluents of the dairy industry with a reported polluting charge of 0.2–2.5 g l–1 biological oxygen demand (Daufin et al., 2001; Perle et al., 1995). In general, biological processes are more efficient and costeffective than the physicochemical methods (Vidal et al., 2000). The high energy requirements associated with aerobic treatment plants have turned the research focus toward anaerobic treatments in recent years, with lower sludge production and no aeration requirements (Demirel et al., 2005). However, the membrane aerobic bioreactor is still quite common for treatment of food-processing wastewater in industry (Abdulgader et al., 2007). Membrane © Woodhead Publishing Limited, 2010

Advances in the effective application of membrane technologies 191 separation substitutes the clarification step used in traditional wastewater treatment, giving several advantages: (1) a significant reduction in the amount of sludge generated, (2) the capacity to treat a major volume of water using less space, and (3) a higher quality of the resulting water (Daufin et al., 2001). 6.5.1 Aerobic membrane bioreactor A membrane bioreactor couples the activated sludge process with membrane separation. An efficient pre-treatment, to avoid excessive fouling of the membrane must precede the biological treatment. In general, either microfiltration or ultrafiltration are used to separate the sludge from the treated water. The membrane can be either submerged or external. The most usual set-up is a submerged hollow fiber or plate membrane ultrafiltration membrane (Daufin et al., 2001; Melin et al., 2006). Once again, fouling is the major factor to assure high performance. Fouling depends, besides other general factors such as membrane type and hydrodynamic conditions, on the presence of compounds that must be produced by microbial metabolism or added to the sludge, e.g. polyelectrolytes (Melin et al., 2006). 6.5.2 Anaerobic membrane bioreactor The main advantage of anaerobic treatment compared with aerobic is the dramatic reduction in the amount of produced sludge, which is at least five times lower. In addition, the anaerobic treatment results in the production of gas that can be used to generate energy to be used elsewhere in the plant and allows a degree of degradation of the organic matter higher than 80% (ArrosAlileche et al., 2008). Some authors reported the need to operate at subcritical fluxes to assure an efficient control of fouling (Hughes and Field, 2006). The selection of the membrane is generally based on a complete retention of the biomass and very low solute rejection, critical for good performance of the membrane (Jefferson et al., 2000). Among the most usual membrane materials for membrane bioreactors, polypropylene and polyethylene are being industrially used (Kubota and Mitsubishi). Because fouling is a major factor in these systems, cleaning is a critical step in these operations and it can be done by chemical or mechanical treatments. Back-flushing has been presented as one of the most innovative and efficient mechanical techniques to remove fouling (see Section 6.7).

6.6 New applications of membrane technology for the food industry: concentration and fractionation of saccharides Production of nutri-functional compounds, highly claimed by consumers over the past decade for being able to promote healthy properties of food, has © Woodhead Publishing Limited, 2010

192 Separation, extraction and concentration processes promoted the development of new techniques for production of high-purity compounds, to be used as supplements or additives in food. Prebiotics are a good example. In recent years, many research efforts have targeted the production of oligosaccharide prebiotics for elaboration of products, e.g. beneficial-health supplements or human-like infant milk formulas (Bode, 2009). In most instances, prebiotics are produced by enzymatic break down of a polysaccharide of vegetal origin, which produces an oligosaccharide with bioactive properties (Beine et al., 2008). Membrane technology is the most feasible strategy for purification in industrial manufacture of enzymatically produced oligosaccharides (Table 6.2). However, the lack of systematic understanding of how properties of the membrane and the oligosaccharide affect membrane filtration has limited to a large extent the industrial development of processes for production of prebiotics (Pinelo et al., 2009). Some of the factors related to design of the most advantageous set-up are described in the next section and the influence of certain operational variables which have to be carefully considered to eventually exploit the process at an industrial level is explored. 6.6.1 Design of separation set-up In most small-scale studies, membrane filtration was performed in a batch dead-end system, which is the simplest set-up to operate (Pinelo et al., 2009). The system consists of a tank for the feed solution, which passes across the membrane module. The permeate is recovered in a proper reservoir (Swennen et al., 2005). A magnetic stirrer is commonly used to reduce fouling and to improve the reaction rates on a laboratory scale where the enzymatic reaction and the membrane filtration occur simultaneously (Sanz et al., 2005). Industrially, further research into other types of efficient mixing is required. In the laboratory, Sarney et al. (2000) used a membrane reactor equipped with a magnetic stirrer for the recovery of oligosaccharides from milk using a combined b-galactosidase treatment with nanofiltration. However, when fouling is a major factor for productivity, a cross-flow filtration system is recommended (Ramaswami et al., 2005). In cross-flow, the feed solution flows parallel to the membrane surface, sweeping the retained particles and helping to reduce the concentration polarization. This is probably the most advisable set-up for industrial purposes. A cross-flow system was used for purification of the products resulting from autohydrolysis of rice hulk in a tubular membrane (Vegas et al., 2006). Goulas et al. (2002) reported the same efficiency for dead-end and cross-flow filtration modes in the removal of glucose from enzymatically obtained oligosaccharides, but concluded that the concentration polarization was a major factor limiting the performance of the dead-end system.



Substrate

Type of separation

Commercial powder of oligosaccharides from chicory rootstock

UF for separation of larger Oligosaccarides from UF cutoff: 20 kDa, impurities and NF for removal of 3 to 10 degrees of NF cutoff: 0.5–5 kDa monosaccharides polymerization (DP)

Kamada et al. (2002)

Arabinoxylan hydrolysates Commercial saccharides

Purification by UF (5, 10 and 30 kDa) Removal of di- and monosaccharides Purification of oligosaccarides and removal of lactose by NF Simultaneous concentration and purification by 1 kDa by NF Separation of di- and monosaccharides by NF

Stirred cell at 4 bar

Swennen et al. (2005)

Stirred cell at 50 °C and 4 bar

Sanz et al. (2005)

Caprine milk Liquors from rice husk autohydrolysis Commercial mixture of galacto-oligosaccharides

Product

Arabinoxylooligosaccharides Oligosaccharides Milk oligosaccharides Xylooligosaccharides Raffinose, sucrose and fructose

Commercial mixture of Concentration and purification by Oligosaccharides of saccharides from yacon UF and NF DP from 3 to 10 rootstock Hemicellulose hydrolyzate Purification by NF Xylose stream Enzymatic hydrolytes from vegetal origin Commercial oligosaccharide mixture Liquors from almond shells autohydrolysis

Concentration by NF Purification by NF Separation of lignin-related impurities by UF

Xylooligosaccharides Fructooligosaccharides Xylooligosaccharides

Filtration conditions

Reference

Feed velocity: 80–120 mL, 20–40 bar Sarney et al. (2000) Vegas et al. (2006) TiO2/ZrO2 ceramic membrane at 30 °C and 2–14 bar Feed concentration varied between Goulas et al. (2002) 0.15–0.08 g mL–1, filtration was performed at 25–60 °C and 5–30 bar UF cutoff 10 kDa (0.52 bar) and NF Olano-Martin et al. (2001) cutoff 5 kDa (5 bar) Concentrated feed solution: 20 wt% Retentate flow: 6 L min–1, filtration performed at 40–60 °C and 20–40 bar Feed velocity: 400 mL min–1 pH 5, 48 °C and 16 bar Feed flow: 1850 L h–1, filtration performed at 45 °C and 1 MPa Cutoffs between 1 and 8 kDa Filtration performed at 25 °C and 2.6–9 bar

Machado et al. (2000)

Huang et al. (2001) Espinoza-Gomez et al. (2004), Grassin et al. (1996) Li et al. (2004)

Advances in the effective application of membrane technologies 193

Table 6.2 Some examples of the use of ultrafiltration (UF) and nanofiltration (NF) membranes for the concentration and purification of oligosaccharides of different nature

194 Separation, extraction and concentration processes 6.6.2 Operational modes The integration of enzyme reactor and membrane is normally advantageous for recovery of prebiotic oligosaccharides from vegetal matrixes, mainly because this disposition diminishes product inhibition (Pinelo et al., 2009). The operational conditions for this particular integrated system depend to a large extent on whether the system operates with immobilized or free enzymes. On some occasions, the membrane itself was used for the immobilization. Nishizawa et al. (2000) reported the chemical immobilization of b-fructofuranosidase with glutaraldehyde to the inner surface of a ceramic membrane for production of fructo-oligosaccharides. Although the use of ceramic membranes has been used for purification of oligosaccharides by ultra- and nanofiltration, it is more common to use polymeric organic membranes for oligosaccharide purification (Artug et al., 2007). Cellulosic membranes seem more suitable to this purpose, being hydrophilic, but the use of polysulfone membranes is also common (Akhtar et al., 1995). A chemical surface treatment was recommended for a polysulfone membrane, in order to make it more hydrophilic (Akhtar et al., 1995). The operating variables that had a major influence on membrane filtration of oligosaccharides were: (1) temperature, (2) pH, (3) concentration of the feed solution, and (4) pressure. Temperature It has been reported that the permeate flux almost doubled when the temperature of saccharide solutions increased from 20 to 50 °C. This is ascribable to the reduction of viscosity, which favors the pass of the flow of the feed solution across the membrane (Goulas et al., 2002; Machado et al., 2000). The favorable effect of the temperature increase on the permeate flux was confirmed by Sjöman et al. (2008), during recovery of xylose from hemicellulose hydrolyzate feeds using various polysulfone membranes. pH For certain types of membrane carrying positive or negative charged groups, e.g. polycarbonate and sulfonated polysulfone membranes, the influence of pH on the saccharide rejection can be dramatic (Lazaridou et al., 2007). When the feed solution includes some charged compounds, flux commonly increases when working close to the isoelectric point of the membrane, as expected (Childress and Elimelech, 1996). Variation of pH can be used as a strategy for more efficient separation of charged saccharides, such as pectin, whose charge density and distribution is a function of the carboxyl groups in the structure.


Advances in the effective application of membrane technologies 195 Concentration of the feed solution Permeate flux is expected to decrease with increasing feed concentration of the solution. As a result, a decrease in the rejection, at constant pressure, is usually observed in these cases (Goulas et al., 2002). Pressure The role of pressure and the recommended levels of pressure depend on the type and properties of the solution to be filtered. Generally, the rejection increases with increasing levels of pressure until reaching a certain level. This is ascribable to the higher flux of solute across the membrane, which decreases the difference in concentration between the solutions at both sides of the membrane. When pressure continues increasing, the concentration polarization and, in turn, the concentration of solute on the surface of the membrane increases too. As a consequence, the concentration of solute on the permeate side increases and the rejection decreases. In purification of xylo-oligosaccharides from lignin impurities, Nabarlatz et al. (2007) observed a decrease in rejection when the pressure was increased from 2.6 to 9 bar. It is then advised to operate at low pressures, within a range in which the concentration polarization is not so high, in order to maximize selectivity.

6.7 Future trends Implementation of membrane technology in food industry has been hampered by the economical limitations derived from fouling over recent decades. Many studies have been devoted to develop techniques and processes to reduce the impact of fouling and it seems that progress in membrane technology will be linked to progress in fouling reduction over the next few years. In particular, there are at least three techniques that have provided promising results for their efficiency and simplicity to be incorporated in a large-scale process: (1) high frequency backflushing, (2) vibrating membrane modules, and (3) electrofiltration. Some of the benefits associated with coupling these techniques in the dairy industry have been discussed in Section 6.3 but the applications in other food processes are increasing 6.7.1 High frequency backflushing Backflushing consists of applying pressure pulses from the permeate to the retentate side, and stripping off the particles causing fouling from the membrane surface into the bulk solution at the permeate side again (Pinelo et al., 2009). Backflushing is commonly achieved by placing a valve in the © Woodhead Publishing Limited, 2010


Flux (L h–1 m–2)

250 With backflush

200 150 100 50 0

Without backflush 0

15

30

45

60 75 Time (min)

90

105

120

Fig. 6.3 Influence of backflushing on the permeate flux versus time applied to the microfiltration of skim milk for removal of bacterial species using a ceramic membrane. Adapted from Guerra et al. (1997).

permeate side. The efficiency of backflushing depends both on the duration of the pulse itself and time between pulses. Guerra et al. (1997) reported a considerable increase of permeate flux when backflushing was incorporated to microfiltration of skim milk, as backflushing contributed to the removal of spores from the membrane surface (Fig. 6.3). Jonsson (2008) narrowed the intervals of fractionation of dextrans in a hollow-fiber ultrafiltration system using times between pulses from 1 to 30 s and backflushing times from 0.1 to 5 s. Application of backflushing in industry is still limited but further research in the coming years will permit a more detailed analysis of the balance between costs associated with implementation and economic benefit from fouling reduction. 6.7.2 Vibrating membrane module A vibrating design consists of applying high shear rates to favor the transmission of macromolecules through the membrane (Beier et al., 2007). This design proved to be particularly efficient for nanofiltration of diluted milk and for enzyme separation applied to food processes (Beier et al., 2007; Jaffrin et al., 2004). This system has been already presented in the section dedicated to fouling reduction in the dairy industry, but it is fair to include it here as a future trend technique, as many studies are currently being conducted to optimize the mechanical characteristics of the system for a feasible high performance of membrane separation in the dairy industry.


Advances in the effective application of membrane technologies 197 6.7.3 Electrofiltration Electrofiltration is used when the particles causing concentration polarization have a net charge and can be attracted or repelled by applying an electric field, in this instance across the membrane (Pinelo et al., 2009). This technique has been successfully employed for separation of industrial solutions of enzymes with a surface charge, resulting in a dramatic increase of the permeate flux (Enevoldsen et al., 2007). The technique also gave good results in ultrafiltration of other proteins (Oussedik et al., 2000) and shows promise for purification of potential food ingredients in which charge can be induced, e.g. modifying the number and methoxyl groups on pectin molecules.

6.8 References Abdulgader M E, Yu Q J, Williams P, Zinatizadeli A A L (2007), ‘A review of the performance of aerobic bioreactors for treatment of food processing wastewater’. Proceedings of the International Conference on Environmental management, Engineering, Planning and Economics, pp. 1131–1136. Skiathos. Akhtar S, Hawes C, Dudley L, Reed P, Strafford P (1995), ‘Coatings reduce the fouling of microfiltration membranes’, J Membr Sci 107, 209–218. Al-Akoum O, Richfield D, Jaffrin M Y, Ding L H, Swart P (2006), ‘Recovery of trypsin inhibitor and soy milk concentration by dynamic filtration’, J Membr Sci 279, 291–300. Álvarez V, Riera F A, Álvarez S, Álvarez R (1998), ‘Permeation of apple aroma compounds in reverse osmosis’, Sep Purif Technol 14, 209–220. Álvarez S, Riera F A, Álvarez R, Coca J (2002), ‘Concentration of apple juice by reverse osmosis at laboratory and pilot-plant scale’, Ind Eng Chem Res 41, 6156–6164. Arros-Alileche S, Merin U, Daufin G, Gesan-Guiziou G (2008), ‘The membrane role in an anaerobic membrane bioreactor for purification of dairy wastewaters: A numerical simulation’. Bioresour Technol 99, 8237–8244. Artug G, Roosmasari K, Richau K, Hapke A (2007), ‘A comprehensive characterization of commercial nanofiltration membranes’, Sep Sci Technol 42, 2947–2986. Bagger-Jørgensen R, Meyer A S, Varming C, Jonsson G (2004), ‘Recovery of volatile aroma compounds from black currant juice by vacuum membrane distillation’, J Food Eng 64, 23–31. Bandini S, Sarti G C (1999), ‘Heat mass transport resistances in vacuum membrane distillation per drop’, AIChE J 45, 1422–1433. Banvolgyi S, Horvath S, Stefanovits-Banyai E, Bekassy-Molnar E, Vatai G (2009), ‘Integrated membrane process for blackcurrant juice concentration’, Desalination 241, 281–287. Barlett M, Bird M R, Howell J A (1995) ‘An experimental study for the development of a qualitative membrane cleaning model’, J Membr Sci, 218, 107–116. Beier S P, Jonsson G (2007), ‘Separation of enzymes and yeast cells with a vibrating hollow fiber membrane module’ Sep Purif Technol 53, 111–118. Beine R, Moraru R, Nimtz M, Na’amnieh S, Pawlowski A, Buchholz K, Seibel J (2008), ‘Synthesis of novel fructooligosaccharides by substrate and enzyme engineering’, J Biotechnol 138, 33–41. Bird M R, Barlett M (1995), ‘CIP optimization for the food industry: Relationships between detergent concentration, temperature and cleaning time’, Trans IChemE, 73, 63–70.


198 Separation, extraction and concentration processes Bode L (2009), ‘Human milk oligosaccharides: prebiotics and beyond’, Nutr Rev 67, 183–191. Bui V A, Nguyen M H, Muller J (2004), ‘A laboratory study on glucose concentration by osmotic distillation in hollow fiber modules’, J Food Eng 63, 237–245. Calabro V, Jiao B L, Drioli E (1994), ‘Theoretical and experimental study on membrane distillation in the concentration of orange juice’, Ind Eng Chem Res 33, 1803– 1808. Cassano A, Conidi C, Timpone R, D’Avella M, Drioli E (2007a), ‘A membrane-based process for the clarification and the concentration of the cactus pear juice’, J Food Eng 80, 914–921. Cassano A, Donato L, Drioli E (2007b), ‘Ultrafiltration of kiwifruit juice: Operating parameters, juice quality and membrane fouling’, J Food Eng 79, 613–621. Childress A E, Elimelech M (1996), ‘Effect of solution chemistry on the surface charge of polymeric reverse osmosis and nanofiltration membranes’, J Membr Sci 119, 253–268. Chou F, Wiley R C, Schlimme D V (1991), ‘Reverse osmosis and flavor retention in apple juice concentration’, J Food Sci 56, 484–487. Chua H T, Rao M A, Acree T E, Cunningham D G (1987), ‘Reverse osmosis concentration of apple juice: flux and flavor retention by cellulose acetate and polyamide membranes’, J Food Proc Eng 9, 231–245. D’Souza N M, Mawson A J (2005), ‘Membrane cleaning in the dairy industry: a review’, Crit Rev Food Sci Nutr, 45, 125–134. Daufin G, Escudier J P, Carrere H, Berot S, Fillaudeau L, Decloux M (2001), ‘Recent and emerging applications of membrane processes in the food and dairy industry’, Trans IChemE, 79, 89–102. Del Rio D, Costa L G, Lean M E J, Crozier A (2010), ‘Polyphenols and health: what compounds are involved?’, Nutr Metab Cardiovasc Dis, 20, 1–6. Demirel B, Yenigun O, Onay T T (2005), ‘Anaerobic treatment of dairy wastewaters: a review’, Process Biochem 40, 2583–2595. Diban N, Voinea O C, Urtiaga A, Ortiz G (2009), ‘Vacuum membrane distillation of the main pear aroma juice compound. Experimental study and mass transfer modelling’, J Membr Sci 326, 64–75. Enevoldsen A D, Hansen E B, Jonsson G (2007), ‘Electro-ultrafiltration of industrial enzyme solutions’, J Membr Sci 28–37. Espinoza-Gomez H, Lin S W, Rogel-Hernandez E (2004), ‘Nanofiltration membrane pore diameter determination’, Rev Soc Quim Mex 48, 15–20. Fernandes G V M, Lopes C G, Correia N (2007) ‘Concentration and rectification of grape must involves combining nanofiltration and electrodialysis of grape must in hybrid process’, World Patent WO2008051100-A2. Flemming S, Skou F (2006), ‘Membrane filtration of a product, e. g. milk, in membrane plant, involves recovering cleaning solution after multi-step cleaning of membrane system in plant; and using recovered cleaning solution for cleaning membrane system’, European Patent EP1726353. Frappart M, Akoum O, Ding L H, Jaffrin M Y (2006), ‘Treatment of dairy process waters modeled by diluted milk using dynamic nanofiltration with a rotating disk module’, J Membr Sci 282, 465–472. Gadea A (1987), ‘Reverse osmosis of orange juice’. In Proceedings of the International fruit juice congress, Orlando, USA. GEA (2009) Report on ‘Evaporation technology for the juice industry’, GEA Wiegand GmbH, www.gea-wiegand.com. Accessed on Dec 15, 2009. Genkin G, Waite A G, Fane S, Chang S (2006), ‘The effect of vibration and coagulant addition on the filtration performance of submerged hollow fiber membranes’, J Membr Sci 281, 726–734. Goulas A K, Kapasakalidid P G, Sinclair H R, Rastall R A, Grandison A S (2002),


Advances in the effective application of membrane technologies 199 ‘Purification of oligosaccharides by nanofiltration’, J Membr Technol 209, 321– 335. Grassin C, Fauquembergue P (1996), ‘Fruit Juices’, in: T. Godfrey, S. West (Eds.), Industrial Enzymology, Macmillan Press. Guerra A, Jonsson G, Rasmussen A, Nielsen E W, Edelsten D (1997), ‘Low cross-flow velocity microfiltration of skim milk for removal of bacterial spores’, Int Dairy J 7, 849–861. Hongvaleerat C, Cabral L M C, Dornier M, Reynes M, Ningsanond S (2008), ‘Concentration of pineapple juice by osmotic evaporation’, J Food Eng 88, 548–552. Huang R Y M, Shao P, Nawawi G, Feng X, Burns C M (2001), ‘Measurements of partition, diffusion coefficients of solvents in polymer membranes using rectangular thin-channel column inverse gas chromatography’, J Membr Sci 188, 205–218. Hughes D, Field R W (2006), ‘Crossflow filtration of washed and unwashed yeast suspensions at constant shear under nominally sub-critical conditions’, J Membr Sci 280, 89–98. Jaffrin M J (2008), ‘Dynamic shear-enhanced membrane filtration: a review of rotating disks, rotating membranes and vibrating systems’, J Membr Sci, 324, 7–25. Jaffrin M Y, Ding L H, Akoum O, Brou A (2004), ‘A hydrodynamic comparison between rotating disk and vibratory dynamic filtration systems’, J Membr Sci 242, 155–167. Jefferson B, Laine A T, Judd S J, Stephenson T (2000), ‘Membrane bioreactors and their role in wastewater reuse’, Water Sci Technol 41, 197–204. Jiao B, Cassano A, Drioli E (2004), ‘Recent advances on membrane processes for the concentration of fruit juices: a review’, J Food Eng 63, 303–324. Johnson J R, Braddock R J, Chen C S (1996), ‘Flavor losses in orange juice during ultrafiltration and subsequent evaporation’, J Food Sci 61, 540–543. Jonsson G (1985), ‘Molecular weight cut-off curves for ultrafiltration membranes of varying pore sizes’, Desalination 53, 3–10. Jonsson G (2008), ‘Tuning of the cut-off curves by dynamic ultrafiltration’, Proceedings of the International Conference on Membranes and Membrane Processes: ICOM2008, Hawaii, July 12–18. Jonsson G, Boesen C E (1975), ‘Water and solute transport through cellulose acetate reverse osmosis membranes’, Desalination 17, 145–165. Kamada T, Nakajima M, Nabetani H, Iwamoto N S (2002), ‘Availability of membrane technology for purifying and concentrationg oligosaccharides’, Eur Food Res Technol 214, 435–440. Khayet M, Godino M P, Mengual J I (2002), ‘Thermal boundary layers in sweeping gas membrane distillation processes’, AIChE J 48, 1488–1497. Koroknai B, Csanadi Z, Gubicza L, Belafi-Bako (2008), ‘Preservation of antioxidant capacity and flux enhancement in concentration of red fruit juices by membrane processes’, Desalination 228, 295–301. Kozak A, Bekassy E, Vatai G (2009), ‘Production of black-currant juice concentrate by using membrane distillation’, Desalination 241, 309–314. Kozak A, Rektor A, Vatai G (2006), ‘Integrated large-scale membrane process for producing concentrated fruit juices’, Desalination 200, 540–542. Lagana F, Barbieri G, Drioli E (2000), ‘Direct contact membrane distillation: modeling and concentration experiments’, J Membr Sci 166, 1–11. Lawson K W, Lloyd D R (1997), ‘Membrane distillation’, J Membr Sci 124, 1–25. Lazaridou A, Biliaderis C G (2007), ‘Molecular aspects of cereal beta-glucan functionality: physical properties, technological applications and physiological effects’, J Cereal Sci 46, 101–118. Li W, Li J, Chen T, Chen C (2004), ‘Study on nanofiltration for purifying fructooligosaccharides II. Extended pore model’, J Membr Sci 258, 8–15. Machado D R, Hasson D, Semiat R (2000), ‘Effects of solvent properties on permeate through nanofiltration membranes. Part II: transport model’, J Membr Sci 166, 6–69.


200 Separation, extraction and concentration processes Makardij-Tossonian A A (2009), ‘Membrane regeneration’, World Patent WO/2009/089587. Melin T, Jefferson B, Bixio D, Thoeye C, De Wilde W, De Koning J, van der Graaf J, Wintgens T (2006), ‘Membrane bioreactor technology for wastewater treatment and reuse’, Desalination 187, 271–282. Mengual J I, Khayet M, Godino M P (2004), ‘Heat and mass transfer in vacuum membrane distillation’, Int J Heat Mass Transfer 47, 865–875. Mickols W, Koreltz M S, Moll D J, Streeter D B, Mickols W, Kobeltz M S (2008), ‘Spiral wound module assembly for e.g. in-situ on-line and real-time measurement of membrane fouling in spring wound module, has membrane envelopes and acoustic transducers which are located adjacent to permeate collection tube’, World Patent WO2008103864. Mulder M (1996), Basic principles of membrane technology, Dordrecht/Boston/London: Kluwer Academic Publishers. Nabarlatz D, Torras C, Garcia-Valls R, Montane D (2007), ‘Purification of xylooligosaccharides from almond shells by ultrafiltration’, Sep Purif Technol 53, 235–243. Nigam M O, Bansal B, Chen X D (2008), ‘Fouling and cleaning of whey protein concentrate fouled ultrafiltration membranes’, Desalination 218, 313–322. Nishizawa K, Nakajima M, Nabetani H (2000), ‘A forced flow membrane reactor for transfructosylation using ceramic membrane’, Biotechnol Bioeng 68, 92–97. Olano-Martin E, Mountzouris K C, Gibson G R, Rastall R A (2001), ‘Continuous production of pectic oligosaccharides in a membrane enzyme reactor’, J Food Sci 66, 966–971. Omprakash M, Bansal B, Cheng D X (2008), ‘Fouling and cleaning of whey protein concentrate fouled ultrafiltration membranes’, Desalination 218, 313–322. Oussedik S, Belhocine D, Grib H, Loucini H, Piron D L, Nameri N (2000), ‘Enhanced ultrafiltration of bovine serum albumin with pulsed electric field and fluidized activated alumina’, Desalination 127, 59–64. Palmieri L, Dalla Rosa M, Dall’Aglio G, Carpi G (1990), ‘Production of kiwifruit concentrate by reverse osmosis process’, Acta Horticulturae 282, 435–439. Perle M, Kimchie S, Shelef G (1995), ‘Some biochemical aspects of the anaerobic degradation of dairy wastewater’, Water Res 29, 1549–1554. Pinelo M, Jonsson G, Meyer A S (2009), ‘Membrane technology for purification of enzymatically produced oligosaccharides: molecular and operational features affecting performance’, Sep Purif Technol, 70, 1–11. Ramaswami H S, Marcotte M (2005), Food processing: principles and applications, CRC Press. Rice G, Barber A, O’Connor A, Stevens G, Kentish S (2009a), ‘Fouling of nanofiltration membranes by dairy ultrafiltration permeates’, J Membr Sci 330, 117–126. Rice G, Kentish S, O’Connor A, Barber A, Pihlajamäki A, Nyström M, Stevens G (2009b), ‘Analysis of separation and fouling behaviour during nanofiltration of dairy ultrafiltration permeates’, Desalination 236, 23–29. Sanz M L, Polemis N, Morales N, Corzo A (2005), ‘In vitro investigation into the potential prebiotic activity of potential oligosaccharides’, J Agric Food Chem 53, 2914–2921. Sarney D B, Hale C, Frankel C, Vulfson E N (2000), ‘A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration’, Biotechnol Bioeng 69, 461–467. Sheu M J, Wiley R C (1983), ‘Preconcentration of apple juice by reverse osmosis’ J Food Sci, 48, 422–429. Sjöman E, Mänttäri M, Nyström M, Koivikko H, Heikkilä H (2008), ‘Xylose recovery by nanofiltration from different hemicellulose hydrolyzate feeds’, J Membr Sci 310, 268–277.


Advances in the effective application of membrane technologies 201 Swennen C M, Coyrtin B, Bruggen C, Vandecasteele C, Gibson G R, Rastall R A (2005), ‘Ultrafiltration and ethanol precipitation for isolation of arabinoxylooligosaccharides with different structures’, Carbohydr Polym 62, 283–292. Tohammadi T, Madaeni S S, Moghadam M K (2002), ‘Investigation of membrane fouling’, Desalination, 153, 155–160. Trägårdh G (1989), ‘Membrane cleaning’, Desalination, 71, 325–335. Valdés H, Romero J, Saavedra A, Plaza A, Bubnovich V (2009), ‘Concentration of noni juice by means of osmotic distillation’, J Membr Sci 330, 205–213. Vegas R, Luque S, Alvarez J R, Alonso J L, Dominguez H, Parajo R C (2006), ‘Membraneassisted processing of xylooligosaccharides-containing liquors’, J Agric Food Chem 54, 5430–5436. Vidal G, Carvalho A, Mendez R, Lema J M (2000), ‘Influence of the content in fats and proteins on the anaerobic biodegradability of dairy wastewaters’, Bioresour Technol 74, 231–239. Wheatley A (1990), Anaerobic digestion: a waste treatment technology. London and New York: Elsevier Applied Science.



7 Electrodialytic phenomena, associated electromembrane technologies and applications in the food, beverage and nutraceutical industries L. Bazinet, A. Doyen and C. Roblet, Laval University, Canada Abstract: Electrodialysis, an electrochemical separation process with charged membranes stacked to separate ionic species from aqueous solutions and uncharged components when an electrical field is applied, is providing new membrane separation processes with numerous applications in the food, nutraceutical and beverage industries. Techniques such as electrolysis with membrane, electrodialysis with ionexchange membranes, electrodialysis with bipolar membrane and electrodialysis with filtration membrane have been used for, among other applications, the coagulation of protein, the electroreduction of the medium and/or the fractionation of several food proteins. Moreover, electrodialysis with filtration membranes has been used for the recovery of molecules with bioactive properties such as antioxidant, anticancer and antihypertensive peptides. Key words: electrodialysis, electromembranes, nutraceuticals, biomolecules, purification, separation.

7.1 Introduction Electrodialysis (ED) is one of a group of membrane-based separation technologies which are finding increasing use, in agri-food industries to concentrate, purify or modify foods. In ED, an electric field provides the driving force and porous or non-porous membranes perform the separation: electrodialysis is a combined method of dialysis and electrolysis (Shaposhnik and Kesore, 1997). ED can be performed with two main cell types: electrolysis (or electro-electrodialysis) cells for oxido-reduction reactions and multimembrane cells for dilution–concentration, water dissociation and purification applications. The electrolysis cell operates with only one membrane that © Woodhead Publishing Limited, 2010

Electrodialytic phenomena and associated electromembrane technologies 203

separates two solutions circulating in each electrode compartment. This application is based on electrode redox reactions that are electrolysis-specific properties. In multi-membrane cells only the membrane transport phenomena intervenes, whereas electrochemical reactions occurring at the electrodes do not interact with the separation process. The principles of electrode and membrane reactions, as well as the technologies associated with these electrodialytic phenomena are reviewed in this chapter. The specific applications of electrodialysis and electrolysis currently used and those under development in the food, beverage and nutraceutical industries are presented.

7.2 Principles of electrodialytic phenomena and associated membrane technologies 7.2.1 Membrane phenomena An ion-exchange membrane is made of a macromolecular material (skeleton) which carries ionizable groups such as ion-exchange resins. The membrane contains fixed ions firmly attached to the skeleton and is electronically neutralized by mobile charges of the opposite sign, called counterions. Counterions, which carry current in the membrane, are positive in the case of cation-exchange membranes (CEM), and negative for anion-exchange membranes (AEM) (Bazinet, 2005). Both of these membranes are monopolar; this means that they are permeable to only one type of ion (Gardais, 1990). The perm-selectivity of the membrane is the result of an electrostatic repulsion called Donnan exclusion (Donnan, 1911). For a cation-exchange membrane (CEM), anions are repulsed from the membrane thus allowing only cations to migrate through the membrane. 7.2.2 Electrode phenomena In membrane electrolysis, membrane selectivity, explained above, and electrolysis phenomenon are both active. In electrolysis, an external potential difference is applied to the cell and chemical reactions occur at the electrode–solution interface. The faradaic reactions, caused by the passage of a current, are characterized by electron transfer at the electrode–solution interface. The oxidation A Æ Az+ + z e– induces the loss of one or more electrons and the reduction Bn+ + n e– Æ B induces the gain of one or more electrons. The anode induces oxidation reactions, and reduction reactions occur at the cathode. This transfer always occurs at the electrode surface (Brett and Oliveira-Brett, 1994; Gardais, 1990).



7.3 Applications of electrodialytic phenomena and associated membrane technologies Only one type of membrane technology is associated with electrode reactions (electrolysis with membrane) whereas three types are associated with membrane phenomena (electrodialysis with ion-exchange membranes, electrodialysis with bipolar membrane, and, recently, electrodialysis with filtration membranes) (Fig. 7.1). 7.3.1 Electrolysis with membrane Two main applications of electrode reactions are electrochemical coagulation (EC) and electroreduction. Both methods are exploratory, but show an interesting potential for applications in the food, beverage and nutraceutical industry (Table 7.1). An example of both applications will be developed. Milk protein electrochemical coagulation Khidirov and Merzametov (1982) used EC to precipitate milk proteins in an electrolysis cell separated by a ceramic diaphragm. The milk is poured into the anodic compartment and an electrolyte or a whey solution is poured into the cathodic compartment. Proteins coagulate on the platinum electrode by forming a highly dense white coagulum. Every type of milk could be treated for protein electrochemical coagulation without the use of rennet. Both the caseins and the whey proteins coagulate at the anode. Janson and Lewis (1994) studied the possible use of electrochemical coagulation to directly separate up to 73.8% of the total cheese whey proteins. The whey was circulated in the anodic compartment of an electrolysis cell with a non-ion-selective membrane, made of cotton, for the acidification–coagulation phase (Janson et al., 1990). Afterwards the solution was separated into the coagulum and an impoverished protein solution. This solution was then circulated in the cathodic compartment of the electrolysis cell to undergo the alkalinization phase. Enhancement of lipid stability of omega-3 enriched commercial milk Consumer demand for specific nutritional qualities is encouraging the dairy industry to develop products supplemented in omega-3 fatty acids. Although these fatty acids are known to have many health benefits, they are extremely susceptible to oxidative deterioration which causes difficulties during their storage. In the study by Haratifar (2008) an electroreduction process was performed to modify the redox state of omega-3 enriched milk. A 4-V electroreduction treatment was applied for 1 h on pasteurized omega-3 enriched milk, at room temperature. The electroreduction treatment reduced the redox potential value of omega-3 enriched milk samples quickly and decreased their dissolved oxygen concentration. The electroreduced and control samples were stored at room temperature for up to 3 weeks in the presence of fluorescent light and


(b)

2H2O +

2e– + 2H2O H+

O2 + 4H+ + 4e– OH–

Anode

H2 +

Desalted solution

AEM CEM

AEM CEM

Cl–

– Cathode

+ Anode


2OH–

Cl

–

Cl Na

Cl–

–

Na+ Na+

+

–

Na+

Cathode n

Feed solution (c)

Anionic/acid fraction

AEM FM

Neutral fraction

FM CEM

FM

P+/– + Anode

A

–

P–

P+ A– C+ P–

P+

A

(d)

Cationic/basic fraction

–

P–

FM CEM P+/–

Cl–

A– P+ P+ C

NaOH solution

AEM CEM BPM AEM Cl–

HCl solution CEM BPM AEM H+

H+

+

C

P–

+

–

+

Cathode

Anode

OH Na+

–

Na+ Cl–

Cl

–

Na+

n Feed solution

OH– Na+

Cl–

CEM

Cl–

–

Na+

Na+ Cathode n

Salt solution

Fig. 7.1 Electrodialytic phenomena and associated membrane configurations: (a) redox reaction; (b)–(d) membrane reactions. (a) Electrolysis with membrane; (b) electrodialysis with ion-exchange membranes; (c) electrodialysis with filtration membranes; and (d) electrodialysis with bipolar membranes. AEM, anion-exchange membrane; CEM, cation-exchange membrane; BPM, bipolar membrane; FM, filtration membrane; A–, anion; C+, cation; P–, anionic peptide; P+, cationic peptide; P+/–, neutral peptide.


(a)

206 Separation, extraction and concentration processes in the dark and the composition in fatty acids of milk samples was measured by gas chromatography with flame ionization detection. It was shown that storage under fluorescent light involved a degradation of the fatty-acids, whereas the electroreduction treatment slowed down the oxidative degradation of electroreduced samples compared with untreated milk samples. Results of this study show that the electroreduction treatment can be a potential method of enhancing the shelf-life of products containing unsaturated fatty acids. 7.3.2 Electrodialysis with ion-exchange membranes Conventional electrodialysis or electrodialysis with ion-exchange membranes consist of a series of cation- and anion-exchange membranes arranged in an alternating pattern between an anode and a cathode to form individual cells. Under the influence of an applied potential gradient between cathode and anode, the positively charged cations migrate towards the cathode and negatively charged anions towards anode (Bazinet, 2005, Bazinet and Firdaous, 2009b). The main applications of ED and dilution–concentration in the food industry consist of the demineralization of milk, sugar and whey. In the 1990s, a new application was developed for tartaric stabilisation of wine. The use of ED in membrane bioreactors, for protein separation and acid production was studied. The main applications of conventional ED are presented in Table 7.1, and the application on tartaric stabilization of wine is described in detail here. The use of ED for tartaric stabilization of wine has been approved by the Council of Europe in 1998 and is presently used worldwide on an industrial scale. Wine contains tartaric acid (H2T), a dicarboxylic acid which dissociates in the tartrate (HT–) and bitartrate (T2–) forms. During alcoholic fermentation, potassium and tartrate ions associate and precipitate to form potassium tartrate crystals (KHT) (Gonçalves et al., 2003). As a consequence, at normal storage temperatures, an undesirable KHT precipitation occurs in wine bottles. The ED process for wine tartaric stabilization was developed by Escudier et al. (1995). The principle is the same as for the conventional ED process with a diluate compartment in which the wine to be treated circulates, under nitrogen bubbling, and a concentrate compartment where salts extracted from the wine are recovered. According to the authors, the level of wine deionization, based on the decrease of the conductivity, should be 5 to 20% to obtain tartaric stabilization of wine. One of the main advantages of this technique is the preservation of the organoleptic properties of wine during the stabilization process. 7.3.3 Electrodialysis with filtration membranes Bazinet et al. (2005) stacked filtration membranes and ion-exchange membranes in a conventional electrodialysis cell. This technology, named electrodialysis


Technology

Substrate

Target compounds Objective

Applications and stage of development

References

Dissolved oxygen

Protection of flavour after spray-drying

Food (in development)

Inoue and Kato, 2003

Fruit juices

Dissolved oxygen

Increase in shelf-life


Hekal, 1983 Swanson and Sommer, 1940

Milk

W-3 fatty acids

Protection against lipid oxidation

Functional food (in development)

Haratifar, 2008

Dissolved oxygen




Enhancement of probiotic bacteria growth


Bolduc et al., 2006


Mondal and Lalvani, 2003

Electrode reactions Electrolysis with Coffee membrane © Woodhead Publishing Limited, 2010

Oil

Trans fatty acid

Decrease trans fatty acid content

Water

Electroreductive compounds

Increase purity and quality Food of tap water (in development)

Crandall et al., 2001 Koseki et al., 2003 Mercier, 1999

Increase microbiological quality of water


Kim et al., 2000 Thompson and Gerson, 1985

Electroreduction of whey protein


Bazinet et al., 1997a

Production of protein isolate


Janson and Lewis, 1994

Whey

Whey proteins


Table 7.1 Electrodialytic phenomena and associated applications in the food, beverage and nutraceutical industries

Table 7.1 Continued Substrate

Membrane reactions Electrodialysis Maple sap with ion-exchange membranes Milk © Woodhead Publishing Limited, 2010



References

Calcium


Bazinet et al., 2007

Food (in development) Food (industrial scale) Food (in development) Food (industrial scale)

Bolzer, 1985

Casein Minerals

Passion fruit Citric acid juice Sugar beet juice, Minerals sugar cane and molasses Soy tofu whey Minerals

Whey

Magnesium or calcium Lactic acid

To avoid sugar sand formation during maple syrup production Production of acid caseinate Demineralization of milk for further applications Deacidification of fruit juice Demineralization of sugar syrups Use of soy tofu whey as bacteria growth medium Recovery of coagulant agent Production of biological preservative agent

Minerals

Demineralization of whey for further applications

Propionic acid

Production of yeast inhibitor Whey protein separation

Whey

Food (in development) Food (in development) Food and biopharmaceutical (in development) Food (industrial scale) Food (in development) Food (in development)

Hiraoka et al., 1979 Vera Calle et al., 2002, 2003 Chaput, 1979 Ben Ounis et al., 2008 Bazinet et al., 1999b Boyaval et al., 1987 Glassner, 1992 Houldsworth, 1980 Pérez et al., 1994 Boyaval et al., 1993 Amundson et al., 1982 Slack et al., 1986; Stack et al., 1995


Technology

Electrodialysis with Apple juice bipolar membranes

Potassium

To avoid potassium bitartrate formation

Polyphenol oxidase Inhibition of enzymatic browning

Food (industrial scale)

Audinos et al., 1979, 1985 Escudier et al., 1995 Guérif, 1993


Quoc et al., 2000, 2006 Tronc et al., 1997, 1998


Chitosan

Chitosanase

Chitosanase inhibition and Nutraceuticals optimization of chitosane (in development) oligomer production

Lin Teng Shee et al., 2008

Milk

Casein

Production of isolate


Bazinet et al., 1999a, 2001

Passion fruit juice

Citric acid

Deacidification of fruit juice


Vera-Calle et al., 2002, 2003

Soybean

Soy proteins



Bazinet et al., 1996, 1997b, 1997c, 1998 Mondor et al., 2004 Skorepova and Moresoli, 2007

11S and 7S

Fractionation of 7S and 11S fractions



Soy proteins

Recovery of soy proteins


Bazinet et al., 1999b

Calcium or magnesium

Recovery of coagulant agent



a-lactalbumin (a-la) Production of a-la and and b-lactoglobulin b-lg enriched fractions (b-lg)


Bazinet et al., 2004a, 2004b

Soy tofu whey

Whey

Phospholipids

Production of phospholipid Food and nutraceuticals Lin Teng Shee et al., 2005, (in development) 2007 and protein enriched fractions


Wine

Technology


Electrodialysis with filtration membranes and electromembrane filtration

Substrate



References

Whey protein

Prodution of isolate



Lactic acid

Production of biologic preservative agent

Food and biopharmaceutical (in development)

Norddahl, 1998 Norddahl et al., 2001

Peptide VW

Production of an ACE inhibitor peptidic fraction

Food, nutraceutical and bio-pharmaceutical (in development)

Firdaous et al., 2009 Bazinet and Firdaous, 2009a

Cranberry juice Proanthocyanidins and anthocyanins

Production of antioxidant enriched fruit juices

Food and nutraceutical (in development)

Bazinet et al., 2009 Bazinet and Firdaous, 2009b

Green tea

Catechin

Production of antioxidants Food, nutraceutical and bio-pharmaceutical (in development)

Labbé et al., 2005

Milk

as2-Casein hydrolysate (fraction 183-207)

Bioactive peptide separation

Bargeman et al., 2000, 2002

Snow crab

Protein hydrolysate Production of an anticancer Nutraceutical and peptidic fraction biopharmaceutical (in development)

Doyen et al., 2010

Whey

ACE inhibitor bioactive b-Lactoglobulin peptide separation hydrolysate (fraction 142–148)

Nutraceutical and biopharmaceutical (in development)

Bazinet et al., 2005 Poulin et al., 2006, 2007

Lactoferrin


Ndiaye et al., 2010

Alfalfa white protein

Separation of lactoferrin from whey



Table 7.1 Continued


with filtration membranes (EDFMs), couples size-exclusion capabilities of porous membranes with the charge selectivity of electrodialysis (ED). Because no pressure is applied in the electrodialysis cell, the electric field is the only driving force (Bazinet and Firdaous, 2009b). This technology has already been tested for the separation/purification of various high-added-value bioactive molecules (Table 7.1) such as anticancer peptides and antioxidant polyphenols that will be discussed in more detail. Production of an anticancer peptidic fraction Protein hydrolysates from marine products have interesting bioactive properties such as antithrombotic, antihypertensive (Kim and Mendis, 2006), antioxidant (Amarowicz and Shahidi, 1997), anticancer (Picot et al., 2006) and antimicrobial activities (Beaulieu et al., 2010). Recently, a snow crab by-product hydrolysate, has demonstrated antibacterial inhibition properties against specific strains such as Aeromonas hydrophila, Vibrio vulnificus and Vibrio parahaemolyticus (Beaulieu et al., 2010). Further investigations on the bioactive properties of polypeptides originating from this specific snow crab by-product hydrolysate were performed by Doyen et al. (2010). They carried out an electroseparation of the mixture at pH 9, 6 and 3. An ultrafiltration membrane was placed in an electrodialysis cell on both side of hydrolysate compartment allowing the simultaneous separation and recovery of anionic and cationic peptides. The pH values of the recovery compartments were also maintained at pH 9, 6 and 3 during all the separation process. The authors concluded that a selective separation was obtained by the recovery of two anionic and three cationic peptides in the adjacent compartment. They also tested the peptidic fractions for their anti-cancer activity. The results showed that only one fraction amongst the six fractions produced demonstrated a significant inhibition of growth cancerous lines especially on A549 (lung), BT549 (breast) and PC3 (prostate) cell lines. Furthermore, no anticancer aspect was observed for the feed hydrolysate fractions before separation. Consequently, the authors concluded that the anticancer activity was obtained after purification by electrodialysis with ultrafiltration membranes. Indeed, at a low concentration of 190 mg mL–1, cellular mortality of 75–85% for the A549 cell line, of 80% for the BT549 and of 95–100% for the PC3 cell line were obtained. Although the mechanism of action is still unknown, the future perspectives are very interesting because it is possible to isolate each peptide of the fractions recovered and to test them to determine the peptide(s) sequence(s) of interest. Antioxidant enrichment of cranberry juice Cranberry fruits are a rich source of phenolic phytochemicals including phenolic acids, flavonoids and ellagic acids (Vattem et al., 2005). Many of these compounds have antioxidant activity, equal to or greater than vitamin E (Yan et al., 2002). Cranberry juice can also prevent gastric ulcers caused


212 Separation, extraction and concentration processes by Helicobacter pylori (Shmuely et al., 2007) and displays potent anticancer activity (Neto et al., 2008). Bazinet et al. (2009) circulated in both compartments on each side of the filtration membrane a volume of cranberry juice (respectively, 450 mL and 200 mL) to concentrate a cranberry juice in its own specific compounds. The total concentrations of proanthocyanidins and anthocyanins increased by 34.8 and 52.9%, respectively in the 200 mL cranberry juice treated by EDFM with a 500 kDa filtration membrane. Moreover, a 18% increase of the antioxidant capacity was measured by means of the oxygen radical absorbance capacity (ORAC) test in the 200 mL of enriched cranberry juice. Moreover, the taste of the enriched cranberry juice was better than the non-treated juice. Based on these results the authors concluded that the production of phenolic antioxidant enriched cranberry juice could be feasible on a large scale and proposed an integrated process flow. According to this process flow, the EDFM process would be directly connected to the bottling process of cranberry juice to produce antioxidant-enriched cranberry juices by a batch process and cranberry juice with very low variation in antioxidant in a continuous process. An advantage of this technology is that compounds of interest would be directly transferred from one juice to another without the need for solvent to extract them first. 7.3.4 Electrodialysis with bipolar membranes A new type of membrane, called a bipolar membrane (BPM), appeared commercially at the end of the 1980s. Bipolar membranes carry out the dissociation of water in the presence of an electric field. These membranes are composed of three parts: an anion-exchange layer, a cation-exchange layer, and a hydrophilic transition layer at their junctions (Mani, 1991). In electrodialysis with bipolar membranes (EDBPMs), cation- and anion-exchange membranes are stacked together with bipolar membranes in an alternating series in an electrodialysis cell and allow its application to numerous products (Table 7.1). Amongst these applications, only the deacidification of fruit juices and the fractionation of 11S-7S soybean protein is developed. Deacidification of passion fruit juices The yellow passion fruit, Passiflora edulis f. flavicarpa, has an intense and special aroma and flavour, which make it a desirable ingredient in the formulation of various food products. However, because of its high acidity, only limited amounts of juice can be added as an ingredient in the formulation of various preparations (Adhikary et al., 1983; Couture and Rousseff, 1992). To overcome this Vera Calle et al. (2002, 2003) tested the deacidification of clarified passion fruit juice by electrodialysis with bipolar membranes (EDBPMs). The stack was equipped with homopolar and bipolar membranes, forming two or three compartments. The reduction of acidity was achieved by increasing the pH from 2.9 to 4.0. This pH limit was chosen to avoid



micro-organism growth and spoilage (Vera Calle et al., 2002). In these EDBPM configurations only anions were able to pass through the anionexchange membranes from the juice to the concentrate compartment. The net effect was the extraction of anions, mainly citrate, and their replacement by hydroxyl ions provided by the BPM. Citric acid was formed in the concentrate compartment by citrate ions extracted from juice and protons provided by the second BPM separating the concentrate compartment from the electrode compartments. The anion concentration was similar for the two processes. The inorganic ions were almost eliminated, and 62% of the citrate ions and 48% of the malate ions were removed from the fresh juice. According to the sensory properties of deacidified juices, no significant differences were observed between the deacidified juices and the fresh juice. 11S–7S fractionation The two major reserve soybean proteins, the globulins 7S or b-conglycinin (37–39% of total protein) and 11S or glycinin (31–44% of total protein) have different intrinsic properties leading to different functional properties. From results presented by Bazinet et al. (2000), it appears that during the EDBPM process, the temperature has a large effect on the selective precipitation of the soybean protein fractions, 11S and 7S. Hence, at 27 °C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of these fractions. However, at 10 °C, the 11S globulin precipitates at a higher pH than it does at 27 °C, respectively, pH 6.7 compared with 5.9, allowing the fractionation of 11S from the other fractions. Using electroacidification, it is possible to obtain a precipitate solution enriched in the 11S fraction (71.8% of 11S and 10.8% of 7S) and a supernatant solution enriched in the 7S fraction (46.6% of 7S and 4.6% of 11S). EDBPM appears to be a promising technology for fractionation of proteins, by adjusting the electroacidification and solution parameters. This technology does not denature protein (Bazinet et al., 1996), does not use any chemical acids or bases during the process, and produces isolates with a low ash content, which results in a final product of high purity.

7.4 Future trends In this chapter, an overwiew of electromembrane technologies and their associated applications has been presented. Numerous studies have already been performed on various raw matrices (milk, soya, whey, fruit juices) using different cell configurations, but new applications await discovery. Very recently electromembrane processes allowed the separation of bioactive peptides from snow-crab by-products (Doyen et al., 2010) and alfalfa protein hydrolyzates (Firdaous et al., 2009), and the enrichment of cranberry juices in antioxidant compounds (Bazinet et al., 2009). In addition, the low energy © Woodhead Publishing Limited, 2010

214 Separation, extraction and concentration processes cost and constant selectivity during separation of EDFM by absence of transmembrane pressure and, consequently, no membrane fouling, represent an improvement over the conventional separation processes used in the agrifood industries. Furthermore, many acid or alkaline solutions traditionally used in conventional processes are generated in situ by the EDBPM cell configuration. Based on these observations, it appears that electromembrane technologies present several advantages in agri-food industries, in particular, enrichment of nutraceutical products to improve their health benefits, in response to customer demands. In the future, numerous aspects will have to be explored to enhance the understanding of the technologies. Hence, studies on (1) protein/peptide interactions with filtration and ion exchange membranes (IEM) interfaces during treatment/separation of hydrolyzate solutions, and (2) the role of hydrolyzate mineral content on the modification of peptide theoretical charges and influence on their migration rates, would be of major interest. The understanding of both phenomena could lead to the development of new membrane materials and applications, and consequently, to promising ways for new products manufacturing.

7.5 References Adhikary, S.K., Harkare, W.P., Gowindan, K.P. and Nanjundaswamy, A.M. (1983) Deacidification of fruit juices by electrodialysis, Indian J. Technol., 21: 120–123. Amarowicz, R. and Shahidi, F. (1997) Antioxidant activity of peptide fractions of capelin protein hydrolysates, Food Chem., 58: 355–359. Amundson, C.H., Watanawanichakorn, S. and Hill, C.G., Jr. (1982) Production of enriched protein fractions of b-lactoglobulin and a-lactalbumin from cheese whey. J. Food Process Preserv, 6: 55–71. Audinos, R., Roson, J.P. and Jouret, C. (1979) Application de l’électrodialyse à l’élimination de certains composants du jus de raisin et du vin. Connaissance Vigne Vin, 13: 229–239. Audinos, R., Lurton, L. and Moutounet, M. (1985) Advantage of electrodialysis to produce sweetening products from grape. Sci. Aliment., 5: 619–637. Bargeman, G., Dohmen-Speelmans, M., Recio, I., Timmer, M. and Van den Horst, C. (2000) Selective isolation of cationic amino acids and peptides by electro-membrane filtration. Lait, 80: 175–185. Bargeman, G., Houwing, J., Recio, I., Koops, G.H. and Van den Horst, C. (2002) Electromembrane filtration for the selective isolation of bioactive peptides from an as2-casein hydrolysate. Biotechnol Bioeng., 80: 599–609. Bazinet, L., Cossec, C., Gaudreau, H. and Desjardins, Y. (2009) Production of a phenolic antioxidant enriched cranberry juice by an electrochemical process. J. Agric. Food Chem., 57: 10245–10251. Bazinet, L. (2005) Electrodialytic phenomena and their applications in the dairy industry: a review. CRC Critical review in Food Science and Nutrition, 45: 307–326.


Electrodialytic phenomena and associated electromembrane technologies 215 Bazinet, L., Amiot, J., Poulin, J.-F. and Labbé, D. and Tremblay, A. (2005) Process and system for separation of organic charged compounds. World Patent 082495A1. Bazinet, L. and Firdaous, L. (2009a) Membrane processes and devices for separation of bioactive peptides. Recent Pat. Biotechnol., 3: 61–72. Bazinet, L. and Firdaous, L. (2009b) Applications of electromembrane processes to the production of nutraceuticals or functional foods. In Handbook of membrane research: properties, performance and applications. Gorley, S.V. (Editor). Chemical engineering methods and technology series, Nova Science Publishers Inc., Hauppauge, NY. Chapitre 8, pp. 291–312. Bazinet, L., Ippersiel, D. and Lamarche, F. (1999b) Recovery of magnesium and protein from soy tofu whey by electrodialytic configurations. J. Chem. Technol. Biotechnol., 74(7): 663–668. Bazinet, L., Lamarche, F. and Ippersiel, D. (1998) Comparison of chemical and bipolar membrane electrochemical acidification for precipitation of soybean proteins. J. Agric. Food. Chem., 46: 2013–2019. Bazinet, L., Lamarche, F., Boulet, M. and Amiot, J. (1997a) Combined effect of pH and temperature during electroreduction of whey proteins. J. Agric. Food Chem., 45: 101–107. Bazinet, L., Lamarche, F., Labrecque, R. and Ippersiel, D. (1997b) Effect of KCl and soya protein concentrations on the performance of bipolar membrane electro-acidification. J. Agric. Food. Chem., 45: 2419–2425. Bazinet, L., Lamarche, F., Labrecque, R. and Ippersiel, D. (1997c) Effect of number of bipolar membranes and temperature on the performance of bipolar membrane electroacidification. J. Agric. Food. Chem., 45: 3788–3794. Bazinet, L., Gaudreau, H., Lavigne, D. and Martin, N. (2007) Partial demineralization of maple sap by electrodialysis: impact on syrup sensory and physicochemical quality. J. Sci. Food Agric., 87: 1691–1698. Bazinet, L., Ippersiel, D., Gendron, C., Mahdavi, B., Amiot, J. and Lamarche, F. (2001) Effect of added salt and increase in ionic strength on skim milk electroacidification performances. J. Dairy Res., 68: 237–250. Bazinet, L., Ippersiel, D., Labrecque, R. and Lamarche, F. (2000) Effect of temperature on the separation of soybean 11S and 7S protein fractions during bipolar membrane electroacidification. Biotechnol. Prog., 16: 292–295. Bazinet, L., Ippersiel, D. and Mahdavi, B. (2004a) Effect of conductivity adjustment on the separation of whey protein by bipolar membrane electroacidification. J. Agric. Food Chem., 52: 1980–1984. Bazinet, L., Ippersiel, D. and Mahdavi, B. (2004b) Fractionation of whey protein by bipolar membrane electroacidification. Innov. Food Sci. Emerg. Technol., 5: 17–25. Bazinet, L., Lamarche, F., Ippersiel, D. and Amiot, J. (1999a) Bipolar membrane electro-acidification to produce bovine milk casein isolate. J. Agric. Food Chem., 47: 5291–5296. Bazinet, L., Lamarche, F., Labrecque, R., Toupin, R., Boulet, M. and Ippersiel, D. (1996) Systematic study on the preparation of a food grade soyabean protein. Rep. Can. Electr. Assoc., 9326, U 987, Research and Development; Montréal. Beaulieu, L., Thibodeau, J., Desbiens, M., Saint-Louis, R. Zatylny, C. and Thibaut, S. (2010) Evidence of antimicrobial activities in peptide fractions originating from snow crab (Chionoecetes opilio) by-products. Probiot. Antimicrob. Prot., DOI 10.1007/ s12602-010-9043-6, in press. Ben Ounis, W., Champagne, C. P., Makhlouf, J. and Bazinet, L. (2008) Utilization of tofu whey pre-treated by electromembrane process as a growth medium for Lactobacillus plantarum LB17. Desalination, 229: 192–203. Bolduc, M.P., Bazinet, L., Lessard, J., Chapuzet, J.M. and Vuillemard, J.C. (2006) Electrochemical modification of the redox potential of pasteurized milk and its evolution during storage. J. Agric. Food Chem., 54: 4651–4657.


216 Separation, extraction and concentration processes Bolzer, R. (1985) Installation and process for the preparation of acid caseinates. United States Patent 4 559 119. Boyaval, P., Corre, C. and Terre, S. (1987) Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotech. Lett., 9: 207–212. Boyaval, P., Seta, J. and Gavach, C. (1993) Concentrated propionic acid production by electrodialysis. Enzyme Microb. Technol.; 15: 683–686. Brett, C.M.A. and Oliveira-Brett, A.M. (1994) Fundamentals of kinetics and mechanism of electrode reactions. In Electrochemistry: principles, methods, and applications; Oxford University Press: New York. Chaput, M. (1979) Le lactose, extraction et hydrolyse et déminéralisation du lactosérum. Rev. Lait. Française, 372: 23–26. Couture, R. and Rouseff, R. (1992) Debittering and deacidifying sour orange (Citrus aurantium) juice using neutral and anion exchange resins. J. Food Sci., 57: 380– 384. Crandall, J.J., Mantz, B.W. and Martz, E.C. (2001) Method for generating oxygenated water, United States Patent 6 284 293 B1. Donnan, F.G. (1911) Theory of membrane equilibrium and membrane potential in the presence of non-dialysing electrolytes: A contribution to physical-chemical physiology. Z. Elektrochem. Angewandte Phys. Chem., 17: 572–581. Doyen, A., Beaulieu, L., Saucier, L., Pouliot, Y. and Bazinet, L. (2010) Peptides from a snow crab by-products hydrolysate demonstrated anticancer properties in vitro after simultaneous separation by electrodialysis with ultrafiltration membranes. Submitted to Sep. Pur. Technol. Escudier, J., Saint-Pierre, B., Batlle, J. and Moutounet, M. (1995) Automatically controlled tartric stabilisation of wine by membrane electrodialysis which reduces its conductivity to the desired level. World Patent 9506110-A1. Firdaous, L., Dhulster, P., Amiot, J., Gaudreau, A., Lecouturier, D., Kapel, R., Lutin, F., Vézina, L.-P. and Bazinet, L. (2009) Concentration and selective separation of bioactive peptides from an alfalfa white protein hydrolysate by electrodialysis with ultrafiltration membranes. J. Membr. Sci., 329: 60–67. Gardais, D. (1990) Les procédés électriques de traitement des rejets industriels. In Environnement et Electricité. Electra, Doppee diffusion, Avon, pp. 200–310. Glassner, D. (1992) ED Applications in biotechnology. In Proceedings 10th annual membrane technology planning conference, Business Communications, Norwalk, pp. 158–165. Gonçalves, F., Fernandes, C., Cameira Dos Santos, P. and De Pinho, M.N. (2003) Wine tartaric stabilization by electrodialysis and its assessment by the saturation temperature. J. Food Eng., 59: 229–235. Guérif, G. (1993) Electrodialysis applied to tartaric stabilisation of wines. Rev. Oenol. Techn. Vitic. Œnol., 69: 39–42. Haratifar, S. (2008) The stability of electro-reduced milk lipids. Master of Science Thesis, Faculty of Agricultural and Food Sciences, Université Laval., Québec, Canada, 94p. Hekal, I.M. (1983) Process for the preservation of color and flavour in liquid containing comestibles. United States Patent 4.374.714. Hiraoka, Y., Itoh, K. and Taneya, S. (1979) Demineralization of cheese whey and skimmed milk by electrodialysis with ion exchange membranes. Milchwissenschaft, 34: 397–400. Houldsworth, D.W. (1980) Demineralization of whey by means of ion exchange and electrodialysis. J. Soc. Dairy Technol., 33: 45–51. Inoue, T.T. and Kato, W.K. (2003). Powdery drinks and process for producing the same. International Patent WO03053153. Janson, H.V. and Lewis, M.J. (1994) Electrochemical coagulation of whey protein. J. Soc. Dairy Technol., 47: 87–90. © Woodhead Publishing Limited, 2010

Electrodialytic phenomena and associated electromembrane technologies 217 Janson, H.V., Kiis, A.A., Alekseev, N.G. and Kirm, A.A. (1990) USSR Patent 1 570 694, 1990. Khidirov, S.S. and Merzametov, M.M. (1982) Electrocoagulation of milk proteins, brief communications, Vol.1, Book 2, XXI International Dairy Congress, Moscow, 12–16 July 1982. Kim, C., Hung, Y.C. and Bracket, R.E. (2000) Roles of oxidation–reduction potential in electrolyzed oxidizing and chemically modified water for the inactivation of foodrelated pathogens. J. Food Prot., 63: 19–24. Kim, S.-K. and Mendis, E. (2006) Bioactive compounds from marine processing byproducts. A review. Food Res. Int., 39: 383–393. Koseki, M., Nakagawa, A., Tanaka, Y., Noguchi, H. and Omochi, T. (2003) Sensory evaluation of taste of alkali-ion water and bottled mineral waters. J. Food Sci., 68: 354–358. Labbé, D., Araya-Farias, M., Tremblay, A. and Bazinet, L. (2005) Electromigration feasability of green tea catechins. J. Membr. Sci., 254: 101–109. Lin Teng Shee, F., Angers, P. and Bazinet, L. (2005) Precipitation of cheddar cheese whey lipids by electrochemical acidification. J. Agric. Food Chem., 53: 5635–5639. Lin Teng Shee, F., Angers, P. and Bazinet, L. (2007) Delipidation of a whey protein concentrate by electroacidification with bipolar membranes (BMEA). J. Agric. Food Chem., 55: 3985–3989. Lin Teng Shee, F., Arul, J., Brunet, S. and Bazinet, L. (2008) Performing a three-step process for conversion of chitosan to its oligomers using an unique bipolar membrane electrodialysis system. J. Agric. Food Chem., 56: 10019–10026. Mani, K.N. (1991) Electrodialysis water splitting technology. J. Membr. Sci., 58: 117–138. Mercier, D. (1999) Electrochemical treatment method and device for softening water. United States Patent 5.897.765. Mondal, K. and Lalvani, S.B. (2003) Electrochemical hydrogenation of canola oil using a hydrogen transfer agent. J. Amer. Oil Chem. Soc., 80: 1135–1141. Mondor, M., Ippersiel, D., Lamarche, F. and Boye, J.I. (2004) Production of soy protein concentrates using a combination of electroacidification and ultrafiltration. J. Agric. Food Chem., 52: 6991–6996. Ndiaye, N., Pouliot, Y., Saucier, L., Beaulieu, L. and Bazinet, L. (2010) Electroseparation of bovine lactoferrin from model and whey solutions. Sep. Pur. Technol., 74: 93–99, DOI 10.1016/j.seppur.2010.05.011. Neto, C.C., Amoroso, J.W. and Liberty, A.M. (2008) Anticancer activities of cranberry phytochemicals: an update. Mol. Nutr. Food Res., 52: S18–S27. Norddahl, B. (1998) Fermentative production and isolation of lactic acid. World Patent 9 828 433. Norddahl, B., Eriksen, S. and Pedersen, F.M. (2001) Method for producing lactic acid. World Patent 01 92555-A1. Pérez, A., Andrés, L.J., Alvarez, R., Coca, J. and Hill, C.G. (1994) Electrodialysis of whey permeates and retentates obtained by ultrafiltration. J. Food Proc. Eng., 17: 177–190. Picot, L., Bordenave, S., Didelot, S., Fruitier-Arnaudin, I., Sannier, F., Thorkelsson, G., Bergé, J.P., Guérard, F., Chabeaud, A. and Piot. J.M. (2006) Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem., 41: 1217–1222. Poulin, J.-F., Amiot, J. and Bazinet, L. (2006) Simultaneous separation of acid and basic bioactive peptides by electrodialysis with ultrafiltration membrane. J. Biotechnol., 123: 314–328. Poulin, J.-F., Amiot, J. and Bazinet, L. (2007) Improved peptide fractionation by electrodialysis with ultrafiltration membrane: influence of ultrafiltration membrane stacking and electrical field strength. J. Membr. Sci., 299: 83–90.


218 Separation, extraction and concentration processes Quoc, A., Lamarche, F. and Makhlouf, J. (2000) Acceleration of pH variation in cloudy apple juice using electrodialysis with bipolar membranes. J. Agric. Food Chem., 48: 2160–2166. Quoc, A.L., Mondor, M., Lamarche, F., Ippersiel, D., Bazinet, L. and Makhlouf, J. (2006) Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice. Food Res. Int., 39: 755–760. Shaposhnik, V.A. and Kesore, K. (1997) An early history of electrodialysis with permselective membranes. J. Membr. Sci., 136: 35–39. Shmuely, H., Yahav, J., Samra, Z., Chodick, G., Koren, R., Niv, Y. and Ofek I. (2007) Effect of cranberry juice on eradication of Helicobacter pylori in patients treated with antibiotics and a proton pump inhibitor. Mol. Nutr. Food Res., 51: 746–751. Skorepova, J. and Moresoli, C. (2007) Carbohydrate and mineral removal during the production of low-phytate soy protein isolate by combined electroacidification and high shear tangential flow ultrafiltration. J. Agric. Food Chem., 55: 5645–5652. Slack, A.W., Amundson, C.H. and Hill, C.G. (1986) Production of enriched b-lactoglobulin and a-lactalbumin whey protein fractions. J. Food Proc. Pres., 10: 19–30. Stack, F.M., Hennessy, M., Mulvihill, D. and O’Kennedy, B.T. (1995) World Patent 9534216-C1. Swanson, A.M. and Sommer, H.H. (1940) Oxidized flavor in milk: II. The relation of oxidation–reduction potentials to its development. J. Dairy Sci., 23: 597–614. Thompson, B.G. and Gerson, D.F. (1985) Electrochemical control of redox potential in batch cultures of Escherichia coli. Biotechnol. Bioeng., 27: 1512–1515. Tronc, J.-S., Lamarche, F. and Makhlouf, J. (1997) Enzymatic browning inhibition in cloudy apple juice by electrodialysis. J. Food Sci. 62: 75–78. Tronc, J.-S., Lamarche, F. and Makhlouf, J. (1998) Effect of pH variation by electrodialysis on the inhibition of enzymatic browning in cloudy apple juice. J. Agric. Food Chem., 46: 829–833. Vattem, D.A., Ghaedian, R. and Shetty, K. (2005) Enhancing health benefits of berries through phenolic antioxidant enrichment: focus on cranberry. Asia Pac. J. Clin. Nutr., 14: 120–130. Vera Calle, E., Dornier, M., Sandeaux, J., Pourcelly, G. (2002) Deacidification of the clarified passion fruit juice using different electrodialysis configurations. Desalination, 149: 357–361. Vera Calle, E., Ruales, J., Sandeaux, J., Dornier, M., Persin, F., Reynes, M. and Pourcelly, G. (2003) Comparison of different methods for deacidification of clarified passion fruit juice. J. Food Eng., 59: 361–367. Yan, X., Murphy, B.T., Hammond, G.B., Vinson, J.A. and Neto, C.C. (2002) Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J. Agric. Food Chem., 50: 5844–5849.


Principles of pervaporation for the recovery of aroma compounds 219

8 Principles of pervaporation for the recovery of aroma compounds and applications in the food and beverage industries S. Sahin, Middle East Technical University, Turkey Abstract: The principles and transport mechanism of pervaporation, a membrane separation process in which the components from a liquid mixture permeate selectively through a dense membrane, are described. Information on membrane materials is given and studies on the recovery of aroma compounds by pervaporation, one of its most important applications in the food industry, are reviewed. Sources of further information are listed and future trends explored. Key words: aroma compounds, food, pervaporation, beverages.

8.1 Introduction Processing, especially at high temperatures, may cause considerable physical and/or chemical changes in the aroma compounds of foods. Physical losses may take place during evaporation and chemical changes of aroma compounds may occur owing to heat-induced oxidation or Maillard reactions. For example, in the fruit juice industry, changes in aroma compounds are inevitable since pasteurization of fruit juice is required in order to increase the shelf life of the product. In addition, aroma compounds are highly volatile so that most of them are lost by evaporation during the production of concentrated juice. The change/loss of aroma compounds may affect the final product quality and consumer’s acceptance. Therefore, the aroma compounds must be recovered either from the loss stream or removed before the raw material is subjected to heat treatment (aroma stripping) and added back to the concentrated juice. There are several methods for the extraction that minimize chemical changes and physical losses of aroma compounds. These methods can be


220 Separation, extraction and concentration processes categorized as: vapor–liquid separations based on distillation/evaporation or partial condensation, separations based on gas injection adsorption and supercritical-fluid extraction. However, these separation techniques have some limitations. Pervaporation, which is a membrane process, is a promising alternative method for aroma recovery because of it has advantages over conventional aroma recovery processes such as high selectivity, low energy consumption, moderate operating temperatures, physical separation mechanism and no additive requirement (Lipnizki et al., 2002a). Compared with other membrane processes like ultrafiltration and reverse osmosis, pervaporation exhibits low fluxes. However, selectivities can be quite high. Therefore, this method is especially suitable for the recovery of highly diluted species. This method can be performed on the juice before the evaporation or pasteurization step thus preventing deterioration or loss of the heat-sensitive aroma compounds during the heat treatment. In addition to aroma recovery, this method can be used for dealcoholization of wine or beer, recovery of high-value-added components or removal of organic pollutants from waste streams, and removal of inhibitors from fermentation broths. It can also be used as an analytical separation technique.

8.2 Principles of pervaporation Pervaporation is a membrane technique in which the components from a liquid mixture are separated by means of partial vaporization through a non-porous permselective membrane. The term ‘pervaporation’ is used in order to emphasize the fact that the permeate undergoes a phase change from liquid to vapor during its transport through the barrier. The pervaporation term was first introduced by Kober (1917). In his study, it was observed that a liquid in a collodion (cellulose nitrate) bag that was suspended in the air, evaporated, although the bag was tightly closed. Kober realized that this phenomenon could be used for separation of liquid mixtures. In pervaporation, a membrane separates an upstream mixture in the liquid state from downstream permeates in the gaseous state. The downstream side is maintained at a reduced pressure to ensure the gaseous state. The liquid feed mixture is circulated in contact with the membrane. The permeate in gaseous state is collected in the liquid state after condensation on a cooled wall. A schematic diagram of a typical experimental set-up for pervaporation is shown in Fig. 8.1. The feed solution is kept in a reservoir inside the temperature-controlled water bath and it is pumped to the upper part of the pervaporation cell. The vacuum is applied to the lower compartment of the cell by use of a vacuum pump in order to provide a pressure gradient across the membrane. The vacuum pressure in the system is controlled by a vacuum controller. The permeate is collected in cold traps by condensation, mostly


Principles of pervaporation for the recovery of aroma compounds 221 Thermocouple

TC Trap 1

Trap 3

Water bath

PC

Trap 2 Peristaltic Pervaporation pump cell

Vacuum pump

Fig. 8.1 Schematic diagram of a typical pervaporation experimental set-up; TC, temperature control; PC, pressure control (reprinted from Isci et al., 2006, with permission from Elsevier).

with liquid nitrogen at –196 °C. There are usually three traps in the set-up; the first two are connected in parallel in order to conduct the experiments continuously and to maintain the steady-state condition during data collection. The third one is connected in series to the parallel traps as a safety trap in order to ensure that practically no permeate reaches the vacuum pump. The components from the liquid feed mixture permeate selectively through a dense membrane driven by a chemical potential gradient obtained by partial pressure reduction on the permeate side. The affinity between the permeant and the polymer material that constitutes the membrane as well as its mobility through the membrane matrix are important for the transport of the compound. Pervaporation is a complex process involving simultaneous heat and mass transfer. Latent heat is required to pervaporate the liquid. The gradient in partial vapor pressure between the feed and the permeate side of the membrane is maintained by reducing the partial vapor pressure in the permeate side. The heat necessary for the evaporation of the permeate has to be transported through the membrane and this transport of energy is coupled to the transport of mass. The evaporation enthalpy is taken from the sensible heat of the liquid feed mixture, leading to a reduction in feed side temperature.

8.3 Transport mechanism in pervaporation for the recovery of aroma compounds Although a phase change from liquid to vapor takes place, this phase change never occurs directly but occurs via a membrane–solute interaction © Woodhead Publishing Limited, 2010

222 Separation, extraction and concentration processes in pervaporation. Therefore, the separation principle is not based on the vapor–liquid equilibrium. Transport through the nonporous membrane is only by diffusion. The solubility and the diffusivities of the constituents in the membrane are important for separation by pervaporation. Therefore, solute transport within the membrane is not directly affected by the external operating conditions. Ideally, the membrane alone determines the solute transport and the characteristics of the separation and so it is important to create the optimum mass transport conditions to and from the membrane (Schafer et al., 2006). The driving force in pervaporation is the gradient of chemical potential of a solute across the selective membrane (Schafer and Crespo, 2002). Thus, the mass transport in a pervaporation membrane can be described as a function of the chemical potential gradient:

Ji = f (D mi)

[8.1]

where Ji and mi are the mass flux and chemical potential of solute i, respectively. The chemical potential can be stated as a function of the state variables; pressure, composition and temperature:

m = f (P, C, T)

[8.2]

Therefore, the differences in the permeate pressure, feed composition and temperature cause differences in chemical potential. For this reason, there are three methods in pervaporation to establish a suitable chemical potential gradient in the membrane as driving force for the mass transport: (a) Vacuum pervaporation: a hydrostatic pressure difference between the feed and the permeate mixture is established by introducing a vacuum on the permeate side. This method is applicable if the volume of permeating vapor is relatively small or the permeate side pressure is not too low. Otherwise, vacuum pumps of extremely large capacities are required and the pumps consume too much energy (Brüschke, 2006). (b) Thermopervaporation: a chemical potential gradient in the membrane is established by providing a temperature difference between the feed and the permeate side (Strathmann and McDonogh, 1993). (c) Sweeping-gas pervaporation: an inert gas stream on the permeate side of the membrane can be used to remove the component and thus establish a concentration gradient driving force. Vacuum pervaporation is the most commonly used method because a chemical potential gradient is readily obtained by reducing partial pressure on the permeate side. Partial pressures of the permeant components are usually lower than their corresponding saturation pressures. Therefore, they are removed as vapor. The principle of solute transport across the membrane in vacuum pervaporation is shown in Fig. 8.2. A nonporous membrane separates a liquid feed, which is usually close to atmospheric pressure, from the downstream compartment where a vacuum is applied. The solutes (denoted by solid circles)


Principles of pervaporation for the recovery of aroma compounds 223 Membrane

Liquid

Membrane

Liquid

Vapor

(a)

Vapor

(b) Membrane

Liquid

Vapor

(c)

Fig. 8.2 The principle of solute transport across the membrane in pervaporation. For explanation of panels see text.

are first absorbed by the membrane surface when they are in contact with the membrane owing to the interactions between the solutes and polymer (Fig. 8.2a). When the chemical potential of these components in the feed and in the selective layer of the membrane are equal, a thermodynamic equilibrium is reached. The solutes that are absorbed by the membrane surface create a chemical potential difference across the membrane, which causes a diffusive net flux of solute through the membrane polymer (Fig. 8.2b). Ideally, all the solute particles that have diffused through the membrane are desorbed suddenly and removed by vacuum applied on the downstream side of the membrane. As a result, the solute concentration on the membrane downstream surface remains practically zero, and a maximum concentration gradient between the two membrane surfaces is maintained. If the vacuum is not low enough to desorb all the solutes reaching the membrane downstream surface, the concentration of the solute at the bottom of the membrane is not zero and, consequently, the concentration gradient decreases, as the net flux across the membrane decreases. For the ideal diffusion of solute i across the membrane toward the membrane downstream surface, Fick’s First law applies:

dC J i = Di ÈÍ i ˘˙ Î dz ˚

[8.3]

where Ji is the flux of the permeant i, Ci is the concentration of the permeant i in the polymer membrane, z is the position in the membrane, measured


224 Separation, extraction and concentration processes from the feed side of the membrane and Di is the diffusion coefficient of the permeant i in the membrane. The diffusion coefficient of a specific component through a polymer can be assumed to be independent of the solute concentration. Then, integration across a membrane, with a thickness Z, yields:

J = Di

(Cimem,f – Cimem,p ) Z

[8.4]

where Cimem,f and Cimem,p are the concentrations of the permeant i inside the membrane at its feed and permeate sides, respectively. If the vacuum applied on the permeate side of the membrane is strong enough, we can assume that all of the solute particles that diffuse through the membrane are removed instantaneously. Then Cimem,p can be taken as zero and equation [8.4] reduces to:

J i = Di

(Cimem,f ) Z

[8.5]

Transport, across the membrane, is generally described by the ‘solutiondiffusion mechanism’ (Strathmann and McDonogh, 1993). Assuming constant solute feed concentration and rapid removal of solute on the membrane downstream, the selective transport of the component ‘i’ from the bulk feed to the downstream compartment can ideally be described by the following steps as: 1. equilibrium partitioning (sorption) of the component i from the liquid phase at the feed solution-membrane upstream surface interphase; 2. diffusion of the absorbed component i through the membrane polymer matrix toward the downstream surface of the membrane; 3. equilibrium partitioning (desorption) of the component i from the membrane downstream surface to the permeate vapor. For the equilibrium partitioning in steps 1 and 3, the sorption (partitioning) coefficient of i in the homogeneous membrane polymer Si can be expressed as: Si =

Cimem,f Cil,bulk

[8.6]

where Cil, bulk is the concentration of component i in the bulk liquid. Combination of equations [8.5] and [8.6] yields the transport model commonly applied in pervaporation:

J i = Si Di

(Cil,bulk ) (C l,bulk ) = Pi i Z Z


[8.7]


where Pi is the permeability of i in the membrane which is the product of the solubility and diffusivity of the solute i in the membrane. It can be concluded from equation [8.7] that, for an ideal case, the flux of a specific component is directly related to the concentration of the component in the bulk feed. In addition, the permeability of the solute can be calculated from the slope of the flux versus feed concentration graph, if the thickness of the membrane is known. The driving force can also be expressed as a partial pressure gradient of the solute i across the membrane. Then, equation [8.7] becomes:

ÈP f – Pip ˘ DP J i = Si Di ÈÍ i ˘˙ = Pi¢ Í i ˙ ÎZ ˚ Î Z ˚

[8.8]

where Pi¢ is the permeability coefficient (kg m–1 s–1 Pa–1), and Pif and Pip are partial pressures of compound i in the feed and permeate sides, respectively. The widely accepted parameters used to describe the performance of the pervaporation process are permeate flux (Ji), selectivity or separation factor (ai/j) and enrichment factor (bi). The permeate flux Ji is defined as the permeate flow rate per unit of membrane area for a given membrane thickness. The separation factor ai/j is a measure of the selectivity and it indicates the preferential permeation of component i compared with component j. It is calculated from the concentrations of the compounds to be separated, i and j, in the feed and the permeate, respectively:

a i /j =

(Cip /Cil,bulk )

(C pj /C l,bulk ) j

[8.9]

The affinity between the permeant and the polymer material that constitutes the membrane and its mobility through the membrane matrix which are responsible for the transport are important parameters for selectivity. Since different species permeate through the membrane at different rates, a substance at a low concentration in the feed stream can be highly enriched in permeate. The enrichment factor bi represents the capacity of the membrane for concentrating component i and is defined as:

bi =

Cip C l,bulk j

[8.10]

If perfect mixing of the feed and sudden removal of the solutes leaving the membrane downstream face can not be provided, boundary layers develop on both sides of the membrane, affecting the solute transport to and away from the respective membrane surface. If the flux of solute i through the membrane is higher than that through the liquid phase toward the membrane, © Woodhead Publishing Limited, 2010

226 Separation, extraction and concentration processes the solute i is depleted in the liquid phase over the membrane upstream surface, resulting in a liquid solute concentration lower than that in the bulk feed. Because the solute concentration in the liquid at the membrane upstream surface determines the partitioning of the solute into the membrane, the concentration of i in the membrane upstream surface is lower with respect to bulk concentration. Therefore, the concentration gradient across the membrane decreases as does the overall flux. This phenomenon is known as ‘concentration polarization’ and affects the fluxes of compounds of high sorption coefficient, even under turbulent hydrodynamic conditions over the membrane. A similar phenomenon can also be found on the membrane downstream face (Schafer and Crespo, 2002). The concentration polarization strongly depends on the feed flow velocity and on the hydraulic diameter. An increase in the feed flow velocity and a decrease in the hydraulic diameter, decrease the effect of concentration polarization (Borjesson et al., 1996). As long as the diffusive flux is sufficiently high compared with the solute flux across the membrane, the boundary layer does not represent an additional transport resistance and the solute concentration at the membrane surface is equal to that in the bulk (Schafer and Crespo, 2007). However, if the diffusive flux across the liquid boundary layer is lower than the maximum achievable transmembrane flux at the respective feed bulk concentration, the boundary layer detrimentally affects the overall transport of solute from the feed bulk to the permeate side of the pervaporation membrane because the membrane surface concentration of solute i is lower than that of the bulk. Overall mass transfer coefficient kov,i can be expressed as the sum of mass transfer resistances:

Ê 1 ˆ Ê 1 ˆ Ê 1 ˆ Ê 1 ˆ ÁË k ˜¯ = ÁË k ˜¯ + ÁË k ˜¯ + ÁË k ˜¯ ov,i bl,i m,i p,i

[8.11]

where kbl,i is the mass transfer coefficient for the boundary layer, km,i is the mass transfer coefficient for the membrane, and kp,i is the mass transfer coefficient for the permeate side. Although in most instances, it is hard to achieve an ideal case, the mass transfer resistance in the feed boundary layer can be neglected compared with the overall mass transfer resistance if the feed flow velocity is maintained sufficiently high during pervaporation (Karlsson and Tragardh, 1993). Moreover, if a good vacuum is applied on the permeate side of the membrane, everything that diffuses through the membrane is instantaneously pumped away and therefore, mass transfer resistance in the permeate side can also be neglected. Then, the only resistance to mass transfer is within the membrane polymer and equation [8.11] simplifies to:

Ê 1 ˆ Ê 1 ˆ ÁË k ˜¯ = ÁË k ˜¯ ov,i m,i

[8.12]



Therefore, overall mass flux of component i from the bulk of the feed to the bulk of the permeate can be described by the following equation:

J i = kov,i (Cil,bulk – Cip ) = km,i (Cil,bulk – Cip )

[8.13]

p

Another phenomenon affecting pervaporation performance is temperature polarization. Owing to the phase change from the feed to the permeate side, the membrane acts as a heat sink and a temperature difference exists between the bulk of the feed and the feed side of the membrane. The resulting reduction in the driving force and hence in performance is called ‘temperature polarization’ and this effect is unique for pervaporation processes (Brüschke, 2006). Flux changes linearly with change in concentration but exponentially with change in temperature which means that temperature polarization is more important than concentration polarization. Therefore, the development of high flux pervaporation membranes requires the development of modules in which temperature polarization is effectively reduced. Concentration polarization does not change significantly with changes in composition of the feed. However, the effect of temperature polarization is more important if the concentration of the component to be removed, and thus the flux, is high.

8.4 Selection of membranes for pervaporation in the recovery of aroma compounds A membrane can be simply defined as a permselective barrier made up of polymers between the feed and permeate. Polymers are very large molecules (about 1000 to 100 000 times larger than water molecule) which are manufactured by chemical processes. Polymers gain different characteristics during their design. Generally, there are two types of polymers: glassy polymers and rubbery polymers or elastomers. Their ground state at room temperature determines their distinction (Koops and Smolders, 1991). Glassy polymers have a glass transition temperature above the room temperature and can be divided into three groups: crystalline, semi-crystalline and amorphous polymers. The presence of crystallites has an important affect on all kinds of polymers in terms of tensile strength, elasticity, impact strength, solubility and diffusivity in the polymer. Generally, glassy polymers are hydrophilic (Böddeker and Bengtson, 1991). They preferentially permit the permeation of water owing to the presence of groups in the polymer chain that are able to interact with water molecules. They are used mainly for removal of water from organic solvents and solvent mixtures with an emphasis on azeotropic mixtures. Membranes for the removal of small alcohol molecules like methanol or ethanol are also hydrophilic. Some examples of glassy polymers are cellulose acetate, polysulfone, and polyvinylalcohol.


228 Separation, extraction and concentration processes Polymers with a glass transition temperature below room temperature are classified as rubbers or elastomers. Elastomers normally present higher chain mobility than glassy polymers and contain rather small, non-polar side groups. Elastomers are flexible and mostly hydrophobic polymers. They preferentially permeate organic substances owing to the lack of strong intermolecular forces, such as hydrogen bonding or dipole–dipole interactions. Since they absorb organic molecules with relatively high fluxes, elastomers are the most suitable polymers for the separation of organic molecules from aqueous solutions (Koops and Smolders, 1991) or from organic–organic mixtures. Some of the examples of hydrophobic polymers are polydimethylsiloxane (PDMS), polyether block polyamide (PEBA), polypropylene (PP), and polyvinylidenefluoride (PVDF) (Koops and Smolders, 1991). Glassy and rubbery membranes differ strongly in terms of flexibility of their polymeric structure. Within the rigid polymeric structure of a glassy membrane, the diffusivity of components is strongly related to their molecular volume, whereas in the flexible region of a rubbery membrane, solute–polymer interactions are much more important than the diffusivities of components. This is why the selectivity of glassy membranes is more diffusion controlled, whereas the selectivity of rubbery membranes is more sorption controlled (Schafer and Crespo, 2002). In pervaporation, nonporous membranes which provide selectivity based on solute–membrane interactions rather than on volatilities are used. The membrane type used in the pervaporation experiments determines the selectivity towards the molecules. Therefore, membrane selection is one of the most important issues in pervaporation. The membrane should have high selectivity and flux for the components to be separated. In addition, it must have chemical, mechanical and thermal stability (Schafer and Crespo, 1997). In pervaporation, the feed side of the membrane is highly swollen in contact with the liquid whereas the permeate side is dry and virtually nonswollen. Thus, a high gradient of swelling exists over the separating layer of the membrane, demanding additional resistance and stability. Pervaporation can be classified as hydrophilic and organophilic pervaporation (Lipnizki et al., 1999). In hydrophilic pervaporation, the target compound water is separated from an aqueous–organic mixture whereas in organophilic pervaporation, the target organic compounds are separated from an aqueous–organic mixture (hydrophobic pervaporation) or from an organic–organic mixture by being preferentially permeated through the membrane (target organophilic pervaporation). Generally, in membrane production for pervaporation processes, composite membrane structure is preferred. Composite membranes are composed of three layers. The first layer is the dense layer responsible for the separation. This layer has to be as thin as possible since the transport through the membrane is by diffusion (0.5–2 mm). The second layer is the porous support layer that has an asymmetric pore structure. It has a thickness of 70–100 mm. Structural polymers with high resistance against chemical attack and good thermal and



mechanical properties such as polyacrylonitrile, polyetherimide, polysulfone, PVDF form the porous support. The final layer, having a thickness of about 100 mm is a woven or nonwoven textile fabric. Polyester, polyethylene, polyphenylene sulfide and similar fibers are used for the textile carrier layer (Brüschke, 2006). The dense separating layer of hydrophilic membranes is made from various polymers that have high affinity towards water. These polymers contain ions, oxygen functions such as hydroxyl-, ester, or carboxylic moieties or nitrogen as imino- or imido- groups. They must be crosslinked in order to render them insoluble after the coating process. For organophilic membranes, the dense separating layer is formed by crosslinked silicones. The most commonly used organophilic membranes are polydimethylsiloxane (PDMS) or polymethyl octyl siloxane (POMS). The monomer of this silicon rubber is: CH3 Si

CH3

O

[8.14] n

POMS is a modified silicon rubber in which one of the methyl groups of the monomer has been replaced by an octyl group. PDMS or POMS membranes with various porous support layers are commercially available. Zeolite-filled silicon membranes have been developed to increase the selectivity and flux. PEBA formulations have elastic segments consisting of thermoplastic polyamides made flexible by elastomeric polyether links. The modification of PDMS membrane by the introduction of rigid organophilic groups has potential in the pervaporation field for the improvement of separation behavior (Luo et al., 2008). Polyphenylmethylsiloxane–cellulose acetate (PPMS–CA) and polydimethylsiloxane–cellulose acetate (PDMS-CA) membranes are used for concentration of volatile organic compounds such as methanol, ethanol and acetone from aqueous solutions by pervaporation. The hydrophobicity of PPMS membranes is stronger than that of PDMS. In the study of Song and Lee (2005), the tube type alumina substrate was modified with silane coupling agent (perfluoroalkylsilane) to obtain hydrophobic membrane. The surface modified membrane showed much higher flux but lower selectivity than nonporous PDMS membrane for the recovery of esters. However, the selectivity of the membrane was sufficiently high. Olsson et al. (2002) studied the effect of three different POMS membranes, a polymethyloctylsiloxane–polyetherimide (POMS-PEI) and two polymethyloctylsiloxane–polyacrylonitrile (POMS-PAN) membranes having different thicknesses for the separation of alcohols, esters and aldehydes. They concluded that the porous support layer could affect the selectivity considerably and membranes should be designed with a low degree of crosslinking to obtain better separation properties, especially for larger permeants. © Woodhead Publishing Limited, 2010


8.5 Recovery of aroma compounds by pervaporation and applications in the food and beverage industries One of the most promising applications of pervaporation is the recovery of aroma compounds from aqueous mixtures. Aroma is a complex mixture of hundreds of different volatile organic compounds present at very low concentrations, typically at ppm or ppb levels. Aroma compounds belong to various chemical groups such as alcohols, aldehydes and esters. Recovery of natural flavor and aroma compounds has received much more attention in the food, biotechnology and cosmetic industries because natural aroma compounds are preferred to chemically synthesized ones. In the food industry, flavor concentrates are widely used to compensate for the loss of aroma compounds during processing. Because aroma compounds are highly heat sensitive and loss of aroma compounds during heat treatments is inevitable, they should be separated from food systems and added back to the final product. This can be accomplished either by recovering the lost aromas from the loss stream or by stripping the aromas from the raw material stream before processing. This eventually increases the quality and acceptance of the final product. There have been a large number of studies in this area. Pervaporation has been successfully applied to the recovery of the aroma compounds of wine (Karlsson et al., 1995; Schafer and Crespo, 2007; Schafer et al., 1999) and several fruit juices, such as apple (Bengtsson et al., 1992; Börjesson et al., 1996; Olsson and Tragardh, 1999 and 2001), cashew apple (De Assis et al., 2007), kiwifruit (Cassano et al., 2006), strawberry (Isci et al., 2006), pineapple (Pereira et al., 2002 and 2005; Sampranpiboon et al., 2000), passion fruit (Pereira et al., 2002; 2005), banana (Sampranpiboon et al., 2000), orange (Aroujalian and Raisi, 2007; Shepherd et al., 2002), blueberry (Peng and Liu, 2003), billberries (Diban et al., 2008; Garcia et al., 2008), grape (Rajagopalan and Cheryan, 1995) and pomegranate (Raisi et al., 2008). In addition, pervaporation has been found to be suitable for concentration of tea (Kanani et al., 2003), cocoa (Kattenberg and Willemsen, 2001) and dairy aroma compounds (Baudot and Marin, 1996; Overington et al., 2008) from aqueous solutions and also for dealcoholization of alcoholic beverages (Takacs et al., 2007). It was successfully applied for the recovery of terpenes from waste waters in essential oil industry (Charbit et al., 1997) and for deodorization of waste streams (Souchon et al., 2002). It can also be used as an analytical separation technique for the continuous determination of volatiles (Amador-Hernandez and Castro, 2000). In most of the studies, model feed solutions have been used because of the complexity of conducting the pervaporation experiments with real multicomponent mixtures (Baudot and Marin, 1999; Bengtsson et al., 1992; Börjesson et al., 1996; Garcia et al., 2008; Isci et al., 2006; Kanani et al., 2003; Olsson and Tragardh, 1999; 2001; Peng and Liu, 2003; Pereira et al., 2002 and 2005; Raisi et al., 2008; Rajagopalan and Cheryan, 1995;



Sampranpiboon et al., 2000). However, there are also some studies in which real food samples are used (Alvarez et al., 2000; Kanani et al., 2003; Pereira et al., 2002 and 2005; Raisi et al., 2008; Rajagopalan and Cheryan, 1995; She and Huang, 2006). Most of the research on aroma recovery by pervaporation has been conducted on a laboratory scale. Studies of scale-up of the pervaporation process for the separation of multi-component mixtures are few. Karlsson et al. (1998) developed a model for the scale-up of pervaporation units for aroma recovery in which the effect of concentration polarization was taken into account with a semi-empirical model. A novel process simulation of pervaporation was developed for multicomponent mixtures (Lipnizki et al., 2002a) and this study was continued with the integration and optimization of hydrophobic pervaporation for the recovery of natural aroma compounds in the food industry (Lipnizki et al., 2002b). Trifunovic et al. (2006) investigated module design aspects of pervaporation using a modified version of an existing pervaporation simulation tool for aroma recovery. By applying the simulation to four aroma compounds: two alcohols (n-butanol, n-hexanol) and two esters (isoamyl acetate and ethyl butyrate), the effect of major process and module design parameters on the performance of a single module has been investigated. In general, for the compounds studied, the permeate composition can be manipulated by changing the module geometry and operating conditions because the effect of these parameters is more significant on esters, which in general have high recovery rates, than for alcohols, which are only marginally affected. 8.5.1 Recovery of fruit juice aromas Apple juice is one of the important fruit juices that pervaporation studies have focused on. Börjesson et al. (1996) investigated the performance of six different pervaporation membranes (PDMS-1060, PDMS-1070 (polydimethyl siloxane + silicalite), PDMS–PT 1100 (polydimethylsiloxane), POMS–PEI (polyoctylmethyl siloxane- polyethermide), POMS–PVDF and PEBA) for the recovery of apple juice aromas. Multicomponent solution containing five esters (ethyl acetate, ethyl butanoate, ethyl- 2-methylbutanoate, isopentyl acetate and hexyl acetate), one aldehyde (trans-2-hexenal) and four alcohols (isobutanol, butanol, isopentanol and hexanol) representing typical aroma compounds of apple juice was used in this study. The best performance was obtained with the PDMS–PT 1100, POMS–PEI and POMS–PVDF membranes. The influence of permeate pressure on the recovery of apple juice aroma compounds by pervaporation had also been examined using the same model solution (Olsson and Tragardh, 2001). A mathematical model, which includes the effect of concentration polarization on the feed side of the membrane and its dependence on permeate pressure, the variation in the separation properties of the membrane with permeate pressure and the properties of the individual permeants, was developed to predict the influence of permeate pressure on separation factor in pervaporation. © Woodhead Publishing Limited, 2010

232 Separation, extraction and concentration processes Moreover, the effect of feed flow velocity on recovery of apple juice aroma compounds by pervaporation was studied (Olsson and Tragardh, 1999). Increasing the feed flow velocity did not increase the recovery of the alcohols significantly because the dominating resistance to mass transfer was in the membrane for the alcohols. However, the recovery of esters and aldehydes was improved by increasing feed flow velocity. The dominating resistance to mass transfer was in the liquid feed boundary layer for the esters studied except ethyl acetate. For the aldehyde and ethyl acetate these two resistances were of the same order. Bengtsson et al. (1992) concentrated the twelve selected flavor compounds in apple juice by pervaporation. It was observed that alcohol had the lowest enrichment factors (2–22), the aldehydes had the medium values (16–67) and the esters had the highest enrichment factors (up to 100). The order of enrichment factors of aroma compounds of various chemical groups was the same in the study of Börjesson et al. (1996). De Assis et al. (2007) found that pervaporation can be used for the concentration of cashew apple juice aroma from cashew pulp. The chromatograms showed an increase in the peak area and also in the number of components in the permeate samples compared with the chromatogram of the cashew juice. Recovery of key flavor components from real flavor concentrates (apple essence, orange aroma and black tea distillate) was achieved by pervaporation in the study of She and Hwang (2006). In this study, both continuous and batch operations were carried out. Generally, the acetates and aldehydes exhibited higher enrichment factors than the alcohols. Significant flavor loss was observed in the pervaporation recovery process. PDMS membrane had higher permeation and loss rates than POMS membrane in batch pervaporation of dilute apple essence. Therefore, it was concluded that POMS membrane with a long operating time should be preferred if highly concentrated products and minimized flavor loss are the major concerns. However, PDMS membrane was found to be better if the time is limited. Rajagopalan and Cheryan (1995) studied pervaporation of model flavor compound of grapes (methyl anthranilate) and of commercial grape essence with several membranes: PDMS–PC (polydimethoxylsiloxane–polycarbonate), PEBA, PDMS-1070). PEBA membrane generally gave higher flux and better selectivity. Flux and selectivity decreased linearly with increase in downstream pressure, but increased with temperature. This relationship was not satisfied when the feed concentration was above 50 ppm where concentration polarization became serious. The presence of ethanol in the feed solution lowered separation factors but increased total flux. Experiments with commercial grape essence confirmed the excellent potential of pervaporation for the production of highly enriched flavors. Pervaporation was found to be a promising technique for the recovery of strawberry aroma compounds (Isci et al., 2006). The experiments were performed using binary aqueous solutions of methyl butyrate (MTB) and



ethyl butyrate (ETB), ternary mixture of MTB, ETB and water and aqueous model strawberry solution containing six aroma compounds (MTB, ETB, butyl butyrate (BTB), methyl caproate (MTC), ethyl caproate (ETC) and linalool). An increase in both flux and selectivity was observed with an increase in feed temperature or decrease in downstream pressure in binary aqueous solution of MTB (Figs 8.3 and 8.4). Selectivity decreased with increase in feed concentration. Enrichment factors of MTB and ETB in binary and ternary solutions were not very different. However, MTB flux was retarded by half by the presence of ETB in ternary solution. In multicomponent mixtures of aqueous strawberry aroma solution, the selectivities of MTB and ETB were adversely affected by the presence of other compounds, owing to coupling effects. It is shown that flux coupling takes place when a permeant of low diffusivity is dragged through the membrane polymer by a permeant of higher diffusivity resulting in higher fluxes of the slower permeant than expected or the reverse. In the study of Sampranpiboon et al. (2000), pervaporation separation was used to recover aroma compounds from ethyl butanoate and ethyl hexanoate mixtures, which are the most important aroma components of pineapple and banana juice, by using POMS and PDMS membranes. The effect of operating conditions on the separation performance was investigated and in general the POMS membrane was found to be more selective to aroma compounds than PDMS membranes. Moreover, a strong interaction was observed between

Total mass flux (kg m–2 h–1)

0.3

0.2

0.1

0 20

30

40 Temperature (°C)

50

60

Fig. 8.3 Effect of feed temperature on total mass flux at different downstream pressures for 100 ppm methyl butyrate solution: ( ) 4 mbar, ( ) 8 mbar (reprinted from Isci et al., 2006, with permission from Elsevier).



100

Selectivity

80

60

40

20

0 20

30


50

60

Fig. 8.4 Effect of feed temperature on selectivity of 100 ppm methyl butyrate solution at different downstream pressures: ( ) 4 mbar, ( ) 8 mbar (reprinted from Isci et al., 2006, with permission from Elsevier).

the two permeating components and permeation of one aroma compound was affected by the presence of the other aroma compound. Pineapple and passion fruit juices were chosen in the study of Pereira et al. (2002) and (2005). Performances of various membrane materials were evaluated with binary and quaternary synthetic aqueous solutions of typical tropical fruit aroma compounds such as ethyl acetate, ethyl butanoate, ethyl hexanoate and 1-octen-3-ol (Pereira et al., 2005). Tests were also carried out using single strength and clarified pineapple juices. Composite flat membranes having selective layers ethylene propylene diene terpolymer (EPDM) or ethylene vinyl acetate copolymer (EVA) and composite hollow fiber membrane having selective layer EPDM were used for comparison. It was concluded that choosing a very selective polymer is advantageous when the organic solute concentration is reduced in the feed. However, more permeable membranes are preferred if the feed concentration is high enough to induce phase separation in the permeate after its condensation. Composite EPDM hollow fiber membrane showed the best performance for pervaporation of synthetic and single-strength pineapple juices. Peng and Liu (2003), evaluated the separation factor of six aroma compounds (1-hexanol, 1-heptanol, trans-2-hexenal, ethyl acetate, linalool and d-limonene) representing typical flavoring ingredients from blueberry juice with a PDMS membrane. The results showed that the separation factor was in the range of 70 to 430, depending on molecule size and polarity of the compounds. No compounds, except 1-heptanol, showed any significant coupling effect in the mixture system. The temperature dependency of component fluxes was © Woodhead Publishing Limited, 2010


expressed by the Arrhenius equation and it was found that the temperature dependency of water flux was the highest whereas that of d-limonene was the lowest. Garcia et al. (2008) studied the concentration of trans-hex-2-en-1-ol, which is one of the major impact aroma compounds of bilberries, from aqueous ethanol using commercial PDMS capillary membranes in pervaporation. The flux of the aroma compound through the membrane was not affected from the variation of the feed flow rate. Therefore, it was concluded that the main mass transfer resistance to trans-hex-2-en-1-ol flux is located in the membrane. The temperature dependency of water, ethanol and trans-hex-2en-1-ol fluxes was expressed by the Arrhenius equation. From the magnitude of calculated activation energies, it was understood that water and ethanol were more influenced by variations in operation temperature than that of the aroma compound. Diban et al. (2008) adapted a previously developed mathematical model to predict the behavior of the separation of a mixture of seven volatile compounds that are characteristic of the aroma of bilberry juice from aqueous solution by pervaporation. It was observed that enrichment factors increased with membrane thickness until an asymptotic value was reached, however, partial fluxes decreased. Shepherd et al. (2002) studied the use of PDMS hollow fibers in orange juice aroma recovery. They used orange juice by product as feed and binary synthetic solutions. The well-spaced module was compared with a hollow fiber not containing spacers and with the transverse flow module with respect to their effect on experimental enrichment factors and mass transfer coefficient values and it was found to be feasible for aroma recovery. Aroujalian and Raisi (2007) also studied the pervaporation of orange juice aroma components. The effects of feed flow rate, feed temperature and permeate pressure on the performance of pervaporation was investigated. Feed flow rate had no significant effect on performance of the process. However, higher flux and enrichment factors were obtained as the feed temperature increased, whereas the increase in permeate pressure resulted in lower flux values. Enrichment factors of ethyl acetate, ethyl butyrate and hexanal increased but that of limonene, linalool and a-terpineol decreased as permeate pressure increased. Aroma compounds of pomegranate juice in multicomponent model solution and real fruit juice have been successfully concentrated by pervaporation (Raisi et al., 2008). The model aroma solution and real pomegranate juice showed similar behavior. The POMS membranes produced higher aroma enrichment factor but lower flux the PDMS membranes. Feed flow rate had no significant effect an either total flux on enrichment factor of aroma compounds, whereas on increase in feed temperature resulted in higher flux and enrichment factor. The effect of permeate pressure on pervaporation performance was related to the properties of the aroma compounds. Alvarez et al. (2000) investigated a combination of membrane processes


236 Separation, extraction and concentration processes (enzymatic membrane reactor, reverse osmosis and pervaporation) for producing clarified juice and apple juice aroma concentrate. Cassano et al. (2006) performed a study to design an integrated membrane process for the production of concentrated kiwi juice and its aroma recovery. Pervaporation was carried out at different stages in the process. They suggested the use of pervaporation for the removal and enrichment of aroma compounds directly from fresh juice before any concentration process. 8.5.2 Recovery of wine aromas In literature, there are some studies on recovery of wine aromas by pervaporation (Karlsson et al., 1995; Schafer and Crespo, 2007; Schafer et al., 1999). Karlsson et al. (1995) carried out pervaporation with Muscat wine in which eight aroma compounds were identified. In this study, the increase in fluxes with feed temperature was very well described by the Arrhenius equation. The effects of temperature on permeation varied for different aroma compounds. Recovery of aroma compounds from a wine-must fermentation by organophilic pervaporation was studied by Schafer et al. (1999). Pervaporation was performed under fermentation conditions. It was shown that organophilic pervaporation can be highly suitable for the continuous recovery of very complex and delicate aromatic profiles produced during microbial fermentation. Schafer and Crespo (2007) determined the degree of concentration polarization in two different flow-cell configurations during recovery of winemust aroma compounds by pervaporation. It was found that concentration polarization could not be overcome even under turbulent feed flow conditions for compounds having high sorption coefficient for the respective membrane polymer. Dealcoholization of alcoholic beverages including beer and wine is another application of pervaporation. The idea behind this process is the separation of ethanol through hydrophobic membranes much more readily than water. There are many publications in this area (Brüschke, 1990; Escoudier et al., 1988; Kimmerle and Gudernatsch, 1991; Takacs et al., 2007). A problem encountered with this application is that the aroma compounds in the wine or beer are generally much more hydrophobic than ethanol and therefore permeate through the membrane even more readily than ethanol. Vankelecom et al. (1997) used a mixture of eight aromatic compounds in water, one of which was ethanol, in their study. Sorption of aromas in PDMS could be explained by the Hildebrandt solubility parameters. Sorption decreased when zeolites were added to the polymer owing to the cross-linking action of the zeolite. Silicalite-filled PDMS membrane was found to be the best for selective removal of ethanol from the aroma mixture. Therefore, high silicalite loading were recommended for preparation of alcohol-free beverages or removal of ethanol from biofermentations.



Takacs et al. (2007) studied the reduction of alcohol content of wine by the pervaporation process using organophilic flat composite membrane, PDMS. Alcohol concentrate was obtained in the permeate stream. At higher temperatures, the flux of the permeate was higher and the membrane surface demand was smaller but the separation efficiency was lower. Lower pervaporation temperatures were more favorable to avoid aroma loss. In another study on the reduction of alcohol content of wine by pervaporation, the flux of the permeate increased but the separation efficiency and the separation ability of the membrane decreased as temperature increased (Takacs et al., 2007). In pervaporation of alcoholic beverages, complicated interactions between ethanol and aroma compounds often exist and affect their pervaporation performance. Tan et al. (2005) studied a series of model solutions containing ethanol and six typical aroma compounds in alcoholic beverages. The results showed that the solubility thermodynamics with the feed solution activity coefficient had a dominant effect on the permeability rather than diffusion factor. The presence of aroma compounds decreased the permeability coefficient and the separation factor of ethanol compared with those in ethanol–water binary solutions whereas its effect on fluxes was less. The effect of ethanol feed concentration on mass transfer of each compound was much related to the solubility properties of the compound in ethanol and water. It was observed that little interaction exists between aroma compounds in the membrane during the pervaporation process for dilute solution having a concentration below 1000 ppm. 8.5.3 Recovery of tea, cocoa and coffee aromas The possibility of using pervaporation to recover the tea aroma compounds from tea extract was studied by Kanani et al. (2003). Two different membrane types (POMS and PDMS) were used to recover eight aroma compounds that make a significant contribution to tea aroma. Pervaporation was performed with binary aqueous solutions of aroma compounds, with multicomponent mixture and with an actual tea extract. The actual tea aroma extract showed quite different behavior from the model aroma mixture. The results indicated that pervaporation is an attractive technology for the recovery of tea aroma compounds from tea aroma condensates. However, a wide range of selectivities for different aroma compounds results in alterations in the aroma profile of the permeate. Pilot plant tests for obtaining aroma extracts from cocoa using pervaporation were reported by Kattenberg and Willemsen (2001). The constructed test unit could process up to 500 L of feed, using hollow fiber membranes with a PDMS skin and two-stage condensation for permeate recovery. Application of pervaporation for recovering aroma concentrate from a caffeine- or theobromine-containing food such as coffee or tea and, in particular, cocoa was investigated by Kattenberg et al. (2002). In this study, first an


238 Separation, extraction and concentration processes aqueous extract was obtained. After incubating the food material at a suitable temperature and for a suitable time, the food extract was pervaporated using a hydrophobic pervaporation membrane to obtain the aroma concentrate. 8.5.4 Recovery of dairy aromas It is also possible to concentrate dairy flavor compounds from aqueous solutions using pervaporation. Baudot and Marin (1996) concentrated two dairy aroma compounds (methylthiobutanoate, diacetyl) in model aqueous solutions by pervaporation through two different membranes (silicalitefilled silicone composite membrane (PDMS 1070) and PEBA homogeneous membrane. Tested membranes showed good selectivity for the extraction of methylthiobutanoate (hydrophobic cheese aroma) at high dilution rate. However, these membranes were less selective for the recovery of diacetyl (2,3-butanedione, hydrophilic butter aroma). For this component, two-stage condensation coupled with pervaporation improved the selectivity of the whole process significantly. Overington et al. (2008) worked with the model feed solution containing nine flavor compounds contributing the flavor of cheese and other dairy products in pervaporation. They studied the effect of membrane type (two types of PDMS and a POMS), feed temperature and permeate pressure. Total flux increased with increase in temperature but decreased with increase in permeate pressure. PDMS membranes gave higher total flux than POMS membrane but the POMS membrane gave higher enrichment factors for the major compounds in the permeate. Esters and ketones passed through the membrane more readily than acids. Diffusion was the controlling mechanism for esters and ketones. Enrichment factors decreased with increasing molecular weight within esters and ketones. However, the effect of molecular weight was more complex for acids and depended on the relative importance of sorption and diffusion mechanisms in the membrane. 8.5.5 Pervaporation as an analytical separation technique Analytical separation techniques play an important role in analytical chemistry. Analytical pervaporation has been successfully used for the continuous determination of volatile analytes or volatile reaction products from complex liquid, semi-solid and solid samples in food and beverage industries (Amador-Hernandez and Castro, 2000). Schafer et al. (2006) investigated the potential of pervaporation combined with an electronic nose based on metal oxide sensors for analyzing wine model solutions. It was shown that target solutes present at minor concentrations can be detected even in the presence of bulk interferents such as ethanol by combining pervaporation with an artificial olfactory system based on metal oxide sensors.



8.5.6 Other applications of pervaporation for the recovery of aromas Pervaporation can also be used for the extraction of aroma compounds from natural matrixes as an alternative to steam distillation and solvent extraction. Enzymatic pretreatment for degradation of cell membrane is necessary in these processes. Figoli et al. (2006) studied the recovery of bergamot peel oil by pervaporation. There are some studies that combine pervaporation with bioprocessing. Bluemke and Schrader (2001) carried out an integrated bioprocess for production and recovery of the synthesized aroma compounds by interlinking a pervaporation membrane (POMS, PEBA) module with a bioreactor. In this study, they used the fungus Ceratocystis moniliformis which produce ethyl acetate, propyl acetate, isobutyl acetate, isoamyl acetate, citronellol and geraniol. In situ product removal using pervaporation leads to decreased product concentrations in the bioreactor and increased microbial growth rates. Total yield of aroma compounds produced was higher in the integrated bioprocess than with batch cultivation. Böddeker et al. (1997) showed that natural vanillin formed by bioconversion of suitable precursors can be recovered directly from the acidified culture broth by pervaporation at elevated temperature. Large volumes of waste water are obtained in most food processes. Since this liquid is generally released into the environment, it represents both pollution and an economical loss because of the cost of the valuable organic molecules involved. Pervaporation can be used to remove these valuable aroma compounds from waste water. Pierre et al. (2001) carried out non-dispersive solvent extraction of three sulfur aroma compounds, dimethyldisulfide, dimethyltrisulfide and S-methyl thiobutanoate, from very dilute aqueous solutions representing real effluent. Mass transfer fluxes obtained experimentally by membrane-based solvent extraction were greater for the three aroma compounds than those obtained by pervaporation. Souchon et al. (2002) used pervaporation process for deodorization of cauliflower blanching effluent in order to reduce its volatile organic compounds content and to recover the valuable food flavoring fraction. Pervaporation has been performed on three sulfur compounds of the cauliflower odor. Pervaporation was successfully applied for the recovery of linalool and eucalyptol which are the most frequent terpenes in wastewaters from the essential oil industry (Charbit et al., 1997).

8.6 Sources of further information and future trends Pervaporation theory and principles, separation characteristics, thermodynamics and polymer materials for membranes are covered in the book edited by Huang (1991), in which the chapter by Böddeker and Bengtson (1991) deals with the separation of organics from water by pervaporation, and that


240 Separation, extraction and concentration processes by Koops and Smolders (1991) gives information about membranes used in pervaporation. Strathmann and McDonogh (1993) studied mass transfer in pervaporation, characterization of pervaporation membranes and use of pervaporation in biotechnology. Brüschke (2006) studied the transport resistances and principles of pervaporation, membranes and the application of pervaporation in the chemical industry. Mass transport phenomena during the recovery of volatile compounds by pervaporation were investigated by Schafer and Crespo (2002). Pereira et al. (2006) reviewed the applications of pervaporation for the recovery of volatile aroma compounds from fruit juices. The studies on scale-up of the pervaporation process are limited and mostly based on the separation of model solutions. Therefore, pervaporation studies using real food systems should be increased in future. In addition, further study is required on scale-up. Studying pervaporation in large-scale systems provides insight into the industrial application of this method. Future trends are towards the combination of pervaporation with other processes in the food process line.

8.7 References Alvarez S, Riera F A, Alvarez R, Coca J, Cuperus F P, Bouwer S T, Boswinkel C, van Gemert RW, Veldsink JW, Giorno L, Donato L, Todisco S, Drioli E, Olsson J, Tragardh G, Gaeta S N and Panyor L (2000), ‘A new integrated membrane process for producing clarified apple juice and apple juice aroma concentrate’, J Food Eng, 46, 109–125. Amador-Hernandez J and de Castro MDL (2000), ‘Pervaporation: a useful tool in food analysis’, Food Chem, 68, 387–394. Aroujalian A and Raisi A (2007), ‘Recovery of volatile aroma components from orange juice by pervaporation’, J Membr Sci, 303, 154–161. Baudot A and Marin M (1996), ‘Dairy aroma compounds recovery by pervaporation’, J Membr Sci, 120, 207–220. Bengtsson E, Tragardh G and Hallstrom B (1992), ‘Concentration of apple juice aroma from evaporator condensate using pervaporation’, Lebensm Wiss Technol, 25, 29–34. Bluemke W and Schrader J (2001), ‘Integrated bioprocess for enhanced production of natural flavors and fragrances by Ceratocystis moniliformis’, Biomol Eng, 17, 137–142. Borjesson J, Karlsson H O E and Tragardh G (1996), ‘Pervaporation of a model apple juice aroma solution: comparison of membrane performance’, J Membr Sci, 119, 229–239. Böddeker K and Bengtson G (1991), ‘Selective pervaporation of organic from water’, in R. Y. M. Huang, Pervaporation membrane separation processes, Elsevier, Amsterdam, The Netherlands, 437–460. Böddeker K W, Gatfield I L, Jahnig J, Schorm C (1997), ‘Pervaporation at the vapor pressure limit: Vanillin’, J Membr Sci, 137, 155–158. Brüschke H E A (1990), ‘Removal of ethanol from aqueous streams by pervaporation’, Desalination, 77, 323–329. Brüschke H E A (2006), ‘State-of-art of pervaporation process in the chemical industry’, in S. P. Nunes and K. V. Peinemann, membrane technology in the chemical industry, Wiley-VCH, USA, 151–202.


Principles of pervaporation for the recovery of aroma compounds 241 Cassano A, Figoli A, Tagarelli A, Sindona G, Drioli E (2006), ‘Integrated membrane process for the production of highly nutritional kiwifruit juice’, Desalination, 189, 21–30. Charbit G, Charbit F and Molina C (1997), ‘Study of mass transfer limitations in the deterpenation of waste waters by pervaporation’, J Chem Eng Jpn, 30, 382–387. De Assis A V R, Bizzo H R, da Matta V M, Cabral L M C (2007), ‘Recovery of aroma compounds of cashew apple fruit (Anacardium accidentale L.) by pervaporation’, Ciencia E Technologia De Alimentos, 27, 349–354. Diban N, Urtiaga A and Ortiz I (2008), ‘Recovery of key components of bilberry aroma using a commercial pervaporation membrane’, Desalination, 224, 34–39. Escoudier J L, Le Bouar M, Moutounet M, Jouret C and Barillere J M (1988), ‘Application and evaluation of pervaporation for production of low alcohol wines’, in R. Bakish, Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corporation, Englewood Cliffs, NJ, USA, 387–397. Figoli A, Tagarelli A, Mecchia A, Trotta A, Cavaliere B, Lavecchia R, Sindona G and Drioli E (2006), ‘Enzyme-assisted pervaporative recovery of concentrated bergamot peel oils’, Desalination, 199, 111–112. Garcia V, Diban N, Gorri D, Keiski R, Urtiaga A and Ortiz I (2008), ‘Separation and concentration of bilberry impact aroma compound from dilute model solution by pervaporation’, J Chem Technol Biotechnol, 83, 973–982. Huang R Y M (1991), Pervaporation membrane separation processes, Elsevier Science Publishers, Amsterdam, The Netherlands. Isci A, Sahin S and Sumnu G (2006), ‘Recovery of strawberry aroma compounds by pervaporation’, J Food Eng, 75, 36–42. Kanani D M, Nikhade B P, Balakrishnan P, Singh G and Pangarkar V G (2003), ‘Recovery of Valuable Tea Aroma Components by Pervaporation’, Ind Eng Chem Res, 42, 6924–6932. Karlsson H O E and Tragardh G (1993), ‘Aroma compound recovery with pervaporationfeed flow effects’, J Membr Sci, 81, 163–171. Karlsson H O E, Loureiro S and Tragardh G (1995), ‘Aroma compound recovery with pervaporation-Temperature effects during pervaporation of a muscat wine’, J Food Eng, 26, 177–191. Karlsson H O E, Tragardh G and Olsson J (1998), ‘The performance of pervaporative aroma recovery units: Process simulations’, Sep Sci Technol, 33, 1629–1652. Kattenberg H R and Willemsen J H A (2001), ‘Aroma extracts from cocoa’, Manuf Confect, 82, 73. Kattenberg H R, Willemsen J H A, Starmans D A J, Hoving H D and Winters MGM (2002), ‘Method for recovering aroma concentrate from caffeine- or theobrominecomprising food base material’, US Patent Office Journal, No. 1139792. Kimmerle K and Gudernatsch W (1991), ‘Pilot dealcoholization of beer by pervaporation’, in R. Bakish, Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corporation, Englewood Cliffs, NJ, USA, pp. 291–307. Kober P A (1917), ‘Pervaporation, perstillation, and percrystallisation’, J Am Chem Soc, 39, 944–948. Koops G H and Smolders C A (1991), ‘Estimation and evaluation of polymeric materials for pervaporation membranes’, in R. Y. M. Huang, Pervaporation membrane separation processes, Elsevier, Amsterdam, The Netherlands, 253–278. Lipnizki F, Hausmanns S, Ten P, Field R W and Laufenberg G (1999), ‘Organophilic pervaporation: prospects and performance’, Chem Eng J, 73, 113–129. Lipnizki F, Olsson J and Tragardh G (2002a), ‘Scale-up of pervaporation for the recovery of natural aroma compounds in the food industry. Part 1: Simulation and performance’, J Food Eng, 54, 183–195.


242 Separation, extraction and concentration processes Lipnizki F, Olsson J and Tragardh G (2002b), ‘Scale-up of pervaporation for the recovery of natural aroma compounds in the food industry. Part 2: Integration and optimization’, J Food Eng, 54, 197–205. Luo Y, Tan S J, Wang H, Wu F W, Liu X M, Li L, Zhang Z B (2008), ‘PPMS composite membranes for the concentration of organics from aqueous solutions by pervaporation’, Chem Eng J, 137, 496–502. Olsson J and Tragardh G (1999), ‘Influence of feed flow velocity on pervaporative aroma recovery from a model solution of apple juice aroma compounds’, J Food Eng, 39, 107–115. Olsson J and Tragardh G (2001), ‘Pervaporation of volatile organic compounds from water. I. Influence of permeate pressure on selectivity’, J Membr Sci, 187, 23–37. Olsson J, Tragardh G and Lipnizki F (2002), ‘The influence of permeant and membrane properties on mass transfer in pervaporation of volatile organic compounds from dilute organic solutions’, Sep Sci Technol, 37, 1199–1223. Overington A, Wong M, Harrison J and Ferreira L (2008), ‘Concentration of dairy flavour compounds using pervaporation’, Int Dairy J, 18, 835–848. Peng M and Liu S X (2003), ‘Recovery of aroma compounds from dilute model blueberry solution by pervaporation’, J Food Sci, 68, 2706–2710. Pereira C C, Ribeiro C P, Nobrega R, Borges C P (2006), ‘Pervaporative recovery of volatile aroma compounds from fruit juices’, J Membr Sci, 274, 1–23. Pereira C C, Rufino J M, Habert A C, Nobrega R, Cabral L M C and Borges C P (2002), ‘Membrane for processing tropical fruit juice’, Desalination, 148, 57–60. Pereira C C, Rufino J R M, Habert A C, Nobrega R, Cabral L M C and Borges C P (2005), ‘Aroma compounds recovery of tropical fruit juice by pervaporation: membrane material selection and process evaluation’, J Food Eng, 66, 77–87. Pierre F X, Souchon I, Marin M (2001), ‘Recovery of sulfur aroma compounds using membrane-based solvent extraction, J Membr Sci, 187, 239–253. Raisi A, Aroujalian A, Kaghazchi T (2008), ‘Multicomponent pervaporation process for volatile aroma compounds recovery from pomegranate juice’, J Membr Sci, 322, 339–348. Rajagopalan N and Cheryan M (1995), ‘Pervaporation of grape juice aroma’, J Membr Sci, 104, 243–250. Sampranpiboon P, Jiraratananon R, Uttapap D, Feng X and Huang R Y M (2000), ‘Separation of aroma compounds from aqueous solutions by pervaporation using polyoctylmethyl siloxane (POMS) and polydimethyl siloxane (PDMS) membranes’, J Membr Sci, 174, 55–65. Schafer T and Crespo J G (1997), ‘Recovery of aromas by pervaporation’, ESMST XIVth Annual Summer School, Membrane integration in clean processes. Lisbon Instituto: Superior Tecnico, IST. Schafer T and Crespo J G (2002), ‘Mass transport phenomena during the recovery of volatile compounds by pervaporation’, in J. Welti-Chanes, J. F. Velez-Ruiz and G.V. Barbosa-Canovas, Transport phenomena in food processing, CRC Press, Florida, USA, 247–263. Schafer T and Crespo J G (2007), ‘Study and optimization of the hydrodynamic upstream conditions during recovery of a complex aroma profile by pervaporation’, J Membr Sci, 301, 46–56. Schafer T, Bentson G, Pingel H, Boddeker K W and Crespo J P S G (1999), ‘Recovery of aroma compounds from a wine-must fermentation by ogranophilic pervaporation’, Biotechnol Bioeng, 62, 412–421. Schafer T, Serrano-Santos M B, Rocchi S and Fuoco R (2006), ‘Pervaporation membrane separation process for enhancing the selectivity of an artificial olfactory system (“electronic nose”)’, Anal Bioanal Chem, 384, 860–866.


Principles of pervaporation for the recovery of aroma compounds 243 She M and Hwang S-T (2006), ‘Recovery of key components from real flavor concentrates by pervaporation’, J Membr Sci, 279, 86–93. Shepherd A, Habert A C and Borges C P (2002) ‘Hollow fiber modules for orange juice aroma recovery using pervaporation’, Desalination, 148, 111–114. Song K H and Lee K R (2005), ‘Pervaporation of flavors with hydrophobic membrane’, Korean J Chem Eng, 22, 735–739. Souchon I, Pierre F X, Athes-Dutour V and Marin M (2002), ‘Pervaporation as a deodorization process applied to food industry effluents: recovery and valorisation of aroma compounds from cauliflower blanching water’, Desalination, 148, 79–85. Strathmann H and McDonogh R M (1993), ‘Use of pervaporation in biotechnology’, in J. A. Howell, V. Sanchez and R. W. Field, Membranes in bioprocessing: theory and applications, Chapman and Hall, Cambridge, UK, 293–329. Takacs L, Vatai G, Korany K (2007), ‘Production of alcohol free wine by pervaporation’, J Food Eng, 78, 118–125. Tan S J, Li L, Xiao Z Y, Wu Y T and Zhang Z B (2005), ‘Pervaporation of alcoholic beverages – the coupling effects between ethanol and aroma compounds’, J Membr Sci, 264, 129–136. Trifunovic O, Lipnizki F and Tragardh G (2006), ‘The influence of process parameters on aroma recovery by hydrophobic pervaporation’, Desalination, 189, 1–12. Vankelecom I F J, Beukelaer S D and Uytterhoeven J B (1997), ‘Sorption and pervaporation of aroma compounds using zeolite-filled PDMS membranes’, J Phys Chem B, 101, 5186–5190.



9 Advances in membrane-based concentration in the food and beverage industries: direct osmosis and membrane contactors E. Drioli and A. Cassano, Institute on Membrane Technology, ITM-CNR, Italy Abstract: The process fundamentals and the role of operating conditions involved in direct osmosis (DO) and membrane contactors, such as osmotic distillation (OD) and membrane distillation (MD) are reviewed. Their main applications in concentration of liquid food and limitations compared with those of conventional de-watering processes – such as thermal evaporation, cryoconcentration and reverse osmosis – are discussed. The advantage lies in allowing very high concentrations (up to 65 °Brix) to be reached under atmospheric pressure and low temperature, thus avoiding thermal and mechanical damage. Key words: membrane contactors, direct osmosis, osmotic distillation, membrane distillation, food industry.

9.1 Introduction Epidemiological studies report that high consumption of fruit and vegetables is associated with a reduced risk of free radical related oxidative damage and diseases, such as various types of cancer, and cardiovascular or neurological diseases (Collins and Harrington, 2002). The world market of these products has remarkably increased in recent years and consumers have addressed their interest towards products with a natural fresh taste, that are high quality and additive-free. In order to meet these demands, the food industry has focused on the development of processed items with increased shelf-life able to retain as much as possible the essence of the fresh fruit, as well as colour, aroma, nutritional value and structural characteristics.


Advances in membrane-based concentration 245 In the traditional methods of processing liquid foods, the concentration operation is particularly important for the production of intermediates that can be easily handled, packaged, stored and shipped before their reutilization for a final transformation (Luh et al., 1986) and also as a step which precedes total dehydration. Moreover, the removal of water reduces the growth of micro-organisms, thus increasing the shelf-life of the liquid foods; fruit juice concentrates, because of their low water activity, have a higher stability than single-strength juices. This chapter briefly describes the existing methods for the concentration of liquid foods (such as thermal evaporation, cryoconcentration and membrane concentration) and then introduces novel membrane processes, including direct osmosis (DO), membrane distillation (MD) and osmotic distillation (OD), which offer improved performances in terms of product quality, reduced energy consumption and environmental impact, and high effectiveness.

9.2 Conventional technologies in the food and beverage industries 9.2.1 Thermal evaporation In thermal evaporation, water is removed from liquid foods as vapour. Falling film evaporators and centrifugal evaporators are most commonly used for this purpose. It is known that the heat required to perform the evaporation results in some ‘cooked’ notes recognized as off-flavours, loss of most volatile aroma compounds, and colour degradation with a consequent remarkable qualitative decline (Maccarone et al., 1996). Partial recovery of aroma compounds may be achieved by cold-water condensation and rectification in a still located on the evaporator, resulting in concentrates that are acceptable to consumers, but still far from fresh products. An additional drawback in the use of thermal evaporation is the high energy demand, despite the use of energy-saving systems. The production of superior quality concentrates, especially fruit juices, is a very important goal and considerable R&D efforts have been devoted to the development of nonthermal concentration techniques. These methods include: freeze concentration systems (cryoconcentration) and membrane processes. 9.2.2 Cryoconcentration Freeze concentration has long been considered a feasible prospect for the selective removal of water from liquid foods. In this instance, water is removed as ice rather than as vapour. The product is cooled below its freezing point allowing water to form crystals which are then removed from the concentrate by centrifugation. This method guarantees an excellent organoleptic quality: the operating temperatures are sufficiently low to avoid chemical and


246 Separation, extraction and concentration processes biochemical reactions and volatile compounds are completely retained. Consequently, the concentrated product is characterized by higher quality than that processed by thermal evaporation (Aider and de Halleux, 2008). However, there are some problems in this process which make it unpractical. The main drawback is that the achievable concentration (40–45 °Brix) is lower than the values obtained by evaporation (60–65 °Brix). Furthermore, fine ice crystals produced by rapid freezing cannot be separated from the residual liquid and the process is not suitable for the treatment of highly pulped liquids. Remarkable energy consumption, high equipment investments and operational costs are additional drawbacks (Jariel et al., 1996). 9.2.3 Pressure-driven membrane processes Pressure-driven membrane processes such as microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) are well established systems in the food industry for concentration, fractionation and purification of liquid foods and for wastewater treatment (Table 9.1). These processes offer significant advantages over traditional food processing technologies in terms of low operation temperatures, no special chemicals required, possibility of automation, and no phase-change involved. In addition, they are simple in concept and operation and characterized by low energy consumption. MF membranes have a symmetric microporous structure with a pore size in the range 0.1–10 mm. They are essentially used for the separation of fine particles, micro-organisms and emulsion droplets from fluids. Particles are rejected mainly by means of sieving mechanisms although the separation is affected also by interactions between the membrane surface and the treated solution. The hydrostatic pressure difference used as a driving force is in the range from 0.5 to 4 bar. Cell harvesting, clarification of fruit juices, must and wine, wastewater treatment, bacteria and particulate turbidity reduction, separation of whey proteins and separation of oil–water emulsions are typical consolidated applications (Cheryan, 1998). UF membranes have a pore size of 1–100 nm and are capable of retaining species of molecular weight 300–500 000 daltons. Typical rejected species include lipids, proteins, polysaccharides, colloids and suspended solids whereas solutes with small molecules such as vitamins, salts and sugars, flow through the membrane together with water. Operating pressures are 2–10 bar (Mulder, 1998). In the food and dairy industry, UF is commonly applied to clarify, concentrate, decolorize and fractionate juices, sugar solutions, proteins and dairy and grain milling products. The membranes used in NF are characterized by a charged surface and equivalent pore diameters in the range 1–3 nm. They are mainly used for the separation of multivalent ions and uncharged organic molecules having molecular weights greater than 1 kDa. Operating


Advances in membrane-based concentration 247 Table 9.1 Commercial application of pressure-driven membrane processes in food processing Unit operation Industrial sector

Application

Microfiltration Fruit and vegetables

Fruit and vegetable juice clarification

Beverages Dairy

Ultrafiltration

Fruit and vegetables Beverages Vegetables Dairy

Fats and oils Nanofiltration

Fruit and vegetables Dairy Beverages

Reverse osmosis

Cold sterilization of beer Clarification of wine, beer and vinegar Bacterial purification of milk and whey Whey defatting and high-quality protein recovery Clarification of fruit juice Clarification of wine Recovery of potato starch and proteins Fractionation/concentration of whey in manufacture of whey protein concentrate Fractionation/concentration of milk in manufacture of processed and natural cheese Recovery of oilseed protein and oil from processing water Fruit and vegetable juice concentration Concentration and demineralization of milk Demineralization of whey Wastewater treatment

Fruit and vegetables

Fruit and vegetable juice concentration Sugar concentration and recovery from rinse water, treatment of water for reuse Recovery of flavours, fragrances, pectins and proteins Beverages Recovery of alcohol from wine Sugar and sweeteners Preconcentration of maple syrup Sugar concentration and recovery from rinse water, treatment of water for reuse Dairy Concentration of milk and whey All possible Treatment of boiler concentrate prior to recycle to boiler

pressures in NF are lower than those in RO therefore separation occurs at low energy consumption (21% less than RO). The concentration of must, the recovery of aromas from fruit juices, the demineralization of whey and the treatment of wastewater from beverage production are typical applications of NF in the food and dairy industry (Warczok et al., 2004). In RO, low molecular weight compounds, such as salts and sugars are separated from a solvent, usually water. The particle size range for application of RO is approximately 0.1–1 nm and a complete separation is achieved with solutes of molecular weight greater than 300 Da. Typical operating pressures are 10–100 bar. Food processing applications of RO include: concentration


248 Separation, extraction and concentration processes of fruit and vegetable juices, concentration of milk and whey for cheese production, pretreatment of boiler water, recovery of sugars and acids from rinse waters, recovery of sweet potato stillage, recovery of flavours, pectins and proteins, removal of alcohol from wine, and water softening (Ho and Sirkar, 1992). The advantages of RO over traditional evaporation are the lower thermal damage to the product, increase in aroma retention, simple system design, lower energy consumption and lower equipment costs. However, RO cannot reach retentate concentrations higher than 25–30 °Brix with a single-stage system owing to the limitation of high osmotic pressure. Furthermore, fouling and concentration polarization are still important drawbacks of RO and other pressure-driven membrane processes in liquid food processing (Jiao et al., 2004).

9.3 Direct osmosis and applications in the food and beverage industries 9.3.1 Process fundamentals In DO, an osmotic pressure gradient across a semi-permeable membrane is established by using an osmotic agent solution in order to remove water from a feed solution. The osmotic agent should be characterized by high solubility in water (low water activity), non-toxicity, high superficial tension, inertia towards the flavour, odour and colour of the foodstuff, low volatility and viscosity, and non-permeability across the membrane. The most frequently employed constituents as osmotic agents are: sodium chloride, calcium chloride, glucose, sucrose, glycerol, cane molasses or corn syrup. Unlike RO the pressure difference across the membrane in DO is negligible and the flux depends on the difference in water activities. The hydraulic pressures required to pump the feed and the osmotic solution over the membrane surface are of about 2 bar (Milleville, 1990). Consequently, fouling phenomena are negligible and only concentration polarization occurs. Additional advantages are low energy consumption, simplicity, modularity, constant permeate flux in time, possibility to treat solutions with a high level of suspended and dissolved solids, and high achievable concentrations. The membranes used in DO are semipermeable membranes similar to those used in RO and NF processes. They are highly selective; however, the diffusion of a small amount of stripping solution cannot be completely avoided. 9.3.2 Effect of operating conditions on the direct osmosis (DO) flux Petrotos et al. (1998, 1999) studied the effect of several membranes and operating parameters on the performance of DO in the concentration of


Advances in membrane-based concentration 249 tomato juice. NaCl, CaCl2, Ca(NO3)2, glucose, sucrose and polyethylene glycol (PEG 400) were used as stripping solutions. The initial concentration of tomato juice was in the range 4.3–11.7 °Brix. These studies suggested that salts, and especially NaCl, produced improved fluxes compared with carbohydrates and PEG 400, owing to their lower viscosity. An increasing of the stripping solution concentration also positively affected the flux (through an increasing of the driving force). On the contrary, an increasing of the feed concentration resulted in lower water flux owing to viscosity and osmotic pressure increase. The lower viscosity of the osmotic agents resulted in reduced polarization phenomena on the stripping solution side and increased fluxes. Increasing the temperature from 26 to 60 °C enhanced permeate flux by 64%, this phenomenon can be attributed to the reduction in the viscosity and to the increase in the diffusion coefficient of both feed and stripping solution. An increase in feed flow rate did not improve the permeate flux during the DO process. The pretreatment of the juice by MF or UF was also found to have a significant effect on the osmotic fluxes (Petrotos et al., 1998). In particular, the ultrafiltered juice gave a 39% increase in flux compared with the untreated juice. Finally, the DO flux is strongly affected by membrane thickness. In particular, a reduction of the membrane support layer determines an increase of the permeate flux (Beaudry and Lampi, 1990). 9.3.3 DO applications Osmotek Inc. (Corvallis, OR, USA) developed the first DO industrial approach as a pretreatment step for RO wastewater treatment. Several studies concerning the fruit juice concentration by DO are reported (Beaudry and Lampi, 1990; Herron et al., 1994; Milleville, 1990; Petrotos and Lazarides, 2001; Petrotos et al., 1998, 1999; Wrolstad et al., 1993). Beaudry and Lampi (1990) reported the performance of the Osmotek DO process in the concentration of fruit juices by using thin-film composite RO membranes with an overall thickness of 25–85 mm and a nominal molecular weight cut off (NMWCO) of 100 Da. These membranes rejected more than 99.9% of the total acidity and colour in orange and red raspberry juice. Herron et al. (1994) developed a simplified DO apparatus for several kinds of liquid food samples including orange and raspberry juice. Maximum osmotic fluxes of 5–6 L m–2 h–1 were obtained through 100-Da membranes by using fructose/glucose solutions as osmotic agent. The concentrated juice showed a better quality than that produced by a conventional vacuum evaporator. Stripping solutions diluted during juice concentration can be reconcentrated by using thermal evaporation: therefore, only the osmotic agent is exposed to high temperatures preserving the quality of concentrated juice. RO was also proposed as a method for the recovery of stripping solutions (Karode et al., 2000). Petrotos and Lazarides (2001) developed a new apparatus for the concentration of fruit juices with an osmotic cell of special configuration


250 Separation, extraction and concentration processes to promote turbulence. The apparatus was equipped with RO flat-sheet membranes to concentrate tomato juice up to 16 °Brix at room temperature. Average fluxes of 4.5 kg m–2h–1 were obtained.

9.4 Membrane contactors and applications in the food and beverage industries Membrane contactors are devices in which a microporous membrane acts as a barrier between two phases permitting gas/liquid or liquid/liquid mass transfer of the components without dispersion of one phase into the other. Fluids to be contacted flow on the opposite side of the membrane and a fluid/fluid interface is located at the mouth of each membrane pore. Mass transfer occurs by diffusion across the interface as in conventional contacting equipment. Unlike conventional membrane operations, the driving force for separation is a concentration or a temperature gradient rather than a pressure gradient and a small pressure drop across the membrane is required to maintain the fluid/fluid interface at the mouth of the pore. Furthermore, the membrane imparts no selectivity to the separation (Gabelman and Hwang, 1999). Membrane contactors offer significant advantages over conventional dispersed-phase contactors such as the absence of emulsions, no density difference required between the fluids, easy scale-up, high interfacial area, no flooding at high flow rates. Within membrane contactors, membrane distillation (MD) and osmotic distillation (OD) seem to be a valid alternative for pressure-driven membrane processes in the concentration of liquid foods. In these processes, schematically represented in Fig. 9.1, the driving force for mass transfer is a vapour pressure difference across the membrane generated by either a temperature gradient (in MD) or a water activity difference (in OD). Their main advantages over traditional pressure-driven membrane processes are in terms of: low fouling, possibility of treatment of highly viscous solutions, high retention of species, and low energy consumption. Furthermore, these processes are not limited by high osmotic pressures and allow concentration levels to be attained that are similar to those obtained in thermal evaporation. 9.4.1 Osmotic distillation Process fundamentals Osmotic distillation is a recent membrane process also known as osmotic evaporation, osmotic concentration by membrane, membrane evaporation, isothermal membrane distillation, transmembrane distillation, thermopervaporation or gas membrane extraction. It can be carried out at room temperature and atmospheric pressure with minimal thermal and mechanical damage of the solutes. Consequently, it represents an attractive process for © Woodhead Publishing Limited, 2010

Advances in membrane-based concentration 251 Evaporation

Condensation

Tf > Tp Vapour

L/G interphase

Hydrophobic membrane

L/G interphase

Feed

Permeate (a)

Evaporation

Condensation

awf > aws Vapour

L/G interphase


Feed

L/G interphase Stripping solution

(b)

Fig. 9.1 Schematic representation of (a) membrane distillation and (b) osmotic distillation. L/G, liquid–gas.

the concentration of solutions containing thermosensitive compounds, such as fruit juices and pharmaceuticals. In OD, a hydrophobic microporous membrane separates two liquid phases having different solute concentrations: a dilute solution on one side and a hypertonic salt solution on the opposite side. The hydrophobic nature of the membrane prevents penetration of the pores by aqueous solutions, creating air gaps within the membrane. The difference in solute concentration and, consequently, in water activity of both solutions, generates, at the vapour–liquid interface, a vapour pressure gradient across the membrane causing a vapour transfer across the pores from the high-vapour pressure phase to the low one (Alves and Coelhoso, 2002; Hogan et al., 1998; Lebfevre, 1988; Mengual et al., 1993). The concentration profile of the OD process is schematically presented in Fig. 9.2. The water transport through the membrane can be summarized in three steps: (1) evaporation of water at the dilute vapour–liquid interface; (2) diffusional vapour transport through the membrane pore; (3) condensation of water vapour at the membrane/brine interface (Schofield et al., 1987; Hogan et al., 1998). © Woodhead Publishing Limited, 2010

252 Separation, extraction and concentration processes High water vapour pressure Low water vapour pressure

Vapour flux

Feed dilutes aqueous solution

Csb Pwf >> Pwp Pore

Csm

Stripping solution (concentrated salt solution)

Cfm Cfb

Air Microporous hydrophobic membrane

Bulk Boundary layer

Bulk Boundary layer

Fig. 9.2 Concentration profile in osmotic distillation.

Because water transport involves condensation and evaporation phenomena, a temperature gradient through the membrane is generated, even if bulk temperatures of solutions separated by the membrane are equal. Consequently, a heat transfer tending to reduce the driving force for the water transport should be considered in addition to a mass transfer (Celere and Gostoli, 2002). The stripping solution after its dilution by water transferred from the feed stream can be reconcentrated by evaporation and reused in the OD operation. Therefore it must be thermally stable and preferably nontoxic, noncorrosive and available at low cost. A number of water-soluble salts such as NaCl, CaCl2, K2HPO4 are suitable. Salts displaying increase in solubility with temperature are preferred because they can be concentrated to high levels avoiding crystallization phenomena. Potassium salts of orthoand pyrophosphoric acid offer several advantages, including low equivalent weight, higher water solubility, steep positive temperature coefficients of solubility and safety in foods and pharmaceuticals (Deblay, 1995; Michaels, 1998). Typical transmembrane pressures encountered in osmotic distillation are in the range of 140 kPa (Hogan et al., 1998). Mass transfer aspects The water transport in the OD process is related to the driving force, represented by the vapour pressure difference at both liquid–vapour interfaces of the membrane (DPwm ), by the following equation:

Jw = Km (Pwf – Pws)

[9.1]

where Jw is the transmembrane flux, Km the membrane mass transfer coefficient, Pwf and Pws are the water vapour pressures of the feed and stripping solution at the membrane surface.


Advances in membrane-based concentration 253 The water vapour pressures of the feed and stripping solutions at the fluid membrane interfaces are calculated as:

* Pwf = Pwf awf

[9.2]

* Pws = Pws aws

[9.3]

* * and Pws where Pwf are the pure water vapour pressures of the feed and stripping solution at the interface, respectively; awf and aws are the water activities of the feed and stripping solution at the interface, respectively. If the temperature is the same on both sides of the membrane equation [9.1] can be written as:

J w = K m Pw* (awf – aws )

[9.4]

The driving force depends on the solute concentration as well as on the temperature conditions prevailing at the vapour–liquid interfaces. A more detailed representation referring to the bulk conditions of both compartments is given by integrating the various mass transfer resistances:

lw = KD Pwb

[9.5]

where

1 1ˆ Ê1 K =Á + + ˜ K K K Ë f m s¯

–1

is defined as the overall mass transfer coefficient which accounts for the resistances opposed by the feed solution (1/Kf), the membrane (1/Km) and the stripping solution (1/Ks). If the concentration polarization is negligible, mass transfer depends only on membrane resistance and consequently:

K = K m

[9.6]

As in other membrane processes, OD is affected by concentration polarization. Owing to the water transport across the membrane, concentration in the bulk phase and, consequently, the water activity, differs from the composition near the membrane interface. As a result, the driving force for mass transfer and water flux are reduced (Ravindra Babu et al., 2006). Independently of the type of driving force (temperature difference, activity difference or both), the water vapour transfer mechanism in the membrane pores can be estimated on the basis of the molecular diffusion model, the Knudsen diffusion model or by a combination of both mechanisms (Geankoplis, 1993). If the mean free path of gas molecules is significantly greater than the membrane pore size, diffusing molecules collide frequently with pore


254 Separation, extraction and concentration processes walls rather than with gas molecules present in the pores. In this instance the molecular transport occurs by Knudsen diffusion: 2e r K mK = M RT 3 cd

8RT pM

[9.7]

where r is the pore radius, e the membrane porosity, c the tortuosity factor, d the membrane thickness, M the molecular weight, R the gas constant and T the temperature. For pore size greater than the mean molecular free path of water vapour, the molecular diffusion is the controlling mechanism and the water mass flux can be written as: De P K mM = M RT cd Plm

[9.8]

where D is the diffusion coefficient and P the pressure. The Knudsen number (Kn) can be used to determine which of the two diffusion models is predominant. It compares the mean molecular free path (l) with the mean pore diameter of the membrane and is defined as: Kn = l 2p

[9.9]

where

l=

kBT P 2p s 2

[9.10]

in which kB is the Boltzmann constant, s is the mean collision diameter of the molecule and r the pore radius. For small pore size, Kn ≥ 10, collisions with pore walls are frequent therefore Knudsen diffusion is the prevailing mechanism. With relatively large pores, Kn ≤ 0.01, more collisions between the gas molecules themselves occur and the molecular diffusion will be predominant. Both mechanisms coexist when Kn is between the two limit values (Courel et al., 2000a). The concentration polarization phenomenon can be reduced by optimization of process parameters as stirring or stripping solution properties. The liquid concentration difference between the membrane and the bulk phase determines a diffusive solute flow according to Fick’s law which counterbalances the convective flow in the opposite direction. A mass balance across the boundary layer of the stripping solution is given by:

ÈÊ C ˆ ˘ J v = kp ln ÍÁ b ˜ ˙ ÎË Cm ¯ ˚

with

kp =

Dsw d

[9.11]


Advances in membrane-based concentration 255 where Jv is the volume flux, Dsw the diffusion coefficient of solute by the membrane, kp the liquid mass transfer coefficient, Cm the salt molar concentration in the membrane, Cb the salt molar concentration in the bulk and d the boundary layer thickness. The liquid mass transfer coefficients depend on the solution physical properties and on the hydrodynamic properties of the system. These coefficients can be estimated by empirical correlations of dimensionless numbers namely Sherwood (Sh), Reynolds (Re) and Schmidt (Sc) numbers, equations [9.12] and [9.13].

Sh = a Reb Scg

[9.12]

with

Sh =

kdh , Dw

Re =

r udh , m

Sc =

m r Dw

[9.13]

where a, b and g are constants, k the feed or osmotic agent side liquid mass transfer coefficient, dh the hydraulic diameter, Dw the diffusion coefficient in water, r the solution density, m the solution dynamic viscosity and u the cross flow velocity of the fluid. Heat transfer OD is essentially a mass transfer process. The water transport through the membrane implies evaporation at the feed side and condensation at the stripping solution side. This phase change at the membrane walls generates a temperature difference across the membrane which reduces the vapour pressure difference decreasing the water transport. Under steady state conditions, the heat transfer equations can be written as:

Q = hf (Tf – Tfm) = hs (Tsm – Ts)

[9.14]

Q = Nw DHv – hm(Tsm – Tfm)

[9.15]

where Q is the heat flux, hf, hs and hm the heat transfer coefficients of the boundary layers (feed and stripping solution, respectively) and of the membrane, Tf and Tm the bulk temperatures of the feed and stripping solution, respectively, Tfm and Tsm the temperatures of the feed and stripping solution at the membrane interfaces, respectively, DHv the water latent heat of vaporization and Nw the molar vapour flux. The relation between heat flux and overall heat transfer coefficient during the OD process can be obtained by substituting Tsm and Tfm from equation [9.14] in equation [9.15], obtaining:

N DH Q = U ÈÍ w v – (Ts – Tf )˘˙ Î hm ˚

where U is the overall heat transfer coefficient given by: © Woodhead Publishing Limited, 2010

[9.16]


1 1ˆ Ê1 U=Á + + Ë hf hm hs ˜¯

–1

[9.17]

The thermal effect is considered negligible by most researchers. For example, Ravindra Babu et al. (2006, 2008) found that the contribution of concentration polarization on driving force reduction, during the concentration of pineapple and sweet-lime juices by OD, was prominent when compared with that of temperature polarization. On the other hand, the thermal effect owing to evaporation and condensation at both liquid–membrane interfaces was considered substantial by other authors (Courel et al., 2000a; Gostoli, 1999). In particular, the results of pure water OD experiments performed by Courel et al. (2000a) with commercial asymmetric porous membranes indicated that a high vapour flux of 12 kg m–2 h–1 generates a transmembrane temperature gradient of about 2 °C inducing a 30% driving force reduction. Osmotic distillation (OD) membranes and modules Hydrophobic polymers with low surface free energy are commonly used to produce membranes with pore sizes and pore size distribution suitable for OD applications. They include both polyolefins, such as polyethylene (PE) and polypropylene (PP), and perfluorocarbons such as polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF). Membranes comprising these polymers, having a pore diameter ranging from 0.1 to 1 mm, allow the gas phase to be maintained in the membrane pores, a fundamental condition required to perform the OD process (Gryta, 2005). However, hydrophilic ceramic membranes can be modified by grafting on the surface molecules containing hydrophobic fluorocarbon chains like fluoroalkylsilanes or by coating the surface of alumina membranes with a thin lipid film. These membranes have been applied successfully in OD (Gabino et al., 2007; Romero et al. 2006). By referring to the configuration, hollow-fiber membranes, characterized by thin walls, are preferred for OD applications because they offer high surface/ volume ratios and do not require supports or spacers. Various parameters have to be considered in the selection of the membrane such as: pore size, porosity, conductivity and thickness. An increase in the pore size enhances the evaporation flux (flux is proportional to the radius); furthermore, membranes with large pore size exhibit a higher retention towards volatile organic flavour/ fragrance components (Barbe et al., 1998) than membranes with smaller pore size. These results can be attributed to differences in feed–membrane and stripper–membrane boundary layer resistances to organic volatiles transport owing to various degrees of liquid intrusion into the pores. Highly porous membranes are preferred because the evaporation flux is proportional to the porosity. Furthermore, an increase in membrane conductivity allows the heat gradient across the membrane to be minimized. Because the evaporation flux is proportional to the reciprocal of the pore length, the membrane thickness should be as low as possible. The thickness © Woodhead Publishing Limited, 2010

Advances in membrane-based concentration 257 is usually limited by the mechanical strength of the membrane: therefore thin membranes are supported by net, e.g. Gelman PTFE membranes. The overall thickness for OD membranes can vary from 80 to 250 mm, depending on the absence or presence of support. The risk of wetting of the hydrophobic membrane, with a consequent reduction in the evaporation flux and separation performance, is the main drawback of the osmotic distillation process. The reduction in the membrane thickness leads to a decrease in thermal resistance that facilitates the heat transfer from the brine to the feed causing a reduction in the temperature gradient (DT) and an increase in the partial pressure gradient (DP) across the membrane. However, thin hydrophobic membranes are more susceptible of wetting with a consequent loss of both evaporation flux and separation performance. If wetting occurs, the liquid can penetrate into pores in the membrane and, consequently, a liquid flux is added to the vapour flux and nonvolatile solutes diffuse across the membrane from one compartment to the other. Some fruit juices, especially citrus juices, contain peel oils and other highly hydrophobic compounds that promote wetting of OD membranes. Furthermore, cleaning solutions often contain surface-active agents that might also promote membrane wetting. Hydrophilic polymer films, which have sufficiently high intrinsic water permeability and are virtually impermeable to macrosolutes and colloids, are suitable as laminated membranes to prevent liquid intrusion without impeding vapour transport (Hogan et al., 1998). They include: esters and ethers of cellulose, crosslinked gelatin, chitin, agar, alginic acid, crosslinked polyacrylamide, crosslinked polyvinyl alcohol (PVA), polyhydroxy 2-ethyl methacrylate (PHEMA). Commercially available cellophane membranes can be suitable for this purpose; the hydrogel-film side of the laminate should be in contact with the solution to be concentrated (Michaels, 1999). Mansouri and Fane (1999) developed modified hydrophobic membranes for OD tolerant to oily feeds (i.e. limonene) by coating the feed side of commercial membranes with a thin layer of PVA. The uncoated membranes were rapidly wetted out by the oily feed even for low concentrations of oil (limonene) dispersed in water; vice versa, the coated membranes were stable for concentrations up to 1 wt.% limonene solution for periods up to 24 h. Commercial membranes for OD applications (termed TF200, TF450 and TF1000) manufactured by Pall-Gelman (East Hills, NY, USA) are characterized by a thin PTFE layer supported by a polypropylene (PP) net. In these asymmetric membranes, the top layer offers a resistance to gas transfer, whereas the membrane support offers an additional resistance to water transfer in the liquid form. The mass transfer resistance in the vapour phase is about 40–70% of the total resistance. The resistance of diluted brine entrapped in the PP support can cover up to 30% of the total resistance and the diluted brine boundary layer up to 60% indicating the sensitivity of the OD system to concentration polarization phenomena (Courel et al., 2000a). The best-known module designed for OD is the Liqui-Cel® Extra-Flow membrane contactor (Membrana-Charlotte, North Carolina, USA) containing


258 Separation, extraction and concentration processes microporous PP hollow fibre membranes approximately 300 mm in external diameter with a wall thickness of about 40 mm; they have a mean pore diameter of about 30 nm and a porosity of about 40%. The fibres are potted into a polyethylene tubesheet and the shell casing is PP, PVDF or 316L stainless steel (Gabelman and Hwang, 1999; Hogan et al., 1998; Sirkar, 1997). The smallest modules are 2.5 ins in diameter with a membrane surface area of 1.4 m2, whereas the largest are 10 ins in diameter and offer a contact area of 130 m2. Commercial membranes commonly used in OD are summarized in Table 9.2. Effect of operating conditions on the OD flux The performance of the OD process in terms of water vapour transport is influenced profoundly by the operating conditions. Feed concentration inversely affected the performance of the OD process. Courel et al. (2000b) observed a decrease in evaporation flux (from 10.3 to 1.1 kg m–2 h–1) when sugar solutions of increasing sucrose content, from 0 to 65% w/w, were dehydrated at 25 °C by using stripping solution of 45.5 w/w% initial CaCl2 content. A transmembrane flux decay by increasing the feed concentration was observed also by Ravindra Babu et al. (2006) in the concentration of sweetlime juice and phycocianin solution by OD. The viscosity of the feed solution increases exponentially with the solute content whereas the diffusion coefficient strongly decreases relative to pure water. The increasing viscosity results in an increase of the concentration polarization effect reducing the driving force and, consequently, the flux rate (Bui et al., 2005). Sheng et al. (1991) studied the effect of operating conditions on the OD flux during the concentration of apple, orange and grape juice through a PTFE membrane with a pore size of 0.2 mm and an overall thickness of 100 mm. The osmotic pressure difference D p between the aqueous streams strongly affected the transmembrane vapour flux; in particular, a 33% decrease in D p determined a five-fold decline in OD flux. The OD flux is significantly affected also by the solute content of the stripping solution. Courel et al. (2000b) observed a 64% vapour flux decay for a 30% mass fraction reduction of the brine solution in extracting pure water at 25 °C by using CaCl2 as extractant. Ravindra Babu et al. (2006) observed a similar trend in the concentration of phycocianin solution and sweet-lime juice. These results may be explained by assuming the strong dependence of the water activity of the stripping solution on salt content. Although a vapour flux improvement is expected when the salt content is reduced (since the density and viscosity of the brine tend to decrease reducing the mass transfer resistance in the salt solution) this improvement is in fact masked by the activity effect. The OD flux is also differently affected by the type of osmotic agent. Calcium chloride produces higher transmembrane fluxes than sodium chloride © Woodhead Publishing Limited, 2010

Table 9.2 Typical membranes used in the OD process (adapted from Gryta, 2005) Manufacturer

Material

Configuration

Thickness (mm)

Porosity (%)

Average pore size (mm)

Durapore GVHP Durapore GVSP Durapore HVHP FHLP Accurel PP Q3/2 Accurel PP S6/2 Celgard 2500 Celgard 2400 Metricel SM35 TF200 TF450 TF1000 Gore-Tex 10387

Millipore Co. Millipore Co. Millipore Co. Millipore Co. Enka A.G. Enka A.G. Celgard LLC Celgard LLC Gelman USA Scimed Life Systems Inc. Pall-Gelman Pall-Gelman Pall-Gelman Gore & Associates

PVDF PVDF PVDF PTFE PP PP PP PP PP PDMS PTFE layer PTFE layer PTFE layer PTFE layer

Flat Flat Flat Flat Capillary Capillary Flat Flat Flat

125 108 125 175 200 400 25 25 90 300 178 178 178 8.5

70 80 75 70 70 70 50 37 55

0.20 0.22 0.45 0.25 0.2 0.2 0.07 0.05 0.1

80 80 80 78

0.2 0.45 1.0 0.2

supported supported supported supported

by by by by

PP PP PP PP

net net net net

Flat Flat Flat Flat

Advances in membrane-based concentration 259


Type

260 Separation, extraction and concentration processes in the concentration of pineapple juice by OD (Nagaraj et al., 2006). This is mainly because of the higher osmotic activity of calcium chloride that results in a higher vapour pressure gradient across the membrane. Aqueous solutions of propylene glycol, glycerol and glycerol–salt mixtures were investigated as an alternative to calcium chloride in order to overcome the problem of corrosion and scaling associated with the use of brines (Celere and Gostoli, 2004). Propylene glycol and glycerol solutions (70–75 wt.%) were less effective than highly concentrated CaCl2 and exhibited a similar extractive power. However, propylene glycol cannot be recommended as an extractant in juice concentration owing to its low penetration pressure through the membrane pores and the not negligible volatility. Ternary mixtures water–glycerol–NaCl are characterized by lower viscosities than glycerol alone, and offer similar fluxes. The transmembrane flux in OD increases with an increase in the flow rate of the osmotic agent (Courel et al., 2000b; Nagaraj et al., 2006). This can be attributed to a stronger shear stress along the condensation side of the membrane leading to a reduction in the hydrodynamic boundary layer thickness and, consequently, to the concentration polarization effect. Ravindra Babu et al. (2008) observed an 8% increase in transmembrane flux when the flow rate of pineapple juice at about 12 °Brix was increased from 25 mL min–1 to 100 mL min–1; the increase in transmembrane flux can be explained by assuming a reduction in the concentration polarization effect on the feed side. The increase in flux, however, was more prominent (about 20%) by increasing the osmotic agent velocity. This phenomenon can be attributed to a lower concentration polarization on the feed side than on the brine side. A minor role of feed velocity on reducing the polarization problem in OD was observed also by Bui and Nguyen (2006) in the concentration of aqueous glucose solutions. They assumed that the heat transfer in OD is only a little involved or not involved at all; on the contrary, feed flow rate is essential to maintain the temperature gradient across the membrane in MD. The evaporation flux in OD is also affected by the feed temperature. Courel et al. (2000b) studied the influence of the temperature under isothermal conditions on the evaporation fluxes of pure water and sugar solutions of 35–65 w/w% sucrose content at 20–35 °C. A calcium chloride solution with an initial concentration of 45 w/w% was used as stripping solution. Evaporation fluxes ranged from 0.5 kg m–2 h–1 for a sugar solution of 65 w/w% (at 20 °C) to 12.8 kg m–2 h–1 for pure water (at 35 °C). The extent of mass transfer increase depended on the solute content: in the range 20–35 °C the evaporation flux increased by 120%, for a 35 w/w% sucrose solution, and only by 32% at 60 w/w% solute content. This phenomenon can be explained by assuming an exponential type relation between the vapour pressure difference across the membrane and the temperature according to Clapeyron’s law. Moreover, an increase in temperature results in a decrease in the feed and brine viscosities and an increase in the solute diffusion


Advances in membrane-based concentration 261 coefficient: the higher the solute content, the stronger the thermal effect. Bui and Nguyen (2006) reported a 200% increase in the evaporation flux for a feed temperature increase of 20 degrees in the concentration of 40 w/w% and 50 w/w% aqueous glucose solutions by means of PVDF hollow fibres. OD applications Because OD can be operated at room temperature and atmospheric pressure, with minimal thermal and mechanical damage of the solutes, it has been successfully applied to the concentration of liquid foods such as milk, fruit and vegetable juices, instant coffee, tea and pharmaceuticals. The low temperature employed avoids the chemical or enzymatic reactions associated with heat treatment. The low operating pressure results in lower energy consumption and capital investment, reduced fouling phenomena and the possibility of using membranes characterized by lower mechanical resistance than those in pressure-driven membrane processes (Courel et al., 2001). OD is particularly suited for fruit juice concentration, producing concentrated juices with a quality and composition very close to fresh ones and higher than conventional products obtained by thermal evaporation (Cassano and Drioli, 2007; Jiao et al., 2004). Plate-and-frame modules with a net-shaped spacer on the extract side and a smooth juice side path were developed for the concentration of whole juice with a high pulp content (Cervellati et al., 1998). This configuration, even if not completely efficient in terms of mass transfer, allows unclarified juices to be processed. Helically wound hollow-fibre modules offer an improvement in the hydrodynamic conditions on the shell-side compared with axial flow modules; consequently, higher concentration of solutes and higher evaporation fluxes can be obtained when viscous feeds are processed (Costello et al., 1997). Rodrigues et al. (2004) evaluated the performance of the OD and RO processes in the concentration of camu-camu juice. RO allowed higher fluxes to be reached (50 kg m–2 h–1) than OD, but lower concentration of soluble solids (25 °Brix). OD allowed the juice to be concentrated up to 63 °Brix with evaporation flux values of 10 kg m–2 h–1. A pre-treatment of the whole juice by UF or MF allows evaporation fluxes to be improved in OD. However, the performance of the OD process is affected by pore diameters of UF membranes. Bailey et al. (2000) observed higher evaporation fluxes when Gordo grape juice was preliminarily ultrafiltered with membranes having pore diameters of 0.1 mm or less. UF membranes with a nominal pore diameter of 0.5 mm did not improve the OD fluxes. UF pretreatment results also in a small increase in juice surface tension with a consequent reduction in the tendency for membrane wet-out to occur. Several kinds of clarified fruit juices (orange, apple, grape, cactus pear, kiwi, passion fruit, and pineapple) were concentrated by OD (Alves and Coelhoso, 2006; Cassano and Drioli, 2007; Cassano et al., 2003, 2004, 2006,


262 Separation, extraction and concentration processes 2007; Durham and Nguyen, 1994; Galaverna et al., 2008; Hongvaleerat et al., 2008; Jiao et al., 1991; Koroknai et al., 2006a, 2006b; Rektor et al., 2006; Rodrigues et al., 2004; Vaillant et al., 2001, 2005). An integrated membrane process was developed by Cisse et al. (2005) to produce concentrated orange juice. The process included a MF pre-treatment followed by OD. The quality of the concentrated juice was similar to the freshly squeezed one. Cassano et al. (2003, 2004, 2006 and 2007) proposed integrated membrane processes including UF, RO and OD to produce concentrated fruit juices such as kiwifruit, orange, lemon and cactus pear juice. Depectinized juices were clarified by UF and then optionally preconcentrated by RO. The OD process was employed as a concentration step to produce concentrated juices with final total soluble solids (TSS) up to 63–65 °Brix. The proposed process employs partial batch recycle on the feed side of the OD step in order to minimize feed viscosity changes through the membrane contactors and continuous counter current recycle plus evaporative reconcentration of the brine strip. The residual fibrous phase coming from the UF treatment (retentate) could be submitted to a stabilizing treatment (pasteurization, ohmic heating, high pressures, electric fields at high voltage) and successively added to the final OD concentrate for the preparation of fibre enriched beverages (Fig. 9.3). The total antioxidant activity (TAA) of the clarified or pre-concentrated juice was kept constant during the OD process independently of the achieved degree of TSS. Both aroma and colour were similar to those of the fresh juice. Ascorbic acid and health-promoting substances were also well preserved. On the contrary, a larger decrease in content of antioxidant compounds was detected in thermally evaporated juices with the same TSS content. In the concentrated orange juice, anthocyanins and hydroxycinnamates (particularly ferulic and p-coumaric acid) underwent a reduction of 36 and 55%, respectively; for the ascorbic acid and flavonones removals were in the order of 30% and 23%, respectively (Galaverna et al., 2008). Cassano and Drioli (2007) also evaluated the quality of kiwifruit juice concentrated by OD and thermal evaporation. The juice concentrated by thermal evaporation at 65 °Brix showed an 87% reduction of the ascorbic acid compared with the clarified juice. The TAA was reduced by about 50% independently of the TSS content achieved. On the contrary, the juice concentrated by OD retained almost all the TAA and the ascorbic acid content of the clarified juice. Blackcurrant juices concentrated by OD, with a final TSS content of 63–72 °Brix, showed a colour intensity, transparent ability and acidic flavour intensity similar to those of the raw juice (Kozák et al., 2008). A hybrid plant consisting of UF and RO pretreatment stages and an OD section (containing two 19.2 m2 Liqui-Cel membrane modules) for the concentration of fruit and vegetables juices was developed by Zenon Environmental (Burlington, Ont.). The plant was designed to produce concentrated juice at 65–70 °Brix at an average flow rate of 50 L h–1. It


Fruit juice 10–11 °Brix

Reconcentrated brine

Feed reservoir Evaporator

Condenser

UF

Condensate

Stripping solution reservoir

OD Pulp

Preconcentrated juice 25–26 °Brix

RO

Water

Pasteurization

Pasteurized pulp

Reconstituted juice Concentrated juice 64–65 °Brix Water

Fig. 9.3 Integrated membrane process for the production of concentrated fruit juices (dashed lines refer to the production of the reconstituted juice).



Clarified juice

264 Separation, extraction and concentration processes was developed primarily for the concentration of grape juices destined for the production of high-quality vintage varietal wines such as Chardonnay, Cabernet Sauvignon and Merlot. Concentrates can be stored for long periods without deterioration and can be used as blending stocks to adjust the sugar content of freshly harvested grapes to minimize variations in alcohol content of the resulting wine between vintages. Additionally, the concentrated juice can be shipped over long distances and used to produce high-priced varietal wines starting from local grapes (Hogan et al., 1998). Evaporation fluxes between 7 and 10 kg m–2 h–1 were obtained by Hongvaleerat et al. (2008) in the concentration of clarified pineapple juice. These values were higher than those obtained with the single strength juice owing to the complete removal of suspended solids in the clarification step. At low TSS the evaporation flux decay during the OD process is more attributable to the dilution of the stripping solution whereas, at higher feed concentrations, the evaporation flux depends mainly on juice viscosity and, consequently, on juice concentration (Cassano and Drioli, 2007; Courel et al., 2000a; Vaillant et al., 2001). Evaporation fluxes in OD can be improved by application of acoustic fields. Narayan et al. (2002) observed an enhancement of the evaporation flux from 0.81 to 0.94 L m–2 h–1 during the OD of sugarcane juice with CaCl2 as stripping solution when ultrasounds were applied to the membrane cell. Concentration and temperature polarizations in OD contribute up to 18% to the flux reduction (Bui et al., 2005). However, concentration polarization gave a larger contribution to the flux reduction than temperature polarization. Furthermore, the flux reduction owing to polarization was smaller on the feed side than on the brine side. Hydrophobic membranes with relatively large pore sizes showed higher organic volatiles retention per unit water removal than those with smaller pores when model aroma aqueous solutions were processed by OD (Barbe et al., 1998). Pores with larger diameter at the membrane surface allow greater intrusion of the feed and brine solutions with a consequent increase in the thickness and resistance of the boundary layer at the pore entrance. Consequently, membranes with large surface pore diameters are preferred for OD applications in which retention of volatile flavour/fragrance components are required. Shaw et al. (2001) investigated the retention of flavours in concentrated orange and passion fruit juices prepared by OD. Losses of volatile components between 32 and 39% were observed in the concentrated products. The transfer of volatiles depends on the nature of compounds and on the operating conditions. A significant reduction in the transfer of juice volatiles was obtained by decreasing the circulation velocity or the temperature (Ali et al., 2002). The management of the diluted brine step is one of the drawbacks associated with the commercial application of the OD in fruit juice processing. Although the regeneration of exhausted brines can be realized by thermal evaporation, this operation is expensive owing to corrosion and scaling phenomena. Solar


Advances in membrane-based concentration 265 ponding, reverse osmosis and pervaporation were proposed as alternatives to re-concentrate the diluted brine solutions (Thompson, 1991). Electrodialysis was also suggested for the regeneration of NaCl brines (Petrotos and Lazarides, 2001). The OD process is also a valid approach for producing low-alcohol beverages because it permits a selective removal of single volatile solutes from aqueous solutions (e.g. ethanol from wine and other ferments) by using water as a stripping solvent. The removal of organic solvents after the extraction of intracellular products (antibiotics, hormones and biologicals) from fermentation broths is a necessity in the drug industry. This operation has to be carried out at low temperature in order to preserve the product from thermal degradation and can be performed by OD in which water is employed as stripping solution (Hogan et al., 1998). The concentration of pharmaceuticals and biological compounds such as vaccines, hormones, recombinant proteins, enzymes, antibiotics, fungicides and nucleic acids is another potential use of OD. These products have to be isolated or recovered in the dry state in order to maintain their activity and shelf stability for a long time; OD can be used as a concentration step, eventually after a pre-concentration unit (by RO or NF), in order to obtain concentrates from which bioactive compounds can be more easily recovered (e.g. by crystallization or extraction). OD can be used also as a pre-concentration step for the lyophilization, reducing the water removal load during freeze drying. 9.4.2 Membrane distillation Process fundamentals Similarly to the OD process, in MD the water vapour transfer is promoted by a vapour pressure difference between two sides of a microporous hydrophobic membrane; however, in MD the physical origin of the vapour pressure difference is a temperature gradient rather than a concentration gradient: the feed is maintained at high temperature while cold water is used as a stripping permeate. Therefore, membrane distillation is a thermaldriven process. The mass transfer in MD can be described as a three-phase sequence: (1) formation of a vapour gap at the warm solution–membrane interface; (2) transport of the vapour phase through membrane pores; (3) its condensation at the cold side membrane–solution interface. Theoretical aspects and potential applications of the MD process have been extensively studied (Bandini et al., 1991; Calabrò et al., 1994; El-Bourawi et al., 2006; Lawson and Lloyd, 1997; Schofield et al., 1987; Tomaszewska, 2000a). The process takes place at atmospheric pressure and at temperatures that may be much lower than the boiling point of the treated solutions. Consequently, it can be used to concentrate solutes sensitive to high


266 Separation, extraction and concentration processes temperatures such as fruit juices and pharmaceuticals. Feed temperatures in MD are typically 60–90 °C, although temperatures as low as 30 °C have been used. Operating pressures are generally of about 0–100 kPa, hence much lower than conventional pressure-driven membrane processes such as RO. Consequently, lower equipment costs and increased process safety can be achieved. Furthermore, the mechanical resistance of the membrane is greatly reduced. Because MD operates on the principles of vapour–liquid equilibrium, another advantage over traditional pressure-driven membrane processes is represented by its high rejection (theoretically 100%) towards ions, macromolecules, colloids, cells and other nonvolatile compounds (Lawson and Lloyd, 1997). Finally, vapour spaces can be reduced compared with conventional distillation processes. As in the OD process, the main drawback of MD is the risk of wetting of the hydrophobic membrane with a consequent reduction in the evaporation flux and separation performance. Therefore, the process solutions must be aqueous and sufficiently dilute. This limits MD to applications such as desalination, removal of trace volatile compounds from wastewater and concentration of nonvolatile aqueous solutions. Membrane distillation (MD) configurations As depicted in Figure 9.4, the MD process can be realized according to four types of configuration. In the direct contact membrane distillation (DCMD), the membrane separates the hot feed from the cold distillate. In this instance, the vapour pressure gradient, which results from the transmembrane temperature difference, is the driving force of the mass transport across the membrane. Volatile molecules evaporate at the hot liquid/vapour interface, cross the membrane pores in the vapour phase, and condense on the cold liquid/vapour interface inside the membrane module (Gryta, 2002; Lawson and Lloyd, 1997; Mengual and Peña, 1997; Tomaszewska, 2000b). DCMD is usually employed for applications in which water is the major fluxing component, such as desalination or concentration of aqueous solutions. In air gap membrane distillation (AGMD), a condensing surface is separated from the membrane by an air gap. Volatile molecules cross both the membrane pores and the air gap and finally condense over a cold surface inside the membrane module. In the sweeping gas membrane distillation (SGMD), a cold inert, gas sweeps the permeate side of the membrane carrying the volatile molecules. In this instance, condensation occurs outside the membrane module (Khayet et al., 2000). In the vacuum membrane distillation (VMD), vacuum is applied on the permeate side of the membrane by means of a vacuum pump and, similarly to SGMD, condensation takes place outside the membrane module (Bandini et al., 1992).


Feed

Retentate

Feed

Retentate Membrane

Membrane

Permeate

Permeate in

Cooling water out

Air gap

Condensing plate

(a)

Feed

(b)

Retentate

Feed

Retentate Membrane

Membrane

Sweep gas in Condenser

Permeate

Sweep gas out

(c)

Cooling water in

Condenser

Permeate

Vacuum

(d)

Fig. 9.4 Membrane distillation configurations: (a) DCMD; (b) AGMD; (c) SGMD; (d) VMD.



Permeate out

268 Separation, extraction and concentration processes Mass transfer and polarization phenomena In a similar way to that of the OD process, classical models of gas diffusion in porous membranes, such as molecular diffusion or Knudsen diffusion, can be used to describe the mass transfer across MD membranes (Courel et al., 2000a; Gryta, 2005). These models suggest a linear relationship between the volume flux per unit surface area of the membrane and the transmembrane water vapour pressure difference at each interface, which depends on composition and temperature of both compartments. The osmotic and thermal contributions can operate either in a synergistic or in an antagonistic way (in which one of them prevails on the other) (Godino et al., 1995). The relationship between the permeate flux (Jw) and the driving force (DP) which causes the mass transfer across the membrane pores is given by:

Jw = Kw DP

[9.18]

where Km is the membrane mass transfer. The driving force in MD is a partial pressure gradient in the vapour phase given by:

DP = Pwf – Pwd

[9.19]

where Pwf and Pwd are water vapour pressures at the feed/membrane and feed/distillate interface, respectively. The mass transfer through the membrane, as in other membrane processes, is caused by the chemical potential difference (Dm) on both sides of the membrane (distillate and feed sides), which, for a ith component is given by:

D mi = D mid – D mif = RT ln

apid a = RT ln id apif ai

[9.20]

where apid and aid are the activity of ith component in the vapour and liquid if of ith component in phase on the distillate side, apif and aif the activity the vapour and liquid phase on the feed side, R the gas constant and T the temperature. The driving force expressed in equation [9.19] requires a knowledge of both temperature and solute composition at the vapour–liquid interfaces. Because the interfacial conditions are not always accessible, the water transport, referred to the bulk conditions, is often described as:

1 1ˆ Ê1 b b J w = K (Pwf – Pwd )=Á + + Ë K f K m K d ˜¯

–1

DP Pwb

[9.21]

where the overall mass transfer coefficient (K) is given by the series of water transport resistances from the bulk of the dilute solution towards the evaporation surface (1/Kf), water vapour transport through the membrane


Advances in membrane-based concentration 269 (1/Km) and from the condensation surface to the bulk of the distillate side (1/Kd). The overall mass transfer coefficient depends on the membrane morphology, module design, solution concentration, temperature and hydrodynamic conditions (Gryta et al., 2005). When the temperatures in the layers near the membrane differ from those measured in the bulk of the feed and permeate side, a temperature polarization phenomenon occurs. However, temperature profiles formed in the DCMD are different from those observed in OD. The temperature gradient results from the evaporation at the feed side and condensation at the permeate side even if the bulk temperatures of two liquids are equal, as in OD. However, the heat conduction from the brine side to the feed side decreases the temperature polarization phenomenon in OD. On the contrary, in the case of DCMD, heat and mass transfer flow through the membrane in the same direction (from the feed to the distillate side) increasing the temperature polarization effect (Gryta, 2005). In all MD variants, as in OD, only water passes through the membrane causing an increase in the solute concentration on the feed side and a decrease in the solute concentration on the distillate side relative to the bulk condition. This phenomenon, called concentration polarization, results in a driving force reduction across the membrane. Several authors assume that the concentration polarization is substantial in OD, whereas the performance of DCMD is affected mainly by the temperature polarization effect, except for the concentration of solutions with a solute content close to the saturated state (Gryta, 2002). Alves and Coelhoso (2006) compared the performance of OD and MD in the concentration of sucrose solution (used as a model fruit juice), in terms of water flux and aroma retention. At the same applied overall driving force, water fluxes in MD were less than half of those observed in OD owing to temperature polarization effects. Moreover a higher retention of aroma compounds was observed for the OD process. MD membranes MD membranes must be porous and hydrophobic with good thermal stability and excellent chemical resistance to feed solutions. They must be characterized by high liquid entry pressure which is defined as the minimum hydrostatic pressure that must be applied to the feed liquid solution before it overcomes the hydrophobic forces and penetrates into the membrane pores. Membranes with high liquid entry pressure can be obtained by using materials with high hydrophobicity and small maximum pore size. The most suitable materials for hydrophobic MD membranes include PVDF, PP, PE and PTFE. These membranes are available in capillary or flatsheet configurations (Ding et al., 2002). PVDF appears of particular interest in MD processes because of its high melting point and good temperature resistance; its resistance to oxidation and gamma radiation, to solvents and to abrasion are also of interest. Hydrophilic membranes, such as cellulose


270 Separation, extraction and concentration processes acetate membranes, can be treated in order to make their surfaces hydrophobic and, consequently, suitable for MD applications (Wu et al., 1992). Composite MD membranes consisting of hydrophobic and hydrophilic layers or a hydrophobic layer sandwiched between two hydrophilic layers have been also realized (Cheng et al., 1982). The size of micropores can range between 100 nm and 1.0 mm. In particular, the pores should be large enough to facilitate the required flux but also small enough in order to prevent liquid penetration through the membrane. For cylindrical pores, the maximum critical pore size at which the liquid penetrates the microporous phase is defined by the Laplace equation:

P = 2g

cos q r

[9.22]

where g is the surface tension of the liquid, q the contact angle between the liquid and the membrane, r the radius of the pore and P the applied pressure. For a given pore size a critical pressure Pc exists. For applied hydrostatic pressures higher than the Pc values, the liquid phase is transported across the membrane. Microporosity can be induced by mechanical stretching or by thermal phase separation technique. The relationship between the transmembrane flux and membrane characteristic parameters is given as:

N µ

ra e dm c

[9.23]

where N is the molar flux, r the mean pore size of the membrane pores, a a factor whose value equals 1 or 2 for Knudsen diffusion and viscous fluxes, respectively, dm the membrane thickness, e the membrane porosity and c the membrane tortuosity. According to equation [9.23], the MD flux increases with the increase in pore size. However, in order to avoid membrane pores wettability, the pore size should be as small as possible. Consequently, an optimum value for the pore size has to be determined for each MD application on the basis of the type of the feed solution to be treated. Schneider et al. (1988) recommended a maximum pore radius of 0.5–0.6 mm in order to avoid membrane wetting owing to fluctuations in process pressure and temperature. As for other membrane processes, the permeate flux in MD is inversely proportional to the membrane thickness: consequently thinner membranes produce higher fluxes. On the contrary, the membrane should be as thick as possible in order to prevent heat loss by conduction through the membrane matrix (Schofield et al., 1987). An optimal membrane thickness, considering the thermal conductivity of commercial membranes, should be within the range of 30–60 mm (Laganà et al., 2000). The porosity is the most influential factor affecting mass transfer rate in MD: membranes with higher porosity produce higher evaporation fluxes (Schneider et al., 1988). Membranes


Advances in membrane-based concentration 271 with high porosity also allows the amount of heat lost by conduction to be reduced, because the heat transfer coefficient of the gas entrapped within the membrane pores is generally an order of magnitude smaller than that of the membrane material. MD membrane modules can be realized with flat-sheet membranes, as plate and frame and spiral-wound configurations, or with capillary membranes in tubular configuration. The design of the MD modules provides a high feed flow rate, to increase turbulence of the feed solution, low pressure drop and high packing density; moreover, because the MD is a nonisothermal process, good heat recovery and thermal stability is also guaranteed. Laboratory-scale modules are usually realized with flat-sheet membranes that are much more versatile than capillary membranes. They can be easily removed from their modules for cleaning, examination or replacement. Consequently, the same membrane module can be used to test different MD membranes. On the other hand, tubular membrane modules are preferred for commercial applications because they do not require a support and allow high membrane surface area/ module volume ratios to be realized. Effect of operating parameters on MD fluxes Evaporation fluxes in MD increase with an increase in the feed temperature. This is because the exponential increase of the vapour pressure of the feed solution with temperature increases the vapour pressure difference across the membrane and, hence, the driving force of the process. In DCMD applications the effect of decreasing the permeate temperature is to increase the evaporation flux (Lawson and Lloyd, 1999). A reduction in the flow rate implies a reduction in the Reynolds number and, consequently, in the transport coefficients. When the mean temperature is kept constant, the permeate flux increases linearly with the temperature difference. On the other hand, when the temperature difference is fixed, the permeate flux increases exponentially with the mean temperature (Mengual and Peña, 1997). When non-volatile solutes are considered, permeate fluxes in all MD configurations decrease by increasing the feed inlet concentration: this phenomenon can be attributed to the reduction of the driving force owing to the decrease in the vapour pressure of the feed solution and to the exponential increase in the viscosity of the feed solution. At high concentration ratios, MD fluxes are higher than those observed in other pressure-driven membrane processes (Cath et al., 2004). Permeate fluxes generally increase by increasing the solute concentration when aqueous solutions containing volatile components (such as alcohols) are processed. This can be explained by assuming that an increase in the volatile compound concentration in the feed side is associated with an increase in its transmembrane partial pressure. The increase in the permeate flow velocity causes an increase in the permeate flux in MD process. The increase in permeate flow velocity increases the heat transfer in the permeate side of the membrane reducing the temperature and concentration polarization phenomena. Consequently, the temperature


272 Separation, extraction and concentration processes at the membrane surface approaches the temperature in the bulk permeate side increasing the driving force and the permeate flux. Finally, a linear increase in the MD flux with the transmembrane vapour pressure difference is typically observed in all MD configurations considered above. MD applications MD configurations previously described can be applied in different areas of interest for the separation of both nonvolatile (ions, colloids, macromolecules) and volatile (benzene, chloroform, trichloroethylene) compounds from water, the extraction of organic compounds such as alcohols from diluted aqueous solutions, the production of distilled water and the concentration of aqueous solutions. Desalination and pure water production from brackish water is the bestknown MD application (Banat and Simandl, 1998; Khayet et al., 2003, 2005). The concentration of radioactive solutions and wastewater treatment in the nuclear industry is another area under investigation (Khayet et al., 2006). In the chemical industry, the separation of azeotropic aqueous mixtures such as alcohol–water mixtures, the removal of volatile organic compounds from water and the concentration of acids, such as sulfuric, hydrochloric and nitric acid, can be achieved by MD (Banat and Simandl, 2000; Duan et al., 2001; Garcia-Payo et al., 2002; Tomaszewska, 2000b; Tomaszewska et al., 1995). Removal of dyes and wastewater treatment in the textile industry have also been reported (Banat et al., 2005; Calabrò et al., 1991). In the pharmaceutical and biomedical areas MD proved attractive for the removal of water from blood and protein solutions and in the wastewater treatment (Ortiz de Zárate et al., 1998; Sakai et al., 1986). MD can be successfully applied in the food industry and in areas where high temperature applications lead to the degradation of process fluids such as concentration of fruit juices and milk processing. A blackcurrant juice concentrate with a high TSS content was produced by MD without degradation of its valuable substances maintaining the juice side temperature at 26 °C and the water side temperature at 11 °C (DT = 15 °C) (Kozák et al., 2009). An increase of a few degrees Celcius in the driving force (DT = 19 °C) influenced significantly the evaporation flux and the operation time of the process. Gunko et al. (2006) used DCMD to investigate the concentration of apple juice. A TSS content of 50 °Brix was obtained when the permeate flux reached about 9 L m–2 h–1. Further concentration to 60–65 °Brix resulted in reduced productivity (up to 3 L m–2 h–1). Highly concentrated apple juices up to 64 °Brix were also produced by using polypropylene hollow fibre DCMD modules with trans-membrane fluxes of 1 kg m–2 h–1 (Curcio et al., 2000; Laganà et al., 2000). Flux rates were dependent essentially upon temperature polarization phenomena located mainly on the feed side, rather than concentration polarization. PVDF membranes used for the concentration of orange juice by MD


Advances in membrane-based concentration 273 showed a very good retention of soluble solids, sugars and organic acids with rejection of sugars and organic acids equal to 100%. The colour and flavour of concentrated juice were satisfactory (Calabrò et al., 1994; Drioli et al., 1992). A protein concentrate containing at least 25% of protein was obtained by Christensen et al. (2006) in the processing of whey protein by DCMD as an alternative to thermal evaporation. The optimal temperature for the whey protein concentrate was 55 °C leading to less protein denaturation than evaporation and therefore a higher quality product. Bandini and Sarti (2002) studied the vacuum membrane distillation (VMD) for the concentration of grape must up to 50 °Brix. The process allowed production of juice concentrates that still retained interesting amounts of the aroma compounds. Bagger-Jørgensen et al. (2004) and Diban et al. (2009) evaluated the potential of VMD to recover aroma compounds from blackcurrant and pear juice, respectively. The highest values of enrichment factor (up to 15) for pear aroma compounds were obtained working at lower temperatures and higher downstream pressures. The highest concentration factors for the blackcurrant aroma compounds (from 21 to 31) were obtained at high feed flow rate (400 L h–1) and low temperatures (10 °C). At 5 vol.% feed volume reduction the recovery of highly volatile compounds was between 68 and 83 vol.% and between 32 and 38 vol.% for the hardly volatile compounds. Also in MD, an improvement in the evaporation flux in fruit juice concentration can be obtained when the juice is preliminarily submitted to a UF treatment. Drioli et al. (1992) found that the UF of the single-strength orange juice resulted in an increase in MD flux that remained almost constant during an approximately two-fold concentration. On the contrary, the MD flux of the unfiltered juice decreases steadily over the same concentration range. The improvement of the MD flux can be attributed to a reduction of juice viscosity as a result of pulp and pectin removal. PVDF membranes showed a very good retention of orange juice components such as total soluble solids, sugars and organic acids; on the other hand, the amount of ascorbic acid decreased by 42%, probably owing to its degradation associated with high temperature and oxidation. By referring to the retention of volatile organic flavour/fragrance components from liquid foods it has been found that membranes having an open fibrous structure rather than discrete pore offer the best volatiles retention for a given amount of removed water. 9.4.3 Coupled operation of osmotic distillation and membrane distillation Table 9.3 summarizes the main advantages and disadvantages of MD and OD processes. Because the water flux in OD is relatively low (usually between 0.07 and 7.2 kg m–2 h–1), the surface area available for flux should be increased. As described above, the thermal effect in OD reduces the driving


274 Separation, extraction and concentration processes Table 9.3 Positive and negative aspects of MD and OD processes Advantages

Disadvantages

Low operating temperature

Low evaporative capacity with a long time of treatment Possibility of wetting of the hydrophobic membrane and consequent loss of flux and separation performance Production costs higher than those of thermal evaporation High cost of membrane replacement

Low operating pressure

No or less degradation of heat-sensitive compounds High TSS concentration in the concentrated products Modularity, easy scale-up Management of diluted brine solutions Possibility to treat solutions with high levels of suspended solids Possibility to concentrate several different products with the same unit No fouling problems Constant permeate flux in time Low investment cost

force of the water transport across the membrane. This phenomenon can be exploited to obtain a coupled process where the brine and feed solutions are thermostatically controlled at different temperatures: the osmotic solution on the cold side and the solution to be concentrated on the warm side. This coupled operation of MD and OD, referred to as membrane osmotic distillation (MOD), allows the water flux across the membrane to be enhanced (Wang et al., 2001). Although the aqueous solution is gently heated during the operation, this coupled method still works under mild conditions, because the temperature difference applied is lower than 15 °C (Bélafi-Bakó and Koroknai, 2006). The effect of varying experimental conditions (solute concentration, stirring rate, mean temperature and bulk temperature difference) on the water flux involved in a coupled process MD/OD was investigated by Godino et al. (1995). Koroknai et al. (2006a and 2006b) obtained concentrated fruit juices (apple, red- and blackcurrant, sour cherry and raspberry) at 60 °Brix in an operation time of 15–20 h, maintaining a temperature difference of 15 °C and using as an osmotic solution CaCl2 6M. In particular, the driving force of the process was greatly enhanced by decreasing the temperature of the osmotic solution, as in the range 15–25 °C only a slight drop in the water vapour pressure, for saturated CaCl2 solution, occurs. Consequently, maintaining the osmotic solution at room temperature, the energy consumption is minimized and the coupled process can be accelerated by increasing the temperature of the solution to be concentrated. Similarly, Bélafi-Bakó and Koroknai (2006) found that the coupled process is more effective than MD © Woodhead Publishing Limited, 2010

Process

Maximum achievable concentration (°Brix)

Product quality

Evaporation rate or flux

Possibility of treating Operating cost different products with the same installation

Thermal evaporation Cryoconcentration Reverse osmosis Direct osmosis Membrane distillation Osmotic distillation

65–75 30–50 25–30 50 60–70 60–70

Poor Very good Very good Good Good Very good

200–300 L h–1 – 5–10 L m–2 h–1 1–5 L m–2 h–1 1–10 L m–2 h–1 1–10 L m–2 h–1

No No No Yes Yes Yes

Moderate High High High High High

Capital investment

Energy consumption

Moderate Very high Moderate Moderate Low Low

Very high Very high High Low Low Low



Table 9.4 Key factors of conventional evaporation and membrane concentration techniques (adapted from Jariel et al., 1996)

276 Separation, extraction and concentration processes or OD alone. The use of short membrane modules in cascade series, with heat exchangers placed between them, was suggested in order to minimize heat losses.

9.5 Conclusions Key factors of conventional evaporation and membrane concentration techniques are summarized in Table 9.4. Several advantages of membranebased processes over conventional evaporation have been successfully demonstrated, including improved product quality, easy scale-up and low energy consumption. Low evaporation fluxes in OD and MD seem to be the main drawbacks of these processes when compared with RO and thermal evaporation. However, when the solution to be concentrated contain solutes sensitive to mechanical or thermal degradation there are serious limitations in using these technologies without significant deterioration in the quality. Currently membrane contactors are emerging technologies in the processing of foods, pharmaceuticals and beverages and will become breakthrough technologies when enhanced effectiveness is attained. Their integration with standard membrane operations is a valid approach for a sustainable industrial growth within the process intensification strategy. The aim of this strategy is to introduce in the productive cycles new technologies characterized by low encumbrance volume, advanced levels of automation capacity, modularity, remote control and reduced energy consumption. In order to improve the competitiveness of membrane contactors towards conventional technologies, efforts should be devoted to the development of new membranes characterized by high selectivity and stability for long-term applications, as well as to improvements in process engineering, including module and process design.

9.6 Nomenclature a a i api C d D h J k B k

water activity activity of the ith component in the liquid phase activity of the ith component in the vapour phase (fugacity) solute molar concentration (mol L–1) diameter (m) diffusion coefficient (m2 s–1) liquid heat transfer coefficient (W m–2 K–1) mass flux (kg m–2 h–1) or volume flux (m3 m–2 s–1) Boltzmann constant (1.3807 ¥ 10–23 J K–1) mass transfer coefficient (m s–1)


Advances in membrane-based concentration 277 K Kn M N P P* Q r R Re Sc Sh T u U Greek g d e q DP Dm l m C r s

mass transfer coefficient (kg m–2 h–1 Pa–1) Knudsen number molecular weight (kg mol–1) molar flux (mol m–2 s–1) water vapour pressure (Pa) pure water vapour pressure (Pa) heat flux (W m–2) pore radius (m) gas constant (8314 J mol–1 K–1) Reynolds number Schmidt number Sherwood number temperature (°C, K) velocity of the fluid (m s–1) overall heat transfer coefficient (W m–2 K–1) symbols liquid surface tension (N m–1) thickness (m) porosity contact angle driving force chemical potential difference mean molecular free path (m) liquid dynamic viscosity (Pa s) tortuosity liquid density (kg m–3) mean collision diameter (m)

Subscripts b bulk d distillate f feed h hydraulic m membrane p pore or permeate s stripping solution or solute w water or vapour Superscripts b bulk location K Knudsen diffusion m membrane location M molecular diffusion


278 Separation, extraction and concentration processes Abbreviations DO direct osmosis MD membrane distillation MF microfiltration OA osmotic agent OD osmotic distillation OMD osmotic membrane distillation PP polypropylene PTFE polytetrafluoroethylene PVDF polyvinylidene fluoride RO reverse osmosis TAA total antioxidant activity TSS total soluble solids UF ultrafiltration

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280 Separation, extraction and concentration processes in osmotic distillation using asymmetric membrane’, J Membrane Sci, 173, 107– 122. Courel M, Tronel-Peyroz E, Rios GM, Dornier M and Reynes M (2001), ‘The problem of membrane characterization for the process of osmotic distillation’, Desalination, 140, 15–25. Curcio E, Barbieri G and Drioli E (2000), ‘Operazioni di distillazione a membrane nella concentrazione dei succhi di frutta’, Industria delle Bevande, XXIX, 113–121. Deblay P (1995), ‘Process for at least partial dehydration of an aqueous composition and devices for implementing the process’, US Patent 5,382,365. Diban N, Voinea OC, Urtiaga A and Ortiz I (2009), ‘Vacuum membrane distillation on the main pear aroma compound: experimental study and mass transfer modelling’, J Membrane Sci, 326, 64–75. Ding Z, Ma R and Fane AG (2002), ‘A new model for mass transfer in direct contact membrane distillation’, Desalination, 151, 217–227. Drioli E, Jiao B and Calabrò V (1992), ‘The preliminary study on the concentration of orange juice by membrane distillation’, Proceedings of VII International Citrus Congress, Acireale (Italy), 3, 1140–1144. Duan SH, Ito A and Ohkawa A (2001), ‘Removal of trichloroethylene from water by aeration, pervaporation and membrane distillation’, J Chem Eng Jpn, 34, 1069–1073. Durham RJ and Nguyen MH (1994), ‘Hydrophobic membrane evaluation and cleaning for osmotic distillation of tomato puree’, J Membrane Sci, 87, 181–189. El-Bourawi MS, Ding Z, Ma R and Khayet M (2006), ‘A framework for better understanding membrane distillation separation process’, J Membrane Sci, 285, 4–29. Gabelman A and Hwang S (1999), ‘Hollow fiber membrane contactors’, J Membrane Sci, 159, 61–106. Gabino F, Belleville MP, Preziosi-Belloy L, Dornier M and Sanchez J (2007), ‘Evaluation of the clearing of a new hydrophobic membrane for osmotic evaporation’, Sep Purif Technol, 55, 191–197. Galaverna G, Di Silvestro G, Cassano A, Sforza S, Dossena A, Drioli E and Marchelli R (2008), ‘A new integrated membrane process for the production of concentrated blood orange juice: effect on bioactive compounds and antioxidant activity’, Food Chem, 106, 1021–1030. Garcia-Payo MC, Rivier CA, Marison IW and Stockar UV (2002), ‘Separation of binary mixtures by thermostatic sweeping gas membrane distillation: II. Experimental results with aqueous formic acid solutions’, J Membrane Sci, 198, 197–210. Geankoplis CJ (1993), ‘Principles of mass transfer’, in Transport processes and unit operation, London, Prentice-Hall, 381–413. Godino MP, Peña L, Ortiz de Zárate JM and Mengual JI (1995), ‘Coupled phenomena membrane distillation and osmotic distillation through a porous hydrophobic membrane’, Sep Sci Technol, 30, 993–1011. Gostoli C (1999), ‘Thermal effects in osmotic distillation’, J Membrane Sci, 163, 75–91. Gryta M (2002), ‘Concentration of NaCl solution by membrane distillation integrated with crystallization’, Sep Sci Technol, 37, 3535–3558. Gryta M (2005), ‘Osmotic MD and other membrane distillation variants’, J Membrane Sci, 246, 145–156. Gunko S, Verbych S, Bryk M and Hilal N (2006), ‘Concentration of apple juice using direct contact membrane distillation’, Desalination, 190, 117–124. Herron JR, Beaudry EG, Jochums CE and Medina LE (1994), ‘Osmotic concentration apparatus and method for direct osmotic concentration of fruit juice’, US Patent 5,281,430. Ho WSW and Sirkar KK (1992), Membrane handbook, Chapman & Hall, New York. Hogan PA, Canning RP, Peterson PA, Johnson RA and Michaels AS (1998), ‘A new option: osmotic distillation’, Chem Eng Prog, 94, 49–61.


Advances in membrane-based concentration 281 Hongvaleerat C, Cabral LMC, Dornier M, Reynes M and Nigsanond S (2008), ‘Concentration of pineapple juice by osmotic evaporation’, J Food Eng, 88, 548–552. Jariel O, Reynes M, Courel M, Durand N, Dornier M and Deblay P (1996), ‘Comparison of some fruit juice concentration techniques’, Fruits, 51, 437–450. Jiao B, Cassano A and Drioli E (2004), ‘Recent advances on membrane processes for the concentration of fruit juices: a review’, J Food Eng, 63, 303–324. Jiao B, Molinari R, Calabrò V and Drioli E (1991), ‘Application of membrane operations in concentrated citru juice processing’, Agro-Ind Hi-Tech, 19–27. Karode SK, Kulkarni SS and Ghorapade MS (2000), ‘Osmotic dehydration coupled reverse osmosis concentration: steady-state model and assessment’, J Membrane Sci, 164, 277–288. Khayet M, Godino MP and Mengual JI (2000), ‘Theory and experiments on sweeping gas membrane distillation’, J Membrane Sci, 165, 261–272. Khayet M, Mengual JI and Matsura T (2005), ‘Porous hydrophobic/hydrophilic composite membranes. Application in desalination using direct contact membrane distillation’, J Membrane Sci, 252, 101–113. Khayet M, Mengual JI and Zakrewska-Trznadel G (2003), ‘Theoretical and experimental studies on desalination using the sweeping gas membrane distillation’, Desalination, 157, 297–305. Khayet M, Mengual JI and Zakrewska-Trznadel G (2006), ‘Direct contact membrane distillation for nuclear desalination. Part II. Experiments with radioactive solutions’, Int J Nuclear Desalination, 56, 56–73. Koroknai B, Gubicza L and Bélafi-Bakó K (2006a), ‘Coupled membrane process applied to fruit juice concentration’, Chem Pap, 60, 399–403. Koroknai B, Kiss K, Gubicza L and Bélafi-Bakó K (2006b), ‘Coupled operation of membrane distillation and osmotic evaporation in fruit juice concentration’, Desalination, 200, 526–527. Kozák A, Bánvölgyi S, Vincze I, Kiss I, Békássy-Molnár E and Vatai G (2008), ‘Comparison of integrated large scale and laboratory scale membrane processes for the production of black currant juice concentrate’, Chem Eng Proc, 47, 1171–1177. Kozák A, Békássy-Molnár E and Vatai G (2009), ‘Production of black-currant juice concentrate by using membrane distillation’, Desalination, 241, 309–314. Laganà F, Barbieri G and Drioli E (2000), ‘Direct contact membrane distillation: modelling and concentration experiments’, J Membrane Sci, 166, 1–11. Lawson KW and Lloyd DR (1997), ‘Membrane distillation’, J Membrane Sci, 124, 1–25. Lebfevre MSM (1988), ‘Method of performing osmotic distillation’, US Patent 4,781,837. Luh BS, Feinberg B, Chung JI and Woodroof JG (1986), ‘Freezing fruits’ in Woodroof JG and Luh BS, Commercial fruit processing, Westport, AVI Publishing Co., 263–351. Maccarone E, Campisi S, Cataldi Lupo MC, Fallico B and Nicolosi Asmundo C (1996), ‘Thermal treatments effects on the red orange juice constituents’, Ind Bevande, 25, 335–341. Mansouri J and Fane AG (1999), ‘Osmotic distillation of oily feeds’, J Membrane Sci, 153, 103–120. Mengual JI, Ortiz de Zárate JM, Peña L and Velázquez A (1993), ‘Osmotic distillation through macroporous hydrophobic membranes’, J Membrane Sci, 82, 129–140. Mengual JI and Peña L (1997), ‘Membrane distillation’, Colloid Interf Sci, 1, 17–29. Michaels AS (1998), ‘Methods and apparatus for osmotic distillation’, US Patent 5,824,223. Michaels AS (1999), ‘Osmotic distillation process using a membrane laminate’, US Patent 5,938,928. Milleville H (1990), ‘Direct osmotic concentrates juices at low temperature’, Food Proc, 51, 70–71. © Woodhead Publishing Limited, 2010

282 Separation, extraction and concentration processes Mulder M (1998), Basic principles of membrane technology, 2nd edn. London, Kluwer Academic Publishers, 280–303. Nagaraj N, Patil G, Babu BR, Hebbar UH, Raghavarao KSMS and Nene S (2006), ‘Mass transfer in osmotic membrane distillation’, J Membrane Sci, 268, 48–56. Narayan AV, Nagaraj N, Hebbar HU, Chakkaravarthi A and Raghavarao KSMS (2002), ‘Acoustic field-assisted osmotic membrane distillation’, Desalination, 147, 149–156. Ortiz de Zárate JM, Rincón C and Mengual JI (1998), ‘Concentration of bovine serum albumin aqueous solutions by membrane distillation’, Sep Sci Technol, 33, 283–296. Petrotos KB, Quantick PC and Petropakis H (1998), ‘A study of the direct osmotic concentration of tomato juice in tubular membrane module configuration. I. The effect of certain basic process parameters on the process performance’, J Membrane Sci, 150, 99–110. Petrotos KB, Quantick PC and Petropakis H (1999), ‘Direct osmotic concentration of tomato juice in tubular membrane module configuration. II. The effect of using clarified tomato juice on the process performance’, J Membrane Sci, 160, 171–177. Petrotos KB and Lazarides HN (2001), ‘Osmotic concentration of liquid foods’, J Food Eng, 49, 201–206. Ravindra Babu B, Rastogi NK and Raghavarao KSMS (2006), ‘Mass transfer in osmotic membrane distillation of phycocyanin colorant and sweet-lime juice’, J Membrane Sci, 272, 58–69. Ravindra Babu B, Rastogi NK and Raghavarao KSMS (2008), ‘Concentration and temperature polarization effects during osmotic distillation’, J Membrane Sci, 322, 146–153. Rektor A, Vatai G and Békássy-Molnár E (2006), ‘Multi-step membrane processes for the concentration of grape juice’, Desalination, 191, 446–453. Rodrigues RB, Menezes HC, Cabral LMC, Dornier M, Rios GM and Reynes M (2004), ‘Evaluation of reverse osmosis and osmotic evaporation to concentrate camu-camu juice (Myrciaria dubia)’, J Food Eng, 63, 97–102. Romero J, Draga H, Belleville MP, Sanchez J, Come-James C, Dornier M and Rios GM (2006), ‘New hydrophobic membranes for contactor processes – applications to isothermal concentration of solutions’, Desalination, 193, 280–285. Sakai K, Muroi T, Ozawa K, Takesawa S, Tamura M and Nakaue T (1986), ‘Extraction of solute-free water from blood by membrane distillation’, Trans Am Soc Artif Intern Organs, 32, 397–400. Schneider K, Holz W and Wollbeck R (1988), ‘Membranes and modules for transmembrane distillation’, J Membrane Sci, 39, 25–42. Schofield RW, Fane AG and Fell CJD (1987), ‘Heat and mass transfer in membrane distillation’, J Membrane Sci, 33, 299–313. Shaw PE, Lebrun M, Dornier M, Ducamp MN, Courel M and Reynes M (2001), ‘Evaluation of concentrated orange and passion fruit juices prepared by osmotic evaporation’, Lebensm Wiss Technol, 34, 60–65. Sheng J, Johnson RA and Lefebvre MS (1991), ‘Mass and heat transfer mechanism in the osmotic distillation process’, Desalination, 80, 113–121. Sirkar KK (1997), ‘Membrane separation technologies: current developments’, Chem Eng Commun, 157, 145–184. Thompson D (1991), ‘The application of osmotic distillation for the wine industry’, Aust Grapegrower Winemaker, 11, 11–14. Tomaszewska M (2000a), ‘Concentration and purification of fluosilicic acid by membrane distillation’, Ind Eng Chem Res, 39, 3028–3041. Tomaszewska M (2000b), ‘Membrane distillation – examples of applications in technology and environmental protection’, Pol J Environ Stud, 9, 27–36. Tomaszewska M, Gryta M and Morawski AW (1995), ‘Study on the concentration of acids by membrane distillation’, J Membrane Sci, 78, 277–282. © Woodhead Publishing Limited, 2010

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10 Separation of value-added bioproducts by colloidal gas aphrons (CGA) flotation and applications in the recovery of value-added food products P. Jauregi and M. Dermiki, The University of Reading, UK Abstract: This chapter describes the application of a flotation method based on the formation of colloidal gas aphrons (CGA), which are surfactant-based microbubbles, to the recovery of value-added food products from fermentation broths and waste streams of food and agricultural industries. Their main properties are described and contrasted with those of conventional foams. The fundamentals of the separation are described and illustrated by several case studies on CGA applications to the recovery of whey proteins, polyphenols and carotenoids. Finally, a critical evaluation of the feasibility of CGA for industrial applications is carried out based on the astaxanthin case study and future applications of CGA are discussed. Key words: colloidal gas aphrons, flotation, bioproducts, surfactants, food ingredients, recovery.

10.1 Introduction Many studies have been conducted in recent years into additional biological functionalities of several food components and ingredients such as the dairy proteins, peptides, polyphenols or carotenoids so that they could be produced as ingredients in functional foods. The development of functional foods poses new challenges to the food industry as new processes and technologies are required. The food industry must adapt to these changing times and to the revolution caused by newly emerging functional foods. In addition, in the last few years reducing the environmental impact of industrial wastes has been a subject of increasing concern. In industrial wastewaters, these compounds considerably increase biochemical and oxygen demands, with detrimental effects on the flora and fauna of discharge zones, whereas in solid residues © Woodhead Publishing Limited, 2010

Separation of value-added bioproducts by colloidal gas aphrons 285 used as fertiliser they may inhibit germination. Furthermore sustainability is given high priority by governments and industries are encouraged to develop practices for the usage and recycling of wastes. Food processing waste streams and agricultural wastes can be exploited as cheap sources of high-value products. For example, proteins and peptides can be extracted from whey which is a by-product in cheese production and polyphenols can be extracted from winemaking waste, such as grape skin and marc. Separation and purification of these products is carried out by a number of steps including liquid–liquid extraction, membrane techniques and/or chromatographic methods. Alternative separations such as flotation may be used for the recovery stage although further processing will be necessary if high purity products are required. Foam fractionation has been applied for the recovery of proteins. Flotation, which is the foam separation of insoluble compounds, has been applied mainly to wastewater treatment and to the recovery of minerals. Separation of biomass has also been successfully carried out by flotation. This chapter deals mainly with a particular type of flotation using microbubbles which are also called colloidal gas aphrons (CGA). CGA were first described by Sebba (1972) as surfactant-stabilised microbubbles (10–100 mm) generated by intense stirring (>8000 rpm) of a surfactant solution. In this chapter, the application of CGA to bioseparations is reviewed particularly in relation to the recovery of value-added food products. First, the structural and dispersion characteristics of CGA are described. Then, the separation fundamentals are described and illustrated with the application of CGA to the separation of proteins. In addition, the feasibility of CGA for industrial applications is considered, focusing on: (i) scalability using a flotation column and (ii) removal and recycling of surfactant. All these aspects are illustrated in several case studies of CGA separations for the recovery of bioactives from plant extracts (norbixin and polyphenols) and recovery of astaxanthin produced by microbial fermentation.

10.2 Colloidal gas aphrons (CGA) properties 10.2.1 Structure of CGA Colloidal gas aphrons (CGA) are surfactant-stabilised microbubbles (10– 100 mm) generated by intense stirring of a surfactant solution at high speeds (>8000 rpm). Sebba (1987) postulated that they are composed of multilayers of surfactant molecules; as depicted in Fig. 10.1 surfactant molecules adsorb at the interface with hydrophilic heads towards the aqueous phase and the hydrophobic tails towards the gas phase. Sebba’s hypothesis on the structure was based on several experimental observations such as delayed coalescence and hence higher stability of CGA than conventional foams. Jauregi et al. (2000) investigated the drainage kinetics of CGA and compared measured drainage rates with those obtained by applying predictive models for foams with and without modifications. The main modifications were in relation to © Woodhead Publishing Limited, 2010

286 Separation, extraction and concentration processes Inner surface of shell

Air core

Viscous water Shell

Electrical double layer

Outer surface of shell

Fig. 10.1 Proposed structure of CGA by Sebba (1987).

structural differences between foams and CGA upon drainage, such as aphrons being surrounded by a liquid film so that they do not adopt a dodecahedral shape. Interestingly, the model giving the best prediction was the one including differences in structural features which further supports Sebbas’s theory. Jauregi and coworkers used for the first time small angle x-ray diffraction in an attempt to determine the thickness of the surfactant film and the number of surfactant layers of CGA generated by the anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT). The analysis of the data proved to be difficult as for the same sample different film thickness values could be obtained. However, an interesting finding was that samples containing aphrons gave similar scattering regardless of the surfactant concentration and these corresponded to 5.4 nm (which is equivalent to seven layers of surfactant assuming the full length of the surfactant molecule arranged in layers and vertically at the interface). Moreover, the same surfactant solutions with no aphrons gave a different scattering signal and, in this instance, differences between different concentrations of surfactant solutions were found; for example, at concentrations around the cmc (critical micellar concentration), the scattering corresponded to a bilayer, which could correspond to micelles whereas, above the cmc, the scattering corresponded to a multilayer of 3–5 molecules, confirming the hypothesis that AOT forms lamellar structures (Jauregi et al., 2000). In a number of research studies predictive models for liquid drainage in CGA were developed based on measurements of liquid drainage rate (Table 10.1). Most of the studies highlight the difference between CGA and conventional foams. Bhatia et al. (2005) studied the effect of stirring speed and stirring time on the stability of CGA generated using a range of


Table 10.1 Summary of studies for the characterisation of CGA (c, cationic; a, anionic; n-i, nonionic) (adapted from Dermiki, 2009) Surfactant

Parameters studied

Main findings


Zhao et al., SDS (a) 2009; Larmignat CTAB (c) et al., 2008 Tween 20 (n-i)

Surfactant type, Csurf, pressure CGA rheology is not affected by pipe shape or hydraulic diameter, CGA drop, T, volumetric and mass can be treated as shear thinning fluid for all surfactant solutions. Increase flow rate, pipe diameters and in Csurf leads to increased shear stress for a given shear rate shape

Feng et al., 2009 Tergitol (n-i) Ramnolipid (a)

Csurf, pH, Csalt

Stability increases with Csurf and decreases with increasing pH, Csalt. Three distinct phases in the liquid drainage

Moshkelani and SDS (a) Amiri, 2008

Electrical conductivity of surfactant solution

Three clear stages of drainage of CGA can be identified, compared with two stages for conventional foams

Tseng et al., 2006

Pressure drop, T, mass and volumetric flow rate of CGA

CGA can be considered shear thinning fluid. Heat transfer coefficients for CGA made of water and surfactant are smaller than that of water, and they are constant and independent of mass flow and heat flux

tstir, rpm, Csurf, mixing two oppositely charged CGA

Development of an empirical correlation for variation of gas hold-up with tstir, rpm Ê t ˆ – 4.025ˆ a = (0.004 + 44.017) Á 0.2407 ln ÊÁ –2.00 ˜¯ ˜¯ Ë2 Ë

Bhatia et al., 2005

SLS (a) CTAB (c)

The mixing of the two oppositely charged CGA showed no effect on drainage Yan et al., 2005 SDS (a) CTAB (c) Tween 80 (n-i)

Type of surfactant, Csurf, T

There was no effect of the type of surfactant on the stability. Mathematical rn model which describes drainage of CGA: Vt = Vmax n . Two distinct K + rn stages of drainage are determined by two independent mechanisms

Jarudilokkul et al., 2004

Tween 20 (n-i)

tstir, pH, rpm, CNaCl, Csurf, protein separation

Increasing Csurf, tstir increases the stability Increase of Csurf leads to decrease of protein separation

Dai and Deng, 2003

HTAC (c)

pH, concentration of silica sol for stabilisation

CGA were stable for up to 12 h at concentration of silica sol 0.15–0.25 mol dm–3 and pH 7–10

Separation of value-added bioproducts by colloidal gas aphrons 287

Reference


Reference

Surfactant

Parameters studied

Main findings

Jauregi et al., 2000

AOT (a)

Csurf, pH, Csalt, T, tstir

X-ray diffraction for the characterisation of CGA showed evidence of the existence of surfactant multilayers


AOT (a)

Csurf, pH, salt, T, tstir

Increase of Csurf increases the stability, whereas addition of salt leads to the opposite effect Significant interactive effects: (i) Csurf · salt, (ii) pH · Csurf, (iii)T · tstir, (iv) Csurf · T Gas hold-up depends on tstir, Csurf, and salt

Bredwell et al., 1995

SDS (a) Csurf, salt CPC (c) Triton X-100 (n-i)

Save and DTAC, CTAC, Pangarkar, 1994 CPC, DMDSAC (c) SDBS, SLS (a) Chaphalkar et al., 1993

pH, tstir, Csurf, Csalt, viscosity, additives, impeller clearance

SDBS (a), CTAB Type and concentration of (c), surfactant, ionic strength Tergitol (n-i)

Amiri and TTAB (c) Woodburn, 1990

Csurf, pH

Increasing Csurf leads to decrease of formation time of CGA and increase in CGA stability. No effect of salt. No effect of surfactant type and surfactant concentration on the gas hold-up pH and impeller clearance had no effect on stability. Type of surfactant, viscosity, addition of enzymes, polymers, nonionic surfactants, solvents and salts affect gas hold-up and stability MDtergitol<MDCTAB<MDDDBS. Increase of Csurf leads to decrease of MD. Addition of salts reduces the MD of ionic surfactants There is agreement between the predicted and observed rise velocities Stability of CGA depends on the Csurf and pH

MD, mean diameter; tstir, stirring time; Csurf, concentration of surfactant; Csalt, concentration of salt; T, temperature; rpm, stirring speed. SDS, sodium dodecyl sulfate; SDBS, sodium dodecylbenzenesulfonate; CTAB, cetyltrimethyl ammonium bromide; SLS, sodium lauryl sulfate; AOT, sodium bis (2-ethyl hexyl) sulfosuccinate. HTAC, hecadecyl trimethyl ammonium chloride; CPC, cetyl piridinium chloride; DTAC, dodecyltrimethylammonium chloride; CTAC, cetyltrimethylammonium chloride; DMDSAC, dimethyl distearyl ammonium chloride; TTAB, tetradecyl trimethyl ammonium bromide.


Table 10.1 Continued

Separation of value-added bioproducts by colloidal gas aphrons 289 surfactants and they developed a first order kinetics model for the drainage. Interestingly, drainage kinetics did not change when they used a mixture of an anionic and a cationic surfactant as opposed to conventional foams (Bhatia et al,. 2005). Yan et al (2005) developed a mathematical kinetic model by studying CGA generated using a range of concentrations of surfactants at varying temperatures. According to this model there were two distinct stages of CGA drainage (Yan et al., 2005). On the other hand, in a more recent study Moshkelani and Amiri (2008) measured the electrical conductivity of CGA dispersions and found that there are three separate stages in the liquid drainage of aphrons as opposed to the two stages in conventional foams (Moshkelani and Amiri, 2008). This was further supported in a more recent study by Feng et al. (2009) where the volume of drained liquid was measured and also photographs of the foams at different stages were taken. At the first stage they showed how drainage increases with time owing to a combination of upflow migration of bubbles and downward liquid drainage under gravity. Then, in the following stage, the dispersion behaves like conventional foams and, at the final stage, the foam becomes water deficient and begins to behave like dry foam. At this stage, the foam drainage is very slow owing to slow liquid release from films under capillary suction , i.e.: liquid is released from the film owing to lower pressure in the capillaries between the bubbles (Feng et al., 2009). Overall these studies support further the hypothesis that there are structural differences between CGA and conventional foams. 10.2.2 Characteristics of CGA Owing to their unique structure CGA possess the following important properties: ∑

∑

Higher stability than conventional foams. Owing to the multilayer structure, CGA exhibit high stability. When two aphrons collide, the momentum may not be enough to break the barrier of six surfactantstabilised interfaces, as opposed to the two surfactant-interfaces when two bubbles of conventional foams collide (Sebba, 1987). Stability is measured in terms of the liquid drainage rate, which is generally considered to follow first order kinetics (Bhatia et al., 2005) consequently, half-life (t) is defined as the time required for half the original volume of liquid to drain. CGA are also characterised in terms of gas hold-up (e), which is defined as the ratio between the gas volume (Vg) and the dispersion final volume after stopping stirring and at time = 0 of drainage (Va0): e = Vg/Va0

[10.1]

The buoyancy of the encapsulated gas leads to easy separation of the aphron phase from the bulk liquid phase. Therefore, no centrifuges are needed to separate the two phases. On the other hand, creaming can be


290 Separation, extraction and concentration processes avoided when necessary by stirring CGA at such a rate that the lateral movement conveyed to the bubbles is greater than the upward buoyancy owing to gravity. ∑ Adherence of particles and molecules to the encapsulating shell. It is possible to modify the surface properties of CGA by using different types of surfactant. Depending on the surfactant used to generate the CGA, the outer surface of the bubble can be negatively (anionic surfactant), positively (cationic surfactant) or non-charged (nonionic surfactant). Consequently, oppositely charged molecules adsorb. Thus, the selectivity of adsorption can be modified by changing the type of surfactant. This is illustrated in experiments carried out in our laboratory with two oppositely charged dyes, the cationic methylene blue, and the anionic methyl orange and CGA generated by the cationic surfactant CTAB (see Plate I, between pages 292 and 293). CGA are contacted with the aqueous solution containing both dyes and then the dispersion is allowed to settle so it separates into two phases (Plate Ic). The top CGA phase contains mainly methyl orange (orange colour top phase) and the liquid phase contains mainly methylene blue (blue colour liquid phase). Basu and Malpani (2001) also reported the effective separation of these dyes using CGA generated by CTAB. ∑ Low viscosity of the system. Flow properties are similar to those of water, as long as the gas hold-up does not exceed 65% (Roy et al., 1995), regardless of the type of the surfactant used to generate them. Consequently, CGA can be pumped easily from the generation point to the point where they are going to be used, whereas, for conventional foams, their characteristics change during pumping owing to the elastic nature of the bubble. This is advantageous when applied to a flotation column for fractionation and flotation in the removal and/or recovery of products. Several researchers have investigated the rheology of CGA, as shown in Table 10.1 (Larmignat et al., 2008; Tseng et al., 2006; Zhao et al., 2009). An interesting application which takes advantage of CGA rheological properties is described in a patent by Brookey (2004) where CGA are used to produce fluids with improved shear thinning properties, and this has led to a successful industrial application for well-drilling fluids by MASI technologies LLC. ∑ Small size of bubbles, resulting in larger interfacial area per unit volume, and thus a large capacity for adsorption of molecules and fine particles. Bubble size depends on the concentration and type of surfactant, ionic strength and the presence of other molecules or particles. Jauregi (1997) found that bubble size of AOT-generated CGA increased with surfactant concentration, ionic strength and stirring time up to 10 min. Size distribution of CGA generated by selected surfactants can be seen in Table 10.2.


Separation of value-added bioproducts by colloidal gas aphrons 291 Table 10.2 Characteristics of CGA generated by ionic and nonionic surfactants (values found at optimum conditions) Surfactant

cmc (mM)

Gas hold-up

Half life (s)

CTAB (c)

0.9 (in H2O)

0.70 1 mm) and natural astaxanthin ( 1.7) leads to a further advantage in terms of cheese yield. A distinction must be made between retentates concentrated to an intermediate level (1.7 < VRF < 5) in which some serum proteins are retained and syneresis still takes place, and concentration to the final composition of the cheese (VRF ~5-7), also called pre-cheeses, where the protein content is similar to the protein content of the final cheese and very little whey drainage is observed (Fig. 12.5). Intermediate concentrated retentates have been applied to numerous cheese varieties, ranging from soft to hard. Feta cheese manufacture, which is the main industrial application of this operation, yield increases of 14% while maintaining good quality products. The pre-cheeses concept was first applied to camembert cheese but many applications have been developed successfully for the manufacture of other cheese varieties, mainly fresh unripened cheeses such as quarg, ricotta and cream cheese, and soft and semi-hard cheeses including mozarella, Saint Paulin and feta, the manufacture of which being unquestionably the greatest success worldwide of the MMV process. The advantages of the MMV process are numerous. The overall cheese yield is about 10–30% higher than in the traditional process owing mainly to the retention of serum proteins and enzyme usage is generally reduced. The MMV process eliminates the need for the large storage tanks traditionally used for heating and cooking the curds, resulting in a saving in both capital investment and energy costs. In addition, it is possible to use the MMV process to convert much of the cheese production in a continuous operation, leading to significant advantages in terms of overall capital costs and operational efficiency. However, during UF, the concentration of serum proteins, with higher water-binding capacities than caseins, and of minerals associated with the casein micelles, that are lost in the whey during coagulation in a conventional process, led to the inherent properties of UF milk cheeses. Thus, rather than duplicating traditional cheese varieties, which requires numerous adaptations (Mistry and Maubois, 2004), UF technology leads to the development of new types of cheeses, well-accepted by consumers. For fifteen years, manufacturing technologies including UF have also emerged and have become important for the production of milk protein concentrates, presenting interesting new technical possibilities in cheesemaking. These concentrates contain both major milk protein groups in proportions similar to milk and are often dried. Because there are no specific standards


354 Separation, extraction and concentration processes of identity for such concentrates, they cover a wide range of compositional and functional characteristics and are manufactured mainly using UF and diafiltration, for the reduction of the lactose content. Milk MF has also recently emerged for the production of protein concentrates with various casein–serum proteins ratio (12.5.2).

12.5 Isolation of whole casein in the dairy industry The characteristics of the two groups of milk proteins (casein and serum proteins) differ significantly, permitting their ready separation from each other by at least three techniques. 12.5.1 Isoelectric precipitation and rennet coagulation There are two principal ways of manufacturing casein on industrial scale: isoelectric precipitation and rennet coagulation, both based on the precipitation/ aggregation of casein. In isoelectric precipitation, destabilization of the casein micelles can be accomplished at pH around 4.6: when milk is acidified, the net charge of the micelles decreases, the calcium and phosphate are removed and the micelles become less and less stable until caseins precipitate. Acidification of the milk may be carried out by one of the following processes: – Inoculation of milk with a mixed or multiple defined strain starters, such as lactic acid-producing bacteria, which degrade some of the lactose to lactic acid during the period of incubation (about 14–18 h). – Direct addition of dilute mineral acid to the milk. Adding acid (HCl or H2SO4) to milk heated at 50 °C results in a casein curd (‘acid casein’) that can be separated by straining and washing several times. The coproduct, which is a mixture of acid whey and part of the washing water, has a high ash and chloride content (where HCl is used), which poses some difficulties during spray-drying. Acid destabilization of casein is exploited in the manufacture of cottage cheese, quarg and fermented milks. – Indirect acidification of the milk. A number of alternative processes have been proposed and patented to improve the quality of the acid whey. The acidification can be performed by contact of all or part of the milk with cation-exchange resins (strong hydrogen ion exchange resin), electrodialysis (Noël, 1992) or using carbon dioxide (Hofland et al., 2003). It is believed that these processes have relatively low commercial significance (Maubois and Ollivier, 1997). Regardless of the processes used, isoelectric caseins may be dried and used as such but they are insoluble in water, which limits their applications. Therefore caseins are usually converted to a soluble form, namely caseinate, © Woodhead Publishing Limited, 2010

Separation technologies in dairy and egg processing 355 by neutralization with alkali, usually NaOH (producing sodium caseinate) and less frequently KOH, NH4OH or CaOH2. Soluble caseinates may then be sprayed or roller dried. Destabilization of casein can also be caused by the proteolysis of the micelle stabilizing protein, namely k-casein. The chymosin, the principal enzyme present in calf rennet, releases the glycomacropeptide (C-terminal segments of glycosylated k-casein), and renders the casein micelles susceptible to precipitation with Ca2+ at the natural concentration in milk. This principle is used in the manufacture of rennet casein and most cheese varieties. 12.5.2 Microfiltration for fractionation of casein micelles and serum proteins The most promising technology for the selective separation of casein micelles is undoubtedly membrane MF using a membrane with a pore size of 0.1–0.2 mm. For the past 15 years, this operation has enjoyed rapid industrial development and is still expected to increase significantly over the next few years owing to the high quality and properties of the two fractions produced: – The retentate, enriched specifically in native casein micelles, improves the rennet coagulability in the cheesemaking process. Such casein enrichment reduces rennet coagulation time, accelerates curd firmness kinetics and increases final curd firmness compared with milk or caseinate. Consequently, casein and fat retentions into the cheese curd are significantly improved (less fines and fat in the drained whey) and that leads to an increase in cheese yield. The fact that a smaller amount of serum proteins end up in the cheese manufacturing process is also advantageous, because there are then fewer flavour and texture defects attributed to serum proteins and also fewer detrimental effects of heat treatment on rennet milk coagulability. The decrease in b-lactoglobulin (b-LG) content evidently lowers the extent of the formation of the complex k-casein/b-LG during heat treatment and, consequently, renneted micelles aggregate well. This property was used to develop a new high-heat milk powder, with a cheesemaking ability similar to that of raw milk (Fig. 12.6, Quiblier et al., 1991). – The permeate, with a composition close to that of a whey, contains soluble proteins in their native state, is sterile, and free of phage particles, cellular debris, fat and glycomacropeptide. It therefore becomes a useful fluid for preparing whey protein concentrates (WPC) and isolates (WPI) (protein ratio up to 97%) with very high functional and nutritional properties, in particular compared with WPC obtained from cheese whey (Foegeding et al., 2009). Despite these advantages, the microfiltrate is sometimes used in the cheesemaking process by reincorporation in the caseinenriched fraction after denaturation under moderate concentration and heat treatment (Fig. 12.6).


356 Separation, extraction and concentration processes Skimmed milk MF 0.1 mm VRF = 3 Microfiltrate

Retentate Cream

UF 10–20 kg mol–1 VRF = 6

Cheese

Permeate

Concentration spray drying HT (95 °C, few minutes) Retentate WPC

Milk powder b-Lactoglobulin High cheese-making ability

Fig. 12.6 Primin process (Quiblier et al., 1991). VRF, volume reduction factor; WPC, whey protein concentrates; HT, heat treatment.

Finally, purified casein micelles obtained by diafiltration of retentate against water, and WPI obtained after UF of milk microfiltrate, are excellent starting fluids for further fractionation of individual caseins or serum proteins. From an engineering point of view, this separation is classically conducted using ceramic membrane either with the UTP system or membranes with linear hydraulic resistance gradient, as is done for bacterial removal (12.3.2). Separation is usually performed at 50–55 °C, at a crossflow velocity of 7 m s–1, TMP ~ 50 kPa and VRF of 2–4. At VRF = 3, serum proteins transmission is 65–80% depending on the milk heat treatment (pasteurization, thermalization or raw skimmed milk). Permeation flux is ª75 L h–1 m–2 for 10 h according to the critical stability criterion, which allows the industry to perform long runs with very moderate fouling and high selectivity (Gésan-Guiziou et al., 1999). Owing to the high running costs and investment required by the tubular ceramic equipment, the industry is starting to operate MF with spiral-wound polymer membranes. These membranes, which can not be used with the UTP system, are operated at low temperature (90 °C and pH 90% w/w) in the desired phosphopeptides. The glycomacropeptide, the C-terminal part of the k-casein released in whey by the action of chymosin was shown to be separated from sodium caseinate using UF membrane or centrifugation. This peptide has numerous uses, such as action on satiety and inhibition of Escherichia coli cells adhesion to intestinal walls and, in particular, it contains no phenylalanine, which makes it suitable for use as a nutritional protein supplement for patients suffering from phenylketonuria, who cannot digest protein containing phenylalanine owing to their lack of the appropriate degrading enzyme. A number of dairy starter cultures, as well as proteolytic enzymes isolated from lactic acid bacteria also lead to the formation of bioactive peptides from milk proteins, in particular during the manufacture of fermented dairy products. For example, several studies have demonstrated that Lactobacillus helveticus strains are capable of releasing antihypertensive peptides. A UHT milk fermented by the GG strain of Lb. rhamnosus and subsequently digested with pepsin and trypsin produced hydrolysate fractions that were immunosuppressive. The occurrence of many bioactive peptides in bovine milk is now well established (Korhonen, 2009), but currently the industrial-scale production of such peptides is limited by a lack of suitable separation technologies. Among them, membrane techniques, such as NF or UF, are used industrially to produce ingredients that contain bioactive peptides based on casein or whey protein hydrolysates (Table 12.7) and seem to be the best technology available for the enrichment of bioactive peptides.

12.8 Treatment of effluents and technical fluids in the dairy industry In many countries, the dairy industry is considered to be one of the largest generators of food-processing wastes. Owing to large water volumes for factory cleaning and disinfection, the dairy industry produces 0.2–11.0 L effluents/L of processed milk. Cleaning-in-place (CIP) operations significantly contribute to water consumption and are responsible for 50 to 95% of the overall volume of waste streams (Daufin et al., 2000, Alvarez et al., 2007). © Woodhead Publishing Limited, 2010

Separation technologies in dairy and egg processing 367 The polluting load of these effluents ranges from 0.2 to 5.0 g L–1 of chemical oxygen demand (COD), and is mainly caused by loss of raw material (0.5 to 2% of milk). CIP systems are also mainly responsible for the high pH value (9–11) of the end-of-pipe wastewaters. Although a high portion of dairy wastewaters is still land spread, the treatment of the effluents mainly takes place in biological treatment plants at most dairies. For a factory of average capacity (106 L of milk a day), the sludge produced is usually used for land spreading (1 to 3 tonne of dry matter) and purified water drained to rivers (0.3 ¥ 106 to 3 ¥ 106 L). Current European regulations relating to landfill management, land spreading and purified water quality along with social pressure, has recently forced the dairy industry to significantly reduce its production of sludge and to improve purified water quality. Over the past few years, the dairy industry has been attempting to find new concentration and separation processes to reduce its effluents production. Several types of effluents are currently treated in the industry, benefiting mainly from the potential of membrane technology: washing waters of rennet casein precipitate were treated, respectively, using NF and dead-end filtration; white (flushing) and pre-rinsing waters, corresponding to the first step of CIP, were treated using UF, NF or RO (Blanchard, 1991; Delbecke, 1981). The outcome is a highly significant improvement of water quality after treatment [suspended solids (SS) < 2 mg L–1; chemical oxygen demand (COD) 80% recovery at a 200 cm h–1 flow rate. Billakanti and Fee (2009) characterized a cryogel monolith chromatography for extraction of minor proteins (LF) by cation-exchange chromatography from whole milk feeds at 550 cm h–1 in a single-step. Yield and purity of LF extracted using this process were >85 and 90%, respectively. Wu and Xu (2009) reported a purification process for isolation of LF and IgG from bovine colostrum using a serial cation–anion exchange chromatography system with 95 and 97% final purities, respectively. Lu et al. (2007) designed a productionscale technology for extraction of LF from bovine colostrum with final yields of 83% and purities of 94% using an ultrafiltration system coupled with a fast-flow strong cation-exchange chromatography column. Fee and Chand (2006) have investigated and optimized a process to isolate LF and LP from


Table 16.6 Author and year

Purification examples for the basic whey proteins Configuration/material

Tu et al. 2002 Anti-LF immunoglobulins (IgYLF) immobilized on Sepharose 4B support Ye et al. 2000 Carboxymethyl cation-exchange chromatography

Target protein

Protein source Description of study/results

LF

Methods for purification of dairy nutraceuticals 465


Colostral whey Final products have purification folds of 103–136 of initial concentration and recoveries of 82–99% Bovine They performed the adsorption process using 50 mM LFa and LFb colostrum phosphate buffer at pH 7.7 LFa was eluted in 0.38 M NaCl and LFb at 0.43 M NaCl buffer solutions. Also investigated variation in the LF concentrations of lactation time and species Li-Chan et al. Immobilized yolk antibody on a LF Milk and Binding capacity was a function of ligand density: at 1998 monoaldehyde-activated support cheese whey 9.2 mg mL–1 of IgYLF, binding capacity was 20% (mol %). This could be increased to 80% by using low ionic strength buffers Camperi et al. Red HE-3B dye affinity chromatography LF Rennet whey This process was achieved with 82% recovery and 98% 2000 purity Jyh-Ping and Microfiltration affinity purification LF and IgG Cheese whey Maximum binding capacity on heparin-Sepharose gel Cheng-Hsin was 124 mg mL–1. LF recovered in this process has 92% activity and 95% purity whereas IgG has 86% activity 1991 and 90% purity Korhonen Review Igs Bovine The latest developments and progress in separation 2004 colostrum, technologies for isolation of Igs in both small and largewhey and milk scale applications Stec et al. Isolation of polyclonal Igs by HPLC Igs Bovine serum Ammonium sulfate precipitation was performed as 2004 a first step of Igs isolation followed by HPLC. This method is applicable for preparative-scale applications DeSilva et al. Conference paper. Novel approaches Whey Dairy whey Recent developments in isolation and purification 2003 to meet the challenges in processing proteins and technologies of various proteins and peptides functional dairy components peptides

Author and year

Continued


Target protein


Xu et al. 2000 Separation of IgG and glycomacropeptides using a polystyrene anion exchanger IRA93 and Amicon YM100 membrane

IgG

Dairy whey

Akita and LiChan 1998

IgG1 and IgG2

Kim and LiChan 1998

Configuration/material

Specific antibody-immobilized immunoaffinity chromatography for isolation of bovine immunoglobulin G subclasses Single-step process for isolation of IgG using avidin-biotinylated IgY chromatography

Konecny et al. Purification of monospecific polyclonal 1994 antibodies from hyper immune bovine whey using immunoaffinity chromatography Labrou and Review Clonis 1994

IgG

Igs

Igs

Successful for concentration of IgG while removing all major whey protein contaminates. A continuous process using IRA 93 resin and 100 kD membranes, designed to produce enriched IgG GMP and WPI ingredients from rennet dairy whey Bovine milk, Gave >98% purity of IgG subclasses. Binding capacities colostrum and of the column were 27 and 38% (molar masses) for whey IgG1 and IgG2, respectively. Antibody columns were stable for more than a year with minimal antibody loss Cheddar Binding capacities achieved in this investigation were cheese whey 50–55% (w/w of IgG and IgY ligands). Bound IgG was eluted at pH 2.8 using glycine–HCl buffer with 99% purity Cheese whey

Various modes of affinity adsorption technologies used for isolation of Igs both in laboratory- and industrialscale applications


Table 16.6

Methods for purification of dairy nutraceuticals 467 whole milk at milking pH and temperatures using SP Sepharose Big Beads as a packed chromatographic resin. In their investigation, they successfully captured both LP and LF (total dynamic capacity of 49 mg mL–1 resin) from raw milk at 300 cm h–1 flow rate with minor leakage (500 cm h–1). Fee and Chand (2005) reported on-farm capture of high-value minor milk proteins (LF and LP) from the raw milk of individual animals using a fully automated protein fractionation robotic system. This method was rapid, used a single step and was designed to minimize microbial contamination, while offering full product traceability to individual animals. Claycomb (2004) holds a patent for on-farm fractionation of high value milk proteins directly from raw milk using an automated milking system (AMS). Dionysius (1991) investigated a CM-Sephadex resin in a stirred tank system, mixing the resin with whey (cheese, rennet and acid) for 60 min at pH 7.0, and captured more than 80% of LP and 90% of LF, although the resin capacity changed with nature of whey. The bound LF was subsequently eluted by NaCl gradient elution. Heeboll-Nielsen et al. (2004) developed a superparamagnetic cation-


468 Separation, extraction and concentration processes exchange chromatographic process for fractionation of bovine whey proteins including LF and LP with 28-fold purification over the starting material. The resin could be recovered magnetically in a high-gradient magnetic fishing system, allowing the resin size to be minimized for fast adsorption, while being recovered without the need for fine particle filtration, settling or centrifugation. Ulber et al. (2001) described a two-step membrane processing method for separation of LF from sweet whey, the first membrane removing lipids and other solids and the permeate then passing through a cation exchange membrane adsorber. Plate et al. (2006) used 2-m2 membrane modules for preparative-scale isolation of LF and LP from sweet whey with >90% recovery yields and purities. Chiu and Etzel (1997) described a method for extraction of LF and LP from cheese whey using microporous (3–5 mm) cationic membranes and achieved 50 and 70% recoveries, respectively. Mitchell et al. (1994) used cation-exchange membranes operated in either dead end or cross-flow configuration to isolate LF and LP from cheese whey. Affinity chromatography Affinity chromatography is the second most popular chromatography method for isolation of minor milk proteins from complex protein mixtures on a laboratory scale. Wolman et al. (2007) used Red HE-3B dye coupled with hollow fibers as a membrane affinity chromatography matrix for isolation of LF in a single step, achieving a higher adsorption capacity (111.0 mg mL–1) than the same ligand immobilized on agarose beads (9.3 mg mL–1 resins). They could achieve >94% purity and 91% recovery in the final product using whey and colostrum as feed materials. Chen et al. (2007) evaluated micrometre-sized monodisperse superparamagnetic polyglycidyl methacrylate (PGMA) particles coupled with heparin (PGMA-heparin) as an affinity method for isolation of LF from bovine whey in a single step. This magnetic affinity method resulted in a very high binding capacity of 164 mg g –1 resin and may hold promise as a fast process tool for industrial-scale purification of high-purity LF, although one must always consider the potential for leaching of toxic compounds from affinity ligands. Noppe et al. (2006; 2007) described immobilized affinity peptides as a new platform for rapid development of alternative affinity chromatography ligands for isolation of target proteins from crude feeds. In their method development, they used selected bacteriophages immobilized on a microporous monolith column for extraction of LF from complex fluids such as milk and blood serum. Bound LF was eluted with 1 M NaCl and fractions were recovered with >95% purity. Kawakami et al. (1987) used an immobilized LF-monoclonal antibody resin for a single-step isolation of LF from both human and bovine skim milk and colostrum, with very high purities and recoveries of >97%. Unlike LF, isolation of LP has attracted limited attention because of its low concentration and low commercial value. Shin et al. (2001) used


Methods for purification of dairy nutraceuticals 469 immunoaffinity chromatography for selective isolation of LP from human whey but such processes have not been extensively studied for large-scale applications. Other chromatographic methods Size-exclusion (gel-filtration) chromatography is not commonly used as an early step in large-volume protein purification because linear velocities are low, but it is often used as a final purification step after other chromatography steps to enhance protein purity. For example, Al-Mashiki and Nakai (1987) applied size-exclusion chromatography for isolation of LF and immunoglobulins from different whey streams. Tomita et al. (2002) reported the possibility of LF and LP isolation from cheese whey or skim milk using a semi-large scale hydrophobic interaction chromatography method. Yoshida (1989) used hydrophobic interaction chromatography followed by DEAE ion exchange chromatography to isolate LF and LP from acid whey. Non-chromatographic methods Noh et al. (2005) selectively purified LF using a cationic surfactant as a micelle foaming agent, manipulating the protein behaviour by changing pH and salt concentration in the aqueous phase and surfactant concentration in the organic phase. LF was partitioned into the aqueous phase whereas all other proteins were solubilized into the organic phase. Fuda (2004) obtained approximately 25-fold enrichment of LF and LP from sweet whey using colloidal aphrons (CGAs), which are surfactant microbubbles generated by intense stirring of the anionic surfactant sodium bis-2ethylhexyl sulfosuccinate (AOT). Noel et al. (2001) investigated a low-cost foam fractionation process as the first step in separating LF from milk and achieved approximately 40% mass recovery at pH 10. Similarly, Saleh and Hossain (2001) developed a semi-batch foaming process to separate LF from a multicomponent mixture of BSA, a-Lac and LF by utilizing protein surface active properties. 16.5.2 Lysozyme purification Owing to its high isoelectric point (pI>11), lysozyme typically co-elutes with other basic proteins (LF and LP), but there have been few studies of the isolation of this protein. However, lysozyme has been successfully purified from milk or acid whey by a combination of affinity chromatography on heparin-Sepharose, followed by gel filtration on Sepharose 4B (Wang and Kloer 1984) or Sephadex-G50 (Boesman-Finkelstein and Finkelstein 1982). Duhaiman (1988) used heparin–Sepharose 4B, Sephadex G-75 and hydroxyapatite for chromatographic extraction of lysozyme from camel milk. Recently, using recombinant technology, human lysozyme was expressed at high concentrations (0.5% dry weight) in transgenic rice seed (Huang et


470 Separation, extraction and concentration processes al. 2002). Wilken and Nikolov (2006) found cation-exchange purification of lysozyme to be optimal at pH 4.5.

16.6 Immunoglobulins in the dairy nutraceutical industry IgG is an important component of bovine milk and comprises the major protein in bovine colostrum. Consumer acceptance of value-added whey protein products that involve Igs began in Asia and migrated to Europe and the US, targeting sports nutrition, infant formulae, dietary supplements and physiologically functional foods (Gapper et al. 2007). Immunoglobulins are g-globulin proteins that are found in blood and other body fluids of human, bovine and all other lactating species. These are used in the immune system to identify and neutralize bacteria, viruses and other antigens. Human immunoglobulins are broadly classified into five different classes: IgA, IgD, IgE, IgG, and IgM, whereas bovine milk and colostrum mainly contain immunoglobulins IgG, IgM and IgA. All immunoglobulin classes share a basic structural unit that comprises four polypeptide chains with two identical heavy (H) chains and two identical light (L) chains, held together with disulfide bonds. Molecular masses of the heavy- and light-chain peptides are approximately 50–70 kDa and 25 kDa, respectively. 16.6.1 Immunoglobulin G Immunoglobulin G is the most abundant class of antibody in the colostrum and milk of several mammalian species, comprising 80–90% of total antibodies. The primary structure of IgG contains two heavy (each of 450–550 amino acids) and two light chains (each of 211–217 amino acids) with a total molecular mass of approximately 150–160 kDa and a pI of 5.5–8.3. Commonly, IgG presents in two-sub classes such as IgG1 (MW 160 kDa, pI 5.5–6.8) being the richest class, at 15–180 g L–1, and IgG2 (MW 150 kDa, pI 7.5–8.3), at 1–3 g L–1. 16.6.2 Immunoglobulin A Similar to other immunoglobulins, the monomeric structure of IgA also contains two heavy and two light chains but it occurs as a monomer or a dimer, the latter joined by a J-chain and a secretory component. This complex structure is called secretory IgA (sIgA) and has a molecular weight of 380–417 kDa. IgA is present in blood, milk and several mucosal surfaces of lungs and gastrointestinal tracts. This antibody acts as a primary defence system against several pathogens. The IgA concentration in bovine colostrum ranges from 1 to 5 g L–1, whereas bovine milk contains only 0.05 to 1 g L–1.


Methods for purification of dairy nutraceuticals 471 16.6.3 Immunoglobulin M The monomeric structure of IgM is similar to IgG but IgM consist of five subunits, linked together to form a pentameric structure in a circular mode by disulfide bonds and a J-chain. The molecular mass of IgM is approximately 900–1000 kDa. In bovine milk, the IgM concentration ranges from 0.04 to 1.0 g L–1, whereas bovine colostrum contains 5 to 10 times greater amounts.

16.7 Purification technologies for immunoglobulins in the dairy nutraceutical industry Immunoglobulins from bovine milk have applications as supplements in infant formulae, hyperimmune foods, functional foods, nutraceutical and pharmaceutical products. Although there are many processing methods available for separation of immunoglobulins from various sources, including colostrum and milk, appropriate technologies for large-scale productions are still lacking. Traditionally, isolation of Igs from colostral whey has been achieved by precipitation with either ammonium sulfate or ethanol, followed by chromatography. Although such methods yield rather pure IgG fractions, most are feasible only in small-scale applications. In particular, they are not appropriate for large-scale production of Igs from bovine colostrum or milk (El-Loly 2007). Because milk is a complex fluid and contains high amounts of fat, it is difficult to isolate Igs using conventional methods. However, rapid developments in separation technologies and particularly in the application of membrane separation methods have made industrial-scale isolation of immunoglobulins from various streams, including whey, colostrum and recombinant cell culture supernatants a possibility. 16.7.1 Ion exchange chromatography Use of ion-exchange chromatography to isolate immunoglobulins from dairy feeds is limited but it is used as an intermediate or final purification step in combination with other chromatographic or membrane processing methods. For example, Wu and Xu (2009) applied cation- and anion-exchange chromatography in series for isolation of IgG and LF from bovine colostral whey. Similarly, Pessela et al. (2006) investigated a simple method for isolation of Igs from whey protein concentrate with >80% recovery and reasonable purity using a combination of DEAE–agarose anion-exchange chromatography followed by low-substitution aminated adsorbents. In the first step, they could remove the main contaminant, BSA, and during the second step, Igs were selectively bound on the aminated resin with minor contamination by a-Lac and b-Lac. Noel (2007) developed a second-generation, robust EBA method for


472 Separation, extraction and concentration processes isolation of Igs from cheese whey, which involved a mixed-mode chemical ligand that has selectivity towards IgG over other whey proteins. The feed whey solution was heated to 50 °C to maintain pasteurization, reduce the viscosity of the solution and maximize flow rate. They could thus load the column at greater than 3000 cm h–1 flow rate without reducing its adsorption capacity. Using a 880-L EBA column, they could produce 13 kg of IgG per day with >90% of final product yields. The eluent from this column was passed through a 160 L anion exchange column for further purification. 16.7.2 Membrane chromatography Since membrane technology was introduced to the dairy industry in the 1970s, many membrane processes have been developed for isolation of Igs from whey and milk using either a single-step process or a combination of several individual steps. For example, UF has been employed either alone or in combination with ion-exchange or gel-filtration chromatography for largescale fractionation of Igs from cheese or colostral whey streams (Abraham 1988; Korhonen 2004; Syväoja et al. 1994). Al-Mashikhi et al. (1988) successfully isolated IgG from acid and cheddar whey using UF, combined with immobilized metal chelate affinity chromatography, obtaining purities of 77 and 53%, respectively. Korhonen (1998) used a multi-step process with UF, microfiltration and reverse osmosis, followed by a cation-exchange resin to achieve 40–75% enrichment of Igs from colostral whey. 16.7.3 Affinity chromatography Affinity adsorption methods have been widely used for isolation of Igs from colostral or cheese whey. Protein-A and Protein-G affinity media are well characterized and used for fractionation of Igs, particularly IgG. Many pilot- and industrial-scale methods using membrane technologies and affinity chromatography have been developed for extraction of Igs from whey and colostrum either alone or in combination with other chromatography techniques (Kochan et al. 1996; Mukkur and Froese 1971; Schmerr et al. 1985). Labrou and Clonis (1994) reviewed various affinity-based purification technologies for Igs. Human and secretory IgA molecules have been isolated by precipitation, ion-exchange and affinity chromatography from human serum (Kondoh et al. 1987; Monteiro et al. 1985; Roque-Barreira et al. 1986) and human milk (Khayam-Bashi et al. 1977). Hutchens et al. (1990) developed a salt-promoted thiophilic adsorption method for simultaneous isolation of all bovine Igs from colostral whey. Mukker and Froese (1971) described isolation of bovine IgM from colostral whey using a combination of molecular sieve and DEAE–cellulose chromatography. Su and Chiang (2003) described a simple non-chromatographic method for isolation of Igs from other whey proteins. They used reverse micelles


Methods for purification of dairy nutraceuticals 473 to extract more than 90% of IgG into an aqueous phase, with 90% purity, whereas all other whey proteins were partitioned into the reverse micelle phase. 16.7.4 Protein-A mimetic (PAM) affinity peptides Protein A and protein G affinity chromatography can achieve capacities of up to 30–40 mg IgG per mL of resin under optimal conditions. However, these affinity ligands suffer from several inherent drawbacks such as high cost, their biological origin, possible contaminants, low stability towards sanitizing agents and the fact that their selectivity is limited to IgG. Recently, with the help of combinatorial chemistry and modern analytical tools, researchers synthesized protein A mimetic (PAM) peptide ligands (D’Agostino et al. 2008; Fassina 1994; Newcombe et al. 2005; Ulbricht and Yang 2005; Yang et al. 2005) for isolation of human immunoglobulins from mammalian cell cultures. Compared with protein A or G, these synthetic peptide ligands have several benefits (D’Agostino et al. 2008), including high stability towards chemical and biological reagents, low production cost and the absence of biological contaminants. Moreover, these synthetic affinity ligands may also have selectivity towards other classes of immunoglobulins (IgG, IgE, IgM and IgA) (Fassina et al. 1998; Yang et al. 2005), which are not recognized by conventional protein A or G ligands. Several synthetic peptides have been proposed as potential replacements for protein A in affinity chromatography (Ulbricht and Yang 2005). However, the lack of selectivity for antibodies from various classes has limited their widespread use and some of these ligands are not compatible with the retention of antibody activities in proposed buffer solutions. However, more recently, Yang et al. (2005) synthesized a hexamer peptide that has a wide-ranging selectivity towards various classes of immunoglobulins at near-neutral pH. By using a gradient elution with 0.2 M sodium acetate buffer, they were able to isolate various classes of immunoglobulins from human serum (Yang et al. 2005). They have also reported the specificity of this ligand towards immunoglobulins of human, bovine and several other species. Although most of this work has been carried out on human immunoglobulins, there has been recent interest in applying this PAM peptide affinity chromatography for isolation of bovine immunoglobulins from early colostral milk, hyperimmunized cows and transgenic animals.

16.8 Future trends In the past few decades, the dairy industry has moved from being solely a commodity food-based industry producing significant waste (whey) volumes to one that has increasingly sought added value and the minimization of waste. The nature of dairy products has expanded from simple nutritional © Woodhead Publishing Limited, 2010

474 Separation, extraction and concentration processes foods to separate fractions sold as individual ingredients or as blends to meet the specific functional and nutritional requirements of secondary producers. This trend will continue, as milk volumes increase to meet the new demand for dairy products from the developing world and greater bulk production efficiencies drive companies to find new products to maintain their competitive advantages. Nutraceutical products, with implicit claims of health benefits, are now commonplace and there are likely to be more such products in the future, with some fractions in the future perhaps even meeting the more stringent proof of efficacy requirements of pharmaceuticals for highly targeted applications, for example around neonatal gut development, anticancer agents or immune system enhancers. The commercial recovery of very minor fractions with useful bioactivities, such as oligosaccharides (including those incorporating sialic acid), the myriad proteins that make up the milk fat globule membrane, and the complex lipids therein (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and glycolipids such as gangliosides) will be a challenge. Recovery of such fractions may require specific affinity techniques and the challenge will be to use techniques to extract minor proteins economically on a large scale, while maintaining the safety and integrity of the residual milk or whey for further processing i.e. without contaminating the flow-through stream with leachates. On-farm extraction of minor products from milk may offer advantages in the production of proteins and other bioactives that are heat-labile and are currently largely lost during the pasteurization required inside the factory gate. On-farm extraction may also offer direct benefit to farmers who select for certain genetic traits or apply unique feeding regimes to enhance the production of specific high-value products in their herds. In summary, higher volumes of milk production and increased global competition in the dairy industry will probably drive further growth in niche bioactive products over the next few decades. High-volume, economically efficient separation technologies with high specificity and compatibility with food production will be required to support this growth.

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Methods of concentration and purification of omega-3 fatty acids 483

17 Methods of concentration and purification of omega-3 fatty acids S. P. J. Namal Senanayake, Danisco USA, Inc., USA

Abstract: An overview is presented of the various methodologies used for producing highly purified omega-3 fatty acids from natural source materials. Omega-3 fatty acids derived from fish, krill and microalgae, consisting of eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3), have beneficial effects in the prevention and management of cardiovascular disease and other chronic disorders. Production of high-purity omega-3 fatty acids is increasingly important in both the nutraceutical and pharmaceutical industries. The physical, chemical and enzymatic methods used include urea adduction, chromatography, low-temperature fractional crystallization, supercritical fluid extraction and distillation. Key words: omega-3 fatty acids, urea adduction, chromatography, crystallization, supercritical fluid extraction, distillation, enzymatic methods.

17.1 Introduction Omega-3 fatty acids are essential fatty acids that have diverse biological effects in human health and disease. They are essential to human health but cannot be manufactured by the body. For this reason, omega-3 fatty acids must be obtained from food. Omega-3 polyunsaturated fatty acids (PUFAs) can be found in fish, such as salmon, tuna, and halibut, other marine organisms such as algae and krill, certain plants, and nut oils. The heart-health benefits of the omega-3 fatty acids are well known. Omega-3 fatty acids are considered to have beneficial effects in the prevention of cardiovascular disease, inflammation, hypertension as well as other chronic disorders (Kris-Etherton et al., 2001; Lands, 2003; Senanayake, 2000; Senanayake and Shahidi, 2000b; Shahidi and Senanayake, 2006; Yokoyama et al. 2007). In 2004, the US Food and © Woodhead Publishing Limited, 2010

484 Separation, extraction and concentration processes Drug Administration gave ‘qualified health claim’ status to eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) found in fish, stating that ‘supportive but not conclusive research shows that consumption of EPA and DHA omega-3 fatty acids may reduce the risk of coronary heart disease’ (United States Food and Drug Administration, 2004). For clinical and nutritional applications, the natural sources of omega-3 fatty acids, as such, may not provide the required amounts of these fatty acids and hence concentration and purification of omega-3 fatty acids may be necessary. A concentrated source of these fatty acids is desired to achieve sustainable benefits. Highly purified omega-3 fatty acids may be produced in the free fatty acid, simple alkyl ester and triacylglycerol forms. To achieve this, physical, chemical and enzymatic techniques may be employed. Methods traditionally employed for the concentration and purification of omega-3 fatty acids include urea adduction, chromatographic methods, low-temperature fractional crystallization, supercritical fluid extraction, distillation, and enzymatic and integrated methods.

17.2 Urea adduction in the concentration and purification of omega-3 fatty acids Urea adduction is one of the most efficient and simplest techniques for concentration and purification of omega-3 fatty acids from natural sources (Senanayake, 2000). The formation of complexes between urea and straightchain saturated fatty acids is a well established and potentially valuable separation technique for fractionation of free fatty acids or esters (Shahidi and Senanayake, 2006). Initially, the triacylglycerols (TAGs) of oil are hydrolyzed into their constituent fatty acids via alkaline hydrolysis using alcoholic KOH or NaOH. The resultant free fatty acids (FFAs) are then mixed with an ethanolic solution of urea for complex formation. Urea molecules readily form solid-phase complexes with saturated and monounsaturated fatty acids and crystallize out on cooling and may be removed by filtration. The liquid fraction is highly enriched with omega-3 fatty acids. In general, the crystallization temperature can be ranged from ambient to –20 °C. Since the process involves relatively mild operating conditions and chemicals (free fatty acids, urea, ethanol, water) that are generally recognized as safe (GRAS) by the US Food and Drug Administration, it can be labeled as eco-friendly. This process can be used as a preliminary fractionation step in conjunction with other purification methods, such as low-temperature fractional crystallization, enzyme-catalyzed methods, and molecular distillation, resulting in a highpurity free-fatty-acid omega-3 product. Urea-based fractionation of fatty acids has been extensively used for enriching omega-3 fatty acids in marine oils (Hayes et al., 2000). Urea adduction of fatty acid ethyl esters of squid (Illex argentinus) visceral oil,



using cyclohexane and methanol, can increase the EPA content from 11.8 to 28.2% and DHA content from 14.9 to 35.6% (Hwang and Liang, 2001). Haagsma et al. (1982) described a urea adduction method for enriching the EPA and DHA levels of cod liver oil from 12 to 28 and 11 to 45%, respectively. Wanasundara and Shahidi (1999) used an experimental design to optimize the conditions on a laboratory scale that led to the maximum concentration of EPA and DHA from seal blubber oil, using the urea-based fractionation. The authors obtained 88.2% of total omega-3 fatty acids at an urea/fatty acid ratio of 4.5, a crystallization time of 24 h and a crystallization temperature of –10 °C. Senanayake and Shahidi (2000a) concentrated and purified DHA from the oil extracted from the microalgae Crypthecodinium cohnii and reported a DHA enrichment from 47.4 to 97.1% with a process yield of 32.5% of the mass of the original algal oil. The fatty acid composition of algal oil and its DHA concentrate obtained by urea adduction are reported in Table 17.1 (Senanayake and Shahidi, 2000a). In another study, mackerel processing waste comprising skins, viscera, and muscle tissue was evaluated by Zuta et al. (2003) for concentrating omega-3 fatty acids by urea adduction. Fish oil was extracted using either chloroform/methanol (2:1 vol/vol) or hexane/isopropanol (3:2 vol/vol). The oil yield and iodine value (which measures the degree of unsaturation in fats and oils) were determined for fresh fish oil extracts. Omega-3 fatty acid concentrates were prepared from saponified fish oil via urea adduction. The mean oil yields were 9.18, 9.2, and 38.1% for viscera, muscle, and skin, respectively. The mean baseline iodine value was 134, which increased to 296 after urea adduction. Hence, it was possible to concentrate omega-3 fatty acids from mackerel processing waste. The type of tissue used did not affect the amount of omega-3 fatty acids concentrated. Mackerel skin was most desirable because of its high oil content. Hayes (2006) investigated the effect of cooling rate on the degree of removal of saturated acyl groups from FFAs derived from canola oil and Table 17.1 Fatty acid composition of algal oil and its DHA concentrate after urea adduction (adapted from Senanayake and Shahidi, 2000a) Fatty acids 10:0 12:0 14:0 16:0 16:1 18:1w-9 18:2 w-6 22:5 w-3 22:6 w-3 Iodine value, calculated

Algal oil (%) 0.6 1.1 15.0 9.0 2.2 19.0 1.0 0.5 47.4 234

DHA concentrate (%) 0.5 0.5 0.1 0.0 0.3 0.2 0.7 0.4 97.1 437


486 Separation, extraction and concentration processes the isolation of di- and polyunsaturated acyl groups from FFAs derived from vegetable and fish oil during urea adduction. Traditionally, slow cooling has been used (–1 °C min–1). A more rapid cooling rate (–47 °C min–1) produced urea inclusion compounds (crystals) of similar morphology and thermodynamic properties, but of a size an order of magnitude smaller than the urea inclusion compounds formed during slow cooling. Fractionations used only renewable materials (urea, FFAs, and 95% ethanol as solvent) and benign operating conditions (ambient pressure, 25–75 °C, and neutral pH). When the recovery of FFAs was relatively high (>60%), the selectivity of urea adduction toward the inclusion of saturated fatty acids and against PUFAs was not affected by the cooling rate. In contrast, when the FFAs recovery was low, representing those instances where an increase in the purity of the PUFAs is a more important economic goal, a slower cooling rate resulted in a significantly greater discrimination against PUFA groups, hence to a FFA product with a measurably greater purity.

17.3 Chromatographic methods for the concentration and purification of omega-3 fatty acids Another method for concentration and purification of omega-3 fatty acids is the use of chromatography. High-performance liquid chromatography, silver resin chromatography and supercritical-fluid chromatography have been used for concentration of omega-3 fatty acids from natural sources. Hayashi and Kishimura (1993) isolated 63–74% pure DHA from skipjack tuna eye orbital oil by stepwise elution with hexane, diethyl ether/hexane and diethyl ether on a silicic acid column. Teshima et al. (1978) employed a silver nitrate-impregnated silica gel column to separate DHA and EPA from squid-liver oil after forming methyl esters. They were able to isolate 95–98% DHA and 85–96% EPA with yields of 48 and 39%, respectively. Guil-Guerrero and Belarbi (2001) purified EPA and DHA from cod liver oil using a silver nitrate-impregnated silica gel column. The oil was saponified and treated with urea, and the non-complexed fatty acids were then converted to methyl esters before chromatography. The column was washed with a sequence of mobile phases. A 64% recovery of DHA with 100% purity was obtained. The recovery of EPA was 29.6%, with a final purity of 90.6%. Perretti et al. (2007) investigated the fractionation of fish oil fatty acid ethyl esters with the aim of obtaining a lipid fraction enriched in omega-3 fatty acids and with a suitable EPA/DHA ratio. They reported the possibility of modifying the original fatty acid ethyl ester concentrations by optimizing the extraction conditions in terms of pressure, temperature, and supercritical CO2 flow rate: 2-h runs, pressures of 100, 140, 150, and 300 bar, and liquid CO2 flow rates of 2.5, 3.5, 5, and 10 kg h–1, at 40, 50, and 60 °C, in the three sections of the column starting from the bottom, respectively. They



stated that supercritical fluid fractionation appears to be a useful processing technique for changing the composition of lipids in order to obtain high added-value functional products. A preparative reversed-phase high-performance liquid chromatography method with gradient elution using acetonitrile–chloroform and evaporative light-scattering detection was used by Mansour (2005) to purify milligram quantities of microalgal PUFAs, separated as methyl esters. PUFA-methyl esters purified included methyl esters of DHA, EPA and the unusual very-longchain highly unsaturated fatty acid, octacosaoctaenoic acid (28:8n-3) from the marine dinoflagellate, Scrippsiella sp. Other fatty acids purified from various microalgae using this method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-methyl esters, and resolution from closely eluting PUFA-methyl esters, which determined the maximum loading. The purity of these fatty acids was determined by electron-impact gas chromatography–mass spectrometry (EI GC–MS) and the chain length and location of double bonds were determined by EI GC– MS of 4,4-dimethyloxazoline derivatives formed using a low-temperature method. The advantages over silver-ion HPLC for purifying PUFA-methyl esters are that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-methyl esters with the same degree of unsaturation but different chain length. In addition, PUFA-methyl esters were not strongly adsorbed, but eluted earlier than their more saturated corresponding PUFA-methyl esters of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. Centrifugal partition chromatography (CPC) is a separation method based on the liquid partition of compounds. Owing to the high centrifugal field force, one phase stays in the rotor (the liquid stationary phase), the other one is the mobile phase as in classical liquid chromatography. CPC has been used for isolation and purification of PUFAs from various natural sources. Several important advantages of centrifugal partition chromatography are reported in Table 17.2. A CPC method to purify DHA from microalgal oil on a laboratory scale, has been developed by Wanasundara and Fedec (2002). The starting microalgal oil contained 39.7% DHA and 15.2% DPA (n-6), along with several other fatty acids. The mobile phase was hexane–methanol–water in a normal phase-ascending mode. A good separation was achieved and recoveries of 84.6% DHA and 84.9% DPA were obtained. Bousquet and Goffic (1995) have examined CPC separation of microalgal oil-based DHA and EPA and were able to isolate pure DHA and EPA from this oil with excellent yields. The first separation used heptane as the stationary phase and aqueous 3% acetonitrile as the mobile phase. The minor fatty acids were eluted, leaving a mixture of four major PUFAs. This mixture was subjected to a second separation, using heptane as a stationary phase and aqueous methanol as the mobile phase. Under these conditions, it was possible to isolate pure DHA and EPA with high yields. High-speed countercurrent chromatography was used by Du et


488 Separation, extraction and concentration processes Table 17.2 Advantages of centrifugal partition chromatography Advantages Cost

Solid supports, which in many cases are costly, are not required but instead a solvent system is used Recovery Elimination of solid supports avoids problems associated with irreversible retention of highly retentive sample components. Almost 100% recovery of the compounds is guaranteed Quantity Compared with conventional liquid chromatography, the volume ratio of the stationary phase to the total column (rotor) volume is greater. Hence, large quantities of materials can be retained in the stationary phase Speed The stationary phase is retained by centrifugal force, enabling the mobile phase to be pumped at high speeds through the apparatus resulting in reduced separation times Stability Mild operating conditions are used. Therefore, decomposition and oxidation of PUFAs are virtually nonexistent under these conditions Versatility Any two-phase solvent system can be used, including many prepared from nontoxic and commonly available solvents

al. (1996) to separate DHA and EPA ethyl esters in a mixture containing 39.3 and 56.4%, respectively, with hexane–dichloromethane–acetonitrile (5:1:4 v/v/v) as mobile phase. However, the separation was not successful with this solvent system because of the low partition coefficients of the esters under the experimental conditions used. Murayama et al. (1988) separated a mixture of stearic, oleic, linoleic and a-linolenic (18:3n-3) acid ethyl esters because their partition coefficients were distributed over a wide range in the mobile phase comprising hexane–acetonitrile (1:1 v/v). The ethyl esters of linoleic acid and a-linolenic acids were successfully separated during the first normal ascending elution whereas the ethyl ester of oleic acid was isolated by switching the elution mode to descending. Despite the developments in chromatographic techniques to concentrate and purify omega-3 oils, the use of very large volumes of solvents, loss of column resolution after repeated use, and potential product solvent residues are likely to hinder scale-up to production scale volumes.

17.4 Low-temperature fractional crystallization for the concentration and purification of omega-3 fatty acids Low-temperature fractional crystallization is one of the simplest methods employed for production of purified omega-3 fatty acids. This process takes advantage of the existing differences in the melting points of different fatty acids as neat oils or in different solvent systems. The more saturated fatty acids have higher melting points and crystallize out of the mixtures



leaving behind the more unsaturated fatty acids (Senanayake, 2000). The melting points of fatty acids are dependent on their degree of unsaturation. For example, EPA and DHA melt at –54 and –44.5 °C compared with 13.4 and 69.6 °C for 18:1 and 18:0, respectively (Merck Index, 1983). As the temperature of a mixture of saturated and unsaturated fatty acids decreases, the saturated fatty acids, having a higher melting point, start to crystallize out first and the liquid phase becomes enriched in the unsaturated fatty acids (Shahidi and Senanayake, 2006). However, as the number and type of fatty acid components in the mixture increases, the crystallization process becomes more complex and repeated crystallization and separation of fractions must be carried out to obtain purified fractions. For fish oils, not only is there a very wide spectrum of fatty acids but the fatty acids exist, not in the FFA form, but esterified in TAGs. However, the principle of low-temperature fractional crystallization can still be applied to marine oils to partially concentrate TAGs rich in omega-3 PUFAs (Shahidi and Wanasundara, 1998). Seal blubber oil in the TAG and FFA forms were subjected to low-temperature fractional crystallization using solvents such as hexane and acetone in order to obtain omega-3 fatty acid concentrates. Preparation of omega-3 fatty acid concentrates from seal blubber oil by low-temperature crystallization was also reported by Wanasundara (1996), who subjected seal blubber oil in the TAG or FFA form to solvent-based fractionation, using hexane and acetone as solvents, at different temperatures. The content of omega-3 fatty acids in the non-crystalline fraction was increased with decreasing the crystallization temperature. Under all temperature conditions, acetone gave rise to the highest concentration of total omega-3 fatty acids. Low-temperature solvent crystallization of seal blubber oil, in the FFA form, at –60 and –70 °C in hexane, resulted in total omega-3 fatty acid content of up to 58.3 and 66.7%, respectively. However, the content of total omega-3 fatty acids in acetone increased up to 56.7 and 46.8%, respectively. In another study, Han et al. (1987) found that alkali salts of less unsaturated fatty acids crystallize more rapidly than those of highly unsaturated fatty acids containing four or more double bonds, when the saponified solution is cooled. They also compared the cooling temperature and the rate of cooling on the enrichment of omega-3 fatty acids of alkali salts of sardine oil fatty acids. The DHA and EPA from sardine oil were concentrated more than 2.3fold with minimum yields of 91 and 87%, respectively. Fatty acid profiles of the prepared concentrates showed that the cooling rate and temperature had little effect on the yield and contents of DHA and EPA. Chen and Ju (2001) utilized a modified low-temperature solvent crystallization process for the enrichment of PUFAs in borage and linseed oil fatty acids. The effects of solvent, operation temperature, and solvent-to-FFA ratio on the concentration of PUFAs were investigated. The best results were achieved when a mixture of 30% acetonitrile and 70% acetone was used as the solvent. With an operating temperature of −80 °C and a solvent-to-FFA ratio of 30 mL g –1, g-linolenic acid (GLA; 18:3n-6) in FFA of saponified borage oil was raised


490 Separation, extraction and concentration processes from 23.4 to 88.9% with a yield of 62.0%. At a yield of 24.9%, a-linolenic acid (ALA; 18:3n-3) in linseed oil was increased from 55.0 to 85.7%.

17.5 Supercritical-fluid extraction for the concentration and purification of omega-3 fatty acids Supercritical-fluid extraction is a relatively novel technique which has found use in food and pharmaceutical applications (Senanayake, 2000). The properties of a supercritical fluid are used to selectively extract a specific compound or to fractionate mixtures by changing the temperature and pressure without any phase change. The most important property of these fluids is the high solvation capacity in their supercritical region (Shahidi and Senanayake, 2006; Shahidi and Wanasundara, 1998). This method is mild and, because it uses CO2, minimizes autoxidation (Senanayake, 2000). A number of gases are known to have good solvent properties at pressures above their critical values. For food applications, CO2 is the solvent of choice because it is inert, inexpensive, non-flammable, environmentally acceptable, non-toxic, relatively safe, completely recoverable, and readily available and has a moderate critical temperature (31.1 °C) and pressure (1070 psig). The fatty acids were most effectively separated on the basis of chain length; hence the method works best for oils with low levels of long-chain fatty acids. Because this method is derived from separation of compounds based on their molecular weight and not their degree of unsaturation, prior concentration steps, such as urea adduction or low-temperature crystallization, may be necessary in order to purify the omega-3 fatty acids. The use of supercritical fluids for extraction and purification of omega-3 fatty acids from fish and krill oils has been reported (Mishira et al., 1993; Yamagouchi, et al., 1986). Fish oils in the form of free fatty acids and fatty acid esters have been extracted with supercritical CO2 to yield concentrates of EPA and DHA. The use of high pressures and high capital costs might limit the widespread use of this technique in large-scale applications (Shahidi and Senanayake, 2006). Alkio et al. (2000) evaluated the technical and economic feasibility of producing ethyl ester concentrates of DHA and EPA from transesterified tuna oil using supercritical fluid chromatography. A systematic experimental procedure was used to find the optimal values for process parameters and the maximal production rate. DHA ester concentrates up to 95 wt% purity were obtained in one chromatographic step with supercritical fluid chromatography, using CO2 as the mobile phase at 65 °C and 145 bar and octadecylsilane-type reversed-phase silica as the stationary phase. DHA ester, 0.85 g/(kg stationary phase/h) and 0.23 g EPA ester/(kg stationary phase/h) can be simultaneously produced at the respective purities of 90 and 50 wt%. The process for producing 1000 kg of DHA concentrate and 410 kg of EPA concentrate per year requires 160 kg of stationary phase



and 2.6 t h–1 of CO2 as recycling mobile phase. The operating cost of the supercritical fluid chromatography was US$550 kg–1 for DHA and EPA ethyl ester concentrate. The US Patent 4,675,132 (Stout and Spinelli, 1987) demonstrated that fish oil esters could be fractionated by supercritical fluid extraction to produce an oil with a DHA content of 60–65%. However, the recovery of omega-3 fatty acids was low during this process. Jachmanián et al. (2007) studied the solubility of various ethyl esters derived from hake liver oil in supercritical CO2. A selectivity factor was used to determine optimal conditions to fractionate the ethyl ester mixture. A strong influence of solvent pressure and temperature was observed at 8.63–18.04 MPa and 40–70 °C. The lowest total solubility of the ethyl ester mixture was obtained when using supercritical CO2 at the lowest density (the lowest pressure and the highest temperature value tested). The highest discrimination against longchain PUFAs (e.g. EPA and DHA) was also obtained under these conditions. Conversely, higher solubility and lower selectivity were obtained when solvent density increased. Considering this inverse correlation between selectivity and solubility, a single-step batch-fractionation process was designed to increase the DHA ethyl ester content from an initial value of 17.5% in the starting material to 55% in the final extract. Davarnejad et al. (2008) examined the solubility of fish oil in supercritical CO2 at temperatures of 40, 50, 60, and 70 °C and pressures of 13.6, 20.4, and 27.2 MPa. The fractionated fish oil samples collected were then esterified using methanol with sodium methoxide catalyst. The samples were analyzed by GC to determine the amount of four fatty acid methyl ester (FAME) components extracted, namely, methyl palmitate, methyl oleate, methyl EPA (5,8,11,14,17-eicosapentanoate), and methyl DHA (4,7,10,13,16,19docosahexenoate). The results showed that the highest solubility of the fish oil (0.921 g of oil in 100 g of CO2) was obtained at optimum conditions of 40 °C and 27.2 MPa. The solubility of fish oil in supercritical CO2 was found to be higher at lower temperature and at lower fractionation time. Furthermore, the average yield obtained for the combined total of the four FAME components was 66%, with methyl palmitate having the highest at 30.5% under extraction conditions of 50 °C and 13.6 MPa whereas methyl EPA has the lowest at 3.24%. Létisse et al. (2006), and Létisse and Comeau (2008) evaluated the enrichment of EPA and DHA from sardine heads, a waste product from the fish canning industry, via supercritical fluid extraction. These studies were done on a laboratory scale. Various parameters, such as pressure, temperature, CO2 rate and time were optimized in order to obtain the highest yield of extracted oil with the highest amount of EPA and DHA in the extraction product. In the first approach, the oil yield was measured. Then, a quadratic model with three variables was employed to maximize the EPA and the DHA concentrations. A multicriteria optimization, using the desirability function, was performed to determine the best level for each parameter. Pressure, temperature and CO2 rate were, respectively, set at 300 bar, 75 °C and © Woodhead Publishing Limited, 2010

492 Separation, extraction and concentration processes 2.5 ml min–1 during the 45 min extraction. A yield of 10.36% of extracted oil (compared with the dry material) was achieved with an amount of 10.9% of EPA and 13.0% of DHA (compared with all fatty acids of the extract). These yields were lower than with a traditional solvent extraction. However, the advantages of supercritical fluid extraction were shorter extraction time, prevention of heating, and better organoleptic properties by excluding the use of toxic organic solvents. Catchpole et al. (2000) reported the countercurrent extraction and fractionation of a range of crude fish oils using supercritical CO2 and CO2– ethanol mixtures. Vitamin A palmitate was extracted from model mixtures of cod liver oil and vitamins using pure CO2. The separation factor was low, owing to similar solubilities of the vitamin ester and the oil. Vitamin A was also recovered from cod liver oil ethyl esters–vitamin A mixtures. The separation factor was substantially improved over the non-esterified oil, owing to large differences in the solubilities of the esters and vitamin A in supercritical CO2. Solubilities of fish oils and squalene are reported using CO2–ethanol mixtures at 333 K, ethanol concentrations of 0 to 12% by mass, and pressures of 200–300 bar. Solubilities of all oils and squalene increased exponentially with linear increases in the ethanol concentration. The solubility of polar components increased more rapidly than non-polar components. Pilot-scale removal of fatty acids from Orange Roughy oil and squalene from deep sea shark liver oil was carried out using CO2–ethanol mixture as the solvent. The extent of fatty acid removal from Orange Roughy oil was higher than with pure CO2, whereas the degree of separation of squalene from shark liver oil was lower. However, throughput was substantially increased relative to pure CO2 in both cases. Temelli et al. (1995) optimized the supercritical CO2 extraction temperature and pressure for oil removal from freeze-dried, fall Atlantic mackerel. The effect of extraction conditions on pH and water-binding potential of the protein residue was evaluated. For the temperature range (35–55 °C) and pressure interval (20.7–34.5 MPa), a combination of 34.5 MPa and 35 °C gave the highest oil yield and concentration of omega-3 fatty acids.

17.6 Distillation methods for the concentration and purification of omega-3 fatty acids Fractional distillation is another simple process for separation of mixtures of omega-3 fatty acids under ultralow pressure. This method takes advantage of differences in the boiling point and molecular weight of fatty acids under the conditions of high temperature (180–250 °C) and reduced pressure (0.1 to 1.00 mm Hg). Fatty acid enrichment is achieved by exploiting the differences in vapour pressure through countercurrent contacting of vapour



and liquid phases in stages using plates or continuously using random or structured packing. Molecular distillation, often also called ‘short path distillation’, may use moderately lower temperatures and short heating intervals. Molecular distillation provides high selectivity, but as a result of its relatively high operating temperatures and reduced pressures, it leads to high operating costs, possible thermal degradation of omega-3 fatty acids and safety concerns. Several advantages and disadvantages of molecular distillation are reported in Table 17.3. Molecular distillation can be used for the concentration of omega-3 fatty acids from fish oils. The content of omega-3 fatty acids in fish oils mainly depends on the fish species itself, their habitat and the season. A typical base material for commercial production of omega-3 fatty acid concentrates is anchovy or sardine oil with a content of approximately 18% EPA and 12% DHA. Tuna oil is also used for the large-scale production of DHA concentrates, as the crude oil contains up to 25% DHA. In general, the concentration of omega-3 fatty acids in the crude oil should not be less than 30% in total. The state-of-the-art method for the concentration of omega-3 fatty acids from fish oil by molecular distillation is by the fractionation of their ethyl esters (Fig. 17.1). The latter can be obtained by interesterification of fish oil with ethyl alcohol. The equipment used in this process consists of two stills (two-stage molecular distillation) with a degasser. The product distillation requires a minimum of three passes through the system. The first, degassing pass, removes any moisture left after interesterification. The residue from this pass is then used as the feed for the second, ‘lights’ removal, pass. The second pass concentrates the DHA and EPA by separating and removing the ‘lights’ from the feed. The second pass removes 20–50% of C10–C18 fatty acid esters. The residue from this pass is then used as the feed for the third, ‘product distillation’, pass. The third pass further concentrates the DHA and EPA (up to 40–80%) by separating the heavier fraction from the feedstock. The heavy fraction (5–10%) consists of longer chain fatty acids, including fatty acids chain length >C24 as residue. Operating pressures and temperatures required are 0.005–0.01 torr and 170–190 °C, respectively. The recoveries can be up to 70% at DHA and EPA concentrations of 55–65 wt%. Table 17.3 Advantages and disadvantages of molecular distillation Advantages

Disadvantages

Stability: the vacuum allows oils to be processed at minimal temperatures, reducing the risk of thermal decomposition and oxidation of PUFAs

Cost: cost is relatively high

Purity: separating the oil components by weight allows contaminants to be reduced far below industry specifications

Natural form: the starting natural triglyceride form is lost in the process

Concentration: weight grouping allows the processor to concentrate specific fatty acids


494 Separation, extraction and concentration processes Crude fish oil

Refining, bleaching and deodorization

Refined fish oil

Interesterification

Fish oil ethyl esters

Molecular distillation

Purified omega-3 fatty acid esters

Fig. 17.1 Purification of crude fish oil into highly concentrated omega-3 fish oil esters.

The DHA to EPA ratio primarily depends on their content in the base material and on the degree of concentration, as EPA ethyl ester, the more volatile component, also accumulates in the mid-chain-length fatty acid ester fraction. Reprocessing of the latter fraction by molecular distillation increases the recovery of omega-3 fatty acids. Stout et al. (1990) pointed out the practical difficulty of purifying omega-3 fatty acids from menhaden oil via molecular distillation in the natural TAG form. The distillation of menhaden oil, in its natural TAG form, concentrated only EPA from an initial value of 16.0% to 19.5%. Hence, this study did not result in a significant improvement in the concentration of omega-3 fatty acids, as they are more or less uniformly distributed in the TAGs. Furthermore, distillation of triglycerides require high temperatures, risking thermal decomposition of unsaturated fatty acids, and a very low operating pressure, which results in an inefficiently low specific feed rate. However, molecular distillation of its ethyl esters increased the EPA content from 15.9% to 28.4% (Stout et al., 1990). The degree of concentration of DHA was even more remarkable. Whereas DHA doubled from 8.4% to 17.3% in the TAG form, in the alkyl ester form it increased from 9.0% to 43.9% (Stout et al. 1990). Although the greater volatility of the fatty acid esters allows the use of lower temperatures (compared with process temperatures required for FFA fractionation), temperatures are still moderately high, and exposure to distillation conditions over a prolonged period of time can be © Woodhead Publishing Limited, 2010


detrimental to the omega-3 fatty acid constituents, causing polymerization, hydrolysis, isomerization and thermal degradation. Methyl esters of Atlantic herring oil containing 62% monoethylenic fatty acids were subjected to batch fractional distillation, under vacuum on a pilot-plant scale, to study the feasibility of fractionating fatty acid esters of marine oils of low iodine value into monounsaturated fractions with increased commercial value for industrial chemical uses (Ackman et al., 1973). A total of 64 methyl ester fractions were collected and recoveries of the major saturated and monounsaturated acids were close to 100%, and some fractions contained over 90% of the desired 22:1 long chain monounsaturated acids. The short-chain polyunsaturated acids were recovered in high yields, but the long-chain highly unsaturated acids were recovered in yields of 60% or less owing to oxidative and thermal decomposition in the particular apparatus employed. If small amounts of unsaturated acids are acceptable, fractional distillation of low iodine value marine oils could inexpensively supply fractions with high concentrations of methyl esters of longer chain (C20 and C22) monounsaturated and shorter chain (C14) saturated acid or (C16) saturated–monounsaturated acid mixture. Liang and Hwang (2000) employed short-path distillation to fractionate EPA and DHA ethyl esters from squid visceral oil. The elimination temperatures of squid visceral oil ethyl esters (SVOEE) ranged from 50 to 140 °C, increasing with the carbon number of ethyl esters. The elimination temperature of cholesterol was higher than those of SVOEE. When SVOEE originally containing 9.0% EPA, 14.7% DHA, and 11.21 mg g–1 of cholesterol was distilled from 50 to 150 °C, the 130 °C distillate contained 15.5% EPA and 34.7% DHA with 0.99 mg g–1 of cholesterol, and the yield was 21.8%. The 150 °C distillate had 43.1% DHA with 4.96 mg g–1 of cholesterol. Furthermore, the distillates collected from 110 to 150 °C contained 24.4 to 50.2% of EPA plus DHA, and their total yield was 58.3%.

17.7 Enzymatic methods for the concentration and purification of omega-3 fatty acids For the concentration of omega-3 fatty acids on a large scale, each of the above physical and chemical separation methods have some drawbacks either in terms of low yield, a requirement for large volumes of solvent or sophisticated equipment, a risk of structural changes in the fatty acid products, or high operational costs (Senanayake, 2000). Lipases work under mild conditions of temperature and pH (Gandhi, 1997), a factor which favors their potential use for the enrichment of omega-3 fatty acids in oils. Lipases are enzymes that catalyze the hydrolysis, esterification, interesterification, acidolysis and alcoholysis reactions (Senanayake and Shahidi, 2000b; Shahidi and Senanayake, 2006; Shimada et al., 2006; Weete et al., 2008). The common


496 Separation, extraction and concentration processes feature among lipases is that they are activated by an interface. Lipases have been used for many years to modify the structure and composition of foods. Lipases which act on neutral lipids generally hydrolyze the esters of PUFAs at a slower rate than those of more saturated fatty acids (Villeneuve and Foglia, 1997). Use has been made of this relative substrate specificity to increase the concentration of omega-3 PUFAs in seal blubber and menhaden oils by subjecting them to hydrolysis by a number of microbial lipases (Wanasundara and Shahidi, 1998). Concentration of omega-3 fatty acids by enzyme-assisted reactions involves benign reaction conditions and provides an alternative to the traditional concentration methods such as distillation and chromatographic separation (Shahidi and Senanayake, 2006). Furthermore, concentration via enzymatic means may also produce omega-3 fatty acids in the acylglycerol form, which is nutritionally preferred. Studies over the past two decades have used microbial lipases to produce purified omega-3 fatty acids via hydrolysis, esterification or transesterification of marine oils. Tanaka et al. (1992) used six types of microbial lipases (lipases derived from Aspergillus niger, Candida cylindracea, Chromobacterium viscosum, Rhizopus javanicus, Rhizopus delemer and Pseudomonas sp.) to hydrolyze tuna oil and found that the lipase from Candida cylindracea was the most effective one in increasing the DHA content in the non-hydrolyzed fraction of the oil. The DHA content in the non-hydrolyzed fraction was increased three-fold compared with the original oil; however, other lipases were not very effective. Various microbial lipases were evaluated for the enrichment of omega-3 fatty acids from cod liver and sardine oils by selective hydrolysis (Hoshino et al., 1990). The best hydrolysis results were obtained for the lipases from Candida cylindracea and Aspergillus niger, but none of the lipases were able to increase the EPA content of the oil considerably. However, over 50% of the total omega-3 fatty acids were produced when these two lipases were used. A Japanese patent (Noguchi and Hibino, 1984) describes a method based on the discrimination of lipases on DHA and EPA for preparation of omega-3 fatty acids. Ethyl esters from sardine and mackerel were hydrolyzed with several lipases derived from Candida cylindracea, Mucor miehei and Aspergillus niger. Selective hydrolysis afforded ethyl ester concentrates of up to 17% DHA and 25% EPA after separation of the hydrolyzed fatty acids. Transesterification of various fish oil TAGs with a stoichiometric amount of ethanol catalyzed by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation of EPA and DHA (Haraldsson and Kristinsson, 1998). When FFAs from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. When tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained. The residual glyceride mixture contained 49% DHA and 6% EPA, with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil FFAs were directly esterified with ethanol, 68% conversion was obtained.



The residual FFAs comprised 74% DHA and only 3% EPA. The recovery of both DHA into the residual FFA fraction and EPA into the ethyl ester product was reported to be very high at 83 and 87%, respectively (Haraldsson and Kristinsson, 1998). Schmitt-Rozieres et al. (2000) studied the enrichment of PUFAs from sardine cannery effluents via enzymatic selective esterification. The sardine canning industry produces vast quantities of effluents that need expensive reprocessing. Their oily component contains EPA and DHA up to 10% each. The author’s goal was to develop a process allowing the recovery of these fatty acids. After the removal of solid particles, proteins, and peptides from the crude effluent, the resultant oil was hydrolyzed and EPA and DHA were enriched by selective enzymatic esterification. Using Lipozyme™, DHA was enriched up to 80%, but no enrichment was observed for EPA. By immobilizing Candida rugosa lipase on Amberlite IRC50 cation-exchange resin, a 30% EPA enrichment was obtained. Shimada et al. (2001) also attempted to purify PUFAs by taking advantage of the enzyme-catalyzed reactions. When FFAs originating from PUFA-containing oil were selectively esterified with lauryl alcohol (LauOH) using a lipase acting on the desired PUFAs very weakly, the PUFA was efficiently enriched in the FFA fraction. In addition, when selective alcoholysis of ethyl esters originating from PUFA-containing oil with LauOH was carried out, the PUFA ethyl ester (EtPUFAs) was enriched to a desired purity in the unreacted ethyl ester fraction. These reaction mixtures contain LauOH, PUFA (EtPUFAs), and lauryl esters, and their molecular weights are different from one another. Hence, PUFA or EtPUFAs can be easily separated by conventional distillation. Selective esterification increased the purity of DHA, GLA, and arachidonic acid (ARA; 20:4n-6) to 91, 98, and 96 wt%, respectively. Selective alcoholysis was also effective for increasing the purity of ethyl docosahexaenoate to 90 wt%. Kojima et al. (2006) studied the enzymatic acidolysis and acylglycerol synthesis using PUFAs with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase). The acidolysis of triolein with EPA or DHA in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 °C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 °C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 °C, respectively. The EPA incorporation rate was even higher at a low temperature (10 °C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, ARA, mead acid (MA), and dihomo-g-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e. MA, ARA, EPA, and DHA, were low for both reactions.


498 Separation, extraction and concentration processes Halldorsson et al. (2003) investigated the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their fatty acid selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40 °C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the fatty acids, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. When FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained. The residual FFA contained 78% DHA and only 3% EPA, with 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration of EPA and DHA.

17.8 Integrated methods for the concentration and purification of omega-3 fatty acids Most of the separation methods described above, when used alone, can only concentrate and purify omega-3 fatty acids to a limited extent. Therefore, two or more procedures are often required to produce highly purified omega-3 fatty acids. A simple and inexpensive method involving saponification of wet biomass, followed by transmethylation, winterization and urea adduction in a sequential manner has been recently developed for concentration of DHA from Crypthecodinium cohnii biomass (Mendes et al., 2007) (Fig. 17.2). The algal biomass grown in shake flasks is concentrated by centrifugation and the wet concentrate is kept frozen until needed. Fatty acids are extracted by direct saponification of wet biomass with KOH-ethanol. Before extracting unsaponifiable matter with hexane, water is added to shift the equilibrium distribution of unsaponifiable matter to the hexane phase. The hydroalcoholic phase, containing the soaps, is acidified with HCl and free fatty acids are extracted with hexane. The organic phase, containing FFAs, is dried with anhydrous sodium sulfate and the solvent is evaporated to recover FFAs. The FFAs are then methylated using transmethylation reagent and sulfuric acid. The mixture containing methyl esters are then winterized at –18 °C. Subsequently, the liquid fraction is separated from the winterized crystals. The liquid fraction is added to a urea-methanol saturated solution and the urea-fatty acids adducts are crystallized; the crystals are separated by centrifugation and the filtrate (non-urea complexing fraction), which contains DHA, is finally recovered. The highest DHA recovery (49.9%) was obtained at 24 °C at a urea/fatty acid ratio of 4.0, corresponding to 89.4% of DHA of the total fatty acids (Mendes et al., 2007).


Methods of concentration and purification of omega-3 fatty acids 499 Microalgal wet biomass Saponification Free fatty acids Transmethylation Fatty acid methyl esters (FAMEs) Winterization DHA methyl esters Urea adduction Highly purified DHA methyl esters

Fig. 17.2 Scheme for the DHA concentration and purification of microalgal biomass from Crypthecodinium cohnii.

Medina et al. (1995) described a two-step process, urea adduction followed by preparative high-performance liquid chromatography, for concentration of omega-3 fatty acids from the marine microalga, Isochrysis galbana. By the urea adduction method, a mixture that contained 94% (w/w) stearidonic (SA), EPA, and DHA acids (4:1 urea/fatty acid ratio and 4 °C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse-phase C18 columns. These isolations were scaled up to a semi-preparative column. A PUFA concentrate was isolated from I. galbana with methanol/water (80:20, w/w) or ethanol/ water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively. Guil-Guerrero and Belarbi (2001) also used an integrated method to purify DHA and EPA from cod liver oil. The process consisted of four main steps: (i) saponification of the oil, (ii) use of urea inclusion adducts method to obtain PUFAs, (iii) PUFA methylation, and (iv) silica gel column chromatography of the methylated PUFAs. Silica gel chromatography yielded highly pure DHA in the process (100% purity, 64% yield). For EPA, the recovery in the combined process was 29.6%, and the final purity was 90.6%, owing to the simultaneous elution of other PUFA esters. The recovery in the urea adduction method was strongly enhanced by application of orbital agitation during the crystallization process, in which EPA yield increased from 60–70% without agitation to 90–97% at 800 rpm; stearidonic acid (18:4n−3) yield ranged from 60–75% without agitation to 87–95% at 800 rpm, and DHA yield varied from 53–73% without agitation to 85–99% at 800 rpm. Chakraborty and Paulraj (2008) used enzymatic hydrolysis followed © Woodhead Publishing Limited, 2010

500 Separation, extraction and concentration processes by urea adduction to purify EPA and ARA from sardine oil. The enzyme used for the hydrolysis of sardine oil was the lipase derived from Bacillus licheniformis MTCC 6824. The enzyme exhibited more hydrolytic resistance toward the ester bonds of EPA and ARA than those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 °C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued. Lipasecatalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided FFAs containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea adduction proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil. Yuzo et al. (2006) employed enzymatic hydrolysis, using lipase from Pseudomonas fluorescens strain HU380, and urea adduction to concentrate EPA and DHA from refined cod oil. The starting oil had 12.2% EPA and 6.9% DHA. Lipase-catalyzed hydrolysis followed by urea adduction provided FFAs with 43.1% EPA and 7% DHA. The resulting yield of concentrated total fatty acids comprised 2.6% of the fatty acids from the cod oil. Thus, EPA was particularly concentrated in the fatty acids derived from refined cod oil as a result of enzymatic hydrolysis followed by urea adduction. On the other hand, hydrolysis of cuttlefish oil with AK-lipase (lipase from Pseudomonas fluorescens strain AK102), followed by urea adduction increased the EPA content from 14.2 to 16.8%, and DHA content from 16.3 to 44.6%. The yield of purified total fatty acids by urea concentrate was 9.4% of the fatty acids from the cuttlefish oil. Thus, DHA was particularly concentrated in this study. A US patent (Zaks and Gross, 1999) disclosed an enzymatic process for preparing an oil-based product significantly enriched in omega-3 fatty acids. The process is a two-step procedure involving lipase-catalyzed transesterification of TAGs followed by low-temperature crystallization. The process yields a mixture of highly pure monoglycerides, at least 60% of which contain omega-3 fatty acids. The process can also be used to produce omega-3 enriched TAG products. Enzymatic methods of enrichment of omega-3 fatty acids have also been investigated under supercritical conditions. Lin et al. (2006) examined the enrichment of omega-3 fatty acids in TAGs of menhaden oil by lipasecatalyzed trans-esterification under supercritical CO2. Before the reaction, menhaden oil was treated by urea adduction to produce 80.1% of omega-3 PUFAs containing 29.4% EPA and 41.8% DHA. Using the sn-1,3-specific lipase from Mucor miehei, the effect of various operating parameters on the reaction was studied, including co-solvent concentration, reaction temperature,



time, pressure, and substrate ratio (free omega-3 fatty acids: TAGs). Water and ethanol were examined as co-solvents, and both fluids exhibited a maximum for omega-3 fatty acid content in TAGs as a function of concentration. Pressure up to 103.4 bar had a significant positive effect on the conversion of omega-3 PUFA onto TAGs. By further increasing pressure, the conversion rate decreased owing to the transformation of the spatial structure of lipase that leads to deactivation. The enzyme exhibited a good performance and stability in the region of 323 K. The optimal substrate molar ratio of TAG to omega-3 PUFA is about 1:4 taking into consideration the TAG inhibition. The conversion in supercritical CO2 appeared 40% higher than in n-hexane at ambient pressure after 5h. In another study, Lin and Chen (2008) used esterification of free omega-3 fatty acids with enriched omega-6 TAGs to produce a desired structured lipid with an omega-3/omega-6 ratio of 4 by using lipases under supercritical CO2. Comparing four different types of lipases, sn-1,3-specific lipase from Mucor miehei had the highest degree of incorporation under 10 wt% loading amount of total substrates. The optimal operating parameters under 10.2 MPa and 323.15 K supercritical CO2 could attain the desired omega-3/ omega-6 ratio in 6 h. Because of the negative effect on the enzyme activity by the enriched omega-6 TAGs, the optimal substrate ratio of the enriched omega-6 TAGs and the omega-3 fatty acids was chosen as 1/4. To enhance the solubility of omega-3 fatty acids in supercritical CO2, ethanol was applied as a co-solvent and reached an optimal input at 10% of total substrates. The enzyme maintained 81% of its initial activity because of moisture removal from the surface of the enzyme after seven cyclic pressurization/ depressurizations.

17.9 Conclusions Production of purified omega-3 fatty acids from natural source materials may be achieved via a number of techniques, namely urea adduction, chromatography, low-temperature crystallization, supercritical fluid extraction, enzymatic splitting, molecular distillation, as well as a combination of any of the above methods. Purified omega-3 fatty acid products thus produced may be in the form of free fatty acids, alkyl esters or acylglycerols. Each method had its own benefits and drawbacks. Of the methods described above, molecular distillation is highly energy consuming and results in a significant distraction of labile highly unsaturated fatty acids. Other methods, which require the use of organic solvents, include low-temperature crystallization, chromatography and supercritical CO2 extraction, among others. These processes have a number of drawbacks. For example, low-temperature crystallization of acylglycerols typically results in only a small omega-3 enrichment of the product, and chromatography and supercritical fluid extraction are expensive and difficult to scale up. Owing to the potential benefits of having the concentrates in the © Woodhead Publishing Limited, 2010

502 Separation, extraction and concentration processes acylglycerol form, enzymatic procedures have become popular. However, there are several limitations to the enzymatic processes as well. These include the necessity for a complex separation of the product from free saturated fatty acids, use of complex multi-enzymatic systems and the low efficiency that results in an insufficient degree of enrichment. Urea adduction has been very successful in enriching omega-3 fatty acids on an industrial scale and the products thus formed are in the free fatty acid or simple ester forms. Efficient and cost-effective methods of enriching the level of omega-3 fatty acids will continue to be needed in order to reduce the cost and to meet the future demand for highly purified omega-3 products.

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18 Extraction of natural antioxidants from plant foods E. Conde, A. Moure, H. Domínguez and J. C. Parajó, University of Vigo, Spain

Abstract: An overview of the most studied vegetal sources of compounds with antioxidant properties and their biological action is firstly presented. The conventional and alternative extraction processes are discussed, special emphasis is given to those using biorenewable, environmentally friendly solvents. Selected examples of integrated processes for extraction, concentration and purification of extracts are included. Key words: antioxidants, plants, extraction, purification.

18.1 Introduction The utilization of phytochemicals in the food and cosmetic industries has attracted public and scientific interest, because of their perceived efficiency, low cost and lack of toxicity. The number of recent publications and patents on antioxidants has increased considerably. The demand for natural antioxidants, which are presumed to be safer, has risen owing to concerns about the long-term safety and negative consumer perception of some synthetic antioxidants. Many natural compounds show antioxidant activity and may act as flavorings, colorants, preservatives and reinforcers of endogenous antioxidant systems (Astley, 2003; Valenzuela et al., 2003; Halliwell, 2002; Halliwell and Gutteridge, 1999). There is epidemiological and clinical evidence relating antioxidant-rich diets with a decreased risk of degenerative diseases, reduced morbidity and mortality. However, the safety limits of many natural antioxidants (which can consist of a mixture of several active substances) are unknown, and their lack of toxicity should be confirmed (Galati and


Extraction of natural antioxidants from plant foods 507 O´Brien, 2004; Pokorny, 2007). The biological activity and bioavailability of natural antioxidants are the subject of much research. Edible vegetals and agroindustrial residues are considered to be abundant and promising sources of natural antioxidants. The extraction method, of crucial importance for both technical and economic reasons, ideally should be non-destructive, time efficient and suitable for producing high quantities of extracts, which should be processed by selective techniques to yield concentrates of enhanced antioxidant capacity. This chapter presents an overview of the most studied vegetal sources of antioxidants and extraction processes (in particular those using biorenewable, environmentally friendly solvents). Selected examples of integrated processes for extraction, concentration and purification of extracts are also included.

18.2 Antioxidant activity in food systems According to a classic definition, an antioxidant is any substance that, when present at low concentrations compared with those of an oxidizable substrate (such as lipids, proteins, DNA or carbohydrates), significantly delays or prevents oxidation of that substrate (Halliwell, 2002; Halliwell and Gutteridge, 1999). In food systems, the term antioxidant is used to designate the inhibitors of lipid peroxidation, whereas in biological systems it usually refers to protection of lipids, proteins and DNA against oxidative damage by processes or reactions involving reactive oxygen and nitrogen species (ROS and RNS, respectively). Antioxidant activity and antioxidant capacity, although often used interchangeably, have different meanings. Activity refers to the rate constant of the reaction between the antioxidant and the oxidant species, and capacity refers to the amount (in moles) of a given free radical scavenged by a sample. The basic mechanism of lipid peroxidation (Frankel, 2005; Laguerre et al., 2007; Pokorny, 1991) is presented in Fig. 18.1. Lipid peroxidation is induced by oxygen in the presence of initiators such as heat, free radicals, light, photosensitizing pigments and metal ions. Once these free radicals are formed, lipid peroxidation progresses at a high rate by a radical chain reaction, and oxidation ends by producing secondary non-radical compounds. In this scheme, antioxidants can act at different levels: ∑ ∑ ∑ ∑ ∑

scavenging species responsible for oxidation initiation or preventive antioxidants; interruptors of the propagation of the autoxidation chain reaction or chain-breaking antioxidants; singlet oxygen quenchers; synergists or compounds increasing the activity of chain-breaking antioxidants in a mixture; reducing agents;


508 Separation, extraction and concentration processes Initiation Formation of radicals: peroxyl (RO2•), alkoxy (RO•) or alkyl (R•) Propagation R• + O2 Æ RO2• RO2• + RH Æ ROOH + R• RO• + RH Æ ROH + R•

ROOH Æ RO• + •OH 2 ROOH Æ RO2• + RO• + H2O Termination 2 R• R• + RO2• Æ Stable products 2 RO2•

Fig. 18.1 Mechanism of lipid oxidation.

∑ ∑

metal chelators, which stabilize metal pro-oxidants (iron or copper cations); inhibitors of specific oxidative enzymes (especially lipoxygenases).

Antioxidants often act by several mixed and co-operative mechanisms. An antioxidant can behave as a pro-oxidant depending on the structure, chemical environment and operational conditions: for example, typical antioxidants such as flavonoids, a-tocopherol or ascorbic acid can act as pro-oxidants in the presence of transition metal ions, whereas carotenoids show pro-oxidant activity at high oxygen pressure (Galati and O’Brien, 2004). Table 18.1 lists representative in vitro tests employed in the assessment of antioxidant properties. The need for developing simple and reliable in vitro analytical antioxidant tests is widely acknowledged, and the convenience of a common standard has been claimed. A mixture of caffeic acid, catechin and epigallocatechin3-gallate, hesperetin, and morin was proposed for this purpose (Luthria and Vinyard, 2008). The problems associated with the determination of antioxidant activity were summarized in several critical reviews (Andrade et al., 2008; Becker et al., 2004; Decker et al., 2005; Fernandez-Panchón et al., 2008; Frankel and Finley, 2008; Gordon, 2001; Halliwell, 2002; Karadag et al., 2009; Kiokias et al., 2008; Magalhães et al., 2008; Moon and Shibamoto, 2009; McDonald-Wicks et al., 2006; Prior et al., 2005; Roginsky and Lissi, 2005; Laguerre et al., 2007; Sánchez-Moreno, 2002; Singh and Singh, 2008; Strube et al., 1997; Verhagen et al., 2003). The antioxidant activity depends on a number of factors, including type of substrate, medium, and initiators; oxidation conditions, partitioning properties of the antioxidant between lipid and aqueous phases, and the experimental method used (Antolovich et al., 2002; Decker et al., 2005; Frankel and Meyer, 2000; Gordon, 2001; Yanishlieva, 2001). Pitfalls of in vitro antioxidant research include the low stability of antioxidants, which can lead to formation and accumulation of oxidation products (Haenen et al., 2006; Knasmüller et al., 2008). © Woodhead Publishing Limited, 2010

Table 18.1 In vitro methods usually employed for testing the antioxidant activity of pure compounds and extracts. The activities considered include antiradical, anti-lipoperoxidation, metal chelation and reducing capacity Antioxidant activity assay

Substrate/initiator

Scavenging, decolorization assay, indirect competition method of chain lipid peroxidation (chain-breaking)


bC (b-carotene bleaching)

ROO˙

Scavenging, chain-breaking, competition method

CAT (conjugated autoxidizable triene)

ROO˙

Chain-breaking

Crocin bleaching

ROO˙


CUPRAC (cupric ion reducing antioxidant activity)

Cu2+

Reducing capacity 2+

Deoxyguanosine oxidation

HO˙, H2O2, Fe

Deoxyribose oxidation

HO˙, H2O2, Fe2+

Scavenging, chain-breaking, chelating Scavenging, chain-breaking, chelating

DMDP

DMDP˙+ (N,N-dimethyl-pphenylenediamine)


DPPH

DPPH˙ (a,a-diphenyl-bpicrylhydrazyl)


FIC (ferrous ion-chelating assay)

Fe2+, Cu2+

Chelating

FRAP (ferric-reducing antioxidant power)

Fe3+-TPTZ

Reducing capacity, indirect competition method of chain lipid peroxidation (chain-breaking)

FTC (antioxidant activity in the linoleic acid system with ROO˙ ferrothiocyanate reagent)

Scavenging, chain-breaking

HOSC (hydroxyl radical scavenging capacity)


HO˙

HPS (hydrogen peroxide scavenging)

H2O 2


Hypochlorous acid scavenging

HOCl


Extraction of natural antioxidants from plant foods 509

ABTS or TEAC (Trolox equivalent antioxidant capacity) ABTS·+ [2, 2¢-azinobis(3ethylbenzothiozoline-6sulfonate)]

Mechanism

Antioxidant activity assay

Substrate/initiator

Mechanism

Liposome oxidation

ROO˙, Fe2+

Scavenging, chain-breaking, chelating

CD (conjugated dienes)

LDL

Oxidation by copper, aldehydes formation


In vitro LDL oxidizability

LDL

Oxidation by copper, conjugated dienes determination

Iodometric assay of lipid peroxides

LDL

Iodide oxidation by lipid peroxides

TBARS (thiobarbituric acid-reacting substances)

LDL, ROO˙

Adduct malondialdehyde–thiobarbituric acid (MDA– TBA) formation, scavenging, chain-breaking

ORAC (oxygen-radical absorbance capacity)

Oxygen radical, ROO˙


Peroxyl radical scavenging

ROO˙


Peroxynitrite scavenging

ONOO–


2 –

PHOTOCHEM (photochemiluminescence)

O˙


PM (phosphomolybdenum method)

Mo6+

Reducing capacity

Reduction of the Fremy’s radical

Fremy’s (potassium nitrosodisulfonate)

Scavenging, indirect competition method of chain lipid peroxidation (chain-breaking)

RP (reducing power)

Fe3+

Reducing capacity

SRS (superoxide radical scavenging)

O 2˙ –


TAR (total antioxidant reactivity)

ROO˙


TOSC (total oxidant scavenging capacity)

ROO˙, O2˙–, HO˙, HOCl, LO(O) , ONOO–, 1O2


TRAA (a-tocopheroxyl radical attenuating ability)

a-Tocopheroxyl

Scavenging

TRAP (total radical-trapping antioxidant parameter)

ROO˙


Trichloromethyl peroxyl scavenging

CCl3O2˙

Scavenging



Extraction of natural antioxidants from plant foods 511 The need to use a variety of antioxidant activity assays is generally agreed, as the results obtained in individual tests can be contradictory. Some recommendations on the in vitro determination of antioxidant activity include: identification and determination of active compounds; evaluation of protection against oxidation in foods or physiological model systems under conditions resembling the real chemical, physical, and environmental conditions in the systems to be protected; detailed understanding of the oxidation mechanisms; measurement of both initial and secondary products, and utilization of reagent concentrations that are physiologically relevant. Low oxidation levels should also be determined, and possible mechanism modification under accelerated oxidation conditions should be considered (Antolovich et al., 2002; Choe and Ming, 2006; Collins, 2005; Decker et al., 2005; Frankel, 1993; Frankel and Meyer, 2000; Frankel and Finley, 2008; Gordon, 2001; Prior et al. 2005; Huang et al., 2005; Schlesier et al., 2002). Several classifications of antioxidants assays have been proposed, based on mechanisms such as the ability to quench free radicals by hydrogen donation, the ability for electron transfer, or a combination of both (Huang et al., 2005; Prior et al. 2005; MacDonald-Wicks et al., 2006). Results from simple in vitro experiments are difficult to extrapolate to the heterogeneous conditions of multifaceted in vivo systems (Haenen et al., 2006), where the possible action on the target tissues is affected by absorption, distribution, metabolism and excretion. The animal model used and a battery of well-validated tests to assess the broad diversity of oxidative damage and antioxidative defence parameters, are crucial for antioxidant research in vivo. Measurement of total antioxidant capacity was proposed as an integrated parameter to consider the balanced action of all antioxidants and their synergistic interactions in plasma and body fluids (Aruoma, 2003; Ghiselli et al., 2000; Halliwell, 2009; Sies, 2007). However, pitfalls and serious limits of applicability of this concept have been manifested (Frankel and Finley, 2008; Sies, 2007) as well as the strong dependency on the analytical method used (Fernández-Panchón et al., 2008).

18.3 Natural compounds with antioxidant activity and major sources 18.3.1 Natural compounds Natural antioxidants are synthesized by plants, micro-organisms, fungi, and animals. The most important groups of natural antioxidants are listed below. Phenolics refer to monomeric, oligomeric or polymeric compounds with an aromatic ring bearing one or more hydroxyl substituents and functional derivatives, such as esters, methyl ethers and glycosides. Phenolics (including


512 Separation, extraction and concentration processes simple phenols, coumarins, flavonoids, stilbenes, lignans, hydrolyzable and condensed tannins, and phlorotannins), have powerful antioxidant activities in vitro (von Gadow et al., 1997; Miller and Ruiz-Larrea, 2002; Tabart et al., 2009) and play a role in signals between plants, plant defense against predation (by micro-organisms, insects and herbivores) or in response to environmental stress (such as air pollution, heavy metal ions and UV-B radiation). Simple phenolics include hydroxybenzoic and hydroxycinnamic acids. The main subclasses of flavonoids are anthocyanins, flavanols, flavanones, flavonols, flavones and isoflavones, and anthocyanidins and their glycosides. Tannins are polyphenolic compounds with varying molecular masses. Plant tannins include hydrolyzable tannins (gallotannins, which yield glucose and gallic acid upon hydrolysis, or ellagitannins, which produce ellagic acid), and condensed tannins (proanthocyanidins). Algae contain phlorotannins, formed by the polymerization of phloroglucinol (Singh and Bharate, 2006). Table 18.2 summarizes the phenolic composition of some plants. Terpenes are a large and diverse class of lipophilic secondary plant metabolites made up of isoprene units, and are classified into hemi-, mono(C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30), and tetraterpenoids (carotenoids), having eight isoprenoid C 5 residues. Monoterpenes, sesquiterpenes and diterpenes are the main components of essential oils, which also contain oxygenated derivatives and other compounds (including aldehydes, ketones, phenolic, acetates and oxides). Antioxidant potential has been reported for terpenes (Escuder et al., 2002; Grassmann, 2005; Grassmann et al., 2002) as well as synergistic effects with phenolics (Milde et al., 2004). Carotenoids (carotenes and xanthophylls) have received attention because of their provitamin and antioxidant roles (Bohm et al., 2002). Vitamin E includes a family of tocopherols and tocotrienols and some of their ester derivatives. Tissues and seeds of plants are the major sources of vitamin E, which is associated with membrane lipids or appears in lipid storage stuctures. Vitamin E is the most important natural antioxidant in vegetable oil-derived foods from rice bran, palm, and wheat germ (Weber and Rimbach, 2002). The richest source is a by-product of soybean processing (‘oil deodorizer distillate’). Vitamin E effectively inhibits the peroxidation of lipids by peroxyl radical scavenging, and shows relevant activities related to the regulation of enzymes and gene expression (Brigelius-Flohé, 2009). Small proteins and peptides isolated from various protein hydrolyzates show antioxidant activity (Chen et al., 2006; Kitts, 2005; Kitts and Weiler, 2003). Their low-molecular-weight (low-MW) and simple structure, easy absorption, stability under different conditions and the lack of immunoreaction have been cited as advantages (Chen et al., 1996). Some peptides from hydrolyzed food proteins exert antioxidant activities against enzymatic and non-enzymatic peroxidation of lipids and essential fatty acids (Chen et al., 2006; Hook et al., 2001). Certain hydrophobic amino acids and their derivatives present high activity, and show synergistic effects with non-peptidic antioxidants


Extraction of natural antioxidants from plant foods 513 Table 18.2 Phenolic contents (mg g–1 dry weight) of some plants Plant (Latin name)

Part Phenolic compounds

Achyrocline F satureioides Anethum graveolens F Averrhoa carambola L B Capparis spinosa L

CA, 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA pAA, Ap, BA, CA, C, EC, CGA, pCouA, PCys, GA, GeA, K, Lt, Myr, Q, SiA CGA, GA, Q CGA, GA CA, pCouA, FeA, Q, R, VA

TPC/TF

145/48** 78.3 30.1 15.9

Cassine orientalis

L

Castanea vulgaris

L

C, EC, EGC, PCyB2, GA, K, 24/24 Myr, Q Ap, FeA, GeA, Ng, Q, R 11.9

Casuarina equisetifolia Cinnamomun zeylanicum Coffea macrocarpa

B L B

CA, FeA, GA, Q K, Q, R CA, FeA, GA, Q

72.1 57.7 48.9

L

24/18

Diospyros neraudii

L

Eugenia tinifolia

L

Ficus microcarpa

B

Geranium purpureum Ilex paraguariensis

L

Monimiastrum acutisepalum Moringa oliefera

L L

C, EC, ECG, EGC, EGCG, PCyB1, PCyB2, GA, K, Q C, EC, ECG, EGCG, PCyB1, K, Q C, EC, EGC,EGCG, PCyB1, PCyB2, GA, Q, K Cat, Cou, pPGu, pVGu, OlA, pPPh, PCA, PR, Syr, Sy, V, iVA HBA, CA, C, EC, pCouA, GeA, Q, R, SyA, VA CA, 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA, 3FQA, 4FQA, 5FQA C, EC, ECG, EGC, PCyB1, GA,Q, K CGA, EA, FeA, GA, Q

Myonima obovata

L

Phytolacca americana Pimpinella anisum

L

L

S

C, ECG, EGC, PCyB1, PCyB2, K, Q HBA, CA, pCouA, FeA, R, VA 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA, 3FQA, 4FQA, 5FQA

75/19 23a/17a 237/6.3*

Reference Marques and Farah, 2009 Shyu et al., 2009 Prakash et al., 2007b Proestos et al., 2006 Soobrattee et al., 2008 Proestos et al., 2006 Prakash et al., 2007b Prakash et al., 2007b Soobrattee et al., 2008 Soobrattee et al., 2008 Neergheen et al., 2006 Ao et al., 2008

28.2

Proestos et al., 2006 Marques and Farah, 2009

10a/9a

Neergheen et al., 2006 Prakash et al., 2007b Neergheen et al., 2006 Proestos et al., 2006 Marques and Farah, 2009

32.9 31a/32a 9.2


514 Separation, extraction and concentration processes Table 18.2 Continued Plant (Latin name)


Ricinus communis

L

EC, EA, GA, GeA, Q, R

Rubus ulmifolius Ruta graveolens

L

Salix aegyptiaca

C

CA, 4CQA, 5CQA, CGA, FeA, KGu, KCouGPy, KCGPy, QGu HBA, CA, C, GeA, FeA, Q, R CA, C, EGCG, pCouA, GA, R, V CA, C, EGCG, pCouA, GA, Myr, V CA, C, EGCG, pCouA, GA, Myr, Q, R, V CA, pCouA, GeA, Lt, Q, VA

L B Spartium junceum

F

Styrax officinalis

L

Syzygium glomeratum Juglans regia

L

Achillea millefolium

TPC/TF

L H L ST

Reference Singh et al., 2009 Dall’Acqua et al., 2008

4.3

Proestos et al., 2006 107/351** Enayat and Banerjee, 2009 64/165** 212/479** 4.8

HBA, CA, C, EC, CouA, GA, 18.4 GeA, FeA, Ng, Q, VA C, EC, ECG, EGCG, EGC, 84a/39a PCyB1, PCyB2, GA, Q, K 5CQA, 3CouQA, 4CouQA, CouA, QA, QGal, QP-d, QR, QX Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi

Proestos et al., 2006 Proestos et al., 2006 Neergheen et al., 2006 Pereira et al., 2007 Benetis et al., 2008

B, bark; C, catkins; F, flowers; H, herbs; L, leaves; S, seeds; ST, stems. a Composition of phenols (mg g–1 fresh weight) of some plants. TF, total flavonoid expressed as mg quercetin equivalent g–1 on a dry weight basis. * Total flavonoid expressed as mg rutin equivalent g–1 on a dry weight basis. ** Total flavonoid expressed as mg catechin equivalent g–1 on a dry weight basis. TPC: total phenolic content expressed as mg gallic acid equivalent g –1 on a dry weight basis. 3,4dCQA, 3,4-dicaffeoylquinic acid; 3,5dCQA, 3,5-dicaffeoylquinic acid; 3CouQA, 3-coumaroylquinic acid; 3FQA, 3-feruloylquinic acid; 4,5dCQA, 4,5-dicaffeoylquinic acid; 4CouQA, 4-coumaroylquinic acid; 4CQA, 4-caffeoylquinic acid; 4FQA, 4-feruloylquinic acid; 5CQA, 5-caffeoylquinic acid; 5FQA, 5-feruloylquinic acid; Ap, apigenin; Apg, apigenin-7-O-glycoside; BA, benzoic acid; C, catechin; CA, caffeic acid; Cat, catechol; CGA, chlorogenic acid; Cou, coumaran; EA, ellagic acid; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate; FeA, ferulic acid; GA, gallic acid; GeA, gentisic acid; HBA, p-hydroxybenzoic acid; iVA, isovanillic acid; K, kaempferol; KCouGPy, kaempferol-3-O-(6≤-p-coumaroyl)-b-d-glucopyranoside; KCGPy, kaempferol-3-O-(6≤-caffeoyl)-b-d-glucopyranoside; KGu, kaempferol-3-O-glucuronide; Lt, luteolin; Ltdg, luteolin-3¢,7-di-O-glycoside; Ltg, luteolin-7-O-glycoside; Myr, myricetin; Ng, naringenin; OlA, oleanolic acid; pAA, p-anisic acid; PCA, protocatechuic acid; pCouA, p-coumaric acid; PCyB1, procyanidin B1; PCyB2, procyanidin B2; PCys, proanthocyanidins; pPGu, p-propylguaiacol; pPPh, p-propylphenol; PR, 4-n-propylresorcinol; pVGu, p-vinylguaiacol; Q, quercetin; QA, quercetin 3-arabinoside; QGal, quercetin 3-galactoside; QGu, quercetin-3-O-glucuronide; QP-d, quercetin-3pentoside derivate; QR, quercetin 3-rhamnoside; QX, quercetin 3-xyloside; R, rutin; SiA, sinapic acid; Sy, syringol; SyA, syringic acid; Syr, syringaldehyde; V, vanillin; VA, vanillic acid; Vi, vicenin-2.


Extraction of natural antioxidants from plant foods 515 such as phenolic compounds (Erdmann et al., 2008). Antioxidant peptides have been obtained from soybean (Pyo and Lee, 2007), soy-fermented foods (Gibbs et al., 2004), soy protein fractions (Moure et al., 2006), barley hordein (Chiue et al., 1997), and potato (Wang and Xiong, 2005). Different hydrolysis conditions result in peptide mixtures with different properties (De Mejia and De Lumen, 2006). Food-derived bioactive peptides from animal or plant proteins have regulatory functions in humans after being released in vitro or in vivo in the gastrointestinal tract (De Mejia and De Lumen, 2006). A number of beneficial health effects have been claimed for food-derived products containing bioactive peptides, including blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption and/or bioavailability, and cyto- or immunomodulatory effects. Maillard reaction products (MRP) such as Schiff bases, premelanoidins and melanoidins are formed by reactions involving the condensation of the carbonyl group of reducing sugars with the amino group of amino acids and proteins. Polyphenols, ascorbic acid and other carbonyl compounds can participate in the Maillard reaction. MRP are naturally formed during food processing and storage. Their characteristics and MW depend on both the type of reagents and the processing conditions (temperature, time, pH, water activity) (Jing and Kitts, 2004). The antioxidant properties of MRP are positively correlated with color when the mechanisms responsible for the formation of antioxidants and color follow the same pathway (Manzocco et al., 2001). Formation of potentially harmful substances (acrylamide and hydroxymethylfurfural) increases rapidly with temperature and time (Morales et al., 2009). The antioxidant capacity of MRP formed from model compounds has been reported (Borrelli et al., 2003; Chen and Kitts, 2008; Dittrich et al., 2003; Kim and Lee, 2008; Morales and Babbel, 2002). Maillardderived antioxidants increase the plasma tocopherol, decrease the plasma thiobarbituric acid reactive substances (TBARS) concentration (Somoza, 2005), prevent low density lipoprotein (LDL) oxidation (Mesa et al., 2008), show reducing activity (Yilmaz and Akgun, 2008) and radical scavenging capacity (Michalska et al., 2008), and may be employed as multifunctional ingredients (Lindenmeier et al., 2002). Carbohydrates including polysaccharides, such as dextran, pullulan, mannan, and lipopolysaccharide, exhibit higher free radical scavenging activity than their constituent sugars, although lower than Trolox™ (Tsiapali et al., 2001). Although glucans are weak free radical scavengers in solution, they stimulate free radical activity in a murine macrophage cell line and modestly augment the generation of free radicals. Arabinoglucogalactan from Panax noto ginseng roots exhibited a, a-diphenyl-b-picrylhydrazyl (DPPH) free radical scavenging activity (Wu and Wang, 2008), whereas porphyran, a sulfated polysaccharide isolated from Porphyra, delayed the aging process in mice by enhancing the amount of antioxidative enzymes and reducing the risk of lipid peroxidation (Zhang et al., 2003). Fucoidans,


516 Separation, extraction and concentration processes sulfated algal polysaccharides made up of fucose as the major component, exhibit antioxidant and biological properties (anticoagulant, anti-inflamatory, antiviral, or antitumoral activities) (Zhao et al., 2008). 18.3.2 Sources The number of potential sources of antioxidant compounds is increasing. Table 18.3 summarizes the composition of some examples of the major vegetal sources of bioactive compounds. Food by-products have the additional advantages of being renewable, widely distributed, largely available and inexpensive. Waste streams from the processing of agricultural or industrial feedstocks are particularly attractive as sources of antioxidants, particularly when they are concentrated and create environmental problems (Balasundram et al., 2006; Moure et al., 2001; Obied et al., 2005). Typical wastes employed for antioxidant production and composition of some representative cases are summarized in Tables 18.4 and 18.5. Table 18.3 Phenolic composition of fruits, vegetables and cereals (% oven-dried substrate) Vegetal source Fruits Acerola (Malpighia emarginata DC.) Andean blackberry (Rubus glaucus Berth.) Blueberry (Vaccinium species) Brazilian mango (Mangifera indica L.) Capulí cherry (Prunus serotina Ehrh. var. Capulí). Grape (Vitis vinifera L.)

Phenolic compounds

Reference

CGA, EC, EGCG, PCyB1, R

Mezadri et al., 2008 pCouA-d, CyG, EA-d, EC, Et, GA, Gs, Vasco et al., PCYs, PeG, Q-d 2009 pHBA, C, CA, CGA, pCouA, Cy, De, Taruscio et al., EC, FeA, Myr, Ma, Peo, Pet, Q 2004 KG,Ma, iMa, Q, QAp, QAf, QG, QGal, Ribeiro et al., QR, QX 2008 C, CGA, CyG, EC, K-d, PCYs, Q-d Vasco et al., 2009

C, CA, HCA, oCouA, pCouA, GA, R, Q Kedage et al., 2007 Lemon (Melissa officinalis CA, mCouA, Er7G, He, Het, Na, Ng, Dastmalchi L.) RA et al., 2008 Plum (Prunus salicina CA-d, nCGA, pCouA-d, Cyg, EC, K-d, Vasco et al., Lindl.) (var. ‘Santa Rosa’ PCYs, Q-d 2009 and ‘Beauty’) Portuguese pear (Pyrus Ar, monC, terC, CQA, pCouMA, Ferreira et al., communis L. var. S. monEC, terEC, extEC 2002 Bartolomeu) Strawberry (Fragaria × CyG, EA, K, K-CouG, Pe, PeG, PeR, Zhang et al., ananassa Duch.) HPhAC, Q 2008 CGA, nCGA, pCouQA, CyG, EC, PeR, Usenik et al., Sweet cherry (Prunus avium L.) PeoR, R 2008 © Woodhead Publishing Limited, 2010

Extraction of natural antioxidants from plant foods 517 Table 18.3 Continued Vegetal source

Phenolic compounds

Reference

Vegetables Broccoli (Brassica oleracea L.) Escarole (Cichorium indivia var. latifolium L.) Lettuce (Lactuca sativa var. capitata L.) Onion (Allium cepa)

nCGA, FeGe, PCA, dSiGe, SiFeGe, SiGe, dSiFeGe, SidFeGe dCQA, CGA, CGA-d, CtA, ChiA, KGl, KMG dCQA,CGA, CGA-d, CtA, ChiA, Q-d, QMG FeA, GA, K, PCA, Q

Vallejo et al., 2003 Degl’innoocenti et al., 2008 Degl’innoocenti et al., 2008 Prakash et al., 2007 Reddivari et al., 2007 Degl’innoocenti et al., 2008 Aehle et al., 2004

Potato (Solanum Axa, C, CA, CGA, Cxa, GA, Lu, Vxa tuberosum L.) Rocket salad (Eruca sativa HCAs-d, K-d Mill.) Spinach (Spinacia Pa, Sp oleracea) Cereals Barley (Hordeum vulgare C, CA, pCouA, EC, FeA, GA, PCA, L.) SyA, VA Corn (Zea mays L.) CyG, aCyG, HCAs, pCouA, PCA, He-d, PeG, aPeG, PeoG, aPeoG, Q-d, VA Millets

pHBA, C, CA, CinA, pCouA, FeA, GA,GeA, L, Or, iOr, PCA, SaA, Sap, SiA, Tr, VA, Vi, Vt, iVt Rice (Oryza sativa L.) HBA, CA, CGA, pCouA, FeA, FS, PCA, SiA, SiS, SyA, VA Rye (Secale cereale L.) ALRs, pHBA, CA, FeA, SiA, SyA, VA, VeA Sorghum (Sorghum Apf, Ap, Api, ApiG, MApi, MApiG, bicolor L.) 5MApi, PApi, pHBA, CA, EC, pCouA, CinA, PCyB1, PDe, Er, Er5G, FeA, GA, PCA, GeA, KRG, Lut, Ltn, LtnG, PLtn, Lt, MLtn, MLtnG, 7MLtn, Ng, Ta, TaG, SaA, SiA, SyA, VA Wheat (Triticum aestivum CA, oCouA, pCouA, FeA, SyA, VA L.)

Zhao et al., 2006 Pedreschi and CisnerosZevallos, 2007 Dykes and Rooney, 2006 Tian et al., 2004 Heiniö et al., 2008 Dykes and Rooney, 2006

Mpofu et al., 2006

5MApi, 5-methoxyapigeninidin; 7-MApiG, 7-methoxyapigeninidin-5-glucoside; 7MLtn, 7-methoxyluteolinidin; aCyG, acylated cyanidin-3-glucoside; ALRs, alkylresorcinols; Ap, apigenin; aPeG, acylated pelargonidin-3-glucoside; aPeoG, acylated peonidin 3-glucoside; Apf, apiforol; Api, apigeninidin; ApiG, apigeninidin-5-glu; Ar, arbutin; Axa, antheraxanthin; C, catechin; CA, caffeic acid; CA-d, caffeic acid derivatives; CGA, chlorogenic acid; CGA-d, chlorogenic acid derivates; ChiA, chicoric acid; CinA, cinnamic acid; CQA, caffeoylquinic acid; CtA, caffeoyltartaric acid; Cxa, canthaxanthin; Cy, cyanidin; Cyg, cyanidin glycosides; CyG, cyanidin-3-glucoside; dCQA, dicaffeoylquinic acid; De, delphinidin; dSiFeGe, 1,2¢-disinapoyl-2-feruloylgentiobiose; dSiGe, 1,2-disinapoylgentiobiose; EA, ellagic acid; EA-d, ellagic acid derivatives; EC, epicatechin; EGCG, epigallocatechin gallate; Er, eriodictyol; Er5G, eriodictyol 5-glucoside; Er7G, eriodictyol-


518 Separation, extraction and concentration processes Table 18.3 Continued 7-O-glucoside; Et, ellagitannins; extEC, extension epicatechin; FeA, ferulic acid; FeGe, 1,2diferuloylgentiobiose; FS, 6¢-O-feruloylsucrose; GA, gallic acid; GeA, gentisic acid; Gs, galloyls; HBA, hydroxybenzoic acid; HCA, hydrocaffeic acid; HCAs, hydroxycinnamic acids; HCAs-d, hydroxycinnamic derivates; He, hesperidin; He-d, hesperidin derivates; Het, hesperetin; HPhAC, 3,4,5trihydroxyphenyl-acrylic acid; iMa, isomangiferin; iOr, isoorientin; iVt, isovitexin; K, kaempferol; K-CouG, kaempferol-3-(6¢-coumaroyl)glucoside; K-d, kaempferol derivatives; KG, kaempferol 3-O-glucoside; KGl, kaempferol-3-O-glucuronide; KMG, kaempferol-3-O-(6-O-malonylglucoside); KRG, kaempferol 3-rutinoside-7-glucuronide; L, lucenin-1; Lt, luteolin; Ltn, luteolinidin; LtnG, luteolinidin-5-glucoside; Lu, lutein; Lut, luteoforol; Ma, mangiferin; MApi, 7-methoxyapigeninidin; mCouA, m-coumaric acid; MLtn, 5-methoxyluteolinidin; MLtnG, 5-methoxyluteolinidin-7-glucoside; monC, monomeric catechin; monEC, monomeric epicatechin; Myr, myricetin; Na, naringin; nCGA, neochlorogenic acid; Ng, naringenin; oCouA, o-coumaric acid; Or, orientin; Pa, patuletin; PApi, proapigeninidin; PCA, protocatechuic acid; pCouA, p-coumaric acid; pCouA-d, p-coumaric acid derivatives; pCouMA, p-coumarylmalic acid; pCouQA, p-coumaroylquinic acid; PCyB1, procyanidin B1; PCYs proanthocyanidins; PDe, prodelphinidin; Pe, pelargonidin; PeG, pelargonidin-3-glucoside; Peo, peonidin; PeoG, peonidin 3-glucoside; PeoR, peonidin 3-rutinoside; PeR, pelargonidin-3rutinoside; Pet, petunidin; pHBA, p-hydroxybenzoic acid; PLtn, proluteolinidin; Q, quercetin; QAf, quercetin 3-O-arabinofuranoside; QAp, quercetin 3-O-arabinopyranoside; Q-d, quercetin derivatives; QG, quercetin 3-O-glucoside; QGal, quercetin 3-O-galactoside; QMG, quercetin-3-O(6-O-malonylglucoside); QR, quercetin 3-O-rhamnoside; QX, quercetin 3-O-xyloside; R, rutin; RA, rosmarinic acid; SaA, salicylic acid; Sap, saponarin; SiA, sinapic acid; SidFeGe, 1-sinapoyl-2,2¢diferuloylgentiobiose; SiFeGe, 1-sinapoyl-2-feruloylgentiobiose; SiGe, 1,2,2¢-trisinapoylgentiobiose; SiS, 6¢-O-sinapoylsucrose; Sp, spinacetin; SyA, syringic acid; Ta, taxifolin; TaG, taxifolin-7-glucoside; terC, terminal catechin; terEC, terminal epicatechin; Tr, tricin; VA, vanillic acid; VeA, veratric acid; Vi, violanthin; Vt, vitexin; Vxa, violaxanthin.

Table 18.4 Residual sources proposed for antioxidant manufacture Feedstock

References

Residues from grape and wine

Cruz et al., 2004; Corrales et al., 2008; Lafka et al., 2007; Makris et al., 2007; Pinelo et al., 2006; Arvanitoyannis et al., 2006; Louli et al., 2004

Apple pomace

Schieber et al., 2003

Olive

Obied et al., 2005; McDonald et al., 2001

Husks and hulls

Moure et al., 2000; Conde et al., 2008; Goli et al., 2005; Takeoka and Dao, 2003; Oliveira et al., 2008; DeliormanOrhan et al., 2009; Rubilar et al., 2007

Woods

Pérez-Bonilla et al., 2006; Castro et al., 2008; Moure et al., 2005

Leaves

Ferreira et al., 2007; Ozsoy et al., 2008

Seeds

Liu and Yao, 2007; Siddhuraju and Becker, 2007

Peels

Berardini et al., 2005; Li et al., 2006

Fruits and vegetables and their processed products and by-products have been extensively investigated. Their activity has been ascribed to the presence of phenolic compounds (Cieślik et al., 2006; Heinonen and Meyer, 2002; Macheix et al., 1990; Peschel et al., 2006; Proteggente et al., 2003; Robards et al., 1999; Sakakibara et al., 2003). Abundant research on fruits © Woodhead Publishing Limited, 2010

Extraction of natural antioxidants from plant foods 519 Table 18.5 Chemical composition of solid agro-industrial by-products (% oven-dried substrate) Solid agro-industrial Phenolic compounds by-product Almond hulls

References

CGA, cCGA, nCGA

Takeoka and Dao, 2003 Almond shells HCinAs, pCouA, VA, SyA Moure et al., 2007 Apple, peach and CA, p-CouA, FeA Gorinstein et al., pear peels 2002 Apple peels CA, C, EC, CGA, FeA, Q, QA, QG, QGal He and Liu, 2008 Schieber et al., 2003 Apple pomace C, EC, CGA, pCouA, pCouQA, PCyB2, FeA, Pht, Phx, Phz, Q, QA, QG, QGal, QR, QRu, QX Banana peel/pulp C, EC, CG Someya et al., 2002 Barley husks dHB, pCouA, GA, SyA, VA, V Conde et al., 2008 Quettier-Deleu Buckwheat hulls/ B2D, B2G, EC, ECG, QGal, QRu, Q et al., 2000 flour Chestnut wood 4HB, Con, EA, FeA, GA, Sin, Sc, SyA, Canas et al., 1999 Syra, V, VA, Um Coconut husks 4HBA, FeA Dey et al., 2003 Corn cobs AV, HMCin, iEu, MEu, Gu, Gua, EGu, Garrote et al., 2007a VGu, VPh, Sy, ASy, Syra, V Citrus peel and seed CA, p-CouA, Er, nEr, FeA, He, nHe, Na, Bocco et al., 1998 Nr, SiA Eucalypt wood 3HB, HMCin, Co, dHEu, MEu, Gu, VGu, Garrote et al., 2007b iEu, V, iVA, HVA Grape skin and seeds C, EC, CA, oCouA, pCouA, CyGl, GA, Lafka et al., 2007 PCA, SyA, Ty, HTy, VA Olive tree leaves ApG, C, CA, Di, DiG, Lt, LtG, Ol, R, Ty, Benavente-García HTy, V, VA, Ve et al., 2000 Olive tree pruning dHB, HMCin, Ol, Sy, Syra, Ty, HTy, V, Conde et al., 2009 HV, VA Potato peel GA, CGA, CA Kanatt et al., 2005 Rice husks AV, 4HB, HMCin, VPh, Gu, Gua, VGu, Garrote et al., 2007 Eu, iEu, MEu, Sy, ASy, Syra, V, HVA Sweetpotato leaves CA, CGA, 3,4dCQA, 3,5dCQA, Islam et al., 2002 4,5dCQA, tCQA Winemaking waste dHB, 3HBA, CinA, pCouA, GA, PCA, Cruz et al., 2004 solids SyA, VA, iVA Wood of olive tree De, iEu, MEu, Gu, Sy, Syra, HSyA, Ty, Castro et al., 2008 branches HTy, Ol, V, HV 3,4dCQA, 3,4-di-O-caffeoylquinic acid; 3,5dCQA, 3,5-di-O-caffeoylquinic acid; 3HB, 3-hydroxybenzaldehyde; 3HBA, 3-hydroxybenzoic acid; 4,5dCQA, 4,5-di-O-caffeoylquinic acid; 4-HB, 4-hydroxybenzaldehyde; 4HBA, 4-hydroxybenzoic acid; ApG, apigenin-7-glucoside; ASy, acetosyringone; AV, acetovanillone; B2D, proanthocyanidin B2; B2G, B2-3´-O-gallate; C, catechin; CA, caffeic acid; cCGA, cryptochlorogenic acid; CG, catechin gallate; CGA, chlorogenic acid; CinA,


520 Separation, extraction and concentration processes Table 18.5 Continued cinnamic acid; Co, coniferol; Con, coniferaldehyde; CYGl, cyanidin glycosides; De, desaspidinol; dHB, 3,4-dihydroxybenzaldehyde; dHEu, dihydroeugenol; Di, diosmetin; DiG, diosmetin-7-glucoside; EA, ellagic acid, EC, epicatechin; ECG, epicatechin gallate; EGu, 4-ethylguaiacol; ER, eriocitrin; Eu, 4-eugenol; FeA, ferulic acid; GA, gallic acid; Gu, guaiacol; Gua, guaiacylacetone; HCinAs, hydroxycinnamic acids; He, hesperidin; HMCin, 4-hydroxy-2-methoxycinnamaldehyde; HSyA, homosyringic acid; HTy, hydroxytyrosol; HV, homovanillyl alcohol; HVA, homovanillic acid; iEu, isoeugenol; iVA, isovanillic acid; Lt, luteolin; LtG, luteolin-7-glucoside; MEu, methoxyeugenol; Na, naringin; nCGA, neochlorogenic acid; nEr, neoeriocitrin; nHe, neohesperidin; Nr, narirutin; oCouA, o-coumaric acid; OL, oleuropein; PCA, protocatechuic acid; pCouA, p-coumaric acid; pCouQA, p-coumaroylquinic acid; PCY-B2, procyanidin B2; Pht, phloretin; Phx, phloretin xyloglucoside; Phz, phloridzin; Q, quercetin; QA, quercetin-3-arabinoside; QG, quercetin-3-glucoside; QGal, quercetin3-galactoside; QR, quercetin-3-rhamnoside; QRu, quercetin-3-rutinoside; QX, quercetin-3-xyloside; R, rutin; Sc, scopoletin; SiA, sinapic acid; Sin, sinapaldehyde; Sy, syringol; SyA, syringic acid; Syra, syringaldehyde; tCQA, 3,4,5-tri-O-caffeoylquinic acid; Ty, tyrosol; Um, umbelliferone; V, vanillin; VA, vanillic acid; Ve, verbascoside;VGu, 4-vinylguaiacol; VPh, 4-vinylphenol.

is available, including citrus (Dastmalchi et al., 2008; Di Majo et al., 2005; Jayaprakasha and Patil, 2007; Tripoli et al., 2007), berry fruits (Szajdek and Borowska, 2008; Vasco et al., 2009; Taruscio et al., 2004; Zhang et al., 2008) and cherries (Piccolella et al., 2008). Results have also been reported for juices (Gardner et al., 2000), teas (Gupta et al., 2008; von Gadow et al., 1997; Wiseman et al., 1997) coffee (Nardini et al., 2002), wines (Burns et al., 2001; Fogliano et al., 1999; Minussi et al., 2003; Sanchez-Moreno et al., 2003; Villaño et al., 2005), and several beverages (Arts et al., 2000; Richelle et al., 2001; Tabart et al., 2009). The potential of aromatic herbs, spices and medicinal plants was first assessed in the 1950s (Chipault et al., 1952). Their major antioxidant components are vitamins, phenolic acids, flavonoids and flavonoid derivatives, terpenoids, carotenoids, phytoestrogens and minerals. Specific compounds from plants include carnosic acid, carnosol, rosmarinic acid, rosmanol, thymol, carvacrol, gingerol-related compounds, curcumins, capsaicin, capsaicinol, and ascorbic acid (Suhaj, 2006; Yanishlieva et al., 2006). Their effectiveness in different substrates and other biological activities have been reviewed (Srinivasan, 2005; Yanishlieva et al., 2006; Zheng and Wang, 2001). The in vitro antioxidant capacity of cereals is significantly correlated with their polyphenol content, except for maize (Decker et al., 2002). During digestion, the antioxidant capacity of cereals is increased, providing a favorable antioxidative environment for the epithelium in the large intestine (Fardet et al., 2008). However, the in vitro antioxidant capacity of cereals is only an approximate reflection of their in vivo antioxidant effect, owing to differences in solubility/bioavailability within the digestive tract and to the metabolism/conjugation with compounds such as polyphenols. About 6000 seaweed species have been identified and they are grouped into green (chlorophytes), brown (pheophytes) and red (rhodophytes) algae.


Extraction of natural antioxidants from plant foods 521 Seaweeds are traditionally exploited mainly for soluble polysaccharides and direct utilization as foods. The soluble fraction of red seaweeds is mostly composed of sulfated galactans (agar, carrageenans), whereas the brown seaweeds (alginates, fucans, and laminarans) correspond mostly to reserve b-glucans. The recovery of valuable biomolecules, including vitamins, minerals, lipids, polyphenols, proteins, sterols, pigments, tocopherol derivatives and related isoprenoids, is a topic of growing interest (Herrero et al., 2006; Kumar et al., 2008). The most potent antioxidants of algae are phlorotannins and sulfated polysaccharides (Rupérez et al., 2002; Wang et al., 2008; Xue et al., 2001; Ye et al., 2008). A recent work has reported the irradiation-dependent production of butylhydroxytoluene (BHT) by green algae and cyanobacteria (Babu and Wu, 2008). Mushrooms are also a source of phenolic antioxidants (Cheung et al., 2003).

18.4 Biological activities of natural antioxidants Reactive oxygen species (ROS) play a dual role: a certain physiological level of ROS is crucial for the regulation of cell functions (Wang and Yi, 2008), but ROS and other radicals are also involved in diabetes, cancer, liver disease, aging, arthritis, AIDS, macular degeneration, and autoimmune, inflammatory, cardiovascular and neurodegenerative diseases. Antioxidants prevent ROS concentrations from reaching a damaging level and have been considered as a promising therapy for the prevention and treatment of these diseases (Seifried et al., 2007). The results from many intervention trials with antioxidants failed to demonstrate benefits in humans, probably because the reactive species may not be important, the pathologies were too advanced, the doses used may be wrong or the administered antioxidants do not decrease the oxidative damage (Halliwell, 2009). Antioxidant properties alone are not sufficient to explain their biological properties, some of which are indicated in Table 18.6. Although an antioxidant is a redox agent that in the presence of metal ions could act as a pro-oxidant, in vivo most transition metal ions are protein-conjugated and not available to catalyze free radical reactions (Hadi et al., 2007). The most studied compounds for controlling various disorders (including cardiovascular, neurological and neoplastic diseases) are plant-derived polyphenols. The use of the cardioprotective properties of dietary antioxidants against cardiovascular diseases has been studied and some conflicting findings have been found (Seifried et al., 2007). Natural polyphenolic compounds possess antioxidant, vasorelaxant and antihypertensive properties (Depeint et al., 2002; Erlund, 2004; Ghosh, 2005; Ullah and Khan, 2008). Some effects of polyphenols (such as anti-atherogenesis) can be attributed to their antioxidant properties (German and Walzem, 2000). Selected polyphenols



Biological activities

Plant or extract

Active compounds

Reference

Antiallergic Antiarthritic

Coronopus didymus Cleome gynandra

Antiasthmatic Antibacterial Anticancer Anticoagulant Antidiabetic Antiglycative Antihyperglycemic

Laurencia undulata Cyperus rotundus Prunus serrulata Cirsium japonicum Psidium guajava L. Helichrysum plicatum Psidium guajava L. Punica granatum

Mantena et al., 2005 Narendhirakannan et al., 2005 Jung et al., 2009 Kilani et al., 2008 Lee et al., 2007 Yin et al., 2008 Hsieh et al., 2007 Aslan et al., 2007 Hsieh et al., 2007 Bagri et al., 2009

Antihyperlipidemic

Coronopus didymus Helichrysum plicatum Punica granatum

Flavonoids, tannins Polyphenolic compounds, tannins, anthroquinones, flavonoids Phlorotannins Tannins, flavonoids, coumarins, sterols Phenolic glucosides, flavonoids, anthocyanins Phenolic acids, flavonoids Phenolic compounds Phenolic acids, flavonoids Phenolic compounds Alkaloids, flavonoids, anthocyanins, hydrolyzable tannins Flavonoids, tannins Phenolic acids, flavonoids Alkaloids, flavonoids, anthocyanins, hydrolyzable tannins Flavonoids and phenolics acids Polyphenols, tannins Bioflavonoids, catechins, procyanidins, phenolic acids Monoterpene glycosides Eugenol Curcuminoids Polyphenols Oligomeric procyanidins, catechins, condensed tannins

Souza et al., 2007 Kim et al., 2007 Hasegawa et al., 2008 Panich et al., 2009 Panich et al., 2009 Ahn et al., 2008 Esquenazi et al., 2002

Anti-inflammatory

Antimelanogenic

Antimicrobial

Geranium robertianum L. Uncaria tomentosa Myracrodruon urundeuva Pycnogenol® Eucalyptus globulus Almina galanga Curcuma aromatic Sasa quelpaertensis Nakai Cocos nucifera L.

Mantena et al., 2005 Aslan et al., 2007 Bagri et al., 2009 Amaral et al., 2009


Table 18.6 Biological activities reported for antioxidant extracts from some plant foods

Pycnogenol® and Ginkgo biloba Hibiscus tiliaceus L.

Antiparkinson Antipyretic Antitumor Antiulcer Antiviral Apoptotic activities Cytotoxic Hepatoprotective

Mucuna pruriens Coronopus didymus Senna alata Myracrodruon urundeuva Cocos nucifera L. Cyperus rotundus Cyperus rotundus Coronopus didymus Commiphora berryi

Polyphenols, procyanidins Polyphenols, carotenoids, tocopherols, flavonoids, anthocyanins, phytosterols Polyphenols Flavonoids, tannins Flavonoids, tannins, polyphenols Polyphenols, tannins Oligomeric procyanidins, catechins, condensed tannins Tannins, flavonoids, coumarins, sterols Tannins, flavonoids, coumarins, sterols Flavonoids, tannins Phenolic compounds, tannins

Decalepis hamiltonii

Phenolic compounds

Ecklonia cava

Polyphenols

Dhanasekaran et al., 2008 Mantena et al., 2005 Pieme et al., 2008 Souza et al., 2007 Esquenazi et al., 2002 Kilani et al., 2008 Kilani et al., 2008 Mantena et al., 2005 Gowri Shankar et al., 2008 Srivastava and Shivanandappa, 2009 Ahn et al., 2008

Morus alba L.

Flavonol glycosides

Katsube et al., 2006

Castanea sativa

Polyphenols

Almeida et al., 2008

Immunomodulatory activity Inhibitor LDL oxidation Skin protector

Križková et al., 2008 Rosa et al., 2006



Antimutagenic

524 Separation, extraction and concentration processes (extracts from grape and wine, resveratrol, genistein, dihydrodaidzeins) relax endothelial function (Shen et al., 2006). Flavonoids can have favorable effects on atherosclerosis. Lipid-lowering activity has been reported for tea flavonoids (Li et al., 2005), a-tocopherol, and b-carotene, which inhibit the oxidation of LDL and the atherosclerotic process (Odeh and Cornish, 1995). Tocopherols show enhanced immune response and regulation of platelet aggregation (Weber and Rimbach, 2002). Protection against coronary heart disease was reported for resveratrol (Szewczuk and Penning, 2004), anthocyanidins, EGCG, and vitamin E (Violi et al., 2006). Biologically active peptides promote diverse activities (antithrombotic, hypocholesterolemic and antihypertensive actions) relevant to cardiovascular health (Erdmann et al., 2008). Inhibitors of angiotensin-converting enzyme (ACE) play an important role in the regulation of blood pressure and fluid and salt balance in mammals. Immunomodulatory activity has also been reported for peptides (Erdmann et al., 2008). Antiallergenic activity has been reported for flavonoids (Das and Rosazza, 2006; Ghosh, 2005; Hodek et al., 2002; McKay and Blumberg, 2006), which may also preserve T-cell-mediated immunity (Horváthová et al., 2001; Strickland, 2001). Bioactive compounds such as flavonoids (Horváthová et al., 2001) and vitamin E protects against cataracts. Vitamin E decreases oxidative stress and the levels of erythrocyte osmotic fragility in patients on dialysis (Uzum et al., 2006) and had beneficial effects on diabetic patients (Levy and Blum, 2007). Epidemiological studies suggested a positive association between a diet rich in fruit and vegetables and a lower incidence of cancer (Ames et al., 1993; Pan et al., 2008a; Ren et al., 2003; Tanaka and Sugie, 2008). The antioxidant properties may only partly explain the antitumor effects of dietary phenolics, which may exert modulatory actions in cells, interfering in the steps leading to the development of malignant tumors (Kanakis et al., 2007). Supplemental antioxidants may help to prevent cancer only in dietdeficient populations or individuals, and antioxidant anticancer agents may affect patients differently according to their health (Vickers, 2007). Although dietary antioxidants have attracted great interest and are considered safe, they have to pass controlled clinical trials for therapeutic or chemopreventive use, and potential toxic flavonoid–drug interactions should be considered (Galati and O’Brien, 2004). Many natural agents have shown potential in bioassays and animal models, and some of them have been selected for ongoing phase I–III clinical trials (Bonfili et al., 2008). An ideal, effective and acceptable agent should be non-toxic in normal and healthy cells, low cost and efficient against multiple cancers, act according to a known mechanism, deserve public acceptance, and be suitable for oral consumption. Potential cancer preventive agents suppress carcinogenesis by several major mechanisms: inhibiting phase I enzymes or blocking carcinogen formation, inducing phase II (detoxification) enzymes, scavenging DNA reactive agents, suppressing the over-expression of pro-oxidant enzymes, modulating hormone homeostasis,


Extraction of natural antioxidants from plant foods 525 suppressing hyper-cell proliferation induced by carcinogens, inducing apoptosis, counteracting angiogenesis, and/or inhibiting certain phenotypic expressions of preneoplastic and neoplastic cells (D’Archivio et al., 2008; Shklar, 1998; Ren et al., 2003; Pan et al., 2008a). Various dietary antioxidants are under investigation for their anticancer properties, including curcumin (Tsvetkov et al., 2005; Maheshwari et al., 2006), oleuropein (Bonfili et al., 2008), and tangeretin, nobiletin and resveratrol (Narayanan, 2006). Green tea inhibits tumor incidence and multiplicity in various organs, and recent phase I and II clinical trials have been conducted to explore its anticancer effects (Dou, 2008). Flavonoids in human diet may reduce the risk of various cancers (Kanadaswami et al., 2005), including hormone-dependent breast and prostate cancers (Hodek et al., 2002), intestinal neoplasia (Hoensch and Kirch, 2005) and skin cancer (Singh and Agarwal, 2002). Lycopene shows both in vitro and in vivo anticancer activities, possibly through ROS scavenging, up-regulation of detoxification systems, interference with cell proliferation, induction of gap-junctional communication, inhibition of cell cycle progression and modulation of signal transduction pathways (Bhuvaneswari and Nagini, 2005). A beneficial role of vitamin C on cancer has been claimed, although further clinical trials are needed (Verrax and Calderón, 2008). Vitamin E and vitamin E-based compounds are potent antioxidants regulating peroxidation, some forms display apoptotic activity against cancer cells and restore multidrug resistant tumor cells sensitivity to chemotherapeutic agents (Kline et al., 2007; Sylvester, 2007). Among soy peptides, lunasin suppresses transformation of cells induced by carcinogens and viral oncogenes (De Mejia and De Lumen, 2006), and the Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase up-regulation (Xiao et al., 2005). Combinations of antioxidant nutrients act synergistically, and their use is proposed to offer a better quality of life (Shklar, 1998), and to present a co-operative action with chemotherapeutic drugs and radiation therapy (Bonfili et al., 2008; Prasad et al., 2002). The nervous system, rich in fatty acids and iron, is vulnerable to free radical generation (Ullah and Khan, 2008). Reactive species are constantly produced in the brain, for example by leakage of electrons from the mitochondrial electron transport chain to generate superoxide radical (O2–). Increased levels of oxidative damage may occur early in the progression of many neurodegenerative diseases (Philips et al., 1993), as oxidative damage may aggravate the accumulation and precipitation of proteins. Epidemiological studies indicate that dietary antioxidants can limit the incidence of neurodegenerative disorders (Morris, 2002). Polyphenols are active agents in neuroprotection owing to their ability to influence and modulate several cellular processes such as signaling, proliferation, apoptosis, redox balance, and differentiation. Their role in neurological disorders has been studied (Dajas et al., 2002; Horváthová et al., 2001; Singh and Singh, 2008). Promising compounds for neuroprotection include polyphenols such as epigallocatechin gallate (EGCG), curcumin, naringenin, extracts of


526 Separation, extraction and concentration processes blueberries and Scutellaria (Ullah and Khan, 2008), and extracts from green tea (Bastianetto et al., 2006). Flavonoids and their derivatives are reported to inhibit the growth and development of HIV by suppressing acute infection and inhibiting protease, integrase and reverse transcriptase (Critchfield et al., 1996), whereas scutellarin showed anti-HIV activity (Zhang et al., 2005). The overall role of reactive species in chronic inflammatory diseases is not clear. Reactive species may help explain why such diseases increase cancer risk, but ironically these species can sometimes be anti-inflammatory. Hence the use of antioxidants to treat chronic inflammatory diseases may not be as simple as it originally sounded (Halliwell, 2009). Natural antioxidants with anti-inflamatory activity include phenolics (Lomnitski et al., 2000), resveratrol, flavonoids (Ghosh, 2005; Das and Rosazza, 2006; Hodek et al., 2002; Kim et al., 2004), terpenoids (De las Heras et al., 2003), and glucans (Tsiapali et al., 2001). Therapeutic potential for combating bronchial asthma has been reported for polyphenolic extracts from Laurencia undulata, an edible red alga (Jung et al., 2009). Tyrosinase inhibitors can ameliorate cutaneous hyperpigmentary disorders. Their use is becoming increasingly important in the cosmetic industry owing to their skin whitening action and preventive effects, and there is a growing interest in their medicinal and cosmetic applications (Parvez et al., 2007). Some carotenoids (a- and b-carotene and b-cryptoxanthin) are precursors of vitamin A and protect against chemical oxidative damage and against several kinds of cancer, age-related macular degeneration and UV-induced erythema.

18.5 Extraction of natural antioxidants from plant foods and residues Solid–liquid extraction is a heterogeneous operation involving transfer of solutes from a solid to a fluid. Extractable compounds of vegetal origin are a complex mixture of solutes that can be extracted at different rates depending on their location (outer surface, pores and vacuoles). Extraction involves the following sequential steps: (i) transport of solvent from the bulk solution to the external surface of the particle, (ii) solvent penetration and diffusion in the solid matrix, (iii) solubilization of the components, (iv) transport of the solute(s) through the solid matrix, and (v) transport of the solute(s) from the external surface of the solid to the bulk solution. 18.5.1 Conventional solvent extraction The type of solvent is one of the most influential variables on both extraction yield and type of extracted solutes. Methanol, ethanol, and water are widely


Extraction of natural antioxidants from plant foods 527 employed for extracting phenols. Methanol exhibits the highest capacity to extract phenolics (Pinelo et al., 2005), but shows toxicity, whereas hot water may extract polyphenols without safety concerns. Ethanol–water mixtures are suited to penetrate the hydrophobic areas of the vegetable matrix and help to precipitate soluble proteins, facilitating further processing. Representative examples of extraction processes involving these solvents are summarized in Table 18.7. Temperature is one of the most influential variables affecting the release of phenols. Increased temperatures favor extraction by enhancing both solubility and diffusion. However, temperature cannot be increased indefinitely, because instability of phenolic compounds and denaturation of membranes may take place at temperatures above 50 °C (Cacace and Mazza, 2003). Partially oxidized polyphenols can exhibit higher antioxidant activity than non-oxidized phenols. Thermal treatment of crude extracts can be performed to modify their composition by oxidation, hydrolysis and isomerization, and can result in the formation of compounds with new antioxidant properties. Therefore, heat-induced antioxidants balance the thermal loss of antioxidants, or the overall antioxidant properties can be increased upon heating (Delgado-Andrade and Morales, 2005). This behavior was reported for catechin, resveratrol, grape extract (Pinelo et al., 2005), and citrus peel (Xu et al., 2007). Steam-processing of broccoli may release more bound phenolic acids from the breakdown of cellular constituents (Roy et al., 2009). Heating vegetal products such as sweet corn increased the total antioxidant activity and the phenolic content (Dewanto et al., 2002). Heat treatment of huyou (Citrus paradisi Changshanhuyou) peel increased the free fraction of phenolic acids and decreased the ester, glycoside, and ester-bound fractions (Xu et al., 2007), whereas the antioxidant capacity of citrus peel [measured by the ABTS and ferric-reducing antioxidant power (FRAP) assays] increased with heating time and temperature owing to the increase of lower MW phenolics (benzoic acids and cinnamic acids). High solvent-to-solid ratios favor phenol yields, but a balance between high costs (derived from solvent usage) and absence of saturation effects has to be found (Pinelo et al., 2005). However, operational variables such as temperature, solvent-to-solid ratio, and type of solvent used only affect solutes weakly linked to the cell wall structure and those contained in vacuoles. Extraction of compounds forming part of the cell walls may require other strategies, such as cell wall breaking or enzymatic treatments. Even though conventional extraction with organic solvents is widely used, it has low efficiency and requires large volumes of solvent, long extraction times and high temperature. This could lead to loss of biological activities and decreased selectivity, as well as to problems derived from organic solvent cost and disposal (Robards 2003). Additionally, organic solvents must be completely removed from the exhausted solids. Recently, there has been an increasing interest in using environmentally friendly extraction technologies for producing extracts of high quality and activity. Some of these techniques



Raw material

Solvent

Solubles yield or Antioxidant activity TEP* (% dry weight)

Reference

Anethum graveolens (flowers)

W

8

DPPH(–)*1; bC(–)*1

Sharififar et al., 2009


E

2.84

TEAC(–)*2; DPPH(–)*2,3; RP(–)*2,3; FIC(–)*4; bC(–)*2

EA

8.28

Hordeum vulgare (seeds) Chamaecyparis obtusa (bark)

E (70%)

4.70

TEAC(–) ; DPPH(–) ; RP(–) ; FIC(–) ; bC(–) RP(–)*1,3; DPPH(–)*1,3; FTC(+)*1(=)*3

E

9.07 (GA)

DPPH(–)*2; TEAC(–)*5; RP(–)*5; bC(–)*5

EA

10.28 (GA)

Cirsium japonicum (leaves) Chukrasia tabularis (leaves)

W

24.13

DPPH(–) ; TEAC(–) ; RP(–) ; bC(–) DPPH(–)*3,6; RP(–)*6; HOSC(–)*6

EA W E (95%)

98.35 (GA) 60.88 (GA) 9.68 (PC)

DPPH(–)*7; RP(–)*7; HOSC(+)*7 DPPH(–)*7; RP(–)*7; HOSC(–)*7 DPPH(+)*1; HOSC(+)*1; RP(+)*1; TA(+)*1; LPPO(+)*1

Kaur et al., 2008

E (96%)

17.74

DPPH(–)*1,8; bC(–)*1,8

Moure et al., 2001

W

17.36

EA E W W

1.09 (GA) 1.94 (GA)

DPPH(–)*1,8; bC(–)*1,8 DPPH(–)*1,(+)*6; FIC(–)*3; RP(–)*1 DPPH(–)*1,(+)*6; FIC(–)*3; RP(–)*1; FTC(+)*1 DPPH(–)*1,6; RP(–)*1 DPPH(–)*2,3; RP(–)*2,3; TEAC(–)*2

Dimocarpus longan Lour. (peel) Gevuina avellana (seeds) Kappaphycus alvarezii

Litchi chinenesis (flowers)

9.92

*2

*2

*2,3

*5

*2,3

*5

*4

Shyu et al., 2009

*2

Liu and Yao, 2007 Marimuthu et al., 2008

*5

Yin et al., 2008

Pan et al., 2008b

Kumar et al., 2008

Liu et al., 2009


Table 18.7 Examples of extraction of plant food antioxidants using conventional solvent extraction. Symbols (+), (–) and (=) indicate higher/ lower/equal activity compared with the reference standard compound

W

1.60

DPPH(–)*1,8; bC(–)*1,8

W

30.96

FTC(+)*6; bX(+)*7(–)*8; DPPH(–)*7,8; SRS(–)*7; HOSC (–)*7,8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4

E

34.46

FTC(+)*6; bC(+)*7(–)*8; DPPH(–)*7,8; HOSC(+)*7(–)*8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4

EA

10.61

E

37 (GA)

FTC(+)*6, bC(+)*7(–)*8, DPPH(–)*7,8; HOSC(+)*7(–) *8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4 DPPH(–)*2,3,5,7,12(+)*1; SRS(+)*2,12; HOSC(–)*1,3,5 (+)*2,12

E

34 (GA)

DPPH(–)*1,2,3,5,7,12; SRS(–)*2,12; HOSC(–)*1,2,3,5,12

E

27 (GA)

DPPH(–)*1,3,7(+)*2,5,12; SRS(–)*2,12; HOSC(–)

W

–

FTC(+)*6; RP(+)*6; SRS(+)*1,6,8; DPPH(–)*5(+)*8; FIC(+)*1,6,8; HPS(–)*1,6,8

W E (50%)

8, 5.4 (GA) 12, 16.8 (GA)

Citrus sinensis

E EA

10, 10.4 (GA) 9.12, 6.43 (C)

Citrus grandis

EA

3.41, 9.72 (C)

Randia echinocarpa W (pulp) Rosa rubiginosa (seeds) E (96%) Smilax excelsa (leaves)

Syzygium cumini (kernel) Syzygium cumini (pulp) Syzygium cumini (seed coat) Urtica dioica (aerial parts)

9.46

*1,2,3,5,12

Ozsoy et al., 2008

Benherlal and Arumughan, 2007 Benherlal and Arumughan, 2007 Benherlal and Arumughan, 2007 Gülçin et al., 2004



1.97

DPPH(–)*3,6, 8,9,10; TBARS(–)*6,8; SRS(–)*11; HOSC(–)*8 Ningappa et al., 2007 DPPH(+)*2,3,8,9,10; TBARS(+)*6,8; SRS(+)*11; HOSC(+)*8; RP(+)*8; FIC(–)*4 DPPH(–)*3,6, 8,9(+)*10; TBARS(+)*6,8; SRS(–)*11; HOSC(–)*8 DPPH(–)*3; ORAC(–)*12; ABTS(–)*3; RP(–)*2; PM(+)*13 Jayaprakasha et al., 2008 *3 *12 *3 *2 *13 Jayaprakasha et al., DPPH(–) ; ORAC(–) ; ABTS(–) ; RP(–) ; PM(+) 2008 *1 Santos–Cervantes bC(–) et al., 2007 *1,8 *1,8 Moure et al., 2001 DPPH(–) ; bC(–)

Murraya koenigii (leaves)


*Total extractable polyphenols. E, ethanol; EA, ethyl acetate; W, water. C, catechin; GA, gallic acid; PC, pyrocatechol. *1 , butylhydroxytoluene (BHT); *2, (+)-catechin; *3, ascorbic acid; *4, ethylene diamine tetra-acetic acid (EDTA); *5, quercetin;*6, a-tocopherol; *7, gallic acid; *8, butylhydroxyanisol (BHA); *9, curcumin; *10, b-carotene;*11, superoxide dismutases (SOD); *12, trolox; *13, propyl gallate. bC, b-carotene bleaching method; DPPH, a,a-diphenyl-b-picrylhydrazyl radical scavenging assay; FIC, ferrous ion-chelating assay; FTC, antioxidant activity in the linoleic acid system with ferrothiocyanate reagent; HOSC, hydroxyl radical scavenging assay; HPS, hydrogen peroxide scavenging assay; LPPO, lipid peroxidation in peanut oil; ORAC, oxygen-radical absorbance capacity; PM, phosphomolybdenum method; RP, reducing power; SRS, superoxide radical scavenging assay; TA, total antioxidant capacity assay; TBARS, thiobarbituric acid-reacting substances; TEAC, Trolox equivalent antioxidant capacity assay.



Extraction of natural antioxidants from plant foods 531 (enzyme-assisted aqueous extraction, some novel methods including subcritical water, pressurized liquid extraction, supercritical fluid extraction (SFE) with CO2, and ultrasound- and microwave-assisted processes) are considered in the following subsections. 18.5.2 Enzyme-aided extraction Enzyme processing is used as a pretreatment for conventional and alternative solvent extraction technologies, owing to the ability of enzymes to disrupt cell walls. Enzyme-aided extraction is considered more environmentally friendly than chemical treatments, and the further utilization of both residue and extract is possible (Mandalari et al., 2006). Enzyme treatments have been considered for a variety of materials, including algae, fruits, herbs and cereals. Edible and other abundant and underutilized seaweeds have been studied for extracting water-soluble compounds with antioxidant activity. Most antioxidant compounds are soluble in polar solvents, but the large amounts of highly viscous polysaccharides act as a physical barrier. Enzymes can break down the cell walls and storage materials to release both free compounds and polysaccharides attached to polyphenols (Heo et al., 2005). Representative studies of antioxidant extraction from algae biomass using hydrolytic enzymes are summarized in Table 18.8. Synergistic effects between different enzyme activities (including endopeptidases, carbohydrases and proteases) enhance the release of antioxidants (Siriwardhana et al., 2008; Heo et al., 2005). The variables affecting the extraction yield and activity include: type of raw material and pre-conditioning stages, temperature, type and concentration of solvent, and type and concentration of enzyme(s). This latter effect is shown in Fig. 18.2. Incubation results in increased extraction efficacy (Siriwardhana et al., 2008). Thermal degradation of high-MW polysaccharides enables higher extraction of target antioxidants. Some enzymatic hydrolysates are stable at 100 °C (Athukorala et al., 2006; Heo et al., 2005) and the DPPH radical scavenging activity increases with heating time (Siriwardhana et al., 2008). Heating followed by enzymatic hydrolysis enhances the access of endopeptidases and b-glucanase to laminarin and proteins. Increased antioxidant activity at alkaline pH was observed in Hizikia fusiformis, owing to hydrolysis, ion exchange, desulfation and prevention of the formation of the protein–polyphenol complex (Siriwardhana et al., 2008). A simple procedure to reduce the viscosity of porphyrans of (sulfated polysaccharide of Porphyra) various MW, consisting of treatments with hydroxyl radical, has been reported (Zhang et al., 2003). Algal enzymatic extracts show a variety of other biological activities. The enzymatic extract of Ecklonia cava inhibited proliferation of cancer cells strongly and selectively (Athukorala et al., 2006), and showed in vivo anti-inflammatory, inmunomodulatory and antiproliferative activity (Ahn et al., 2008). Highly sulfated fucose and galactose polysaccharides from


Table 18.8 Examples of enzyme-aided extraction of plant food antioxidants

Algae Ecklonia cava Hizikia fusiformis © Woodhead Publishing Limited, 2010

Ishige okamurae Sargassum fullvelum Sargassum horneri Sargassum coreanum Sargassum thunbergii Scytosipon lomentaria Undaria pinnatifida

Terrestrial plants Buckwheat (Fagopyrum esculentum) Caroleo (Olea europaea)

Enzymes

Extraction yield (total phenolics)

Antioxidant activity

Reference

Viscozyme Alcalase 2.4L FG Alcalase and Ultraflo

1352 mg GAE g–1 1095 mg GAE g–1

DPPH; HO˙; O2˙; H2O2

Heo et al., 2005

DPPH; H2O2

Ultraflo L Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Viscozyme Protamex Viscozyme Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Alcalase, Flavourzyme, Promozyme, Viscozyme, Celluclast

275 mg GAE g–1 420 mg GAE g–1 313 mg GAE g–1 366 mg GAE g–1 384 mg GAE g–1 533 mg GAE g–1 1123 mg GAE g–1 996 mg GAE g–1 386 mg GAE g–1 416 mg GAE g–1 149 mg GAE g–1 207 mg GAE g–1

DPPH; HO˙; O2–; H2O2

Siriwardhana et al., 2008 Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005

DPPH; HO–; O2–

Je et al., 2009

Alcalase

0.371 mg R g–1

DPPH; reducing power; inhibition of LA peroxidation

Tang et al., 2009

Bioliva

234.1 mg CA kg–1

Oxidative stability; peroxide value Ranalli et al., 2003b


Vegetal material (Latin name)

Leccino (Olea europaea) Leccino (Olea europaea) Lemon peel (Citrus limon cv. Meyer) Lemon peel (Citrus limon cv. Yen Ben) Mandarin peel (Citrus reticulara cv. Ellendale)

Bioliva

362.2 mg CA kg–1

Oxidative stability; peroxide value Ranalli et al., 2003a

Rapidase adex D

128 mg CA kg–1


Cytolase

170 mg CA kg–1

Oxidative stability; peroxide value Ranalli et al., 1999

Cytolase

79 mg CA kg–1


Rapidase adex D

92 mg CA kg–1


Cellulase

220 mg F g–1

Huang et al., 2009

Pectinases and cellulases

89 mg CA g–1

HO˙; O2–; inhibition of lipid peroxidation; reducing power DPPH; oxidative stability

Cellulase MX Cellulase CL Kleerase AFP Bioliva

1.52 mg GAE g–1 1.30 mg GAE g–1 1.25 mg GAE g–1 121.2 mg CA kg–1

FRAP

Li et al., 2006

Cytolase

121 mg CA kg–1

Oxidative stability

Ranalli et al., 1999

Cellulase MX Cellulase CL Kleerase AFP Cellulase MX Cellulase CL Kleerase AFP Cellulase MX Cellulase CL Kleerase AFP

0.4429 mg GAE g–1 0.4895 mg GAE g–1 0.4375 mg GAE g–1 1.131 mg GAE g–1 0.99 mg GAE g–1 1.108 mg GAE g–1 1.39 mg GAE g–1 1.15 mg GAE g–1 1.19 mg GAE g–1

FRAP

Li et al., 2006

FRAP

Li et al., 2006

FRAP

Li et al., 2006

Ramadan et al., 2008




Coratina (Olea europaea) Coratina (Olea europaea) Coratina (Olea europaea) Dritta (Olea europaea) Dritta (Olea europaea) Eucommia leaf (Folium eucommiae) Goldenberry (Physalis peruviana) Grapefruit peel (Citrus x paradisi)



Enzymes



Moringa concanensis seeds

1.283 mg T kg–1 1.236 mg T kg–1 1.176 mg T kg–1

Oxidative stability; peroxide value; Latif and Anwar, conjugated diene/triene 2009

Sunflower (Helianthus annuus)

Kemzyme Natuzyme Feedzyme Protex 7L Kemzyme Alcalase 2.4L Viscozyme L Natuzyme

Sweet orange peel (Citrus sinensis cv. Navel)

Cellulase MX Cellulase CL Kleerase AFP

0.80 mg GAE g–1 0.79 mg GAE g–1 0.90 mg GAE g–1

FRAP

Li et al., 2006

Macer8 Fj (M) Grindamyl pectinase (G) (M) + (G) Olivex and Celluclast

358 mg GAE L–1 237 mg GAE L–1 383 mg GAE L–1 5.2 g C kg–1

Conjugated diene

Landbo and Meyer, 2001

DPPH

Soto et al., 2008

Grindamyl pectinase (G) Celluclast (C) (G) + (C) Ultrazym 100G and Cellubrix Ferulic acid esterase

3072 mg GAE L–1 2773 mg GAE L–1 3017 mg GAE L–1 0.25 g C g–1

Conjugated diene

Meyer et al., 1998

DPPH

Collao et al., 2007

6937 mg SAE g–1

DPPH; conjugated diene; hydroperoxide; hexanal; liposome model system

Vuorela et al., 2004

Residues Blackcurrant pomace (Ribes nigrum) Borage seeds (Borago officinalis) Grape seeds pomace (Vitis vinifera) Evening primrose seeds (Oenothera biennis) Rapeseed (Brassica rapa)

13 14 13 15 13

mg mg mg mg mg

GAE GAE GAE GAE GAE

g–1 g–1 g–1 g–1 g–1

Reference

DPPH; inhibition of LA; oxidation Latif and Anwar, 2009

C, catechin; CA, caffeic acid; F, flavonoid; GAE, gallic acid equivalents; R, rutin; SAE, sinapic acid equivalents; T, tocopherol.



Extraction of natural antioxidants from plant foods 535 Extractable compounds (mg/100 g)

4.5

1

4.0 3.5

2

3

4

3.0 2.5 5

2.0 1.5

7

1.0

9

0.5 0

6

8

10 0

2

4 6 Enzyme concentration (%)

8

10

Fig. 18.2 Effect of enzyme concentration on the yield of extractable compounds. 1: Bilberry (Biopectinase CCN) (Puupponen-Piniä et al., 2008). 2: Bilberry (Pectinex BE 3L) (Puupponen-Piniä et al., 2008). 3: Blackcurrant (Biopectinase CCN) (PuupponenPiniä et al., 2008). 4: Blackcurrant (Pectinex BE 3L) (Puupponen-Piniä et al., 2008). 5: Pigeonpea (Fu et al., 2008); lutein (mg/100 g) ¥ 10–1; enzyme concentration ¥ 10–2. 6: Grape pomace (Maier et al., 2008); enzyme concentration ¥ 10–1; 7: Apple pomace (Pinelo et al., 2008). 8: Pigeonpea (Fu et al., 2008); apigenin ¥ 10–1; enzyme concentration ¥ 10–2. 9: Meyer lemon (Li et al., 2006). 10: Yen Ben lemon (Li et al., 2006).

Ecklonia cava showed antiproliferative action (Athukorala et al., 2006). The phlorotannin derivatives of this alga exhibited inhibitory effect on human immunodeficiency virus type 1 reverse transcriptase and protease (Ahn et al., 2004). Enzymatic extracts of Stellaria dichotoma inhibited the in vitro hydroxyl radical-induced DNA damage (Lim et al., 2008). A peptide with ACE-inhibitory activity was isolated from the pepsin hydrolyzate of Chlorella vulgaris waste (Sheih et al., 2009), whereas some glucans (hexaose, schizophyllan, and laminarin) showed inhibition against AAPH (Tsiapali et al., 2001). The major antioxidants of fruits and juices are phenolic compounds. Phenols may be found in cell walls, bound to polysaccharides by hydrophobic interactions and hydrogen bonds, linked to the protein matrix of vacuolar inclusions, confined inside the cellular vacuoles, and near or associated with the cell nucleus (Pinelo et al., 2006). Plant cell walls are a complex network of cellulose, hemicelluloses (mainly of xyloglucans, mannans, xylans, and arabinogalactans), pectins and lignin (a hydrophobic polymer derived from p-coumaryl, coniferyl, and sinapyl alcohols). Degradation of the cell-wall polysaccharide structure is a fundamental step in the release of phenols linked to the cell wall or associated with cell vacuoles. Ferulic and p-coumaric acids, the major lignin monomers that link hemicellulose sugars and lignin, are potent in vitro antioxidants (Meyer et al., 1998; Nardini et al., 1995). Enzymatic degradation of cell-wall polysaccharides is assumed to increase © Woodhead Publishing Limited, 2010

536 Separation, extraction and concentration processes the overall substrate porosity, facilitating solvent penetration and extraction efficiency, but lignin and tannins may block the enzyme action. The use of enzymes that degrade plant cell walls to enhance the extraction of phenolic antioxidant compounds has been reported for food products and by-products. Although no interactions between proteases and pectinases was confirmed for blackcurrant juice production, degradation of the protein network enhanced both substrate porosity and phenol recovery (Landbo and Meyer, 2001). In grape skins, cellulases, hemicellulases, pectinases, and other hydrolytic enzymes can be used (Pinelo et al., 2006). Despite the high cellulose content of the grape cell wall, cellulolytic activity did not influence the extraction yield of phenols from pomace (Meyer et al., 1998), but pectinases, cellulases and proteases increased the extraction yields from apple skin (Pinelo et al., 2008). Preferential enzymecatalyzed solubilization of pectin and cellulose from bergamot peel caused the selective release of glucose and galacturonic acid (Mandalari et al., 2006). Glucanases and pectinases were used for processing citrus peels (Li et al., 2006). Neutral protease, papain and alkaline protease have been assayed on the fruit of Physalis alkekengi (Ge et al., 2009), whereas culture broths of Aspergillus niger enriched in cinnamoyl esterases were used to release hydroxytyrosol from olive oil by-products (Bouzid et al., 2005). The combined effect of the major operational variables (including particle size, time, temperature, and enzyme to substrate ratio) is evaluated using experimental designs (Meyer et al., 1998). The particle size is relevant for degrading plant cell wall carbohydrates, as well as for solvent penetration and solute recovery. A favorable effect of the reduction of particle size on the extraction of phenolics was reported for spent coffee grounds (Pinelo et al., 2007), blackcurrant pomace (Landbo and Meyer, 2001), apple skin (Pinelo et al., 2008), and citrus peels (Li et al., 2006). Reduced particle size favored the degradation of polysaccharides by pectinase without affecting the recovery of phenols (Meyer et al., 1998). Although a beneficial effect of high enzyme concentration (5%) was reported for citrus peel, the optimal value for this material was 1–3% (Li et al., 2006). Increased enzyme dosage enabled higher juice yields and phenol concentrations from blackcurrant mash. However, the non-enzyme-treated juices exhibited higher antioxidant activity than the enzyme-treated ones, presumably because of differences in their phenolic profiles (Bagger-Jørgensen and Meyer, 2004). The effects of enzymatic treatments were different for various citrus fruits (lemon, mandarin, grapefruit and orange) (Li et al., 2006), and various effects of temperature, particle size reduction and ethanol concentration were reported for the extraction of individual compounds from apple skins (Pinelo et al., 2008). Optimal temperatures of 50–60 °C were reported for citrus peel (Li et al., 2006), apple skins (Pinelo et al., 2008), and elderberry (Landbo et al., 2007). Prolonged incubation time (24 h) with pectinolytic and cellulolytic enzymes was necessary to solubilize carbohydrates and flavonoids from bergamot peel (Mandalari et al., 2006), but a negative effect of long reaction


Extraction of natural antioxidants from plant foods 537 times was observed for grape owing to degradation of phenolic compounds (Meyer et al., 1998). Enzyme-assisted aqueous of citrus peels required an incubation period of 3 h, compared with 6 h for water extraction without enzymes (Li et al., 2006). The amount of quercetin in enzyme-treated juices increased with incubation time, but the quercetin content did not involve a higher antioxidant activity (Sun et al., 2005). The yields of soluble carbohydrates were higher in enzyme-treated samples than in untreated ones. A positive linear correlation between extent of cell wall degradation and phenol extraction was observed for grape pomace (Meyer et al., 1998), elderberry (Bagger-Jørgensen and Meyer, 2004), and blackcurrants (Bagger-Jørgensen and Meyer, 2004; Landbo and Meyer, 2001), but no significant differences were found in carbohydrate degradation, distribution of phenolics and release of anthocyanins and total phenols (Landbo et al., 2007). Similarly, pectinolysis affected both juice yield and contents of phenolic acids, flavonoids and anthocyanins (Kaack et al., 2008). Even though the amount of phenols extracted from the pomace is enhanced, specific compounds may decrease: for example, enzyme-mediated anthocyanin degradation may occur owing to enzymatic hydrolysis of glycosylated anthocyanins by polyphenol oxidases. Pectinase preparations produced by Aspergillus niger, containing b-glucosidase, b-galactosidase, and a-l-arabinosidase, affect anthocyanin pigments and color quality (Landbo and Meyer, 2001). Despite b-galactosidase and b-glucosidase causing color degradation, benefits for extraction of potent antioxidants from grape pomace were reported (Meyer et al., 1998). Enzymatic release of esterified hydroxycinnamic acids can be achieved by synergistic action of cell-wall-degrading enzymes (esterases and xylanases). Commercial pectinases facilitate the release of ferulic acid and other phenolic acids from ground rye grain (Andreasen et al., 1999). A cold pressing process was used for defatting borago before extracting phenolics with conventional solvents. Enzymatic pretreatment of seeds enhanced the extraction of phenolic compounds with DPPH radical scavenging capacity from the meal; the tocopherol content of the resulting oil was maintained by cold pressing (Soto et al., 2008). Treatments with cell-wall-degrading enzymes were proposed for releasing ferulic acid from agro-industrial byproducts such as sugar beet pulp or maize bran (Bouzid et al., 2005). Other less studied materials include microbial sources. Saccharomyces cerevisiae b-glucan, isolated by hot water and enzyme treatment, exhibited antioxidant activity (Jaehrig et al., 2007). Protease treatments can be included within the enzyme-assisted extraction methods. Relatively short bioactive peptides (2–9 amino acids) can be obtained from sources such as wheat, corn, soybean and mushrooms (Dziuba et al., 2004). Commercial proteases have been used to process a number of substrates (Tang et al., 2009; Wang et al., 2007). Hydrolysis increased ACE inhibition and the radical-scavenging activity of protein hydrolyzates from potato isolates and by-products, possibly related to peptides and /or


538 Separation, extraction and concentration processes free amino acids liberated during digestion (Pihlanto et al., 2008). Rapeseed protein hydrolyzates present DPPH radical-scavenging activity, provide protection against the peroxidation of lipids and/or fatty acids (Amarowicz and Shahidi, 1997; Peña-Ramos and Xiong, 2002; Sakanaka et al., 2004) and show reducing power and Fe2+ chelating ability (Moure et al., 2006; Tang et al., 2009). Esterified phenolic acids (p-coumaric acid, ferulic acid) present in sugarcane bagasse were released by alkaline hydrolysis (Ou et al., 2009). Ferulic acid (FA), a scavenger of free radicals approved in certain countries as a food additive to prevent lipid peroxidation (Srinivasan et al., 2007), can be extracted using enzymatic, alkaline or acidic extractions. 18.5.3 Pressurized-liquid extraction Pressurized-liquid extraction (PLE) is an efficient, innovative and environmentally clean technique, which is performed at high temperature and pressure to maintain the solvent in the liquid state. Compared with traditional extraction techniques, it is faster and requires lower amounts of solvent. Alternatively, it is known as accelerated solvent extraction (ASE), or high-pressure liquid extraction (HPE) (Adil et al., 2008). The structure, activity and properties of the extracts are usually unaffected in an oxygenfree and light-free environment, and micro-organisms and enzymes may be inactivated (Qadir et al., 2009). The influence of HPE on the antioxidant capacity of food products was recently reviewed by Oey et al. (2008). PLE was proposed for the extraction of a variety of natural products and wastes, including the production of anthocyanins from grape skins (Ju and Howard 2003), flavonoids from spinach (Howard and Pandjaitan, 2008) and processing of berry substrates (King et al., 2003). The use of non-toxic solvents (water and ethanol) offers environmental advantages. Ethanol–water mixtures gave high extraction yields from Phormidium species (Rodríguez-Meizoso et al., 2008), sour cherry pomace (Adil et al., 2008), and dried spinach (Howard and Pandjaitan, 2008); whereas methanol was used for extracting catechin and epicatechin from tea leaves and grape seeds (García-Marino et al., 2006; Piñeiro et al., 2004), and flavonoids from apple extracts (Alonso-Salces et al., 2001). The compounds recovered and their extraction efficiency differed with the solvent, providing different properties to the extracts, including color and odor compounds (Howard and Pandjaitan, 2008). The amount of carotenoids extracted from Dunaliella salina with hexane was more than seven times higher than the value extracted with ethanol, but the antioxidant activity was only twice as high owing to structural differences (Herrero et al., 2006). Combinations of pressure, temperature, solid/solvent ratio and extraction time can be addressed by experimental designs (Adil et al., 2008; Herrero et al., 2006). Temperature, vapor pressure of the most volatile compounds and improved mass transfer have been reported as the major variables involved in


Extraction of natural antioxidants from plant foods 539 PLE regardless the solvent used (Rodríguez-Meizoso et al., 2008). Temperature was the most influential factor in the extraction of antioxidant compounds from Dunaliella salina (Herrero et al., 2006). The optimal temperature for water and ethanolic extraction of Spinacia oleracea flavonoids was 50–130 °C for water, and 50–150 °C for ethanol (Howard and Pandjaitan, 2008), compared with 100–130 °C for PLE processing of grape by-products (Ju and Howard 2003; Piñeiro et al., 2004) and berries (King et al., 2003). The optimal temperature depends on the solvent considered. Optimal antioxidant activity of Phormidium species in hexane extracts was obtained operating at 50 °C, whereas in ethanol and water extracts it was observed at 150 and 200 °C, respectively (Rodríguez-Meizoso et al., 2008). Thermal degradation of apple polyphenols was observed at extraction temperatures higher than 60 °C in methanol, whereas activity decreased at 110–190 °C in water and ethanol (Alonso-Salces et al., 2001). Thermal degradation of flavonoids was reported for extraction temperatures >130 °C (Howard and Pandjaitan, 2008). The reduction in antioxidant activity observed at prolonged extraction times during PLE of Dunaliella salina with hexane and ethanol suggested some kind of carotenoid degradation (Herrero et al., 2006). HPE extraction can give a moderately high extraction efficiency of bioactive compounds in a short time, but treatments for prolonged periods could result in degradation (Qadir et al., 2009). The development of brown, highly polar, odoriferous compounds with high antioxidant capacity at high extraction temperatures suggested the participation of Maillard reactions in ethanolic (Howard and Pandjaitan, 2008) and in water extracts (Rodríguez-Meizoso et al., 2008). Extracts of propolis obtained by high hydrostatic pressure extraction in 1 min presented similar properties to the ethanolic extracts obtained by leaching at room temperature for a few days (Jun, 2006). No degradation of compounds present in ethanolic extracts occurred after exposure to light and air at room temperature for 2 days (Herrero et al., 2006). Other reported biological activities include cytotoxic effects on the growth of human promyelocytic leukemia cells (HL-60) of sweet potato extracts (Rabah et al., 2005), toxicity on preformed monolayers of Vero cells (African green monkey kidney cell line) of Phormidium extracts from PLE (Rodríguez-Meizoso et al., 2008). Antimicrobial activity of Phormidium sp. extracts has been ascribed to terpenes (i.e., b-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) (Rodríguez-Meizoso et al., 2008). Subcritical-water extraction (SWE) is an environmentally friendly technology for the selective extraction of bioactive compounds from plant materials with advantages derived from its simplicity, reduced extraction time, high quality of the extract, low cost of the extracting solvent and possibility of scaling-up (Rodríguez-Meizoso et al., 2006). Extract with water under pressure (between 100 °C and the critical temperature of 374 °C) is also known as superheated water extraction, subcritical water extraction, high-temperature water extraction, pressurized hot water extraction, or hot


540 Separation, extraction and concentration processes water extraction. Most studies have been performed in batch mode on a small scale, but continuous flow equipment has also been described (Budrat and Shotipruk, 2008). The feedstocks studied include fruits or vegetables and their processing wastes, seeds, herbs and algae (Table 18.9). SWE was more efficient than conventional solvents for extracting flavanols from grape seeds (García-Marino et al., 2006) or antioxidant phenolics from bitter melon (Budrat and Shotipruk, 2008), and was sucessfully employed to concentrate and isolate antioxidant compounds from oregano leaves (Rodríguez-Meizoso et al., 2006). Temperature has a marked effect on yield and selectivity. The dielectric constant of water decreases with temperature, enabling the extraction of compounds having different polarities at different temperatures. High temperature also enhances diffusivity, facilitating the transport of solutes from the solid matrix, and a compromise to avoid thermal degradation must be reached. The release of hydroxycinnamates from cell walls is favored by temperature, but anthocyanins undergo degradation reactions. Lignin decomposes in sub- and supercritical water to give phenol, which are degraded at high temperatures (Budrat and Shotipruk, 2008; Garrote et al., 2003). Sequential extraction with stepwise pressure increase was proposed for processing black tea leaves (Chambers et al., 1984) as well as to recover quercetin glycosides from onion waste (Turner et al., 2006), and catechins and proanthocyanidins from winery by-products (García-Marino et al., 2006). The major variable is temperature, because at 150 °C or above, substrates are hydrolyzed, and the solvent polarity decreases. Optimization of operational conditions has to be carried out for each material. The yield of total phenolics from sweet potato and bitter melon increased with temperature (Budrat and Shotipruk, 2008; Rabah et al., 2005). Although higher temperatures resulted in higher amounts of extracted phenolics, their antioxidant activity was higher operating at lower temperatures (Budrat and Shotipruk, 2008). Carnosic acid, the most potent antioxidant in rosemary, was preferentially extracted at 200 °C (Rodríguez-Meizoso et al., 2006). Extraction yields of oregano leaves increased with temperature: at the lowest temperature considered, the more polar compounds (flavanones and dihydroflavonol structures such as dihydroquercetin, eriodictyol and dihydrokaempferol) were preferentially extracted, whereas less polar compounds could also be extracted at 200 °C (Rodríguez-Meizoso et al., 2006). The phenolic content was a maximum at 25 °C and a minimum at 200 °C, but the highest antioxidant activities were achieved at 150–200 °C. Boiling ground coffee beans in water at 110 °C under elevated pressure gave an efficient extraction of antioxidants (Budryn and Nebesny, 2008). The total phenolic contents and the antioxidant activities of HPE extracts from longan fruit were higher than those obtained with conventional solvent extract (Prasad et al., 2009), whereas enhanced extraction of onion flavonols was observed at 100–400 MPa (Roldán-Marín et al., 2009). The conditions of SWE influence the degree of polymerization and structure


Table 18.9 Examples of extraction of plant food antioxidants using high pressure systems Raw material

Operational conditions

Elderberry leaves (Sambucus nigra L.) Elderberry berries (Sambucus nigra L.) Elderberry flowers (Sambucus nigra L.) Phormidium species

Red grape skin (Vitis vinifera)

Antioxidant activity Reference

0.32 mg GAE g–1

DPPH

Bonoli et al., 2004

Fiber: 14.99 g GAE kg–1 Flour: 20.93 g GAE kg–1

DPPH

Papagiannopoulos et al., 2004

28.91 mg GAE g–1

DPPH ABTS FRAP DPPH bC bleaching

Güçlü-Üstündağ and Mazza, 2009

DPPH bC bleaching DPPH bC bleaching TEAC Antimicrobial activity

Dawidowicz et al., 2006 Dawidowicz et al., 2006 Rodríguez-Meizoso et al., 2008

ORAC

Ju and Howard, 2003

1 g; ECV(22); 17 (g F/100 g ¥ 10–2) SE: E:W (80:20); -; 10 min; 60 bar; 200 ºC; PLE: FV( 60% ECV); P(120) 20.18 (g F/100 g ¥ 10–2) 214.2 (g F/100 g ¥ 10–2) 1 g sample H: 20 min; 200 ºC; E: 20 min; 200 ºC; W: 20 min; 200 ºC; 0.2 g + 30 g sea sand; ECV(22); SE: acidified W; 3 cycles; 5 min; 10.1 MPa; 50 ºC PLE; FV (50% ECV); P(90)

6.54% 40.91% 22.81% 179.9 mg GAE g–1

Dawidowicz et al., 2006



Pressurized liquid extraction (PLE) Barley flour 2 g + 4 g Hydromatrix; ECV(33); (Hordeum vulgare L.) SE: E:W (4:1); 2 cycles; 5 min, 20 MPa 60 °C PLE: FV (60% ECV); P(60) Carob fruits 1 g + 2 g diatomaceous earth; (Ceratonia siliqua L.) SE: A:W (1:1); 2 cycles; 5 min; -; 60 °C; PLE: 50% FV; Cow Cockle seeds 2 g; (Saponaria vaccaria) W; 3 h; 125–175 °C; F(2)

Solubles yield or TEP*

Raw material


Spinach (Spinacia oleracea)

0.5 g + 30 g Ottawa sand 42 mmol GAE kg–1 SE: E:W (7:3); 3 cycles; 5 min; 13.8 MPa; -; PLE: R (70% ECV) 2.5 g; ECV(11) PE; 9 min; 170 ºC; 77.0 mg extract H; 15 min; 170 ºC; 95.5 mg extract E; 9 min; 170 ºC; 270.1 mg extract W; 15 min; 170 ºC; 290.0 mg extract 2.5 g; ECV(11) PE; 9 min; 170 ºC; 2.94% H; 9 min; 170 ºC; 4.28% E; 9 min; 170 ºC; 19.70%

Spirulina plantensis © Woodhead Publishing Limited, 2010

Spirulina plantensis


Antioxidant activity Reference ORAC

DPPH

DPPH

Howard and Pandjaitan, 2008 Herrero et al., 2004

Jaime et al., 2005

High pressure extraction (HPE) Korean barberry bark (Berberis koreana)

1 g mL–1; 15 min; 500 MPa; RT

50% E; 0.02 g mL–1; 30 min; 500 MPa; 50 ºC E; 0.06–0.07 g mL–1; 25 min; 176–193 MPa; 60 °C High pressurized fluid extraction (HPFE) Brazilian propolis RSL 1:10; 20% E; 40 g surfactant; 50 psig; 393 K Longan fruit pericarp (Arillus longan) Sour cherry pomace

11.04% (317.35 mg GAE g–1)

DPPH Inhibition of xanthine oxidase

Qadir et al., 2009

30% (23 mg GAE g–1)

Prasad et al., 2009

3.80 mg GAE g–1

DPPH O2– DPPH

Adil et al., 2008

25.2%

DPPH

Chen et al., 2007



Taiwan Red Pine (Pinus taiwanensis)

70% E; 180 min; 689 kPa; 343 K; F (10)

26.3 mg g–1

Taiwan White Pine (Pinus morrisonicola)

70% E; 180 min; 689 kPa; 343 K; F (10)

39.8 mg g–1

Propolis crude

RSL 1:35, E 75%; 1 min; 500 MPa; RT

Accelerated solvent extraction (ASE) Spirulina plantesis 2.5 g; ECV (11); 15 min; -; 170 °C

–

DPPH

Rieger et al., 2008

–

DPPH

Rieger et al., 2008

317 mmol GAE g–1 dm

ABTS

Corrales et al., 2008

–

DPPH

Rieger et al., 2008

TPA: 0.20, TF: 7.10, TOD: DPPH ORAC 63.5 mg GAE g–1

Súarez et al., 2009

290 mg GAE g–1

bC bleaching DPPH

Xi, 2006

6.43 g GAE g–1

bC bleaching DPPH

Xi and Shouqin, 2007

H: 4.3%; PE: 4.01% E: 17.14%; W: 10.12%

DPPH

Herrero et al., 2005



High hydrostatic pressure (HPP) Bilberry ECV(11); (Vaccinium myrtillus) SE: 3 cycle, 7 min, 60 bar, 80 ºC; PLE: FV (100%), P (100) Elderberry ECV (11); (Sambucus nigra) SE: 3 cycle; 5 min; 68.9 bar; 60 °C; PLE: FV (100%), P (60) Grape skins RSL 1:4.5; E:W (1:1); 1 h; 600 MPa; (Dornfelder V. vinifera) 70° C; Heather ECV (11); (Calluna vulgaris) SE: 3 cycle, 5 min; 60 bar; 60 ºC PLE: FV (100%), P (60) Olive cake 30 g OC; ECV (100) E:W (80:20), FV (60% ECV), 2 cycles; 5 min Propolis extract RSL 1:35, E 75%; 1 min; 500 MPa; RT

DPPH, Fe Lin et al., 2009 chelating, reducing ability, O2–, NO DPPH, Fe Lin et al., 2009 chelating, reducing ability, O2– NO

Raw material





Hot water extraction (HWE) Canola meal RSL 1:16.7; W; 30 min; -; 80 °C (Brassica napus)

0.15 g g–1; 7.83 g SA g–1

Citrus peels

RSL 1:20; bddW; 30 min; -; 100 ºC

~32.5 mg GAE g–1 dw

Formosa koa leaves (Acacia confusa)

bdd H2O; 4 h; -; -

7.3 g

Green tea

RSL 1:100; dW; 5–30 min; -; 80 ºC

Herbal tea (Anagallis arvensis) Japanese horse chestnut (Aesculus turbinata ) Kaffir lime fruit peel (Citrus hystrix) Lettuce extract (Lactuca sativa) Mallotus japonicus leaves

RSL 1:166.7; bW; 5 min;- ; -

TP: 2050 mg GAE L–1 TF: 1700 mg GAE L–1 1.47 mmol Trolox g–1

RSL 1:100; bW; 2 h

1560 mg (280 mg EE)

DPPH TEAC Reducing power bC bleaching DPPH FRAP DPPH NBT Reducing power FRAP ABTS CUPRAC ABTS DPPH

0.2 g; ECV (10.8); 15 min; 200 ºC

23.7 mg GAE mL–1

DPPH

RSL 1:2; W; 80 ºC; 10 min

–

RSL 1:50 mL; bdW; 20 min

7.5 mg GAE mL–1

Morinda citrifolia roots

0.5g, ECV(10); 4 MPa; 2 h; 170 °C F (5)

92.55%

Peroxidation of liposomes DPPH O2– HO· DPPH

Hassas-Roudsari et al., 2009 Xu et al., 2008 Tung et al., 2009 Rusak et al., 2008 Apak et al., 2006 Ogawa et al., 2008 Khuwijitjaru et al., 2008 Altunkaya et al., 2008 Tabata et al., 2009 Pongnaravane et al., 2006



Samor thai fruits (Terminalia chebula Retz.) Sasa palmata leaf

15.96 mg TP g–1 dw

ABTS

Rangsriwong et al., 2009

SE: 50 g; steam; 0.5–20 min; 180–260 °C (1.0–4.9 MPa) HWE: RSL 1:100; dW; 2 h; 98 °C RSL 1:100; dW; 2 h; 98 ºC

128.24 mg GAE g–1

DPPH

Kurosumi et al., 2007

12.08 mg GAE g–1

DPPH

Kurosumi et al., 2007 Spigno and De Faveri, 2009 Rusak et al., 2008

Tea

RSL 1:100; bW; 30–210 s

White tea

RSL 1:100; dW; 5–30 min; 80 ºC

Hot pressurized water extraction (HPWE) Boldo RSL 1:10; W; 3 h; 110 ºC (Peumus boldus M.) Sage 0.15 g + sea sand; 60 min; 100 kg cm–2; 100 ºC; F(1) (Salvia officinalis) Yam (Dioscorea alata)

LSR: 20 kg kg–1; ECV(1000); W; 3 h; 1.34 MPa; 120 ºC; F(10)

Subcritical water extraction (SWE) Bitter melon 1 g; ECV (10); (Momordica charantia) 3 mL min–1; 60 min, 200 °C, 10 MPa Canola meal 30 min; 6.89 MPa; 160 °C; F(1)

ABTS TP: 1300 mg GAE L–1 TF: 900 mg GAE L–1

FRAP ABTS

51.4% 5.2 mg B kg–1

ABTS DPPH

Del Valle et al., 2005 Ollanketo et al., 2002

2.143 g kg–1

DPPH

Chen et al., 2004

53 mg GAE g–1

ABTS

0.45 g g–1 15.42 g SA g–1

TEAC bC bleaching DPPH Reducing power

Budrat and Shotipruk, 2008 Hassas-Roudsari et al., 2009



RSL 1:150; 100 °C; 120 min


Raw material




Eucalyptus leaves (Eucalyptus globulus) Licorice roots (Glycyrrhiza glabra) Oregano (Origanum vulgare)

60 g; ECV (300) 200 mL W; 30 min; 4.91 MPa; 200 ºC 0.1 g; ECV (18.57) 10 mL W; 60 min; 0.002–5 MPa; 300 °C 0.75 g; ECV (11) SE:W; 15–30 min; 1500 psi; 25–200 °C; SWE: R (70% ECV) 0.40 g + 30 g sea sand; ECV (22) SE: 1 cycle; 40 s; – 110 ºC; SWE: R (70% ECV), P (90) 1.0 g; ECV (10) 37.5 min; 4 MPa; 180 °C; F (4) RSL 1:50; 10–60 min; 0.47–4.76 bar; 80–150 °C

4.8 mg g–1

Peroxynitrite

1521.3 ppm GAE 54% (0.149 g GAE g–1)

Radical scavenging Baek et al., 2008 Reducing power DPPH Rodriguez-Meizoso et al., 2006

52.3 mg TP g–1

ORAC

Ju and Howard, 2005

21.43 mg TP g–1

ABTS

0.73 mg TC g–1 dm 5.1 mg TF g–1 dm

ABTS

Rangsriwong et al., 2009 Prommuak et al., 2008

Red grape skin Samor thai fruits (Terminalia chebula) Thai silk waste *

Kulkarni et al., 2008

Total extractable polyphenols. B, boldine; bdW, boiled distilled water; bddW, boiled double distilled water; bW, boiled water; bC, b-carotene bleaching; dm, dry matter; dW, distilled water; E, ethanol; ECV, extraction cell volume; EE, epicatechin equivalent; F, flux (mL/min); FV, flush volume; GAE, gallic acid equivalent; H, hexane; LSR, liquid–solid ratio; P, purge (s); PE, petroleum ether; PLE, pressurized liquid extraction; pm, plant material; R, rising; RT, room temperature; SE, static extraction; TC, total carotenoids; TF, total flavonoids; TP, total phenolics; W, water.



Extraction of natural antioxidants from plant foods 547 of molecules, as reported for procyanidin extraction (García-Marino et al., 2006). Enhanced recoveries of flavanol dimers and trimers, and gallic acid was obtained in greater quantities by a single extraction at 150 °C, in which gallic acid accounted for 61% of total phenolics. Higher temperatures favored the extraction of oligomeric fractions. When the galloylated moieties were situated in the terminal sub-unit, sequential extraction (50–100 °C) was preferable, whereas a single extraction at 150 °C was better for the rest of the galloylated derivatives (García-Marino et al., 2006). Treatment of lignocellulosic substrates with water or steam at 160–240 °C (autohydrolysis processing) results in depolymerization of hemicelluloses and in breakage of lignin-carbohydrate bonds, leading to solubilization of lignin fragments of low MW. The aqueous treatments of lignocellulosics for both the hydrolytic degradation of hemicelluloses and the solubilization of antioxidant compounds have been reviewed (Garrote et al., 2004; Meireles, 2009). Other related technologies, based on the utilization of chemicals (for example, solvents, oxygen or acids) added to water, were proposed to provide higher extraction yields. Steam treatments were efficient in releasing hydroxytyrosol and 3,4-dihydroxyphenylglycol (DHPG), from the semisolid waste of olive oil extraction systems (alperujo) (Fernández-Bolaños et al., 2002; Rodríguez et al., 2009). The amount of solubilized hydroxytyrosol increased with temperature and time, reaching 1.4−1.7 g/100 g of dry alperujo (FernándezBolaños et al., 2002). 18.5.4 Supercritical-fluid extraction Supercritical-fluid extraction (SFE), based on the utilization of a fluid under supercritical conditions, is suitable for the extraction and purification of natural compounds with bioactive and antioxidant properties. The interest in SFE is promoted by increasingly restrictive environmental, toxicological, and health regulations. Carbon dioxide shows favorable properties, including environmental safety, high availability at low cost, high purity, and suitability for extracting heat-labile, natural compounds with low volatility and polarity. The physicochemical properties of supercritical CO2 (high diffusivity, low viscosity and low surface tension compared with conventional solvents) facilitate mass transfer, enabling an environmentally friendly operation. The properties of the final product are improved by high selectivity, low processing temperatures, absence of light and oxygen, and absence of solvent in extracts. The extracts are regarded as natural, and do not require additional sterilization. The major disadvantages of supercritical CO2 are the high critical pressure (requiring expensive equipment), and the poor solvent power derived from its low polarity. The use of cosolvents enhances the solubility of some compounds and may increase the extraction selectivity, allowing operation at a lower pressure. Representative antioxidant compounds extracted by SFE are summarized


548 Separation, extraction and concentration processes in Table 18.10. SFE successfully recovered phenolic compounds from liquid solutions (Gamse, 2004), and countercurrent contact has been employed for processing propolis (Wang et al., 2004), B. kaoi (Wang et al., 2005) and orange juice (Señoráns et al., 2001). Studies on the SFE of bioactive compounds with antioxidant activity are available (Del Valle et al., 2005; Díaz-Reinoso et al., 2006; Herrero et al., 2006; Meireles, 2003; Mukhopadhyay, 2000; Reverchon, 1997). The most influential variables in SFE of compounds with antioxidant properties from natural sources are summarized here. The drying technique (Mechanical and thermal conditioning) affects both the structure of the solid matrix and the thermal stability of the active compounds. Mass transfer is facilitated by a higher degree of grinding, but the performance of fixed beds during leaching could be limited by too fine particles. Pressure and temperature are the variables that determine the solubility equilibrium, an understanding of which is essential for process design. Results are mostly available for a single solute in the supercritical solvent, but the presence of other solutes may affect the equilibrium, and the natural products are a complex mixture of compounds. Studies dealing with the solubility of solid mixtures of phenolic compounds in supercritical CO2 have been reported (Del Valle et al., 1998; Díaz-Reinoso et al., 2006; Fornari et al., 2005; Lucien and Foster, 2000; Reverchon, 1997). Solvent density increases with pressure, but beyond a certain threshold, the increased solvent viscosity reduces the diffusion coefficients. When the target compounds are lipophilic, the operational conditions may be milder than those required for phenolic antioxidants. Highly labile compounds require mild temperatures (below 50 °C) to avoid alteration. A stepwise increase in the extraction pressure allows selective extraction of different compounds. The presence of a modifier increases the solvent density resulting in a higher interaction of solutes with the solvent, causing alterations and swelling of the vegetal matrix. A suitable cosolvent may improve the extraction yield and selectivity. However, cosolvents of hazardous nature or resulting in decreased selectivity should be avoided. The common modifiers used in SFE are alcohols, which induce dipole/dipole interactions and hydrogen bonding with polar functional groups. Ethanol has been employed to increase the solubility of ginsenoids (Wang et al., 2001), antioxidants from tamarind seed coat (Luengthanaphol et al., 2004), Eucalyptus (El-Ghorab et al., 2003) and olive leaves (Tabera et al., 2004), lipophilic compounds from marigold (Baumann et al., 2004), and flavonoids and terpenoids from Ginko biloba (Yang et al., 2002). Methanol was used for the extraction of magnolol (Dean et al., 1998), soy isoflavones (Rostagno et al., 2002), phenolics from chamomille (Scalia et al., 1999), pistachio hulls (Goli et al., 2005) or aloe extracts (Hu et al., 2005). Isopropanol was used for the extraction of ginger oleoresin (Zancan et al., 2002). Water is the cosolvent employed in the industrial extraction of vanilla. The extracts resulting from the unselective extraction of compounds at high pressure can be fractionated in multiple-stage separators. The pressure and


Table 18.10 Examples of extraction of plant food antioxidants using SFE Extraction yield (total extract, TE and active compounds)

Antioxidant activity1

Reference

Aloe vera leaf skin (Aloe barbadensis) Baical skullcap root (Scutellaria baicalensis) Boldo (Peumus boldus) Boldo (P. boldus) Coriander seeds (Coriandrum sativum) Eucalyptus leaf oil (Eucalyptus camaldulensis) Eucalyptus leaves (E. camaldulensis)

TF = 1.50 TF = 0.27

DPPH DPPH

TE = 2.9; B (0.0031)e TE = 4.9; B (7.4)e TE = 1.92 MTp H (0.242); STp (0.0825) pCl (1.16); oHC (0.17); MEP (0.34); Thy (0.98)a TE = 16.6

TEAC ABTS DPPH Inhibition of LA oxidation Inhibition of LA oxidation

Hu et al., 2005 Bimbato and Tamanini, 2003 Del Valle et al., 2005 Del Valle et al., 2005 Yépez et al., 2002 Fadel et al., 1999

Ginger (Zingiber officinale)

Monoterpenes, sesquiterpenes and gingerols

b-C, LA

Leal et al., 2003

Ginger (Z. officinale)

TE = 2.31

b-C, LA

Zancan et al., 2002

Helichrysum dried flower heads (Helichrysum italicum) Hop (Humulus lupulus) Lemon balm (Melissa officinalis subs. officinalis) (M. officinalis subs. inodora) Marjoram (Origanum vulgare) Hungarian marjoram (O. majorana) Egyptian marjoram Nigella seeds (Nigella sativa)

Flavonoids; TE = 4.5–4.9

O2–;

Marongiu et al., 2003

TP = 0.52–3.79/TF = 0.2–0.92 TE = 1.9 TE = 0.7

LA oxidation LA oxidation

Lermusieau et al., 2001 Marongiu et al., 2004

TE = 3.2 TE = 3.76 TE = 5.39 T.O. (1.14)b; Tq (0.49); p-Cy (0.23)

Peroxide value Inhibition of oil oxidation

Uy et al., 1991 Vági et al., 2005

b-C, LA

Machmudah et al., 2005

DPPH; b-C, LA

El-Ghorab et al., 2003






Extraction yield (total extract, TE and active compounds)


Reference

Black pepper oleoresin (Piper nigrum)

RER = 5.43 (Pp: 39.4)

Tipsrisukond et al., 1998

Rosemary (Rosmarinus officinalis) Rosemary (R. officinalis)

TE = 5.2 TE = 7.15

Hexanal formation; TBARS Peroxide value

Rosemary (R. officinalis) Rosemary (R. officinalis)

Carnosic acid solubility TE = 4.5; Cm (39.6); Vbn (20.3); t-C (10.3)a

Rosemary (R. officinalis)

Sage (Salvia officinalis) Sage (S. officinalis)

F1: Rsl (3.6); Gk (11.9); Cr (5.6); CrA (66.0); MCr(1.6)a F2: Rsl (8.0); Gk (0.13); Cr (6.4); CrA (60.7); MCr(4.6)a TE = 1.6; Cm (0.025 ); Cn (0.001); CrA (0.236); RA(0.065)c TE = 5.7 TE = 5.02

Sage (S. officinalis)

TE = 46.26; F1 (024)/F2 (27.50)/F3 (18.52)


Black sesame seed (Sesamum indicum) Black sesame seed (S. indicum) Summer savory (Satureja hortensis)

TE = 51.83 Lignans (sesamin, sesamolin) S1: Crv (32); Lnl (24); Myc (22); Myl (21)c S2: Crv (44); Lnl (12); Myl (11); g-Tpnn (20)c S3: Crv (55); g-Tpnn (38)c Tamarind seed coat (Tamarindus indica) 0.13 mg EC g–1

b-C, LA Peroxide value; TBARS

Uy et al., 1991 Dapkevicius et al., 1998 Ramírez et al., 2005 Leal et al., 2003

b-C, LA DPPH

Cavero et al., 2005

b-C, LA

Carvalho et al., 2005

Peroxide value

Uy et al., 1991 Dapkevicius et al., 1998

AAb-c PF

Daukšas et al., 2001

DPPH; Lipid oxidation DPPH; lipid oxidation Inhibition oil oxidation

Hu et al., 2004 Xu et al., 2005a Esquível et al., 1999

Peroxide value

Luengthanaphol et al., 2004



Tamarind seed coat (T. indica)

Thyme leaves (T. vulgaris) Thyme (T. vulgaris)

TE = 4.92 TE = 0.7; Thy (0.17)d

Turmeric oil (Curcuma longa) Turmeric (C. longa)

TE = 5.5 (STp H)

Green tea (Camelia sinensis)


1

Zg A (40.1); Eg A (18.2); arT (15.7); Zg (3.1)

Peroxide value

Tsuda et al., 1995

Lipid oxidation

Chang et al., 2000

IPDEBTA Peroxide value

Ko et al., 2002 Uy et al., 1991 Dapkevicius et al., 1998

b-C, LA Rancimat Rancimat AOP

a

b-C, LA

Simándi et al., 2001 Zeković et al., 2001 and 2003 Gopalan et al., 2000 Leal et al., 2003

Antioxidant activity reported in the referenced work or, if not indicated, other works referencing antioxidant activity of the plant extracts. Relative percentage of the normalized area detected by LC–MS or GC. b Total oil (essential oil). c Relative weight content in the extract; d mg (100 g)–1. e mg g–1. TE, total extraction yield (%); TF, total flavonoids extraction yield (%); TP, total phenolics extraction yield (%); RER, relative extraction rate. AAb-c = antioxidant activity during bleaching of b-carotene–linoleate solution, scale (0:Low–5:High). AOP = antioxidant potency with linoleic acid in an iron/ascorbate system. b-C, LA = absorbance of the b-carotene–linoleic acid system. Hexanal = percentage of reduction in the formation of hexanal respect to control. IP DEBTA = inhibition of linoleic acid peroxidation by the DEBTA (diethyl-2-thiobarbituric acid) method. PF = protection factor calculated as the ratio between the induction period of the sample with additive and the control sample during vegetable oil oxidation. TBARS = percentage of reduction in the formation of TBARS with respect to control. TEAC = Trolox equivalent antioxidant capacity. O 2– = superoxide radical scavenger capacity of superoxide dismutase. Simple phenolics, derivatives and flavonoids: B, boldine; Crv, carvacrol; DPA, 3,4-dihydroxyphenyl acetate; EgA, E-g-atlantone; EC, epicatechin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate; GA, gallic acid; Gk, genkwanin; HDA, 2-hydroxy-3¢,4¢-dihydroxyacetophenone; MDB, methyl 3,4-dihydroxybenzoate; MEP, 2-methyl-6-ethylphenol; RA, rosmarinic acid; Thy, thymol. Terpenoids: t-C, trans-caryophylene; Cm, camphor; Cn, cineole; Cr, carnosol; CrA, carnosic acid; p-Cy, p-cymene; MCr, methyl carnosate; Lnl, linalool; MTp H, monoterpenes hydrocarbons; Myc, myrcene; Myl, myrtenol; Rsl, rosmanol; STp, sesquiterpenes; STp H, sesquiterpene hydrocarbons; ar-T, ar-turmerone; g-Tpnn, g-terpinene; Vbn, verbenone; ZgA, Z-g-atlantone; Zg, zingiberene. Other compounds: pCl, p-cymen-7-ol; oHC, O-hydroxycumine; Pp, piperine; Tq, thymoquinone. a


Terminalia catappa leaves, seeds Thyme (Thymus vulgaris) Thyme (T. vulgaris)

TE = 0.29; HDA (0.043); MDB (0.068); DPA (0.885); EC (0.164)d EGC (290); EGCG (510); ECG (105); EC (90); GA (7)e Tannin, flavonoid glycosides TE = 2.0 TE = 5.46

552 Separation, extraction and concentration processes temperature in the separators influence the fractionation of extracts. Series of two or three separation stages are used to recover the desirable components. Although the extraction yields are increased under harsh conditions, the selectivity is reduced, leading to extracts with poor antioxidant activity. These instances require optimization of the extraction stage (Gelmez et al., 2009) and/or separation steps. The yield can be similar to the use of a stepwise increase in the extraction pressure, but with a lower consumption of solvent. 18.5.5 Other novel extraction technologies The fundamentals and application of alternative techniques using solvents at low pressure for the extraction of bioactive compounds have been comprehensively reviewed (Meireles, 2009). Some of these techniques have been developed for analytical purposes, but they could be scaled up. Studies dealing with the antioxidant activity of extracts obtained with ultrasonic- and microwave-assisted technologies are summarized in Table 18.11. Ultrasound-assisted extraction (UAE) is a cheap, scalable technique allowing reduced extraction times, decreased extraction temperature, and increased extraction yields. Its effects are mainly attributed to cavitation forces upon the propagation of the acoustic waves. Ultrasounds result in physical, chemical, and mechanical effects promoting the release of soluble compounds from the plant body, enhancing mass transfer and facilitating the access of solvent to cell contents (Knorr et al., 2002; Ma et al., 2008). UAE was used to extract valuable compounds such as phenolic acids and flavanone glycosides (Ma et al., 2008). In UAE, both treatment time and (particularly) temperature have significant effects on the properties of extracts. However, UAE must be applied carefully to avoid degradation of susceptible solutes (Ma et al., 2008). Degradation of phenolics depends on their substitution pattern. High comparative stability has been reported for sinapic and vanillic acids, which have methoxyl-type substituents in their aromatic rings, whereas benzoic acids are more stable than cinnamic acids. In previously dried samples, ultrasound accelerated rehydration and swelling, without significant chemical degradation (Qadir et al., 2009). The ultrasonic power had a positive effect on the contents of phenolic acids from mandarin peels (Ma et al., 2008), tannins from myrobalan nuts (Sivakumar et al., 2008), and anthraquinones from Morinda citrifolia roots (Hemwimol et al., 2006). Differences in the effects of acoustic treatments can be ascribed to the utilization of different equipment, effects of the nature of the solid matrix (hardness, compactness, and solute distribution), and cavitation. Some of the general trends outlined above were confirmed in the extraction of active components from Hypericum perforatum L. (Smelcerovic et al., 2006). Other technologies enhancing mass transfer in plant tissues and permeability of cytoplasmatic membranes include microwave-assisted extraction (MAE), pulsed electric fields (PEF) and high hydrostatic pressure (HHP) (Corrales


Table 18.11 Other extraction technologies applied to the recovery of antioxidants from food plants Operational conditions

Extraction yield


Reference

Ultrasound Almond shells

LSR 1:10; 5% NaOH; RT; 10 min

TEP (%)10.1

DPPH

Bearberry leaves

LSR 1:25; W:E; 60 kHz; 25 ºC; 40 min

2.15 %

Cow cockle seeds (Saponaria vaccaria)

LSR 1:50; (W, 50% E, ACN); RT; 1 h

W: 0.78 g GAE g–1 dw 50%E: 1.58 g GAE g–1 dw ACN: 0.20 g GAE g–1 dw

Dornfelder skins (Vitis vinífera) Ginseng (Panax ginseng) Ginseng (P. ginseng) Indian mulberry (Morinda citrifolia) Indian mulberry roots (M. citrifolia) Mandarin peels (Citrus unshiu)

35 kHz; 70 °C; 1 h

360 mmol GAE g–1 dw

Inhibition of oil oxidation DPPH ABTS FRAP ABTS

Ebringerová et al., 2008 Gribova et al., 2008


LSR 1:50; 70% E; 60 ºC; 40 min; 250 W; 42 kHz LSR 1:50; 70% E; 70 °C; 2 h; 42 kHz; 284 W RLS 100; E; 60 °C; 60 min; 15.7 W

TS: 3.89 %

DPPH

Chen et al., 2010

G: 13.7 mg g–1 TP: 7.1 mg g–1 62.23%

DPPH

Kim et al., 2007

DPPH

79.62%

DPPH –

Pistachio (Pistachia vera) Plum (Prunus domestica)

LSR 1:8; W; M; 45 min

TCE:1935.12 mg g–1 dw FG: 1374.35 mg g–1 dw W: 34.2 mg TAE g–1 dw M: 32.8 mg TAE g–1 dw TP:1.74–3.75 mg GAE g–1 TP:1.18–2.37 mg CE g–1

Hemwimol et al., 2006 Pongnaravane et al., 2006 Ma et al., 2008

Peroxide value

Goli et al., 2005

ABTS

Kim et al., 2003

LSR 1:10; E; 60 ºC; 2 h; 38.5 kHz; 270 W LSR 1:80; 80% M; 30 °C; 10 min; 8 W

LSR 1:10; 80% M; -; 20 min




Raw material


Raw material


Extraction yield


Reference

Sage (Salvia officinalis) Sea-buckthorn (Hippophae rhamnoides) Sempervivum (Sempervivun marmoreum) Wheat bran (Triticum aestivum) Microwave

LSR 1:4; M; RT, 20 min

–

DPPH

LSR 1:10; E; 30 °C; 60 min

15.6 (0.39) mg GAE g–1

DPPH TEAC

Ollanketo et al., 2002 Sharma et al., 2008

LSR 1:10; dW; 25 °C; 60 min; 40 kHz

2.5 mg g–1

Hydroxyl DPPH

Stojičević et al., 2008

LSR 1:10; 5%NaOH; 60 °C; 60 min; 100 W

24.3% polysaccharides

DPPH

Hromádková et al., 2008

Sappan wood (Caesalpinia sappan)

540 W; 20 min

0.747 g

DPPH Nitric oxide

Shrishailappa et al., 2007

Cranberry (Vaccinium macrocarpon)

LSR 1:5.7; 10 min; 125 °C W E A LSR 1:50; 70% M; 50 W; 10 min

Ginseng (Panax ginseng) Ginseng (Panax ginseng) Indian mulberry (Morinda citrifolia)

LSR 1:50; 70% E; 250 W; 10 min 2450 MHz LSR 1:100; E; 720 W; 30 min; 60 °C

17.6% (9.42 mg Q g–1) 2% (1272 mg Q g–1) 3.25% (960 mg Q g–1) G: 9.8 mg TAE g–1 dw TP: 5.8 mg TAE g–1 dw TS: 3.30% 65.88%

Inhibition of lipid Raghavan and oxidation Richards, 2007 TBARS DPPH

Kim et al., 2007

DPPH

Chen et al., 2010

DPPH

Hemwimol et al., 2006



141 W; 83 s; 9.8 mL; 4 cycles

5.94 mg A mg–1

DPPH

Zhao et al., 2009

LSR 1:10; E 95%; 30 min; 500 W; 2450 MHz, 80 °C

96.78 mg TP g–1

Pan et al., 2008b

30 min; 800 W

0.12% oil

LSR 1:3; I (a) T gradually increased for 5 min, 800 W, and (b) 15 min; 500 W; 40–120 ºC LSR 1:10; E; 20 min; 150 W; 60 ºC

0.15 g oil g–1 125.3 mg Vit E g–1

DPPH Hydroxyl, reducing power DPPH, ABTS, FRAP, lipid peroxidation DPPH

DPPH TEAC Scavenging capability

Sharma et al., 2008

Sea-buckthorn (Hippophae rhamnoides) Wheat bran -

23.5 mg GAE g–1 489.5 mg CE

Phutdhawong et al., 2007 Zigoneanu et al., 2008

Oufnac et al., 2007

ACN, acetonitrile; E, ethanol; LSR, liquid:solid ratio; M, methanol; RT, room temperature; W, water; dW, distilled water. A, astaxanthin; CE, catechin equivalent; FG, flavanones glycosides; G, ginsenosides; GAE, gallic acid equivalent; Q, quercetin; TAE, tannic acid equivalent; TCE, total content of extract; TP, total phenolics; TEP, total extractable phenolics; TS, total saponins.



Green algae (Haematococcus pluvialis) Longan peel (Dimocarpus longan Lour.) Teja (Cinnamomum iners Reinw.) Rice bran (Oryza sativa)

556 Separation, extraction and concentration processes et al., 2008). PEF enhances mass transfer by improving softness and texture (Eshtiaghi and Knorr, 2002), and increasing the contents of some solutes, including total phenolics. However, the antioxidant activity varies significantly with the treatment used (Corrales et al., 2008). PEF inactivates degrading enzymes. HHP increases the extraction yields by deprotonation of charged groups, disruption of salt bridges and hydrophobic bonds in cell membranes and decreasing the dielectric constant of water (Corrales et al., 2008). Combinations of the different extraction methods are used. High-pressure extraction combined with sonication was used to extract compounds from Berberis koreana bark, improving the extraction of bioactive compounds compared with the high-pressure process (Qadir et al., 2009).

18.6 Integration of extraction processes and purification Several technologies with different degrees of sophistication (including solvents, resins and membranes) were used to obtain purified compounds or fractions with biological activity from crude extracts. Some examples of their individual and combined application to the concentration and purification of natural antioxidants from several sources are included here. Combinations of conventional and emerging extraction technologies were proposed to extract and/or to purify natural compounds with antioxidant and biological activity. The extraction of oil and/or lipophylic fractions with supercritical fluids at low pressures and the subsequent solvent extraction of the solid residue was proposed to recover essential oils from aniseed (Rodrigues et al., 2003), Melissa officinalis (Marongiu et al., 2004), rosemary, sage (Nakatsu and Yamasaki, 2000), summer savory (Esquível et al., 1999), and oils from borage seeds (Soto et al., 2008). A sequence of SFE and hydrothermal extraction has been proposed to obtain antioxidants from bamboo (Quitain et al., 2004). Alternatively, a first extraction of the raw material with conventional solvents and further purification of the crude extracts by SFE offer operational advantages, including reduction in CO2 consumption, increased extraction yields, and processing of less substrate owing to its enhanced content of bioactive compounds. Two options are used to purify the bioactive compounds from the crude extract: relatively mild SFE to extract non-polar compounds (desirable when antioxidant compounds of a phenolic nature have to be recovered from the residue) (Hadolin et al., 2004), and SFE under harsh conditions to recover polar compounds in the extract (Díaz-Reinoso et al., 2006). A simple purification process based on the sequential extraction of the raw materials or extracts with solvents of different polarity was applied to the extraction of antioxidant compounds from plant foods. Cheung et al. (2003) reported on the processing of edible mushrooms, leading to similar yields as those obtained with methanol and water on large and small scales. The


Extraction of natural antioxidants from plant foods 557 chemical components of the methanol extract were fractionated by solvents of increasing polarity (dichloromethane, ethyl acetate, butanol and water) (Cheung and Cheung, 2005). The dichloromethane subfraction of the methanol extract of Volvariella volvacea had a high antioxidant activity against lipid peroxidation of rat brain homogenate, whereas the ethyl acetate subfraction of the methanol extract of V. volvacea has antioxidant activity against the oxidation of human low density lipoprotein (LDL) similar to that of caffeic acid. Combined extraction involving pressurized hot water extraction as a first stage has been reported for a variety of purposes (Chambers et al., 1984; Wang and Weller, 2006). The advantages of membrane processing include a low energy requirement, no additives, mild operating conditions, separation efficiency and easy scaling up. The utilization of membrane technologies for concentrating and purifying bioactive phenolic compounds from aqueous streams is a topic of growing interest. For example, fractionation of phenolics into low- and high- MW compounds was reported for conventional solvent crude extracts from edible mushrooms (Cheung and Cheung, 2005), grape (Nawaz et al., 2006), grape pomace (Díaz-Reinoso et al., 2009), partially purified extracts from persimmon pulp (Ga et al., 2008), and mulberry root cortices (Yu et al., 2007). Ultrafiltration (UF) membranes are easy and fast to use in the separation of phenolics according to their MW. At the analytical scale, almond skin phenolics were fractionated to separate low MW compounds in permeate and proanthocyanidin oligomers (up to decamers) in retentate. Permeates from a 50 kDa membrane contained proanthocyanidin pentamers (Prodanov et al., 2008). UF membranes can be used for tailoring grape anthocyanins (containing about 60% monomers, 20% polymers, and 20% other forms) from grapes, having benzoic acids, hydroxycinnamates, anthocyanins, flavan-3-ols, and flavonols as major phenolic groups. The membrane (83% of total egg white proteins. The major egg yolk proteins take the form of lipoprotein complexes, which are divided into the plasma and granule fractions comprising lipovitellin, phosvitin, livetin and lipovitellenin. A number of egg proteins and some of their peptide sequences that have biological activities have been identified. For example, lysozyme is the egg protein that has probably attracted the most attention because of its potential use as an antimicrobial agent in foods. Chicken lysozyme contains a peptide sequence that is potently antimicrobial against both Gram-positive and Gram-negative bacteria. Biologically active proteins and peptides occur at relatively low concentrations in foods and pre-concentration of purification © Woodhead Publishing Limited, 2010

Fractionation of egg proteins and peptides 597 is needed to obtain a dose level to produce beneficial effects in situ. It was therefore necessary to develop technologies for fractionation and purification of bioactive molecules of interest for nutraceutical applications. This chapter mainly focuses on the chemistry and biological activities of proteins and peptides derived from egg components and the various technologies applied for fractionation/purification of proteins/peptides.

19.2 Composition and physicochemical characteristics of egg proteins and applications in the nutraceutical industry 19.2.1 Egg white The physicochemical characteristics of some egg white proteins are listed in Table 19.3. Egg white proteins are predominantly globular proteins having an acidic isoelectric point (pI), the exceptions being lysozyme and avidin. Except for lysozyme, egg white proteins are glycoproteins that are rich in sulfur amino acids and very sensitive to heat denaturation. Ovalbumin Ovalbumin has a molecular mass of 45 kDa and is composed of 385 amino acids. This protein belongs to the serpins family and possesses two genetic variants which differ at residue 289 and 311 by a substitution of Glu and Asn, respectively, by Gln and Asp (Ishihara et al., 1981; Wiseman et al., 1972). The primary sequence reveals one disulfide bond between Cys74 and Cys121. Table 19.3 Physicochemical characteristics of egg white proteins [adapted from LiChan and Nakai (1989)]

Amino acid residues

MW (kDa)

pI

T d*

Number of cystein residues [disulfide bridge]

Ovalbumin Ovotransferrin Ovomucoid Ovomucin Lysozyme G2 globulin G3 globulin Ovoinhibitor Ovoglycoprotein Ovoflavoprotein Ovomacroglobulin Avidin Cystatin

385 686 186 8300 129 – – – – 219 – 128 –

45 77.7 28 5500–8300 14.3 30–45 – 49 24.4 32 760–900 68.3 12.7

4.5 6.0 4.1 4.5–5.0 10.7 5.5 4.8 5.1 3.9 4.0 4.5 10 5.1

84 61 79 – 75 92.5 – – – – – 85 –

6 [1] 30 [15] 18 [9] – 8 [4] – – – – 18 [9] – 2 [1] 4 [2]

(–) Data not available. * Denaturation temperature.


598 Separation, extraction and concentration processes Approximately one half of ovalbumin amino acids residues are hydrophobic and one third are acidic, which confer a pI of 4.5 (Li-Chan and Nakai, 1989). The secondary structure of ovalbumin consists of 30% a-helix and 32% b-sheet structure using high resolution x-ray diffraction (Stein et al., 1990, 1991). Three components A1, A2 and A3 have two, one and no phosphate, respectively, and are found in purified ovalbumin (Perlman, 1952). In solution, ovalbumin can be denatured and aggregated by thermal denaturation or by exposure to the air–water surface (Mine et al., 1990). Partial thermal denaturation of ovalbumin occurs at 78 to 86 °C. The protein adopts a more stable form, termed S-ovalbumin to denote its increased stability. At a heating rate of 10 °C min–1 at pH 9, the denaturation temperature of ovalbumin is 84.5 °C compared with 92.5 °C for S-ovalbumin (Donovan and Mapes, 1976). The greater stability, compactness and hydrophobicity of the S-form contrast with that of ovalbumin (Nakamura and Ishimaru, 1981). Ovotransferrin Ovotransferrin, also called conalbumin, belongs to the transferrin family. Ovotransferrin, with a molecular mass of 77.7 kDa and 686 amino acids, contains no phosphorus or free sulfhydryl group. Ovotransferrin has a pI of 6.0 and has the capacity to bind two Fe3+ ions per molecule with two CO2–3 or HCO3– ions (Mine, 2007). Ovotransferrin is folded into two lobes which show similar structural elements (Williams et al., 1982). Each lobe contains a site for iron binding and is further divided into two distinct domains. Ovotransferrin is the most heat-sensitive protein of egg white. The protein easily binds metallic ions, such as Fe3+, Al3+, Cu2+ or Zn2+ forming heatstable complexes. Ovomucoid Ovomucoid is a glycoprotein with a molecular weight of 28 kDa and a pI of 4.1. Its amino acid sequence is composed of 186 residues and it possesses no tryptophan residues. Ovomucoid has nine disulfide bridges and no sulfhydryl groups. Ovomucoid has three domains defined by the amino acid sequences of 1–68, 69–130 and 131–186, each domain is cross linked by three disulfide bridges (Kato et al., 1987). Its secondary structure is composed of 26% a-helix, 46% b-structure, 10% b-turn and 18% random coil structure (Watanabe et al., 1981). Ovomucoid is highly resistant to heat owing to its high cystine content and, consequently, to the high number of disulfide linkages. Under acidic conditions ovomucoid can resist to heat treatments up to 100 °C, but it is rapidly (89 °C) denaturated in alkaline solutions (pH 9) (Matsuda et al., 1982). Ovomucin Ovomucin is a highly glycosylated protein displaying a molecular weight in the range of 5500–8300 kDa depending on the method of isolation and environmental conditions (Li-Chan and Nakai, 1989). Ovomucin differs from


Fractionation of egg proteins and peptides 599 other egg white proteins because it is extremely large. Its structure comprises sulfate esters, large amounts of cystine and 50% of the total sialic acid contents of egg white (Stadelman and Coterril, 1973). Ovomucin is composed of two subunits: a- and b-ovomucin, which differ by a carbohydrate-poor fraction (15% of dry matter) and a carbohydrate-rich fraction (50% of dry matter), respectively, and amino acids composition. Watanabe et al. (2004) estimated the molecular weight of a-ovomucin at 254 kDa whereas that of b-ovomucin has been estimated, using SDS-PAGE, to be between 400 and 720 kDa (Itoh et al., 1987). Ovomucin is heat stable over the pH range between 7.1 and 9.4. Cunningham and Lineweaver (1965) showed that the viscosity and absorbance of ovomucin solutions did not change upon heat treatment at 90 °C for 2 h. Lysozyme Lysozyme is a small enzyme molecule of 14.3 kDa capable of hydrolyzing specific polysaccharides that cleaves the b 1-4 linkages between N-acetylneuraminic and N-acetylglucosamine in bacteria cell. The single polypeptide chain consisting of 129 amino acid residues is cross-linked by four disulfide bridges. Because of its strong basic character (pI = 10.7), lysozyme binds to ovomucin, ovotransferrin and ovalbumin, via electrostatic interactions between positively charged lysozyme and negatively charged residues of sialic acid in these glycoproteins. Young et al. (1994) analyzed the three-dimensional structure of hen egg white lysozyme. The lysozyme molecule has two domains: the N-terminal domain (residues 40 to 88) with a hydrophobic core is composed of antiparallel b-sheets whereas the second hydrophilic domain, residues 1 to 39 and 89 to 129, is made up of a-helix. The enzyme is much more heat sensitive in egg albumen than when present alone. Minor egg white proteins The remaining proteins listed in Table 19.2 and 19.3 (G2 and G3 globulin, ovoinhibitor, ovoglycoprotein, ovoflavoprotein, ovomacroglobulin, cystatin, avidin) account for 400 mM) but exerted a potent vasorelaxant and antihypertensive effect upon oral administration to SHR rats at a dosage of 10 mg kg–1 (Matoba et al., 1999). Non-hydrolyzed whole egg white did not have ACE-inhibitory property whereas its digestion with pepsin resulted in high ACE-inhibitory activity with IC50 = 55.3 mg mL–1 after hydrolysis for 3 h. Treatment with trypsin and chymotrypsin induced very poor ACE-inhibitory activity values, indicating the importance of the enzyme specificity in producing peptides with ACEinhibitory activities (Miguel et al., 2004). Furthermore, these workers showed that the active peptides could be enriched from the hydrolysate using ultrafiltration with a 3 kDa molecularweight-cut-off (MWCO) membrane. The permeate generated contained peptides with molecular mass 90%) was observed under optimized conditions and moderately pure lysozyme (80–90%) could be obtained by carrying out a diafiltration step (Ghosh et al., 2000). Production of ACE-inhibitory peptides from egg white proteins with various proteolytic enzymes was carried out using a membrane reactor. Egg white proteins hydrolyzed with thermolysin produced the highest ACE-inhibitory potential (IC50 = 54.1 mg mL–1). Thermolysin hydrolysate was fractionated using UF membrane of MWCO of 10, 3 and 1 kDa. A lower IC50 (17.2 mg mL–1) was obtained in the UF permeate with 1 kDa MWCO (Chiang et al., 2008). By a modification of polymeric forms of lysozyme under optimal conditions of UF, a preparation comprising 53.3% of lysozyme polymeric forms, i.e. 33.2% of dimer and 20.1% of trimer, was obtained. Modified lysozyme preparation by UF showed the highest bacteriostatic activity against Gramnegative bacteria selected (Lesnierowski et al., 2009). A water-soluble plasma protein from egg yolk granules was obtained with various simple water dilutions, followed by centrifugation or filtration. Two factors were critical, pH and egg yolk dilution, for IgY recovery. Optimum recovery of IgY (93–96%) was obtained by a six-fold water dilution at pH 5.0–5.2 with incubation for 6 h at 4 °C. A 100 kDa MWCO extracted IgY from water-soluble plasma upon the separation of crude IgY by ammonium sulfate precipitation. A maximal recovery of IgY (> 98%) was obtained and this value was similar to the degree of purity obtained by gel filtration (Akita and Nakai, 1992). A serial filtration approach, involving dilutions, paper filtration and delipidation using hydrophobic filters or using different UF membranes, was developed by Kim and Nakai (1996; 1998). The delipidated water-soluble fraction was thereafter purified using 100 kDa UF membranes and both high recoveries (72–89%) and purity (74–99%) were obtained. Although the fractionation of proteins through a membrane depends


612 Separation, extraction and concentration processes on physicochemical parameters such as pH and salt concentration (Kim and Nakai, 1996; 1998), the surface properties of the membrane are also important for the successful protein fractionation. Hernandez-Campos et al. (2010) purified IgY from egg yolk water-soluble protein by UF–diafiltration with different membranes. The best selectivity and purification factors were obtained without salt at pH 5.7 and 6.7 using polyethersulfone and modified polyethersulfone of 100 MWCO membrane, respectively. Compared with chromatographic methods, membrane separation techniques offer the advantage of lower cost and ease of scale-up for commercial production. Membrane-based separation techniques are powerful tools for fractionation/purification of proteins and bioactive peptides. UF membranes can be used as preliminary step for the removal of enzymes and non-hydrolyzed proteins, and to further fractionate the peptide mixture. Various combinations of precipitation and UF using selective membranes can be applied for the fractionation of food proteins. In addition to selective membranes, chromatographic methods can be employed successfully to isolate specific bioactive compounds from a complex mixture. UF membranes were successfully used to enrich peptide fractions. Membranes consisting of negatively charged materials such as NF were used to desalt or to fractionate acidic peptides from hydrolysate mixture (Chay Pak Ting et al., 2007; Wijers et al., 1998). 19.4.4 Other separation methods A new isolation method was described for IgY. Use of food-grade products such as sodium alginate or l-carrageenan was investigated for precipitation of yolk lipoproteins from the water-soluble fraction to purify IgY. Polysaccharides exhibit satisfactory yields of IgY (Hatta et al., 1988; 1990). In another study, the optimal separation of IgY was achieved with 0.15% pectin and at pH 5.0, implying that the interactions between polysaccharides and lipoproteins were mainly ionic bonds, hydrophobic interactions and hydrogen bonds (Chang et al., 2000).

19.5 Conclusion and perspectives Considering that the discovery of novel bioactive peptides and their possible functions and health benefits are constantly increasing, there is a growing need to develop new technologies for the production of specific bioactive peptides from hydrolysate mixtures. Membrane-based separation techniques have traditionally been used to separate molecules of different sizes and lowmolecular-weight components from proteins. UF, alone and in combination with other techniques such as NF and chromatography has shown its potential to separate peptide mixtures on a large scale. However, the further research into membrane areas (such as membrane materials and chemistry, © Woodhead Publishing Limited, 2010

Fractionation of egg proteins and peptides 613 module configurations) for the complete separation/purification of proteins/ peptides is still needed. The addition of affinity ligands to chelate proteins/ peptides could be useful in obtaining maximal selectivity by combining with membrane processes.

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Supercritical-fluid extraction of lycopene from tomatoes 619

20 Supercritical-fluid extraction of lycopene from tomatoes J. Shi and S. Jun Xue, Agriculture and Agri-Food Canada, Canada, Y. Jiang, The Chinese Academy of Sciences, China and X. Ye, Zhejiang University, China

Abstract: Several process parameters for supercritical CO2 fluid extraction – such as pressure, temperature, flow rates, co-solvent or modifier concentrations, resident time, moisture content, particle sizes, and particle size distribution – have individual or combined effects on the recovery of lycopene from tomatoes. The solubility and bioactivity of lycopene, and the composition of the extract and extraction yield can be affected. Improved processing conditions and reduced cost are required if the extraction of lycopene from tomato materials using the supercritical CO 2 fluid extraction process is to become more economical at low throughputs. Key words: bioactivity, lycopene, supercritical fluid extraction, tomato.

20.1 Introduction Lycopene is a phytochemical responsible for the red pigments found in plants. It is a non-provitamin A carotenoid that plays an important role in the biosynthesis of many carotenoids. Recently, the extraction of lycopene and other carotenoids has attracted attention owing to their biological and physiochemical properties, particularly those which possess natural antioxidant activities. Antioxidants have been associated with disease prevention and reduction (Negre-Salvayre et al., 2006; Papas, 1999; Wu, et al., 1999). Structurally, lycopene is an acyclic, open-chain, C40 polyisoprenoid unsaturated carotenoid having 13 double bonds, of which 11 are conjugated, arranged in a linear array, and it has a molecular formula of C40H56 (Fig. 20.1). Because of this unique molecular structure, lycopene has high antioxidant activity and singlet oxygen quenching ability that is thought to be beneficial



Fig. 20.1 Structure of lycopene.

to human health. The antioxidant protective effect of lycopene and tomatoes has been shown in vitro as well as in vivo (Breinholt et al., 2000; Hadley et al., 2003; Rao and Agarwal, 1998; Suganuma and Inakuma, 1999; Willcox et al., 2003). Therefore, foods containing lycopene are of considerable interest. The singlet oxygen quenching ability of lycopene has been found to be three times greater than that of b-carotene and a-tocopherol (Stahl and Sies, 1992, 1996). Epidemiological studies and investigations have shown important roles of lycopene and other carotenoids in free radical inactivation and fat peroxidation inhibition (Krinsky and Rock, 1998). The health benefits of lycopene-rich diets and its effect on minimizing the risk of cardiovascular ailments and various forms of cancers have been reported (Palozza, 1998; Olson, 1986). More recent studies have demonstrated the important functions of lycopene and other carotenoids in the pathophysiology of chronic diseases (Rao and Agarwal, 1999; Rao and Rao, 2007). Increasingly, in vivo and in vitro clinical studies have shown its protective effect against the growth of tumor cells and its ability to protect against cardiovascular (Arab and Steck, 2000) and coronary heart diseases, and cancer (Clinton, 1998). With its 11 conjugated and two non-conjugated double bonds, lycopene is a more efficient antioxidant (singlet oxygen quencher) than b-carotene, a-carotene, and a-tocopherol (Mascio et al., 1989; Shi, 2002; Shi and Le Maguer, 2000). As the most abundant carotenoid in tomatoes, lycopene is the dominant pigment responsible for the color of tomatoes. Lycopene occupies the largest portion, about 80–90%, of the carotenoids in ripe tomatoes, followed by b-carotene, phytoene, and the other minor carotenoids. Concentrations of lycopene in tomatoes approach 50–100 mg kg–1, and it has a higher colour intensity than b-carotene (Shi and Le Maguer, 2000). Other sources of lycopene include pink grapefruit, guava, watermelon, autumn olive, and apricots. Ripe tomato skins contain approximately five times more lycopene than the pulp. The industrial processing of tomato products produces wastes such as seeds and skins. However, reuse of those wastes is limited to animal feed. Even though ripe tomatoes are the most abundant source of lycopene, over 90% is located in the skin, which constitutes the greater part of the waste, and is a potential natural source for lycopene extraction (Shi and Le Maguer, 2000). Lycopene content in plant materials is dependent on the species and the temperature at which growth and maturation occurs. Factors such as climate trends significantly alter the amount of lycopene in the material, thus affecting the yield. Lycopene is the last pigment to appear during maturation and its


Supercritical-fluid extraction of lycopene from tomatoes 621 development is inhibited by temperatures higher than 30–32 °C, whereas other carotenoids degrade at these temperatures. As a result, the pigment concentrations in plant materials may vary between crop cycles (Shi et al., 1999b). The concentration of lycopene also changes during ripening and storage. Liu et al. (2009) reported that the accumulated lycopene contents of tomatoes did not change significantly during the first four days of storage. However, between days 4 and 21, the lycopene content increased. A growing interest in and demand for healthy, environmentally safe, and cost-efficient products has driven the research and application of new technologies in the food, pharmaceutical and cosmetic industries. Extraction processes are commonly used to enrich and detoxify food through the removal of targeted components from natural products. Organic solvents are used in conventional methods for the extraction of bio-compounds such as lycopene from plant materials. However, these solvents not only generate environmentally hazardous problems and requests for expensive disposal procedures for the chemical extraction solvents, but also chemical residues remaining in the final products become a major safety concern. With increasing government restrictions reflecting consumer concerns on food safety, alternative and reliable extraction techniques are of great interest. Extraction processes are needed for ‘additive-free natural’ products. It is clear that the current concern for safety in food products has increased interest in ‘green’ extraction techniques, instead of the conventional organic solvent extraction processes. The determination of lycopene in food products and development of a safe ‘green’ extraction process to complement the fortification of functional foods is of great public interest. One possible environmentally friendly alternative is supercritical fluid extraction (SFE), in particular using supercritical CO2 ‘green technology’ because it is physiologically harmless, environmentally safe, non-explosive, exhibits high selectivity as a result of low viscosity, high diffusivity, and liquid-like density, as well as being readily available and easily removed from products (Simandi et al., 2002). Because of the CO2 supercritical state (31 °C and 7.38 MPa), procedures should allow supercritical operation of thermally labile compounds that would be easily degraded at high temperatures. SFE has attracted growing interest for the recovery of natural compounds for large-scale industrial production over recent decades (O’Day and Rosenau, 1982). SFE is successfully and widely used for the extraction of lycopene from ripe tomatoes (Cadoni et al., 2000) and tomato processing wastes (Baysal et al., 2000; Kassama et al. 2008; Ollanketo et al., 2001; Rozzi et al., 2002; Sabio et al., 2003; Topal et al., 2006; Vasapollo et al., 2004). One significant thermodynamic advantage of using supercritical fluid is its ease of separation from the extracted solutes by simply modifying the operation conditions, either pressure or temperature. Supercritical fluids have liquidlike densities that give superior mass transfer characteristics compared with organic solvents, and are characterized as low-viscosity and high-diffusivity fluids. In addition, supercritical fluids have low surface tension leading to


622 Separation, extraction and concentration processes easy penetration into the porous biological matrix of plant material while releasing the targeted components.

20.2 Supercritical-fluid extraction (SFE) of lycopene Supercritical fluids are, by definition, at a temperature and pressure greater than or equal to the critical temperature and pressure of the fluid. They actually have physical properties somewhere between those of a liquid and a gas. Supercritical fluids are able to spread out along a surface more easily than a true liquid because they have lower surface tensions than liquids. The supercritical CO2 is fed at high pressure by means of a pump, which pressurizes the extraction tank and also circulates the supercritical fluid throughout the system. Figure 20.2 demonstrates an example of a typical single-stage supercritical CO2 extraction system. Once the supercritical CO2 reaches the equilibrium state in the extraction vessel, the extraction process proceeds with the manipulation of pressure and temperature to achieve the ideal operating conditions. The mobile phase, consisting of the supercritical CO2 and the solubilized lycopene component, are transferred to the separator where the solvating power of the fluid is decreased by increasing the temperature and or decreasing the pressure of the system. The lycopene extract precipitates in the separator whereas the supercritical CO2 is either released or recycled back to the extractor. As the solution containing the extracts of lycopene compounds leave the extractor and flow to the first separation vessel via the pressure regulator, a paste of lycopene oleoresins settles to the bottom as they separate and this is collected, whereas the remaining solution goes to the second-stage separator where the fractionation of the lycopene components occurs. A third stage of separation might be required for the complete isolation of pure lycopene

Back pressure valve

Pressure valve

Separator

Cooling bath

Extract column

CO2 outlet Heat exchanger

Heat exchanger

CO2 pump

Extracts outlet

CO2 source

Fig. 20.2 Schematic diagram of a typical single-stage supercritical fluid extraction system with CO2 (modified from Shi et al., 2006).


Supercritical-fluid extraction of lycopene from tomatoes 623 components. In commercial-scale processing, multiple extraction vessels are normally used to enhance operation efficiency and throughput (Fig. 20.3). The process requires an intermittent batch system because it is interrupted at the end of the extraction period, when the pressure must be released so the extraction vessel can be switched out of the extractor loop. By having two or more interchangeable extraction vessels, the unloading and reloading of a vessel can occur while extraction of a previously charged vessel is in progress. This reduces the downtime and improves the overall production efficiency.

20.3 Factors affecting lycopene yield Many studies have successfully extracted lycopene from tomato materials by SFE. The technology has been continuously developed and users have established environmentally sound and complimentary safe techniques for extracting food-grade, bioactive components used as natural ingredients from agricultural materials. There have been numerous studies on the effects of various independent variables – such as pressure, temperature, flow rates, and co-solvent or modifier concentrations – on lycopene yield using SFE (Baysal et al., 2000; Ollanketo et al., 2001; Ciurlia et al., 2009; Kassama et al., 2008; Yi et al., 2009, Shi et al., 2009a; 2009b; Vaughn et al., 2008). Two-stage separation vessels

Storage tank

Three-stage extraction columns

CO2 inlet

Co-solvent inlet Pumps

Pressure valve Heat exchanger

Fig. 20.3 Schematic diagram of commercial scale multi-stage supercritical fluid extraction system used to fractionate bioactive components.


624 Separation, extraction and concentration processes The optimization of pressure, temperature, and flow rate is necessary to obtain a high recovery of lycopene. Baysal et al. (2000) observed the highest recovery (54%) of lycopene obtained at 55 °C, 30 MPa, and 4 kg h–1 with 5% ethanol as modifier. Ollanketo et al. (2001) recovered 94% of lycopene in 15 min at 40 MPa and 110 °C. Rozzi et al. (2002) obtained a maximum recovery (61%) at 80 °C, 35 MPa, and 2.5 mL min–1. Yi et al. (2009) studied the effects of SFE parameters on the recovery of lycopene and antioxidant activity. In this study, the maximum total lycopene content of 31.25 mg g–1 of raw tomato was extracted at the highest temperature of 100 °C, 40 MPa and 1.5 mL min–1. Process parameters such as solvent flow rate, residence 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, bioactivity, and composition of the extract. For example, the residence time can have an important influence on the composition of the extracted compound. 20.3.1 Effects of temperature, pressure and flow rate on recovery of lycopene Effect of pressure Plate IIa (between pages 292 and 293) shows that pressure significantly influences the rate of extraction. Higher density causes a double effect that increases the solvation power and reduces the interaction between the fluids and matrix, resulting in a decrease in 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 (Tonthubthimthong et al., 2001). It is commonly considered that an increase in pressure results in an increase in CO2 density, increasing the solvating power of the supercritical fluid. Thus, higher pressure is responsible for quantitative recoveries and stronger interactions between the fluid and the matrix (Shi et al., 2009a; Topal et al., 2006). Effect of temperature Temperature is a parameter with significant influence on SFE yield; thus manipulating it could have an adverse implication on the process and yield. Plate IIb shows the general trend of increased extraction yield as temperature increases relative to the pressure. The extraction yields increased both with temperature and pressure, whereas the effect of temperature is more significant than that of pressure. Vági et al. (2007) maximized the recovery of lycopene at 46 MPa and 80 °C by SFE. At 40–100 °C, maximum yields were obtained for extraction of lycopene. The highest temperature gave the maximum lycopene content (Rozzi et al., 2002). Cadoni et al. (2000) reported that at 85 °C a maximum of 65% of lycopene was recovered from the pulp


Supercritical-fluid extraction of lycopene from tomatoes 625 and skins of ripe tomatoes. However, with further elevation of temperature, degradation and isomerization of lycopene was often encountered (Shi et al., 1999a, 2009b, 2002; Shi and Le Maguer, 2000; Yi et al., 2009). It is well-known that increasing the temperature reduces the solvent density and consequently decreases the recovery of lycopene at constant pressure. At the same time, higher temperature promotes the solubility of lycopene and increases the recovery by increasing mass transfer of lycopene in the matrix, and from the matrix to the CO2 extraction medium (Marsili and Callahan, 1993; Shi et al., 2007d). Therefore, the increase in temperature could have either a positive or a negative effect, as a result of the balance between CO2 density (r) and solute vapour pressure. The increase in the recovery of lycopene by SFE depends more on the solute’s vapour effect. Although lycopene is not stable during long heating times and was approximately 53.5% degraded after 60 min at 100 °C through isomerization or auto-oxidation (Boskovic, 1979; Mayeaux et al., 2006; Shi and Le Maguer, 2000; Shi et al., 1999a). However, in the thermal processing of tomato products, the total lycopene contents still increased in the products owing to the elevated release of lycopene from the tomato tissue matrix (Dewanto et al., 2002; Seybold et al., 2004; Toor and Savage, 2006). For SFE, the enhanced release of lycopene from the insoluble fibre portion of tomato skin may also contribute to the increased recovery. Effect of CO2 flow rate In an extraction, the flow rate of supercritical CO2 has an influence on the extraction efficiency. The flow rate controls the amount of solvent (e.g. CO2) to be used and the extraction time. Extraction is a diffusion-based process, with the solvent required to diffuse into the matrix, and the extracted component to diffuse out of the matrix into the solvent at equilibrium state. The flow rate also greatly affects solubility of extracted components in fluid and equilibrium of diffusion between solvent and extracted components during extraction. If the concentration of extracted components is much higher than the solvent, the solubility of extracted components in the solvent could be limited and slow down the extraction process. Therefore, optimization of flow rate is important for extraction time and cost efficiency. For example, to maximize the rate of extraction, the flow rate should be high enough for the extraction to be completely diffusion limited, but this is very wasteful of solvent. In contrast, the lower flow rate would minimize the amount of solvent used, but the extraction should be completely solubility limited, and thus would take a very long time. Therefore, the optimum flow rate is probably somewhere in the region where both solubility and diffusion are significant factors. At optimum temperature and pressure, the extraction process is performed under conditions in which the CO2 flow rate is variable. The linear correlation is observed for the flow rates and the extraction rates. The extraction rate increased with increasing flow rate. When the intra-particle diffusion


626 Separation, extraction and concentration processes resistance is considered as a factor in the material matrix, more CO2 is required to make contact with the targeted components. Thus, the extraction rate, is a function of CO2 consumed: the greater the amount of supercritical gas used, the higher the yield obtained (Friedrich and Pryde, 1984). When the temperature, pressure, and overall extraction time are kept constant, the effect of various solvent flow rates on the carotenoid yield is minor. Plate IIc shows the effect of flow rate on total lycopene yield at constant pressure (30 MPa) or temperature (70 °C). An increase in flow rate from 1.0 to 2.0 mL min–1 did not show a significant change in the yield of lycopene (Yi et al., 2009). Results indicated that the higher flow rate could produce faster extractions and higher recoveries since the higher flow rate could overcome the interface resistance for lycopene transport from the tomato skin tissue to the CO2 fluid (Hawthorne et al., 1995). However, excessively high flow rates would produce undesirable results owing to a decrease in contact times between the solvent and the solute (Topal et al., 2006). This may force the solvent to leave the system without reaching its solubility limit (Anitescu et al., 1997). High flow rates may also cause the sample to compact and restrict the fluid flow, thus reducing the amount of solvent that comes in contact with the solute (Franca and Meireles, 2000). Increasing the CO2 flow rate would increase fluid diffusivity and improve the heat and mass transfer within the system, because the resulting fluid temperature approaches the wall temperature more quickly (Shi et al., 2007a). The effect of solvent flow rate on mass transfer rate and total yield in the constant extraction rate period is significant. Effect of operating time Residence time is an important factor that influences yield and the economic viability of the process. When optimizing extraction conditions, the extraction time of the process is an important parameter. For the extraction of lycopene by SFE Baysal et al. (2000) found that the highest carotene yield was obtained at an extraction time of 2 h, as opposed to 1 or 3 h. A 1-h extraction may not suffice for the maximum amount of carotenoids to be dissolved in the solvent, whereas over 3 h there is an increase in the occurrence of degradation. The duration and intensity of thermal processing are directly correlated to the degree of isomerization and degradation of the targeted compounds (Baysal et al., 2000). Therefore, to minimize the degradation effect, it is advisable to reduce the extraction time as much as possible. In addition, optimizing extraction conditions towards the shortest extraction time possible with the maximum extraction recovery is ideal from the point of view of the extract quality and processing cost. Effect of moisture content of raw materials Water represents approximately 80–90% of the total weight of fresh plant materials and interferes with the effectiveness of the SFE (Hopper and King, 1991). Thus, the initial moisture content may improve or impede the


Supercritical-fluid extraction of lycopene from tomatoes 627 yield results depending on the polarity of the compound. The non-polar compounds such as lycopene produce smaller yields with higher moisture content (Anitescu et al, 1997; Chao et al., 1993). Water has a small but finite solubility in supercritical CO2, therefore, it can also be extracted with the targeted components. The presence of a little water (less than 10%) aided in the extraction of lycopene, particularly at the beginning of the process (Shi et al., 2006). A moisture content of 7–18% had negligible effect on extractability of oil. However, above an 18% moisture content, the presence of water affected the extraction and the efficiency decreased (Nagy and Simándi, 2008). The high water content in the matrix inhibited the flow of SCF by changing surface tension and contact angles as a result of phase interaction. Therefore, water removal in most cases frees the internal pores and thus increases the mass transport intensity. Higher moisture contents cause a higher probability of formation of a thin film of water between the matrix and the SCF phase. Possible effects of water include an increase in distance that carotenoids must travel to reach the solvent and swelling of the cell matrix, which have negative and positive effects on the process, respectively. Effect of particle size Particle size is a factor that influences the extraction recovery, having a significant impact on the flow behaviour of SCF in the sample matrix. The extraction process and diffusivity of the extraction medium are hindered by large particle sizes (>550 mm) because of the distance which the lycopene travels from the inside of the particle to the surface (Shi et al., 2007a). By reducing the size of the particles, components such as carotenoids have less distance to travel. Thus, the collective amount of surface area with which the solvent is in contact is increased, an important factor during the initial stages of extraction (Ollanketo et al., 2001). The particle size is affected by sample pre-treatment. For example, the methods of drying (air, oven, vacuum, or freeze drying), milling, and other mechanical, ultrasonic, high-pulsed electronic field, and non-mechanical treatments can change the particle size by attrition or size reduction. Numerous experimental results have indicated that smaller particle sizes are ideal for extraction processes, and the manner in which the solid material is crushed and powdered also affect the results (Ferreira and Meireles, 2002; Spanos et al., 1993). The degree to which the material is crushed determines the amount of cells that are broken, and this has a positive correlation with recovery. The smaller the particle size, the larger is the surface area, and the bioactive components are released more easily. However, if the particle size is exceedingly small that can favour channelling which commonly results in a decrease in recovery (Ziémons et al., 2005). Only 40% lycopene recovery was obtained from powdered tomato skins crushed with a household blender, relative to the amount achieved when sea sand was used to assist the grinding procedure (Ollanketo et al., 2001). Therefore, optimizing particle


628 Separation, extraction and concentration processes size significantly improves the yield of extraction. Sun and Temelli (2006) noted that the optimal range of particle size was 0.25–0.5 mm for lutein, b-carotene, and a-carotene. However, for certain processes, it is important to be cautious of the degree of crushing. Gouveia et al. (2006) studied the effects of slight, moderate and complete crushing, which gave 20%, 40%, and 65% carotenoid recovery, respectively, and also found that samples that have been extremely crushed may not be affected by the addition of entrainers.

20.4 Effects of pressure and temperature on the antioxidant activity of lycopene Recovery is increased with increasing temperature owing to increasing solubility of the targeted compounds; therefore, for economic reasons, food industries prefer to increase the temperature to achieve higher yields. However, the bioactivity of the lycopene may be compromised by heat because of the instability of lycopene at higher temperatures (Mayer-Miebach et al., 2005; Shi et al., 2002b, 2004b, 2008, 2009b; Vega et al., 1996; Yi et al., 2009). Yi et al. (2009) reported that the recovery and bioactivity of lycopene increased when the temperature increased from 20 to 60 °C. The recovery of lycopene increased slowly and its antioxidant activity declined above 80 °C. Pol et al. (2004) made a remarkable observation that lycopene recovery was increased with increasing temperature, but the bioactivity of the extracted lycopene did not follow the trend of the recovery of lycopene extracts at high temperature. No significant effects of pressure or flow rate on the antioxidant activity were observed (Yi et al., 2009). Cadoni et al. (2000) and Shi and Zhou (2006) reported that the results obtained from SFE of lycopene from tomatoes are poorly reproducible because of the decomposition and isomerization of lycopene during the extraction process. Yi et al. (2009) investigated the effects of the two major supercritical fluid extraction parameters (temperature and pressure) on the antioxidant activities of the extracts as shown in Fig. 20.4. The recovery increased with the increase in pressure, whereas antioxidant activity decreased slightly. The recovery of total lycopene increased with temperature, but the antioxidant activity of the lycopene-rich extract decreased with increasing temperature (Hackett et al., 2004; Yi et al., 2009). At 25 and 50 °C, lycopene in an oleoresin is degraded predominately through oxidation, whereas isomerization increases at 75 and 100 °C. If lycopene is isomerized, the proportion of lycopene isomers is changed, that then further influences the antioxidant activity. In general, the change in antioxidant activity in lycopene-rich extracts is mainly influenced by their lycopene content, proportions of the isomers of lycopene, and the synergistic effects of carotenoids in the extract (Shi et al., 2004a,b, 2004c, 2007c; Shixian et al., 2005). The total antioxidant activity of


0.30

30

0.25

25

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15

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60 70 80 Temperature (°C) (a)

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35

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20 0.15

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30 Pressure (MPa) (b)

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Torlox of lycopene (mM mg–1)

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0.05 40

Fig. 20.4 Changes of lycopene yield (mg –1g raw material) (d) and antioxidant activity (mM mg–1 Torlox) of the extract (m) (a) at 40 to 100 °C, 30 MPa and flow rate of 1.5 mL min–1, and (b) at 20–40 MPa, 70 °C and flow rate of 1.5 mL min–1 (modified from Yi et al., 2009).

the extract, that is the antioxidant activity of each unit of lycopene multiplied by the total lycopene amount in the extract, firstly significantly increases as the temperature increases, and then slightly decreases as temperature is kept at a high temperature. The antioxidant activity of extracts first increased with increasing lycopene contents in the extracts between 40 and 80 °C, and then started to decline thereafter. The slight decrease above 90 °C might be affected by the degradation of other carotenoids (Cocero et al., 2003). Dewanto et al. (2002) found that thermal processing increased the total antioxidant activity of tomato from 4.13±0.36 to 6.70±0.25 mmol g–1 of vitamin C equivalents after 30 min of heating at 88 °C. The total antioxidant activity probably increases because larger amounts of lycopene are released from the matrix during thermal processing. HPLC chromatograms of the extracts


630 Separation, extraction and concentration processes obtained at 50 and 90 °C are shown in Fig. 20.5. All-trans lycopene is the major configuration eluting with a retention time of 15.87 min under both conditions. Three major cis-isomer peaks are detected around 6.8, 8.9, and 16.1 min of elution time in extracts prepared at 50 °C. When the extraction temperature is elevated to 90 °C, total lycopene increased 1.18 fold, whereas all-trans lycopene contents increased only 1.03 fold and total cis-isomers increased more than 2.13 fold, respectively. Moreover, more than three major peaks of cis-isomers are detected (Fig. 20.5) because of new cis-isomer generations. The higher increase in cis-isomers than total and all-trans lycopene indicates the transformation of all-trans to cis-isomers. As extraction vessel temperature increased from 50 to 90 °C, the ratio of all-trans to total cislycopene isomers changed from 1.67 to 1.40 (Table 20.1). Further increases in the operating temperature resulted in dramatic isomerization. The lower ratio of trans- to cis-isomers obtained at high temperature indicated higher lycopene bioavailability owing to the cis-isomers generated (Boileau et al., 1999). The isomerization of lycopene in tomato samples during thermal processing above 75 °C, and the decrease in the trans- to cis-isomer ratios from 9:1 to 2:3 after heating at 140 °C, were demonstrated (Cocero et al.,

4 cis isomers mAU

3

trans-lycopene

2 5-cis

1 0

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15 20 Time (min) (a)

25

30

25

30

trans-lycopene

4 mAU

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0

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Fig. 20.5 Chromatograph of SFE extract obtained at (a) 50 °C and (b) 90 °C at 30 MPa and flow rate of 1.5 mL min–1 (modified from Yi et al., 2009). mAu, milli absorbance unit. © Woodhead Publishing Limited, 2010

Supercritical-fluid extraction of lycopene from tomatoes 631 Table 20.1 All-trans and cis-isomers contents and ratio in the SFE extract obtained at 40–100 °C, 30 MPa and a flow rate of 1.5 mL min–1 (modified from Yi et al., 2009) Temperature Total content All-trans (mg g–1) content (mg g–1) 100 90 80 70 60 50 40

29.46 26.12 22.69 14.66 13.85 12.00 10.35

16.75 15.26 13.76 9.04 8.74 7.50 6.52

Total cis Ratio Ratio Ratio* content (trans:total) (cis:total) (trans:cis) (mg g–1) 12.71 10.86 8.93 5.62 5.11 4.50 3.83

0.57 0.58 0.61 0.62 0.63 0.63 0.63

0.43 0.42 0.39 0.38 0.37 0.37 0.37

1.32c 1.40c 1.54bc 1.62ab 1.71a 1.67a 1.70a

*The different letters shows the values that are significantly different (P 500 L by region. (Source: Schuetz Consulting/Uhde HPT.) 800 700

Pressure (bar)

600 500 400 300 200 100 0 1980

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Fig. 1.18 Maximum pressure of SFE processes. (Source: Schuetz Consulting/Uhde HPT.)

by pressure- and/or temperature-dependent precipitation. In Fig. 1.19 an ultra high pressure extraction unit operating up to 2500 bar is presented. The evaluation of costs for several plant materials processed by ultra high pressure extraction using SC CO2 is shown in Fig. 1.20. It is clear that with © Woodhead Publishing Limited, 2010


E2

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Fig. 1.19 Ultra high pressure extraction unit (operating pressure 2500 bar). (E1 and E2, extractors; S1 and S2, separators). (Photo: courtesy of Uhde HPT, Hagen, Germany.) 2.50

Specific processing costs (7/kg)

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Fig. 1.20 Specific processing costs versus operating pressure (Lütge et al., 2007)

an increased operating pressure in a SC extraction plant (retaining the same capacity of equipment), the total processing costs decrease. In the future, further limitations on the use of organic solvents, new applications of several substances, changing customer requirements, sustainable © Woodhead Publishing Limited, 2010

36 Separation, extraction and concentration processes production and processing of substances, will all lead to new developments in high-pressure processing. We can be sure that advances in the field of high-pressure research into cheap and environmental friendly solvents, such as CO2 and some other gases and sub- or supercritical water, will open up new pathways for substances and products produced at high pressure.

1.7 References Y. Adachi and H. Sugie, Fluid Phase Equilib. 28(2) (1986) 103–118. A. Anderko, Fluid Phase Equilib. 61 (1990) 145–225. A. Anderko, in: J.V. Sengers, R.F. Kayser, C.J. Peters, H.J. White (Eds.), Equations of state for fluids and fluid mixtures, Elsevier, Amsterdam, 2000, pp. 75–126. J.D.S. Arons and G.A.M. Diepen, Rec. Trav. Chim. Pays-Bas, 82 (1963) 249–256. A. Bertuco and G. Vetter, High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2001. G. Brunner, Counter-current separations, J. Supercrit. Fluids 47 (2009) 574–582. G. Brunner, Gas extraction. An introduction to fundamentals of supercritical fluids and the application to separation processes, Darmstadt: Steinkopff; New York: Springer, 1994. O.J. Catchpole, S.J. Tallon, W.E. Eltringham, J.B. Grey, K.A. Fenton, E.M. Vagi, M.V. Vyssotski, A.N. MacKenzie, J. Ryan, Y. Zhu, The extraction and fractionation of specialty lipids using near critical fluids, J. Supercrit. Fluids 47 (2009) 591–597. R.P. Danner and P.A. Gupte, Fluid Phase Equilib. 29 (1986) 415–430. B. Diaz-Reinoso, A. Moure, H. Dominguez, and J.C. Parajó, Antioxidant extraction by supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 275–304. R. Dohrn, Berechnung von Phasengleichgewichten, Friedr. Vieweg & Sohn Verlagsgesellschaft mbH, Braunschweig, 1994. R. Eggers, Supercritical fluid technology in oil and lipid chemistry, AOCS Press, 1996, 35–62. W. Eltringham and O.J. Catchpole, Processing of fish oils by supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 141–188. T. Fang, M. Goto, M. Sasaki, and D. Yang, Extraction and purification of natural tocopherols by supercritical CO2, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 103–140. U. Fleck, G. Brunner and R. Karge, Purification of synthetic crude tocophenol acetate by means of supercritical fluid extraction, in: Proceedings of the 5th International Symposium on Supercritical Fluids, Atlanta, April 2000. T. Gamse, I. Rogler and R. Marr, 1999, Supercritical CO2 extraction for utilisation of excess wine of poor quality, J. Supercrit. Fluids 14 (1999) 123–128. T. Gamse and R. Marr in A. Bertucco, G. Vetter. High pressure process technology: fundamentals and applications, Industrial Chemistry Library, volume 9, 2001, 383. D.S. Gardner, Commercial scale extraction of alpha acids and hop oils with compressed CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 84–100. J.-H. Hsu and C.-S. Tan, Separation of ethanol/water solution with supercritical CO2 in the presence of a membrane, in S.S.H. Rizvi, Supercritical fluid processing of food and biomaterials, Chapman & Hall, 1994, 114–122. M.J. Huron and J. Vidal, Fluid Phase Equilib. 3 (1979) 255.


Principles of supercritical fluid extraction and applications 37 P.G. Jessop and W. Leitner, Chemical synthesis using supercritical fluids, Wiley-VCH, July 1999. J.W. King and K. Srinivas, Multiple unit processing using sub- and supercritical fluids, J. Supercrit. Fluids 47 (2009) 598–610. Ž. Knez, Separation of components of citrus oils – industrial project, unpublished data, 1989. Ž. Knez, F. Posel and I. Krmelj, High pressure extraction of organics from water, in S.S.H. Rizvi, Supercritical fluid processing of food and biomaterials, Chapman & Hall, 1994, 181–186. Ž. Knez and M. Škerget, Phase equilibria of the vitamins D2, D3 and K3 in binary systems with CO2 and propane, J. Supercrit. Fluids 20 (2001) 131–144. P.H. van Konynenburg and R.L. Scott, Critical lines and phase equilibria in binary van der Waals mixtures, Phil. Trans. Roy. Soc. London, Ser. A 298 (1980) 495. E. Lack and H. Seidlitz, Commercial scale decaffeination of coffee and tea using supercritical CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 101–139. E. Lack and B. Simandy in A. Bertucco and G. Vetter. High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2001, 537–575. S. Li, Application of supercritical fluids in traditional chinese medicines and natural products, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 215–242. T.W. de Loos, On the phase behaviour of asymmetric systems: The three-phase curve solid–liquid–gas. J. Supercrit. Fluids 39 (2006) 154–159. D. Luedecke and J.M. Prausnitz, Fluid Phase Equilib. 22(1) (1985) 1–19. C. Lütge and E. Schuetz, Market trends and technical developments in high pressure technology, in: Ž. Knez and M.J. Cocero, Proceedings of the 5th international symposium on high pressure process technology and chemical engineering, June 24–27, 2007, Segovia, Spain. C. Lütge, M. Bork, Ž. Knez, M. Knez Hrn�i�, M. Krainer, Ultra high pressure dense gas extraction and fractionation, in: Ž. Knez and M.J. Cocero, 5th International symposium on high pressure process technology and chemical engineering, June 24–27, 2007, Segovia, Spain. R. Marr and T. Gamse, High pressure technology: fundamentals and application, Industrial Chemistry Library, volume 9, 2000, 396–402. M.A. McHugh and V.J. Krukonis, Supercritical fluid extraction: principles and practice, Butterworths, Stoneham, 1986. M.A. Meireles, Extraction of bioactive compounds from Latin American Plants, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 243–274. R. Mendes, Supercritical fluid extraction of active compounds from algae, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 189–214. D.A. Moyler, Extraction of flavours and fragrances with compressed CO2, in M.B. King and T.R. Bott, Extraction of natural products using near-critical solvents, Chapman & Hall, 1993, 140–183. M. Mukhopadhyay, Processing of spices using supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 337–366. J.P. O’Connell, W. Weber and G. Brunner, Measurement and thermodynamics of triglyceride melting in near-critical fluids, in: Paper Presented at the AIChE Annual Meeting, Indianapolis, USA, 2003. A.Z. Panagiotopoulos, R.C. Reid, S. Watanasiri, Fluid Phase Equilib. 29 (1986) 525.


38 Separation, extraction and concentration processes J.M. Prausnitz, R.N. Lichtenthaler and E. Gomes de Azevedo, Molecular thermodynamics of fluid phase equilibria. Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 1986. E. Reverchon and I. De Marco, Essential oils extraction and fractionation using supercritical fluids, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 305–336. E. Reverchon, R. Adami, S. Cardea, G. D. Porta, Supercritical fluids processing of polymers for pharmaceutical and medical applications, J. Supercrit. Fluids 47 (2009) 484–492. R.J. Sadus, High pressure phase behavior of multicomponent fluid mixtures, Elsevier Amsterdam, 1992. S.I. Sandler and H. Orbey, Mixing and combining rules; in: J.V. Sengers, R.F. Kayser, C.J. Peters, H.J. White (Eds), Equations of state for fluids and fluid mixtures, Elsevier, Amsterdam, 2000, pp. 321–357. R. Sandoval, G. Wilczek-Vera, J.H. Vera, Fluid Phase Equilib. 52 (1989) 119. J. Schwartzentruber and H. Renon, Ind. Eng. Chem. Res. 28 (1989a) 1049. J. Schwartzentruber, H. Renon, Fluid Phase Equilib. 52 (1989b) 127. H. Sovova, Mathematical model for supercritical fluid extraction of natural products and extraction curve evaluation, J. Supercrit. Fluids 33 (2005) 35–52. E. Stahl, K.-W. Quirin, D. Gerard, Verdichtete Gase zur Extraktion und Raffination, Springer-Verlag, 1987, 82–225. M Škerget, Z. Novak-Pintari�, Ž Knez and Z. Kravanja, Estimation of solid solubilities in supercritical carbon dioxide, Peng–Robinson adjustable binary parameters in the near critical region. Fluid Phase Equilib. 5086 (2002) 1–22. L.T. Taylor, Supercritical fluid chromatography for the 21st century, J. Supercrit. Fluids 47 (2009) 566–573. F. Temelli, M.D.A Saldaña, P.H.L. Moquin, and M. Sun, Supercritical fluid extraction of specialty oils, in J.L. Martinez, Supercritical fluid extraction of nutraceuticals and bioactive compounds, CRC Press, 2007, 51–102. F. Temelli, Perspectives on supercritical fluid processing of fats and oils, J. Supercrit. Fluids 47 (2009) 583–590. W.H. Tuminello, G.T. Dee and M.A. McHugh, Macromolecules 28 (1995) 1506. E. Weidner, V. Wiesmet, Z. Knez, M. Skerget, Phase equilibrium (solid–liquid–gas) in polyethylene glycol–carbon dioxide systems, J. Supercrit. Fluids 10 (1997) 139–147. E. Weidner, High pressure micronization for food applications, J. Supercrit. Fluids 47 (2009) 556–565. D.S.H. Wong and S.I. Sandler, AIChE J. 38 (1992) 671. K. Zosel, Process for the decaffeination of coffee, US Patent 4247570, January, 1981.



2 Principles of pressurized fluid extraction and environmental, food and agricultural applications C. Turner and M. Waldebäck, Uppsala University, Sweden

Abstract: The main principles of pressurized fluid extraction (PFE), including basic extraction theory and the effects of solvent selection and temperature variations are discussed. Methods for achieving selectivity during the extraction are described, and future trends explored. Examples of applications include extraction methods developed for environmental and food analysis. Some advice on building equipment in the laboratory is given. Key words: food analysis, pressurized fluid extraction, PFE, environmental analysis, solvent extraction.

2.1 Introduction When developing new products, processes and technologies, it is important to strive for sustainable development. This is especially important because chemists, chemicals, and the chemical industry as a whole are commonly regarded to be the cause of many of the current environmental problems. To ensure sustainability, new processes and new techniques should be studied from a life cycle point of view. Life cycle assessment (LCA) is a methodology in which the entire life cycle of a product or utility effect is analyzed, such as extraction and processing of raw materials, production, distribution, use, consumption and disposal as well as the potential ecological effects. In addition, energy conversions occurring in a life cycle and the resulting burden on the environment are assessed. The indoor environment and health perspective is as important as the outdoor environment. Chemicals should be used in smaller amounts and the ones used should be less hazardous.


40 Separation, extraction and concentration processes One process in which large volumes of organic solvents are used is the procedure of extraction. Traditional liquid–liquid extraction (LLE), also known as solvent extraction, and liquid–solid extraction (LSE) are techniques in which different compounds can be separated from each other based on their relative solubility. For a solid sample, the separation of a substance from the mixture occurs by dissolving that substance in an appropriate solvent. The extraction process usually requires several hours or even several days to perform, depending on the extraction temperature. These processes are slowly being replaced by more attractive alternatives. The most widely used extraction techniques today are still Soxhlet (developed in 1879) and sonication extraction (from 1960). These classical techniques are usually multi-step procedures based on exhaustive extractions from a sample matrix followed by successive clean-up steps before analysis. Such sample preparation procedures require large amounts of sample, sorbents and organic solvents, which are often hazardous and/or toxic, resulting in high costs of both purchase and disposal (Ramos et al., 2002). These methods also demand extensive manual handling, which often creates work-related health problems. During the past few decades new techniques have been developed and among the more successful ones are those employing high-diffusion liquids. Highdiffusion liquids are in this chapter defined as liquids of elevated temperature and pressure. By using high-diffusion liquids, the diffusion coefficient of the liquid is increased. This is the most effective way to increase the rate of the extraction process and decrease the required amount of organic solvent. Diffusion rates in liquids have been shown to increase about 2–20 fold upon increasing the temperature from 25 to 150 °C (Perry et al., 1984). Thus, the mass transfer rate can be increased and the whole extraction process becomes faster. Techniques that employ high-diffusion liquids with raised temperature and pressure are microwave-assisted extraction (MAE) and pressurized fluid extraction (PFE) (Majors, 1996). Another related extraction technique is supercritical fluid extraction (SFE), which usually employs carbon dioxide at pressures and temperatures above 74 atm and 31 °C, respectively, resulting in a liquid-like density in combination with high diffusion rates and low viscosity. In PFE, the solvent is kept in a liquid phase even at temperatures much above the atmospheric boiling point as a result of the applied pressure. This technique is also known as pressurized liquid extraction (PLE“), pressurized solvent extraction (PSE™), accelerated solvent extraction (ASE“) and enhanced solvent extraction (ESE). PFE was originally introduced at the Pittsburgh conference (Pittcon) in 1995 as ASE“ by the Dionex Corporation. The PFE technique utilizes the same basic principles as traditional liquid solvent extractions, but the extractions are carried out at higher temperatures and pressures. An increased temperature during the extraction gives more efficient extraction, and results in both time savings and lowered solvent consumption (Richter et al., 1996). The principle of PFE is similar to


Principles of pressurized fluid extraction and applications 41 MAE, where the solvent is heated by microwave energy, although in the PFE technique both higher temperatures and pressures can be obtained, independently. In this chapter, the basic instrumentation of dynamic (continuous flow) and static (batch mode) PFE as well as its principles of operation are described. Furthermore, fundamental extraction theory is explained including illustrative examples and classical extraction models from literature. A guide to solvent selection is included, which on an elementary level guides the reader to an appropriate choice of solvent in various applications, depending on the chemistry of the sample matrix and analytes to be extracted. Effects of changing the extraction temperature, time and pressure in PFE are described, in addition to some remarks on how to obtain selectivity as well as accuracy and precision. One section is devoted to describing common applications for this technique. Finally, future trends for PFE in terms of sustainability, ‘like-nature’ applications, hyphenated techniques, downscaling and transfer to industry are depicted. The final paragraph also lists commercial vendors of PFE equipment and some advice for building equipment in the laboratory. The next subsection is largely based on a doctoral thesis written by one of the authors of this chapter (Waldeback, 2005).

2.2 Instrumentation and principles of pressurized fluid extraction 2.2.1 Basic instrumentation Extractions using a pressurized liquid above its atmospheric boiling point require open/close valves and/or pressure restrictors, in order to maintain pressure during the extraction. Two main types of instrumentation can be used: static PFE, which is a batch process with one or several extraction cycles with replacement of solvent in between; and dynamic PFE, in which the extraction solvent is continuously pumped through the extraction vessel containing the sample. A schematic of a static PFE instrument is shown in Fig. 2.1. Temperatures applied usually range from room temperature to 200 °C and pressures are generally between 35 and 200 bar. Filter paper is inserted into a stainlesssteel extraction cell followed by the sample, if necessary mixed with a drying agent. The cell is either loaded on a carousel and automatically placed in the oven, or, for simpler equipment, manually placed into the oven. Most equipment has a pump that fills the extraction cell with solvent. Commonly, a static valve in combination with a pressure relief valve control the pressure in the sample vessel during the static extraction; either by adding more solvent to the cell or by opening the static valve, whichever is needed to maintain the desired pressure. The first part of the extraction is a pre-heating step to reach thermal equilibrium. During this heating, thermal expansion of the


42 Separation, extraction and concentration processes Purge valve

Pressure relief valve Extraction cell Oven

Pump

Solvent A Solvent B Static valve

Vent Solvent C Nitrogen

Waste vial

Solvent D Collection vial

Fig. 2.1 Schematic diagram of a PFE system (courtesy of Dionex Corporation).

solvent occurs and causes an increase in pressure within the cell. When the set values are achieved static extraction is performed during a selected time, typically 5–10 min. After the static time, part of the solvent in the extraction cell is replaced with fresh solvent, to start the next extraction cycle. After the last cycle, the sample cell is purged with an inert gas such as nitrogen to remove the remaining solvent from the cell and the lines to a vial that contains the extract. In some equipment, sequential extractions can easily be performed by repeating the procedure with a new solvent, and purging in the same or a different collection vial. Dynamic PFE is quite similar to static PFE, but requires a more sophisticated high-pressure or HPLC pump as well as a pressure restrictor rather than a static open/close valve. The equipment is similar to that used for ‘superheated water chromatography’ (Smith et al., 1999), but the column is replaced with an extraction cell and the tubing has slightly wider inner-diameter. Currently, there is no dynamic PFE equipment available on the market. In fact, there are only a few different types of commercial instruments available (Section 2.5.1), but many examples of home-made instrumentation have been described. Some of them perform both dynamic and static extractions at temperatures above 200 °C (Bautz et al., 1998; Hawthorne et al., 2000; Lou et al., 1997; Vandenburg et al., 1998). A short paragraph about building


Principles of pressurized fluid extraction and applications 43 your own equipment can be found in Section 2.5.2, including Fig. 2.8 that shows a schematic of what a dynamic PFE system may look like. 2.2.2 Extraction strategy In order to perform efficient and smart extractions, an understanding of the matrix characteristics and the different steps of the extraction is of great importance. The nature of the sample matrix (water and organic/inorganic content) and its physical characteristics (homogeneity, porosity, particle size) should also be considered. To better understand the extraction process, two models showing the distribution of analytes in different types of sample matrices are illustrated below. Figure 2.2 is a conceptualization of an aggregate of matrix particles from a source-separated household waste, and the possible sites where analytes in this instance chlorinated paraffins, are expected to be found (Nilsson et al., 2001). Figure 2.2 works as a model showing the variety of possible (and likely) positions and status of analytes in many different types of sample matrices, i.e. the analyte can be: 1. 2. 3. 4. 5.

adsorbed at the surface of the matrix, dissolved in the pore solvent and/or adsorbed at the pore surface, dissolved/adsorbed in a micro/nano pore, chemically bonded to the matrix, or dissolved in the bulk solution.

A simpler extraction model can be described for extracting small organic compounds from polymer particles, for instance the antioxidant Irganox

5 1 2

4 3

Household waste particle Stagnant solvent layer

Fig. 2.2 Schematic of a household waste particle and some possible sites (for explanation of numbers see text) where the analyte (chlorinated paraffin) might be adsorbed or chemically bonded (Waldeback, 2005).


44 Separation, extraction and concentration processes 1076 from the polymer LLDPE (Waldeback et al., 1998), Fig. 2.3. The extraction process of compounds from polymer particles, or other similar sample matrices, generally follows the following steps: 1. diffusion of the solvent into the matrix, 2a. desorption of the analytes from the matrix (including breaking of chemical bonds), 2b. solvation of the analyte into the extraction solvent, 3. diffusion of the analyte out from the matrix, and 4. diffusion of the analyte through the stagnant solvent layer and into the bulk solvent. Knowledge about distribution coefficients and distribution ratios are useful tools to provide guidance to the selection of solvent for the extractive separation process. Solubility of the analytes, their diffusivity in the solvent and matrix characteristics are the main factors to consider, when choosing a solvent for a successful extraction process. It is also important to understand the mass transfer mechanism across chemical/physical interfaces in order to design liquid/liquid and liquid/solid extraction processes. Toxicity and sustainability aspects of the solvent should also be considered. Additionally, in most of the analytical-scale applications, the concentration of the target molecule is very low, and thus the rate of the extraction is not limited by the analyte concentration in the extraction solvent, but rather determined by the rate of mass transfer out of the matrix. In order to perform a fast and complete extraction, a solvent has to be chosen that has the right chemical properties to dissolve and release the analyte, but should preferably not dissolve other solutes in the sample, i.e. the solvent power should not be higher than needed. Solubility theory has been discussed and proposed in classic works by J. Hildebrand, who combined the correlation between vaporization and intermolecular forces, van der Waal forces and hydrogen bonding, to the correlation between vaporization and solubility behaviour (Hildebrand and Scott, 1962; 1964). This model assumes that the same intermolecular attractive forces have to be overcome to vaporize a liquid as to dissolve an analyte. The term ‘solubility parameter’ d was described by Hildebrand as the square root of the cohesive energy density c, giving a numerical value indicating the analyte behaviour in a specific solvent, equation [2.1], where DH is the heat of vaporization (J mol–1) of the solvent, R the gas constant (J K–1 mol–1), T the temperature (K) and V is the molar volume of the analyte.

d = c = DH – RT [2.1] V Hansen (1967) took Hildebrand’s work further and assumed that the total cohesive energy is a linear addition of three components; dh (hydrogen bonding ability contribution), dd (dispersion coefficient contribution), and


Principles of pressurized fluid extraction and applications 45 dp (polarity contribution). They are linked by equation [2.2], where dt is the total solubility parameter (Fitzpatrick and Dean, 2002).

d t2 = d h2 + d d2 + d p2

[2.2]

Fitzpatrick and Dean (2002) predicted a suitable solvent to extract persistent organic pollutants (POPs) from contaminated soil and certified reference material using the Hildebrand solubility parameter and confirmed the results by experiments. The ideal extraction solvent from the calculations was a mixture of acetonitrile and dichloromethane (1:1 v/v), perhaps the best solvent from the view of solvent power, but not from a health and environmental view. When choosing a solvent it is a good start to have the Hildebrand and Hansen’s theories in mind. However, the choice of solvent in a particular situation involves other factors apart from the solvent power. As described in Fig. 2.2 and 2.3, the solvent has to penetrate the matrix thoroughly, break the bonds between the matrix and the analytes of interest, help the dissolved analytes to diffuse out from the matrix and finally be dissolved in the extraction solvent. The process of solute transfer across an interface between two liquid phases may be rate-controlled by molecular diffusion, by motion of eddies, by irregular surface disturbances or even by chemical reactions in the bulk of a phase or in the interface region. Local velocities in the interface region could be of importance as well as other factors affecting the local conditions, not least the presence of surface-active agents. The dependence of matrix geometry has been reported for SFE of mineral oils applied to metal devices (Bjorklund et al., 1996). Irganox 1076

1

2 3

Polymer particle Stagnant solvent layer

4

Fig. 2.3 Schematic showing the extraction of Irganox 1076 from an LLDPE polymer particle (Waldeback, 2005).


46 Separation, extraction and concentration processes Partitioning processes have a central role of concern in the extraction procedure. These involve partitioning of the analytes between the surface of the matrix and the solvent, as well as chemisorption of the analytes on active surface sites and within the solvent. Different matrices behave somewhat differently, e.g. polymeric samples usually build up a layer of stagnant liquid around the polymeric particles, as seen in Fig. 2.3, through which the analyte has to transfer into the extracting solvent. In this case, the partitioning of the analyte between the stagnant liquid and the extraction solvent has to be considered. Soils differ strongly in surface physicochemical properties and grain-size characteristics. Sediments, on the other hand, contain water having various types of bonding, from free water available for the plants, to water strongly bound to the particles, therefore a variety of equilibria take place. In milk powder, hard shells of lactose are formed during the short crystallization time when the milk is spraydried (Walstra and Jenness, 1984), and this may lead to difficult extraction of lipids and fat soluble vitamins. If the extraction solvent is nonpolar, it may be necessary to add a more polar alcohol such as methanol or ethanol to the sample before extraction (Turner and Mathiasson, 2000). There are only a few theoretical models suggested for the PFE technique. For instance, Vandenburg et al. applied the ‘hot ball’ model, originally described for SFE, for the extraction of additives from polymeric samples using PFE (Vandenburg et al., 1998). By plotting ln(m1/m0), where m1 is the mass of analyte remaining in the particle of radius r at time t, m0 is the initial amount of analyte and D the diffusion coefficient of the analyte in the solvent, a linear portion is given in equation [2.3] (Cotton et al., 1993). In this instance, when ln(m1/m0) is plotted against time, the line falls steeply initially and shortly after becomes linear where it follows equation [2.3] (Vandenburg et al., 1998):

ln(m1/m0) = – 0.4977 – (p2Dt/r2)

[2.3]

The physical explanation of the shape of the curve is that the analyte near the surface is rapidly extracted until a smooth falling concentration gradient is established across the particle. The extraction rate is then completely controlled by the rate at which the analyte diffuses to the surface. By plotting the amount of extracted analyte versus the extraction time for different solvents at different temperatures, the resulting curves showed a good fit to the ‘hot ball’ model. With a good kinetic model of the extraction process it would be possible to predict experimental parameters, and find out where the extraction is expected to be only diffusion dependent. However, the models mentioned here can at most be considered to give useful hints when developing new extraction methods based on PFE. There is no theoretical model that includes strong solute-matrix interactions caused by chemical bonding between the solute molecules and active sites on the matrix. In addition, sample matrices are seldom homogeneous, thus the penetration of the solvent is difficult to foresee. As a result, accurate descriptions are by


Principles of pressurized fluid extraction and applications 47 no means easy to provide because of experimental difficulties, as well as the chemical and mathematical complexity of the total extraction process. Empirical approaches, in combination with multivariate chemometric methods, are the most widely used. 2.2.3 Solvent selection As mentioned in Section 2.2.2, a solvent has to have the right chemical properties to desorb and dissolve the analyte preferably without dissolving other solutes in the sample, i.e. the solvent power should not be higher than needed. Richter et al. (1996) have suggested the conventional LLE solvent to be used when developing a new PFE method. Generally, when choosing a solvent the rule-of-thumb is ‘like dissolves like’, i.e. polar solvents dissolve polar analytes, and nonpolar solvents dissolve nonpolar analytes. In addition, the dipole moment and/or dielectric constants of solvents are useful for selecting an appropriate extraction solvent, see Table 2.1. Other important aspects to consider when facing a new extraction problem/challenge is in how the analyte of interest is dissolved or attached Table 2.1 Chemical properties of common extraction solvents (from: Handbook of chemistry and physics, CRC press, 52nd Edition, 1971–1972; and Handbook of chemistry and physics, CRC Press, 60th Edition, 1979–1980) Solvent Boiling Density point (°C) (g mL–1)

Vapour Dipole pressure moment at 20 °C (debye) (mbar)

Dielectric constant at 20 °C (mbar)

Polar, protic solvents Water 100.0 Methanol 64.9 Ethanol 78.5 n-Propanol 97.1 2-Propanol 82.4 n-Butanol 117.7

23 128 44 – 43 –

1.85 1.70 1.69 1.68 1.66 1.66

80.2 32.2 24.3 20.1 18.3 17.8

3.96

47.2

1.000 0.791 0.789 0.803 0.785 0.810

Polar, aprotic solvents Dimethyl sulfoxide 189 (DMSO) Acetonitrile 81.6 Acetone 56.2 Dichloromethane 40.0 Tetrahydrofuran 66 Ethyl acetate 77.1

0.786 0.790 1.326 0.886 0.894

93 233 453 200 97

3.92 2.88 1.60 1.63 1.78

37.5 20.7 9.1 7.4 6.0

Nonpolar solvents Chloroform Diethyl ether Toluene Benzene n-Hexane

1.498 0.714 0.867 0.879 0.660

211 507 29 108 160

1.0 1.15 0.4 0 0

4.8 4.3 2.4 2.3 1.9

61.7 34.5 110.6 80.1 68.9

1.092

0.56


48 Separation, extraction and concentration processes to the matrix, see Fig. 2.2 and 2.3, and also the type and strength of the intermolecular forces between the solvent molecules, which partly can be elucidated by the ΔHvap value, alternatively by the vapour pressure of the solvent (Table 2.1). When dissolving a solute, the intermolecular forces, i.e. hydrogen bonding, dipole–dipole and/or van der Waals interactions between the solvent molecules have to break and form new bindings/interactions with the solute. In instances when the solute is chemically bonded to the matrix, as in Fig. 2.2 (site 4), the extraction solvent has to overcome such bindings and then form new solute–solvent interactions, which altogether will lead to a more stable system of lower energy. Methanol and acetonitrile, two organic solvents commonly used in extraction and separation processes, have somewhat similar dielectric constants, 32 and 37, respectively, but differ strongly with regard to intermolecular forces as methanol forms hydrogen bonds, whereas acetonitrile relies on dipole–dipole interactions. Hence, acetonitrile, which is a less environmentally friendly solvent than methanol, should only be used in cases where such dipole–dipole interactions are more suitable. What are the partitioning processes or equilibrium reactions taking place during the extraction? For example, when extracting samples of high water content, such as wet sediments, vegetables or animal muscles, using organic solvents, there is always a risk that a nonpolar solvent will not be able to penetrate water-sealed pores that contain the analyte, and thus result in a lower extraction yield and unrepeatable results. The more knowledge about the chemistry of the target solutes, pertinent coextractable (unwanted) compounds and chemical and physical properties of the sample matrix and solute–matrix interactions, the easier it is to make a good selection of extraction solvent for instance from the ones listed in Table 2.1. One interesting aspect with PFE is the possibility of choosing a solvent, apart from very low or high pH solvents that may not be applicable in all kinds of equipment owing to possible corrosion of the tubings. In a collaborative study with an oil company, the objective was to predict the amount of process chemicals that could migrate into the sea if there was an oil discharge. By using a water solution of the same salinity as seawater, oil samples containing added process chemicals were extracted at different temperatures and times. The results could be used to predict what would probably happen in a real world situation (data not presented – only used internally at the company). This type of investigation is of course more environmentally relevant than a study performed with traditional extraction techniques based on organic solvents. How does the PFE technique and the solvent selection fit to a more sustainable chemistry? One of the advantages with PFE is that owing to the higher temperature and higher mass transfer during the extraction, more environmentally friendly solvents or solvent mixtures can be used according to Nilsson et al., 2001). As described above, the solvation power of a liquid increases with higher temperature and higher pressure. Furthermore, the


Principles of pressurized fluid extraction and applications 49 dielectric constant decreases with increasing temperature, which implies that the solvent polarity can be tuned by changing the temperature. This is especially true for water, the dielectric constant of which decreases from e = 78 at 25 °C to e = 56 at 100 °C; e = 36 at 200 °C; and e = 8 at 400 °C, since hydrogen bonding between the water molecules become less and less substantial at higher temperatures (Hawthorne et al., 1994). This can be compared to other polar, protic solvents and nonpolar solvents in Table 2.1. Hence, water is a very interesting alternative as an extraction solvent, and can potentially replace many of the organic solvents conventionally used in solvent extraction. The first work describing the use of water at elevated temperature and pressure for extraction of nonpolar solutes is one by Hawthorne et al. (1994). In this work, polar organics (e.g., chlorinated phenols), low-polarity organics (e.g., PAH), and nonpolar organics (alkanes) were extracted from soils and sediments using water at temperatures ranging from 50 to 400 °C. In our own work, water at 120 °C and 50 bar was used as a solvent for the extraction of polyphenolic glycosides from onions, as compared with using a conventional water/methanol (1:1) mixture at 80 °C (Turner et al., 2006). The developed method was also faster, 15 min compared with 2 h for the conventional. In a continuous study, a life cycle assessment was conducted to assess environmental impacts of the two methodologies, showing that the production and use of methanol compared with water has a large impact in terms of carbon dioxide emissions and energy usage, whereas the difference in energy usage for heating the solvents in the two extraction methods was much smaller (Lindahl et al., 2010). If more nonpolar solutes are to be extracted, and these solutes are slightly thermolabile, then ethanol is an interesting alternative to water since not as high a temperature is needed to obtain the same solvent strength. For instance, betulin was extracted from birch bark using ethanol at 120 °C and 50 bar in only 10 min (Co et al., 2009). Initially, the study tested to see if water at elevated temperature close to 200 °C could be an appropriate solvent for betulin, because pressurized liquid water at this temperature has a dielectric constant of around 36, and this is similar to that of methanol at room temperature (e = 32, see Table 2.1), which is indeed a good solvent for betulin. However, it turned out that it is not sufficient to use the dielectric constant as a measure of solubility of a solute in a solvent, as other chemical properties are also important. For this reason, the total (sometimes called ‘relative’) solubility parameter taking dh, dd and dp into account (equation [2.2], Hansen, 2007) was plotted as a function of temperature for betulin, water and ethanol, showing that even at 250 °C, the solubility of betulin in water is estimated to be very low (Fig. 2.4), whereas ethanol at temperatures between 50 and 150 °C should be appropriate, which was confirmed by experiment (Co et al., 2009).



Solubility parameter (MPa0.5)

45 40 35 30 25 20 15 Water

10

Betulin

5

Ethanol

0 0

50

100 150 Temperature (°C)

200

250

Fig. 2.4 Solubility parameters as a function of temperature for betulin, ethanol and water (Co et al., 2009).

2.2.4 Temperature effects An increase in temperature usually tends to promote solubility, as the thermal kinetic energy rises. Increasing the temperature also facilitates analyte diffusion and/or reduces interactions between analytes and the matrix by disrupting intermolecular forces such as van der Waal’s forces, hydrogen bonding and dipole attractions. Higher temperatures also decrease the viscosity of a liquid solvent, thus enabling better penetration of matrix particles. An increased temperature will also decrease the surface tension of the solvent, allowing the solvent to better ‘wet’ the sample matrix (Richter et al., 1996). Both lower viscosity and lower surface tension facilitate better contact of the solvent with the solutes and thereby enhance the extraction. Besides the selection of an extraction solvent, temperature can be considered as the second most important parameter in PFE. The advantages of using higher temperatures are described in every study published on PFE. As a general rule of thumb, a higher temperature gives a better extraction yield. It needs to be remembered that the range of solvents applicable in PFE is much wider than that of Soxhlet extraction. A poor solvent for Soxhlet extraction can, however, be a good solvent in PFE owing to the higher extraction temperatures used (Lou et al., 1997). Hence, as described below, by using a higher temperature, a more environmentally friendly and/or less toxic solvent can be used, for instance water or ethanol. However, other factors might hinder the use of the highest temperature even if it should give the best yield, e.g. as predicted in an experimental design. For example, Waldeback et al. (1998) found that the sample matrix (granules of LLDPE) started to dissolve/melt and block the tubings of the instrument depending on solvent and temperature, e.g. at temperatures above 75 °C © Woodhead Publishing Limited, 2010

Principles of pressurized fluid extraction and applications 51 using tetrahydrofuran as a solvent. Pihlstrom et al. (2002) found that, where pesticides from canola seed were extracted, the results from the screening study showed that the interpretation of the chromatograms was difficult owing to the large number of co-extracted compounds. It was obvious that higher temperatures gave more matrix peaks (Nemoto and Lehotay, 1998), which were most probably derived from co-extracted lipids. In another study (Waldeback et al., 2004), where squalene was extracted from olive biomass, the thermostability of the analyte was a limiting factor in the choice of extraction temperature. To determine the concentration of squalene in the olive biomass, an optimization study using experimental design was performed for the variables extraction temperature, extraction time and concentration of acetone in a mixture of acetone and 2-propanol. Significant factors were determined to be temperature, extraction time, the interaction between temperature and extraction time, and the square of the extraction time. This study showed that, at temperatures above 100 °C, the yield of squalene decreased with extraction times longer than 12–15 min, indicating that squalene either decomposed or reacted with other sample components or with the solvent. An example of extremely thermolabile compounds are anthocyanins, which are antioxidants found in vegetables and fruits, such as grapes, red cabbage, red onions, blueberries and red beets. PFE has been used to extract anthocyanins from red cabbage and red onions using water/ethanol/formic acid (94:5:1, vol%) at 99 °C and 50 bar (Arapitsas and Turner, 2008; Petersson et al., 2008, 2010). However, preliminary results show that anthocyanins extracted from red onion start to degrade after only a few minutes at a set temperature. Hence, extraction and degradation naturally occurs simultaneously in PFE. The kinetics of extraction and degradation depend on the nature of the sample matrix, on the chemical properties of the solutes to be extracted, and on temperature. Theoretical extraction curves were calculated using experimental extraction curves (with simultaneous degradation) combined with experimental degradation curves, as schematically described in Fig. 2.5 below. The conclusion was that, theoretically, a 20 to 30% higher yield of anthocyanins could be obtained from red onions if there were no degradation problems. This would require an extraction time of around 30 min (plus 8 min pre-heating time). In another study (Co et al., 2009), an increasing extraction temperature resulted in higher antioxidant activity in both water and ethanol extracts of birch bark. Furthermore, the antioxidant activities did not level out at high extraction temperature; hence, it appeared that some types of reactions occurred, and that these resulted in higher antioxidant activity. For instance, hydrolysis of polyphenolic glycosides leads to aglycons, which results in compounds of higher antioxidant activity because of the higher number of ‘reactive’ hydroxyl groups. Hence, an important conclusion is that a higher temperature may result in higher extraction yield of the target molecules, but also degradative reactions as well as extraction of more unwanted compounds, i.e. a less selective extraction.



25

Peak area (a.u.)

20

15

10

5

0

0

20

40 60 Time (min)

80

100

Fig. 2.5 Calculation of an ideal extraction curve (continuous lines on top) from experimentally obtained data on degradation (dashed line) and extraction with degradation (continuous line with circles), respectively, for cyanidin-3-(6≤ malonoylglucoside) in red onion (Petersson et al., 2010).

Another type of degradative reaction that may occur in food and agricultural samples is caramelization of sugars. When water is used as a solvent at temperatures of around 160 °C and above, sugars such as glucose caramelize (Montilla et al., 2006). Depending on the length of the extraction time, this obstacle may cause erratic results as well as plugging of tubings and filters. If such high temperatures are to be used, it is advisable to carry out the extraction as fast as possible, to minimize the occurance of caramelization. Maillard reactions may also occur during PFE, involving reducing sugars and amino acids or proteins, but also flavonoids, ascorbic acid and other carbonyl compounds (Manzocco et al., 2000). Maillard polymers can be a major contributor to antioxidant capacity at higher temperature (>170 °C), when using for instance water or water/ethanol mixtures as extraction solvents in food applications (Howard and Pandjaitan, 2008). Hence, in food applications, to be on the safe side, it is advisable to use an extraction temperature of less than 160 °C. 2.2.5 Extraction time The third most important parameter after extraction solvent and temperature, and maybe the absolutely most interesting one, is extraction time. Several studies have shown that PFE offers faster extraction methods relative to those based on Soxhlet, sonication and SFE, and similar to MAE (Dean, 1996;


Principles of pressurized fluid extraction and applications 53 Focant et al., 2004; Vandenburg et al., 1999). For instance, a comparison study was made for the extraction of PAHs from contaminated soil using Soxhlet, two different MAE methods, SFE and PFE (Saim et al., 1997). The Soxhlet method needed 24 h, the SFE method 35 min, the two MAE methods 20 min and the PFE method only 10 min, and Soxhlet extraction gave the highest recovery and the worst precision. In another study, Heemken et al. (1997) made a comparison of PFE and SFE with Soxhlet extraction of PAHs, aliphatic hydrocarbons and chlorinated hydrocarbons from marine samples. Statistical evaluations of accuracy and precision showed that equivalent results were achieved. The extraction time needed for the PFE method was 15 min, whereas SFE needed 90 min and Soxhlet 24 h. In general, during method development, extraction time and temperature are the most important parameters that need to be optimized once the solvent or solvent mixture has been chosen, as well as the mode of sample pre-treatment. Typically, extraction times are in the range of 10–15 min, plus a preheating step of 5–10 min depending on the temperature used and instrumental capabilities. However, if a dynamic extraction system is used (Section 2.5.2), the solvent can be preheated and continuously pumped through the sample without needing to preheat the cell. In commercially available PFE systems, dynamic extraction is mimicked using several short cycles of static extraction, replacing a certain volume of extraction solvent in between each cycle. The most recent Dionex PFE system, ASE-350®, can accomplish semi-dynamic extractions by step-wise replacement of small volumes of solvent continuously during the extraction. 2.2.6 Extraction pressure An increased pressure in the PFE technique is mainly applied to keep the solvents as liquids, at temperatures above their atmospheric boiling point. Most PFE studies observe no difference in extraction yield when the pressure is varied in the range of 34–204 bar (Lundstedt et al., 2000), although it has been reported (Richter et al., 1996) that a higher pressure rendered a higher extraction yield, when a standard mixture of PAH and polychlorinated hydrocarbons from a reference material was spiked onto wet silica of different pore sizes, using methylene chloride/acetone (1:1 v/v) at 100 °C as a solvent. Higher pressure gave higher extraction yield, when the pore size of the wet silica was 300 Å, but no difference in recovery was observed with the dry silica. It was suggested that higher pressure could probably facilitate the extraction of analytes trapped inside matrix pores because the pressure would force the solvent into the pores of the matrix that normally would not be contacted by solvents at atmospheric pressure. Pressurized flow would also aid in the solubilization of air bubbles so that the solvent could more easily penetrate the sample matrix. Kremer et al. (2004) also obtained higher yield of acidic herbicides in soil when pressure was increased from 69 to 138 bar, at 100 °C with dichloromethane as solvent.


54 Separation, extraction and concentration processes 2.2.7 Selectivity during the extraction Selective extractions are in general obtained by changing the solvent or the solvent power. Under ambient conditions (room temperature and atmospheric pressure) the solvent power of a liquid is essentially constant regardless of extraction conditions. In PFE, selectivity can mainly be obtained by varying the nature of the solvent or the solvent mixture (Majors, 1999; Ramos et al., 2002). It has been pointed out that a potential disadvantage with PFE is that the extraction tends to be exhaustive, therefore leading to nonselective extractions (Reighard and Olesik, 1996). Hawthorne et al. (2000) compared the difference in color of the extracts, when soils contaminated with PAH were extracted by Soxhlet, SFE, PFE and pressurized hot water extraction (PHWE). The Soxhlet and the PFE extracts were much darker mainly owing to the greater amount of extracted compounds, i.e. the extractions were less selective. Hence, to avoid coextracting compounds with maintained high yield of the target analytes, it is essential to choose the most selective solvent for the analyte combined with an appropriate temperature, i.e. high enough for efficient extraction of the target compounds, but not too high to avoid coextraction of unwanted molecules. Extraction time is also an important factor: the minimum time necessary should be used to achieve targeted extraction rather than exhaustive extraction of other matrix solutes. However, by using adsorbents in the extraction cell as an in-line clean-up step, more selective PFE extractions can be obtained. For instance, activated acidic alumina has been employed to adsorb fat when PCBs were extracted from spiked freeze-dried fish tissue, and in this way better chromatogram separations were achieved (Ezzell et al., 1996). Sulfuric acid impregnated silica has similarly been used to adsorb the lipids from fat-containing food and feed when extracting PCBs to obtain lipid-free extracts (Sporring and Björklund, 2004). In our own study (Nilsson et al., 2001), when extracting chlorinated paraffins from household waste, clean extracts and chromatograms were obtained when the drying agents Hydromatrix and sodium sulfate were used, a result which is in agreement with other studies (Hawthorne et al., 2000; Ramos et al., 2002). On the other hand, the use of adsorbents such as XAD-2, XAD-4, and XAD-16 did not provide the removal of unidentified interfering compounds. In another work of ours, squalene and a-tocopherol have been selectively extracted from olive oil without coextraction of triglycerides when utilizing Amberlite XAD-16 as adsorbent (data not published). A liquid chromatogram of the olive oil before and after PFE is illustrated in Fig. 2.6. 2.2.8 Accuracy and precision Accuracy and precision obtained with the various PFE methods are similar to those accomplished with other extraction techniques such as Soxhlet, sonication, SFE and MAE (Helaleh et al., 2005; Saim et al., 1997). As with any extraction technique, accuracy and precision of the chemical analysis



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Fig. 2.6 Liquid chromatograms of (a) olive oil diluted with ethanol/acetone and (b) a PFE extract of olive oil, obtained using XAD-16 as adsorbent and methanol as extraction solvent.

method heavily depend on how well the sample is homogenized, and if the collected sub-sample is an accurate representation of the entire sample under study. These problems with sample preparation can of course be minimized by taking out a larger number of sub-samples, i.e. replicates, for analysis. Another important variable is the particle size, i.e. too large a particle size leads to inefficient extraction and inaccurate results. This was demonstrated by Björklund et al. (1999) for sediment samples of different particle size distributions. Similarly, Isaac et al. (2005) in a study of the extraction of lipids from homogenized cod tissue obtained a higher yield compared with extracting intact wet cod muscle. The conclusion was that the proteins in the sample denaturated to a hard pellet during the extraction making the diffusion of the solvent into the matrix not efficient enough to extract all the lipids. On the other hand, too small a particle size led to a relatively compact sample, which in turn may result in channeling inside the sample and again inefficient extraction.



2.3 Applications of pressurized fluid extraction Most of the studies on PFE deal with either environmental analysis or food and agricultural samples, targeting either pollutants or health-beneficial compounds such as antioxidants. Figure 2.7 shows the publication record from 1995 to 2008, taken from Web of Science. There is not enough space in this chapter to discuss in detail the wide range of different applications for PFE, hence, the reader is referred to a number of reviews of the field: on applications for environmental analysis (Giergielewicz-Mozajska et al., 2001; Nieto et al., 2008); food and drugs (Beyer and Biziuk, 2008; Carabias-Martinez et al., 2005; Herrero et al., 2006; Mendiola et al., 2007); and medicinal plants, herbs and agricultural (Huie, 2002; Wang and Weller, 2006). 2.3.1 Environmental applications Pesticides were extracted from fruits and vegetables (Adou et al., 2001; Barriada-Pereira et al., 2007; Blasco et al., 2005; Herrera et al., 2002; Pihlstrom et al., 2002; Tanaka et al., 2007; Wennrich et al., 2001) and from agricultural crops and soils (Hildebrandt et al., 2007; Otake et al., 2008; Popp et al., 1997; Schreck et al., 2008) using solvents and solvent mixtures such as acetone, acetone/dichloromethane, acetone/hexane, methanol, hexane saturated with acetonitrile, and acetonitrile and water, at temperatures between 60 and 130 °C. For polyhalogenated persistant organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), it is more common to use hexane, toluene/acetone and hexane/ acetone, at temperatures between 100 and 150 °C (Antunes et al., 2008; Bjorklund et al., 1999; Brandl et al., 2006), but water has also been used at temperatures between 250 and 300 °C (Yang et al., 1995). Number of publications

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Fig. 2.7 Publication record (Web of Science 2009-08-02, search term: ‘pressurized liquid extraction’ OR ‘accelerated solvent extraction’ OR ‘pressurized solvent extraction’ OR ‘subcritical water extraction’ OR ‘pressurized fluid extraction’). In total 1469 publications between 1995 and 2008.


Principles of pressurized fluid extraction and applications 57 Organometallic compounds containing tin, arsenic and mercury have been extracted from sediments and soils (Beichert et al., 2000; Chiron et al., 2000), vegetables (Marcic et al., 2005) and seafood (Mato-Fernandez et al., 2007; Wahlen and Catterick, 2004) using methanol, water, methanol/water and methanol/ethyl acetate mixtures, at temperatures between ambient and 160 °C. There are also a few studies on the extraction of explosives from soil samples (Ragnvaldsson et al., 2007) and biological samples (Pan et al., 2005); and antibiotics from soil (Jacobsen et al., 2004; Schlusener et al., 2003) and meat samples (Berrada et al., 2008). Pharmaceutical applications are less common than environmental ones, and most of them consider the extraction of pharmaceutical drugs and their metabolites in environmental samples such as sediments, sludge and fish/ shell fish. For instance, pharmaceutical residues have been extracted from sludge using methanol/water (1:1) at 60 °C as solvent (Barron et al., 2008). There is also an increasing number of studies on endocrine disruptors in the environment, for instance, 17-b-estradiol has been extracted from soil using acetone/hexane (1:1) at 100 °C (Chun et al., 2005); and alkylphenolic compounds have been extracted from river sediment using methanol/acetone (1:1) at 50 °C as a solvent (Petrovic et al., 2002). 2.3.2 Food and agricultural applications Food and agricultural applications considering endogeneous compounds commonly involve the use of more environmentally friendly solvents such as water and ethanol, because these applications often aim at isolating healthbeneficial compounds for later use as food additives, or alternatively, the compounds that are extracted are fairly polar, hence stronger organic solvents are not needed. Exceptions are in applications concerning the extraction of lipids such as acylglycerols, sterols, terpenoids and essential oils, for which stronger organic solvents are generally employed, e.g. isopropanol, ethyl acetate and hexane. Flavonoids such as quercetin, kaempferol, catechin and anthocyanidins have been extracted from vegetables and herbal plants using pressurized hot water, ethanol or water/ethanol mixtures as solvents at temperatures between 50 and 160 °C (Arapitsas and Turner, 2008; Howard and Pandjaitan, 2008; Ibanez et al., 2003; Ollanketo et al., 2002; Turner et al., 2006). For instance, water and a 70:30 mixture of ethanol and water at temperatures of 50 to 190 °C were used to extract flavonoids from dried spinach (Howard and Pandjaitan, 2008). The results showed that the total phenolic content as well as the antioxidant capacity were the highest in extracts obtained with the highest extraction temperatures (170–190 °C) for both water and the water/ethanol mixture, although the solvent mixture was somewhat more efficient than pure water in extracting flavonoids. However, when separating the extracts into low (1000 Da) molecular weight fractions, it turned out that at temperatures above 130 °C for water and


58 Separation, extraction and concentration processes above 150 °C for ethanolic solvents, polymeric Maillard reaction products were responsible for the increase in antioxidant capacity. Howard and Pandjaitan (2008) concluded that, in order to extract flavonoids effectively, water should be used at temperatures between 50–130 °C and for ethanolic solvents, 50–150 °C. In one of our own studies, quercetin and its glycosides as well as isorhamnetin and kaempferol were extracted from yellow and red onion using water at 120 °C and 50 bar, employing three consecutive 5-min extraction cycles (Turner et al., 2006). Thermostable b-glucosidase was used to convert the polyphenolic glucosides into their respective aglycons within only 10 min of reaction, using water at 90 °C and pH 5 as a reaction media. Results obtained compared well with a conventional extraction method employing a 1:1 mixture of methanol and 2.4 M HCl at 80 °C and 2 h combined extraction/ reaction, followed by filtration. Phenolic compounds such as rosmarinic and carnosic acids, carnosol and methyl carnosate have been extracted by PHWE, ultrasonication-assisted methanol extraction, hydrodistillation, and maceration with 70% ethanol from sage (Ollanketo et al., 2002). It turned out that PHWE, performed at 100 °C, gave the highest antioxidant activity of the extracts. Furthermore, PHWE was faster than the conventional techniques used in this work. In another study (Ibanez et al., 2003), antioxidative compounds such as carnosol, rosmanol, carnosic acid, methyl carnosate, cirsimaritin and genkwanin have been extracted from rosemary leaves using subcritical water at temperatures between 25 and 200 °C, showing that the selectivity of the extraction can be tuned by varying the temperature. At 25 °C a high concentration of rosmanol was obtained in the extract, while at 200 °C, the dominant compound was carnosic acid. The antioxidant activity was high in all of the extracts obtained at 100, 150 and 200 °C, respectively (Ibanez et al., 2003). Phenolic acids have been extracted from fruits and vegetables using water, methanol and water/methanol mixtures, at temperatures ranging between 20 and 100 °C (Alonso-Salces et al., 2001; Chen et al., 2007a; Mukhopadhyay et al., 2006; Waksmundzka-Hainos et al., 2007). In general, the main concern for the extraction of phenolic acids as well as many of the flavanoids is the thermal stability, or rather the tendency to degrade during the extraction, as discussed in Section 2.2.4. Terpenoids including limonene, pinene, artemisinin, retinol, taxol, squalene, lycopene and carotene, have only limited water solubility even at elevated temperatures, hence these are extracted with, for instance, 2-propanol or methanol/ethyl acetate/light petroleum (1:1:1) at temperatures ranging from 40 to 190 °C (Breithaupt, 2004; Fojtova et al., 2008; Schaneberg and Khan, 2002; Waldeback et al., 2004). There are, however, also studies in which pressurized hot water is used as a solvent; Yang et al. (2007) used water at temperatures between 100 and 250 °C to extract a-pinene, limonene, camphor, citronellol, and carvacrol terpenoids from oregano and basil leaves. However, it was shown that thermal degradation occurred at all temperatures tested,


Principles of pressurized fluid extraction and applications 59 although least severe at 100 °C – between 10 and 25% for a 30-min treatment in static mode. In a work by Breithaupt (2004), carotenoids were extracted by PFE and compared with a conventional solvent extraction, in both cases using a solvent mixture of methanol/ethyl acetate/light petroleum (1:1:1). The PFE extraction conditions were optimized by varying the temperature (25–80 °C) and the pressure (70–140 bar), giving the highest yield at 40 °C and 70 bar. Both methods were used for the extraction of a large number of different carotenoids from beverages, pudding mixes, cereals, cookies and sausages (Breithaupt, 2004). Acylglycerols and sterols have been extracted from eggs and egg-containing food products, using chloroform/methanol 2:1 (v/v) and hexane/isopropanol 3:2 (v/v), at various extraction temperatures and pressures (60 °C and 150 bar, 100 °C at 150 bar, and 120 °C and 200 bar (Boselli et al., 2001). Results showed that the hexane/isopropanol mixture employed at 60 °C and 150 bar could successfully be used to extract lipids from egg products. In another study, lipids were extracted from freshly ground corn kernels and ground rolled oats, using four different organic solvents: hexane, dichloromethane, isopropanol, and ethanol, at two temperatures, 40 and 100 °C (Moreau et al., 2003). The results showed that oat oil with the highest levels of digalactosyldiacylglycerol (DGDG) was obtained using ethanol at 100 °C. Comparison of the extraction effects with seeds of two different species of grains indicated that the extractability is greatly dependent on the polarity and temperature of the solvent. Tocopherols and tocotrienols are fat-soluble compounds extractable using solvents such as methanol and acetone. For instance, tocopherols and tocotrienols have been extracted from cereals using methanol as extraction solvent at 50 °C and 110 bar, using only one 5-min extraction cycle (Bustamante-Rangel et al., 2007). Other food and agricultural applications include for instance alkaloids (Mroczek and Mazurek, 2009); saponins (Chen et al., 2007b); coumarins (Waksmundzka-Hajnos et al., 2004); oligosaccharides (Bansleben et al., 2008); lignans (Smeds et al., 2007); and various essential oils (JimenezCarmona et al., 1999; Ozel et al., 2003; Schaneberg and Khan, 2002).

2.4 Future trends The authors believe that some of the most important future trends for PFE are: (i) switching to more environmentally friendly solvents; (ii) hyphenation and automation; (iii) miniaturization; (iv) mimic nature; and (v) transfer to industry.


60 Separation, extraction and concentration processes All these trends are somewhat connected with the growing push for a more sustainable development. In order to minimize the use of organic solvents that need to be produced and then disposed of, both switching to more environmentally friendly solvents and miniaturizing the extraction equipment is vital. The use of solvents at elevated temperatures implies that more efficient extractions can be obtained, even when using more environmentally friendly solvents such as water and ethanol rather than the classical ones such as hexane and ethyl acetate. Hence, future trends will most probably involve switching from conventional organic solvent based extraction methods to PFE using water, ethanol and other ‘green’ alternatives. This has already been demonstrated (Burkhardt et al., 2005; Co et al., 2009; Crescenzi et al., 1999; Curren and King, 2001; Fernandez-Perez and de Castro, 2000; Hawthorne et al., 1998; Hyotylainen et al., 2000; Morales-Munoz et al., 2002; Smith, 2002; Turner et al., 2006; Yang and Li, 1999), but more applications are expected in the near future. Regarding miniaturization of PFE, we found only one example (Ramos et al., 2000), in which PAHs were extracted from soil and sediment using an extraction vessel constructed by a 10 mm ¥ 3 mm inner diameter stainlesssteel tubing. The only extraction solvent used was 100 mL of toluene at 200 °C and 150 bar, in an extraction process taking 10 min, with subsequent direct injection into large-volume injection gas chromatography–mass spectrometry (GC–MS). Future trends will probably include more examples of miniaturized PFE systems, probably also combined with analytical separation and detection systems. It has been recognized that in order to minimize the production of waste and emissions of carbon dioxide, it is advantageous to use as few steps in a process as possible, i.e. combined (hyphenated) methods and automation are relevant. There are several good examples showing that PFE can be coupled on-line with separation by HPLC (Hyotylainen et al., 2000; Li et al., 2000), but also with GC using a miniaturized PFE system (Ramos et al., 2000). Smith showed that subcritical water extraction can be coupled on-line with chromatography using subcritical water as the only mobile phase (Smith, 2002; Tajuddin and Smith, 2002). In the future, technology development enabling combination with several other separation and detection techniques is expected, as well as in situ analysis instrumentation. In order to better understand processes occurring in real life or in the environment, laboratory-scale methods that mimic nature in an efficient way are important. Thus, by using PFE, an interesting possibility is to simulate what could happen in a real life situation (in the human body, in the environment or within an industrial process). This can be accomplished using a suitable solvent, similar to the surroundings in question, for example water with appropriate pH and ion concentrations (such as seawater). Anaerobic conditions can be simulated using a nitrogen purge between sequential extractions. Hence, it is possible to predict what would happen if a specific chemical is used by a specific company in a specific environment


Principles of pressurized fluid extraction and applications 61 and, in this way, obtain detailed information for an environmental regulatory impact analysis. Another challenge would be to follow the migration of any hazardous compound and their degradation products in soil, sediment, sludge or biota. Ongoing research in this area is expected to address important environmental issues. A final note on future trends of PFE is that several of the smaller-scale processes will be transferred to industry to replace conventional solvent extraction methods in chemical analysis applications, as well as scaled up for industrial processes in applications regarding isolation of interesting (valuable) compounds from the plant kingdom. For instance, a standard method (US EPA SW-846 Method 3545) for the extraction of PCBs from sediments using hexane/acetone (1:1) as a solvent at 150 °C has replaced a traditional Soxhlet method. Both new applications and replacement of technology in old applications are expected to be developed in the near future.

2.5 Sources of further information and advice 2.5.1 Commercially available equipment There is equipment available on the market that performs static PFE, with full or semi-automation. Currently, the following vendors have been identified: Dionex (www.dionex.com) products include ASE® 150 (one extraction vessel at the time, of sizes 1, 5, 10, 22, 34, 66 or 100 mL) and ASE® 350 (carousel with up to 24 extraction vessels of the same sizes as for the 150-system). A solvent controller allows mixing and delivery of up to three solvents. Temperature range applicable is 40–200 °C and the pressure is fixed to 1500 psi (103 bar). pH hardened pathway with Dionium™ components and extraction vessels make the instrument compatible with acid or alkaline pretreated sample matrices. ∑ Applied Separations (www.appliedseparations.com) has two extraction systems, one PSE™ and fast-PSE™, which operate with one and six in-parallel extraction vessels, respectively. Available vessel sizes are 11, 22 and 33 mL. An automated solvent dispenser is optional, and up to four different solvents can be handled. Temperature range is 50– 200 °C and maximum pressure is 150 bar. ∑ Fluid Management Systems (www.fmsenvironmental.com) market PFE™ systems that handle one to six samples (six modules) at the time in parallel, with the option of in-line clean up by column chromatography. Temperature and pressure ranges obtainable are 70–200 °C and 1500– 3000 psi (103–207 bars), respectively. Extraction vessels come in sizes ranging between 5–250 mL. ∑


62 Separation, extraction and concentration processes 2.5.2 Designing your own equipment In our laboratory, we usually build our own equipment, since the ones commercially available are all based on static PFE and, in addition, operate within a strictly limited temperature range. Hence, in order to run dynamic PFE, even at temperatures above 200 °C, it is necessary to custom build PFE instrumentation. What we recommend is to use the following parts: (a) an optional heater plate set to a temperature of around 20–30 °C below the boiling point of the solvent; (b) a bottle containing the extraction solvent or solvent mixture; (c) an ordinary HPLC pump; (d) a long stainless-steel tubing that enables the solvent to reach desired temperature before passing through the extraction vessel; (e) a type K thermocouple temperature probe for measuring the temperature of the solvent just before entering the extraction vessel; (f) a stainless-steel extraction vessel (for instance a preparative HPLC column) in which the sample is placed before extraction; (g) a simple GC oven (only temperature control is needed, hence no need for injector or detector); (h) a cooling bath to bring down the temperature of the extractant to below the boiling point of the solvent; (i) a needle valve or a back pressure regulator; (j) collection vial(s); and (k) optional nitrogen gas to flush out the entire system after extraction. It is advisable to install an in-line filter to protect the needle valve/back pressure regulator. It is also recommended to install a pressure gauge for control of the pressure as well as a burst disc in case the pressure rises too high in the system. There are burst discs available for different pressures and solvents. Figure 2.8 shows a schematic of a home built dynamic PFE system. (c)

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Fig. 2.8 Schematic of a home-built dynamic PFE system. (a) Heater plate; (b) extraction solvent; (c) HPLC pump; (d) stainless steel tubing; (e) temperature probe; (f) extraction vessel; (g) oven; (h) cooling bath; (i) needle valve; (j) collection vial; (k) nitrogen gas.


Principles of pressurized fluid extraction and applications 63 A factor to consider is which solvents and temperatures are allowable by the different parts of the extraction system. For instance, an HPLC pump cannot take too high a temperature, and especially not with some of the organic solvents. If water is used as a solvent, caution must be taken if temperatures above ca. 200 °C are used, since water becomes more and more corrosive at higher temperature. Hence, higher-quality nickel steel alloy, such as Hastelloy® that exhibits high resistance to corrosion, should be used. However, owing to obstacles such as caramelization and Maillard reactions occurring in food and agricultural samples, temperatures applied are rarely above 160 °C. Finally, if an organic solvent is used, safety arrangements around your homebuilt device, such as ventilation and burst discs connected to stainless-steel tubings leading to a secure waste container, should be provided.

2.6 Conclusions In industrial and governmental analytical and research laboratories, PFE is a promising extraction technique that easily replaces conventional techniques such as Soxhlet extraction – especially since the same solvents can be used. Compared with conventional extraction techniques, PFE is faster; more automated; and enables a ‘friendlier’ work environment. Furthermore, new PFE methods are fairly easy to develop by optimizing temperature and extraction time. Since PFE operates at elevated temperatures, it is possible to use less harmful solvents than in Soxhlet with maintained extraction efficiency. However, caution has to be taken regarding degradation of thermolabile target compounds during the extraction, even though the problems are usually minor if the extraction time and temperature are carefully optimized. Finally, selectivity can be obtained in PFE by firstly selecting an appropriate extraction solvent, secondly by varying the temperature of the solvent, which is especially effective for water, or thirdly by using adsorbents in the extraction cell. How does PFE fit into a sustainable development? PFE uses smaller amounts of organic solvent, thereby preventing unnecessary waste, because less organic solvent needs to be manufactured and less waste is thereby produced by the manufacturer. In addition, less organic solvent waste is produced at the laboratory and, in total, there will be a decrease in volatile organic carbon emissions and a reduced risk for photochemical smog formation. Accidental emissions to municipal wastewater plants will do less harm. By being able to substitute hazardous solvents with more environmentally friendly solvents such as ethanol and water, the toxicity is reduced with positive effects on both the environment and health in general. Creating high diffusion liquids requires a significant amount of energy, which is a disadvantage. However, because the extraction times are shorter, minutes compared with hours in Soxhlet and modified Soxhlet techniques, and, as these traditional solvent extraction techniques are usually performed © Woodhead Publishing Limited, 2010

64 Separation, extraction and concentration processes just below the boiling point, a significant amount of energy is saved. Lifecycle assessment should be conducted to calculate the overall energy usage in different methodologies.

2.7 References Adou, K., Bontoyan, W. R. and Sweeney, P. J. (2001), ‘Multiresidue method for the analysis of pesticide residues in fruits and vegetables by accelerated solvent extraction and capillary gas chromatography’, J. Agric. Food Chem., 49, 4153–60. Alonso-Salces, R. M., Korta, E., Barranco, A., Berrueta, L. A., Gallo, B. and Vicente, F. (2001), ‘Determination of polyphenolic profiles of Basque cider apple varieties using accelerated solvent extraction’, J. Agric. Food Chem., 49, 3761–67. Antunes, P., Viana, P., Vinhas, T., Capelo, J. L., Rivera, J. and Gaspar, E. (2008), ‘Optimization of pressurized liquid extraction (PFE) of dioxin–furans and dioxin-like PCBs from environmental samples’, Talanta, 75, 916–25. Arapitsas, P. and Turner, C. (2008), ‘Pressurized solvent extraction and monolithic columnHPLC/DAD analysis of anthocyanins in red cabbage’, Talanta, 74, 1218–23. Bansleben, D., Schellenberg, I. and Wolff, A. C. (2008), ‘Highly automated and fast determination of raffinose family oligosaccharides in Lupinus seeds using pressurized liquid extraction and high-performance anion-exchange chromatography with pulsed amperometric detection’, J. Sci. Food Agric., 88, 1949–53. Barriada-Pereira, M., Gonzalez-Castro, M. J., Muniategui-Lorenzo, S., Lopez-Mahia, P., Prada-Rodriguez, D. and Fernandez-Fernandez, E. (2007), ‘Comparison of pressurized liquid extraction and microwave assisted extraction for the determination of organochlorine pesticides in vegetables’, Talanta, 71, 1345–51. Barron, L., Tobin, J. and Paull, B. (2008), ‘Multi-residue determination of pharmaceuticals in sludge and sludge enriched soils using pressurized liquid extraction, solid phase extraction and liquid chromatography with tandem mass spectrometry’, J. Environ. Monit., 10, 353–61. Bautz, H., Polzer, J. and Stieglitz, L. (1998), ‘Comparison of pressurised liquid extraction with Soxhlet extraction for the analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans from fly ash and environmental matrices’, J. Chromatogr A, 815, 231–41. Beichert, A., Padberg, S. and Wenclawiak, B. W. (2000), ‘Selective determination of alkylmercury compounds in solid matrices after subcritical water extraction, followed by solid-phase microextraction and GC–MS’, Appl. Organometal. Chem., 14, 493–98. Berrada, H., Borrull, F., Font, G. and Marce, R. M. (2008), ‘Determination of macrolide antibiotics in meat and fish using pressurized liquid extraction and liquid chromatography–mass spectrometry’, J. Chromatogr. A, 1208, 83–89. Beyer, A. and Biziuk, M. (2008), ‘Applications of sample preparation techniques in the analysis of pesticides and PCBs in food’, Food Chem., 108, 669–80. Bjorklund, E., Bowadt, S., Nilsson, T. and Mathiasson, L. (1999), ‘Pressurized fluid extraction of polychlorinated biphenyls in solid environmental samples’, J. Chromatogr. A, 836, 285–93. Bjorklund, E., Turner, C., Karlsson, L., Mathiasson, L., Sivik, B. and Skogsmo, J. (1996), ‘The influence of oil extractability and metal part geometry in degreasing processes using supercritical carbon dioxide’, J. Supercrit Fluids, 9, 56–60. Blasco, C., Font, G. and Pico, Y. (2005), ‘Analysis of pesticides in fruits by pressurized liquid extraction and liquid chromatography-ion trap-triple stage mass spectrometry’, J. Chromatogr. A, 1098, 37–43.


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3 Principles of physically assisted extractions and applications in the food, beverage and nutraceutical industries E. Vorobiev, Compiègne University of Technology, France and F. Chemat, University of Avignon and Pays de Vaucluse, France Abstract: The physical methods used to enhance pressure and solute extraction methods in the food industry are reviewed. The quality of extracted products (e.g. purity, colour, texture, flavour and nutrients) may be degraded by conventional mechanical, thermal or chemical pretreatments. Several physical treatments (pulsed electric fields, power ultrasound, microwaves, ohmic heating, arc discharges) have been shown to be important and of great interest for the food industry in terms of enhancing solute and pressure extraction, and dehydration processes. Emerging extraction technologies that show promise for commercial food processing are discussed. Key words: plants, food, extraction, pulsed electric field, ultrasonic extraction, microwave extraction.

3.1 Introduction Pressure and solvent extractions are widely used in the food industry for the production of juices, wine, sugar and vegetable oil; these methods are also frequently applied in the extraction of various target compounds, such as colorants, antioxidants, essential oils, and aromas, from raw plant materials. Pressing, hot water and organic solvent extractions are long established processes that have excellent efficacy when applied in optimal conditions. These extractions can be done concurrently but more often they compliment each other and are technologically combined (e.g. the hot water extraction of sugar from sugar beets is combined with the subsequent pressing of pulps; the pressing of oilseeds is combined with the subsequent solvent extraction of oil from press-cake; the pressing of apples or grapes can be combined © Woodhead Publishing Limited, 2010

72 Separation, extraction and concentration processes with the subsequent solvent extraction of bioactive compounds from mash). The yield of extracted compounds can be very high in optimal conditions. Unfortunately, the quality of solutions and extracted products (e.g. purity, turbidity, colour, texture flavour and nutrients) may be degraded in the course of the raw material treatments that are necessary to increase the yield (grinding, heating, chemicals/enzyme addition). Moreover, a significant quantity of waste is often produced from the purification of impure extraction solutions, when undesirable loss of solvents and other additives occurs. These problems need to be resolved or minimised for the future development of environmentally sustainable food technologies. In recent decades there has been a growing interest in alternative food technologies that allow for nonthermal or mild thermal food preservation. Several emerging technologies are prominent and of great interest to the food industry, in particular pulsed electric fields (PEF), power ultrasound (PU), microwave (MW), pulsed light (PL), ohmic heating (OH), irradiation (IR), radio-frequency (RF) heating, high pressure processing (HP), high voltage electric discharges (HVED), amongst others (Barbosa-Cánovas et al., 1998; Zeuthen and Bogh-Sorensen, 2000; Povey and Mason, 1998). Such success in the development of novel preservation technologies has encouraged further research and has renewed the industrial interest in extractions assisted by nonthermal or mild thermal physical treatments (PU, PEF, HVED, OH, MW). Recently, these treatments were found to be effective for the enhancement of solute and pressure extractions, and dehydration processes (Knorr et al., 2001; Kulshrestha and Sastry, 2003; Li et al., 2004; Virot et al., 2007; Vorobiev and Lebovka, 2006, 2008). The potential benefits of physically assisted extractions are important. For instance, they can lead to the future substitution of hot water or organic solvent extractions with cold, or mild thermal, aqueous or pressure extractions. Moreover, the alternative physical treatments are found to be less invasive methods for the processing of plant foods, making it possible to avoid many undesirable changes in products, pigments, vitamins, and flavouring agents, which are typical of other extraction techniques, including thermal, chemical and enzymatic techniques. Some of these novel technologies remain very much in the research arena, whereas some others are on the brink of commercialisation. This chapter provides an overview of the emerging extraction technologies that are promising in terms of their potential applications in commercial food processing.

3.2 Pulsed electric field-assisted extractions in the food industry 3.2.1 Principles of pulsed electric field (PEF) treatment Pulsed electric field processing is a technique in which a food is placed between two electrodes in a batch or continuous treatment chamber and © Woodhead Publishing Limited, 2010

Principles of physically assisted extractions and applications 73 exposed to a pulsed voltage (typically 15–80 kV cm–1 with several pulses of 1–5 ms for the killing of micro-organisms; and 0.1–5 kV cm–1 with pulses of 50–1000 ms for the electroporation of plant cells and nonthermal extraction from solid foods). To generate such short pulses, various pulse-forming networks are used, with main components that include power supply at the selected voltage, one or several capacitor banks, inductors and/or resistors (Barbosa-Cánovas et al., 1998; Bluhm, 2006). Various different pulse shapes can be generated, including the simplest exponential decay pulses and squarewave pulses (mono-and bipolar). The duration and/or number of pulses are limited in order to prevent any significant temperature elevation, which is usually less than 3–5 °C. Mechanism of cell membrane electroporation When the intensity of an applied electric field increases, the potential difference across a cell membrane (transmembrane potential um) also increases. If um exceeds a stated threshold value (typically 0.2–1 V), a temporary loss of membrane semipermeability occurs. This phenomenon of membrane damage is named electroporation (electropermeabilisation) (Fig. 3.1). A number of models have been proposed in the biophysical literature to describe the electroporation mechanism (Konduser and Miklavcic, 2008; Zimmermann,

E < Ecr

Compression

Compressed membrane

E > Ecr

(a) Repulsion

Pore

Membrane

2r

(b)

Fig. 3.1 Membrane electroporation. With application of electric field E, the cell membrane is compressed owing to the electric charges attraction (a). (b) When the electric field exceeds some critical value (E > Ecr), the transmembrane potential increases to a threshold value and electroporation occurs (from Vorobiev et al., 2005).


74 Separation, extraction and concentration processes 1986). The transmembrane potential um of a spherical cell depends on the angle q between the external field E direction and the normal vector on the membrane surface. The transmembrane potential may be given by the following equation (Zimmermann, 1986):

um = 0.75fdcEcosq [1 – exp (– t/tc)]

[3.1]

where dc is the cell diameter, tc is the time of membrane charging, and f is a parameter depending on the electrophysical and dimensional properties of the cell, membrane, and surrounding media. The values of f and tc vary for different cell sizes and ratios of extracellular/intracellular conductivities se/ si (Lebovka et al., 2000; Vorobiev and Lebovka, 2008). At complete cell damage, it follows that se/si = 1 and f ≈ 1. The critical value of transmembrane potential required for biological membrane electroporation at ambient temperature is estimated as um ≈ 1 V (Zimmerman, 1986) and can vary in plant and animal cells from 0.7 to 2.2 V (Knorr et al., 2001). The typical size of a cell in plant tissue is dc ≈ 50–100 mm. Therefore, the required threshold PEF intensity for the electroporation of food plants tissues (for f = 1 and tc 7.5 kV cm–1) (Schrive et al., 2006;


Principles of physically assisted extractions and applications 83 Zhang et al., 1994), though smaller fields can also affect the structure of cells. For instance, the early stages of damage in the yeast cells were observed at E < 7.5 kV cm–1, in a PEF treatment of long duration (El Zakhem et al., 2006a,b). The electroextraction of proteins from suspensions of S. cerevisiae was also observed at 3.2 kV cm–1 (Ganeva et al., 2003), but high efficiency (yield of 85%) required long extraction following PEF treatment (>4 h at 30 °C). Recently, Shynkaryk et al. (2009) demonstrated that a rather long application of PEF treatment (1000 trains, 200 pulses of 100 ms in each train) was needed to result in a high level of membrane disintegration (Z > 0.8), even at E = 10 kV cm–1. Moreover, the quantity of released high molecular weight intracellular components extracted with PEF was lower than that extracted with other methods (HVED and high-pressure homogenisation) (Shynkaryk et al., 2009).

3.3 Ohmic heating-assisted extractions in the food industry 3.3.1 Principles of ohmic heating (OH) treatment Ohmic heating (OH) processing is a technique in which a food is placed between two electrodes in a batch or continuous treatment chamber and exposed to a dc, ac or pulsed voltage (typically 20–80 V cm–1) to heat the food and kill any micro-organisms. OH generators, which use ac and highfrequency electric fields permit reduced electrolysis and product contamination, compared with dc designs (Lima et al., 1999; Sastry, 2005). The basic principle of OH is the dissipation of electrical energy in the form of heat, resulting in the generation of internal energy (Sastry, 2005):

q = sE2

[3.3]

where q is the heat power per unit volume, s is the electrical conductivity of food and E is the electric field intensity. OH processing is currently commercialised for the pasteurisation and sterilisation of particulate foods (Biss et al., 1989; Sastry, 2005). However, the effects of OH are not purely thermal. Several studies have noted that electropermeabilisation is also induced by OH (Kemp and Fryer, 2007; Kulshrestha and Sastry, 2003; Lima et al., 2001; Praporscic et al., 2006; Schreier et al., 1993). Lebovka et al. (2005) have studied the membrane damage of ohmically heated potato and apple tissues. The OH experiments conducted under ac treatment were started from room temperature T = 22 °C at different electric field strengths E, and the treatment was stopped when the temperature reached T = Tf = 50 °C. The total treatment time te was higher with lower E (Fig. 3.5), but the samples were not kept in the warm juice (at 40–50 °C) for more than 15 min. The thermal damage and softening of potato and apple tissues at such mild thermal treatment conditions can be neglected (Lebovka et al., 2004a; 2004b). But the conductivity disintegration index Z © Woodhead Publishing Limited, 2010

84 Separation, extraction and concentration processes increased considerably at the electric fields E > 20 V cm–1, both for apples and potatoes (Fig. 3.5), and these data provide evidence for the significance of electrically induced tissue damage. More recently, Lebovka et al. (2007) estimated the variation of Z values of sugar beet tissue during OH. They obtained electrical conductivity graphs for intact, maximally damaged and ohmically treated tissues at various temperatures and calculated Z(T) from equation [3.2] for various electric fields E. The results provide evidence for the significance of the electroporation effect in OH. This effect becomes more significant as E is increased from 40 to 100 V cm–1. Imai et al. (1995) have found that the OH behaviour and impedance values of Japanese white radish depended on the frequency of the electric field, which varied from 50 Hz to 10 kHz. At a constant voltage of 40 V cm–1, the time required to heat the sample to the desired temperature was longer for higher frequencies of electric field. It was concluded that the initial rapid heating of white radish at low frequency (50 Hz) is caused by the electroporation effect. 3.3.2 Juice expression and solutes extraction enhanced by OH The first studies of extractions enhanced by OH were completed in the former Soviet Union, and summarised by Rogov and Gorbatov (1974) and by Lazarenko et al. (1977). In Soviet publications, electroporation was known as ‘electroplasmolysis’, and the combined thermal and electric effects caused by OH were called ‘thermo-electroplasmolysis’ or ‘low-gradient ac, Tf = 50 °C

Potato Apple 103

0.6

te (s)

Conductivity disintegration index, Z

0.8

0.4 102

0.2

0

0

20

40 60 80 Electric field strength, E (V cm–1)

100

101

Fig. 3.5 The conductivity disintegration index Z and time of treatment te versus electric field strength E for potato and apple tissues ohmically heated to 50 °C by ac treatment.


Principles of physically assisted extractions and applications 85 plasmolysis’ to underline the relatively low electric fields (typically 10–100 V cm–1) used for OH. Research interest in extractions assisted by OH processing was renewed in Europe and the USA during the 1990s and 2000s (Sastry, 2005; Schreier et al., 1993; Wang and Sastry, 2002) probably due to the successful commercialisation of OH systems for food sterilisation (Biss et al., 1989). This interest in OH extractions focused on the synergy of electrical and thermal effects on cell tissue, and the lower temperatures needed for effective membrane damage compared with conventional heating. On the other hand, the electrical field applied in OH-assisted extraction is lower than that for nonthermal PEF treatment. Therefore, electrical equipment can be simplified to deliver a lower output voltage than typical high voltage PEF generators. Various studies have recently demonstrated the effectiveness of OH pretreatment for the intensification of pressing and solute extraction (Kemp and Fryer, 2007; Kulshrestha and Sastry, 2003; Lima et al., 1999; Praporscic et al., 2005, 2006; Sensoy and Sastry, 2004; Wang and Sastry, 2002). For instance, Lima et al. (1999) ohmically heated apple cuts up to 40 °C with a voltage of E = 40 V cm–1 and wave frequencies of 4 and 60 Hz. Subsequent pressing at a constant rate (3 cm min–1) resulted in a higher juice yield than that obtained for untreated cuts, and the 4 Hz pretreatment was more effective than the 60 Hz pretreatment. More recently, Wang and Sastry (2002) have demonstrated that OH pretreatments of 40 or 50 °C by ac with 40 V cm–1 and 60 Hz enhanced the juice yield from apple samples. Compared with untreated samples, or those preheated by microwaves, the juices extracted from ohmically preheated apple tissue were visually similar in quality, and the yields were higher. The lower frequency of OH compared with MW heating may be the important factor that caused the greater increase in juice yield (Wang and Sastry, 2002). There may be other mechanisms involved in intra- and extracellular moisture flow during OH. In addition to its thermal effects and electrical breakdown, electro-osmosis may also be the reason for moisture flowing inside of cell tissue. Bazhal and Vorobiev (2000) have demonstrated that electro-osmotic flow was induced in apple tissue by a dc electric field of low voltage, which resulted in a significant increase in juice yield, even at ambient temperature. Solute extraction can also be enhanced by OH pretreatment. Kemp and Fryer (2007), Kulshrestha and Sastry (2003), and Schreier et al. (1993), showed that there is an increased diffusion of betanine dye from red beets treated by OH. In their interesting study, Kulshrestha and Sastry (2003) treated red beet cuts at 45 °C over a range of voltages from 0 to 23.9 V cm–1, and at frequencies of 0 (dc), 10, 50, 250 and 5000 Hz. As the electric field frequency decreased, the betanine content in the solution increased. This was explained by the increased time for which the cell membrane was charged at lower field frequencies. An exception to this tendency was detected for those tissues treated by dc, which was less effective than OH treatment at 10 Hz. All the mechanisms involved in tissue modification by


86 Separation, extraction and concentration processes OH treatment have not yet been elucidated. Electrolysis phenomena might also be important in terms of process optimisation. Other fresh materials, for example, mint leaf (Sensoy and Sastry, 2004), potato (Praporscic et al., 2006), and sugar beets (Praporscic et al., 2005) can also be processed by OH-assisted extraction.

3.4 Extraction assisted by high-voltage electrical discharges and applications in the food industry 3.4.1 Principles of underwater high-voltage electrical discharges (HVED) Underwater (electrohydraulic) HVED is a technique in which solid particles are placed in a dielectric liquid (typically tap water) inside a chamber, containing a high-voltage (HV) needle electrode and a plated grounded electrode, and exposed to pulsed shockwaves (typically 40–60 kV cm–1, 2–5 ms) to produce a liquid breakdown and particle fragmentation. This technique has various applications (defragmentation of solid materials, such as concrete, treatment of municipal solid waste, and drilling) (Bluhm, 2006). Some publications propose the use of the HVED application for the deactivation of micro-organisms and for the enhancement of extractions (Grémy-Gros et al., 2008). Figure 3.6 shows a schematic diagram of a HVED generator, as well as typical voltage and current curves measured during a HVED (Boussetta et al., 2009a). Effects of HVED The mechanisms of HVED are very complex and not yet well understood. The phenomenon is based on the electrical breakdown of water (Bluhm, 2006). Air bubbles that are already present in the water, or formed as a result of local heating, participate in and accelerate this phenomenon. If the electrical field is intense enough, the avalanche of electrons becomes a starting point for streamer propagation from the HV needle electrode to the grounded one (Fig. 3.7). A crack can be attracted from the discharge channel into the solid inclusion if their dielectric properties are different. The electrical breakdown is accompanied by a number of secondary phenomena (high-amplitude pressure shock waves, bubbles cavitation, and creation of liquid turbulence). For instance, the pressure exerted by the expanding channel almost always exceeds the tensile strength of the solid inclusions, and leads to the formation of cracks in the solids. Several hundred to thousands of discharges are sufficient to fragment the stones into small pieces of less than 1 mm (Bluhm, 2006). For biological particles, the cell structure can be damaged by HVED, which accelerates solute extraction (Grémy-Gros et al., 2008). If electrical breakdown does not occur, water carries electricity and behaves


Principles of physically assisted extractions and applications 87 HVED generator

Vent hole

Insulation

Air Water + sample

Needle electrode Plate electrode

Metal

HVED treatment cell (a) 15

40

10

20 5

0

0

Current (kA)

Voltage (kV)

tf ª 0.5 ms

–5 0

2

4 6 Time (ms) (b)

8

10

Fig. 3.6 HVED treatment (a) experimental setup and (b) typical voltage and current graphs.

like a good electrical conductor; then electrical signals have exponential form. When HVED are produced, electrical signals look like damped oscillations, as presented in Fig. 3.6 (Boussetta et al., 2009a; Grémy-Gros et al., 2008). 3.4.2 Solute extraction enhanced by HVED HVED have been used to accelerate the extraction of solutes from soybeans, potato, tea leaves, peat, and fennel (Barskaya et al., 2000; El-Belghiti, 2005; El-Belghiti et al., 2007) as well as from other food materials.



(a)

(b)

(c)

HV electrode

Shock wave

Charging unit

Ground Solid (d)

Fig. 3.7 Type of electric discharge in aqueous solution: (a) streamer, E = 4.4 kV cm–1; (b) streamer and spark, E = 13.3 kV cm–1; (c) spark, E = 33.3 kV cm–1 (Sugiarto et al., 2001). (d) Schematic diagram of electro-hydraulic disintegration process.

Oil The application of HVED can enhance the aqueous extraction of oil from oilseeds (Grémy-Gros et al., 2008; Gros et al., 2003). Linseeds were crushed and pressed with a hydraulic press, and then the press-cake obtained was reduced to powder and dissolved in demineralised water at ambient temperature. The mixture was treated with HVED (1–1640 discharges) and centrifuged (Gros et al., 2003). Approximately 26% of oil remained in the residue after 1640 pulses. The process was then optimised by varying the number of pulses, pH, water/press-cake ratio and temperature (Grémy-Gros et al., 2008). The HVED treatment also made it possible to enhance mucilage extraction from whole linseed (Gros et al., 2003). Seeds were dissolved in demineralised water and treated for 10 min (i.e. 300 pulses of 0.5 Hz frequency). A centrifuge separation was then performed to obtain solution and residue. The residue was then treated a second and a third time with fresh water under the same conditions. During the HVED treatment, linseeds were defragmented under the action of shock waves. Three successive 10-min treatments were sufficient to extract the mucilage almost entirely. The extraction of proteins began during the third treatment. However, the oil–protein–water emulsions obtained by HVED treatment then had to be separated. The following research study


Principles of physically assisted extractions and applications 89 resulted in the development of membrane separation of emulsions created by HVED (Li et al., 2009). Grape pomace Grape pomace (composed of stems, seeds and skins) has a high polyphenol content. These polyphenols have attracted great interest as they exhibit antibacterial, antiviral, and antioxidant properties, and can help prevent cardiovascular diseases (Dugand, 1980). Boussetta et al. (2009b) studied the application of HVED in the extraction of polyphenols from the pomace obtained as a residue from pressed white grapes (Vitis vinifera L., cultivar ‘Chardonnay’, vintage 2007). The treatment chamber (Fig. 3.6) was initially filled with pomace mixed with distilled water (the liquid-to-pomace ratio was 3:1, the water temperature was 20, 40 or 60 °C), and 80 successive discharges were applied (40 kV, pulse repetition rate 0.5 Hz). Afterwards, the total solute and polyphenol diffusion from the untreated and treated pomace mixtures were studied at the desired temperatures. Figure 3.8 shows that both temperature and HVED treatment improved the extraction of polyphenols. The difference between the yield of solutes for experiments with HVED treatment and without HVED treatment decreased with temperature elevation (Boussetta et al., 2009b). On the contrary, the opposite tendency was observed for the yield of polyphenols. The application of HVED enhanced the polyphenol yield, not just for fresh grape pomace, but also for sulfured

1

20

T (°C) 40

60 With HVED treatment

0.8

Ypolyphenols (%)

0.6

0.4

Without HVED treatment 0.2

0.0034

0.0032 1/T (K)

0.003

Fig. 3.8 Effect of treatment temperature on final yield of polyphenols after 1 h of extraction of fresh grape pomace with and without HVED treatment.


90 Separation, extraction and concentration processes and frozen pomaces. The final yields of solutes, reached after HVED and subsequent diffusion for 40 min were more than twice those reached after 240 min without HVED. Yeast cells Shynkaryk et al. (2008) studied the efficiency of HVED-assisted extraction from wine yeast cells (S. cerevisiae) in aqueous suspension (1% w/w). HVED induced damage in yeast membranes, leakage in cytoplasmic ions, and the release of intracellular bioproducts; it also resulted in an increase in the disintegration index Z. For HVED treatment at 40 kV, the high level of membrane disintegration (Z > 0.8) required more than 100 discharges, which corresponded to the effective time of HVED treatment between 100 and 200 ms. HVED treatment decreased the average cell size somewhat, owing to cell disruption. However, the quantity of high molecular weight intracellular components released, controlled by the absorbance analysis, was lower than that obtained by high-pressure homogenisation (Shynkaryk et al., 2008).

3.5 Ultrasound-assisted extraction (UAE) in the food industry 3.5.1 Principles of UAE Power ultrasound, which has frequencies between 20 kHz and 1 MHz, is now well known to have significant effects on the rate of various physical and chemical processes. Cleaning and solubilisation are its more developed applications, and a large variety of ultrasound baths exist for chemical laboratory use. The effect of ultrasonic waves on solid samples is widely used for the extraction of aromas from plant materials, or metal impurities from soils. Degassing and stripping are widely used for flavour analysis, and in environmental and polymer research. Other interesting ultrasound applications include homogenisation, emulsification, sieving, filtration, and crystallisation. The most interesting effect of ultrasound-based operational units is the reduction of processing time and the increase in product quality. All these effects are attributed to acoustic cavitation: when a liquid is irradiated by ultrasound, micro-bubbles form, grow and oscillate extremely fast, and eventually collapse powerfully (if the acoustic pressure is high enough). When the size of these bubbles reaches a critical point, they collapse during a compression cycle and release large amounts of energy. The temperature and pressure at the moment of collapse have been estimated to be up to 5000 K and 5000 atmospheres. This creates hotspots that are able to dramatically accelerate chemical reactivity in the medium. When these bubbles collapse onto the surface of a solid material, the high pressure and


Principles of physically assisted extractions and applications 91 temperature released generate microjets directed towards the solid surface. These microjets are responsible for the degreasing effect of ultrasounds on metallic surfaces, which is widely used for cleaning materials. Another application of microjets in the food industry is the extraction of vegetal compounds (Mason, 1990; Suslick, 1988). As shown in Fig. 3.9, a cavitation bubble can be generated close to the surface of the plant material. Then, during a compression cycle, this bubble collapses and creates a microjet (400 km h–1) directed toward the plant matrix. The high pressure and temperature involved in this process will destroy the cell walls of the plant matrix, and its content can be released into the medium. Power ultrasound involves the mechanical and chemical effects of cavitation. The mechanism can be explained by two competing theories. The hot spot theory assumes that the high pressures and temperatures generated in the bubbles during the last nearly adiabatic compression, just before collapse, are responsible for the breakage of molecular bonds and the formation of radicals. On the other hand, the electrical theory describes micro-discharges, produced by the high electrical fields generated by the deformation and fragmentation of the bubbles. The most commonly used frequencies in sono-extraction are between 20 and 40 kHz. With higher frequencies, cavitation would be more difficult to induce because the cavitation bubbles need a slight delay to be initiated during the rarefaction cycle. The higher the frequency, the shorter the rarefaction cycle, so the less time available for the bubble to be created (Povey and Mason, 1998).

Cavitation bubble with negative pressure

Maximum and critical bubble size 2

1

A new cavitation bubble appears and cycle is repeated

3

4

Implosion of bubble by compression

Fig. 3.9 Ultrasonic cavitation phenomenon and its action on extraction of natural products.


92 Separation, extraction and concentration processes 3.5.2 UAE reactors The two most common types of ultrasound equipment that are used for extraction are the ultrasonic cleaning bath and the more powerful probe system. For small extraction volumes, an ultrasound horn with its tip submerged in the fluid can be sufficient. Large volumes of fluids have to be sonicated in an ultrasound bath or in continuous or recycled-flow sono-reactors. There are also ultrasonic reactors conceived especially for solvent extraction on a laboratory scale (3 l), in a pilot plant (30 l) and on an industrial scale (150–1000 l). Pump systems are coupled to the ultrasonic reactor to stir the mixture and to empty the system at the end of the experiment. The intensity of the ultrasound is about 1 W cm–2, with a frequency of 25 kHz. In order to maintain constant temperature, the reactor is made of a double mantle into which cooling water can circulate. The main advantage of this type of apparatus is that the natural products and extraction solvent are mixed into a container, and the ultrasound is directly applied to the mixture (Fig. 3.10). Recently, the new methodology of continuous-flow systems has been used in analytical chemistry. Most UAE applications have been developed in discrete systems, using a bath or an ultrasonic probe, particularly in the extraction of food samples. Less frequent has been the design of on-line UAE systems in the same field. However, it is noted that the latter approach is considerably faster. It consists of an open system, in which fresh solvent flows continuously through the sample. This induces the displacement of mass transfer equilibria toward the solubilisation of analytes into the liquid phase (Priego-Capote and Luque de Castro, 2007). 3.5.3 Factors affecting UAE All the research carried out over the last 50 years strongly supports the importance of each identified factor in the ultrasound process, where frequency, intensity, treatment time, temperature, pressure, and treatment

(a)

(b)

Fig. 3.10 (a) Laboratory (3 l) and (b) industrial (150 l) ultrasound extraction reactors (with permission, www.etsreus.com).


Principles of physically assisted extractions and applications 93 media define the extraction and the separation kinetics of ingredients from natural products (De Gennaro et al., 1999). Generally, the highest efficiency of UAE, in terms of the yield and composition of the extracts, can be achieved by increasing the ultrasound power, reducing the moisture of food matrices to enhance solvent–solid contact, and optimising the temperature to allow a shorter extraction time. Solvent choice is dictated by the solubility of the ingredients of interest, the interactions between the solvent and matrix, and the intensity of ultrasound cavitation phenomena in the solvent. The impact of ultrasound is attributed to intracellular cavitation, that is, micro-mechanical shocks that disrupt cellular structural and functional components up to the point of cell lysis (Butz and Tauscher, 2002). Figure 3.11 shows a proposed mechanism for ultrasound-induced cell damage. Cavitation is the formation, growth and, sometimes, the implosion of microbubbles created in a liquid when ultrasound waves propagate through it. The collapse of the bubbles leads to energy accumulation in hot spots, where temperatures of above 5000 °C and pressures of approximately 500 MPa have been measured. This phenomenon can cause enzyme inactivation through three mechanisms, which can act alone or combined. The first one is purely thermal, owing to the enormous temperatures achieved during cavitation. The second is caused by free radicals, generated by water sonolysis; and the third is the result of the mechanical forces (shear forces) created by microstreaming and shock waves (Raso et al., 1999). 3.5.4 UAE: main applications Essential oils and aromas UAE has also been developed for the extraction of essential oils from aromatic plants, such as peppermint leaves (Shotipruk et al., 2001), artemisia (Asfaw et al., 2005) and lavender (Da Porto et al., 2009), or from other vegetal matrices, such as garlic (Kimbaris et al., 2006) and citrus flowers (Alissandrakis et al., 2003). Increased yields of essential oil were found for peppermint leaves (up to 12%) and for artemisia when using UAE; and yields of the main compounds of lavandula essential oil were found to increase by Ultrasound (US)

US

H 2O

Cell

US

Cell

Pore initiation

US

H2O influx and swelling

Cell

Membrane rupture and cell lysis

Fig. 3.11 Mechanism of ultrasound-induced cell damage.


94 Separation, extraction and concentration processes 2- to 3-fold when comparing UAE with conventional distillation. Moreover, UAE not only improved yields, but also showed less thermal degradation in the final extracts. Several studies have been conducted into the extraction of the main aroma compounds from spices. For example, vanillin was extracted from vanilla pods (Jadhav et al., 2009) carvone from caraway seeds (Chemat et al., 2004) and safranal from Greek saffron (Kanakis et al., 2004). Yields of vanillin obtained after 1 h using UAE were similar to those obtained after 8 h using conventional extraction methods. Wines and other alcoholic beverages contain many volatile compounds, which play an important role in organoleptic characteristics. The ultrasound-assisted maceration of fruits and herbs in alcoholic beverages to extract volatile compounds is an important process in the food industry (Fig. 3.12). Antioxidants A wide variety of fruits and vegetables have been studied by UAE because antioxidants are present in different amounts in different varieties of plants, and these antioxidants come from different families. One of the most common antioxidants is the lycopene extracted from tomatoes (Lianfu and Zelong, 2008). In this particular work, authors not only worked on UAE, but also on coupling ultrasounds with microwaves, which produces high lycopene extraction in only 6 min. Herrera and Luque de Castro (2005) showed that the amount of phenolic compounds that could be extracted from strawberries in 2 min, was similar to the amount extracted over 20 h using the conventional method, and 3 h using supercritical fluid extraction. Another example is the extraction of anthocyanins from raspberries, developed by Chen et al. (2007). In this work, the same yields of anthocyanins were achieved in 3 min when using UAE, compared with 53 min when using the conventional extraction system. The difference between ultrasound and conventional methods will be more or less significant, depending on the part of the plant studied. This has been highlighted by studies of grapes and grape seeds (Palma and Barroso, Sono-extraction

0.100

Conventional extraction

Yield (%)

0.075 0.050

0.025 0.000

0

30

60

90

120 150 180 210 240 270 Time (min)

Fig. 3.12 Rapid sono-extraction of orange peels in alcoholic beverages.


Principles of physically assisted extractions and applications 95 2002). In this work, authors found that the use of ultrasound instead of maceration was more interesting when working on seeds than on the whole fruit (Table 3.1). Oil and fat Many papers have reported on the UAE of oil and fat from various food samples. According to Luque-Garcia and Luque de Castro (2004), extraction from oleaginous seeds is difficult. Indeed, only 75–85% of the oil is solubilised in the solvent. The rest of the oil content is strongly bound to the matrix and cannot be extracted without additional treatments. For instance, Li et al. (2004) described the UAE of soybean oil in an optimised yield and reduced operating time, compared with conventional maceration. Luque-Garcia and Luque de Castro (2004) have reported a device consisting of an ultrasoundassisted Soxhlet apparatus, which is an attractive alternative to traditional Soxhlet extraction. Using this device, it takes only 90 minutes to obtain the same yield (99% of fat recovery) as can be obtained over 12 h with the traditional Soxhlet extraction method (Table 3.1). 3.5.5 Hazard analysis critical control point (HACCP) for UAE processing operation The hazard analysis critical control point (HACCP) system is a process that identifies and assesses the hazards and risks associated with the manufacture, Table 3.1 Ultrasound-assisted extraction of food ingredients Matrix (reference)

Extracts

Essential oils and aromas Citrus flowers (Alissandrakis et al., 2003) Caraway seeds (Chemat et al., 2004) Garlic (Kimbaris et al., 2006) Lavender flowers (Da Porto et al., 2009) Greek saffron (Kanakis et al., 2004) Antioxidant extracts Strawberries (Herrera and Luque de Castro, 2005) Raspberries (Chen et al., 2007) Tomatoes (Liangfu and Zelong, 2008) Grape (Palma and Barroso, 2002) Fat and oil extraction Olive seeds (Luque-Garcia and Luque de Castro, 2004) Soybean germ and seaweed (Li et al., 2004) Cocoa powder and nibs (Luque- Garcia and Luque de Castro, 2004) Bakery products (Luque-Garcia and Luque de Castro, 2004)

Linalool Carvone and limonene Essential oil (organosulfur compounds) Essential oil (1,8-cineol, camphor and linalyl acetate) Safranal Polyphenolic compounds Anthocyanins Lycopene Phenolic Oil Oil Fat content Fat content


96 Separation, extraction and concentration processes storage and distribution of foods and implement the appropriate controls, aiming at the elimination or reduction of these hazards at specific points of the production line. In UAE processing, parameters such as the temperature and physical properties of the product, the timescale of treatment in the ultrasound chamber, the frequency and power of the ultrasound, and the nature of the probe are points which need to be monitored. For each part of the food process, including ultrasound processing, the evaluation and classification of hazards are completed. In this way, a grade is given to each hazard that has been identified according to its gravity, its risk of occurrence and the ability to detect it. For each hazard, preventive measures are set up, with procedures that indicate who will be responsible for them, as well as how, when and where. Preventive measures are aimed primarily at avoiding the occurrence of the hazard. For example, a good maintenance plan decreases the risk of metal contamination from the ultrasound horn (Table 3.2).

3.6 Microwave-assisted extraction (MAE) in the food industry 3.6.1 Principles of MAE Microwaves are electromagnetic waves with a frequency range of between 100 MHz and 3 GHz that comprise electric and magnetic field components and thus constitute propagating electromagnetic energy. This energy acts as a non-ionising radiation, which causes molecular motion of ions and rotation of the dipoles, but does not affect molecular structure. Table 3.2 Possible critical control point limits and associated corrective actions in an ultrasound-assisted extraction processing Critical control point

Danger

Target

Deviation

Processing Temperature Adequate Does not conform operation to the requirements Ultrasound Adequate Does not conform frequency to the requirements Ultrasound Adequate Does not conform power to the requirements Flow rate Below Does not conform specifications to the requirements Probe Absence Detected spoilage

Corrective actions

Adjust temperature reject or reprocess the product Adjust frequency, laboratory controls, or reprocess the product Adjust the power, laboratory controls, or reprocess the product Adjust flow, laboratory controls, or reprocess the product Reject


Principles of physically assisted extractions and applications 97 When dielectric materials containing either permanent or induced dipoles are placed in a MW field, the rotation of the dipoles in the alternating field produces heat. More precisely, the applied MW field causes the molecules to spend slightly more time, on average, orienting themselves in the direction of the electric field rather than in other directions. When the electric field is removed, thermal agitation returns the molecules to a disordered state in the relaxation time and thermal energy is released. Thus, MW heating results from the dissipation of the electromagnetic waves in the irradiated medium (Fig. 3.13). The amount of dissipated power in the medium depends on the complex permittivity of the material and the local time-averaged electric field strength (Metaxas and Meredith, 1993). In conventional heating, heat is transferred from the heating medium to the interior of the sample, whereas in MW heating; heat is dissipated volumetrically inside the irradiated medium, and thus heat transfers occur from the sample to the colder environment. This causes a significant difference Moisture (%)

Classical heating

Microwave heating

Microwave heating Heat transfer

1 2

1. Solid friction desorption 2. Internal diffusion 3. External diffusion

Mass transfer

3

Conventional heating Heat transfer Mass transfer

Fig. 3.13 Microwave versus conventional extraction.


98 Separation, extraction and concentration processes between conventional and MW heating. In conventional heating, heat transfer depends on thermal conductivity, on the temperature difference across the sample, and, for fluids, on convection currents. As a result, the temperature increase is often rather slow. In contrast, the volumetric heating effect in MW heating enables much faster temperature increases to be obtained, depending on the MW power and the dielectric loss factor of the material being irradiated. The influence of MW energy on chemical or biochemical reactions is strictly thermal. The MW energy quantum is given by the standard equation W = hn. Within the frequency domain of microwaves and hyper-frequencies (300 MHz–300 GHz), the corresponding energies are respectively 1.24 ¥ 10–6 eV to 1.24 ¥ 10–3 eV. These energies are much lower than the usual ionisation energies of biological compounds (13.6 eV), of covalent bond energies like OH (5 eV), hydrogen bonds (2 eV), and van der Waals intermolecular interactions (lower than 2 eV) and even lower than the energy associated with Brownian motion at 37 °C (2.7 ¥ 10–3 eV). From a scientific point of view, the direct molecular activation of microwaves should be excluded. 3.6.2 MAE reactors It is now possible to use a wide range of vessels and instrumentation when working with microwaves, depending on the intended purpose and the analytes to be extracted. Two types of MW ovens can be found in laboratories and they differ in terms of their cavity type: single-mode (also called monomode or focused mode) and multi-mode. A single-mode cavity permits the generation of one type of frequency, allowing only one mode of resonance to be excited. Thus, the sample is placed in such a way as to receive the maximum amount of MW energy, ensuring a direct and efficient application of a high density of MW energy to the sample matrix, and inducing a rapid heating of the medium with a fast extraction of the analyte. However, monomode systems are quite limited in terms of the volume and quantity of the sample that is ultimately useable. On the other hand, multi-mode ovens are larger and allow several modes of resonance. Thus, a large number of incident waves can be applied to the sample being studied. This kind of oven allows for a large quantity of matrix, and can be used with several types and forms of vessels. There are also MW reactors conceived especially for solvent extraction in the laboratory (0.1 to 1 l) and on an industrial scale (100 l h–1) (Fig. 3.14). 3.6.3 MAE: main applications Microwaves are very useful for rapid extraction and obtaining high quality aromatic compounds from garlic, lavender flowers, orange peel and rosemary leaves. The extraction of essential oils from orange peels with solvent-free MW extraction is better in terms of energy saving, extraction time (30 min



(a)

(b) –1

Fig. 3.14 (a) Laboratory (1 l) and (b) industrial (100 l h ) MAE reactors (with permission, www.milestonesrl.com and www.rchimex.com).

versus 3 h), product yield (0.42 versus 0.39%) and product quality (AbertVian et al., 2008). Hemwimon et al. (2007) made a comparison between the MAE of antioxidative anthraquinones from the roots of Morinda citrifolia, and extraction with other conventional techniques (maceration and Soxhlet). The efficiency of extraction using MAE (15 min) was much higher than that using maceration (3 days) and was also similar to that of Soxhlet (4 h). Similarly, the best recoveries of antioxidants in short time periods, and with a lower consumption of polar solvent were also observed from sweet grass by Grigonis et al. (2005). As well as obtaining a higher yield, Pan et al. (2008) extracted phenolic compounds with scavenging ability, compared with synthetic antioxidants from longan peel with MAE. Elkhori et al. (2007) described the MAE of cocoa powder with hexane/ isopropanol, which resulted in the rapid determination of fat contents, with recoveries similar to, or better than, those of the conventional method, and with the added advantages of low solvent consumption, short extraction time, low energy consumption and excellent reproducibility. Focused MW-assisted Soxhlet extraction was used by Pérez-Serradilla et al. (2007) for acorn oil determination, which resulted in trans fatty acid-free oil, probably because of the reduced exposure to drastic conditions in just 30 min, which is much less than the time required by the Soxhlet (8 h) and stirring (56 h) reference methods. Lianfu and Zelong (2008) described the extraction of lycopene from tomatoes with the combined innovative techniques of ultrasound and MW-assisted extraction (UMAE) in comparison with ultrasound-assisted


100 Separation, extraction and concentration processes extraction (UAE). Similarly, a higher yield of anthocyanins in red raspberries was also observed by Sun et al. (2007) with optimal conditions of MAE. MAE proved to be more rapid and efficient as it extracts various types of anthocyanins, without any destruction of their chemical structure, in a very short time, owing to the intensive disruption of tissue structure under MW irradiation. Table 3.3 clearly shows some applications of MAE for various useful metabolites, carried out using several vegetal matrices. 3.6.4 Safety considerations The MAE process is simple and can be readily understood in terms of the operating steps to be performed. However, in inexperienced hands, the application of MW energy can pose serious hazards. All persons dealing with microwaves must exercise a high level of safety and attention to detail when planning and performing such experiments. They have to ensure that they seek proper information from knowledgeable sources and that they do not attempt to implement this technique unless proper guidance is provided. Only approved equipment and scientifically sound procedures should be used.

3.7 Combination of physical treatments for extraction in the food industry Various physical treatments can induce various effects on cell and tissue structure, which may be more or less beneficial for the extraction of intracellular components. Unfortunately, the studies comparing or combining two or more Table 3.3 Microwave-assisted extraction of food ingredients Matrix (reference)

Extracts

Antioxidant extracts Green tea leaves (Pan et al., 2003) Noni plant roots (Hemwimon et al., 2007) Sea buckthorn (Sharma et al., 2008) Longan peel (Pan et al., 2008)

Polyphenols Anthraquinones Phenolic constituents Flavonoids

Fat and oil extraction Olive seeds (Virot et al., 2007) Oleaginous seeds (Pérez-Serradilla et al., 2007) Soybean germ and seaweed (Cravotto et al., 2008) Cocoa powder and nibs (Elkhori et al., 2007)

Oil Oil Oil Fat content

Natural food colour extraction Tomato paste (Lianfu and Zelong, 2008) Curcuma (Mandal et al., 2008) Red raspberries (Sun et al., 2007) Paprika (Csiktusnádi Kiss et al., 2000)

Lycopene Curcumin Anthocyanins Carotenoids


Principles of physically assisted extractions and applications 101 physically assisted extractions are rather rare. El-Belghiti (2005) compared the application of PEF (680 V cm–1, 1000 pulses of 100 ms) and HVED (40 kV, 100 discharges) for the cold (20 °C) aqueous extraction of solutes from two dried products: tea leaves and Datura innoxia (common name: moonflower) roots. PEF treatment did not have any influence on the process of extracting from dried and rehydrated products, probably because of the absence of the cell membrane element, which had been disrupted by previous drying. The non-efficacy of moderate electric treatment for the dried cell tissue was reported earlier in Sensoy and Sastry (2004). In contrast to PEF, the HVED smashed products to small fragments and the extraction kinetics were significantly enhanced for both tea leaves and Datura innoxia. El-Belghiti et al. (2007) compared PEF, HVED and PU as treatments to enhance cold (20 °C) aqueous extraction from fennel gratings. The objective was to obtain extracts used as natural food preservatives (antioxidants). The three assisted extractions led to the same final yield of solutes, and all preserved the antioxidant substances. However, their kinetics were different. The most rapid and the slowest kinetics were offered by HVED and PU, respectively. The final yield of 98% was reached in 20 min with HVED, in 40 min with PEF, and in more than 180 min with PU. The amounts of energy needed for these treatments were also different: the PEF treatment appeared to be the most economic energetically. Recently, Shynkaryk et al. (2008) compared PEF, HVED, and high-pressure homogenisation (HPH) for disruption of wine yeast cells (S. cerevisiae bayanus). The PEF and HVED were applied at electric field strengths of 10 kV cm–1 and 40 kV cm–1, respectively. The HPH was applied within a pressure range of 30–200 MPa for a different number of passes (1–20). It was shown that the releasing efficiency of ionic components, enzymes, proteins and other bioproducts dramatically depended on the applied method of disruption. Extraction efficiency was rather high for ionic components and small for high molecular weight components in PEF or HVED pretreated suspensions. The PEF treatment removes membrane barriers and accelerates the release of ionic contents, but has practically no influence on the cell walls. The efficiency of releasing high molecular weight content is questionable and may depend on the cell strain, the age of culture, the time of extraction, temperature and many other factors. For example, high levels of PEF extraction of proteins for baker’s yeast was previously reported (Ganeva et al., 2003), but the efficiency of the PEF extraction of proteins was found to be rather low for wine yeast in the study by Shynkaryk et al. (2008). On the contrary, the application of the combined HVED–HPH technique resulted in a material loss of integrity in the yeast cell walls. Even a small disintegration (Zi = 0.15±0.05), initially induced by HVED pretreatment, resulted in noticeable acceleration of HPH disruption kinetics. Although most of the research effort in UAE has concentrated on ultrasound itself, some studies have also examined the coupling between ultrasound and other techniques. For instance, UAE is currently being employed in


102 Separation, extraction and concentration processes combination with MW energy (Lagha et al., 1999), supercritical fluid extraction (Hu, 2007) or simply with conventional methods such as Soxhlet extraction (Luque-García and Luque de Castro, 2004). When combined with supercritical fluid extraction, UAE enhances the mass transfer of the species of interest, from the solid phase to the solvent used for extraction. Soxhlet extraction can also be improved by ultrasound when applied at the cartridge zone before siphoning, thus permitting the removal of lipid fractions from very compact matrices. The efficiency of combining MW and ultrasound has been clearly shown in applications such as the extraction of food ingredients. Studies combining assisted extractions present undeniable interest as synergistic effects of different treatments may be discovered.

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104 Separation, extraction and concentration processes stages of Saccharomyces cerevisiae yeast suspensions damage in moderate pulsed electric fields’, Colloids and Surfaces B: Biointerfaces, 479(2), 189–197. Eshtiaghi, MN and Knorr D, (2002), ‘High electric field pulse pretreatment: Potential for sugar beet processing’, Journal of Food Engineering, 52, 265–272. Fincan M, DeVito F and Dejmek P (2004), ‘Pulsed electric field treatment for solid–liquid extraction of red beetroot pigment’, Journal of Food Engineering, 64(3), 381–388. Fincan M and Dejmek P (2002), ‘In situ visualization of the effect of a pulsed electric field on plant tissue’, Journal of Food Engineering, 55, 223–230. Ganeva V, Galutzov B and Teissié J (2003), ‘High yield electroextraction of proteins from yeast by a flow process’, Analytical Biochemistry 315(1), 77–84. Grémy-Gros C, Lanoisellé J-L and Vorobiev E (2008) ‘Application of high-voltage electrical discharges for the aqueous extraction from oilseeds and other plants’, In: E. Vorobiev and N. Lebovka (Eds), Electrotechnologies for extraction from food plants and biomaterials, Springer, New York, 217–236. Grigonis D, Venskutonis P, Sivik B, Sandahl M, Eskilsson C (2005), ‘Comparison of different extraction techniques for isolation of antioxidants from sweet grass’, Journal of Supercritical Fluids, 33, 223–233. Grimi N, Lebovka M, Vorobiev E, Vaxelaire J (2009a), ‘Compressing behaviour and texture evaluation for potatoes pretreated by pulsed electric field’, Journal of Texture Studies, 40, 208–224. Grimi N, Lebovka M, Vorobiev E and Vaxelaire J (2009b), ‘Effect of a pulsed electric field treatment on expression behaviour and juice quality of Chardonnay grape’, Food Biophysics, 4, 1557–1858. Grimi N, Praporscic I, Lebovka N and Vorobiev E (2007), ‘Selective extraction from carrot slices by pressing and washing enhanced by pulsed electric fields’, Separation and Purification Technology, 58(2), 267–273. Grimi N, Vorobiev E and Vaxelaire J (2008), ‘Juice extraction from sugar beet slices by belt filter press: effect of pulsed electric field and operating parameters’, 14th World Congress of Food Science and Technology, 19–23 October 2008, Shanghai, China, TS24–29, 554. Gros C, Lanoisellé J-L and Vorobiev E (2003), ‘Towards an alternative extraction process for linseed oil’, Chemical Engineering Research and Design, 81(9), 1059–1065. Hemwimon S, Pavasant P and Shotipruk A (2007), Microwave-assisted extraction of antioxidative anthraquinones from roots of Morinda citrifolia, Separation and Purification Technology, 54, 44–50. Herrera M and Luque de Castro M D (2005), Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection, Journal of Chromatography A, 1100, 1–8. Hu A (2007), ‘Ultrasound assisted supercritical fluid extraction of oil and coixenolide from adlay seed’, Ultrasonics Sonochemistry, 14, 219–222. Imai T, Uemura K, Ishida N, Yoshizaki S, and Noguchi A (1995), ‘Ohmic heating of Japanese white radish Rhaphanus sativus L’, International Journal of Food Science and Technology, 30(4), 461–472. Jadhav D, Rekha B N, Gogate P R, Rathod V K (2009), ‘Extraction of vanillin from vanilla pods: A comparison study of conventional soxhlet and ultrasound-assisted extraction’, Journal of Food Engineering, 93, 421–426. Jaeger H, Balasa A, and Knorr D (2008), ‘Food industry applications for pulsed electric fields’, In: E Vorobiev and N Lebovka (Eds), Electrotechnologies for extraction from food plants and biomaterials, Springer, 181–216. Jemai A B and Vorobiev E (2002), ‘Effect of moderate electric field pulse (MEFP) on the diffusion coefficient of soluble substances from apple slices’, International Journal of Food Science and Technology, 37, 73–86. Jemai A B and Vorobiev E (2003), ‘Enhancing leaching from sugar beet cossettes by pulsed electric field’, Journal of Food Engineering, 59, 405–412.


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106 Separation, extraction and concentration processes Lima M, Heskitt B and Sastry S (1999), ‘The effect of frequency and wave form on the electrical conductivity–temperature profiles of turnip tissue’, Journal of Food Process Engineering, 22(1), 41–54. Lima M, Heskitt B F and Sastry S K (2001), ‘Diffusion of beet dye during electrical and conventional heating at steady-state temperature’, Journal of Food Process Engineering, 24, 331–340. Lopez N, Puertolas E, Condon S, Raso J and Alvarez I (2009a), ‘Enhancement of the extraction of betanine from red beetroot by pulsed electric fields’, Journal of Food Engineering, 90(1), 60–66. Lopez N, Puertolas E, Hernández-Orte P, Alvarez I and Raso J (2009b), ‘Effect of a pulsed electric field treatment on the anthocyanins composition and other quality parameters of Cabernet Sauvignon freshly fermented model wines obtained after different maceration times’, LWT – Food Science and Technology, 42(7), 1225–1231. Luque-García J L and Luque de Castro M D (2004), ‘Ultrasound-assisted Soxhlet extraction: an expeditive approach for solid sample treatment: application to the extraction of total fat from oleaginous seeds’, Journal of Chromatography A, 1034, 237–241. Mandal V, Mohan Y and Hemalatha S (2008), ‘Microwave assisted extraction of curcumin by sample-solvent dual heating mechanism using Taguchi L9 orthogonal design’, Journal of Pharmaceutical and Biomedical Analysis, 46, 322–327. Mason T J (1990), Chemistry with ultrasound, Elsevier Applied Science, New York. McLellan M R, Kime R L and Lind K R (1991), ‘Electroplasmolysis and other treatments to improve apple juice yield’, Journal of Science of Food and Agriculture, 57(2), 303–306. Metaxas A C and Meredith R J (1993), Industrial microwave heating, IEEE, London. Palma M and Barroso C (2002), ‘Ultrasounds-assisted extraction and determination of tartaric and malic acids from grapes and winemaking by-products’, Analitica chimica acta, 458, 119–125. Pan X, Niu G, Liu H (2003), ‘Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves’, Chemical Engineering and Processing, 42, 129–133. Pan Y, Wang K, Huang S, Wang H, Mu X, He C, Ji X, Zhang J, Huang F (2008), ‘Antioxidant activity of microwave-assisted extract of longan peel’, Food Chemistry, 106, 1264–1270. Pérez-Serradilla J, Ortiz M, Sarabia L, Luque de Castro M (2007), ‘Focused microwaveassisted soxhlet extraction of acorn oil for determination of the fatty acid profile by GC-MS’, Analytical and Bioanalytical Chemistry, 388, 451–462. Povey M and Mason T J (1998), Ultrasound in Food Processing, Blackie Academic & Professional, London. Praporscic I, Ghnimi S and Vorobiev E (2005), ‘Enhancement of pressing of sugar beet cuts by combined pulsed electric field and ohmic heating’, Journal of Food Processing and Preservation, 29(5-6), 378–389. Praporscic I, Lebovka N I, Ghnimi S and Vorobiev E (2006), ‘Ohmically heated, enhanced expression of juice from apple and potato tissues’, Biosystems Engineering, 93(2), 199–204. Praporscic I, Lebovka NI, Vorobiev E, Mietlon-Peuchot M (2007), ‘Pulsed electric field enhanced expression and juice quality of white grapes’, Separation and Purification Technology, 52(3), 520–526. Praporscic I, Shynkaryk M, Lebovka N and Vorobiev E (2007b), ‘Analysis of juice colour and dry matter content during pulsed electric field enhanced expression of soft plant tissues’, Journal of Food Engineering, 79(2), 662–670. Priego-Capote F and Luque de Castro M D (2007), Ultrasound-assisted digestion: a useful alternative in sample preparation, Journal of Biochemical and Biophysical Methods, 70, 299–310.


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4 Advances in process chromatography and applications in the food, beverage and nutraceutical industries M. Ottens and S. Chilamkurthi, Delft University of Technology, The Netherlands Abstract: The basics of process chromatography and its application to separation and purification in the food, beverage and nutraceutical industry are discussed. Basic principles and modes of chromatography are described together with guidelines for appropriate modeling; all illustrated with examples. New developments are described in the area of novel ligands, new materials, and modes of operation and control. Finally, future trends with respect to the production of high-value nutraceuticals, regulation and process control are given. Key words: process chromatography, nutraceuticals, proteins, model, simulated moving bed.

4.1 Introduction 4.1.1 Chromatography as a unit operation in food, beverage and nutraceutical processing The global food, beverage, and agriculture industry caters to the population of the entire world. It is a complex, global collective of diverse businesses that together supply much of the food energy consumed by the world population. Global market forces are driving the continuous evolution of the food and beverage industry. Consolidation, changing consumer preferences and increasing government regulations are significantly impacting manufacturing and business strategy. Producing quality goods at the lowest possible cost is a major aim. Consumers are increasingly interested in the health benefits of foods and have begun to look beyond the basic nutritional benefits of food to the


110 Separation, extraction and concentration processes disease-prevention and health-enhancing compounds contained in many foods. This, combined with a more widespread understanding of how diet affects disease, health-care costs and aging populations, has created a market for functional foods and natural health products. There is a growing interest in functional foods, which are dietary supplements that increase a person’s well being. A functional food contains one or more ingredients that are bioactive, that is: they promote desired biological activity in the human body. Process-scale chromatography is an indispensable unit operation in biotechnology and finds wide application in the food and beverage industry (Fig. 4.1). Even though the food industry does not rely on process scale chromatography to the same extent as perhaps other industries such as the pharmaceutical sector, the sheer size of the industry results in a considerable application for chromatography. Other than process-scale applications of chromatography, many applications are related to safety, quality control, and research of new food ingredients and products. Many product streams in the food industry contain components that may have applications for, for example, functional foods. These product streams are typically large in volume and contain only a small amount of the component (or mixture of components) of interest. For economical application of large-scale preparative protein, peptide or oligosaccharide chromatography from agricultural/biotechnology streams, there is a need for ‘cheap’ resins (the costs for resins may be as high as 70% of total processing costs). The standard technology for fractionation or enrichment of food streams is using a packed bed. The close contact between interstitial liquid and carrier beads ensures effective mass transfer. For the treatment of large process streams

Fermentation Cell disruption Separation of cell debris

Removal of ‘like’ molecules such as mis-folds, multimers, degradation products via: Crystallization (extraction) Chromatography

Concentration Purification

Physical adsorption Ion exchange Affinity, size (sorption processes)

Formulation

(membrane separations) (electrokinetic separations)

Market

Fig. 4.1 Positioning of chromatography in a general layout of a bioprocess. For food processes, the fermentation step may not be present.


Advances in process chromatography and applications 111 and/or process streams where suspended solids or fouling components are present, problems may arise: the pressure drop over the column might be too high. The workhorse of preparative protein purification is ion exchange chromatography (IEC). For dilute feeds, IEC can be used in a capture step. Another way of pre-concentration is precipitation followed by resolubilization and IEC fractionation. Large-scale preparative chromatography can be done more efficiently using counter-current chromatography, which has larger driving forces for mass transfer and more efficient use of desorbent and (expensive) resin (see Section 4.4). As an example of the fractionation of protein hydrolyzates, simulated moving bed (SMB) chromatography has been recently applied (Ottens et al. 2006b). An example of large-scale purification of biomolecules being applied in industry is, for example, the purification of lysine (an amino acid) in SMB carousels (Van Walsum and Thomson, 1997). However, using a larger number of protein fractions, more side streams are needed, or a different approach will be needed (similar to distillation with different side streams). Resin screening is an important aspect of the development of an economical process (with respect to resin capacity, regeneration cycles, resin lifetime, costs of resins, food grade resins: some resin is always lost in the feed and the need for ‘water-soluble elution liquids’, as many food companies are not used to processing organic solvents). Last but not least, fouling characteristics are important as well as a proper cleaning program. The current challenges that are associated with the use of chromatography on a large scale in the food industry are the following (see also section 4.4): Large diluted stream with minor component (mg m–3) from large product streams (m3 h–1). – For high throughputs, expanded bed adsorption (EBA) or SMB chromatography might be considered, as indicated above. Considerable effort in the application and development of EBA was made by Kula and co-workers (e.g. Hubbuch et al., 2006). ∑ Pressure drop. Possible solutions include: – use of micro-structured materials, multimodal pores, perfusion resins; monoliths, structured woven packing, all allowing for a large convective flow and creating an interfacial area in the (functionalized) micropores, – (partly) fluidization, as in EBA, – radial flow chromatography. ∑ Fouling: – removal of debris, proteins and fats from the feed by membrane filtrations preferably in combination with a pretreatment. ∑ An understanding of the required purity of the targeted molecule. Mixtures with enriched fractions or pure targeted component. ∑

These considerations are addressed in this chapter.


112 Separation, extraction and concentration processes Several examples of functional food products that can be enriched or purified by chromatography are briefly introduced next. Peptides are known to have a versatility of bioactivity, e.g. ranging from a metal binding function, through influencing mood functions, to a reduction of blood pressure. The peptides can be obtained from various (food) proteins; dairy liquids such as milk and whey have often been investigated (e.g. Meisel and Schlimme, 1996). Typically, the production of a bioactive ingredient for functional food is obtained by enzymatic hydrolysis of the native protein. From the resulting complex mixture, the peptide(s) of interest need to be isolated. Currently, this is often done by membrane filtration or chromatography. Membrane filtration is regarded as a cheap technology, but the selectivity is rather low. Electromembrane filtration has been suggested as an alternative isolation route for charged components. In this membrane-based separation, the selectivity is increased by applying an electrical field as the (selective) driving force for migration over the membrane (Bargeman et al., 2002a; 2002b), which contributes to separation costs and complexity in comparison to regular membrane filtration. On the other hand, chromatography is highly selective, but is often regarded as expensive, as a result of the relatively high costs of the column media and eluents. The use of SMB technology is investigated as an alternative separation route, as this counter-current chromatographic separation method has been shown to be able to efficiently reduce sorbent inventory and the use of buffers by one order of magnitude in comparison to regular (fixed bed) column chromatography (e.g. Ballanec and Hottier, 1993; Cavoy et al., 1997). Furthermore, the use of chromatography for glucose–fructose separation (Azevedo and Rodrigues, 2005; Luz et al., 2008) sugar recovery from molasses (Hannu and Jarmo, 2000), stabilization of beers (Rehmanji et al., 2002), separation of proteins from various sources, such as milk (Kim et al., 2003a) and rapeseed, are only a few of the process scale applications of chromatography in the food and beverage industry. In addition to this several other functional components such as amino acids (Kostova and Bart, 2007a,b), flavors and fragrances (Gunther and Armin, 1993), nutraceuticals (Genovese et al., 2007), organic acids (Yoshikawa, et al., 2007), and oligosaccharides (Berensmeier and Buchholz, 2004) have also been separated on process and analytical scales. All these separations involve various interaction mechanisms, including ligand-exchange chromatography, LEC (Azevedo and Rodrigues, 2005, Luz et al., 2008, Steffenson and Westerlund, 1996), size-exclusion chromatography, SEC (Thurl et al., 1993), IEC (Kim et al., 2003a), and reversed-phase highperformance liquid chromatography, RP-HPLC (Stah et al., 1994; Thurl et al., 1991). The various interaction modes are described in the next section. Sanitation and hygienic design are key characteristics of food, beverage and nutraceutical processing. Major chromatographic companies that operate in the field are amongst others Universal Oil Products (UOP) and Novasep, and they can readily assist in designing large-scale chromatographic separations.


Advances in process chromatography and applications 113 4.1.2 Main chapter themes In the remaining part of this chapter first the basic principles of chromatography are described and the various types and basic modeling outlined. Then some applications are presented to indicate how chromatography is currently employed and its future potential in the food field. Subsequently, a section on recent developments in the chromatographic field is given with implications and applications for use in the food industry. This section is followed by an overview of future trends in the food chromatography field and finally some conclusions and suggestions for further reading are given.

4.2 Basic principles of process chromatography 4.2.1 Types of chromatography used The different modes or types of chromatography that can be used are categorized and depicted in Fig. 4.2 (see also GE website for further information; GE Healthcare is one of the major suppliers of chromatography equipment and resins: http:\\www.gehealthcare.com). The various methods are ranked according to their interaction strength, with the weakest on top. Size-exclusion chromatography is based on differences in the size of the solute molecules, by using a porous resin with distinct pore sizes (Horneman et al., 2004, 2006). Hydrophobic interaction chromatography and reversed-phase chromatography involve separation based on the differences in (surface) hydrophobicity of the solutes. The most powerful and most often used in food processing is ion-exchange chromatography, which separates solutes based on the differences in charge, which can be modulated by the pH and ionic strength of the solution. The strongest and most selective method is Sorption mechanisms

Weak

∑ Size

∑ Gel filtration (GF)

∑ Van der Waals

∑ Hydrophobic interaction (HIC) and reversed phase (RPC)

∑ Polar

d+

d–

d+ d+ +

∑ Ionic Strong ∑ Affinity

+

+

∑ Ion exchange (IEC)

+ ∑ Affinity chromatography (AC)

Fig. 4.2 Illustration of the basic interaction modes of chromatographic separation.


114 Separation, extraction and concentration processes affinity chromatography, and it is most often used in the pharmaceutical field, because of its selectivity and because its high cost can be met here. The purpose of process chromatography is to separate a target component from a mixture. Liquid chromatographic separations are based upon the different degrees of interaction of solutes dissolved in a mobile phase with a chromatographic medium or resin (the stationary phase), resulting in differential migration of the solutes through the column as illustrated in Fig. 4.3. The feed to the chromatography column is a binary mixture of solutes A and B, moving with concentration (wave) velocities wA and wB, respectively, through the column, with wB > wA. That is, solute B is more strongly retained or retarded by the resin than solute A. Each solute band becomes more dispersed as the chromatographic process progresses (depicted with fainter shading in Fig. 4.3b). Therefore, a chromatographic purification that is mainly based on adsorption and desorption of the target compound consists of several steps as follows. Equilibration of the resin For equilibration of the resin, the adsorbent material is prepared for the chromatographic separation. Depending on the mechanism of adsorption the equilibration step may include equilibration with the desired ionic strength, pH, and solvent concentration. Usually the equilibration solvent is chosen such that upon adsorption of the target compound and its impurities no change in composition (pH, ionic strength, and solvent composition) of the mobile phase occurs. In some instances it may be necessary to change the composition of the mobile phase to enhance the adsorption process. Loading In the loading process step, the target compound (and some impurities) is adsorbed onto the media. The capacity of the resin and/or the difference in affinity between target compound and its impurities determine the loading volume and loading conditions. It may be necessary to change or alter the feed stream properties to optimize the adsorption process. Washing In the wash phase, a washing buffer or solvent is used to desorb impurities from the resin. The washing buffer may be similar to the equilibration buffer, but this is not necessarily the case. Elution of the target compound needs to be avoided in this step. Elution In the elution phase the target compound is desorbed into an elution buffer solution. This can be done both in isocratic mode (constant mobile phase composition) and in gradient mode in which a gradient in the eluting buffer is applied to achieve optimal purity and/or minimal elution volume.


Advances in process chromatography and applications 115 Feed Mobile liquid phase

WB

Mobile phase

WA

WB

Mobile phase

WA

WA

WB

More strongly retained Less strongly retained

Stationary solid phase

t = 0 min

t = 10 min

t = 20 min Concentration

tR,B-tR,A

A

(a)

B A

B A

tR,A

tR,B

wA

wB

Time

Regenerant (eluent)

Feed B A

B

Flush

A

A B

B

B

A

A

B

A

A

A

A

Time

(b)

Fig. 4.3 (a) Illustration of the basic principle of chromatographic separation. Chromatogram showing the exit concentrations of A and B as a function of time (arbitrary times are used to show the progress of the chromatographic separation). (b) Adsorption, flush and regeneration.

Sanitization After elution a sanitization step may be applied in which compounds that show a higher affinity to the resin than the target compound are desorbed. For polymeric media, often a solvent is used to remove very apolar compounds from the resin. For hydrophilic media, a caustic solution is often used to © Woodhead Publishing Limited, 2010

116 Separation, extraction and concentration processes hydrolyze any irreversibly bound proteins. Acidic washes are applied to remove salts with low solubility (often calcium salts). Varying the salt concentration, pH or solvent composition can also be used to deliberately shrink and swell the resin which may enhance sanitization. After sanitization, the process starts again from equilibration. For processes in which there is very little affinity difference between the target compound and a limited number of impurities, it is often decided to use a continuous mobile phase with constant composition. The compounds are collected sequentially at the exit of the column and no intermediate equilibration, washing and sanitization is applied. A typical example of such a process is the separation of glucose and fructose on strong cationexchange resins in the Ca form. The requirements for sorbents to be used in chromatography and the different basic types are outlined in Fig. 4.4. 4.2.2 Modes of operation There are three different modes of operating a chromatographic process: frontal, elution and displacement. These stem from the three different ways of desorbing bound solutes from the chromatographic medium. Requirements ∑ Insoluble

∑ Hydrophilic Compromise

∑ Macroporous ∑ Mechanically stable

∑ Proper shape ∑ (Bio-)chemical stability

Main types

Composite

Organic ‘polymers’

Inorganic ‘pellets’

(a) ∑ Inorganic

∑ Organic polymers

Activated carbon

Polystyrene

Silica

Poly(meth)acryl

Alumina

Polysaccharides (dextran, agarose, etc.)

Pellet

Dense gel

Apolar

Macroreticular

Polar

Open gel

(b)

Fig. 4.4 (a) Requirements for sorbents and the main types available. (b) Examples of inorganic and organic polymers used as sorbents.


Advances in process chromatography and applications 117 In frontal chromatography, the sample is also used for elution. In this case, the sample feed is continuously applied to the column and the sample components displace each other in order of decreasing affinity for the chromatographic medium. The least retained solute is obtained in a pure form (i.e. depleted of the more strongly retained components) until the other solutes break through. Eventually, the column is saturated with the strongest retained component and the effluent has the same composition as the feed. In elution chromatography, the solutes are desorbed from the chromatographic medium by the action of a competing or modifying agent in the mobile phase, such as a salt. The concentration of the modifying agent is a key parameter. Three situations can be distinguished: isocratic, gradient or step change in the mobile phase composition. By keeping the composition (e.g. ionic strength) of the eluting buffer constant, the capacity factor is also kept constant; this is called isocratic elution and it is primarily used for separation of small molecules for which the variation of retention factor with mobile phase composition is not as large as for larger molecules. In gradient elution, the composition of the mobile phase is continuously changed (e.g. increasing ionic strength as in ion exchange chromatography), resulting in a continuous change in the retention factor. Instead of using a continuous gradient, the mobile phase composition may be changed in a stepwise fashion, resulting in discontinuous changes or ‘shifts’ in the retention factor. Gradient or step elution may be necessary for separation of large biomolecules for which the binding strengths may be too strong for isocratic elution to be feasible. In displacement chromatography, the affinity of the competing agent for the chromatography media is much higher than that of the solute, such that the agent effectively displaces the solute. This takes place irrespective of the concentration of the displacing agent (or displacer), in contrast to elution chromatography. Displacement chromatography has the advantage of being able to concentrate samples. However, selecting an appropriate displacer may not be easy. Several important aspects of using chromatography in food separations are outlined in Fig. 4.5. 4.2.3 Basic modeling The use of mathematical modeling is helpful in the design of chromatographic separators to size the chromatographic piece of equipment and to determine its optimal operation conditions. There is a wealth of information available on modeling of chromatography in different levels of detail. Guiochon gives a good basic overview (see section 4.8), and some recent modeling efforts using the general rate model and the linear driving force model for mass transfer are given by Nilsson (2005) and Horneman et al. (2006), respectively. Although the general rate model is the more accurate one, a less computationally involved but still accurate one is the linear driving force


118 Separation, extraction and concentration processes Eluent recycle ∑ Operating conditions and short-cut design Raffinate product Feed

∑ Complex phase behavior (azeotropic separations) ∑ Non-equilibrium fractionation ∑ Reactive separations

Extract product

Eluent make-up

∑ Multiple side/product streams ∑ Column dynamics

Sorbent recycle

Fig. 4.5 Schematic diagram of the operation of a large-scale chromatographic fractionator, such as a simulated moving bed, and the main factors for successful operation.

model, as shown in Fig. 4.6. It contains two partial differential equations describing the concentration of solutes in the bulk liquid and solid phase as function of time and axial position in the column. The equations are connected by a mass transfer term containing the isotherm, which gives the ratio of partitioning over the solid and liquid phase of a solute under certain environmental conditions. Several isotherms can be used, the most common being the Langmuir one, which gives a linear relation between the concentration in the solid and liquid phase at low concentrations and levels off at a constant saturated value at higher concentration at the maximum loading capacity of the specific resin used. Any appropriate PDE solver can be used (e.g. PDESol, Comsol) to solve the set of obtained partial differential equations. Commercial packages that are available on the market are for example ASPEN Chrom, gPROMS, and for flowsheeting SuperProDesign (Intelligen).

4.3 Applications of process chromatography in the food, beverage and nutraceutical industries Process scale chromatography has been effectively used in the food, beverage and nutraceutical industry (Fig. 4.7 and Table 4.1). Examples of these applications are presented here and by doing so a brief overview is provided of the industrial areas where process chromatography is applied. The various sources and products that can be concentrated, fractionated and purified by chromatography are given in Fig. 4.7 and Table 4.1, as well as scales of some chromatographic separations actually in industrial practice with its operation mode (SMB, and fixed bed chromatography, FB).


Advances in process chromatography and applications 119 4.3.1 Glucose–fructose separation The technology used previously (up to the middle 1970s) in the preparation of fructose was based on the application of a scarce and expensive raw material, inulin, which is a fructose polymer. This prevented the wide-scale use of fructose in the food and medical industry. Only in the 1980s did the application of chromatographic methods permit the large-scale manufacture of fructose by its isolation from glucose–fructose syrups obtained either as ∑ ∑ ∑ ∑

Which phases? Which convective streams? Is there dispersion? Mass transfer between phases?

Void fraction f

(Interstitial) velocity v

Feed flow Q MT

Cross-section A

Q = v fA

Mass transfer

Dispersion

(a) Mass balance over a slice Convection

(fA)vc

Dispersion

Ê ˆ (fA)Á – E ∂c ˜ Ë ∂z ¯

Mass transfer Accumulation

z + Dz

– [(1 – f)A] MTDz (fADz ) ∂c ∂t

Change = in – out ∂c = – ∂t Equation 1

z

Per volume solid

Dz (vc ) |z+ z

Dz

+E

Ê ∂c ˆ ÁË ∂z ˜¯

∂c = – v ∂c + E ∂2c – 1 – f MT ∂t ∂z f ∂z 2

z+Dz z

Dz

–

1–f MT f

E = 2vdp

(b)

Fig. 4.6 Mathematical modeling for chromatography (a). Different (partial differential) equations to be used (b) and (c), with ‘closing relations’ for mass transfer (d). Symbols: z axial length scale, D difference, c concentration, t time, k mass transfer coefficient, a interfacial area, K partition coefficient, Sh Sherwood number, Re Reynolds number, Sc Schmidt number, D diffusion coefficient, d particle diameter, n kinematic viscosity, v velocity, A cross- sectional area, Q flow rate, f void fraction, MT mass transfer, E dispersion coefficient. Subscripts: f film, p particle. Superscripts: * at equilibrium, vinculum solid phase.


120 Separation, extraction and concentration processes z + Dz

z

Mass balance over a slice of solid Mass transfer MTDz = ka (c* – c) Dz Overall coefficient Area per volume of solids Equilibrium concentration Accumulation

∂c Dz ∂t

Equation 2

∂c = ka (c* – c) = MT ∂t

Overall MT coefficient k–1 = (Kkf)–1 + k–1 p

Solve two coupled PDEs (c) dp = 10–3 m

In the ‘film’ Shf = 4 + 1.1Re0.6 Scf0.33 kf dp

Shf ∫ Re ∫

Df

; Scf ∫

nf ; Df

vdp nf

Df = 10–9 m2 s–1 v = 10–3 m s–1 Re = 0.5 Scf = 1000 Shf = 10 kf = 10

Df dp

= 10–5 m s–1

In the ‘particle’ Dp = 10–10 m2 s–1

Shp = 10 Shp ∫

k pd p Dp

; Dp π Df

kp = 10

Dp dp

= 10–6 m s–1

(d)

Fig. 4.6 Continued

a result of the inversion of sucrose or by converting glucose with the aid of glucose isomerase. The most common type of fructose syrup, usually called high-fructose syrup, contains 42% fructose, 52% glucose, and 6% oligosaccharides on a dry basis, and is used in place of sucrose in some foods and beverages (Kishihara et al., 1992). For some purposes, because fructose is sweeter and more soluble in water at low temperatures than glucose, syrup with 55 to 90% fructose, called higher-fructose syrup, is desirable. Therefore, to produce syrup containing more than 50% fructose a process to separate fructose from the equilibrium mixture is desirable. This mixture can be separated by chemical and physical means. The most widely used method for this kind of separation is chromatographic separation using cation exchange resins in Ca2+ form (Barker et al., 1984). In this instance, SMB is a widely used technology (see Section 4.4.3) (Bubnik et al., 2002). © Woodhead Publishing Limited, 2010


Dairy industry

Marine

Milk Eggs

Fish Algae Peptides Proteins

Omega-3, -6 fatty acids

Agriculture Sugars Flavors

Fig. 4.7 Source and product areas where process chromatography is being used or has potential. Table 4.1 Industrial scale chromatographic separations. Taken from Lecture notes from the Advanced Course on Downstream Processing, Delft, 2009, and Ganetsos and Barker (1992) (see Section 4.8) System

Process*

Scale (tonne a–1)

Xylene isomers Glucose–fructose Lactic acid, citric acid Amino acids Chiral compounds Peptides Milk proteins Flavors

SMB SMB FB/SMB FB/SMB SMB SMB FB FB

400 000 100 000 10 000 1500 1–10 0.1–1 0.1 0.01–01

*FB, fixed bed; SMB, simulated moving bed.

When large-scale separations are required, the SMB process is often the technology of choice in order to reduce separation costs. Compared with batch chromatography, SMB technology uses less eluent; in addition the separation productivity is higher thus using less adsorbent (Broughton, 1984; McCulloch et al., 1994). In recent years, fructose and glucose were separated from complex mixtures such as cashew apple juice using SMB technology (Azevedo and Rodrigues, 2005; Luz et al., 2008). 4.3.2 Recovery of sucrose from molasses Chromatographic separations are used to separate valuable components from beet molasses in beverage industries. Characteristic products that are © Woodhead Publishing Limited, 2010

122 Separation, extraction and concentration processes currently produced in addition to sugar include betaine, inositol, peptides, amino acid mixtures and several individual amino acids. The recovery of the other components can significantly improve the economy of sucrose recovery, but still sucrose remains as the most important product controlling the overall economy of the process. Chromatographic separation can also be applied to treat intermediate juices in the beet sugar industry. Thick juice separation has also been suggested. The resolution of the peaks may change owing to the changing composition of the feed material. Industrial batch systems were first built in the 1960s and the 1970s. In the mid-1980s the continuous simulated moving bed process was applied in molasses separation. The sequential SMB process, which can simultaneously recover multiple product fractions, was introduced in the late 1990s (Hyöky, 1999; Lancrenon, 1997; Paananen, 1996; Rousset, 1997). Hyöky et al. (1998) described the multi-profile FAST separation, which effectively doubled the efficiency. Further development includes a patented two-stage process (Hyöky et al., 1998). It is a combination of two chromatographic fractionators to improve the recovery and the purity of overlapping components, such as sucrose and betaine. It is uniquely suitable for recovering multiple value-added products from the process streams of the sugar industry (Paananen and Kuisma, 1999). Chromatographic systems are used in production of sweeteners based on beet, cane and starch as described by Paillat (1999). Sugar beet produces sucrose, betaine, raffinose, invert and many other water-soluble components. These components are extracted from the beets as juice. Part of these watersoluble components are removed or destroyed in the juice purification, but a large part of them will end up in the by-product: molasses. Molasses is the most common raw material in chromatographic desugarization plants. The main product is sucrose, but in addition betaine, inositol, amino acid mixtures and individual amino acids have been commercially separated from molasses (Hyöky et al., 1998; Paananen, 1996; Paananen and Kuisma, 1999). Low green or B molasses separation is already in industrial use. Kearney has suggested thick juice separation (Kearney, 1997; Kearney and Rearick, 1995). The positioning of the chromatographic step in the beet process has distinct effects on the recovery of sucrose and especially on the recovery of the other components. 4.3.3 Stabilization of beers Brewery production of some corns and hops for beer is a universal process in the beer industry. The quality of beer depends on its taste, clearness, color and foam retention. One of the key processes is the procedure of removing non-micro-organisms impurities. Fixed-bed chromatography with several resins like silica gel (SiO2nH2O) are in use to remove proteins that make beer muddy. Beers are known to contain a wide variety of phenolic compounds, most of which originate from the raw materials of brewing, i.e., barley and hops


Advances in process chromatography and applications 123 (Gardner and McGuinness, 1977). Of these phenolic compounds, the flavanoids are of particular interest to brewers, as they have long been thought to be precursors of non-biological haze in unstabilized beers (McMurrough et al., 1983). The flavanols have been classified into three groups on the basis of their chromatographic behavior. The first group, the simple flavanols, comprise flavanol monomers [e.g. (+)-catechin and (–)-epicatechin)], dimers (e.g. prodelphinidin B3 and procyanidin B3), and trimers. The second groups, the polymeric flavanols, are formed by oxidation and polymerization of simple flavanols. The complexed flavanols, the third group, result from the interaction of polyphenols with proteins to form complex structures (McMurrough, 1979). Stabilization of beer against haze formation may be achieved by decreasing the simple flavanol content, thereby limiting further flavanol polymerization and complexation. Increasingly, this stabilization is being achieved in breweries by treatment of beer with polyvinylpolypyrrolidone (PVPP) before packaging (McMurrough, 1993; McMurrough et al., 1992). Stabilization is achieved by sorption of phenolics on PVPP, which is subsequently removed by sheet filtration. This procedure can be carried out in conjunction with silica hydrogel treatment to remove haze-forming proteins (McMurrough and Madigan, 1996). These amorphous, non-additive stabilizers offer selective removal of haze forming proteins, improving colloidal stability of beer with no adverse effects on head or taste. Typically these silicas have been used with body feed diatomaceous earth filter aid (D.E.). However, by changing the conditions in the manufacturing process, it is possible to vary permeability (filterability) of silica hydrogel products. An innovation in the stabilization of beer is the ‘combined stabilization system’ (CSS) which is capable of a combined removal of turbidity forming protein and polyphenols in a single step (Janey and Katzke, 2002). As the economic alternative to PVPP and silica stabilization, the CSS is a compact rig mounted fully automated stabilization system, which can be integrated into any existing filter line. The adsorbent is permanently retained between an inlet and an outlet screen, making the dosing of precoat and feed suspension before each filtration unnecessary. A CSS adsorber has a particle size of 100– 300 mm. The solid phase adsorber resin is based on a high-grade, cross-linked, insoluble agarose (polysaccharide). Protein and polyphenols are adsorbed and then removed from the agarose adsorbent during the regeneration. Neither substance is dissolved in the beer nor is the beer quality affected in a negative way. The beer’s organoleptic properties, its foam stability, color and bitterness units remain unaltered with this kind of approach. 4.3.4 Separation of lysozyme from egg white Lysozyme has a number of possible applications; for example it can be used as an additive to baby milk or ophthalmic preparations; for treatment of ulcers, wounds and infections, as a potentiator of some antibiotics; as an


124 Separation, extraction and concentration processes antioxidant; and for its antimicrobial properties (Owen and Chase, 1997). The expanding potential for application of lysozyme in many fields of science and technology dictates the development of efficient and simple methods for lysozyme purification. Several chromatographic techniques that have been used for the separation of hen egg white lysozyme include ion exchange chromatography (Banka et al., 1993; Li et al., 1999; McCreath et al., 1997), affinity chromatography (Chiang et al., 1993; Yamada et al., 1985; Yamasaki and Eto, 1981), dye-binding chromatography (Tejeda-Mansir et al., 2003), affinity membrane separation (Arica and Bayramoglu, 2005; Bayramoglu et al., 2003; Ruckenstein and Zeng, 1997), ultrafiltration (Ghosh and Cui, 2000; Ghosh et al., 2000), PEG/salt aqueous two-phase system (Su and Chiang, 2006), reverse micelles (Noh and Imm, 2005), metal-affinity precipitation (Roy et al., 2003) or adsorption to plant residues (Hou and Lin, 1997). Commercial cation-exchange resins such as Duolite C-464, Amberlite, CM Sephadex have been used for the separation of lysozyme on process scales with recoveries of 90–95% (Li-Chan et al., 2006). Nonetheless, the lengthy steps and dilution of product at the end of the process have hindered their application at process scales. More recently this problem has been overcome by the use of magnetic separation techniques for the separation of egg white lysozyme and several other proteins (Safarik and Safarikova, 1993; 2004). These techniques usually enable simple, onestep separation of target proteins, and in most instances magnetic affinity adsorbents can be used. However, large-scale application may be difficult to achieve. 4.3.5 Separation of whey protein from milk Milk proteins are the most important source of bioactive peptides. Typical bovine milk contains 13% solids, with 4% fat present as an emulsion of globules with diameters up to 10 mm and caseins present as a colloidal suspension of particles with diameters up to 0.1 mm (Bylund, 2003). Fat globules normally cause problems for chromatographic separations, as they block packed columns as soon as the feed is introduced. Raw whole milk contains larger suspended particles than whole (full fat) processed milk, as the latter is homogenized to produce a uniform consistency. The composition of bovine milk includes water, fat, lactose and minerals, and up to 6% of the mass is made up by proteins and peptides, among them a number of high-value substances (Lourdes et al., 2000). In particular, milk contains two major protein groups, caseins and whey proteins, which differ greatly with regard to their physicochemical and biological properties. Normal milk contains 30–35 g L–1 proteins, approximately 80% of which are caseins with the remainder being the whey proteins (Korhonen et al., 1998). Whey proteins can be acquired as a by-product in cheese manufacturing process. The required long-term stability in functional performance of these proteins is often lacking. In general, whey is dilute liquid composed of lactose, a variety


Advances in process chromatography and applications 125 of proteins, minerals, vitamins and fat. Whey contains about 6% solids of which 70% or more is lactose and about 0.7% is proteins (Gerberding and Byers, 1998). Whey protein components are a-lactalbumin, b-lactoglobulin, immunoglobulins A, M and G, bovine serum albumin (BSA), lactoferrin and lactoperoxidase. b-Lactoglobulin is the major whey protein in bovine milk; it has a molecular weight of 18.4 kDa, possesses 162 amino acid residues and its concentration is 2–4 g L–1. a-Lactalbumin is an albumin which has 123 amino acid residues. It possesses a molecular weight of 14.2 kDa and its concentration in milk is 0.6–1.7 g L–1 (Ye et al., 2000). Bovine whey proteins have potential applications in veterinary medicine, food industry and as supplements for cell culture media. Immunoglobulin G (IgG) and immunoglobulin A (IgA), present in bovine whey, have high pharmaceutical value (Hahn et al., 1998). a-Lactalbumin can be used in infant formula and as a nutraceutical because of its high tryptophan content. b-Lactoglobulin is used in the production of confections (Zydney, 1998). Oral administration of bovine IgG is known to be an effective treatment of various infections of newborn infants (Hutchens et al., 1990). Typical concentrations, molecular weights and isoelectric points of whey proteins are given in Table 4.2 (Al-Mashikhi et al., 1988). Several techniques are used for the partitioning of whey proteins by e.g. aqueous two-phase systems (Jose et al., 2000; da Silva and Meirelles, 2000; Rito-Palomares and Miguel, 1998). Also, some attempts have been made to isolate whey proteins by using membrane filtration (Lucas et al., 1998; Gerd et al., 2000). The separation of whey proteins by using ion exchange chromatography on a process scale has been investigated by many researchers and several methods have been reported (Gerberding and Byers, 1998); Ionexchange separations take advantage of electrostatic interaction between surface charges on biomolecules, such as amino acids or proteins, and clusters of charged groups on the resin phase. An adsorbing biomolecule displaces counterions associated with the surface, discharging a complementary buffer salt in the process. Adequate buffering is required to shield native protein structures from changes in pH adjacent to exchange surfaces (Donnan effect) and pH effects were induced by sorption. Selection of an appropriate buffer Table 4.2 Typical concentration of whey proteins and their isoelectric points (pI) Protein

Approximate MW (kD) concentration (w/w%)

Isoelectric point (–)

b-Lactoglobulin a-Lactalbumin Immunoglobulins (A, M, G) BSA Protease-peptones Lactoferrin (LF) Lactoperoxidase (LP)

0.3 0.07 0.06 0.03 0.14 0.003 0.002

5.35–5.49 4.2–4.5 5.5–8.3 5.13 3.3–3.7 7.8–8.0 9.2–9.9

18.4 14.2 150–900 69 37–55 78 78


126 Separation, extraction and concentration processes is critical to the success of ion-exchange separations (Keith and Edwin, 1995). 4.3.6 Separation of lactoferrin and lactoperoxidase In addition to the caseins and whey proteins, which are generally isolated from milk, some specialty proteins are also identified as byproducts in these separations. Two very important such proteins of commercial interest, which are part of the whey protein fraction, are lactoferrin (LF) and lactoperoxidase (LP). Although production of such high-value whey proteins is a commercial reality, two aspects of dairy processing may not be optimal for their production. First, the proteins are subjected to a series of processing steps before being extracted. It is a generally accepted principle of bioseparation process design that proteins should be separated from a source material as fast and in as few steps as possible to avoid loss of activity and yield (Ladisch, 2001; Harrison et al., 2003; Ottens et al., 2006a). Lactoferrin (LF) and lactoperoxidase (LP) being basic proteins are usually captured from whey or skim milk by cationexchange chromatography and sold as specialty ingredients (Lonnerdal and Carlsson, 1977). The costs associated with the separation of these types of proteins is high, but the value and wide range applications in various functional food and nutraceutical products outweigh the cost. Lactoferrin, a 70–80 kDa single subunit glycoprotein, consist of two domains, each with one iron-binding site that requires synergistic binding of a bicarbonate anion (Al-Mashikhi and Li-Chan, 1988). A conformational change in the binding site, between open or closed forms, accompanies iron binding and release. The affinity of lactoferrin for iron is much greater than of transferrin which functions as an iron carrier in serum. It is present in human milk, and many exocrine fluids (saliva, mucous secretion) as well as in mammalian. Lactoferrin is a complex molecule with a number of interesting functional properties. Based upon these, lactoferrin may enter the composition in a wide range of products. Lactoperoxidase belongs to the category of peroxidase and acts as a biopreservative. Both these products find several applications based on their antimicrobial and antiviral activities such as in baby foods, food supplements, cosmetics, oral care, and preservation of meat. The production of high-value dairy proteins such as lactoferrin and lactoperoxidase normally requires extensive pre-treatments of milk to remove fat and caseins by centrifugation, precipitation, Ca2+ chelation and/ or filtration. Similarly, fat and caseins are normally removed before capture of recombinant proteins from the milk of transgenic animals (Morison and Joyce, 2005). Such pre-treatments can result in significant loss of protein yield and/or activity. Currently, high-value dairy proteins are viewed as by-products, with the major income of the industry coming from commodity dairy foods such as milk powder, cheese and butter. Economies of scale for production of commodity


Advances in process chromatography and applications 127 dairy products mean that centralized processing is the industry norm. Milk is typically cooled and held at 4 °C in vessels on the farm for up to 2 days before being transported to a dairy factory. There, it is pumped to holding tanks and then undergoes a series of unit operations such as cream (fat) separation, pasteurization, homogenization and blending for standardization before further processing into individual products (Bylund, 2003). After a number of such operations (which varies from factory to factory), LF and LP may be extracted from skim (low fat) milk or, more commonly, from whey, which is produced as permeate during membrane concentration of milk or after precipitation of caseins much further down the processing chain as a by-product of casein production or cheese making (Tomita et al., 2002). Extensive pre-treatments of milk and whey before ion-exchange capture of proteins are by no means restricted to industry but are also used in the laboratory. Many authors have examined the capture and analysis of whey proteins by chromatography (Al-Mashikhi and Li-Chan, 1988; Al-Mashikhi and Nakai, 1987; Andrews et al., 1985; Chaplin, 1986; de Frutos et al., 1992; Donnelly, 1991; Doultani et al., 2004; Elgar et al., 2000; Etzel et al., 2000; Felipe and Law, 1997; Francis and Regester, 1995; Geberding and Byers, 1998; Hahn et al., 1998; Humphrey and Newsome, 1984; Konecny et al., 1994; Lonnerdal and Carlsson, 1997; Morr and Ha, 1993; Noppe et al., 1999; Torre and Cohen, 1996; Visser et al., 1991; Xu et al., 2000; Ye et al., 2000; Yoshida, 1988). Hahn et al. (1998) examined the performance of a range of commercially available pharmaceutical grade cation exchangers for protein capture from acid whey. Doultani et al. (2004) used cationexchange chromatography to produce a number of protein products from mozzarella cheese whey. Ye et al. (2000) used both anion and cation exchange chromatography to isolate a-lactalbumin, b-lactoglobulin, lactoferrin and lactoperoxidase from rennet whey. Cation exchange membrane chromatographic systems have been successfully used for the separation of lactoferrin and lactoperoxidase on an industrial scale from sweet whey concentrate with yields of more than 90% for lactoferrin. It has also been demonstrated that these system can be scaled up to handle 1 ¥ 105 tonne/year of whey (Plate et al., 2006). 4.3.7 Separation of napin from rapeseed meal Preparative chromatography of napin and other proteins from rapeseeds was successfully carried out with a combination of ion exchange followed by hydrophobic interaction and size-exclusion chromatography. The yield of this combined process was 18% for napin and 40% for cruciferin (Berot et al., 2005). Rapeseed proteins were underexploited as it was being used only as animal feed until its non-food applications were demonstrated in a European project (Green chemicals and biopolymers from rapeseed meal with enhanced end-user performance. European contract Enhance QLK5 CT 199901442). Hence large quantities were needed to demonstrate its potential © Woodhead Publishing Limited, 2010

128 Separation, extraction and concentration processes in functional additives. Preparative purification processes of seed storage proteins were demonstrated for 11S and 7S globulin-type proteins in pea and soy (Crevieu et al., 1996; Gueguen et al., 1984; Howard et al., 1983; Lehnhardt et al., 1983; Larre and Gueguen, 1986; Nielsen, 1985; Thanh and Shibasaki, 1976). But with rapeseed storage proteins, which are composed of 11S globulin and 2S albumin-type proteins, only analytical purifications were developed, except procedures based on ammonium sulfate selective precipitation of proteins (Raab and Schwenke, 1984). SEC was successfully used to achieve the separation of 11S and 2S proteins (Dalgalarrondo et al., 1986), and cation-exchange chromatography (CEC) to separate 2S isoforms (Monsalve and Rodriguez, 1990). More recently a new procedure for fractional enrichment and purification of plant (e.g. soy beans) and milk-based proteins was demonstrated by the use of volatile electrolytes for isoelectric precipitations (Hofland et al., 2003; Golubovic et al., 2005). Rapeseed protein meal has two major classes of seed storage proteins: 12S globulin (cruciferin), which represents 25–65% of its protein content (Raab et al., 1992) and 2S albumin (napin). In addition to these two proteins it also contains some minor proteins of interest, such as thionins, trypsin inhibitors and a lipid transfer protein (LTP). From the structure and physicochemical properties of these proteins it is evident that cruciferin shows very different characteristics from other proteins including: high molecular weight, neutral isoelectric point pI; but aggregation could occur during the purification process. On the contrary, napin and LTP show rather close characteristics in their molecular weights and pIs, which could complicate the purification process. Separation of napin and cruciferin can be obtained by a combination of nanofiltration in combination with several chromatographic steps of cationexchange, size-exclusion and hydrophobic interaction chromatography on process scales.

4.4 Recent developments in process chromatography Figure 4.8 shows some of the areas where there is potential for development in process scale chromatography. The innovations can be categorized in several fields, materials, modes of operation, and equipment. In the subsequent subsections some of the innovation in these areas will be highlighted. 4.4.1 Structured matrices and monoliths Monoliths for enhancing reactions have been around for several decades. For example, PolyHipe is a highly porous macrocellular cross-linked styrene–divinylbenzene (DVB) copolymer, prepared by polymerization of a high internal phase emulsion (HIPE) of water droplets dispersed in a styrene/DVB continuous phase (Ottens et al., 2000). This type of monolith © Woodhead Publishing Limited, 2010

Advances in process chromatography and applications 129 ∑ Materials – optimization of selectivity, MT, and hydrodynamics, magnetic beads, monoliths, structured matrices ∑ Mode of operation – exploiting non-linear effects of coupled multi-component equilibria ∑

Equipment – CPC: centrifugal partitioning chromatography – Radial flow: annular chromatography – SMB (counter-current chromatography) – EBA (expanded bed adsorption)

∑ Chromatographic reactors – coupled reaction – separation

I Feed A,B

Waste

II III IV

Solids B Raffinate A Extract

Desorbent

Fig. 4.8 Areas of innovation in chromatography and a schematic diagram of a simulated moving bed.

was used to enhance a multiphase reaction, but had structural properties that also made it suitable for high capacity, low-pressure drop chromatographic separations. Structured monoliths from Corning that have been used in chemical conversions in the work of Kreuzer et al. (2005) would also suit chromatographic separations purposes. Pictures of monoliths are given in Fig. 4.9. For more than a century, chromatography practitioners have been separating the components of chemical mixtures by using columns packed with various types of particulate matter. Monoliths are a relatively new class of stationary phases, completely different from conventional stationary phases. A good overview of this relatively new stationary chromatographic phase in the biotechnology area is given by Jungbauer and Hahn (2004). The material is cast into a chromatography column as a continuous block of one piece interlaced with channels (Jungbauer et al., 2002a). The ramified channels do not have dead ends. Owing to this structure, the transport of the solute to the surface is solely by convection instead of diffusion as observed in conventional media (Hahn and Jungbauer, 2000; Hahn et al., 2002;). These nontraditional column materials have recently been commercialized and are being recommended by manufacturers and users for the enhanced speed and thoroughness with which they can separate complex mixtures of biological molecules. The most important characteristics of monolithic media are the excellent mass transfer properties and the low pressure drops. The large channel diameter makes monoliths excellent stationary phase materials for chromatographic separations. The first monoliths were developed by Hjerten et al. (Hjerten and Liao, 1988; Hjerten et al., 1989, 1992) and Tennikova et al. (1990). Hjerten and coworkers compressed polyacrylamide gels and observed excellent resolution. Scale up was initially difficult but recently was successfully performed (Podgornik et al., 2000). Another important development is in the use of monoliths as a support for solid-phase synthesis (Jungbauer et al., 2002c). An interesting application is the direct synthesis of peptides on © Woodhead Publishing Limited, 2010


(a)

(b)

Fig. 4.9 Photographs of monoliths: (a) SEM photograph (15 kV; scale, 1 cm = 32.7 mm) of PolyHipe-type X20PV90 on an ISI-DS-130 SEM apparatus (Ottens et al., 2000); (b) Corning monoliths, as used in catalytic multiphase conversions by Kreutzer et al. (2005), with potential for use in large-scale food, beverage and nutraceutical purification.

monolithic columns. Because the synthesis is performed on polymethacrylate monoliths, the directly grown peptide can be used as an affinity ligand without any further treatment (Pflegerl et al., 2002a; 2002b; 2002c). This strategy provides an excellent screening platform for affinity ligands. The screening of the ligand can be performed by microtitration and the same resin can be used for screening and large-scale separation. Monoliths are also interesting supports for enzyme reactors. In packed-bed reactors, the efficiency is often limited by pore diffusion. The high porosity of monoliths ensures that the enzymatic process is not mass transfer limited (Jungbauer et al., 2002b). © Woodhead Publishing Limited, 2010

Advances in process chromatography and applications 131 A relatively new stationary phase is the combination of chromatographic resin beads and a membrane. Mixed matrix membranes (MMMs), which incorporate adsorptive particles during membrane casting, can be prepared simply and have performances that are competitive with other membrane chromatography materials. The application of MMM chromatography for fractionation of b-lactoglobulin from bovine whey was recently shown (Saufi and Fee, 2009). The dynamic binding capacity of b-lactoglobulin in whey solution was about 80 mg g–1 membrane (24 mg mL–1 of MMM), which is promising for whey fractionation using this technology. 4.4.2 Novel ligands Affinity separations are normally very expensive; therefore, application in the food industry requires either very high value products or a lower ligand cost. Affinity ligands are the basic moieties on which the various affinity chromatographic separations run. Affinity chromatography has been used widely in biomedical research and biotechnology (Wilchek and Chaiken, 2000). It is based on molecular recognition where one recognition partner is immobilized on a base matrix, and soluble target molecules can be retained from a crude mixture. The target molecule can then be released and recovered in a functional form. The basis of elution is to reduce the affinity between immobilized ligand and analyte. This is most often accomplished by use of a bond-breaking buffer (i.e., by changing pH, ionic strength, and solvent) or by use of competitive elution (Firer, 2001). In either instance, the diffusion of immobilized ligand is always strictly limited, and therefore, the local concentration of immobilized ligand is constant. The environment surrounding the immobilized ligand changes to modulate the ligand–analyte affinity. Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. Although affinity chromatography is used extensively on a laboratory scale, its widespread acceptance has been limited on the preparative scale because of the high cost of the affinity ligands and their biological and chemical instability (Lowe et al., 2001). Only recently, the development of new methods for screening, selection and design of stable synthetic ligands, has opened the opportunity of exploitation of such materials on a large-scale (Ladner and Ley, 2001). The rapid growth of bioinformatics and molecular docking techniques and the introduction of combinatorial methods for systematic generation and screening of large numbers of novel compounds, has made feasible the rapid and efficient generation of ligands for affinity chromatography (Clonis, 1990; Labrou, 2002; 2003; Labrou et al., 2004; Spalding, 1991). Immobilized lectin affinity chromatography (Endo, 1996; Kobata, 1994) using Ricinus communis agglutinin (RCA) has been used for separation of several glycopeptides and oligosaccharides (Merkle and Cummings, 1987;


132 Separation, extraction and concentration processes Shinohara et al., 1987). RCA binds specifically to nonreducing-end galactose residues (Wei and Koh, 1978). Competitive elution using lactose, a hapten sugar, is generally used to elute the target molecule from the RCA immobilized column. Separation of bioactive peptides from defatted sunflower meal using immobilized lung extracts or immobilized angiotensin converting enzyme (ACE) (Megıas et al., 2004; 2006a; 2006b; 2007a; 2007b) has been used. Peptides that have angiotensin converting enzyme (ACE)-inhibitory activity are of great interest because of their potential antihypertensive effect, and have been found in hydrolysates of plant and animal origin (Murray and FitzGerald, 2007; Ottens et al., 2006a; 2006b). These are a few examples of using novel ligands in the separation of several valuable products from food sources. Enrichment by affinity chromatography generally allows further purification using classical chromatographic techniques. 4.4.3 Mode of operation Counter-current chromatography: simulated moving bed In conventional chromatography, the resin is fixed in a column, and the liquid flows through the column. As in extraction and distillation processes, the counter-current movement of the two phases improves the efficiency of the separation process, by establishing a higher driving force for mass transfer. A typical moving bed system is shown in Fig. 4.10b. It is equivalent to a distillation column. The liquid flows from section I to section IV, the sorbent moves from section IV to section I. A feed mixture (F) is introduced in the middle of the system. It is fractionated in an extract stream (E) containing the more retained component(s), and a raffinate stream (R), containing the less retained component(s). Similar to distillation, the movement of the components depends on the flow rates of the liquid and solid phases and the equilibrium distribution coefficient over both phases. Commonly, a four section SMB is used, the middle sections II and III serving to achieve the actual separation and section I cleaning the sorbent for reuse while section IV cleans the liquid for (partial) reuse. A real continuous movement of the solid phase is difficult to achieve in chromatography without losing resolution, owing to attrition of the solid phase. In practice, the movement of the solid phase is ‘simulated’ by periodic switching of a carousel of fixed chromatographic columns (Fig. 4.10a). The efficient use of the resin and liquid (desorbent D) is an important contribution to the efficiency of the SMB compared with that of fixed bed (FB) chromatography. Normally a reduction by a factor of 2–10 in resin and buffer use per amount of product produced can be achieved in an SMB compared with a FB. Chiral separation was the main driving force for the establishment of SMB separations in the fine chemicals and pharmaceutical industry over the past two decades (e.g. Cavoy et al., 1997; Pais et al., 1997; Rodriques and Pais, 2004). It is now a well-known and regularly applied chiral binary separation


Advances in process chromatography and applications 133 Feed Liquid flow cycle

Liquid flow direction

Raffinate

Extract Liquid flow direction

Desorbent

(a) Sorbent recycle q4

q3

q2

IV c4

c3

II c2 c¢2

R

q0

q1

III

cF F

I c1

c0

D

E

N species: 11 N unknowns (c, q)0, …, 4 + c¢2 11 N independent equations (all linear)

4N concentration ratio 4N overall mass balances 3N balances: feed, desorbent, sorbent [A] x = b (b)

Fig. 4.10 (a) Schematic of the concept and operation of continuous multi-column simulated moving bed chromatography. Different tints indicate the extent of concentration of the two solutes. (b) A simple steady-state model for mathematical modeling of four-zone linear SMB chromatography: q solute solid-phase concentration, c solute liquid-phase concentration, R the raffinate stream, F the feed stream, E the extract stream, D the desorbent stream. The different zones are indicated by I–IV and 1–4.

technique. SMB is also a well-studied operation. However, some areas of application need to be developed. Separations of biologicals (Houwing, 2003; Houwing et al., 2002, 2003a; 2003b; Paredes et al., 2005) as well as multi-component (MC) separations in SMBs (e.g. Abel et al., 2004; Wang and Ching, 2005) are important research fields with a large industrial relevance. Multi-component (MC) separations can be performed in SMBs and some studies are devoted to it, mainly by serial SMB systems. Multiplezone (or section) SMBs have been reported (up to nine zones, Wooley © Woodhead Publishing Limited, 2010

134 Separation, extraction and concentration processes et al., 1998). Five-zone SMBs are being investigated and design criteria have been presented, for example, by Kim et al. (2003b). Wang and Ching (2005) presented a straightforward extension of a four-section SMB to a five-section SMB. An extra raffinate or extract port was added to the standard four-section SMB to perform a ternary separation. The pros and cons of two raffinate or two extract streams were discussed. However, in all these cases, a single component was purified from a three-component mixture. An equal or larger volume of industrial separations, particularly in the food industry, concern enrichment of a (MC) product in a particular functionality, for instance from a health or nutritional point of view. When using chromatography, the functionality can be associated to a particular MC fraction of an (also) MC feedstock. Developing and optimizing an efficient separation has to deal with the inherent MC nature of the feedstock and product(s). Also in chromatographic separations in food and biotechnology, consumption of costly resin and salty buffers is high, which are reasons to use SMB technology for these applications as well. As yet, few biotechnology applications of SMB technology have been described in open literature. The few examples found mainly concern small biomolecules, such as amino acids (e.g. Maki et al., 1987; Van Walsum and Thompson, 1997; Wu et al., 1998) disaccharides (Geisser et al., 2005) polypeptides (e.g. Mun et al., 2003) but also protein separations and purifications (e.g. Adachi, 1994; Gottschlich et al., 1996; Gottschlich and Kasche, 1997; Hashimoto et al., 1988; Horneman et al., 2006, 2007a; Houwing, 2003; Houwing et al., 2002, 2003a; Huang et al., 1988), plasmid purifications (Paredes et al., 2005) and viral clearance (Horneman et al., 2007b). In biotechnology, a whole range of chromatographic techniques is available for separation of proteins. SMB is applicable to any of these systems. Considering size exclusion; several forms have been investigated: separation of dilute mixtures (i.e., the linear isotherm case), surfactant-aided gel filtration (Horneman et al., 2004, 2006, 2007a; 2007b), separation of concentrated mixtures (the non-linear isotherm case) (Houwing, 2003), and separation of multicomponent mixtures (Ottens et al., 2006a). In the latter work, the technological and economic feasibility of SMB technology for multi-component product (and feedstock) separations is described, using the fractionation of peptides from protein hydrolysates as functional food ingredients as an example. The impact on the environment was also assessed. The model system consisted of an enzymatic hydrolysate of casein, which was fractionated using gel filtration (size-exclusion chromatography). The technical feasibility of isolating peptides, capable of inhibiting the ‘angiotensinconverting enzyme’ (ACE), from a casein hydrolysate by means of size-exclusion chromatography was investigated experimentally. In another study, the successful separation of the disaccharide lactose from a complex mixture of human milk oligosaccharides (HMOS) with the continuous chromatography of SMB technique is described (Geisser et al., 2005).


Advances in process chromatography and applications 135 Expanded bed adsorption A major concern when operating a chromatographic column is fouling and, clogging of the column causing high pressure drop and preferential flow, or channeling, and thereby loss of capacity. To overcome this problem a technique was developed over recent decades that overcomes this problem, by operating the adsorption and washing step in the fluidized mode. Liquid enters the column from below at sufficient velocity to drag the sedimented solid stationary material with it, thus creating a fluidized or expanded bed. This mode of operation allows processing of crude feed streams with a high load of particulates, and can be used as a combined solid–liquid separation step and an initial capture step (e.g. Hubbuch et al., 2006). A recent application of expanded bed adsorption (EBA) in the food industry is the recovery fractionation and purification of valuable proteins from potato juice (Løkra et al., 2009). Horneman et al. (2008) used EBA in SMB mode to optimally recover and purify potato proteins to obtain an economically viable process.

4.5 Process control in chromatography In order to have an optimal separation at high productivity, yield and purity, it is necessary to operate under the appropriate conditions. The conditions may change owing to fouling or loss of capacity, a fluctuating feed composition or the inherent unsteady state of the operational procedure (i.e. with SMB chromatography). To operate at optimal conditions, necessitates control of the operation of the industrial-scale chromatography column(s). For SMB a safe regime is usually 10% below the optimal boundary. Proper control strategies may allow higher productivity, while maintaining the production constraints of purity and yield (Grossmann et al., 2008). With the availability of massive, cheap computer power and detailed mathematical models (as outlined in section 4.2.3), process scale chromatography will benefit more and more in the near future from proper control of the operation of the unit.

4.6 Future trends 4.6.1 High-value nutraceuticals Functional foods and nutraceuticals provide an opportunity to improve health while reducing health care costs. Functional foods involve the broad class of prebiotics and probiotics. Probiotics are viable microbial dietary supplements that influence the host and have a beneficial effect in the gastro-intestinal tract (Jardine, 2009; Salminen et al., 1998), whereas prebiotics are nondigestible food ingredients that benefit the host organism by stimulating the growth or activity of one or limited number of bacteria in the colon (Gibson and Roberfroid, 1995; Jardine, 2009). © Woodhead Publishing Limited, 2010

136 Separation, extraction and concentration processes Nutraceuticals refers to extracts of foods claimed to have a medicinal effect on human health. An important trend in food technology is the production of these ingredients from sources such as aromatic plants or spices. Among the nutraceuticals, antioxidants receive much attention in the food industry (Madhavi et al., 1996), not only as preservatives in food products to prevent or retard oxidation of fats and oils, but also because of their beneficial effects on human health. Supercritical-fluid chromatography (SFC) is one of the widely used process options in nutraceutical separations. Preparative-scale SFC is an environmentally clean technology whose main advantage, compared with preparative LC, is the easy recovery of the isolated compounds by a simple decompression of the supercritical fluid (Coleman et al., 1999). Fish oil preparation and separation of polyunsaturated fatty acid esters such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) (Alkio et al., 2000) is a well-documented example of preparative SFC and has been used in nutraceutical production since the early 1990s. Novel methods such as continuous membrane chromatography reactor system (CMCRS) are used for the synthesis and separation of galacto oligosaccharides (GOS) (Engela et al., 2008). Membrane separations using ion-exchange chromatography is another widely used technique for functional components from milk sources (Goodall et al., 2008). 4.6.2 Good manufacturing practice (GMP), quality assurance (QA) and quality control (QC) and Food and Drug Administration (FDA) regulation Good manufacturing practice (GMP) is a term that is recognized worldwide for the control and management of manufacturing and quality control of foods, pharmaceutical products, and medical devices. GMPs are guidelines that outline the aspects of production that affect the quality of a product. The guiding principle of GMP is that quality is built into a product, and not just tested into a product. Therefore, the assurance is that the product not only meets the final specifications, but that it has been made by the same procedures, called standard operating procedures (SOP), under the same conditions each and every time it is made. In the food industry GMPs address all factors involved in a manufacturing process: personnel, building, premises, machinery and apparatus, documentation, and quality control, that generally affect product quality or impact quality monitoring. Besides these factors, an important theme that is part of GMP is validation. It is that part of GMP that ensures that facility systems, equipment, processes, and test procedures are under control and therefore consistently produce quality product. This is a special quality assurance method, which is explicitly required by GMP codes and mandated by law. GMPs are enforced in the United States by the FDA (http://www.fda.gov); within the European Union, GMP inspections are performed by National


Advances in process chromatography and applications 137 Regulatory Agencies, e.g. GMP inspections are performed in the United Kingdom by the Medicines and Healthcare products Regulatory Agency (MHRA)(http://www.mhra.gov.uk), in the Republic of Korea (South Korea) by the Korea Food and Drug Administration (KFDA), and by similar national organizations worldwide. 4.6.3 Process analytical techniques (PAT) and control Process analytical techniques (PAT) are now being implemented in the pharmaceutical industry to monitor and control proper operation of these processes. Also in chromatographic fractionating and purification in the food industry such an application is seen. It allows for GMP, QC, better operation, better control, higher productivity and on spec production/higher number of on spec batches.

4.7 Conclusions Process chromatography is being applied in food, beverage and nutraceutical processing. Chromatography has a high resolving power which can give highpurity food products. With the advent of the nutraceutical field, also more expensive forms of chromatography, based on affinity, may find application, as the potential higher sales revenues balance the higher operating costs. Chromatography is a flexible purification method that is likely to play a role in food, beverage and nutraceutical processing for years to come.

4.8 Sources of further information and advice ∑

Ganetsos, G., Barker, P.E. Preparative and production scale chromatography. Marcel Dekker, Inc., 1992. ∑ Guiochon, G., Shirazi, S.G., Katti, A.M. Fundamentals of preparative and nonlinear chromatography. Academic Press, Inc., 2nd Edition, 1994. ∑ Sofer, G., Hagel, L. Handbook of process chromatography – a guide to optimization, scale-up and validation; Academic Press, London, 3rd Edition, 2001.

4.9 List of abbreviations ACE CEC CMCRS

angiotensin converting enzyme cation-exchange chromatography continuous-membrane chromatography reactor © Woodhead Publishing Limited, 2010

138 Separation, extraction and concentration processes CSS DHA DVB EBA EPA FB FDA GMP GOS HIPE HMOS IEC IgG LEC LF LP LTP MC MHRA MMM MT PAT PDE QA QC RCA RP-HPLC SEC SFC SMB SOP

combined stabilization system docosahexaenoic acid divinylbenzene expanded bed adsorption eicosapentaenoic acid fixed bed Food and Drug Administration good manufacturing practice galacto oligosaccharides high internal phase emulsion human milk oligosaccharides ion-exchange chromatography immunoglobulin G ligand-exchange chromatography lactoferrin lactoperoxidase lipid transfer protein multi-component Medicines and Healthcare products Regulatory Agency mixed matrix membranes mass transfer process analytical techniques partial differential equation quality assurance quality control ricinus communis agglutin reversed-phase high-performance liquid chromatography size-exclusion chromatography supercritical-fluid chromatography simulated moving bed standard operating procedure

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5 Novel adsorbents and approaches for nutraceutical separation B. W. Woonton, CSIRO Food and Nutritional Sciences, Australia and G. W. Smithers, Food Industry Consultant, Australia

Abstract: In this chapter, an overview is provided of novel chromatographic adsorbents that are perhaps on or just over the horizon but hold promise as the basis for future industrial technologies in the cost-effective extraction of nutraceuticals from agri-food streams. Such adsorbents include molecular imprinted polymers, organic monoliths, stimuli-responsive resins, mesoporous molecular sieves, peptide affinity ligands, and membrane adsorbers. These adsorbents have the potential to improve specificity, selectivity, simplicity, robustness, and productivity, and to reduce the environmental impact of nutraceutical manufacture. Key words: nutraceuticals, separation, molecular imprinted polymers, monoliths, stimuli-responsive resins, molecular sieves, peptide affinity ligands, membrane adsorbers.

5.1 Introduction Nutraceuticals have entered the mainstream of foods and beverages. By definition, these components derived from agricultural and food raw materials have a positive influence on human health through a specific prophylactic or therapeutic physiological effect against chronic disease when consumed (Hardy, 2000). Nutraceuticals have usually been delivered to the consumer in a medicinal vehicle such as a tablet, capsule or in powdered form. More recently, the functional foods market, driven by consumer demands for healthpromoting foods and drinks, has seen nutraceuticals enter the food industry by way of the active component(s) in ‘functional’ varieties of everyday food and beverage products like yoghurt, bread, margarine, juice, and even water. This market is becoming a lucrative outlet for new and novel nutraceuticals, and © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 149 food companies are keen to capture a slice of this market. These companies are thus exploring new sources of nutraceuticals, and the means to isolate, characterize and substantiate the bioactivity of such components to support the development of successful functional foods and beverages. In an earlier chapter (Chapter 4), more traditional approaches (e.g. fixed and moving bed chromatography) to the extraction, separation and concentration of nutraceuticals have been addressed. Chromatography is a powerful technique for the cost-effective separation and isolation of nutraceuticals from agrifood streams, including food processing co-product streams. Developments in continuous chromatography have enabled the commercial separation and isolation of biologically active proteins from milk and whey. For example, continuous separation (CSEP) technology is being used to isolate valuable dairy-derived molecules, including nutraceuticals (De Silva et al., 2003). Central to the chromatographic process is the relevant adsorbent that is responsible for effecting the separation. In this chapter, we introduce novel chromatographic adsorbents and approaches for the isolation of nutraceuticals, those that are perhaps on/over the horizon but hold promise as future industrial technologies for the cost-effective isolation of nutraceuticals, and the potential to deliver on consumer demands for effective functional foods and beverages. This overview is not intended to be exhaustive or inclusive of all novel adsorbents and technologies, but rather to provide a summary of the most promising adsorbents and approaches, that with further research and development can be employed for large-scale nutraceutical manufacture. The chapter provides an account of developments in: (i) (ii) (iii) (iv) (v) (vi)

molecular imprinted polymers; organic monoliths; stimuli-responsive resins; mesoporous molecular sieves; peptide affinity ligands; and membrane adsorbers.

5.2 Molecular imprinted polymers and applications in the nutraceutical industry Molecular recognition is an important process in many biological systems, for example the specific recognition of an antigen by an antibody. Such recognition mechanisms have been utilized in the development of biological affinity-based separation media (e.g. mono- or poly-clonal antibodies and affinity peptide resins) (Tharakan et al., 1992). Molecular imprinted polymers (MIPs) differ from these biological affinity systems in that they are synthetic organic materials designed with cavities specific to a template molecule employed during synthesis. In effect, they are tailored synthetic affinity materials that can be used in a range of applications including the separation © Woodhead Publishing Limited, 2010

150 Separation, extraction and concentration processes and analysis of molecules, the sensing of molecules, as artificial enzymes, and as antibody mimetics. MIPs are worthy of discussion as they have the potential to reduce the complexity of nutraceutical separation processes, thus reducing processing costs and the environmental impact associated with such processes. The concept of molecular imprinting was first reported by Kikuchi et al. (1972) and Wulff & Sarhan (1972), and since these early reports, there has been a significant amount of research undertaken to improve MIPs using alternative polymer chemistries and synthesis methodologies. There are a large number of publications reporting the development and application of MIPs for a range of target molecules including small organic molecules such as pharmaceuticals, pesticides, amino acids, peptides, high molecular weight proteins, polypeptides, steroids, sugars and cells (Andersson et al., 1995; Bolisay et al., 2006; Bossi et al., 2007; Nicholls et al., 1995; Rachkov & Minoura, 2001; Sellergren, 1994; Shi et al., 1999; Steinke et al., 1995). 5.2.1 Synthesis The synthesis of MIPs involves the organization of a cross-linked polymer matrix around a template molecule, followed by removal of the template, leaving recognition sites complementary to the template or similar molecules (Fig. 5.1). There are three main steps involved in the synthesis of a MIP, and these include: (i) pre-arrangement of selected monomers (such as vinyl or acrylic) around the target molecule; (ii) polymerization of the monomers (e.g. radical polymerization) with the addition of a cross-linker; and (iii) extraction of the target molecule leaving binding sites that recognize the target molecule (Gupta & Kumar, 2008). Two different approaches may be employed to manufacture MIPs, these are covalent and non-covalent imprinting. Non-covalent imprinting is the most common and involves the interaction of the template molecule with the monomers via hydrogen bonding, ionic bonding, hydrophobic interactions and van der Waals forces (Mosbach, 1994). After polymerization and removal of the template, the functional groups of the polymer are positioned to rebind the target molecule using the same non-covalent interactions. The non-covalent approach can only be employed where template molecules have functional groups capable of strong non-covalent interactions with the monomers. However, this can be overcome by using the covalent imprinting method, which involves covalently attaching the template molecule to a monomer followed by polymerization, cleavage of the covalent bond to liberate the template, and removal of the template. The reactive groups are positioned within the polymer cavities to allow covalent bonds to be reformed with the target molecule and provide selectivity. As there is a relatively small number


(i) Complexation Template

(ii) Polymerization

(iii) Washing

+ cross-linker

Binding site

Template

Fig. 5.1 Schematic illustration of the method for preparing molecular imprinted polymers (Takeuchi & Haginaka, 1999). Reproduced with permission. Copyright Elsevier (1999).

Novel adsorbents and approaches for nutraceutical separation 151


Functional monomer

152 Separation, extraction and concentration processes of suitable reversible covalent bonding reactions that can be employed, the covalent imprinting method has limited applications (Gupta & Kumar, 2008). MIPs are often manufactured using bulk polymerization methods to produce monolithic materials which can be used for in situ separations, or ground and sieved to produce particles suitable for column packing and separation applications (Haginaka, 2009). MIPs can also be manufactured using suspension, seed and dispersion/precipitation polymerization methods to produce micro- or nano-spheres (Haginaka, 2009; Kempe & Kempe, 2006; Ye & Mosbach, 2001). Using such suspension polymerization methods, MIPs can be generated in aqueous environments; however, hydrogen bonding between the template molecule and the functional monomer, and therefore MIP selectivity, can be reduced when using aqueous solvents (Haginaka, 2009). Suspension polymerization techniques in liquid perfluorocarbon or mineral oil do not reduce hydrogen bonding and electrostatic interactions and the resultant MIP microspheres have been shown to be selective for biologically active molecules (Perez-Moral & Mayes, 2006). MIPs are usually packed into columns of various dimensions to allow (a) sample preparation before analysis; (b) removal of valuable molecules; or (c) removal of undesirable molecules from various raw material streams. 5.2.2 Advantages and disadvantages The benefit of MIPs is their stability in various chemical and physical environments for an extended period of time without any loss in template recognition (Ciardelli et al., 2006; Kriz & Mosbach, 1995). In addition, they are physically robust, resistant to elevated temperatures and pressures, and inert towards acids, bases, metals ions and organic solvents. The reported limitations of MIPs include binding site heterogeneity and slow mass transfer, which subsequently leads to peak broadening and tailing. In addition, MIPs prepared by bulk polymerization often have irregular sized and shaped particles which can lead to poor chromatographic performance and large pressure drops (Turiel et al., 2005). These limitations may be overcome by: (i) optimizing the MIP loading solvent; (ii) optimizing the MIP washing regimen to remove contaminant molecules; and (iii) finer control over the polymerization reaction leading to more regularly shaped particles. 5.2.3 Applications The characteristics of MIPs make them ideal for use in separation applications including chromatography, capillary electrophoresis, solid phase extraction and membrane separations. They also have potential to be used as microreactors, in immunoassays, as antibody mimics and artificial enzymes. Owing to their © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 153 selectivity and robustness, MIPs have been applied in the selective isolation, concentration and analysis of food-derived molecules such as theophylline, caffeine, catechin, cholesterol, glycyrrhetinic, theanine, a-tocopherol and flavonoids (Haginaka, 2009). For example, Puoci et al. (2007) developed a MIP and optimized conditions for the selective extraction and analysis of a-tocopherol from bay leaf (Fig. 5.2). This method recovered approximately 60% of the a-tocopherol with high purity as measured by HPLC. Many other MIPs have been reported for the separation and analysis of agricultural herbicides, pesticides and fungicides, toxins, antibiotics and other chemicals in food (Haginaka, 2009). The ability to produce MIPs for the selective isolation of proteins and peptides is improving (Bossi et al., 2007; Rachkov & Minoura, 2001; Shi et al., 1999), and being employed to selectively target bacteria through their surface expressed proteins (Xue et al., 2009). 5.2.4 Commercialization MIPs can be employed in analytical applications as solid-phase extraction media to selectively increase the concentration of analyte, decrease sample preparation time and increase method sensitivity. MIPs for the solid phase extraction and analysis of food contaminants such as antibacterials (chloramphenicol, nitroimidazoles, fluoroquinolones) and herbicides (triazines) have been manufactured by MIP Technologies AB (Lund, Sweden) and distributed by Sigma-Alridch (St Louis, USA). MIPs for solid phase extraction and analysis of vitamins in food such as riboflavin have recently become available (MIP Technologies AB, Sweden). There are few reports in the public domain verifying large-scale applications of MIPs in the food, pharmaceutical and chemical industries. However, given the reported benefits of MIPs including stability and specificity, with further research and development into large-scale manufacture of these adsorbents together with scaled applications, it is likely that they will find targeted use in the isolation of nutraceuticals.

5.3 Organic monoliths and applications in the nutraceutical industry In contrast to columns packed with small particles to facilitate separation (e.g. silica or agarose beads), monoliths are continuous solid phases which can be synthesized in situ in confined spaces such as columns, capillaries, and microfluidic and microchip devices. Monoliths are unique as they have an interconnected skeletal structure with flow-through paths that provide very high permeability to a mobile phase, high flow rates, and low back pressures during chromatography. These characteristics make monoliths promising for the large-scale cost-effective purification of nutraceuticals


154 Separation, extraction and concentration processes 0.9 0.8

Absorbance (292 nm)

0.7 0.6 0.5 a-TP 0.4 0.3 0.2 0.1 0 0

2

4

6 Time (min) (a)

8

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12

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Fig. 5.2 HPLC chromatograms of (a) crude bay leaf extract and (b) bay leaf extract after isolation and concentration of the a-tocopherol (a-TP) (Puoci et al., 2007). Reproduced with permission. Copyright Elsevier (2007). © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 155 in the food industry. Monoliths can be synthesized using either inorganic materials such as silica or organic materials such as methacrylate polymers. As silica has poor stability and operational robustness during the caustic cleaning procedures employed in the food industry, this chapter focuses on the development and application of organic polymeric monoliths. 5.3.1 Synthesis Polymeric monoliths were first introduced in the late 1980s (Hjerten et al., 1989; Svec & Fréchet, 1992). Since then there have been several publications detailing developments in their manufacture and use in liquid chromatography, capillary electrochromatography and microscale separations (Nordborg & Hilder, 2009). Polymeric monoliths are synthesized by mixing an initiator, organic monomers (including a cross-linking monomer) such as styrene, methacrylate, acrylamide-based materials and pore forming solvents (porogens). The reagents are placed within a suitable mould, such as a column, capillary or channel of a microfluidic device and polymerized using radical polymerization initiated by either thermal treatment, ultraviolet, gamma or microwave radiation (Nordborg & Hilder, 2009). As polymerization proceeds, the polymer chains formed become insoluble in the polymerization mixture, phase separation takes place and the polymer chains precipitate out of solution, forming the monolith. The properties of the monolith synthesized, including the size and distribution of macro-, meso- and micropores are determined by the composition of the polymer mixture and the polymerization conditions. For example, the mode of initiation, type and amount of cross-linker, porogen, and the polymerization temperature. The large number of monomers that can be chosen to impart charged groups, other monomers, cross-linking agents and porogens results in a very large number of variations that can be examined and optimized to produce the desired monolith (Nordborg & Hilder, 2009). Organic monoliths can also be synthesized using sub-zero temperatures, where ice-crystals act as the pore-forming elements. After formation of the monolith, the ice crystals are allowed to thaw, the resulting water removed, leaving behind macropores between 10 and 100 mm in diameter. Such monoliths are known as cryogels (Arvidsson et al., 2002, 2003). Solid granules such as sodium sulfate can also be employed during the synthesis to improve bi-model pore formation and the hydrolytic characteristics of the resulting polymeric monoliths (Fig. 5.3) (Du et al., 2007). 5.3.2 Functionalization To improve the applicability of polymeric monoliths for separation applications, functionalization either during or after polymerization is required. In the first approach, the choice of monomers with charged functional groups results in material having charged groups throughout the monolith, including its surface.


156 Separation, extraction and concentration processes (a)


10 mm


10 mm

(b)

Fig. 5.3 SEM image of a glycidyl methacrylate-co-ethylene glycol dimethacrylate monolith prepared in the presence of the organic porogens dodecanol and cyclohexanol, (a) without and (b) with sodium sulfate granules (Du et al., 2007). Reproduced with permission. Copyright Elsevier (2007).

Examples of charged monomers that can be employed include methacrylic acid and ethylene glycol dimethacrylate. Optimization of monolith chemistry must take into account that the charged monomers participate in the polymerization process and will not all be available for separation interactions on the surface © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 157 of the monolith. The approach of post-polymerization modification has been examined and reported to be advantageous as the number of variables is reduced and the monolith chemistry can be optimized followed by modification of the surface with the desired functionality. Monoliths can be modified to achieve separation based on ion-exchange, affinity recognition, reversedphase, and hydrophobic interaction. 5.3.3 Advantages and disadvantages Polymer monoliths have similar benefits to their polymeric particles. Their pH and pressure stability are two advantages that allow them to be employed in a range of environments where high flow rates may lead to large pressure drops across the bed. However, as monoliths are made from one piece of material with large interconnected pores (Fig. 5.3), diffusion mass transfer is minimized and convective mass transfer is maximized. As convective mass transfer is faster than diffusive transport, higher mobile phase flow rates can be employed and faster separation of molecules can be achieved. Breakthrough curves of lysozyme and immunoglobulin G using a polymethacrylate monolithic column (Connective Interaction Media, BIA Separations; Ljubljana, Slovenia) and a column packed with Source 30S particles (GE Healthcare Life Sciences Uppsala, Sweden) have been studied (Hahn et al., 2002). As expected, mass transfer in the particle packed column was very dependent on the linear velocity of the mobile phase, molecule size, and feed concentration, whereas mass transfer in the monolith was not significantly affected by velocity, molecule size or feed concentration (Hahn et al., 2002). A disadvantage of monoliths is their low surface area as a result of their high porosity. Thus, monoliths have been reported to be more suitable for the isolation of large biomolecules (e.g. proteins) rather than small molecules. Monoliths also have a tendency to be hydrophobic and thus adsorb proteins via both ion-exchange and hydrophobic interaction. This characteristic makes proteins difficult to elute from the monolith without solvent which can lead to protein denaturation (Nordborg & Hilder, 2009). Research to develop polymeric monoliths with less hydrophobic characteristics or modification of monolith surfaces with hydrophilic coatings is being undertaken to improve their effectiveness in the isolation of biological molecules. 5.3.4 Applications Monoliths have been employed to separate a range of food-derived biomolecules and nutraceuticals. These include enzymes such as pectin methylesterase, endo-polygalacturonase (Vovk et al., 2005; Vovk & Simonovska, 2007), lysozyme (Svec & Fréchet, 1995), and proteins such as trypsin inhibitor (Svec & Fréchet, 1995), bovine serum albumin (Du et al., 2007), lactoferrin (Adam et al., 2008), casein peptides (Gu et al., 2006), and virus particles


158 Separation, extraction and concentration processes (Kramberger et al., 2004). Hilder et al. (2004) has also demonstrated that monoliths coated with quaternary amine-functionalized latex particles can effectively separate sugars (Fig. 5.4), and separate glucose and maltose from amylase-hydrolyzed corn starch. There are a number of studies indicating that monolithic adsorbents have potential for the preparative-scale separation and purification of biomolecules, such as immunoglobulins (Brne et al., 2007), a-lactalbumin, enzymes, plasmid DNA, viruses and cells (Jungbauer & Hahn, 2008). In addition, monolithic columns have been evaluated for their potential to isolate immunoglobulins using simulated moving bed chromatography (Pennings et al., 2008). 5.3.5 Commercialization Currently, a number of companies are manufacturing and distributing polymeric monolithic columns for analytical applications. These include BIA Separations (Villach, Austria), Dionex (Sunnyvale, USA), and BioRad (Hercules, USA). BIA Separations are one of only a few companies designing and supplying organic monoliths for both small- and large-scale purification. Their disk format monoliths are manufactured for small-scale separations using axial flow design characteristics, and their tube format monoliths are prepared for 3 1

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Fig. 5.4 Separation of carbohydrates within 10 min using an optimized anionexchange latex-coated polymeric monolithic capillary column. Peaks: (1) d(+) galactose, (2) d(+)glucose, (3) d(+)xylose, (4) d(+)mannose, (5) maltose, (6) d(–) fructose, (7) sucrose (Hilder et al., 2004). Reproduced with permission. Copyright Elsevier (2004).


Novel adsorbents and approaches for nutraceutical separation 159 larger scale separation using radial flow design characteristics. The latter design is employed for large columns (e.g. up to 8 L) and is reported to allow for a significant increase in the linear velocity through the monolithic bed, without adversely affecting performance or pressure characteristics. Optimization of surface chemistry and adsorption capacity is a prerequisite for the application of organic monoliths in large-scale isolation of nutraceuticals, together with a determination of the economic benefit of using such adsorbents over traditional particle-packed columns. As manufacturing processes are optimized, and costs reduced, larger monolithic columns will deliver cost-efficient nutraceutical separation and isolation processes in the food industry.

5.4 Stimuli-responsive resins and applications in the nutraceutical industry Polymers that undergo a significant change in their structure and behavior (e.g. solubility) in response to an external physical, chemical or electrical stimulus are termed ‘stimuli-responsive’, ‘smart’ or ‘intelligent’ polymers. A number of polymers have this property and various stimuli, including temperature, pH and light can be applied to induce a change in polymer conformation and subsequent properties. Stimuli-responsive polymers with temperature responsiveness have been extensively studied and characterized. These polymers have been grafted onto chromatographic supports to produce stimuli-responsive resins. These novel resins have the potential to reduce solvent usage, processing costs and the environmental impact of nutraceutical isolation in the food industry. The best characterized temperature-responsive polymer is poly(Nisopropylacrylamide (poly-NIPAAm). This polymer differs from traditional polymers as its solubility decreases as the temperature of the poly-NIPAAm solution increases. When a critical temperature is reached (32 °C for polyNIPAAm), hydrogen bonding of water to the amide group is reduced and the poly-NIPAAm becomes unstable, contracts and enters a globular state (Fig. 5.5). The temperature at which this occurs is called the lower critical solution temperature (LCST) (Ayano & Kanazawa, 2006). The LCST of temperature-responsive polymers including poly-NIPAAm can be modified by incorporating hydrophilic or hydrophobic monomers into the polymer (Ayano & Kanazawa, 2006). Hydrophilic monomers generally increase the LCST of the polymer whereas incorporation of hydrophobic monomers generally decrease the LCST of the polymer. 5.4.1 Synthesis Temperature-responsive resins are synthesized by grafting temperatureresponsive polymers to the surface of solid chromatographic supports. Silica © Woodhead Publishing Limited, 2010


H 2O PNIPAAm

+ Stimulus – Temperature

Below LCST

Above LCST

Fig. 5.5 Coil to globule transition and subsequent solution turbidity change when poly-NIPAAm (PNIPAAm) is heated above or cooled below the lower critical solution temperature (LCST) (Ayano & Kanazawa, 2006). Reproduced with permission. Copyright Wiley-VCH Verlag GmbH & Co. KGaA.

has been the predominant support employed using radical co-polymerization of initiator immobilized silica or activated ester amine coupling to aminopropylmodified silica (Maharjan et al., 2008). Temperature-responsive co-polymers that have been grafted onto silica beads for chromatography include polyNIPAAm, poly-NIPAAm-co-butyl methacrylate, poly-NIPAAm-co-acrylic acid, and poly-NIPAAm-co-acrylic acid-co-N-tert-butylacrylamide (Maharjan et al., 2008). The use of silica matrices for the separation of nutraceuticals in the food industry is limited owing to their lack of operational robustness, including their instability at the high pH employed during cleaning processes (Maharjan et al., 2008). A temperature-responsive resin employing a porous crosslinked agarose support was recently reported. This resin was synthesized by functionalizing agarose beads with the initiator 4,4-azobis(4-cyanovaleric acid), followed by polymerization of N-isopropylacrylamide, tert-butylacrylamide, acrylic acid and the cross-linking agent, N,N-methylenebisacrylamide onto the surface using free radical polymerization (Maharjan et al., 2009). 5.4.2 Applications Ion-exchange chromatography There have been a number of studies examining the effect of pH and temperature-responsive resins on retention and separation of biomolecules by ion-exchange chromatography. Temperature-responsive poly-NIPAAmco-acrylic acid-co-N-tert-butylacrylamide silica beads with cation-exchange functionality have been synthesized and employed to separate bioactive peptides and amino acids. This temperature-responsive silica resin showed greater retention and separation of basic bioactive peptides at higher temperatures


Novel adsorbents and approaches for nutraceutical separation 161 than at lower temperatures when using aqueous conditions (Kobayashi et al., 2002; 2003). Similarly, silica beads grafted with poly-NIPAAm-cobutylmethacrylate-co-dimethylaminopropyl acrylamide show better separation of small bioactive molecules (anti-inflammatory compounds) at 50 °C than at 10 °C when using an isocratic aqueous mobile phase (Ayano et al., 2006). Although changes in the retention and separation behavior of molecules have been demonstrated with temperature responsive silica resins, there have been a minimal number of reports showing the effective capture and release of biological molecules. Research recently published using porous cross-linked agarose resins has demonstrated the potential of temperature-responsive cation-exchange agarose-based resins to selectively capture and isolate charged cationic proteins such as lactoferrin from mixed protein systems (Maharjan et al., 2009). The temperature-responsive agarose resin had a 3-fold higher lactoferrin adsorption capacity at 50 °C than at 20 °C (Fig. 5.6), and was able to selectively adsorb lactoferrin from a mixture of lactoferrin, a-lactalbumin and b-lactoglobulin. In addition, dynamic studies indicated that approximately 50% of the adsorbed lactoferrin could be eluted from the agarose by simply reducing the temperature. The remaining lactoferrin was eluted using low concentrations of NaCl (Maharjan et al., 2009). Hydrophobic-interaction chromatography A significant amount of research has been undertaken to develop and understand temperature-responsive resins for hydrophobic-interaction chromatography. For example, temperature-responsive poly-NIPAAm-co-butyl methacrylate functionalized silica beads have been employed to separate a mixture of insulin chains A and B, and b-endorphin fragment 1–27 (Fig. 5.7). Temperature Adsorbed lactoferrin (mg mL–1)

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Fig. 5.6 Equilibrium adsorption isotherms of lactoferrin onto poly-NIPAAm-coacrylic acid-co-N-tert-butylacrylamide agarose resin at 20 °C (continuous line) and 50 °C (dashed line) (error bars represent the standard deviation from the mean) (Maharjan et al., 2009). Reproduced with permission. Copyright Elsevier (2009).


162 Separation, extraction and concentration processes 1, 2, 3 2

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Fig. 5.7 Separation of a mixture of insulin chains A and B, and endorphin fragment 1–27 on a poly-NIPAAm-co-butyl methacrylate modified silica resin. Column temperature: (a) 0 °C, (b) 40 °C. Mobile phase: aqueous 0.17M NaCl. Flow-rate: 0.5 ml min–1. Detection: 215 nm (Kanazawa et al., 1997). Reproduced with permission. Copyright Elsevier (1997).

gradients were able to alter the hydrophobicity of the resin, resulting in temperature-modulated peptide elution from the column (Kanazawa et al., 1997). In addition, temperature-responsive poly-NIPAAm silica beads have been employed to separate steroids with an aqueous mobile phase (Kanazawa et al., 1996). The steroids were unresolved at temperatures below the LCST and well resolved above the LCST. The improved resolution at temperatures above the LCST was most probably the result of increased hydrophobic interaction of the steroids with the poly-NIPAAm stationary phase at the higher temperatures. Size-exclusion chromatography Temperature-responsive polymers have also been applied to synthesize temperature-responsive size-exclusion resins. For example, poly-NIPAAm was grafted onto porous glass beads, the poly-NIPAAm end functionalized with mercaptopropionic acid, and the resulting resin employed for gel permeation size-exclusion chromatography (Gewehr et al., 1992). The elution time of the dextrans was substantially altered between 25 °C and 32 °C, possibly owing to a change in the effective pore size of the resin via transition of the poly-NIPAAm chains from coils to globules on the surface of the pores of the glass beads. Porous polystyrene beads grafted with poly-NIPAAm have also shown temperature-responsive size-exclusion characteristics (Hosoya et al., 1994). In this example, an increase in temperature prolonged the elution time of higher molecular weight dextrans, possibly owing to collapse of the poly-NIPAAm causing the pore size to expand, and thus permitting the dextrans to penetrate the pores.


Novel adsorbents and approaches for nutraceutical separation 163 Affinity separations Temperature-responsive polymers can also be conjugated to proteins and employed as affinity separation materials. Cycling below and above the LCST of the temperature-responsive polymer induces reversible precipitation/ solubilization of the bioconjugates in the aqueous solution, thereby allowing separation of the bioconjugate and the target molecule. Proteins such as a-chymotrypsin (Kim & Park, 1998) and a-glucosidase (Hoshino et al., 1998) were purified using this method. In addition, temperature-dependent isolation of lactate dehydrogenase from porcine muscle was achieved using a dye-affinity agarose modified by physically adsorbing the smart polymer poly-N-vinylcaprolactam onto the surface. Using this method, the lactate dehydrogenase was adsorbed to the column at 40 °C and eluted by reducing the temperature to 23 °C and adding KCl (Galaev et al., 1994). 5.4.3 Commercialization Temperature-responsive polymers can be employed to separate a wide spectrum of molecules using a range of separation mechanisms (e.g. charge, size, affinity). However, temperature-responsive resins are not currently available commercially for large-scale separation applications. With further research and development into specific applications and scale-up, temperature-responsive resins with silica or other matrices could be employed commercially to isolate hydrophobic and charged nutraceutical molecules. Such processes should require less solvent (e.g. salt, ethanol), which would reduce the cost and environmental impact of such separations, and potentially improve retention of the biological activity of such isolated molecules.

5.5 Mesoporous molecular sieves and applications in the nutraceutical industry Mesoporous molecular sieves, also termed mobile crystalline materials (MCMs), are a family of inorganic solids with regular nanostructure, large internal surface area (>1000 m2 g–1) and void volume, and narrow pore size distribution. These remarkable materials, exhibiting defined and tailored pore diameters from approximately 2 nm through to 50 nm, offer new possibilities for designing highly effective adsorption media based upon their pore size and large internal surface area. Mesoporous molecular sieves, notably the MCM-4X family, were first reported in the early 1990s (Beck, 1991; Beck et al., 1992; Kresge et al., 1992), and have attracted considerable research attention since then as novel catalysts, guest supports in chemical reactions, and potential adsorbents for the isolation of biological components (Beck & Vartuli, 1996; Brady et al., 2008; Kisler et al., 2001a; 2001b; Selvam et al., 2001).


164 Separation, extraction and concentration processes 5.5.1 Synthesis Mesoporous molecular sieves are prepared using a technique called liquidcrystal ‘templating’, illustrated schematically for MCM-41 in Fig. 5.8. In this process, mesoporous solids are synthesized by thermal treatment and ultimate calcination of aluminosilicate gels in the presence of suitable surfactants. Unlike other mesoporous inorganic materials, such as silica, the MCM family of mesoporous molecular sieves comprise regular arrays of non-intersecting uniform hexagonal channels (Fig. 5.8 and 5.9), the dimensions of which can be tailored within 1.5–50 nm through the selection of different surfactants (‘template molecules’) and reaction conditions (Gusev et al., 1996; Beck, 1991; Kresge et al., 1992). The pore size can be tailored during synthesis to accommodate biological molecules of almost any size (Fig. 5.9). Hexagonal array

Silicate

Calcination

MCM-41

Fig. 5.8 Schematic illustration of the liquid-crystal ‘templating’ technique for manufacture of mesoporous molecular sieves such as MCM-41 (Kresge et al., 1992). Reproduced with permission of Macmillan Publishers Ltd: Nature. Copyright Macmillan Publishers Ltd (1992).

Fig. 5.9 Model of the molecular sieve MCM-41 illustrating methane and ethane inside one of the hexagonal pores (pore diameter ~3 nm).


Novel adsorbents and approaches for nutraceutical separation 165 5.5.2 Advantages and disadvantages The high internal surface area, tailored pore size, and very narrow pore size distribution of these mesoporous molecular sieves are characteristics that make them ideal candidates as ‘designed media’ for size-exclusion and adsorptionbased separation of biological molecules, notably proteins, peptides and other functional ingredients (Brady et al., 2008; Kisler et al., 2001a,b). For these types of applications, the best studied mesoporous molecular sieves include MCM-41 and MCM-48 (Melo et al., 1999; Zhao et al., 1996). Although the uniform mesoporous pore size makes these materials attractive candidates for adsorption and separation of biological macromolecules, their stability in aqueous systems is limited (Kisler et al., 2001b). Such instability has been a major limitation in the widespread use of these materials for waterbased extractions, and in the separation and isolation of targeted bioactive components for use as nutraceuticals. In order to overcome this shortcoming, many researchers have modified the mesoporous materials with a hydrophobic coating to assist in stabilizing the material in water (Brady et al., 2008; Kisler et al., 2001b). Such modification of MCM-41, for example, has enhanced the utility of this mesoporous molecular sieve in biochemical applications (Brady et al., 2008; Kisler et al., 2001a; 2001b). These materials have also been studied as processing aids in water treatment and in the generation of potable water (Cooper & Burch, 1999). 5.5.3 Applications Size-based separations The utility of mesoporous molecular sieves as size-exclusion materials in the isolation and separation of proteins was first examined by Diaz and Balkus (1996) and subsequently by Washmon-Kriel et al. (2000) in studies of cytochrome C permeation and adsorption. This research showed a correlation between protein adsorption and the pore size of the molecular sieve being examined, as would be expected, and retention of biological activity, an important consideration when designing a separation protocol for isolation of bioactive components like nutraceuticals. Takahashi et al. (2000; 2001) and Vinu et al. (2004) also showed strong correlation between enzyme immobilization and pore size in a study of several macroporous molecular sieves including MCM-41. The isolation of both large and small biological molecules using MCM-41 and MCM-48 has been explored (Brady et al., 2008; Kisler et al., 2001a; 2001b; Xue et al., 2004). Trypsin, lysozyme and riboflavin have been studied as model biological solutes in order to examine the size-exclusion characteristics of these mesoporous materials and their potential as bioseparation media (Kisler et al., 2001a). Similarly, Brady et al. (2008) explored the potential of hierarchical mesoporous silica sieves in the isolation of valuable dairy bioactive components as functional food ingredients, and the feasibility of such materials as industrial-scale adsorbents.


166 Separation, extraction and concentration processes Protein stabilization Stabilization of labile enzyme activity, both in aqueous and organic environments, may well be an important application for mesoporous molecular sieves. MCM-48 was used to immobilize penicillin G acrylase and to stabilize the activity of this enzyme (Xue et al., 2004), and was shown to be superior to MCM-41 for this application. Kim et al. (2006) provided a comprehensive review of the probable mechanisms involved in stabilization of enzymes by various nanostructures, including mesoporous molecular sieves, which include confinement, charge and hydrophobic interaction, and multipoint attachment to the protein. Wang & Caruso (2005) reported the usefulness of mesoporous silica and modified forms thereof as supports for immobilization and encapsulation of enzymes, and stabilization of biological activity, demonstrating success of the technology with catalase. Mesoporous sieves may also provide utility in expanding the application scope of enzymes for processing in organic solvents by stabilizing their catalytic activity in such environments (Takahashi et al., 2001; Yan et al., 2002). 5.5.4 Commercialization The highly regular structure, tailored and tuneable properties, uniform pore size distribution and high internal surface area of selected mesoporous sieves represent attractive properties for the design of modern separation materials and protocols based upon size-exclusion and surface-interaction chemistry. However, before their industrial potential can be realized and these characteristics exploited, the long-term stability and safety, effectiveness in processing agrifood streams, and scaleability and process economics must be established.

5.6 Peptide affinity ligands and phage display methodology and applications in the nutraceutical industry Affinity chromatography is a method for separating biochemical components, often macromolecules (proteins, peptides, nucleic acids), from complex mixtures based on a highly specific biological interaction such as that between an antigen and antibody, substrate and enzyme, or ligand and receptor. The rationale for this process was first reported in the early part of the 20th century (Starkenstein, 1910). However, it was not until the late 1960s, associated with developments in modern chromatographic resins and support materials, and chemistry/linking reactions for immobilization of functional ligands, that affinity chromatography emerged as a viable industrial technology for the isolation of valuable biological components, including nutraceuticals (Cuatrecasas & Anfinsen, 1971; Wilchek & Miron, 1999). Although affinity chromatography is a very powerful technique and has revolutionized the fields of modern biochemistry, biotechnology and medical science over the past 40 years, it is not without its challenges. These relate © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 167 to the preparation of the affinity adsorbent (ligand and resin support) and the use of the adsorbent in processing, and include considerations of selectivity, functionality, stability, efficiency, capacity, repeatability, and productivity. The choice of functional ligand to be immobilized and to effect the affinity separation is vast, and is dictated by the characteristics of the molecule targeted for isolation. Such functional ligands include proteins, enzymes, peptides, nucleic acids, sugars, vitamins, lipids, synthetic organic molecules (e.g. dyes), and metal ions. Once the ligand is selected, immobilization chemistry, maintenance of biological functionality, stability, avoidance of steric effects associated with the resin support, and effectiveness of the adsorbent under processing conditions, among others, must be considered (Wilchek & Miron, 1999). In the design of modern affinity adsorbents, the selection of ligands with broad applicability that meet most of these requirements is important in addressing the cost-effectiveness consideration critical for industrial processes. In this regard, peptides offer an attractive option (Amatschek et al., 2000; Pflegerl et al., 2002). 5.6.1 Advantages and disadvantages The main advantage of small peptides is their high affinity and selectivity. In addition, they are usually stable to mild chemical, physical and biological conditions, and not prone to degradation by protease enzymes found in most agri-food streams. Peptides are also amenable to a variety of linkage chemistries that provide for a very stable covalent bond between peptide and resin support. Tailored peptides of almost any composition and sequence can be readily synthesized through modern solid-phase techniques. Parallel and combinatorial approaches to directed peptide synthesis can readily result in large peptide libraries which can be rapidly screened for their affinity to a target compound (Pflegerl et al., 2002). Although peptide affinity resins have high affinity and selectivity, they can have relatively low binding capacity, increasing the overall cost of nutraceutical manufacture and the cost of the final product. In addition, manufacturing specific peptides on a large scale can be time consuming and expensive. Research groups are currently working on methodologies to improve the binding capacity of peptide adsorbents and significant competition in the field of peptide production is rapidly improving manufacturing technologies and reducing costs. There are also concerns regarding the toxicity and biocompatibility of novel affinity peptides when employed to produce products for human consumption. Before peptide affinity ligands can be employed in the food industry, detailed studies to ensure that they are stable during processing and non-toxic will be required. 5.6.2 Peptide identification A technique developed recently that will perhaps revolutionize the development and selection of peptide ligands for affinity adsorbtion and other applications


168 Separation, extraction and concentration processes is called phage display, based on a concept reported in the mid-1980s (Smith, 1985). Phage display is a powerful technique for the study of protein–protein, protein–peptide, and protein–nucleic acid interactions. This approach uses bacteriophage vectors to express a protein or peptide on the surface of the virus particle coded by genetic material residing on the inside of the particle. Phage display thus creates a connection between genotype and phenotype, and facilitates the creation of a library of variants of a peptide (or protein) expressed on the outside of the virus, and allows for rapid in vitro screening of these variants based upon their binding affinity to a target molecule (e.g. enzyme, antibody, receptor, cell structural protein). This in vitro selection process is called panning, and allows for the high-throughput screening of protein interactions (Kay et al., 2001; Koivunen et al., 1999). In its simplest form, panning is carried out by incubating a library of phage-expressed peptides with the target molecule, usually in a 96-well plate, washing away any unbound phage, and then eluting the specifically bound phage. The eluted phage is then taken through additional binding and amplification cycles to enrich the pool in favor of the specific binding peptide sequence (Kay et al., 2001). In this way, phage display/panning allows for the rapid discovery of useful peptide ligands with specific affinity for the target protein or other molecules (nutraceuticals), and thus the development of effective peptide affinity adsorbents for research and industrial applications. The phage display/panning technique has been widely used in drug discovery, notably for the identification of promising new ligands (e.g. enzyme inhibitors, and receptor agonists and antagonists) to target proteins. Perhaps the most successful commercial outcome from phage display/panning technology has been the therapeutic antibody drug adalimumab (Bain & Brazil, 2003; Scheinfeld, 2003). This antibody, developed by Cambridge Antibody Technology (Cambridge, UK), targets the inflammatory cytokine TNF-a and is marketed commercially by Abbott Laboratories as HUMIRA (‘human monoclonal antibody in rheumatoid arthritis’) (Scheinfeld, 2003). 5.6.3 Commercialization The use of phage display/panning in the discovery of specific peptide– biomolecule interactions that may lead to effective affinity adsorbents was simplified through the development and sale of commercial ‘kits’ containing pre-made random peptide libraries, as well as a cloning vector for construction of custom libraries. Such ‘kits’ and related reagents are available from a number of commercial biotechnology/analytical companies, including for example New England BioLabs (Ipswich, USA), V-Biolabs (Sawston, UK), Mimotopes (Melbourne, Australia), and Cambridge Antibody Technology (Cambridge, UK) (now part of AstraZeneca). Development and refinement of phage display technology by the pharmaceutical industry provides an example of a biotechnology that can be applied in food processing applications and nutraceutical isolation. Peptide


Novel adsorbents and approaches for nutraceutical separation 169 affinity technologies are not yet commercially available for the isolation of nutraceuticals at commercial scale. However, the phage display technology represents a robust tool for research into novel peptide affinity adsorbents. With further research and development to optimize capacity and manufacturing procedures, peptide affinity resins aimed at cost-effective isolation and separation of nutraceuticals should become available in the near future.

5.7 Membrane adsorbers, membrane chromatography and applications in the nutraceutical industry A recent development in the arena of chromatographic adsorbents is that of membrane adsorbers aimed at improving the efficiency and simplicity of the chromatographic process by allowing the targeted separation to occur on and within a macroporous membrane support (see Charcosset, 1998; Klein, 2000; Przybycien et al., 2004 for reviews of alternative bioseparation processes including the use of membrane adsorbers). This process has also been termed membrane chromatography (Charcosset, 1998; Thömmes and Kula, 1995). Membrane adsorbers are porous membranes with functional ligands (such as ion-exchange or affinity groups) covalently attached to the membrane (Demmer et al., 1989) or immobilized within the membrane structure. 5.7.1 Advantages and disadvantages Membrane adsorbers have several potential advantages over traditional particle matrices and adsorbents (Charcosset, 1998). Ion-exchange and other functionalized membranes avoid problems with swelling or packing and can easily be scaled up. Membrane adsorbers have low compressibility characteristics and also tend to demonstrate lower back pressures than traditional chromatographic resin materials thus allowing for the rapid processing of large volumes of fluid. Large targeted molecules, such as proteins and large peptides, are convected through the membrane, rather than having to diffuse into a resin bead before adsorbing to the functional ligand, and thus diffusion limitations are minimized. Overall mass transfer is improved, leading to shorter cycle times and higher overall throughput (Brandt et al., 1988). These properties facilitate the use of processing, maintenance and cleaning protocols more diverse and adaptable to existing procedures in the food processing industry. Based on the advantages of membrane adsorbers over traditional chromatographic resins (Charcosset, 1998), these adsorbents, once scaled to industrial size, would appear to be best suited for the separation and isolation of targeted components, notably proteins and peptides, found in low concentrations in large volumes of liquid (Gebauer et al., 1997). Membrane adsorbers have similar disadvantages to traditional membrane technologies. For example, when processing protein


170 Separation, extraction and concentration processes containing fluids, a secondary boundary layer can form, reducing flux rates and increasing membrane cleaning requirements. In addition, as membrane adsorbers are made from organic materials, they are sensitive to the elevated temperatures and cleaning regimes often used in the food industry. These membranes must therefore be cleaned using mild conditions and replaced when their adsorption capacity or flux rates fall outside critical process specification. As with traditional membranes, large pressure drops across the membrane should also be avoided as they can cause deformation and irreversible changes in pore-size characteristics. 5.7.2 Functionalization The functional ligand chemistry available with membrane adsorbers is almost endless and is similar to that for traditional chromatographic resins, including strong and weak anion and cation exchange, affinity interaction, reversed phase, and hydrophobic interaction. Selection of the appropriate ligand chemistry, and membrane support material and configuration, will depend upon the specific targeted component of interest and separation strategy, source and ultimate end-use application of the isolate. 5.7.3 Configurations Improvements in membrane materials, equipment for conducting membrane chromatography, and the means by which the targeted fluid is presented to the membrane give the user choice in designing the best combination of membrane adsorber, device, and geometric configuration to achieve the most cost-effective separation of the targeted compound. Macroporous functionalized membranes are available as single or stacked flat sheets, hollow fibers, spiral-wound sheets, dead-end/single-use filters, and cassettes from several established commercial manufacturers (e.g. Pall, Sartorius) and some new entrants in the field (e.g. Mosaic Systems, Natrix Separations). These configurations allow for simple dead-end filtration applications, but also the more commercially relevant cross-flow processing for the treatment of large volumes of liquid in a short timeframe. The benefits of membrane adsorbers are most apparent in flow-through applications because capacity constraints in retention mode, particularly at high loading rates, make resin chromatography a more attractive option for capture steps. 5.7.4 Applications Ion-exchange separations Ion-exchange (anion and cation) functionalized membrane adsorbers have been the most extensively researched as cost-effective alternatives to traditional resin chromatography for the isolation of valuable minor protein and peptide constituents from agri-food streams, notably dairy whey (Adisaputro et al.,


Novel adsorbents and approaches for nutraceutical separation 171 1996; Mitchell et al., 1994; Yang et al., 1999; Zietlow & Etzel, 1995). Attention has been devoted to the use of strongly acidic membrane adsorbers in the isolation of lactoferrin and lactoperoxidase from cheese whey (Chiu & Etzel, 1997; Mitchell et al., 1994), although some attention has also been paid to the growth factor constituents (Mitchell et al., 1994). Glycosylated and non-glycosylated forms of the glycomacropeptide have been isolated from whey using anion-exchange membrane chromatography (Kreub & Kulozik, 2009; Kreub et al., 2008). In the former study, the authors describe a direct-capture method for the isolation of glycosylated glycomacropeptide at pilot scale using a membrane adsorption process. Membrane ion-exchange chromatography has also been described for the isolation of bovine serum albumin (He & Ulbricht, 2008; Sarfert & Etzel, 1997), soybean trypsin inhibitor (He & Ulbricht, 2008; Josic & Strancar, 1999), patatin from potato (Alt et al., 2004), a-lactalbumin (Yang et al., 2002), a large protein, thyroglobulin (Yang et al., 2002), and in the large-scale production of antibodies (Zhou & Tressel, 2005; 2006; Zhou et al., 2006). Affinity separations Integration of membrane processing and affinity chromatography by way of affinity membrane adsorbers provides a number of advantages over traditional affinity chromatography with resin packed columns, notably in respect to time and recovery of bioactivity (Brandt et al., 1988; Klein, 2000; Zou et al., 2001). Such membrane adsorbents have been applied to the isolation of pepsin and chymosin using an immobilized pepstatin A membrane (Suen & Etzel, 1994), and to the separation of specific antibodies using membraneimmobilized bovine serum albumin, soybean trypsin inhibitor, Protein G, and immunoglobulin G (Kochan et al., 1996; Ostryanina et al., 2002). Affinity membranes can be dominated by slow sorption kinetics thus potentially impacting the performance of the separation (Suen & Etzel, 1994). Many of the protein and peptide examples shown have the potential to serve as valuable nutraceuticals in functional food applications (Etzel, 2004; McIntosh et al., 1998), and as natural antibacterial and bioactive agents and thus enter areas of both human and veterinary medicine, and biotechnology (Dionysius et al., 1993; Perraudin, 1991; Pouliot & Gauthier, 2006; Regester & Belford, 1999; Smithers, 2004; 2008). Micro-organism removal Membrane adsorbers provide an attractive alternative to traditional resinbased chromatography columns used to remove trace impurities and microbial contaminants (e.g. virus particles) in downstream processing applications, notably those requiring good manufacturing practice (GMP) compliance (Etzel & Riordan, 2006; Zhou et al., 2008). These membranes have been reported to reproducibly achieve greater than 4-log removal of mammalian viruses and greater than 3-log removal of endotoxin and contaminant DNA (Zhou et al., 2008).


172 Separation, extraction and concentration processes 5.7.5 Commercialization Pall Filtration (Port Washington, USA) and Sartorius (Goettingen, Germany) are perhaps the best known manufacturers of membrane adsorbers for research and small industrial use. These manufacturers have targeted agrifood, biotechnology and biopharmaceutical processing applications for their membrane adsorber products, primarily the ion-exchange offerings. There are a number of citations in the scientific literature on the effective use of membrane ion exchangers in the isolation of biological molecules (He & Ulbricht, 2008; Mitchell et al., 1994; Przybycien et al., 2004; Yang et al., 1999). Several new manufacturers have introduced further innovations into the field that improve the inherent efficiency and simplicity of membrane adsorbers. For example, Mosaic Systems (Ba Breda, The Netherlands) is a biotechnology enterprise with a specialization in commercial-scale separation and isolation of components for the food ingredient and biopharmaceutical industries. This company has developed a novel mixed-matrix membrane adsorber that comprises functionalized resin beads (5–10 mm) immobilized in a macroporous membrane. The company claims that the embedded resin beads retain full chromatographic functionality resulting in the products’ selectivity and binding capacity, but that the macroporous membrane decouples pressure drop from the resin particle size leading to enhanced hydrodynamic performance. This mixed-matrix system leads to improved throughput and productivity compared with an equivalent traditional chromatographic system, minimal fouling, reduced water and buffer use, and retention of efficiency and specificity through multiple adsorption cycles. Mosaic Systems claims their mixed-matrix adsorbers have been proven in trials with customers for the manufacture of food ingredients, biopharmaceuticals, and the isolation of valuable components from dairy streams. Natrix Separations (Burlington, Canada) (formerly Nysa Technologies) has developed a patented polymeric hydrogel technology that combines the high binding capacity, selectivity and specificity associated with traditional chromatographic resins with the high throughput and ease of use of macroporous membranes (Childs et al., 2008). The membrane adsorber consists of a polymeric hydrogel formed within a flexible porous support matrix. The matrix provides mechanical strength, whereas the hydrogel characteristics determine the separation chemistry of the product. The hydrogel polymer provides high binding site density, a large surface area for binding, and rapid mass transfer supporting high flow rates while providing highly efficient capture of the target molecule (Childs et al., 2008).

5.8 Conclusions and sources of further information and advice There appear to be many advantages to the application of ‘next-generation’ separation materials, such as those covered in this chapter, in the isolation © Woodhead Publishing Limited, 2010

Novel adsorbents and approaches for nutraceutical separation 173 of nutraceuticals. These advantages include improved specificity, selectivity, simplicity, robustness, productivity and reduced environmental impact. However, several of these materials do remain on or over the horizon from an industrial-scale perspective. Further research is needed to establish: (i) large-scale manufacturing procedures for a number of these novel adsorbents; (ii) specific scaled applications in nutraceutical manufacture; (iii) their processing advantages over traditional chromatographic materials and devices; and (iv) perhaps of most importance from an industrial perspective, process economics and commercial feasibility. The reader is directed to the following websites for further information about the novel adsorbents and approaches covered in this chapter. ∑ ∑ ∑ ∑ ∑ ∑

Molecular imprinted polymers: www.MIPtechnologies.com www.molecular-imprinting.com http://www.sigmaaldrich.com/analytical-chromatography/samplepreparation/spe/supelmip.html Organic monoliths: www.bio-labs.com www.BIAtechnologies.com www.dionex.com Stimuli-responsive polymers: http://macromolecules.case.edu/research_stimuli-responsive.htm Mesoporous molecular sieves: www.mrs.org Peptide affinity ligands and phage display methodology: www.neb.com www.vbiolabs.com www.mimotopes.com www.cambridgeantibody.com Membrane adsorbers and membrane chromatography: www.pall.com www.sartorius.com www.mosaicsystems.nl www.natrixseparations.com

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6 Advances in the effective application of membrane technologies in the food industry M. Pinelo, G. Jonsson and A. S. Meyer, Technical University of Denmark, Denmark Abstract: This chapter focuses on the recent advances in the use of membrane technology for efficient separation and concentration of solutes in the dairy and fruit juice industry, as well as advances in the purification of bioactive compounds to be used as food additives. The importance of fouling reduction is emphasized because this is necessary for membrane processes to become economically feasible. Key words: membranes, dairy industry, fruit juice, fouling, bioactive compounds, membrane bioreactors.

6.1 Introduction Membrane separation technologies have attracted much attention in the food processing industries over recent decades. The main reason for the interest is that membranes allow efficient separation and concentration of solutes without changing phase, maintaining worthy chemical and physical properties of food components and systems with particular economical relevance. This chapter primarily focuses on the use of membrane technology in the dairy and fruit juice industries. The incorporation of new, advanced membrane filtration techniques generally occurred first in the dairy industry, and was only later incorporated into other food manufacturing processes. Fouling is still the main challenge to overcome before use of membrane technologies becomes economically feasible in industrial processes. Various techniques to reduce fouling, related to the dairy industry, are discussed in the first section of the chapter. In the fruit juice industry, membrane technology is mainly used for concentration and clarification purposes. The main challenge is to


Advances in the effective application of membrane technologies 181 reduce the volume of juice maintaining the aroma compounds, which are otherwise lost when concentration methods such as evaporation are employed. The most recent advances in reverse osmosis and membrane distillation are reviewed, as these techniques are the most promising ones to maintain aroma compounds and juice quality. Wastewater treatment in food processing is also discussed in the chapter, as regulation regarding purified water quality and landfill management are becoming more and more restrictive. We pay special attention to wastewater treatment by using membrane bioreactors (MBR), particularly anaerobic MBR, on which research has focused on the recent years. Membrane separation is also a promising down-stream technique for purification of food ingredients and additives. We present the current challenges and limitations of applying membrane separation, coupled or not with enzyme technology, to purification and fractionation of oligosaccharides, as an example of an integrated process where membrane technology can act in synergy with other unit operations and processes.

6.2 Theoretical fundamentals of membrane separation Implementation of membrane technology in the dairy and fruit juice industry is determined by the economical feasibility of the process, which is directly associated with the separation efficiency and impact of fouling. Several theories and models have been developed in order to relate separation efficiency to the separation set-up, molecular features of solute, solvent and membrane, and operational conditions during filtration. One of these models (Jonsson and Boesen, 1975; Jonsson et al., 2008) is based on the transport of solvent and solute through the membrane pores and the friction of the solute particles and the pore wall. The model relates separation efficiency to several physical parameters: bˆ cm Ê t l Jv ˆ Ê [6.1] = 1 = b + 1 – ˜ exp Á – cp 1 – R K ÁË K¯ Ë e Di ˜¯ where cm and cp are the solute concentrations on the membrane surface and at the permeate, respectively. R is the rejection factor, b is the friction coefficient (dependent on solute and pore diameter), K is the distribution factor between pore fluid and bulk solution, t is a tortuosity factor, l is the real thickness of the fouling layer, e is the fractional pore area, Jv is the volumetric flow per unit membrane area and Di is the diffusion coefficient for a component of the solute. In certain conditions, e.g. high fluxes, concentration polarization can have a particular relevance on membrane performance. There are equations to quantify the influence of concentration polarization (e.g. Jonsson, 1985):

cm – cp ˆ Ê = exp Á J v d ˜ Ë Di ¯ cb – cp


[6.2]

182 Separation, extraction and concentration processes and

Di =

kT 3 ph di

[6.3]

where c b represents the solute concentration in the bulk solution, d the polarization layer thickness, k the Boltzmann’s constant, and h the viscosity. These models and equations can be used as a tool to determine preliminarily, within a certain error interval, the efficiency of a particular process that could eventually be implemented at a large scale.

6.3 Membrane technology in the dairy industry Use of membrane technology in the dairy industry represents ~40% of the total applications of membranes in food processes (Daufin et al., 2001). Both polymeric and ceramic membranes are used for concentration purposes in the dairy industry. Among the polymeric ones, spiral cartridges of polysulfone or polyethersulfone are usually chosen, and tubular ceramic membranes of zirconium or aluminum oxide are also widely used. Ultrafiltration is the most widely used membrane separation process in the dairy industry (D’Souza et al., 2005). Ultrafiltration is used, for example, for concentration of milk before the cheese-making process or to reduce shipping volumes as well as for whey concentration, resulting in a protein-enriched stream which is being examined for novel applications, and which may have potential antiinflammatory and anti-cancer properties. 6.3.1 Fouling as a major concern in the dairy industry Fouling is the major concern for the dairy industry nowadays. In recent years, research efforts have aimed at the development of ultrafiltration systems with enhanced life time that can efficiently reduce the concentration polarization and fouling (Nigam et al., 2008; Rice et al., 2009a). Concentration polarization is provoked by an increasing solute concentration on the membrane surface owing to the transport of solute during convective solvent flux, which is rejected and accumulated on the membrane surface. The accumulation results in a back-diffusion of solute into the solution in the retentate side, hence creating a concentration gradient. As a consequence, an initial drop of permeate flux is observed, followed by a further progressive decrease caused by fouling. During microfiltration of skim milk for removal of bacterial spores, Guerra et al. (1997) observed a peak of permeate flux at 0.25 bar of transmembrane pressure, followed by a continuous decrease of permeate flux at increasing pressures (Fig. 6.1). After reaching 1 bar, the pressure was diminished, resulting in lower permeate fluxes than the ones obtained during


Advances in the effective application of membrane technologies 183 the increasing pressure phase. This confirmed the influence of fouling on the permeate flux. Although theoretically the flux stabilizes when the steady-state is reached, in practice, the flux continues to decrease slowly with continued filtration. Also, fouling reduces dramatically the useful life of membranes (Rice et al., 2009b). The cleaning treatment is a key factor influencing the whole plant productivity. 6.3.2 Cleaning of membranes in the dairy industry D’Souza et al. (2005) made a complete study about the causes of membrane fouling as well as the main cleaning processes used in the dairy industry. They classified the cleaning agents into six different categories: (1) alkalis, which hydrolyze proteins and carbohydrates; (2) acids, for dissolving salts and oxides; (3) enzymes, particularly proteases and lipases; (4) surfactants, which decrease surface tension and charge; (5) sequestrants, for removal of minerals; and (6) disinfectants, since one of the main targets of the cleaning process is also the reduction of microbial load. A cleaning process generally involves three steps: product removal from the membrane and water rinsing; use of a single or a combination of appropriate cleaning agents followed by water rising; and disinfection (Trägårdh, 1989). In most cases, an acid–alkali treatment is used, the concentration of each cleaning agent being a critical parameter for each cleaning situation (Tohammadi et al., 275 250 225 Flux (L h–1 m–2)

200 175 150 125 100 75 50 25 0 0.0

0.1

0.2

0.3

0.4 0.5 0.6 0.7 0.8 Transmembrane pressure (bar)

0.9

1.0

1.1

Fig. 6.1 Permeate flux versus transmembrane pressure during microfiltration of skim milk for removal of bacterial spores using a ceramic membrane.The arrows show the direction in which the transmembrane pressure was changed. Adapted from Guerra et al. (1997).


184 Separation, extraction and concentration processes 2002). Moreover, temperature and mechanical aspects i.e. flow rate of the cleaning solution and applied pressure during the cleaning process have to be carefully selected for each particular situation (Bartlett et al., 1995; Bird et al., 1995). The election of a proper cleaning method is also an important factor to consider in nanofiltration and reverse osmosis processes, which also have a major presence in the dairy industry. Nanofiltration is currently used, for example, to remove salt from whey as well as for the recovery of lactose from the permeate of the ultrafiltration step. Reverse osmosis has proved to be useful for whey concentration and for obtaining concentrated lactose by removing salt and minerals. 6.3.3 Recent advances for fouling reduction in the dairy industry In recent years, patents and publications on membrane technology applied to the dairy industry have focused, almost exclusively, on fouling reduction by (a) proposing new cleaning processes, (b) by optimizing the existing ones or (c) by coupling devices to the membrane set-up particularly designed to reduce the fouling, e.g. rotation/vibration and electric fields (see Section 6.7). Some have reported the advantage of using rotating or vibrating systems to enhance permeate flux and membrane selectivity (Frappart et al., 2006; Genkin et al., 2006; Jaffrin, 2008), providing promising results ready to be extrapolated to large-scale processes. Al-Akoum (2006) found a strong correlation between the permeate flux and the vibration speed in ultrafiltration of soy milk at 25 °C. The permeate flux increased ~10 times when using disks with 6-mm vanes at 11,000 rpm compared with smooth disks at 2500 rpm. Genkin et al. (2006) doubled the critical flux when using transverse vibration on submerged hollow fiber membranes, and with combined axial and transverse vibration, a five-fold increase of critical flux was observed. Frappart et al. (2006) increased the permeate flux from 130 to 230 L h–1 m–2 during recovery of lactose from diluted milk when using a rotating disk with vanes at 2000 rpm compared with a smooth disk at 1000 rpm. The same authors compared the performance of a vibrating system to the rotating disk, concluding that the rotating system is more efficient in reducing the fouling, owing to its higher shear rate. A recent patent (Makardij-Tossonian, 2009) proposes the use of an enzyme solution propelled by injecting gas to synergistically combine the effect of the enzyme solution on the breakdown of the particles causing fouling with the mechanical de-fouling effect promoted by the gas. The patent was especially developed for dairy food and beverage processing. Other inventions are more focused on the use of cleaning solutions than on the use of mechanical systems to prevent fouling. Flemming and Skou (2006) patented a system to reuse the cleaning solution by filtering it through the membrane, providing good results for ultrafiltration of dairy products. Omprakash et al. (2008) found that a 0.2% caustic solution provided the best performance for cleaning of fouled membranes used for concentration of whey protein. Measurement of fouling is another critical factor which


Advances in the effective application of membrane technologies 185 has also been the subject of some patents. Mickols et al. (2008) conceived a system for on-line measurement of fouling in spiral wound membranes with particular potential for the dairy industry. It is based on the difference between acoustic impedances on the outer surface for a cylindrical wall of a permeate collection tube. Other systems applied other physical principles to measure the degree and nature of fouling, but the high costs associated with their implementation on a large scale is still hampering utilization at an industrial level. It can be concluded that the implementation and degree of profusion of membrane technology into the dairy industry over the coming years will be dependent on the economic impact that the several presented strategies for fouling reduction can have on the overall economy of the process. The most promising strategies are: (1) mechanical removal of fouling by using, for example, vibrating systems; (2) optimization of the conditions of use of specific chemical agents; and (3) use of enzymes able to breakdown the structure of the particular compounds causing the fouling, e.g. proteases for whey solutions (Nigam et al., 2008).

6.4 Membrane technology in the fruit juice industry Membrane technology is mainly used for clarification and for concentration of fruit juices, replacing the traditional evaporation (Table 6.1). There are several advantages of membrane processes over evaporation in fruit juices manufacture: (1) a higher quality of the final juice because of the reduction in the loss of aroma and other compounds with nutritional value; (2) lower energy consumption; and (3) versatility of the equipment which can be used to treat different products (Jiao et al., 2004). Use of traditional vacuum evaporation for concentration of fruit juices usually results in generation of off-flavours, the loss of aroma compounds and, in some cases, color degradation. Despite the drawbacks mentioned, vacuum evaporation is still the most widely used technique for concentration of juices in industry (GEA, 2009). This is probably ascribable to the fact that evaporation permits higher concentration efficiency than membrane treatments, which also in general, have, higher operational costs. 6.4.1 Reverse osmosis Amongst the membrane separation techniques used for juice concentration, reverse osmosis is the one of major interest for industry. The main advantage


186 Separation, extraction and concentration processes Table 6.1 Some examples of utilization of membrane technology for concentration and clarification of fruit juices Application

Type of filtration

Conditions

Reference


Reverse osmosis

Spiral wound cellulose acetate and polyamide membranes 21.1–26.7 °C

Chua et al., 2007


Reverse osmosis and ultrafiltration

Plate and frame cellulose acetate membrane 20 °C

Sheu and Wiley, 1983

Clarification of cactus pear juice

Ultrafiltration

Hollow fiber polysulfone membrane 25 °C

Cassano et al., 2007a

Concentration of cactus pear juice


Microporous polypropylene Cassano et al., hollow fiber 28 °C 2007a

Clarification of kiwifruit juice

Ultrafiltration

Hollow fiber polysulfone membrane 20–30 °C

Cassano et al., 2007b

Concentration of pineapple juice

Osmotic evaporation

Polytetrafluoroethylene membrane 25 °C

Hongvaleerat et al., 2008

Concentration of Reverse osmosis blackcurrant juice

Tubular polyethersulfone membrane 25 °C

Banvolgyi et al., 2009

Concentration of noni juice


Hollow fiber polypropylene Valdes et al., 30 °C 2009

Concentration of Membrane blackcurrant juice distillation

Hollow fiber polypropylene Kozak et al., membrane 15 and 19 °C 2009

Recovery of the Vacuum main pear aroma membrane compound (ethyl distillation 2,4-decadienoate)

Hollow fiber module of polypropylene 25 °C

Diban et al., 2009

is that the process is carried out at low temperatures, which results in higher retention of juice components and reduction in the consumption of energy. The efficiency of the process is highly determined by the operational variables and type of membrane. Polyamide and cellulose acetate are the most common materials used for reverse osmosis of fruit juices. Polyamide membranes used for concentration of apple juice have been found to be more resistant, and have provided higher retention of flavors and higher flux than the cellulose acetate ones, and similar results were obtained for other fruit juices (Chua et al., 1987; Sheu et al., 1983; Palmieri et al., 1990). A different configuration also seems to play a main role in the performance of reverse osmosis. The plate and frame configuration have, in general, a higher retention capacity for flavor aromas than the spiral wound, owing to the lower membrane package density and area (Chou et al., 1991). Increases in transmembrane pressure and decreasing temperatures favored the retention of flavor compounds such as ethyl-2-methyl butanoate, hexanol and hexanal in concentration of apple juice (Alvarez et al., 1998; Chou et al., 1991). There are no results


Advances in the effective application of membrane technologies 187 of advances or a significant research focus on reverse osmosis applied to concentration of fruit juices in recent years. This is probably ascribable to the limited capacity of reverse osmosis to achieve high concentration levels. Jiao et al. (2004) set 25–30 °Brix as the maximum achievable concentration of fruit juice when reverse osmosis is used, the low efficiency ascribable to the osmotic pressure, versus ~80 °Brix that can be reached by multistep vacuum evaporation. This suggests the use of reverse osmosis as a pre-step in the concentration process, which is coupled, for instance, to a subsequent evaporation. This is already being implemented in commercial plants (Gadea, 1987), being energy-saving and resulting in a higher production capacity. 6.4.2 Membrane distillation Membrane distillation can be defined as the transfer of a target solute between two solutions subjected to different temperatures and separated by a hydrophobic membrane. The temperature gradient creates a vapor pressure difference between the two interfaces, resulting in a water flux from the high-temperature to the low-temperature side. The volatile compounds evaporate and diffuse and/or convect across the membrane from the feed side, and are condensed in the other side (Khayet et al., 2002; Lawson and Lloyd, 1997). The maximum achievable concentration when using membrane distillation was reported to be 60–70 °Brix, which is very close to the one reachable by traditional evaporation (Jiao et al., 2004). The selectivity of the membrane for each of the components in the feed solution is determined by the vapor–liquid equilibrium. Therefore, the highest permeation rate will correspond to the component with the highest partial pressure (Mulder, 1996; Bandini and Sarti, 1999). The pore size of the membrane used for membrane distillation are usually between 0.2 and 1.0 mm and hydrophobic materials such as polyvinyldifluoride and polytetrafluoroethylene are the most used polymers (Mulder, 1996). The material of the membrane must be carefully selected. If the affinity between the material of the membrane and the properties of the feed solution is too high, an undesired wetting of the membrane can occur (Mengual et al., 2004; Mulder, 1996). As expected, the flux rate across the membrane will increase with higher porosities and thinner membranes. A higher temperature difference between juice and water results in a flux increase, however, temperature cannot be increased indefinitely, as some of the solute components of fruit juice are thermosensitive. As expected, permeate flux only slightly decreases with increasing juice feed concentration compared with reverse osmosis and, in general, flux is higher than for reverse osmosis at high concentration ratios (Calabro et al., 1994). Depending on the method by which the vapor is recovered from the membrane pores, the membrane distillation process can be performed by using several configurations (Fig. 6.2): (a) direct-contact membrane distillation (DCMD) uses an aqueous solution for vapor recovery; © Woodhead Publishing Limited, 2010

188 Separation, extraction and concentration processes Membrane

Aqueous solution

Flow Aqueous solution

Sweep gas Vapor

Vacuum or Air gap

(a) Vacuum Aqueous solution

Aqueous solution

Sweeping gas

(b)

(c) Boundary layers

Cfeed

Cfm

Cpermeate Cpm

Tfeed

Tfm Tpm Tpermeate

Membrane (d)

Fig. 6.2 Membrane distillation processes employed for concentration of juices: (a) cross-sectional view of a hydrophobic membrane in contact with an aqueous solution illustrating the vapor–liquid interfaces in MD; (b) configuration of SGMD; (c) configuration of VMD; (d) schematic illustration of temperature (T) and concentration (C) profiles in VMD and SGMD during the concentration process; membrane–feed boundary layer (fm), membrane–permeate boundary layer (pm).

(b) osmotic membrane distillation (OMD), an osmotic medium; (c) vacuum membrane distillation (VMD) couples to a vacuum system; (d) sweeping gas membrane distillation (SGMD) uses a sweeping gas; and


Advances in the effective application of membrane technologies 189 (e) air-gap membrane distillation (AGMD), a stagnant air gap plus a cold plate. Of these, DCMD has probably been the most studied configuration. About 5 years ago, a very limited number of studies on application of membrane distillation for juice concentration or aroma recovery were available (Calabro et al., 1994; Khayet et al., 2002; Lagana et al., 2000). However, the number of studies on this topic has dramatically increased recently. Calabro et al. (1994) succeed in concentrating orange juice by membrane distillation with total retention of sugars and organic acids, but a considerable degradation of vitamin C caused by oxidation and high temperature. Temperature seems to be the most important factor for process improvement in membrane distillation. Bui et al. (2004) reported a flux increase of 3–4 times when feed temperature was 15 degrees higher in glucose solutions of 30 to 60%. A reduction of the flux is normally caused by viscosity increase when the concentration is higher than 50%. In all the studies, however, membrane distillation is still coupled to a prior evaporation or membrane filtration step. Kozak et al. (2006) used OMD as the final step for concentration of must and apple juice in laboratory and in a large-scale installation. After reverse osmosis, grape and apple juice reached a concentration of 23 °Brix, which increased up to ~62 °Brix by using OMD at 20 °C. No advances in the use of new membrane materials or setups are being investigated for improving membrane distillation performance. Instead, most of the studies are focused on the optimization of the operating variables for each particular fruit, in an attempt to enhance the total soluble solids concentration in the particular juice. Temperature was the major factor in the concentration of ethyl 2,4decadienoate, a pear aroma compound, over feed flow rate, feed concentration and pressure when a commercial hollow-fiber polypropylene membrane was used for VMD (Diban et al., 2009). Changes in temperature were also reported to have a higher effect than feed flux in the concentration of volatile aroma compounds from blackcurrant juice (Bagger-Jørgensen et al., 2004). The low temperature (10 °C) favored the concentration of blackcurrant aroma esters compared with 45 °C by VMD. Kozak et al. (2009) reported an increase of 80% of the permeate flux by increasing the temperature by only four degrees (from 15 to 19 °C) between both sides of the membrane, confirming the dramatic effect of this variable in blackcurrant juice concentration. In this case, a microfiltration treatment was used for pre-concentration, and the membrane distillation contribute to concentrate the juice from 22 to 58.2 °Brix. Many recent works have also measured antioxidant capacity and content of phenolic compounds, which are reported to exert beneficial health effects (Del Rio et al., 2010), in juices subjected to membrane distillation to prove the advantages of this technique over the traditional methods. The antioxidant capacity and the content of phenols remained constant after the membrane distillation treatment in cactus pear juice, noni juice and other red fruit juices (Cassano et al., 2007a; Valdés et al., 2009; Koroknai et al., 2008). The practically non-existent patents on the application of membrane © Woodhead Publishing Limited, 2010

190 Separation, extraction and concentration processes distillation for concentration of fruit juices suggest that interest in this topic is still confined to academic circles. However, the higher concentration degree attainable by using membrane distillation compared with reverse osmosis suggests a major use of this technique for the future. Integrated processes As stated above, reverse osmosis and membrane distillation processes are commonly coupled to pre-evaporation or, to a micro/ultrafiltration step at industrial level. Micro- or ultrafiltration has been proved to be useful not only for clarification purposes, but also to reduce the viscosity of the juice by removing solids and pectin, easing the posterior membrane processes by increasing the permeate flux (Jiao et al., 2004). Johnson et al. (1996) developed a method for concentrating orange juice, combining ultrafiltration for clarification with traditional evaporation, reaching a concentration higher than 80 °Brix. A more complex method has been proposed by Álvarez et al. (2002) for concentrating apple juice, including a membrane bioreactor for clarification, reverse osmosis for pre-concentration, pervaporation to recover aromas and traditional evaporation as a final step. Enzyme treatment and ultrafiltration has become the most usual clarification treatment. It has been used for concentration of blackcurrant juice coupled with reverse osmosis in an integrated process (Banvolgyi et al., 2009). In turn, membrane filtration techniques themselves are sometimes coupled to other techniques with the aim, in most instances of reducing fouling. Fernandes et al. (2007) patented a hybrid method that combines nanofiltration and electrodialysis for concentration and fouling removal of grape must, respectively.

6.5 Membrane technology for treatment of wastewater in the food industry Again, the dairy industry is the first food processing industry incorporating the new advances in membrane technology applied to wastewater treatment. It has been estimated that the dairy industry generates 0.2–11 l of waste water per liter of milk (Daufin et al., 2001). Dairy industry effluents have been reported as one of the highest volume industrial effluents (Wheatley, 1990). Carbohydrates, lipids and proteins are the most abundant organic compounds in the effluents of the dairy industry with a reported polluting charge of 0.2–2.5 g l–1 biological oxygen demand (Daufin et al., 2001; Perle et al., 1995). In general, biological processes are more efficient and costeffective than the physicochemical methods (Vidal et al., 2000). The high energy requirements associated with aerobic treatment plants have turned the research focus toward anaerobic treatments in recent years, with lower sludge production and no aeration requirements (Demirel et al., 2005). However, the membrane aerobic bioreactor is still quite common for treatment of food-processing wastewater in industry (Abdulgader et al., 2007). Membrane © Woodhead Publishing Limited, 2010

Advances in the effective application of membrane technologies 191 separation substitutes the clarification step used in traditional wastewater treatment, giving several advantages: (1) a significant reduction in the amount of sludge generated, (2) the capacity to treat a major volume of water using less space, and (3) a higher quality of the resulting water (Daufin et al., 2001). 6.5.1 Aerobic membrane bioreactor A membrane bioreactor couples the activated sludge process with membrane separation. An efficient pre-treatment, to avoid excessive fouling of the membrane must precede the biological treatment. In general, either microfiltration or ultrafiltration are used to separate the sludge from the treated water. The membrane can be either submerged or external. The most usual set-up is a submerged hollow fiber or plate membrane ultrafiltration membrane (Daufin et al., 2001; Melin et al., 2006). Once again, fouling is the major factor to assure high performance. Fouling depends, besides other general factors such as membrane type and hydrodynamic conditions, on the presence of compounds that must be produced by microbial metabolism or added to the sludge, e.g. polyelectrolytes (Melin et al., 2006). 6.5.2 Anaerobic membrane bioreactor The main advantage of anaerobic treatment compared with aerobic is the dramatic reduction in the amount of produced sludge, which is at least five times lower. In addition, the anaerobic treatment results in the production of gas that can be used to generate energy to be used elsewhere in the plant and allows a degree of degradation of the organic matter higher than 80% (ArrosAlileche et al., 2008). Some authors reported the need to operate at subcritical fluxes to assure an efficient control of fouling (Hughes and Field, 2006). The selection of the membrane is generally based on a complete retention of the biomass and very low solute rejection, critical for good performance of the membrane (Jefferson et al., 2000). Among the most usual membrane materials for membrane bioreactors, polypropylene and polyethylene are being industrially used (Kubota and Mitsubishi). Because fouling is a major factor in these systems, cleaning is a critical step in these operations and it can be done by chemical or mechanical treatments. Back-flushing has been presented as one of the most innovative and efficient mechanical techniques to remove fouling (see Section 6.7).

6.6 New applications of membrane technology for the food industry: concentration and fractionation of saccharides Production of nutri-functional compounds, highly claimed by consumers over the past decade for being able to promote healthy properties of food, has © Woodhead Publishing Limited, 2010

192 Separation, extraction and concentration processes promoted the development of new techniques for production of high-purity compounds, to be used as supplements or additives in food. Prebiotics are a good example. In recent years, many research efforts have targeted the production of oligosaccharide prebiotics for elaboration of products, e.g. beneficial-health supplements or human-like infant milk formulas (Bode, 2009). In most instances, prebiotics are produced by enzymatic break down of a polysaccharide of vegetal origin, which produces an oligosaccharide with bioactive properties (Beine et al., 2008). Membrane technology is the most feasible strategy for purification in industrial manufacture of enzymatically produced oligosaccharides (Table 6.2). However, the lack of systematic understanding of how properties of the membrane and the oligosaccharide affect membrane filtration has limited to a large extent the industrial development of processes for production of prebiotics (Pinelo et al., 2009). Some of the factors related to design of the most advantageous set-up are described in the next section and the influence of certain operational variables which have to be carefully considered to eventually exploit the process at an industrial level is explored. 6.6.1 Design of separation set-up In most small-scale studies, membrane filtration was performed in a batch dead-end system, which is the simplest set-up to operate (Pinelo et al., 2009). The system consists of a tank for the feed solution, which passes across the membrane module. The permeate is recovered in a proper reservoir (Swennen et al., 2005). A magnetic stirrer is commonly used to reduce fouling and to improve the reaction rates on a laboratory scale where the enzymatic reaction and the membrane filtration occur simultaneously (Sanz et al., 2005). Industrially, further research into other types of efficient mixing is required. In the laboratory, Sarney et al. (2000) used a membrane reactor equipped with a magnetic stirrer for the recovery of oligosaccharides from milk using a combined b-galactosidase treatment with nanofiltration. However, when fouling is a major factor for productivity, a cross-flow filtration system is recommended (Ramaswami et al., 2005). In cross-flow, the feed solution flows parallel to the membrane surface, sweeping the retained particles and helping to reduce the concentration polarization. This is probably the most advisable set-up for industrial purposes. A cross-flow system was used for purification of the products resulting from autohydrolysis of rice hulk in a tubular membrane (Vegas et al., 2006). Goulas et al. (2002) reported the same efficiency for dead-end and cross-flow filtration modes in the removal of glucose from enzymatically obtained oligosaccharides, but concluded that the concentration polarization was a major factor limiting the performance of the dead-end system.



Substrate

Type of separation

Commercial powder of oligosaccharides from chicory rootstock

UF for separation of larger Oligosaccarides from UF cutoff: 20 kDa, impurities and NF for removal of 3 to 10 degrees of NF cutoff: 0.5–5 kDa monosaccharides polymerization (DP)

Kamada et al. (2002)

Arabinoxylan hydrolysates Commercial saccharides

Purification by UF (5, 10 and 30 kDa) Removal of di- and monosaccharides Purification of oligosaccarides and removal of lactose by NF Simultaneous concentration and purification by 1 kDa by NF Separation of di- and monosaccharides by NF

Stirred cell at 4 bar

Swennen et al. (2005)

Stirred cell at 50 °C and 4 bar

Sanz et al. (2005)

Caprine milk Liquors from rice husk autohydrolysis Commercial mixture of galacto-oligosaccharides

Product

Arabinoxylooligosaccharides Oligosaccharides Milk oligosaccharides Xylooligosaccharides Raffinose, sucrose and fructose

Commercial mixture of Concentration and purification by Oligosaccharides of saccharides from yacon UF and NF DP from 3 to 10 rootstock Hemicellulose hydrolyzate Purification by NF Xylose stream Enzymatic hydrolytes from vegetal origin Commercial oligosaccharide mixture Liquors from almond shells autohydrolysis

Concentration by NF Purification by NF Separation of lignin-related impurities by UF

Xylooligosaccharides Fructooligosaccharides Xylooligosaccharides

Filtration conditions

Reference

Feed velocity: 80–120 mL, 20–40 bar Sarney et al. (2000) Vegas et al. (2006) TiO2/ZrO2 ceramic membrane at 30 °C and 2–14 bar Feed concentration varied between Goulas et al. (2002) 0.15–0.08 g mL–1, filtration was performed at 25–60 °C and 5–30 bar UF cutoff 10 kDa (0.52 bar) and NF Olano-Martin et al. (2001) cutoff 5 kDa (5 bar) Concentrated feed solution: 20 wt% Retentate flow: 6 L min–1, filtration performed at 40–60 °C and 20–40 bar Feed velocity: 400 mL min–1 pH 5, 48 °C and 16 bar Feed flow: 1850 L h–1, filtration performed at 45 °C and 1 MPa Cutoffs between 1 and 8 kDa Filtration performed at 25 °C and 2.6–9 bar

Machado et al. (2000)

Huang et al. (2001) Espinoza-Gomez et al. (2004), Grassin et al. (1996) Li et al. (2004)

Advances in the effective application of membrane technologies 193

Table 6.2 Some examples of the use of ultrafiltration (UF) and nanofiltration (NF) membranes for the concentration and purification of oligosaccharides of different nature

194 Separation, extraction and concentration processes 6.6.2 Operational modes The integration of enzyme reactor and membrane is normally advantageous for recovery of prebiotic oligosaccharides from vegetal matrixes, mainly because this disposition diminishes product inhibition (Pinelo et al., 2009). The operational conditions for this particular integrated system depend to a large extent on whether the system operates with immobilized or free enzymes. On some occasions, the membrane itself was used for the immobilization. Nishizawa et al. (2000) reported the chemical immobilization of b-fructofuranosidase with glutaraldehyde to the inner surface of a ceramic membrane for production of fructo-oligosaccharides. Although the use of ceramic membranes has been used for purification of oligosaccharides by ultra- and nanofiltration, it is more common to use polymeric organic membranes for oligosaccharide purification (Artug et al., 2007). Cellulosic membranes seem more suitable to this purpose, being hydrophilic, but the use of polysulfone membranes is also common (Akhtar et al., 1995). A chemical surface treatment was recommended for a polysulfone membrane, in order to make it more hydrophilic (Akhtar et al., 1995). The operating variables that had a major influence on membrane filtration of oligosaccharides were: (1) temperature, (2) pH, (3) concentration of the feed solution, and (4) pressure. Temperature It has been reported that the permeate flux almost doubled when the temperature of saccharide solutions increased from 20 to 50 °C. This is ascribable to the reduction of viscosity, which favors the pass of the flow of the feed solution across the membrane (Goulas et al., 2002; Machado et al., 2000). The favorable effect of the temperature increase on the permeate flux was confirmed by Sjöman et al. (2008), during recovery of xylose from hemicellulose hydrolyzate feeds using various polysulfone membranes. pH For certain types of membrane carrying positive or negative charged groups, e.g. polycarbonate and sulfonated polysulfone membranes, the influence of pH on the saccharide rejection can be dramatic (Lazaridou et al., 2007). When the feed solution includes some charged compounds, flux commonly increases when working close to the isoelectric point of the membrane, as expected (Childress and Elimelech, 1996). Variation of pH can be used as a strategy for more efficient separation of charged saccharides, such as pectin, whose charge density and distribution is a function of the carboxyl groups in the structure.


Advances in the effective application of membrane technologies 195 Concentration of the feed solution Permeate flux is expected to decrease with increasing feed concentration of the solution. As a result, a decrease in the rejection, at constant pressure, is usually observed in these cases (Goulas et al., 2002). Pressure The role of pressure and the recommended levels of pressure depend on the type and properties of the solution to be filtered. Generally, the rejection increases with increasing levels of pressure until reaching a certain level. This is ascribable to the higher flux of solute across the membrane, which decreases the difference in concentration between the solutions at both sides of the membrane. When pressure continues increasing, the concentration polarization and, in turn, the concentration of solute on the surface of the membrane increases too. As a consequence, the concentration of solute on the permeate side increases and the rejection decreases. In purification of xylo-oligosaccharides from lignin impurities, Nabarlatz et al. (2007) observed a decrease in rejection when the pressure was increased from 2.6 to 9 bar. It is then advised to operate at low pressures, within a range in which the concentration polarization is not so high, in order to maximize selectivity.

6.7 Future trends Implementation of membrane technology in food industry has been hampered by the economical limitations derived from fouling over recent decades. Many studies have been devoted to develop techniques and processes to reduce the impact of fouling and it seems that progress in membrane technology will be linked to progress in fouling reduction over the next few years. In particular, there are at least three techniques that have provided promising results for their efficiency and simplicity to be incorporated in a large-scale process: (1) high frequency backflushing, (2) vibrating membrane modules, and (3) electrofiltration. Some of the benefits associated with coupling these techniques in the dairy industry have been discussed in Section 6.3 but the applications in other food processes are increasing 6.7.1 High frequency backflushing Backflushing consists of applying pressure pulses from the permeate to the retentate side, and stripping off the particles causing fouling from the membrane surface into the bulk solution at the permeate side again (Pinelo et al., 2009). Backflushing is commonly achieved by placing a valve in the © Woodhead Publishing Limited, 2010


Flux (L h–1 m–2)

250 With backflush

200 150 100 50 0

Without backflush 0

15

30

45

60 75 Time (min)

90

105

120

Fig. 6.3 Influence of backflushing on the permeate flux versus time applied to the microfiltration of skim milk for removal of bacterial species using a ceramic membrane. Adapted from Guerra et al. (1997).

permeate side. The efficiency of backflushing depends both on the duration of the pulse itself and time between pulses. Guerra et al. (1997) reported a considerable increase of permeate flux when backflushing was incorporated to microfiltration of skim milk, as backflushing contributed to the removal of spores from the membrane surface (Fig. 6.3). Jonsson (2008) narrowed the intervals of fractionation of dextrans in a hollow-fiber ultrafiltration system using times between pulses from 1 to 30 s and backflushing times from 0.1 to 5 s. Application of backflushing in industry is still limited but further research in the coming years will permit a more detailed analysis of the balance between costs associated with implementation and economic benefit from fouling reduction. 6.7.2 Vibrating membrane module A vibrating design consists of applying high shear rates to favor the transmission of macromolecules through the membrane (Beier et al., 2007). This design proved to be particularly efficient for nanofiltration of diluted milk and for enzyme separation applied to food processes (Beier et al., 2007; Jaffrin et al., 2004). This system has been already presented in the section dedicated to fouling reduction in the dairy industry, but it is fair to include it here as a future trend technique, as many studies are currently being conducted to optimize the mechanical characteristics of the system for a feasible high performance of membrane separation in the dairy industry.


Advances in the effective application of membrane technologies 197 6.7.3 Electrofiltration Electrofiltration is used when the particles causing concentration polarization have a net charge and can be attracted or repelled by applying an electric field, in this instance across the membrane (Pinelo et al., 2009). This technique has been successfully employed for separation of industrial solutions of enzymes with a surface charge, resulting in a dramatic increase of the permeate flux (Enevoldsen et al., 2007). The technique also gave good results in ultrafiltration of other proteins (Oussedik et al., 2000) and shows promise for purification of potential food ingredients in which charge can be induced, e.g. modifying the number and methoxyl groups on pectin molecules.

6.8 References Abdulgader M E, Yu Q J, Williams P, Zinatizadeli A A L (2007), ‘A review of the performance of aerobic bioreactors for treatment of food processing wastewater’. Proceedings of the International Conference on Environmental management, Engineering, Planning and Economics, pp. 1131–1136. Skiathos. Akhtar S, Hawes C, Dudley L, Reed P, Strafford P (1995), ‘Coatings reduce the fouling of microfiltration membranes’, J Membr Sci 107, 209–218. Al-Akoum O, Richfield D, Jaffrin M Y, Ding L H, Swart P (2006), ‘Recovery of trypsin inhibitor and soy milk concentration by dynamic filtration’, J Membr Sci 279, 291–300. Álvarez V, Riera F A, Álvarez S, Álvarez R (1998), ‘Permeation of apple aroma compounds in reverse osmosis’, Sep Purif Technol 14, 209–220. Álvarez S, Riera F A, Álvarez R, Coca J (2002), ‘Concentration of apple juice by reverse osmosis at laboratory and pilot-plant scale’, Ind Eng Chem Res 41, 6156–6164. Arros-Alileche S, Merin U, Daufin G, Gesan-Guiziou G (2008), ‘The membrane role in an anaerobic membrane bioreactor for purification of dairy wastewaters: A numerical simulation’. Bioresour Technol 99, 8237–8244. Artug G, Roosmasari K, Richau K, Hapke A (2007), ‘A comprehensive characterization of commercial nanofiltration membranes’, Sep Sci Technol 42, 2947–2986. Bagger-Jørgensen R, Meyer A S, Varming C, Jonsson G (2004), ‘Recovery of volatile aroma compounds from black currant juice by vacuum membrane distillation’, J Food Eng 64, 23–31. Bandini S, Sarti G C (1999), ‘Heat mass transport resistances in vacuum membrane distillation per drop’, AIChE J 45, 1422–1433. Banvolgyi S, Horvath S, Stefanovits-Banyai E, Bekassy-Molnar E, Vatai G (2009), ‘Integrated membrane process for blackcurrant juice concentration’, Desalination 241, 281–287. Barlett M, Bird M R, Howell J A (1995) ‘An experimental study for the development of a qualitative membrane cleaning model’, J Membr Sci, 218, 107–116. Beier S P, Jonsson G (2007), ‘Separation of enzymes and yeast cells with a vibrating hollow fiber membrane module’ Sep Purif Technol 53, 111–118. Beine R, Moraru R, Nimtz M, Na’amnieh S, Pawlowski A, Buchholz K, Seibel J (2008), ‘Synthesis of novel fructooligosaccharides by substrate and enzyme engineering’, J Biotechnol 138, 33–41. Bird M R, Barlett M (1995), ‘CIP optimization for the food industry: Relationships between detergent concentration, temperature and cleaning time’, Trans IChemE, 73, 63–70.


198 Separation, extraction and concentration processes Bode L (2009), ‘Human milk oligosaccharides: prebiotics and beyond’, Nutr Rev 67, 183–191. Bui V A, Nguyen M H, Muller J (2004), ‘A laboratory study on glucose concentration by osmotic distillation in hollow fiber modules’, J Food Eng 63, 237–245. Calabro V, Jiao B L, Drioli E (1994), ‘Theoretical and experimental study on membrane distillation in the concentration of orange juice’, Ind Eng Chem Res 33, 1803– 1808. Cassano A, Conidi C, Timpone R, D’Avella M, Drioli E (2007a), ‘A membrane-based process for the clarification and the concentration of the cactus pear juice’, J Food Eng 80, 914–921. Cassano A, Donato L, Drioli E (2007b), ‘Ultrafiltration of kiwifruit juice: Operating parameters, juice quality and membrane fouling’, J Food Eng 79, 613–621. Childress A E, Elimelech M (1996), ‘Effect of solution chemistry on the surface charge of polymeric reverse osmosis and nanofiltration membranes’, J Membr Sci 119, 253–268. Chou F, Wiley R C, Schlimme D V (1991), ‘Reverse osmosis and flavor retention in apple juice concentration’, J Food Sci 56, 484–487. Chua H T, Rao M A, Acree T E, Cunningham D G (1987), ‘Reverse osmosis concentration of apple juice: flux and flavor retention by cellulose acetate and polyamide membranes’, J Food Proc Eng 9, 231–245. D’Souza N M, Mawson A J (2005), ‘Membrane cleaning in the dairy industry: a review’, Crit Rev Food Sci Nutr, 45, 125–134. Daufin G, Escudier J P, Carrere H, Berot S, Fillaudeau L, Decloux M (2001), ‘Recent and emerging applications of membrane processes in the food and dairy industry’, Trans IChemE, 79, 89–102. Del Rio D, Costa L G, Lean M E J, Crozier A (2010), ‘Polyphenols and health: what compounds are involved?’, Nutr Metab Cardiovasc Dis, 20, 1–6. Demirel B, Yenigun O, Onay T T (2005), ‘Anaerobic treatment of dairy wastewaters: a review’, Process Biochem 40, 2583–2595. Diban N, Voinea O C, Urtiaga A, Ortiz G (2009), ‘Vacuum membrane distillation of the main pear aroma juice compound. Experimental study and mass transfer modelling’, J Membr Sci 326, 64–75. Enevoldsen A D, Hansen E B, Jonsson G (2007), ‘Electro-ultrafiltration of industrial enzyme solutions’, J Membr Sci 28–37. Espinoza-Gomez H, Lin S W, Rogel-Hernandez E (2004), ‘Nanofiltration membrane pore diameter determination’, Rev Soc Quim Mex 48, 15–20. Fernandes G V M, Lopes C G, Correia N (2007) ‘Concentration and rectification of grape must involves combining nanofiltration and electrodialysis of grape must in hybrid process’, World Patent WO2008051100-A2. Flemming S, Skou F (2006), ‘Membrane filtration of a product, e. g. milk, in membrane plant, involves recovering cleaning solution after multi-step cleaning of membrane system in plant; and using recovered cleaning solution for cleaning membrane system’, European Patent EP1726353. Frappart M, Akoum O, Ding L H, Jaffrin M Y (2006), ‘Treatment of dairy process waters modeled by diluted milk using dynamic nanofiltration with a rotating disk module’, J Membr Sci 282, 465–472. Gadea A (1987), ‘Reverse osmosis of orange juice’. In Proceedings of the International fruit juice congress, Orlando, USA. GEA (2009) Report on ‘Evaporation technology for the juice industry’, GEA Wiegand GmbH, www.gea-wiegand.com. Accessed on Dec 15, 2009. Genkin G, Waite A G, Fane S, Chang S (2006), ‘The effect of vibration and coagulant addition on the filtration performance of submerged hollow fiber membranes’, J Membr Sci 281, 726–734. Goulas A K, Kapasakalidid P G, Sinclair H R, Rastall R A, Grandison A S (2002),


Advances in the effective application of membrane technologies 199 ‘Purification of oligosaccharides by nanofiltration’, J Membr Technol 209, 321– 335. Grassin C, Fauquembergue P (1996), ‘Fruit Juices’, in: T. Godfrey, S. West (Eds.), Industrial Enzymology, Macmillan Press. Guerra A, Jonsson G, Rasmussen A, Nielsen E W, Edelsten D (1997), ‘Low cross-flow velocity microfiltration of skim milk for removal of bacterial spores’, Int Dairy J 7, 849–861. Hongvaleerat C, Cabral L M C, Dornier M, Reynes M, Ningsanond S (2008), ‘Concentration of pineapple juice by osmotic evaporation’, J Food Eng 88, 548–552. Huang R Y M, Shao P, Nawawi G, Feng X, Burns C M (2001), ‘Measurements of partition, diffusion coefficients of solvents in polymer membranes using rectangular thin-channel column inverse gas chromatography’, J Membr Sci 188, 205–218. Hughes D, Field R W (2006), ‘Crossflow filtration of washed and unwashed yeast suspensions at constant shear under nominally sub-critical conditions’, J Membr Sci 280, 89–98. Jaffrin M J (2008), ‘Dynamic shear-enhanced membrane filtration: a review of rotating disks, rotating membranes and vibrating systems’, J Membr Sci, 324, 7–25. Jaffrin M Y, Ding L H, Akoum O, Brou A (2004), ‘A hydrodynamic comparison between rotating disk and vibratory dynamic filtration systems’, J Membr Sci 242, 155–167. Jefferson B, Laine A T, Judd S J, Stephenson T (2000), ‘Membrane bioreactors and their role in wastewater reuse’, Water Sci Technol 41, 197–204. Jiao B, Cassano A, Drioli E (2004), ‘Recent advances on membrane processes for the concentration of fruit juices: a review’, J Food Eng 63, 303–324. Johnson J R, Braddock R J, Chen C S (1996), ‘Flavor losses in orange juice during ultrafiltration and subsequent evaporation’, J Food Sci 61, 540–543. Jonsson G (1985), ‘Molecular weight cut-off curves for ultrafiltration membranes of varying pore sizes’, Desalination 53, 3–10. Jonsson G (2008), ‘Tuning of the cut-off curves by dynamic ultrafiltration’, Proceedings of the International Conference on Membranes and Membrane Processes: ICOM2008, Hawaii, July 12–18. Jonsson G, Boesen C E (1975), ‘Water and solute transport through cellulose acetate reverse osmosis membranes’, Desalination 17, 145–165. Kamada T, Nakajima M, Nabetani H, Iwamoto N S (2002), ‘Availability of membrane technology for purifying and concentrationg oligosaccharides’, Eur Food Res Technol 214, 435–440. Khayet M, Godino M P, Mengual J I (2002), ‘Thermal boundary layers in sweeping gas membrane distillation processes’, AIChE J 48, 1488–1497. Koroknai B, Csanadi Z, Gubicza L, Belafi-Bako (2008), ‘Preservation of antioxidant capacity and flux enhancement in concentration of red fruit juices by membrane processes’, Desalination 228, 295–301. Kozak A, Bekassy E, Vatai G (2009), ‘Production of black-currant juice concentrate by using membrane distillation’, Desalination 241, 309–314. Kozak A, Rektor A, Vatai G (2006), ‘Integrated large-scale membrane process for producing concentrated fruit juices’, Desalination 200, 540–542. Lagana F, Barbieri G, Drioli E (2000), ‘Direct contact membrane distillation: modeling and concentration experiments’, J Membr Sci 166, 1–11. Lawson K W, Lloyd D R (1997), ‘Membrane distillation’, J Membr Sci 124, 1–25. Lazaridou A, Biliaderis C G (2007), ‘Molecular aspects of cereal beta-glucan functionality: physical properties, technological applications and physiological effects’, J Cereal Sci 46, 101–118. Li W, Li J, Chen T, Chen C (2004), ‘Study on nanofiltration for purifying fructooligosaccharides II. Extended pore model’, J Membr Sci 258, 8–15. Machado D R, Hasson D, Semiat R (2000), ‘Effects of solvent properties on permeate through nanofiltration membranes. Part II: transport model’, J Membr Sci 166, 6–69.


200 Separation, extraction and concentration processes Makardij-Tossonian A A (2009), ‘Membrane regeneration’, World Patent WO/2009/089587. Melin T, Jefferson B, Bixio D, Thoeye C, De Wilde W, De Koning J, van der Graaf J, Wintgens T (2006), ‘Membrane bioreactor technology for wastewater treatment and reuse’, Desalination 187, 271–282. Mengual J I, Khayet M, Godino M P (2004), ‘Heat and mass transfer in vacuum membrane distillation’, Int J Heat Mass Transfer 47, 865–875. Mickols W, Koreltz M S, Moll D J, Streeter D B, Mickols W, Kobeltz M S (2008), ‘Spiral wound module assembly for e.g. in-situ on-line and real-time measurement of membrane fouling in spring wound module, has membrane envelopes and acoustic transducers which are located adjacent to permeate collection tube’, World Patent WO2008103864. Mulder M (1996), Basic principles of membrane technology, Dordrecht/Boston/London: Kluwer Academic Publishers. Nabarlatz D, Torras C, Garcia-Valls R, Montane D (2007), ‘Purification of xylooligosaccharides from almond shells by ultrafiltration’, Sep Purif Technol 53, 235–243. Nigam M O, Bansal B, Chen X D (2008), ‘Fouling and cleaning of whey protein concentrate fouled ultrafiltration membranes’, Desalination 218, 313–322. Nishizawa K, Nakajima M, Nabetani H (2000), ‘A forced flow membrane reactor for transfructosylation using ceramic membrane’, Biotechnol Bioeng 68, 92–97. Olano-Martin E, Mountzouris K C, Gibson G R, Rastall R A (2001), ‘Continuous production of pectic oligosaccharides in a membrane enzyme reactor’, J Food Sci 66, 966–971. Omprakash M, Bansal B, Cheng D X (2008), ‘Fouling and cleaning of whey protein concentrate fouled ultrafiltration membranes’, Desalination 218, 313–322. Oussedik S, Belhocine D, Grib H, Loucini H, Piron D L, Nameri N (2000), ‘Enhanced ultrafiltration of bovine serum albumin with pulsed electric field and fluidized activated alumina’, Desalination 127, 59–64. Palmieri L, Dalla Rosa M, Dall’Aglio G, Carpi G (1990), ‘Production of kiwifruit concentrate by reverse osmosis process’, Acta Horticulturae 282, 435–439. Perle M, Kimchie S, Shelef G (1995), ‘Some biochemical aspects of the anaerobic degradation of dairy wastewater’, Water Res 29, 1549–1554. Pinelo M, Jonsson G, Meyer A S (2009), ‘Membrane technology for purification of enzymatically produced oligosaccharides: molecular and operational features affecting performance’, Sep Purif Technol, 70, 1–11. Ramaswami H S, Marcotte M (2005), Food processing: principles and applications, CRC Press. Rice G, Barber A, O’Connor A, Stevens G, Kentish S (2009a), ‘Fouling of nanofiltration membranes by dairy ultrafiltration permeates’, J Membr Sci 330, 117–126. Rice G, Kentish S, O’Connor A, Barber A, Pihlajamäki A, Nyström M, Stevens G (2009b), ‘Analysis of separation and fouling behaviour during nanofiltration of dairy ultrafiltration permeates’, Desalination 236, 23–29. Sanz M L, Polemis N, Morales N, Corzo A (2005), ‘In vitro investigation into the potential prebiotic activity of potential oligosaccharides’, J Agric Food Chem 53, 2914–2921. Sarney D B, Hale C, Frankel C, Vulfson E N (2000), ‘A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration’, Biotechnol Bioeng 69, 461–467. Sheu M J, Wiley R C (1983), ‘Preconcentration of apple juice by reverse osmosis’ J Food Sci, 48, 422–429. Sjöman E, Mänttäri M, Nyström M, Koivikko H, Heikkilä H (2008), ‘Xylose recovery by nanofiltration from different hemicellulose hydrolyzate feeds’, J Membr Sci 310, 268–277.


Advances in the effective application of membrane technologies 201 Swennen C M, Coyrtin B, Bruggen C, Vandecasteele C, Gibson G R, Rastall R A (2005), ‘Ultrafiltration and ethanol precipitation for isolation of arabinoxylooligosaccharides with different structures’, Carbohydr Polym 62, 283–292. Tohammadi T, Madaeni S S, Moghadam M K (2002), ‘Investigation of membrane fouling’, Desalination, 153, 155–160. Trägårdh G (1989), ‘Membrane cleaning’, Desalination, 71, 325–335. Valdés H, Romero J, Saavedra A, Plaza A, Bubnovich V (2009), ‘Concentration of noni juice by means of osmotic distillation’, J Membr Sci 330, 205–213. Vegas R, Luque S, Alvarez J R, Alonso J L, Dominguez H, Parajo R C (2006), ‘Membraneassisted processing of xylooligosaccharides-containing liquors’, J Agric Food Chem 54, 5430–5436. Vidal G, Carvalho A, Mendez R, Lema J M (2000), ‘Influence of the content in fats and proteins on the anaerobic biodegradability of dairy wastewaters’, Bioresour Technol 74, 231–239. Wheatley A (1990), Anaerobic digestion: a waste treatment technology. London and New York: Elsevier Applied Science.



7 Electrodialytic phenomena, associated electromembrane technologies and applications in the food, beverage and nutraceutical industries L. Bazinet, A. Doyen and C. Roblet, Laval University, Canada Abstract: Electrodialysis, an electrochemical separation process with charged membranes stacked to separate ionic species from aqueous solutions and uncharged components when an electrical field is applied, is providing new membrane separation processes with numerous applications in the food, nutraceutical and beverage industries. Techniques such as electrolysis with membrane, electrodialysis with ionexchange membranes, electrodialysis with bipolar membrane and electrodialysis with filtration membrane have been used for, among other applications, the coagulation of protein, the electroreduction of the medium and/or the fractionation of several food proteins. Moreover, electrodialysis with filtration membranes has been used for the recovery of molecules with bioactive properties such as antioxidant, anticancer and antihypertensive peptides. Key words: electrodialysis, electromembranes, nutraceuticals, biomolecules, purification, separation.

7.1 Introduction Electrodialysis (ED) is one of a group of membrane-based separation technologies which are finding increasing use, in agri-food industries to concentrate, purify or modify foods. In ED, an electric field provides the driving force and porous or non-porous membranes perform the separation: electrodialysis is a combined method of dialysis and electrolysis (Shaposhnik and Kesore, 1997). ED can be performed with two main cell types: electrolysis (or electro-electrodialysis) cells for oxido-reduction reactions and multimembrane cells for dilution–concentration, water dissociation and purification applications. The electrolysis cell operates with only one membrane that © Woodhead Publishing Limited, 2010


separates two solutions circulating in each electrode compartment. This application is based on electrode redox reactions that are electrolysis-specific properties. In multi-membrane cells only the membrane transport phenomena intervenes, whereas electrochemical reactions occurring at the electrodes do not interact with the separation process. The principles of electrode and membrane reactions, as well as the technologies associated with these electrodialytic phenomena are reviewed in this chapter. The specific applications of electrodialysis and electrolysis currently used and those under development in the food, beverage and nutraceutical industries are presented.

7.2 Principles of electrodialytic phenomena and associated membrane technologies 7.2.1 Membrane phenomena An ion-exchange membrane is made of a macromolecular material (skeleton) which carries ionizable groups such as ion-exchange resins. The membrane contains fixed ions firmly attached to the skeleton and is electronically neutralized by mobile charges of the opposite sign, called counterions. Counterions, which carry current in the membrane, are positive in the case of cation-exchange membranes (CEM), and negative for anion-exchange membranes (AEM) (Bazinet, 2005). Both of these membranes are monopolar; this means that they are permeable to only one type of ion (Gardais, 1990). The perm-selectivity of the membrane is the result of an electrostatic repulsion called Donnan exclusion (Donnan, 1911). For a cation-exchange membrane (CEM), anions are repulsed from the membrane thus allowing only cations to migrate through the membrane. 7.2.2 Electrode phenomena In membrane electrolysis, membrane selectivity, explained above, and electrolysis phenomenon are both active. In electrolysis, an external potential difference is applied to the cell and chemical reactions occur at the electrode–solution interface. The faradaic reactions, caused by the passage of a current, are characterized by electron transfer at the electrode–solution interface. The oxidation A Æ Az+ + z e– induces the loss of one or more electrons and the reduction Bn+ + n e– Æ B induces the gain of one or more electrons. The anode induces oxidation reactions, and reduction reactions occur at the cathode. This transfer always occurs at the electrode surface (Brett and Oliveira-Brett, 1994; Gardais, 1990).



7.3 Applications of electrodialytic phenomena and associated membrane technologies Only one type of membrane technology is associated with electrode reactions (electrolysis with membrane) whereas three types are associated with membrane phenomena (electrodialysis with ion-exchange membranes, electrodialysis with bipolar membrane, and, recently, electrodialysis with filtration membranes) (Fig. 7.1). 7.3.1 Electrolysis with membrane Two main applications of electrode reactions are electrochemical coagulation (EC) and electroreduction. Both methods are exploratory, but show an interesting potential for applications in the food, beverage and nutraceutical industry (Table 7.1). An example of both applications will be developed. Milk protein electrochemical coagulation Khidirov and Merzametov (1982) used EC to precipitate milk proteins in an electrolysis cell separated by a ceramic diaphragm. The milk is poured into the anodic compartment and an electrolyte or a whey solution is poured into the cathodic compartment. Proteins coagulate on the platinum electrode by forming a highly dense white coagulum. Every type of milk could be treated for protein electrochemical coagulation without the use of rennet. Both the caseins and the whey proteins coagulate at the anode. Janson and Lewis (1994) studied the possible use of electrochemical coagulation to directly separate up to 73.8% of the total cheese whey proteins. The whey was circulated in the anodic compartment of an electrolysis cell with a non-ion-selective membrane, made of cotton, for the acidification–coagulation phase (Janson et al., 1990). Afterwards the solution was separated into the coagulum and an impoverished protein solution. This solution was then circulated in the cathodic compartment of the electrolysis cell to undergo the alkalinization phase. Enhancement of lipid stability of omega-3 enriched commercial milk Consumer demand for specific nutritional qualities is encouraging the dairy industry to develop products supplemented in omega-3 fatty acids. Although these fatty acids are known to have many health benefits, they are extremely susceptible to oxidative deterioration which causes difficulties during their storage. In the study by Haratifar (2008) an electroreduction process was performed to modify the redox state of omega-3 enriched milk. A 4-V electroreduction treatment was applied for 1 h on pasteurized omega-3 enriched milk, at room temperature. The electroreduction treatment reduced the redox potential value of omega-3 enriched milk samples quickly and decreased their dissolved oxygen concentration. The electroreduced and control samples were stored at room temperature for up to 3 weeks in the presence of fluorescent light and


(b)

2H2O +

2e– + 2H2O H+

O2 + 4H+ + 4e– OH–

Anode

H2 +

Desalted solution

AEM CEM

AEM CEM

Cl–

– Cathode

+ Anode


2OH–

Cl

–

Cl Na

Cl–

–

Na+ Na+

+

–

Na+

Cathode n

Feed solution (c)

Anionic/acid fraction

AEM FM

Neutral fraction

FM CEM

FM

P+/– + Anode

A

–

P–

P+ A– C+ P–

P+

A

(d)

Cationic/basic fraction

–

P–

FM CEM P+/–

Cl–

A– P+ P+ C

NaOH solution

AEM CEM BPM AEM Cl–

HCl solution CEM BPM AEM H+

H+

+

C

P–

+

–

+

Cathode

Anode

OH Na+

–

Na+ Cl–

Cl

–

Na+

n Feed solution

OH– Na+

Cl–

CEM

Cl–

–

Na+

Na+ Cathode n

Salt solution

Fig. 7.1 Electrodialytic phenomena and associated membrane configurations: (a) redox reaction; (b)–(d) membrane reactions. (a) Electrolysis with membrane; (b) electrodialysis with ion-exchange membranes; (c) electrodialysis with filtration membranes; and (d) electrodialysis with bipolar membranes. AEM, anion-exchange membrane; CEM, cation-exchange membrane; BPM, bipolar membrane; FM, filtration membrane; A–, anion; C+, cation; P–, anionic peptide; P+, cationic peptide; P+/–, neutral peptide.


(a)

206 Separation, extraction and concentration processes in the dark and the composition in fatty acids of milk samples was measured by gas chromatography with flame ionization detection. It was shown that storage under fluorescent light involved a degradation of the fatty-acids, whereas the electroreduction treatment slowed down the oxidative degradation of electroreduced samples compared with untreated milk samples. Results of this study show that the electroreduction treatment can be a potential method of enhancing the shelf-life of products containing unsaturated fatty acids. 7.3.2 Electrodialysis with ion-exchange membranes Conventional electrodialysis or electrodialysis with ion-exchange membranes consist of a series of cation- and anion-exchange membranes arranged in an alternating pattern between an anode and a cathode to form individual cells. Under the influence of an applied potential gradient between cathode and anode, the positively charged cations migrate towards the cathode and negatively charged anions towards anode (Bazinet, 2005, Bazinet and Firdaous, 2009b). The main applications of ED and dilution–concentration in the food industry consist of the demineralization of milk, sugar and whey. In the 1990s, a new application was developed for tartaric stabilisation of wine. The use of ED in membrane bioreactors, for protein separation and acid production was studied. The main applications of conventional ED are presented in Table 7.1, and the application on tartaric stabilization of wine is described in detail here. The use of ED for tartaric stabilization of wine has been approved by the Council of Europe in 1998 and is presently used worldwide on an industrial scale. Wine contains tartaric acid (H2T), a dicarboxylic acid which dissociates in the tartrate (HT–) and bitartrate (T2–) forms. During alcoholic fermentation, potassium and tartrate ions associate and precipitate to form potassium tartrate crystals (KHT) (Gonçalves et al., 2003). As a consequence, at normal storage temperatures, an undesirable KHT precipitation occurs in wine bottles. The ED process for wine tartaric stabilization was developed by Escudier et al. (1995). The principle is the same as for the conventional ED process with a diluate compartment in which the wine to be treated circulates, under nitrogen bubbling, and a concentrate compartment where salts extracted from the wine are recovered. According to the authors, the level of wine deionization, based on the decrease of the conductivity, should be 5 to 20% to obtain tartaric stabilization of wine. One of the main advantages of this technique is the preservation of the organoleptic properties of wine during the stabilization process. 7.3.3 Electrodialysis with filtration membranes Bazinet et al. (2005) stacked filtration membranes and ion-exchange membranes in a conventional electrodialysis cell. This technology, named electrodialysis


Technology

Substrate



References

Dissolved oxygen




Fruit juices

Dissolved oxygen

Increase in shelf-life


Hekal, 1983 Swanson and Sommer, 1940

Milk

W-3 fatty acids

Protection against lipid oxidation


Haratifar, 2008

Dissolved oxygen




Enhancement of probiotic bacteria growth


Bolduc et al., 2006


Mondal and Lalvani, 2003

Electrode reactions Electrolysis with Coffee membrane © Woodhead Publishing Limited, 2010

Oil

Trans fatty acid

Decrease trans fatty acid content

Water

Electroreductive compounds

Increase purity and quality Food of tap water (in development)

Crandall et al., 2001 Koseki et al., 2003 Mercier, 1999

Increase microbiological quality of water


Kim et al., 2000 Thompson and Gerson, 1985

Electroreduction of whey protein



Production of protein isolate


Janson and Lewis, 1994

Whey

Whey proteins


Table 7.1 Electrodialytic phenomena and associated applications in the food, beverage and nutraceutical industries

Table 7.1 Continued Substrate

Membrane reactions Electrodialysis Maple sap with ion-exchange membranes Milk © Woodhead Publishing Limited, 2010



References

Calcium



Food (in development) Food (industrial scale) Food (in development) Food (industrial scale)

Bolzer, 1985

Casein Minerals

Passion fruit Citric acid juice Sugar beet juice, Minerals sugar cane and molasses Soy tofu whey Minerals

Whey

Magnesium or calcium Lactic acid

To avoid sugar sand formation during maple syrup production Production of acid caseinate Demineralization of milk for further applications Deacidification of fruit juice Demineralization of sugar syrups Use of soy tofu whey as bacteria growth medium Recovery of coagulant agent Production of biological preservative agent

Minerals

Demineralization of whey for further applications

Propionic acid

Production of yeast inhibitor Whey protein separation

Whey

Food (in development) Food (in development) Food and biopharmaceutical (in development) Food (industrial scale) Food (in development) Food (in development)

Hiraoka et al., 1979 Vera Calle et al., 2002, 2003 Chaput, 1979 Ben Ounis et al., 2008 Bazinet et al., 1999b Boyaval et al., 1987 Glassner, 1992 Houldsworth, 1980 Pérez et al., 1994 Boyaval et al., 1993 Amundson et al., 1982 Slack et al., 1986; Stack et al., 1995


Technology

Electrodialysis with Apple juice bipolar membranes

Potassium

To avoid potassium bitartrate formation

Polyphenol oxidase Inhibition of enzymatic browning

Food (industrial scale)

Audinos et al., 1979, 1985 Escudier et al., 1995 Guérif, 1993


Quoc et al., 2000, 2006 Tronc et al., 1997, 1998


Chitosan

Chitosanase

Chitosanase inhibition and Nutraceuticals optimization of chitosane (in development) oligomer production

Lin Teng Shee et al., 2008

Milk

Casein



Bazinet et al., 1999a, 2001

Passion fruit juice

Citric acid

Deacidification of fruit juice


Vera-Calle et al., 2002, 2003

Soybean

Soy proteins



Bazinet et al., 1996, 1997b, 1997c, 1998 Mondor et al., 2004 Skorepova and Moresoli, 2007

11S and 7S

Fractionation of 7S and 11S fractions



Soy proteins

Recovery of soy proteins



Calcium or magnesium

Recovery of coagulant agent



a-lactalbumin (a-la) Production of a-la and and b-lactoglobulin b-lg enriched fractions (b-lg)


Bazinet et al., 2004a, 2004b

Soy tofu whey

Whey

Phospholipids

Production of phospholipid Food and nutraceuticals Lin Teng Shee et al., 2005, (in development) 2007 and protein enriched fractions


Wine

Technology


Electrodialysis with filtration membranes and electromembrane filtration

Substrate



References

Whey protein

Prodution of isolate



Lactic acid

Production of biologic preservative agent

Food and biopharmaceutical (in development)

Norddahl, 1998 Norddahl et al., 2001

Peptide VW

Production of an ACE inhibitor peptidic fraction

Food, nutraceutical and bio-pharmaceutical (in development)

Firdaous et al., 2009 Bazinet and Firdaous, 2009a

Cranberry juice Proanthocyanidins and anthocyanins

Production of antioxidant enriched fruit juices

Food and nutraceutical (in development)

Bazinet et al., 2009 Bazinet and Firdaous, 2009b

Green tea

Catechin

Production of antioxidants Food, nutraceutical and bio-pharmaceutical (in development)

Labbé et al., 2005

Milk

as2-Casein hydrolysate (fraction 183-207)

Bioactive peptide separation

Bargeman et al., 2000, 2002

Snow crab

Protein hydrolysate Production of an anticancer Nutraceutical and peptidic fraction biopharmaceutical (in development)

Doyen et al., 2010

Whey

ACE inhibitor bioactive b-Lactoglobulin peptide separation hydrolysate (fraction 142–148)


Bazinet et al., 2005 Poulin et al., 2006, 2007

Lactoferrin


Ndiaye et al., 2010

Alfalfa white protein

Separation of lactoferrin from whey



Table 7.1 Continued


with filtration membranes (EDFMs), couples size-exclusion capabilities of porous membranes with the charge selectivity of electrodialysis (ED). Because no pressure is applied in the electrodialysis cell, the electric field is the only driving force (Bazinet and Firdaous, 2009b). This technology has already been tested for the separation/purification of various high-added-value bioactive molecules (Table 7.1) such as anticancer peptides and antioxidant polyphenols that will be discussed in more detail. Production of an anticancer peptidic fraction Protein hydrolysates from marine products have interesting bioactive properties such as antithrombotic, antihypertensive (Kim and Mendis, 2006), antioxidant (Amarowicz and Shahidi, 1997), anticancer (Picot et al., 2006) and antimicrobial activities (Beaulieu et al., 2010). Recently, a snow crab by-product hydrolysate, has demonstrated antibacterial inhibition properties against specific strains such as Aeromonas hydrophila, Vibrio vulnificus and Vibrio parahaemolyticus (Beaulieu et al., 2010). Further investigations on the bioactive properties of polypeptides originating from this specific snow crab by-product hydrolysate were performed by Doyen et al. (2010). They carried out an electroseparation of the mixture at pH 9, 6 and 3. An ultrafiltration membrane was placed in an electrodialysis cell on both side of hydrolysate compartment allowing the simultaneous separation and recovery of anionic and cationic peptides. The pH values of the recovery compartments were also maintained at pH 9, 6 and 3 during all the separation process. The authors concluded that a selective separation was obtained by the recovery of two anionic and three cationic peptides in the adjacent compartment. They also tested the peptidic fractions for their anti-cancer activity. The results showed that only one fraction amongst the six fractions produced demonstrated a significant inhibition of growth cancerous lines especially on A549 (lung), BT549 (breast) and PC3 (prostate) cell lines. Furthermore, no anticancer aspect was observed for the feed hydrolysate fractions before separation. Consequently, the authors concluded that the anticancer activity was obtained after purification by electrodialysis with ultrafiltration membranes. Indeed, at a low concentration of 190 mg mL–1, cellular mortality of 75–85% for the A549 cell line, of 80% for the BT549 and of 95–100% for the PC3 cell line were obtained. Although the mechanism of action is still unknown, the future perspectives are very interesting because it is possible to isolate each peptide of the fractions recovered and to test them to determine the peptide(s) sequence(s) of interest. Antioxidant enrichment of cranberry juice Cranberry fruits are a rich source of phenolic phytochemicals including phenolic acids, flavonoids and ellagic acids (Vattem et al., 2005). Many of these compounds have antioxidant activity, equal to or greater than vitamin E (Yan et al., 2002). Cranberry juice can also prevent gastric ulcers caused


212 Separation, extraction and concentration processes by Helicobacter pylori (Shmuely et al., 2007) and displays potent anticancer activity (Neto et al., 2008). Bazinet et al. (2009) circulated in both compartments on each side of the filtration membrane a volume of cranberry juice (respectively, 450 mL and 200 mL) to concentrate a cranberry juice in its own specific compounds. The total concentrations of proanthocyanidins and anthocyanins increased by 34.8 and 52.9%, respectively in the 200 mL cranberry juice treated by EDFM with a 500 kDa filtration membrane. Moreover, a 18% increase of the antioxidant capacity was measured by means of the oxygen radical absorbance capacity (ORAC) test in the 200 mL of enriched cranberry juice. Moreover, the taste of the enriched cranberry juice was better than the non-treated juice. Based on these results the authors concluded that the production of phenolic antioxidant enriched cranberry juice could be feasible on a large scale and proposed an integrated process flow. According to this process flow, the EDFM process would be directly connected to the bottling process of cranberry juice to produce antioxidant-enriched cranberry juices by a batch process and cranberry juice with very low variation in antioxidant in a continuous process. An advantage of this technology is that compounds of interest would be directly transferred from one juice to another without the need for solvent to extract them first. 7.3.4 Electrodialysis with bipolar membranes A new type of membrane, called a bipolar membrane (BPM), appeared commercially at the end of the 1980s. Bipolar membranes carry out the dissociation of water in the presence of an electric field. These membranes are composed of three parts: an anion-exchange layer, a cation-exchange layer, and a hydrophilic transition layer at their junctions (Mani, 1991). In electrodialysis with bipolar membranes (EDBPMs), cation- and anion-exchange membranes are stacked together with bipolar membranes in an alternating series in an electrodialysis cell and allow its application to numerous products (Table 7.1). Amongst these applications, only the deacidification of fruit juices and the fractionation of 11S-7S soybean protein is developed. Deacidification of passion fruit juices The yellow passion fruit, Passiflora edulis f. flavicarpa, has an intense and special aroma and flavour, which make it a desirable ingredient in the formulation of various food products. However, because of its high acidity, only limited amounts of juice can be added as an ingredient in the formulation of various preparations (Adhikary et al., 1983; Couture and Rousseff, 1992). To overcome this Vera Calle et al. (2002, 2003) tested the deacidification of clarified passion fruit juice by electrodialysis with bipolar membranes (EDBPMs). The stack was equipped with homopolar and bipolar membranes, forming two or three compartments. The reduction of acidity was achieved by increasing the pH from 2.9 to 4.0. This pH limit was chosen to avoid



micro-organism growth and spoilage (Vera Calle et al., 2002). In these EDBPM configurations only anions were able to pass through the anionexchange membranes from the juice to the concentrate compartment. The net effect was the extraction of anions, mainly citrate, and their replacement by hydroxyl ions provided by the BPM. Citric acid was formed in the concentrate compartment by citrate ions extracted from juice and protons provided by the second BPM separating the concentrate compartment from the electrode compartments. The anion concentration was similar for the two processes. The inorganic ions were almost eliminated, and 62% of the citrate ions and 48% of the malate ions were removed from the fresh juice. According to the sensory properties of deacidified juices, no significant differences were observed between the deacidified juices and the fresh juice. 11S–7S fractionation The two major reserve soybean proteins, the globulins 7S or b-conglycinin (37–39% of total protein) and 11S or glycinin (31–44% of total protein) have different intrinsic properties leading to different functional properties. From results presented by Bazinet et al. (2000), it appears that during the EDBPM process, the temperature has a large effect on the selective precipitation of the soybean protein fractions, 11S and 7S. Hence, at 27 °C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of these fractions. However, at 10 °C, the 11S globulin precipitates at a higher pH than it does at 27 °C, respectively, pH 6.7 compared with 5.9, allowing the fractionation of 11S from the other fractions. Using electroacidification, it is possible to obtain a precipitate solution enriched in the 11S fraction (71.8% of 11S and 10.8% of 7S) and a supernatant solution enriched in the 7S fraction (46.6% of 7S and 4.6% of 11S). EDBPM appears to be a promising technology for fractionation of proteins, by adjusting the electroacidification and solution parameters. This technology does not denature protein (Bazinet et al., 1996), does not use any chemical acids or bases during the process, and produces isolates with a low ash content, which results in a final product of high purity.

7.4 Future trends In this chapter, an overwiew of electromembrane technologies and their associated applications has been presented. Numerous studies have already been performed on various raw matrices (milk, soya, whey, fruit juices) using different cell configurations, but new applications await discovery. Very recently electromembrane processes allowed the separation of bioactive peptides from snow-crab by-products (Doyen et al., 2010) and alfalfa protein hydrolyzates (Firdaous et al., 2009), and the enrichment of cranberry juices in antioxidant compounds (Bazinet et al., 2009). In addition, the low energy © Woodhead Publishing Limited, 2010

214 Separation, extraction and concentration processes cost and constant selectivity during separation of EDFM by absence of transmembrane pressure and, consequently, no membrane fouling, represent an improvement over the conventional separation processes used in the agrifood industries. Furthermore, many acid or alkaline solutions traditionally used in conventional processes are generated in situ by the EDBPM cell configuration. Based on these observations, it appears that electromembrane technologies present several advantages in agri-food industries, in particular, enrichment of nutraceutical products to improve their health benefits, in response to customer demands. In the future, numerous aspects will have to be explored to enhance the understanding of the technologies. Hence, studies on (1) protein/peptide interactions with filtration and ion exchange membranes (IEM) interfaces during treatment/separation of hydrolyzate solutions, and (2) the role of hydrolyzate mineral content on the modification of peptide theoretical charges and influence on their migration rates, would be of major interest. The understanding of both phenomena could lead to the development of new membrane materials and applications, and consequently, to promising ways for new products manufacturing.

7.5 References Adhikary, S.K., Harkare, W.P., Gowindan, K.P. and Nanjundaswamy, A.M. (1983) Deacidification of fruit juices by electrodialysis, Indian J. Technol., 21: 120–123. Amarowicz, R. and Shahidi, F. (1997) Antioxidant activity of peptide fractions of capelin protein hydrolysates, Food Chem., 58: 355–359. Amundson, C.H., Watanawanichakorn, S. and Hill, C.G., Jr. (1982) Production of enriched protein fractions of b-lactoglobulin and a-lactalbumin from cheese whey. J. Food Process Preserv, 6: 55–71. Audinos, R., Roson, J.P. and Jouret, C. (1979) Application de l’électrodialyse à l’élimination de certains composants du jus de raisin et du vin. Connaissance Vigne Vin, 13: 229–239. Audinos, R., Lurton, L. and Moutounet, M. (1985) Advantage of electrodialysis to produce sweetening products from grape. Sci. Aliment., 5: 619–637. Bargeman, G., Dohmen-Speelmans, M., Recio, I., Timmer, M. and Van den Horst, C. (2000) Selective isolation of cationic amino acids and peptides by electro-membrane filtration. Lait, 80: 175–185. Bargeman, G., Houwing, J., Recio, I., Koops, G.H. and Van den Horst, C. (2002) Electromembrane filtration for the selective isolation of bioactive peptides from an as2-casein hydrolysate. Biotechnol Bioeng., 80: 599–609. Bazinet, L., Cossec, C., Gaudreau, H. and Desjardins, Y. (2009) Production of a phenolic antioxidant enriched cranberry juice by an electrochemical process. J. Agric. Food Chem., 57: 10245–10251. Bazinet, L. (2005) Electrodialytic phenomena and their applications in the dairy industry: a review. CRC Critical review in Food Science and Nutrition, 45: 307–326.


Electrodialytic phenomena and associated electromembrane technologies 215 Bazinet, L., Amiot, J., Poulin, J.-F. and Labbé, D. and Tremblay, A. (2005) Process and system for separation of organic charged compounds. World Patent 082495A1. Bazinet, L. and Firdaous, L. (2009a) Membrane processes and devices for separation of bioactive peptides. Recent Pat. Biotechnol., 3: 61–72. Bazinet, L. and Firdaous, L. (2009b) Applications of electromembrane processes to the production of nutraceuticals or functional foods. In Handbook of membrane research: properties, performance and applications. Gorley, S.V. (Editor). Chemical engineering methods and technology series, Nova Science Publishers Inc., Hauppauge, NY. Chapitre 8, pp. 291–312. Bazinet, L., Ippersiel, D. and Lamarche, F. (1999b) Recovery of magnesium and protein from soy tofu whey by electrodialytic configurations. J. Chem. Technol. Biotechnol., 74(7): 663–668. Bazinet, L., Lamarche, F. and Ippersiel, D. (1998) Comparison of chemical and bipolar membrane electrochemical acidification for precipitation of soybean proteins. J. Agric. Food. Chem., 46: 2013–2019. Bazinet, L., Lamarche, F., Boulet, M. and Amiot, J. (1997a) Combined effect of pH and temperature during electroreduction of whey proteins. J. Agric. Food Chem., 45: 101–107. Bazinet, L., Lamarche, F., Labrecque, R. and Ippersiel, D. (1997b) Effect of KCl and soya protein concentrations on the performance of bipolar membrane electro-acidification. J. Agric. Food. Chem., 45: 2419–2425. Bazinet, L., Lamarche, F., Labrecque, R. and Ippersiel, D. (1997c) Effect of number of bipolar membranes and temperature on the performance of bipolar membrane electroacidification. J. Agric. Food. Chem., 45: 3788–3794. Bazinet, L., Gaudreau, H., Lavigne, D. and Martin, N. (2007) Partial demineralization of maple sap by electrodialysis: impact on syrup sensory and physicochemical quality. J. Sci. Food Agric., 87: 1691–1698. Bazinet, L., Ippersiel, D., Gendron, C., Mahdavi, B., Amiot, J. and Lamarche, F. (2001) Effect of added salt and increase in ionic strength on skim milk electroacidification performances. J. Dairy Res., 68: 237–250. Bazinet, L., Ippersiel, D., Labrecque, R. and Lamarche, F. (2000) Effect of temperature on the separation of soybean 11S and 7S protein fractions during bipolar membrane electroacidification. Biotechnol. Prog., 16: 292–295. Bazinet, L., Ippersiel, D. and Mahdavi, B. (2004a) Effect of conductivity adjustment on the separation of whey protein by bipolar membrane electroacidification. J. Agric. Food Chem., 52: 1980–1984. Bazinet, L., Ippersiel, D. and Mahdavi, B. (2004b) Fractionation of whey protein by bipolar membrane electroacidification. Innov. Food Sci. Emerg. Technol., 5: 17–25. Bazinet, L., Lamarche, F., Ippersiel, D. and Amiot, J. (1999a) Bipolar membrane electro-acidification to produce bovine milk casein isolate. J. Agric. Food Chem., 47: 5291–5296. Bazinet, L., Lamarche, F., Labrecque, R., Toupin, R., Boulet, M. and Ippersiel, D. (1996) Systematic study on the preparation of a food grade soyabean protein. Rep. Can. Electr. Assoc., 9326, U 987, Research and Development; Montréal. Beaulieu, L., Thibodeau, J., Desbiens, M., Saint-Louis, R. Zatylny, C. and Thibaut, S. (2010) Evidence of antimicrobial activities in peptide fractions originating from snow crab (Chionoecetes opilio) by-products. Probiot. Antimicrob. Prot., DOI 10.1007/ s12602-010-9043-6, in press. Ben Ounis, W., Champagne, C. P., Makhlouf, J. and Bazinet, L. (2008) Utilization of tofu whey pre-treated by electromembrane process as a growth medium for Lactobacillus plantarum LB17. Desalination, 229: 192–203. Bolduc, M.P., Bazinet, L., Lessard, J., Chapuzet, J.M. and Vuillemard, J.C. (2006) Electrochemical modification of the redox potential of pasteurized milk and its evolution during storage. J. Agric. Food Chem., 54: 4651–4657.


216 Separation, extraction and concentration processes Bolzer, R. (1985) Installation and process for the preparation of acid caseinates. United States Patent 4 559 119. Boyaval, P., Corre, C. and Terre, S. (1987) Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotech. Lett., 9: 207–212. Boyaval, P., Seta, J. and Gavach, C. (1993) Concentrated propionic acid production by electrodialysis. Enzyme Microb. Technol.; 15: 683–686. Brett, C.M.A. and Oliveira-Brett, A.M. (1994) Fundamentals of kinetics and mechanism of electrode reactions. In Electrochemistry: principles, methods, and applications; Oxford University Press: New York. Chaput, M. (1979) Le lactose, extraction et hydrolyse et déminéralisation du lactosérum. Rev. Lait. Française, 372: 23–26. Couture, R. and Rouseff, R. (1992) Debittering and deacidifying sour orange (Citrus aurantium) juice using neutral and anion exchange resins. J. Food Sci., 57: 380– 384. Crandall, J.J., Mantz, B.W. and Martz, E.C. (2001) Method for generating oxygenated water, United States Patent 6 284 293 B1. Donnan, F.G. (1911) Theory of membrane equilibrium and membrane potential in the presence of non-dialysing electrolytes: A contribution to physical-chemical physiology. Z. Elektrochem. Angewandte Phys. Chem., 17: 572–581. Doyen, A., Beaulieu, L., Saucier, L., Pouliot, Y. and Bazinet, L. (2010) Peptides from a snow crab by-products hydrolysate demonstrated anticancer properties in vitro after simultaneous separation by electrodialysis with ultrafiltration membranes. Submitted to Sep. Pur. Technol. Escudier, J., Saint-Pierre, B., Batlle, J. and Moutounet, M. (1995) Automatically controlled tartric stabilisation of wine by membrane electrodialysis which reduces its conductivity to the desired level. World Patent 9506110-A1. Firdaous, L., Dhulster, P., Amiot, J., Gaudreau, A., Lecouturier, D., Kapel, R., Lutin, F., Vézina, L.-P. and Bazinet, L. (2009) Concentration and selective separation of bioactive peptides from an alfalfa white protein hydrolysate by electrodialysis with ultrafiltration membranes. J. Membr. Sci., 329: 60–67. Gardais, D. (1990) Les procédés électriques de traitement des rejets industriels. In Environnement et Electricité. Electra, Doppee diffusion, Avon, pp. 200–310. Glassner, D. (1992) ED Applications in biotechnology. In Proceedings 10th annual membrane technology planning conference, Business Communications, Norwalk, pp. 158–165. Gonçalves, F., Fernandes, C., Cameira Dos Santos, P. and De Pinho, M.N. (2003) Wine tartaric stabilization by electrodialysis and its assessment by the saturation temperature. J. Food Eng., 59: 229–235. Guérif, G. (1993) Electrodialysis applied to tartaric stabilisation of wines. Rev. Oenol. Techn. Vitic. Œnol., 69: 39–42. Haratifar, S. (2008) The stability of electro-reduced milk lipids. Master of Science Thesis, Faculty of Agricultural and Food Sciences, Université Laval., Québec, Canada, 94p. Hekal, I.M. (1983) Process for the preservation of color and flavour in liquid containing comestibles. United States Patent 4.374.714. Hiraoka, Y., Itoh, K. and Taneya, S. (1979) Demineralization of cheese whey and skimmed milk by electrodialysis with ion exchange membranes. Milchwissenschaft, 34: 397–400. Houldsworth, D.W. (1980) Demineralization of whey by means of ion exchange and electrodialysis. J. Soc. Dairy Technol., 33: 45–51. Inoue, T.T. and Kato, W.K. (2003). Powdery drinks and process for producing the same. International Patent WO03053153. Janson, H.V. and Lewis, M.J. (1994) Electrochemical coagulation of whey protein. J. Soc. Dairy Technol., 47: 87–90. © Woodhead Publishing Limited, 2010

Electrodialytic phenomena and associated electromembrane technologies 217 Janson, H.V., Kiis, A.A., Alekseev, N.G. and Kirm, A.A. (1990) USSR Patent 1 570 694, 1990. Khidirov, S.S. and Merzametov, M.M. (1982) Electrocoagulation of milk proteins, brief communications, Vol.1, Book 2, XXI International Dairy Congress, Moscow, 12–16 July 1982. Kim, C., Hung, Y.C. and Bracket, R.E. (2000) Roles of oxidation–reduction potential in electrolyzed oxidizing and chemically modified water for the inactivation of foodrelated pathogens. J. Food Prot., 63: 19–24. Kim, S.-K. and Mendis, E. (2006) Bioactive compounds from marine processing byproducts. A review. Food Res. Int., 39: 383–393. Koseki, M., Nakagawa, A., Tanaka, Y., Noguchi, H. and Omochi, T. (2003) Sensory evaluation of taste of alkali-ion water and bottled mineral waters. J. Food Sci., 68: 354–358. Labbé, D., Araya-Farias, M., Tremblay, A. and Bazinet, L. (2005) Electromigration feasability of green tea catechins. J. Membr. Sci., 254: 101–109. Lin Teng Shee, F., Angers, P. and Bazinet, L. (2005) Precipitation of cheddar cheese whey lipids by electrochemical acidification. J. Agric. Food Chem., 53: 5635–5639. Lin Teng Shee, F., Angers, P. and Bazinet, L. (2007) Delipidation of a whey protein concentrate by electroacidification with bipolar membranes (BMEA). J. Agric. Food Chem., 55: 3985–3989. Lin Teng Shee, F., Arul, J., Brunet, S. and Bazinet, L. (2008) Performing a three-step process for conversion of chitosan to its oligomers using an unique bipolar membrane electrodialysis system. J. Agric. Food Chem., 56: 10019–10026. Mani, K.N. (1991) Electrodialysis water splitting technology. J. Membr. Sci., 58: 117–138. Mercier, D. (1999) Electrochemical treatment method and device for softening water. United States Patent 5.897.765. Mondal, K. and Lalvani, S.B. (2003) Electrochemical hydrogenation of canola oil using a hydrogen transfer agent. J. Amer. Oil Chem. Soc., 80: 1135–1141. Mondor, M., Ippersiel, D., Lamarche, F. and Boye, J.I. (2004) Production of soy protein concentrates using a combination of electroacidification and ultrafiltration. J. Agric. Food Chem., 52: 6991–6996. Ndiaye, N., Pouliot, Y., Saucier, L., Beaulieu, L. and Bazinet, L. (2010) Electroseparation of bovine lactoferrin from model and whey solutions. Sep. Pur. Technol., 74: 93–99, DOI 10.1016/j.seppur.2010.05.011. Neto, C.C., Amoroso, J.W. and Liberty, A.M. (2008) Anticancer activities of cranberry phytochemicals: an update. Mol. Nutr. Food Res., 52: S18–S27. Norddahl, B. (1998) Fermentative production and isolation of lactic acid. World Patent 9 828 433. Norddahl, B., Eriksen, S. and Pedersen, F.M. (2001) Method for producing lactic acid. World Patent 01 92555-A1. Pérez, A., Andrés, L.J., Alvarez, R., Coca, J. and Hill, C.G. (1994) Electrodialysis of whey permeates and retentates obtained by ultrafiltration. J. Food Proc. Eng., 17: 177–190. Picot, L., Bordenave, S., Didelot, S., Fruitier-Arnaudin, I., Sannier, F., Thorkelsson, G., Bergé, J.P., Guérard, F., Chabeaud, A. and Piot. J.M. (2006) Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines. Process Biochem., 41: 1217–1222. Poulin, J.-F., Amiot, J. and Bazinet, L. (2006) Simultaneous separation of acid and basic bioactive peptides by electrodialysis with ultrafiltration membrane. J. Biotechnol., 123: 314–328. Poulin, J.-F., Amiot, J. and Bazinet, L. (2007) Improved peptide fractionation by electrodialysis with ultrafiltration membrane: influence of ultrafiltration membrane stacking and electrical field strength. J. Membr. Sci., 299: 83–90.


218 Separation, extraction and concentration processes Quoc, A., Lamarche, F. and Makhlouf, J. (2000) Acceleration of pH variation in cloudy apple juice using electrodialysis with bipolar membranes. J. Agric. Food Chem., 48: 2160–2166. Quoc, A.L., Mondor, M., Lamarche, F., Ippersiel, D., Bazinet, L. and Makhlouf, J. (2006) Effect of a combination of electrodialysis with bipolar membranes and mild heat treatment on the browning and opalescence stability of cloudy apple juice. Food Res. Int., 39: 755–760. Shaposhnik, V.A. and Kesore, K. (1997) An early history of electrodialysis with permselective membranes. J. Membr. Sci., 136: 35–39. Shmuely, H., Yahav, J., Samra, Z., Chodick, G., Koren, R., Niv, Y. and Ofek I. (2007) Effect of cranberry juice on eradication of Helicobacter pylori in patients treated with antibiotics and a proton pump inhibitor. Mol. Nutr. Food Res., 51: 746–751. Skorepova, J. and Moresoli, C. (2007) Carbohydrate and mineral removal during the production of low-phytate soy protein isolate by combined electroacidification and high shear tangential flow ultrafiltration. J. Agric. Food Chem., 55: 5645–5652. Slack, A.W., Amundson, C.H. and Hill, C.G. (1986) Production of enriched b-lactoglobulin and a-lactalbumin whey protein fractions. J. Food Proc. Pres., 10: 19–30. Stack, F.M., Hennessy, M., Mulvihill, D. and O’Kennedy, B.T. (1995) World Patent 9534216-C1. Swanson, A.M. and Sommer, H.H. (1940) Oxidized flavor in milk: II. The relation of oxidation–reduction potentials to its development. J. Dairy Sci., 23: 597–614. Thompson, B.G. and Gerson, D.F. (1985) Electrochemical control of redox potential in batch cultures of Escherichia coli. Biotechnol. Bioeng., 27: 1512–1515. Tronc, J.-S., Lamarche, F. and Makhlouf, J. (1997) Enzymatic browning inhibition in cloudy apple juice by electrodialysis. J. Food Sci. 62: 75–78. Tronc, J.-S., Lamarche, F. and Makhlouf, J. (1998) Effect of pH variation by electrodialysis on the inhibition of enzymatic browning in cloudy apple juice. J. Agric. Food Chem., 46: 829–833. Vattem, D.A., Ghaedian, R. and Shetty, K. (2005) Enhancing health benefits of berries through phenolic antioxidant enrichment: focus on cranberry. Asia Pac. J. Clin. Nutr., 14: 120–130. Vera Calle, E., Dornier, M., Sandeaux, J., Pourcelly, G. (2002) Deacidification of the clarified passion fruit juice using different electrodialysis configurations. Desalination, 149: 357–361. Vera Calle, E., Ruales, J., Sandeaux, J., Dornier, M., Persin, F., Reynes, M. and Pourcelly, G. (2003) Comparison of different methods for deacidification of clarified passion fruit juice. J. Food Eng., 59: 361–367. Yan, X., Murphy, B.T., Hammond, G.B., Vinson, J.A. and Neto, C.C. (2002) Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon). J. Agric. Food Chem., 50: 5844–5849.



8 Principles of pervaporation for the recovery of aroma compounds and applications in the food and beverage industries S. Sahin, Middle East Technical University, Turkey Abstract: The principles and transport mechanism of pervaporation, a membrane separation process in which the components from a liquid mixture permeate selectively through a dense membrane, are described. Information on membrane materials is given and studies on the recovery of aroma compounds by pervaporation, one of its most important applications in the food industry, are reviewed. Sources of further information are listed and future trends explored. Key words: aroma compounds, food, pervaporation, beverages.

8.1 Introduction Processing, especially at high temperatures, may cause considerable physical and/or chemical changes in the aroma compounds of foods. Physical losses may take place during evaporation and chemical changes of aroma compounds may occur owing to heat-induced oxidation or Maillard reactions. For example, in the fruit juice industry, changes in aroma compounds are inevitable since pasteurization of fruit juice is required in order to increase the shelf life of the product. In addition, aroma compounds are highly volatile so that most of them are lost by evaporation during the production of concentrated juice. The change/loss of aroma compounds may affect the final product quality and consumer’s acceptance. Therefore, the aroma compounds must be recovered either from the loss stream or removed before the raw material is subjected to heat treatment (aroma stripping) and added back to the concentrated juice. There are several methods for the extraction that minimize chemical changes and physical losses of aroma compounds. These methods can be


220 Separation, extraction and concentration processes categorized as: vapor–liquid separations based on distillation/evaporation or partial condensation, separations based on gas injection adsorption and supercritical-fluid extraction. However, these separation techniques have some limitations. Pervaporation, which is a membrane process, is a promising alternative method for aroma recovery because of it has advantages over conventional aroma recovery processes such as high selectivity, low energy consumption, moderate operating temperatures, physical separation mechanism and no additive requirement (Lipnizki et al., 2002a). Compared with other membrane processes like ultrafiltration and reverse osmosis, pervaporation exhibits low fluxes. However, selectivities can be quite high. Therefore, this method is especially suitable for the recovery of highly diluted species. This method can be performed on the juice before the evaporation or pasteurization step thus preventing deterioration or loss of the heat-sensitive aroma compounds during the heat treatment. In addition to aroma recovery, this method can be used for dealcoholization of wine or beer, recovery of high-value-added components or removal of organic pollutants from waste streams, and removal of inhibitors from fermentation broths. It can also be used as an analytical separation technique.

8.2 Principles of pervaporation Pervaporation is a membrane technique in which the components from a liquid mixture are separated by means of partial vaporization through a non-porous permselective membrane. The term ‘pervaporation’ is used in order to emphasize the fact that the permeate undergoes a phase change from liquid to vapor during its transport through the barrier. The pervaporation term was first introduced by Kober (1917). In his study, it was observed that a liquid in a collodion (cellulose nitrate) bag that was suspended in the air, evaporated, although the bag was tightly closed. Kober realized that this phenomenon could be used for separation of liquid mixtures. In pervaporation, a membrane separates an upstream mixture in the liquid state from downstream permeates in the gaseous state. The downstream side is maintained at a reduced pressure to ensure the gaseous state. The liquid feed mixture is circulated in contact with the membrane. The permeate in gaseous state is collected in the liquid state after condensation on a cooled wall. A schematic diagram of a typical experimental set-up for pervaporation is shown in Fig. 8.1. The feed solution is kept in a reservoir inside the temperature-controlled water bath and it is pumped to the upper part of the pervaporation cell. The vacuum is applied to the lower compartment of the cell by use of a vacuum pump in order to provide a pressure gradient across the membrane. The vacuum pressure in the system is controlled by a vacuum controller. The permeate is collected in cold traps by condensation, mostly


Principles of pervaporation for the recovery of aroma compounds 221 Thermocouple

TC Trap 1

Trap 3

Water bath

PC

Trap 2 Peristaltic Pervaporation pump cell

Vacuum pump

Fig. 8.1 Schematic diagram of a typical pervaporation experimental set-up; TC, temperature control; PC, pressure control (reprinted from Isci et al., 2006, with permission from Elsevier).

with liquid nitrogen at –196 °C. There are usually three traps in the set-up; the first two are connected in parallel in order to conduct the experiments continuously and to maintain the steady-state condition during data collection. The third one is connected in series to the parallel traps as a safety trap in order to ensure that practically no permeate reaches the vacuum pump. The components from the liquid feed mixture permeate selectively through a dense membrane driven by a chemical potential gradient obtained by partial pressure reduction on the permeate side. The affinity between the permeant and the polymer material that constitutes the membrane as well as its mobility through the membrane matrix are important for the transport of the compound. Pervaporation is a complex process involving simultaneous heat and mass transfer. Latent heat is required to pervaporate the liquid. The gradient in partial vapor pressure between the feed and the permeate side of the membrane is maintained by reducing the partial vapor pressure in the permeate side. The heat necessary for the evaporation of the permeate has to be transported through the membrane and this transport of energy is coupled to the transport of mass. The evaporation enthalpy is taken from the sensible heat of the liquid feed mixture, leading to a reduction in feed side temperature.

8.3 Transport mechanism in pervaporation for the recovery of aroma compounds Although a phase change from liquid to vapor takes place, this phase change never occurs directly but occurs via a membrane–solute interaction © Woodhead Publishing Limited, 2010

222 Separation, extraction and concentration processes in pervaporation. Therefore, the separation principle is not based on the vapor–liquid equilibrium. Transport through the nonporous membrane is only by diffusion. The solubility and the diffusivities of the constituents in the membrane are important for separation by pervaporation. Therefore, solute transport within the membrane is not directly affected by the external operating conditions. Ideally, the membrane alone determines the solute transport and the characteristics of the separation and so it is important to create the optimum mass transport conditions to and from the membrane (Schafer et al., 2006). The driving force in pervaporation is the gradient of chemical potential of a solute across the selective membrane (Schafer and Crespo, 2002). Thus, the mass transport in a pervaporation membrane can be described as a function of the chemical potential gradient:

Ji = f (D mi)

[8.1]

where Ji and mi are the mass flux and chemical potential of solute i, respectively. The chemical potential can be stated as a function of the state variables; pressure, composition and temperature:

m = f (P, C, T)

[8.2]

Therefore, the differences in the permeate pressure, feed composition and temperature cause differences in chemical potential. For this reason, there are three methods in pervaporation to establish a suitable chemical potential gradient in the membrane as driving force for the mass transport: (a) Vacuum pervaporation: a hydrostatic pressure difference between the feed and the permeate mixture is established by introducing a vacuum on the permeate side. This method is applicable if the volume of permeating vapor is relatively small or the permeate side pressure is not too low. Otherwise, vacuum pumps of extremely large capacities are required and the pumps consume too much energy (Brüschke, 2006). (b) Thermopervaporation: a chemical potential gradient in the membrane is established by providing a temperature difference between the feed and the permeate side (Strathmann and McDonogh, 1993). (c) Sweeping-gas pervaporation: an inert gas stream on the permeate side of the membrane can be used to remove the component and thus establish a concentration gradient driving force. Vacuum pervaporation is the most commonly used method because a chemical potential gradient is readily obtained by reducing partial pressure on the permeate side. Partial pressures of the permeant components are usually lower than their corresponding saturation pressures. Therefore, they are removed as vapor. The principle of solute transport across the membrane in vacuum pervaporation is shown in Fig. 8.2. A nonporous membrane separates a liquid feed, which is usually close to atmospheric pressure, from the downstream compartment where a vacuum is applied. The solutes (denoted by solid circles)


Principles of pervaporation for the recovery of aroma compounds 223 Membrane

Liquid

Membrane

Liquid

Vapor

(a)

Vapor

(b) Membrane

Liquid

Vapor

(c)

Fig. 8.2 The principle of solute transport across the membrane in pervaporation. For explanation of panels see text.

are first absorbed by the membrane surface when they are in contact with the membrane owing to the interactions between the solutes and polymer (Fig. 8.2a). When the chemical potential of these components in the feed and in the selective layer of the membrane are equal, a thermodynamic equilibrium is reached. The solutes that are absorbed by the membrane surface create a chemical potential difference across the membrane, which causes a diffusive net flux of solute through the membrane polymer (Fig. 8.2b). Ideally, all the solute particles that have diffused through the membrane are desorbed suddenly and removed by vacuum applied on the downstream side of the membrane. As a result, the solute concentration on the membrane downstream surface remains practically zero, and a maximum concentration gradient between the two membrane surfaces is maintained. If the vacuum is not low enough to desorb all the solutes reaching the membrane downstream surface, the concentration of the solute at the bottom of the membrane is not zero and, consequently, the concentration gradient decreases, as the net flux across the membrane decreases. For the ideal diffusion of solute i across the membrane toward the membrane downstream surface, Fick’s First law applies:

dC J i = Di ÈÍ i ˘˙ Î dz ˚

[8.3]

where Ji is the flux of the permeant i, Ci is the concentration of the permeant i in the polymer membrane, z is the position in the membrane, measured


224 Separation, extraction and concentration processes from the feed side of the membrane and Di is the diffusion coefficient of the permeant i in the membrane. The diffusion coefficient of a specific component through a polymer can be assumed to be independent of the solute concentration. Then, integration across a membrane, with a thickness Z, yields:

J = Di

(Cimem,f – Cimem,p ) Z

[8.4]

where Cimem,f and Cimem,p are the concentrations of the permeant i inside the membrane at its feed and permeate sides, respectively. If the vacuum applied on the permeate side of the membrane is strong enough, we can assume that all of the solute particles that diffuse through the membrane are removed instantaneously. Then Cimem,p can be taken as zero and equation [8.4] reduces to:

J i = Di

(Cimem,f ) Z

[8.5]

Transport, across the membrane, is generally described by the ‘solutiondiffusion mechanism’ (Strathmann and McDonogh, 1993). Assuming constant solute feed concentration and rapid removal of solute on the membrane downstream, the selective transport of the component ‘i’ from the bulk feed to the downstream compartment can ideally be described by the following steps as: 1. equilibrium partitioning (sorption) of the component i from the liquid phase at the feed solution-membrane upstream surface interphase; 2. diffusion of the absorbed component i through the membrane polymer matrix toward the downstream surface of the membrane; 3. equilibrium partitioning (desorption) of the component i from the membrane downstream surface to the permeate vapor. For the equilibrium partitioning in steps 1 and 3, the sorption (partitioning) coefficient of i in the homogeneous membrane polymer Si can be expressed as: Si =

Cimem,f Cil,bulk

[8.6]

where Cil, bulk is the concentration of component i in the bulk liquid. Combination of equations [8.5] and [8.6] yields the transport model commonly applied in pervaporation:

J i = Si Di

(Cil,bulk ) (C l,bulk ) = Pi i Z Z


[8.7]


where Pi is the permeability of i in the membrane which is the product of the solubility and diffusivity of the solute i in the membrane. It can be concluded from equation [8.7] that, for an ideal case, the flux of a specific component is directly related to the concentration of the component in the bulk feed. In addition, the permeability of the solute can be calculated from the slope of the flux versus feed concentration graph, if the thickness of the membrane is known. The driving force can also be expressed as a partial pressure gradient of the solute i across the membrane. Then, equation [8.7] becomes:

ÈP f – Pip ˘ DP J i = Si Di ÈÍ i ˘˙ = Pi¢ Í i ˙ ÎZ ˚ Î Z ˚

[8.8]

where Pi¢ is the permeability coefficient (kg m–1 s–1 Pa–1), and Pif and Pip are partial pressures of compound i in the feed and permeate sides, respectively. The widely accepted parameters used to describe the performance of the pervaporation process are permeate flux (Ji), selectivity or separation factor (ai/j) and enrichment factor (bi). The permeate flux Ji is defined as the permeate flow rate per unit of membrane area for a given membrane thickness. The separation factor ai/j is a measure of the selectivity and it indicates the preferential permeation of component i compared with component j. It is calculated from the concentrations of the compounds to be separated, i and j, in the feed and the permeate, respectively:

a i /j =

(Cip /Cil,bulk )

(C pj /C l,bulk ) j

[8.9]

The affinity between the permeant and the polymer material that constitutes the membrane and its mobility through the membrane matrix which are responsible for the transport are important parameters for selectivity. Since different species permeate through the membrane at different rates, a substance at a low concentration in the feed stream can be highly enriched in permeate. The enrichment factor bi represents the capacity of the membrane for concentrating component i and is defined as:

bi =

Cip C l,bulk j

[8.10]

If perfect mixing of the feed and sudden removal of the solutes leaving the membrane downstream face can not be provided, boundary layers develop on both sides of the membrane, affecting the solute transport to and away from the respective membrane surface. If the flux of solute i through the membrane is higher than that through the liquid phase toward the membrane, © Woodhead Publishing Limited, 2010

226 Separation, extraction and concentration processes the solute i is depleted in the liquid phase over the membrane upstream surface, resulting in a liquid solute concentration lower than that in the bulk feed. Because the solute concentration in the liquid at the membrane upstream surface determines the partitioning of the solute into the membrane, the concentration of i in the membrane upstream surface is lower with respect to bulk concentration. Therefore, the concentration gradient across the membrane decreases as does the overall flux. This phenomenon is known as ‘concentration polarization’ and affects the fluxes of compounds of high sorption coefficient, even under turbulent hydrodynamic conditions over the membrane. A similar phenomenon can also be found on the membrane downstream face (Schafer and Crespo, 2002). The concentration polarization strongly depends on the feed flow velocity and on the hydraulic diameter. An increase in the feed flow velocity and a decrease in the hydraulic diameter, decrease the effect of concentration polarization (Borjesson et al., 1996). As long as the diffusive flux is sufficiently high compared with the solute flux across the membrane, the boundary layer does not represent an additional transport resistance and the solute concentration at the membrane surface is equal to that in the bulk (Schafer and Crespo, 2007). However, if the diffusive flux across the liquid boundary layer is lower than the maximum achievable transmembrane flux at the respective feed bulk concentration, the boundary layer detrimentally affects the overall transport of solute from the feed bulk to the permeate side of the pervaporation membrane because the membrane surface concentration of solute i is lower than that of the bulk. Overall mass transfer coefficient kov,i can be expressed as the sum of mass transfer resistances:

Ê 1 ˆ Ê 1 ˆ Ê 1 ˆ Ê 1 ˆ ÁË k ˜¯ = ÁË k ˜¯ + ÁË k ˜¯ + ÁË k ˜¯ ov,i bl,i m,i p,i

[8.11]

where kbl,i is the mass transfer coefficient for the boundary layer, km,i is the mass transfer coefficient for the membrane, and kp,i is the mass transfer coefficient for the permeate side. Although in most instances, it is hard to achieve an ideal case, the mass transfer resistance in the feed boundary layer can be neglected compared with the overall mass transfer resistance if the feed flow velocity is maintained sufficiently high during pervaporation (Karlsson and Tragardh, 1993). Moreover, if a good vacuum is applied on the permeate side of the membrane, everything that diffuses through the membrane is instantaneously pumped away and therefore, mass transfer resistance in the permeate side can also be neglected. Then, the only resistance to mass transfer is within the membrane polymer and equation [8.11] simplifies to:

Ê 1 ˆ Ê 1 ˆ ÁË k ˜¯ = ÁË k ˜¯ ov,i m,i

[8.12]



Therefore, overall mass flux of component i from the bulk of the feed to the bulk of the permeate can be described by the following equation:

J i = kov,i (Cil,bulk – Cip ) = km,i (Cil,bulk – Cip )

[8.13]

p

Another phenomenon affecting pervaporation performance is temperature polarization. Owing to the phase change from the feed to the permeate side, the membrane acts as a heat sink and a temperature difference exists between the bulk of the feed and the feed side of the membrane. The resulting reduction in the driving force and hence in performance is called ‘temperature polarization’ and this effect is unique for pervaporation processes (Brüschke, 2006). Flux changes linearly with change in concentration but exponentially with change in temperature which means that temperature polarization is more important than concentration polarization. Therefore, the development of high flux pervaporation membranes requires the development of modules in which temperature polarization is effectively reduced. Concentration polarization does not change significantly with changes in composition of the feed. However, the effect of temperature polarization is more important if the concentration of the component to be removed, and thus the flux, is high.

8.4 Selection of membranes for pervaporation in the recovery of aroma compounds A membrane can be simply defined as a permselective barrier made up of polymers between the feed and permeate. Polymers are very large molecules (about 1000 to 100 000 times larger than water molecule) which are manufactured by chemical processes. Polymers gain different characteristics during their design. Generally, there are two types of polymers: glassy polymers and rubbery polymers or elastomers. Their ground state at room temperature determines their distinction (Koops and Smolders, 1991). Glassy polymers have a glass transition temperature above the room temperature and can be divided into three groups: crystalline, semi-crystalline and amorphous polymers. The presence of crystallites has an important affect on all kinds of polymers in terms of tensile strength, elasticity, impact strength, solubility and diffusivity in the polymer. Generally, glassy polymers are hydrophilic (Böddeker and Bengtson, 1991). They preferentially permit the permeation of water owing to the presence of groups in the polymer chain that are able to interact with water molecules. They are used mainly for removal of water from organic solvents and solvent mixtures with an emphasis on azeotropic mixtures. Membranes for the removal of small alcohol molecules like methanol or ethanol are also hydrophilic. Some examples of glassy polymers are cellulose acetate, polysulfone, and polyvinylalcohol.


228 Separation, extraction and concentration processes Polymers with a glass transition temperature below room temperature are classified as rubbers or elastomers. Elastomers normally present higher chain mobility than glassy polymers and contain rather small, non-polar side groups. Elastomers are flexible and mostly hydrophobic polymers. They preferentially permeate organic substances owing to the lack of strong intermolecular forces, such as hydrogen bonding or dipole–dipole interactions. Since they absorb organic molecules with relatively high fluxes, elastomers are the most suitable polymers for the separation of organic molecules from aqueous solutions (Koops and Smolders, 1991) or from organic–organic mixtures. Some of the examples of hydrophobic polymers are polydimethylsiloxane (PDMS), polyether block polyamide (PEBA), polypropylene (PP), and polyvinylidenefluoride (PVDF) (Koops and Smolders, 1991). Glassy and rubbery membranes differ strongly in terms of flexibility of their polymeric structure. Within the rigid polymeric structure of a glassy membrane, the diffusivity of components is strongly related to their molecular volume, whereas in the flexible region of a rubbery membrane, solute–polymer interactions are much more important than the diffusivities of components. This is why the selectivity of glassy membranes is more diffusion controlled, whereas the selectivity of rubbery membranes is more sorption controlled (Schafer and Crespo, 2002). In pervaporation, nonporous membranes which provide selectivity based on solute–membrane interactions rather than on volatilities are used. The membrane type used in the pervaporation experiments determines the selectivity towards the molecules. Therefore, membrane selection is one of the most important issues in pervaporation. The membrane should have high selectivity and flux for the components to be separated. In addition, it must have chemical, mechanical and thermal stability (Schafer and Crespo, 1997). In pervaporation, the feed side of the membrane is highly swollen in contact with the liquid whereas the permeate side is dry and virtually nonswollen. Thus, a high gradient of swelling exists over the separating layer of the membrane, demanding additional resistance and stability. Pervaporation can be classified as hydrophilic and organophilic pervaporation (Lipnizki et al., 1999). In hydrophilic pervaporation, the target compound water is separated from an aqueous–organic mixture whereas in organophilic pervaporation, the target organic compounds are separated from an aqueous–organic mixture (hydrophobic pervaporation) or from an organic–organic mixture by being preferentially permeated through the membrane (target organophilic pervaporation). Generally, in membrane production for pervaporation processes, composite membrane structure is preferred. Composite membranes are composed of three layers. The first layer is the dense layer responsible for the separation. This layer has to be as thin as possible since the transport through the membrane is by diffusion (0.5–2 mm). The second layer is the porous support layer that has an asymmetric pore structure. It has a thickness of 70–100 mm. Structural polymers with high resistance against chemical attack and good thermal and



mechanical properties such as polyacrylonitrile, polyetherimide, polysulfone, PVDF form the porous support. The final layer, having a thickness of about 100 mm is a woven or nonwoven textile fabric. Polyester, polyethylene, polyphenylene sulfide and similar fibers are used for the textile carrier layer (Brüschke, 2006). The dense separating layer of hydrophilic membranes is made from various polymers that have high affinity towards water. These polymers contain ions, oxygen functions such as hydroxyl-, ester, or carboxylic moieties or nitrogen as imino- or imido- groups. They must be crosslinked in order to render them insoluble after the coating process. For organophilic membranes, the dense separating layer is formed by crosslinked silicones. The most commonly used organophilic membranes are polydimethylsiloxane (PDMS) or polymethyl octyl siloxane (POMS). The monomer of this silicon rubber is: CH3 Si

CH3

O

[8.14] n

POMS is a modified silicon rubber in which one of the methyl groups of the monomer has been replaced by an octyl group. PDMS or POMS membranes with various porous support layers are commercially available. Zeolite-filled silicon membranes have been developed to increase the selectivity and flux. PEBA formulations have elastic segments consisting of thermoplastic polyamides made flexible by elastomeric polyether links. The modification of PDMS membrane by the introduction of rigid organophilic groups has potential in the pervaporation field for the improvement of separation behavior (Luo et al., 2008). Polyphenylmethylsiloxane–cellulose acetate (PPMS–CA) and polydimethylsiloxane–cellulose acetate (PDMS-CA) membranes are used for concentration of volatile organic compounds such as methanol, ethanol and acetone from aqueous solutions by pervaporation. The hydrophobicity of PPMS membranes is stronger than that of PDMS. In the study of Song and Lee (2005), the tube type alumina substrate was modified with silane coupling agent (perfluoroalkylsilane) to obtain hydrophobic membrane. The surface modified membrane showed much higher flux but lower selectivity than nonporous PDMS membrane for the recovery of esters. However, the selectivity of the membrane was sufficiently high. Olsson et al. (2002) studied the effect of three different POMS membranes, a polymethyloctylsiloxane–polyetherimide (POMS-PEI) and two polymethyloctylsiloxane–polyacrylonitrile (POMS-PAN) membranes having different thicknesses for the separation of alcohols, esters and aldehydes. They concluded that the porous support layer could affect the selectivity considerably and membranes should be designed with a low degree of crosslinking to obtain better separation properties, especially for larger permeants. © Woodhead Publishing Limited, 2010


8.5 Recovery of aroma compounds by pervaporation and applications in the food and beverage industries One of the most promising applications of pervaporation is the recovery of aroma compounds from aqueous mixtures. Aroma is a complex mixture of hundreds of different volatile organic compounds present at very low concentrations, typically at ppm or ppb levels. Aroma compounds belong to various chemical groups such as alcohols, aldehydes and esters. Recovery of natural flavor and aroma compounds has received much more attention in the food, biotechnology and cosmetic industries because natural aroma compounds are preferred to chemically synthesized ones. In the food industry, flavor concentrates are widely used to compensate for the loss of aroma compounds during processing. Because aroma compounds are highly heat sensitive and loss of aroma compounds during heat treatments is inevitable, they should be separated from food systems and added back to the final product. This can be accomplished either by recovering the lost aromas from the loss stream or by stripping the aromas from the raw material stream before processing. This eventually increases the quality and acceptance of the final product. There have been a large number of studies in this area. Pervaporation has been successfully applied to the recovery of the aroma compounds of wine (Karlsson et al., 1995; Schafer and Crespo, 2007; Schafer et al., 1999) and several fruit juices, such as apple (Bengtsson et al., 1992; Börjesson et al., 1996; Olsson and Tragardh, 1999 and 2001), cashew apple (De Assis et al., 2007), kiwifruit (Cassano et al., 2006), strawberry (Isci et al., 2006), pineapple (Pereira et al., 2002 and 2005; Sampranpiboon et al., 2000), passion fruit (Pereira et al., 2002; 2005), banana (Sampranpiboon et al., 2000), orange (Aroujalian and Raisi, 2007; Shepherd et al., 2002), blueberry (Peng and Liu, 2003), billberries (Diban et al., 2008; Garcia et al., 2008), grape (Rajagopalan and Cheryan, 1995) and pomegranate (Raisi et al., 2008). In addition, pervaporation has been found to be suitable for concentration of tea (Kanani et al., 2003), cocoa (Kattenberg and Willemsen, 2001) and dairy aroma compounds (Baudot and Marin, 1996; Overington et al., 2008) from aqueous solutions and also for dealcoholization of alcoholic beverages (Takacs et al., 2007). It was successfully applied for the recovery of terpenes from waste waters in essential oil industry (Charbit et al., 1997) and for deodorization of waste streams (Souchon et al., 2002). It can also be used as an analytical separation technique for the continuous determination of volatiles (Amador-Hernandez and Castro, 2000). In most of the studies, model feed solutions have been used because of the complexity of conducting the pervaporation experiments with real multicomponent mixtures (Baudot and Marin, 1999; Bengtsson et al., 1992; Börjesson et al., 1996; Garcia et al., 2008; Isci et al., 2006; Kanani et al., 2003; Olsson and Tragardh, 1999; 2001; Peng and Liu, 2003; Pereira et al., 2002 and 2005; Raisi et al., 2008; Rajagopalan and Cheryan, 1995;



Sampranpiboon et al., 2000). However, there are also some studies in which real food samples are used (Alvarez et al., 2000; Kanani et al., 2003; Pereira et al., 2002 and 2005; Raisi et al., 2008; Rajagopalan and Cheryan, 1995; She and Huang, 2006). Most of the research on aroma recovery by pervaporation has been conducted on a laboratory scale. Studies of scale-up of the pervaporation process for the separation of multi-component mixtures are few. Karlsson et al. (1998) developed a model for the scale-up of pervaporation units for aroma recovery in which the effect of concentration polarization was taken into account with a semi-empirical model. A novel process simulation of pervaporation was developed for multicomponent mixtures (Lipnizki et al., 2002a) and this study was continued with the integration and optimization of hydrophobic pervaporation for the recovery of natural aroma compounds in the food industry (Lipnizki et al., 2002b). Trifunovic et al. (2006) investigated module design aspects of pervaporation using a modified version of an existing pervaporation simulation tool for aroma recovery. By applying the simulation to four aroma compounds: two alcohols (n-butanol, n-hexanol) and two esters (isoamyl acetate and ethyl butyrate), the effect of major process and module design parameters on the performance of a single module has been investigated. In general, for the compounds studied, the permeate composition can be manipulated by changing the module geometry and operating conditions because the effect of these parameters is more significant on esters, which in general have high recovery rates, than for alcohols, which are only marginally affected. 8.5.1 Recovery of fruit juice aromas Apple juice is one of the important fruit juices that pervaporation studies have focused on. Börjesson et al. (1996) investigated the performance of six different pervaporation membranes (PDMS-1060, PDMS-1070 (polydimethyl siloxane + silicalite), PDMS–PT 1100 (polydimethylsiloxane), POMS–PEI (polyoctylmethyl siloxane- polyethermide), POMS–PVDF and PEBA) for the recovery of apple juice aromas. Multicomponent solution containing five esters (ethyl acetate, ethyl butanoate, ethyl- 2-methylbutanoate, isopentyl acetate and hexyl acetate), one aldehyde (trans-2-hexenal) and four alcohols (isobutanol, butanol, isopentanol and hexanol) representing typical aroma compounds of apple juice was used in this study. The best performance was obtained with the PDMS–PT 1100, POMS–PEI and POMS–PVDF membranes. The influence of permeate pressure on the recovery of apple juice aroma compounds by pervaporation had also been examined using the same model solution (Olsson and Tragardh, 2001). A mathematical model, which includes the effect of concentration polarization on the feed side of the membrane and its dependence on permeate pressure, the variation in the separation properties of the membrane with permeate pressure and the properties of the individual permeants, was developed to predict the influence of permeate pressure on separation factor in pervaporation. © Woodhead Publishing Limited, 2010

232 Separation, extraction and concentration processes Moreover, the effect of feed flow velocity on recovery of apple juice aroma compounds by pervaporation was studied (Olsson and Tragardh, 1999). Increasing the feed flow velocity did not increase the recovery of the alcohols significantly because the dominating resistance to mass transfer was in the membrane for the alcohols. However, the recovery of esters and aldehydes was improved by increasing feed flow velocity. The dominating resistance to mass transfer was in the liquid feed boundary layer for the esters studied except ethyl acetate. For the aldehyde and ethyl acetate these two resistances were of the same order. Bengtsson et al. (1992) concentrated the twelve selected flavor compounds in apple juice by pervaporation. It was observed that alcohol had the lowest enrichment factors (2–22), the aldehydes had the medium values (16–67) and the esters had the highest enrichment factors (up to 100). The order of enrichment factors of aroma compounds of various chemical groups was the same in the study of Börjesson et al. (1996). De Assis et al. (2007) found that pervaporation can be used for the concentration of cashew apple juice aroma from cashew pulp. The chromatograms showed an increase in the peak area and also in the number of components in the permeate samples compared with the chromatogram of the cashew juice. Recovery of key flavor components from real flavor concentrates (apple essence, orange aroma and black tea distillate) was achieved by pervaporation in the study of She and Hwang (2006). In this study, both continuous and batch operations were carried out. Generally, the acetates and aldehydes exhibited higher enrichment factors than the alcohols. Significant flavor loss was observed in the pervaporation recovery process. PDMS membrane had higher permeation and loss rates than POMS membrane in batch pervaporation of dilute apple essence. Therefore, it was concluded that POMS membrane with a long operating time should be preferred if highly concentrated products and minimized flavor loss are the major concerns. However, PDMS membrane was found to be better if the time is limited. Rajagopalan and Cheryan (1995) studied pervaporation of model flavor compound of grapes (methyl anthranilate) and of commercial grape essence with several membranes: PDMS–PC (polydimethoxylsiloxane–polycarbonate), PEBA, PDMS-1070). PEBA membrane generally gave higher flux and better selectivity. Flux and selectivity decreased linearly with increase in downstream pressure, but increased with temperature. This relationship was not satisfied when the feed concentration was above 50 ppm where concentration polarization became serious. The presence of ethanol in the feed solution lowered separation factors but increased total flux. Experiments with commercial grape essence confirmed the excellent potential of pervaporation for the production of highly enriched flavors. Pervaporation was found to be a promising technique for the recovery of strawberry aroma compounds (Isci et al., 2006). The experiments were performed using binary aqueous solutions of methyl butyrate (MTB) and



ethyl butyrate (ETB), ternary mixture of MTB, ETB and water and aqueous model strawberry solution containing six aroma compounds (MTB, ETB, butyl butyrate (BTB), methyl caproate (MTC), ethyl caproate (ETC) and linalool). An increase in both flux and selectivity was observed with an increase in feed temperature or decrease in downstream pressure in binary aqueous solution of MTB (Figs 8.3 and 8.4). Selectivity decreased with increase in feed concentration. Enrichment factors of MTB and ETB in binary and ternary solutions were not very different. However, MTB flux was retarded by half by the presence of ETB in ternary solution. In multicomponent mixtures of aqueous strawberry aroma solution, the selectivities of MTB and ETB were adversely affected by the presence of other compounds, owing to coupling effects. It is shown that flux coupling takes place when a permeant of low diffusivity is dragged through the membrane polymer by a permeant of higher diffusivity resulting in higher fluxes of the slower permeant than expected or the reverse. In the study of Sampranpiboon et al. (2000), pervaporation separation was used to recover aroma compounds from ethyl butanoate and ethyl hexanoate mixtures, which are the most important aroma components of pineapple and banana juice, by using POMS and PDMS membranes. The effect of operating conditions on the separation performance was investigated and in general the POMS membrane was found to be more selective to aroma compounds than PDMS membranes. Moreover, a strong interaction was observed between

Total mass flux (kg m–2 h–1)

0.3

0.2

0.1

0 20

30


50

60

Fig. 8.3 Effect of feed temperature on total mass flux at different downstream pressures for 100 ppm methyl butyrate solution: ( ) 4 mbar, ( ) 8 mbar (reprinted from Isci et al., 2006, with permission from Elsevier).



100

Selectivity

80

60

40

20

0 20

30


50

60

Fig. 8.4 Effect of feed temperature on selectivity of 100 ppm methyl butyrate solution at different downstream pressures: ( ) 4 mbar, ( ) 8 mbar (reprinted from Isci et al., 2006, with permission from Elsevier).

the two permeating components and permeation of one aroma compound was affected by the presence of the other aroma compound. Pineapple and passion fruit juices were chosen in the study of Pereira et al. (2002) and (2005). Performances of various membrane materials were evaluated with binary and quaternary synthetic aqueous solutions of typical tropical fruit aroma compounds such as ethyl acetate, ethyl butanoate, ethyl hexanoate and 1-octen-3-ol (Pereira et al., 2005). Tests were also carried out using single strength and clarified pineapple juices. Composite flat membranes having selective layers ethylene propylene diene terpolymer (EPDM) or ethylene vinyl acetate copolymer (EVA) and composite hollow fiber membrane having selective layer EPDM were used for comparison. It was concluded that choosing a very selective polymer is advantageous when the organic solute concentration is reduced in the feed. However, more permeable membranes are preferred if the feed concentration is high enough to induce phase separation in the permeate after its condensation. Composite EPDM hollow fiber membrane showed the best performance for pervaporation of synthetic and single-strength pineapple juices. Peng and Liu (2003), evaluated the separation factor of six aroma compounds (1-hexanol, 1-heptanol, trans-2-hexenal, ethyl acetate, linalool and d-limonene) representing typical flavoring ingredients from blueberry juice with a PDMS membrane. The results showed that the separation factor was in the range of 70 to 430, depending on molecule size and polarity of the compounds. No compounds, except 1-heptanol, showed any significant coupling effect in the mixture system. The temperature dependency of component fluxes was © Woodhead Publishing Limited, 2010


expressed by the Arrhenius equation and it was found that the temperature dependency of water flux was the highest whereas that of d-limonene was the lowest. Garcia et al. (2008) studied the concentration of trans-hex-2-en-1-ol, which is one of the major impact aroma compounds of bilberries, from aqueous ethanol using commercial PDMS capillary membranes in pervaporation. The flux of the aroma compound through the membrane was not affected from the variation of the feed flow rate. Therefore, it was concluded that the main mass transfer resistance to trans-hex-2-en-1-ol flux is located in the membrane. The temperature dependency of water, ethanol and trans-hex-2en-1-ol fluxes was expressed by the Arrhenius equation. From the magnitude of calculated activation energies, it was understood that water and ethanol were more influenced by variations in operation temperature than that of the aroma compound. Diban et al. (2008) adapted a previously developed mathematical model to predict the behavior of the separation of a mixture of seven volatile compounds that are characteristic of the aroma of bilberry juice from aqueous solution by pervaporation. It was observed that enrichment factors increased with membrane thickness until an asymptotic value was reached, however, partial fluxes decreased. Shepherd et al. (2002) studied the use of PDMS hollow fibers in orange juice aroma recovery. They used orange juice by product as feed and binary synthetic solutions. The well-spaced module was compared with a hollow fiber not containing spacers and with the transverse flow module with respect to their effect on experimental enrichment factors and mass transfer coefficient values and it was found to be feasible for aroma recovery. Aroujalian and Raisi (2007) also studied the pervaporation of orange juice aroma components. The effects of feed flow rate, feed temperature and permeate pressure on the performance of pervaporation was investigated. Feed flow rate had no significant effect on performance of the process. However, higher flux and enrichment factors were obtained as the feed temperature increased, whereas the increase in permeate pressure resulted in lower flux values. Enrichment factors of ethyl acetate, ethyl butyrate and hexanal increased but that of limonene, linalool and a-terpineol decreased as permeate pressure increased. Aroma compounds of pomegranate juice in multicomponent model solution and real fruit juice have been successfully concentrated by pervaporation (Raisi et al., 2008). The model aroma solution and real pomegranate juice showed similar behavior. The POMS membranes produced higher aroma enrichment factor but lower flux the PDMS membranes. Feed flow rate had no significant effect an either total flux on enrichment factor of aroma compounds, whereas on increase in feed temperature resulted in higher flux and enrichment factor. The effect of permeate pressure on pervaporation performance was related to the properties of the aroma compounds. Alvarez et al. (2000) investigated a combination of membrane processes


236 Separation, extraction and concentration processes (enzymatic membrane reactor, reverse osmosis and pervaporation) for producing clarified juice and apple juice aroma concentrate. Cassano et al. (2006) performed a study to design an integrated membrane process for the production of concentrated kiwi juice and its aroma recovery. Pervaporation was carried out at different stages in the process. They suggested the use of pervaporation for the removal and enrichment of aroma compounds directly from fresh juice before any concentration process. 8.5.2 Recovery of wine aromas In literature, there are some studies on recovery of wine aromas by pervaporation (Karlsson et al., 1995; Schafer and Crespo, 2007; Schafer et al., 1999). Karlsson et al. (1995) carried out pervaporation with Muscat wine in which eight aroma compounds were identified. In this study, the increase in fluxes with feed temperature was very well described by the Arrhenius equation. The effects of temperature on permeation varied for different aroma compounds. Recovery of aroma compounds from a wine-must fermentation by organophilic pervaporation was studied by Schafer et al. (1999). Pervaporation was performed under fermentation conditions. It was shown that organophilic pervaporation can be highly suitable for the continuous recovery of very complex and delicate aromatic profiles produced during microbial fermentation. Schafer and Crespo (2007) determined the degree of concentration polarization in two different flow-cell configurations during recovery of winemust aroma compounds by pervaporation. It was found that concentration polarization could not be overcome even under turbulent feed flow conditions for compounds having high sorption coefficient for the respective membrane polymer. Dealcoholization of alcoholic beverages including beer and wine is another application of pervaporation. The idea behind this process is the separation of ethanol through hydrophobic membranes much more readily than water. There are many publications in this area (Brüschke, 1990; Escoudier et al., 1988; Kimmerle and Gudernatsch, 1991; Takacs et al., 2007). A problem encountered with this application is that the aroma compounds in the wine or beer are generally much more hydrophobic than ethanol and therefore permeate through the membrane even more readily than ethanol. Vankelecom et al. (1997) used a mixture of eight aromatic compounds in water, one of which was ethanol, in their study. Sorption of aromas in PDMS could be explained by the Hildebrandt solubility parameters. Sorption decreased when zeolites were added to the polymer owing to the cross-linking action of the zeolite. Silicalite-filled PDMS membrane was found to be the best for selective removal of ethanol from the aroma mixture. Therefore, high silicalite loading were recommended for preparation of alcohol-free beverages or removal of ethanol from biofermentations.



Takacs et al. (2007) studied the reduction of alcohol content of wine by the pervaporation process using organophilic flat composite membrane, PDMS. Alcohol concentrate was obtained in the permeate stream. At higher temperatures, the flux of the permeate was higher and the membrane surface demand was smaller but the separation efficiency was lower. Lower pervaporation temperatures were more favorable to avoid aroma loss. In another study on the reduction of alcohol content of wine by pervaporation, the flux of the permeate increased but the separation efficiency and the separation ability of the membrane decreased as temperature increased (Takacs et al., 2007). In pervaporation of alcoholic beverages, complicated interactions between ethanol and aroma compounds often exist and affect their pervaporation performance. Tan et al. (2005) studied a series of model solutions containing ethanol and six typical aroma compounds in alcoholic beverages. The results showed that the solubility thermodynamics with the feed solution activity coefficient had a dominant effect on the permeability rather than diffusion factor. The presence of aroma compounds decreased the permeability coefficient and the separation factor of ethanol compared with those in ethanol–water binary solutions whereas its effect on fluxes was less. The effect of ethanol feed concentration on mass transfer of each compound was much related to the solubility properties of the compound in ethanol and water. It was observed that little interaction exists between aroma compounds in the membrane during the pervaporation process for dilute solution having a concentration below 1000 ppm. 8.5.3 Recovery of tea, cocoa and coffee aromas The possibility of using pervaporation to recover the tea aroma compounds from tea extract was studied by Kanani et al. (2003). Two different membrane types (POMS and PDMS) were used to recover eight aroma compounds that make a significant contribution to tea aroma. Pervaporation was performed with binary aqueous solutions of aroma compounds, with multicomponent mixture and with an actual tea extract. The actual tea aroma extract showed quite different behavior from the model aroma mixture. The results indicated that pervaporation is an attractive technology for the recovery of tea aroma compounds from tea aroma condensates. However, a wide range of selectivities for different aroma compounds results in alterations in the aroma profile of the permeate. Pilot plant tests for obtaining aroma extracts from cocoa using pervaporation were reported by Kattenberg and Willemsen (2001). The constructed test unit could process up to 500 L of feed, using hollow fiber membranes with a PDMS skin and two-stage condensation for permeate recovery. Application of pervaporation for recovering aroma concentrate from a caffeine- or theobromine-containing food such as coffee or tea and, in particular, cocoa was investigated by Kattenberg et al. (2002). In this study, first an


238 Separation, extraction and concentration processes aqueous extract was obtained. After incubating the food material at a suitable temperature and for a suitable time, the food extract was pervaporated using a hydrophobic pervaporation membrane to obtain the aroma concentrate. 8.5.4 Recovery of dairy aromas It is also possible to concentrate dairy flavor compounds from aqueous solutions using pervaporation. Baudot and Marin (1996) concentrated two dairy aroma compounds (methylthiobutanoate, diacetyl) in model aqueous solutions by pervaporation through two different membranes (silicalitefilled silicone composite membrane (PDMS 1070) and PEBA homogeneous membrane. Tested membranes showed good selectivity for the extraction of methylthiobutanoate (hydrophobic cheese aroma) at high dilution rate. However, these membranes were less selective for the recovery of diacetyl (2,3-butanedione, hydrophilic butter aroma). For this component, two-stage condensation coupled with pervaporation improved the selectivity of the whole process significantly. Overington et al. (2008) worked with the model feed solution containing nine flavor compounds contributing the flavor of cheese and other dairy products in pervaporation. They studied the effect of membrane type (two types of PDMS and a POMS), feed temperature and permeate pressure. Total flux increased with increase in temperature but decreased with increase in permeate pressure. PDMS membranes gave higher total flux than POMS membrane but the POMS membrane gave higher enrichment factors for the major compounds in the permeate. Esters and ketones passed through the membrane more readily than acids. Diffusion was the controlling mechanism for esters and ketones. Enrichment factors decreased with increasing molecular weight within esters and ketones. However, the effect of molecular weight was more complex for acids and depended on the relative importance of sorption and diffusion mechanisms in the membrane. 8.5.5 Pervaporation as an analytical separation technique Analytical separation techniques play an important role in analytical chemistry. Analytical pervaporation has been successfully used for the continuous determination of volatile analytes or volatile reaction products from complex liquid, semi-solid and solid samples in food and beverage industries (Amador-Hernandez and Castro, 2000). Schafer et al. (2006) investigated the potential of pervaporation combined with an electronic nose based on metal oxide sensors for analyzing wine model solutions. It was shown that target solutes present at minor concentrations can be detected even in the presence of bulk interferents such as ethanol by combining pervaporation with an artificial olfactory system based on metal oxide sensors.



8.5.6 Other applications of pervaporation for the recovery of aromas Pervaporation can also be used for the extraction of aroma compounds from natural matrixes as an alternative to steam distillation and solvent extraction. Enzymatic pretreatment for degradation of cell membrane is necessary in these processes. Figoli et al. (2006) studied the recovery of bergamot peel oil by pervaporation. There are some studies that combine pervaporation with bioprocessing. Bluemke and Schrader (2001) carried out an integrated bioprocess for production and recovery of the synthesized aroma compounds by interlinking a pervaporation membrane (POMS, PEBA) module with a bioreactor. In this study, they used the fungus Ceratocystis moniliformis which produce ethyl acetate, propyl acetate, isobutyl acetate, isoamyl acetate, citronellol and geraniol. In situ product removal using pervaporation leads to decreased product concentrations in the bioreactor and increased microbial growth rates. Total yield of aroma compounds produced was higher in the integrated bioprocess than with batch cultivation. Böddeker et al. (1997) showed that natural vanillin formed by bioconversion of suitable precursors can be recovered directly from the acidified culture broth by pervaporation at elevated temperature. Large volumes of waste water are obtained in most food processes. Since this liquid is generally released into the environment, it represents both pollution and an economical loss because of the cost of the valuable organic molecules involved. Pervaporation can be used to remove these valuable aroma compounds from waste water. Pierre et al. (2001) carried out non-dispersive solvent extraction of three sulfur aroma compounds, dimethyldisulfide, dimethyltrisulfide and S-methyl thiobutanoate, from very dilute aqueous solutions representing real effluent. Mass transfer fluxes obtained experimentally by membrane-based solvent extraction were greater for the three aroma compounds than those obtained by pervaporation. Souchon et al. (2002) used pervaporation process for deodorization of cauliflower blanching effluent in order to reduce its volatile organic compounds content and to recover the valuable food flavoring fraction. Pervaporation has been performed on three sulfur compounds of the cauliflower odor. Pervaporation was successfully applied for the recovery of linalool and eucalyptol which are the most frequent terpenes in wastewaters from the essential oil industry (Charbit et al., 1997).

8.6 Sources of further information and future trends Pervaporation theory and principles, separation characteristics, thermodynamics and polymer materials for membranes are covered in the book edited by Huang (1991), in which the chapter by Böddeker and Bengtson (1991) deals with the separation of organics from water by pervaporation, and that


240 Separation, extraction and concentration processes by Koops and Smolders (1991) gives information about membranes used in pervaporation. Strathmann and McDonogh (1993) studied mass transfer in pervaporation, characterization of pervaporation membranes and use of pervaporation in biotechnology. Brüschke (2006) studied the transport resistances and principles of pervaporation, membranes and the application of pervaporation in the chemical industry. Mass transport phenomena during the recovery of volatile compounds by pervaporation were investigated by Schafer and Crespo (2002). Pereira et al. (2006) reviewed the applications of pervaporation for the recovery of volatile aroma compounds from fruit juices. The studies on scale-up of the pervaporation process are limited and mostly based on the separation of model solutions. Therefore, pervaporation studies using real food systems should be increased in future. In addition, further study is required on scale-up. Studying pervaporation in large-scale systems provides insight into the industrial application of this method. Future trends are towards the combination of pervaporation with other processes in the food process line.

8.7 References Alvarez S, Riera F A, Alvarez R, Coca J, Cuperus F P, Bouwer S T, Boswinkel C, van Gemert RW, Veldsink JW, Giorno L, Donato L, Todisco S, Drioli E, Olsson J, Tragardh G, Gaeta S N and Panyor L (2000), ‘A new integrated membrane process for producing clarified apple juice and apple juice aroma concentrate’, J Food Eng, 46, 109–125. Amador-Hernandez J and de Castro MDL (2000), ‘Pervaporation: a useful tool in food analysis’, Food Chem, 68, 387–394. Aroujalian A and Raisi A (2007), ‘Recovery of volatile aroma components from orange juice by pervaporation’, J Membr Sci, 303, 154–161. Baudot A and Marin M (1996), ‘Dairy aroma compounds recovery by pervaporation’, J Membr Sci, 120, 207–220. Bengtsson E, Tragardh G and Hallstrom B (1992), ‘Concentration of apple juice aroma from evaporator condensate using pervaporation’, Lebensm Wiss Technol, 25, 29–34. Bluemke W and Schrader J (2001), ‘Integrated bioprocess for enhanced production of natural flavors and fragrances by Ceratocystis moniliformis’, Biomol Eng, 17, 137–142. Borjesson J, Karlsson H O E and Tragardh G (1996), ‘Pervaporation of a model apple juice aroma solution: comparison of membrane performance’, J Membr Sci, 119, 229–239. Böddeker K and Bengtson G (1991), ‘Selective pervaporation of organic from water’, in R. Y. M. Huang, Pervaporation membrane separation processes, Elsevier, Amsterdam, The Netherlands, 437–460. Böddeker K W, Gatfield I L, Jahnig J, Schorm C (1997), ‘Pervaporation at the vapor pressure limit: Vanillin’, J Membr Sci, 137, 155–158. Brüschke H E A (1990), ‘Removal of ethanol from aqueous streams by pervaporation’, Desalination, 77, 323–329. Brüschke H E A (2006), ‘State-of-art of pervaporation process in the chemical industry’, in S. P. Nunes and K. V. Peinemann, membrane technology in the chemical industry, Wiley-VCH, USA, 151–202.


Principles of pervaporation for the recovery of aroma compounds 241 Cassano A, Figoli A, Tagarelli A, Sindona G, Drioli E (2006), ‘Integrated membrane process for the production of highly nutritional kiwifruit juice’, Desalination, 189, 21–30. Charbit G, Charbit F and Molina C (1997), ‘Study of mass transfer limitations in the deterpenation of waste waters by pervaporation’, J Chem Eng Jpn, 30, 382–387. De Assis A V R, Bizzo H R, da Matta V M, Cabral L M C (2007), ‘Recovery of aroma compounds of cashew apple fruit (Anacardium accidentale L.) by pervaporation’, Ciencia E Technologia De Alimentos, 27, 349–354. Diban N, Urtiaga A and Ortiz I (2008), ‘Recovery of key components of bilberry aroma using a commercial pervaporation membrane’, Desalination, 224, 34–39. Escoudier J L, Le Bouar M, Moutounet M, Jouret C and Barillere J M (1988), ‘Application and evaluation of pervaporation for production of low alcohol wines’, in R. Bakish, Proceedings of the Third International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corporation, Englewood Cliffs, NJ, USA, 387–397. Figoli A, Tagarelli A, Mecchia A, Trotta A, Cavaliere B, Lavecchia R, Sindona G and Drioli E (2006), ‘Enzyme-assisted pervaporative recovery of concentrated bergamot peel oils’, Desalination, 199, 111–112. Garcia V, Diban N, Gorri D, Keiski R, Urtiaga A and Ortiz I (2008), ‘Separation and concentration of bilberry impact aroma compound from dilute model solution by pervaporation’, J Chem Technol Biotechnol, 83, 973–982. Huang R Y M (1991), Pervaporation membrane separation processes, Elsevier Science Publishers, Amsterdam, The Netherlands. Isci A, Sahin S and Sumnu G (2006), ‘Recovery of strawberry aroma compounds by pervaporation’, J Food Eng, 75, 36–42. Kanani D M, Nikhade B P, Balakrishnan P, Singh G and Pangarkar V G (2003), ‘Recovery of Valuable Tea Aroma Components by Pervaporation’, Ind Eng Chem Res, 42, 6924–6932. Karlsson H O E and Tragardh G (1993), ‘Aroma compound recovery with pervaporationfeed flow effects’, J Membr Sci, 81, 163–171. Karlsson H O E, Loureiro S and Tragardh G (1995), ‘Aroma compound recovery with pervaporation-Temperature effects during pervaporation of a muscat wine’, J Food Eng, 26, 177–191. Karlsson H O E, Tragardh G and Olsson J (1998), ‘The performance of pervaporative aroma recovery units: Process simulations’, Sep Sci Technol, 33, 1629–1652. Kattenberg H R and Willemsen J H A (2001), ‘Aroma extracts from cocoa’, Manuf Confect, 82, 73. Kattenberg H R, Willemsen J H A, Starmans D A J, Hoving H D and Winters MGM (2002), ‘Method for recovering aroma concentrate from caffeine- or theobrominecomprising food base material’, US Patent Office Journal, No. 1139792. Kimmerle K and Gudernatsch W (1991), ‘Pilot dealcoholization of beer by pervaporation’, in R. Bakish, Proceedings of the Fifth International Conference on Pervaporation Processes in the Chemical Industry, Bakish Materials Corporation, Englewood Cliffs, NJ, USA, pp. 291–307. Kober P A (1917), ‘Pervaporation, perstillation, and percrystallisation’, J Am Chem Soc, 39, 944–948. Koops G H and Smolders C A (1991), ‘Estimation and evaluation of polymeric materials for pervaporation membranes’, in R. Y. M. Huang, Pervaporation membrane separation processes, Elsevier, Amsterdam, The Netherlands, 253–278. Lipnizki F, Hausmanns S, Ten P, Field R W and Laufenberg G (1999), ‘Organophilic pervaporation: prospects and performance’, Chem Eng J, 73, 113–129. Lipnizki F, Olsson J and Tragardh G (2002a), ‘Scale-up of pervaporation for the recovery of natural aroma compounds in the food industry. Part 1: Simulation and performance’, J Food Eng, 54, 183–195.


242 Separation, extraction and concentration processes Lipnizki F, Olsson J and Tragardh G (2002b), ‘Scale-up of pervaporation for the recovery of natural aroma compounds in the food industry. Part 2: Integration and optimization’, J Food Eng, 54, 197–205. Luo Y, Tan S J, Wang H, Wu F W, Liu X M, Li L, Zhang Z B (2008), ‘PPMS composite membranes for the concentration of organics from aqueous solutions by pervaporation’, Chem Eng J, 137, 496–502. Olsson J and Tragardh G (1999), ‘Influence of feed flow velocity on pervaporative aroma recovery from a model solution of apple juice aroma compounds’, J Food Eng, 39, 107–115. Olsson J and Tragardh G (2001), ‘Pervaporation of volatile organic compounds from water. I. Influence of permeate pressure on selectivity’, J Membr Sci, 187, 23–37. Olsson J, Tragardh G and Lipnizki F (2002), ‘The influence of permeant and membrane properties on mass transfer in pervaporation of volatile organic compounds from dilute organic solutions’, Sep Sci Technol, 37, 1199–1223. Overington A, Wong M, Harrison J and Ferreira L (2008), ‘Concentration of dairy flavour compounds using pervaporation’, Int Dairy J, 18, 835–848. Peng M and Liu S X (2003), ‘Recovery of aroma compounds from dilute model blueberry solution by pervaporation’, J Food Sci, 68, 2706–2710. Pereira C C, Ribeiro C P, Nobrega R, Borges C P (2006), ‘Pervaporative recovery of volatile aroma compounds from fruit juices’, J Membr Sci, 274, 1–23. Pereira C C, Rufino J M, Habert A C, Nobrega R, Cabral L M C and Borges C P (2002), ‘Membrane for processing tropical fruit juice’, Desalination, 148, 57–60. Pereira C C, Rufino J R M, Habert A C, Nobrega R, Cabral L M C and Borges C P (2005), ‘Aroma compounds recovery of tropical fruit juice by pervaporation: membrane material selection and process evaluation’, J Food Eng, 66, 77–87. Pierre F X, Souchon I, Marin M (2001), ‘Recovery of sulfur aroma compounds using membrane-based solvent extraction, J Membr Sci, 187, 239–253. Raisi A, Aroujalian A, Kaghazchi T (2008), ‘Multicomponent pervaporation process for volatile aroma compounds recovery from pomegranate juice’, J Membr Sci, 322, 339–348. Rajagopalan N and Cheryan M (1995), ‘Pervaporation of grape juice aroma’, J Membr Sci, 104, 243–250. Sampranpiboon P, Jiraratananon R, Uttapap D, Feng X and Huang R Y M (2000), ‘Separation of aroma compounds from aqueous solutions by pervaporation using polyoctylmethyl siloxane (POMS) and polydimethyl siloxane (PDMS) membranes’, J Membr Sci, 174, 55–65. Schafer T and Crespo J G (1997), ‘Recovery of aromas by pervaporation’, ESMST XIVth Annual Summer School, Membrane integration in clean processes. Lisbon Instituto: Superior Tecnico, IST. Schafer T and Crespo J G (2002), ‘Mass transport phenomena during the recovery of volatile compounds by pervaporation’, in J. Welti-Chanes, J. F. Velez-Ruiz and G.V. Barbosa-Canovas, Transport phenomena in food processing, CRC Press, Florida, USA, 247–263. Schafer T and Crespo J G (2007), ‘Study and optimization of the hydrodynamic upstream conditions during recovery of a complex aroma profile by pervaporation’, J Membr Sci, 301, 46–56. Schafer T, Bentson G, Pingel H, Boddeker K W and Crespo J P S G (1999), ‘Recovery of aroma compounds from a wine-must fermentation by ogranophilic pervaporation’, Biotechnol Bioeng, 62, 412–421. Schafer T, Serrano-Santos M B, Rocchi S and Fuoco R (2006), ‘Pervaporation membrane separation process for enhancing the selectivity of an artificial olfactory system (“electronic nose”)’, Anal Bioanal Chem, 384, 860–866.


Principles of pervaporation for the recovery of aroma compounds 243 She M and Hwang S-T (2006), ‘Recovery of key components from real flavor concentrates by pervaporation’, J Membr Sci, 279, 86–93. Shepherd A, Habert A C and Borges C P (2002) ‘Hollow fiber modules for orange juice aroma recovery using pervaporation’, Desalination, 148, 111–114. Song K H and Lee K R (2005), ‘Pervaporation of flavors with hydrophobic membrane’, Korean J Chem Eng, 22, 735–739. Souchon I, Pierre F X, Athes-Dutour V and Marin M (2002), ‘Pervaporation as a deodorization process applied to food industry effluents: recovery and valorisation of aroma compounds from cauliflower blanching water’, Desalination, 148, 79–85. Strathmann H and McDonogh R M (1993), ‘Use of pervaporation in biotechnology’, in J. A. Howell, V. Sanchez and R. W. Field, Membranes in bioprocessing: theory and applications, Chapman and Hall, Cambridge, UK, 293–329. Takacs L, Vatai G, Korany K (2007), ‘Production of alcohol free wine by pervaporation’, J Food Eng, 78, 118–125. Tan S J, Li L, Xiao Z Y, Wu Y T and Zhang Z B (2005), ‘Pervaporation of alcoholic beverages – the coupling effects between ethanol and aroma compounds’, J Membr Sci, 264, 129–136. Trifunovic O, Lipnizki F and Tragardh G (2006), ‘The influence of process parameters on aroma recovery by hydrophobic pervaporation’, Desalination, 189, 1–12. Vankelecom I F J, Beukelaer S D and Uytterhoeven J B (1997), ‘Sorption and pervaporation of aroma compounds using zeolite-filled PDMS membranes’, J Phys Chem B, 101, 5186–5190.



9 Advances in membrane-based concentration in the food and beverage industries: direct osmosis and membrane contactors E. Drioli and A. Cassano, Institute on Membrane Technology, ITM-CNR, Italy Abstract: The process fundamentals and the role of operating conditions involved in direct osmosis (DO) and membrane contactors, such as osmotic distillation (OD) and membrane distillation (MD) are reviewed. Their main applications in concentration of liquid food and limitations compared with those of conventional de-watering processes – such as thermal evaporation, cryoconcentration and reverse osmosis – are discussed. The advantage lies in allowing very high concentrations (up to 65 °Brix) to be reached under atmospheric pressure and low temperature, thus avoiding thermal and mechanical damage. Key words: membrane contactors, direct osmosis, osmotic distillation, membrane distillation, food industry.

9.1 Introduction Epidemiological studies report that high consumption of fruit and vegetables is associated with a reduced risk of free radical related oxidative damage and diseases, such as various types of cancer, and cardiovascular or neurological diseases (Collins and Harrington, 2002). The world market of these products has remarkably increased in recent years and consumers have addressed their interest towards products with a natural fresh taste, that are high quality and additive-free. In order to meet these demands, the food industry has focused on the development of processed items with increased shelf-life able to retain as much as possible the essence of the fresh fruit, as well as colour, aroma, nutritional value and structural characteristics.


Advances in membrane-based concentration 245 In the traditional methods of processing liquid foods, the concentration operation is particularly important for the production of intermediates that can be easily handled, packaged, stored and shipped before their reutilization for a final transformation (Luh et al., 1986) and also as a step which precedes total dehydration. Moreover, the removal of water reduces the growth of micro-organisms, thus increasing the shelf-life of the liquid foods; fruit juice concentrates, because of their low water activity, have a higher stability than single-strength juices. This chapter briefly describes the existing methods for the concentration of liquid foods (such as thermal evaporation, cryoconcentration and membrane concentration) and then introduces novel membrane processes, including direct osmosis (DO), membrane distillation (MD) and osmotic distillation (OD), which offer improved performances in terms of product quality, reduced energy consumption and environmental impact, and high effectiveness.

9.2 Conventional technologies in the food and beverage industries 9.2.1 Thermal evaporation In thermal evaporation, water is removed from liquid foods as vapour. Falling film evaporators and centrifugal evaporators are most commonly used for this purpose. It is known that the heat required to perform the evaporation results in some ‘cooked’ notes recognized as off-flavours, loss of most volatile aroma compounds, and colour degradation with a consequent remarkable qualitative decline (Maccarone et al., 1996). Partial recovery of aroma compounds may be achieved by cold-water condensation and rectification in a still located on the evaporator, resulting in concentrates that are acceptable to consumers, but still far from fresh products. An additional drawback in the use of thermal evaporation is the high energy demand, despite the use of energy-saving systems. The production of superior quality concentrates, especially fruit juices, is a very important goal and considerable R&D efforts have been devoted to the development of nonthermal concentration techniques. These methods include: freeze concentration systems (cryoconcentration) and membrane processes. 9.2.2 Cryoconcentration Freeze concentration has long been considered a feasible prospect for the selective removal of water from liquid foods. In this instance, water is removed as ice rather than as vapour. The product is cooled below its freezing point allowing water to form crystals which are then removed from the concentrate by centrifugation. This method guarantees an excellent organoleptic quality: the operating temperatures are sufficiently low to avoid chemical and


246 Separation, extraction and concentration processes biochemical reactions and volatile compounds are completely retained. Consequently, the concentrated product is characterized by higher quality than that processed by thermal evaporation (Aider and de Halleux, 2008). However, there are some problems in this process which make it unpractical. The main drawback is that the achievable concentration (40–45 °Brix) is lower than the values obtained by evaporation (60–65 °Brix). Furthermore, fine ice crystals produced by rapid freezing cannot be separated from the residual liquid and the process is not suitable for the treatment of highly pulped liquids. Remarkable energy consumption, high equipment investments and operational costs are additional drawbacks (Jariel et al., 1996). 9.2.3 Pressure-driven membrane processes Pressure-driven membrane processes such as microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and reverse osmosis (RO) are well established systems in the food industry for concentration, fractionation and purification of liquid foods and for wastewater treatment (Table 9.1). These processes offer significant advantages over traditional food processing technologies in terms of low operation temperatures, no special chemicals required, possibility of automation, and no phase-change involved. In addition, they are simple in concept and operation and characterized by low energy consumption. MF membranes have a symmetric microporous structure with a pore size in the range 0.1–10 mm. They are essentially used for the separation of fine particles, micro-organisms and emulsion droplets from fluids. Particles are rejected mainly by means of sieving mechanisms although the separation is affected also by interactions between the membrane surface and the treated solution. The hydrostatic pressure difference used as a driving force is in the range from 0.5 to 4 bar. Cell harvesting, clarification of fruit juices, must and wine, wastewater treatment, bacteria and particulate turbidity reduction, separation of whey proteins and separation of oil–water emulsions are typical consolidated applications (Cheryan, 1998). UF membranes have a pore size of 1–100 nm and are capable of retaining species of molecular weight 300–500 000 daltons. Typical rejected species include lipids, proteins, polysaccharides, colloids and suspended solids whereas solutes with small molecules such as vitamins, salts and sugars, flow through the membrane together with water. Operating pressures are 2–10 bar (Mulder, 1998). In the food and dairy industry, UF is commonly applied to clarify, concentrate, decolorize and fractionate juices, sugar solutions, proteins and dairy and grain milling products. The membranes used in NF are characterized by a charged surface and equivalent pore diameters in the range 1–3 nm. They are mainly used for the separation of multivalent ions and uncharged organic molecules having molecular weights greater than 1 kDa. Operating


Advances in membrane-based concentration 247 Table 9.1 Commercial application of pressure-driven membrane processes in food processing Unit operation Industrial sector

Application

Microfiltration Fruit and vegetables

Fruit and vegetable juice clarification

Beverages Dairy

Ultrafiltration

Fruit and vegetables Beverages Vegetables Dairy

Fats and oils Nanofiltration

Fruit and vegetables Dairy Beverages

Reverse osmosis

Cold sterilization of beer Clarification of wine, beer and vinegar Bacterial purification of milk and whey Whey defatting and high-quality protein recovery Clarification of fruit juice Clarification of wine Recovery of potato starch and proteins Fractionation/concentration of whey in manufacture of whey protein concentrate Fractionation/concentration of milk in manufacture of processed and natural cheese Recovery of oilseed protein and oil from processing water Fruit and vegetable juice concentration Concentration and demineralization of milk Demineralization of whey Wastewater treatment

Fruit and vegetables

Fruit and vegetable juice concentration Sugar concentration and recovery from rinse water, treatment of water for reuse Recovery of flavours, fragrances, pectins and proteins Beverages Recovery of alcohol from wine Sugar and sweeteners Preconcentration of maple syrup Sugar concentration and recovery from rinse water, treatment of water for reuse Dairy Concentration of milk and whey All possible Treatment of boiler concentrate prior to recycle to boiler

pressures in NF are lower than those in RO therefore separation occurs at low energy consumption (21% less than RO). The concentration of must, the recovery of aromas from fruit juices, the demineralization of whey and the treatment of wastewater from beverage production are typical applications of NF in the food and dairy industry (Warczok et al., 2004). In RO, low molecular weight compounds, such as salts and sugars are separated from a solvent, usually water. The particle size range for application of RO is approximately 0.1–1 nm and a complete separation is achieved with solutes of molecular weight greater than 300 Da. Typical operating pressures are 10–100 bar. Food processing applications of RO include: concentration


248 Separation, extraction and concentration processes of fruit and vegetable juices, concentration of milk and whey for cheese production, pretreatment of boiler water, recovery of sugars and acids from rinse waters, recovery of sweet potato stillage, recovery of flavours, pectins and proteins, removal of alcohol from wine, and water softening (Ho and Sirkar, 1992). The advantages of RO over traditional evaporation are the lower thermal damage to the product, increase in aroma retention, simple system design, lower energy consumption and lower equipment costs. However, RO cannot reach retentate concentrations higher than 25–30 °Brix with a single-stage system owing to the limitation of high osmotic pressure. Furthermore, fouling and concentration polarization are still important drawbacks of RO and other pressure-driven membrane processes in liquid food processing (Jiao et al., 2004).

9.3 Direct osmosis and applications in the food and beverage industries 9.3.1 Process fundamentals In DO, an osmotic pressure gradient across a semi-permeable membrane is established by using an osmotic agent solution in order to remove water from a feed solution. The osmotic agent should be characterized by high solubility in water (low water activity), non-toxicity, high superficial tension, inertia towards the flavour, odour and colour of the foodstuff, low volatility and viscosity, and non-permeability across the membrane. The most frequently employed constituents as osmotic agents are: sodium chloride, calcium chloride, glucose, sucrose, glycerol, cane molasses or corn syrup. Unlike RO the pressure difference across the membrane in DO is negligible and the flux depends on the difference in water activities. The hydraulic pressures required to pump the feed and the osmotic solution over the membrane surface are of about 2 bar (Milleville, 1990). Consequently, fouling phenomena are negligible and only concentration polarization occurs. Additional advantages are low energy consumption, simplicity, modularity, constant permeate flux in time, possibility to treat solutions with a high level of suspended and dissolved solids, and high achievable concentrations. The membranes used in DO are semipermeable membranes similar to those used in RO and NF processes. They are highly selective; however, the diffusion of a small amount of stripping solution cannot be completely avoided. 9.3.2 Effect of operating conditions on the direct osmosis (DO) flux Petrotos et al. (1998, 1999) studied the effect of several membranes and operating parameters on the performance of DO in the concentration of


Advances in membrane-based concentration 249 tomato juice. NaCl, CaCl2, Ca(NO3)2, glucose, sucrose and polyethylene glycol (PEG 400) were used as stripping solutions. The initial concentration of tomato juice was in the range 4.3–11.7 °Brix. These studies suggested that salts, and especially NaCl, produced improved fluxes compared with carbohydrates and PEG 400, owing to their lower viscosity. An increasing of the stripping solution concentration also positively affected the flux (through an increasing of the driving force). On the contrary, an increasing of the feed concentration resulted in lower water flux owing to viscosity and osmotic pressure increase. The lower viscosity of the osmotic agents resulted in reduced polarization phenomena on the stripping solution side and increased fluxes. Increasing the temperature from 26 to 60 °C enhanced permeate flux by 64%, this phenomenon can be attributed to the reduction in the viscosity and to the increase in the diffusion coefficient of both feed and stripping solution. An increase in feed flow rate did not improve the permeate flux during the DO process. The pretreatment of the juice by MF or UF was also found to have a significant effect on the osmotic fluxes (Petrotos et al., 1998). In particular, the ultrafiltered juice gave a 39% increase in flux compared with the untreated juice. Finally, the DO flux is strongly affected by membrane thickness. In particular, a reduction of the membrane support layer determines an increase of the permeate flux (Beaudry and Lampi, 1990). 9.3.3 DO applications Osmotek Inc. (Corvallis, OR, USA) developed the first DO industrial approach as a pretreatment step for RO wastewater treatment. Several studies concerning the fruit juice concentration by DO are reported (Beaudry and Lampi, 1990; Herron et al., 1994; Milleville, 1990; Petrotos and Lazarides, 2001; Petrotos et al., 1998, 1999; Wrolstad et al., 1993). Beaudry and Lampi (1990) reported the performance of the Osmotek DO process in the concentration of fruit juices by using thin-film composite RO membranes with an overall thickness of 25–85 mm and a nominal molecular weight cut off (NMWCO) of 100 Da. These membranes rejected more than 99.9% of the total acidity and colour in orange and red raspberry juice. Herron et al. (1994) developed a simplified DO apparatus for several kinds of liquid food samples including orange and raspberry juice. Maximum osmotic fluxes of 5–6 L m–2 h–1 were obtained through 100-Da membranes by using fructose/glucose solutions as osmotic agent. The concentrated juice showed a better quality than that produced by a conventional vacuum evaporator. Stripping solutions diluted during juice concentration can be reconcentrated by using thermal evaporation: therefore, only the osmotic agent is exposed to high temperatures preserving the quality of concentrated juice. RO was also proposed as a method for the recovery of stripping solutions (Karode et al., 2000). Petrotos and Lazarides (2001) developed a new apparatus for the concentration of fruit juices with an osmotic cell of special configuration


250 Separation, extraction and concentration processes to promote turbulence. The apparatus was equipped with RO flat-sheet membranes to concentrate tomato juice up to 16 °Brix at room temperature. Average fluxes of 4.5 kg m–2h–1 were obtained.

9.4 Membrane contactors and applications in the food and beverage industries Membrane contactors are devices in which a microporous membrane acts as a barrier between two phases permitting gas/liquid or liquid/liquid mass transfer of the components without dispersion of one phase into the other. Fluids to be contacted flow on the opposite side of the membrane and a fluid/fluid interface is located at the mouth of each membrane pore. Mass transfer occurs by diffusion across the interface as in conventional contacting equipment. Unlike conventional membrane operations, the driving force for separation is a concentration or a temperature gradient rather than a pressure gradient and a small pressure drop across the membrane is required to maintain the fluid/fluid interface at the mouth of the pore. Furthermore, the membrane imparts no selectivity to the separation (Gabelman and Hwang, 1999). Membrane contactors offer significant advantages over conventional dispersed-phase contactors such as the absence of emulsions, no density difference required between the fluids, easy scale-up, high interfacial area, no flooding at high flow rates. Within membrane contactors, membrane distillation (MD) and osmotic distillation (OD) seem to be a valid alternative for pressure-driven membrane processes in the concentration of liquid foods. In these processes, schematically represented in Fig. 9.1, the driving force for mass transfer is a vapour pressure difference across the membrane generated by either a temperature gradient (in MD) or a water activity difference (in OD). Their main advantages over traditional pressure-driven membrane processes are in terms of: low fouling, possibility of treatment of highly viscous solutions, high retention of species, and low energy consumption. Furthermore, these processes are not limited by high osmotic pressures and allow concentration levels to be attained that are similar to those obtained in thermal evaporation. 9.4.1 Osmotic distillation Process fundamentals Osmotic distillation is a recent membrane process also known as osmotic evaporation, osmotic concentration by membrane, membrane evaporation, isothermal membrane distillation, transmembrane distillation, thermopervaporation or gas membrane extraction. It can be carried out at room temperature and atmospheric pressure with minimal thermal and mechanical damage of the solutes. Consequently, it represents an attractive process for © Woodhead Publishing Limited, 2010

Advances in membrane-based concentration 251 Evaporation

Condensation

Tf > Tp Vapour

L/G interphase


L/G interphase

Feed

Permeate (a)

Evaporation

Condensation

awf > aws Vapour

L/G interphase


Feed

L/G interphase Stripping solution

(b)

Fig. 9.1 Schematic representation of (a) membrane distillation and (b) osmotic distillation. L/G, liquid–gas.

the concentration of solutions containing thermosensitive compounds, such as fruit juices and pharmaceuticals. In OD, a hydrophobic microporous membrane separates two liquid phases having different solute concentrations: a dilute solution on one side and a hypertonic salt solution on the opposite side. The hydrophobic nature of the membrane prevents penetration of the pores by aqueous solutions, creating air gaps within the membrane. The difference in solute concentration and, consequently, in water activity of both solutions, generates, at the vapour–liquid interface, a vapour pressure gradient across the membrane causing a vapour transfer across the pores from the high-vapour pressure phase to the low one (Alves and Coelhoso, 2002; Hogan et al., 1998; Lebfevre, 1988; Mengual et al., 1993). The concentration profile of the OD process is schematically presented in Fig. 9.2. The water transport through the membrane can be summarized in three steps: (1) evaporation of water at the dilute vapour–liquid interface; (2) diffusional vapour transport through the membrane pore; (3) condensation of water vapour at the membrane/brine interface (Schofield et al., 1987; Hogan et al., 1998). © Woodhead Publishing Limited, 2010

252 Separation, extraction and concentration processes High water vapour pressure Low water vapour pressure

Vapour flux

Feed dilutes aqueous solution

Csb Pwf >> Pwp Pore

Csm

Stripping solution (concentrated salt solution)

Cfm Cfb

Air Microporous hydrophobic membrane

Bulk Boundary layer

Bulk Boundary layer

Fig. 9.2 Concentration profile in osmotic distillation.

Because water transport involves condensation and evaporation phenomena, a temperature gradient through the membrane is generated, even if bulk temperatures of solutions separated by the membrane are equal. Consequently, a heat transfer tending to reduce the driving force for the water transport should be considered in addition to a mass transfer (Celere and Gostoli, 2002). The stripping solution after its dilution by water transferred from the feed stream can be reconcentrated by evaporation and reused in the OD operation. Therefore it must be thermally stable and preferably nontoxic, noncorrosive and available at low cost. A number of water-soluble salts such as NaCl, CaCl2, K2HPO4 are suitable. Salts displaying increase in solubility with temperature are preferred because they can be concentrated to high levels avoiding crystallization phenomena. Potassium salts of orthoand pyrophosphoric acid offer several advantages, including low equivalent weight, higher water solubility, steep positive temperature coefficients of solubility and safety in foods and pharmaceuticals (Deblay, 1995; Michaels, 1998). Typical transmembrane pressures encountered in osmotic distillation are in the range of 140 kPa (Hogan et al., 1998). Mass transfer aspects The water transport in the OD process is related to the driving force, represented by the vapour pressure difference at both liquid–vapour interfaces of the membrane (DPwm ), by the following equation:

Jw = Km (Pwf – Pws)

[9.1]

where Jw is the transmembrane flux, Km the membrane mass transfer coefficient, Pwf and Pws are the water vapour pressures of the feed and stripping solution at the membrane surface.


Advances in membrane-based concentration 253 The water vapour pressures of the feed and stripping solutions at the fluid membrane interfaces are calculated as:

* Pwf = Pwf awf

[9.2]

* Pws = Pws aws

[9.3]

* * and Pws where Pwf are the pure water vapour pressures of the feed and stripping solution at the interface, respectively; awf and aws are the water activities of the feed and stripping solution at the interface, respectively. If the temperature is the same on both sides of the membrane equation [9.1] can be written as:

J w = K m Pw* (awf – aws )

[9.4]

The driving force depends on the solute concentration as well as on the temperature conditions prevailing at the vapour–liquid interfaces. A more detailed representation referring to the bulk conditions of both compartments is given by integrating the various mass transfer resistances:

lw = KD Pwb

[9.5]

where

1 1ˆ Ê1 K =Á + + ˜ K K K Ë f m s¯

–1

is defined as the overall mass transfer coefficient which accounts for the resistances opposed by the feed solution (1/Kf), the membrane (1/Km) and the stripping solution (1/Ks). If the concentration polarization is negligible, mass transfer depends only on membrane resistance and consequently:

K = K m

[9.6]

As in other membrane processes, OD is affected by concentration polarization. Owing to the water transport across the membrane, concentration in the bulk phase and, consequently, the water activity, differs from the composition near the membrane interface. As a result, the driving force for mass transfer and water flux are reduced (Ravindra Babu et al., 2006). Independently of the type of driving force (temperature difference, activity difference or both), the water vapour transfer mechanism in the membrane pores can be estimated on the basis of the molecular diffusion model, the Knudsen diffusion model or by a combination of both mechanisms (Geankoplis, 1993). If the mean free path of gas molecules is significantly greater than the membrane pore size, diffusing molecules collide frequently with pore


254 Separation, extraction and concentration processes walls rather than with gas molecules present in the pores. In this instance the molecular transport occurs by Knudsen diffusion: 2e r K mK = M RT 3 cd

8RT pM

[9.7]

where r is the pore radius, e the membrane porosity, c the tortuosity factor, d the membrane thickness, M the molecular weight, R the gas constant and T the temperature. For pore size greater than the mean molecular free path of water vapour, the molecular diffusion is the controlling mechanism and the water mass flux can be written as: De P K mM = M RT cd Plm

[9.8]

where D is the diffusion coefficient and P the pressure. The Knudsen number (Kn) can be used to determine which of the two diffusion models is predominant. It compares the mean molecular free path (l) with the mean pore diameter of the membrane and is defined as: Kn = l 2p

[9.9]

where

l=

kBT P 2p s 2

[9.10]

in which kB is the Boltzmann constant, s is the mean collision diameter of the molecule and r the pore radius. For small pore size, Kn ≥ 10, collisions with pore walls are frequent therefore Knudsen diffusion is the prevailing mechanism. With relatively large pores, Kn ≤ 0.01, more collisions between the gas molecules themselves occur and the molecular diffusion will be predominant. Both mechanisms coexist when Kn is between the two limit values (Courel et al., 2000a). The concentration polarization phenomenon can be reduced by optimization of process parameters as stirring or stripping solution properties. The liquid concentration difference between the membrane and the bulk phase determines a diffusive solute flow according to Fick’s law which counterbalances the convective flow in the opposite direction. A mass balance across the boundary layer of the stripping solution is given by:

ÈÊ C ˆ ˘ J v = kp ln ÍÁ b ˜ ˙ ÎË Cm ¯ ˚

with

kp =

Dsw d

[9.11]


Advances in membrane-based concentration 255 where Jv is the volume flux, Dsw the diffusion coefficient of solute by the membrane, kp the liquid mass transfer coefficient, Cm the salt molar concentration in the membrane, Cb the salt molar concentration in the bulk and d the boundary layer thickness. The liquid mass transfer coefficients depend on the solution physical properties and on the hydrodynamic properties of the system. These coefficients can be estimated by empirical correlations of dimensionless numbers namely Sherwood (Sh), Reynolds (Re) and Schmidt (Sc) numbers, equations [9.12] and [9.13].

Sh = a Reb Scg

[9.12]

with

Sh =

kdh , Dw

Re =

r udh , m

Sc =

m r Dw

[9.13]

where a, b and g are constants, k the feed or osmotic agent side liquid mass transfer coefficient, dh the hydraulic diameter, Dw the diffusion coefficient in water, r the solution density, m the solution dynamic viscosity and u the cross flow velocity of the fluid. Heat transfer OD is essentially a mass transfer process. The water transport through the membrane implies evaporation at the feed side and condensation at the stripping solution side. This phase change at the membrane walls generates a temperature difference across the membrane which reduces the vapour pressure difference decreasing the water transport. Under steady state conditions, the heat transfer equations can be written as:

Q = hf (Tf – Tfm) = hs (Tsm – Ts)

[9.14]

Q = Nw DHv – hm(Tsm – Tfm)

[9.15]

where Q is the heat flux, hf, hs and hm the heat transfer coefficients of the boundary layers (feed and stripping solution, respectively) and of the membrane, Tf and Tm the bulk temperatures of the feed and stripping solution, respectively, Tfm and Tsm the temperatures of the feed and stripping solution at the membrane interfaces, respectively, DHv the water latent heat of vaporization and Nw the molar vapour flux. The relation between heat flux and overall heat transfer coefficient during the OD process can be obtained by substituting Tsm and Tfm from equation [9.14] in equation [9.15], obtaining:

N DH Q = U ÈÍ w v – (Ts – Tf )˘˙ Î hm ˚

where U is the overall heat transfer coefficient given by: © Woodhead Publishing Limited, 2010

[9.16]


1 1ˆ Ê1 U=Á + + Ë hf hm hs ˜¯

–1

[9.17]

The thermal effect is considered negligible by most researchers. For example, Ravindra Babu et al. (2006, 2008) found that the contribution of concentration polarization on driving force reduction, during the concentration of pineapple and sweet-lime juices by OD, was prominent when compared with that of temperature polarization. On the other hand, the thermal effect owing to evaporation and condensation at both liquid–membrane interfaces was considered substantial by other authors (Courel et al., 2000a; Gostoli, 1999). In particular, the results of pure water OD experiments performed by Courel et al. (2000a) with commercial asymmetric porous membranes indicated that a high vapour flux of 12 kg m–2 h–1 generates a transmembrane temperature gradient of about 2 °C inducing a 30% driving force reduction. Osmotic distillation (OD) membranes and modules Hydrophobic polymers with low surface free energy are commonly used to produce membranes with pore sizes and pore size distribution suitable for OD applications. They include both polyolefins, such as polyethylene (PE) and polypropylene (PP), and perfluorocarbons such as polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF). Membranes comprising these polymers, having a pore diameter ranging from 0.1 to 1 mm, allow the gas phase to be maintained in the membrane pores, a fundamental condition required to perform the OD process (Gryta, 2005). However, hydrophilic ceramic membranes can be modified by grafting on the surface molecules containing hydrophobic fluorocarbon chains like fluoroalkylsilanes or by coating the surface of alumina membranes with a thin lipid film. These membranes have been applied successfully in OD (Gabino et al., 2007; Romero et al. 2006). By referring to the configuration, hollow-fiber membranes, characterized by thin walls, are preferred for OD applications because they offer high surface/ volume ratios and do not require supports or spacers. Various parameters have to be considered in the selection of the membrane such as: pore size, porosity, conductivity and thickness. An increase in the pore size enhances the evaporation flux (flux is proportional to the radius); furthermore, membranes with large pore size exhibit a higher retention towards volatile organic flavour/ fragrance components (Barbe et al., 1998) than membranes with smaller pore size. These results can be attributed to differences in feed–membrane and stripper–membrane boundary layer resistances to organic volatiles transport owing to various degrees of liquid intrusion into the pores. Highly porous membranes are preferred because the evaporation flux is proportional to the porosity. Furthermore, an increase in membrane conductivity allows the heat gradient across the membrane to be minimized. Because the evaporation flux is proportional to the reciprocal of the pore length, the membrane thickness should be as low as possible. The thickness © Woodhead Publishing Limited, 2010

Advances in membrane-based concentration 257 is usually limited by the mechanical strength of the membrane: therefore thin membranes are supported by net, e.g. Gelman PTFE membranes. The overall thickness for OD membranes can vary from 80 to 250 mm, depending on the absence or presence of support. The risk of wetting of the hydrophobic membrane, with a consequent reduction in the evaporation flux and separation performance, is the main drawback of the osmotic distillation process. The reduction in the membrane thickness leads to a decrease in thermal resistance that facilitates the heat transfer from the brine to the feed causing a reduction in the temperature gradient (DT) and an increase in the partial pressure gradient (DP) across the membrane. However, thin hydrophobic membranes are more susceptible of wetting with a consequent loss of both evaporation flux and separation performance. If wetting occurs, the liquid can penetrate into pores in the membrane and, consequently, a liquid flux is added to the vapour flux and nonvolatile solutes diffuse across the membrane from one compartment to the other. Some fruit juices, especially citrus juices, contain peel oils and other highly hydrophobic compounds that promote wetting of OD membranes. Furthermore, cleaning solutions often contain surface-active agents that might also promote membrane wetting. Hydrophilic polymer films, which have sufficiently high intrinsic water permeability and are virtually impermeable to macrosolutes and colloids, are suitable as laminated membranes to prevent liquid intrusion without impeding vapour transport (Hogan et al., 1998). They include: esters and ethers of cellulose, crosslinked gelatin, chitin, agar, alginic acid, crosslinked polyacrylamide, crosslinked polyvinyl alcohol (PVA), polyhydroxy 2-ethyl methacrylate (PHEMA). Commercially available cellophane membranes can be suitable for this purpose; the hydrogel-film side of the laminate should be in contact with the solution to be concentrated (Michaels, 1999). Mansouri and Fane (1999) developed modified hydrophobic membranes for OD tolerant to oily feeds (i.e. limonene) by coating the feed side of commercial membranes with a thin layer of PVA. The uncoated membranes were rapidly wetted out by the oily feed even for low concentrations of oil (limonene) dispersed in water; vice versa, the coated membranes were stable for concentrations up to 1 wt.% limonene solution for periods up to 24 h. Commercial membranes for OD applications (termed TF200, TF450 and TF1000) manufactured by Pall-Gelman (East Hills, NY, USA) are characterized by a thin PTFE layer supported by a polypropylene (PP) net. In these asymmetric membranes, the top layer offers a resistance to gas transfer, whereas the membrane support offers an additional resistance to water transfer in the liquid form. The mass transfer resistance in the vapour phase is about 40–70% of the total resistance. The resistance of diluted brine entrapped in the PP support can cover up to 30% of the total resistance and the diluted brine boundary layer up to 60% indicating the sensitivity of the OD system to concentration polarization phenomena (Courel et al., 2000a). The best-known module designed for OD is the Liqui-Cel® Extra-Flow membrane contactor (Membrana-Charlotte, North Carolina, USA) containing


258 Separation, extraction and concentration processes microporous PP hollow fibre membranes approximately 300 mm in external diameter with a wall thickness of about 40 mm; they have a mean pore diameter of about 30 nm and a porosity of about 40%. The fibres are potted into a polyethylene tubesheet and the shell casing is PP, PVDF or 316L stainless steel (Gabelman and Hwang, 1999; Hogan et al., 1998; Sirkar, 1997). The smallest modules are 2.5 ins in diameter with a membrane surface area of 1.4 m2, whereas the largest are 10 ins in diameter and offer a contact area of 130 m2. Commercial membranes commonly used in OD are summarized in Table 9.2. Effect of operating conditions on the OD flux The performance of the OD process in terms of water vapour transport is influenced profoundly by the operating conditions. Feed concentration inversely affected the performance of the OD process. Courel et al. (2000b) observed a decrease in evaporation flux (from 10.3 to 1.1 kg m–2 h–1) when sugar solutions of increasing sucrose content, from 0 to 65% w/w, were dehydrated at 25 °C by using stripping solution of 45.5 w/w% initial CaCl2 content. A transmembrane flux decay by increasing the feed concentration was observed also by Ravindra Babu et al. (2006) in the concentration of sweetlime juice and phycocianin solution by OD. The viscosity of the feed solution increases exponentially with the solute content whereas the diffusion coefficient strongly decreases relative to pure water. The increasing viscosity results in an increase of the concentration polarization effect reducing the driving force and, consequently, the flux rate (Bui et al., 2005). Sheng et al. (1991) studied the effect of operating conditions on the OD flux during the concentration of apple, orange and grape juice through a PTFE membrane with a pore size of 0.2 mm and an overall thickness of 100 mm. The osmotic pressure difference D p between the aqueous streams strongly affected the transmembrane vapour flux; in particular, a 33% decrease in D p determined a five-fold decline in OD flux. The OD flux is significantly affected also by the solute content of the stripping solution. Courel et al. (2000b) observed a 64% vapour flux decay for a 30% mass fraction reduction of the brine solution in extracting pure water at 25 °C by using CaCl2 as extractant. Ravindra Babu et al. (2006) observed a similar trend in the concentration of phycocianin solution and sweet-lime juice. These results may be explained by assuming the strong dependence of the water activity of the stripping solution on salt content. Although a vapour flux improvement is expected when the salt content is reduced (since the density and viscosity of the brine tend to decrease reducing the mass transfer resistance in the salt solution) this improvement is in fact masked by the activity effect. The OD flux is also differently affected by the type of osmotic agent. Calcium chloride produces higher transmembrane fluxes than sodium chloride © Woodhead Publishing Limited, 2010

Table 9.2 Typical membranes used in the OD process (adapted from Gryta, 2005) Manufacturer

Material

Configuration

Thickness (mm)

Porosity (%)

Average pore size (mm)

Durapore GVHP Durapore GVSP Durapore HVHP FHLP Accurel PP Q3/2 Accurel PP S6/2 Celgard 2500 Celgard 2400 Metricel SM35 TF200 TF450 TF1000 Gore-Tex 10387

Millipore Co. Millipore Co. Millipore Co. Millipore Co. Enka A.G. Enka A.G. Celgard LLC Celgard LLC Gelman USA Scimed Life Systems Inc. Pall-Gelman Pall-Gelman Pall-Gelman Gore & Associates

PVDF PVDF PVDF PTFE PP PP PP PP PP PDMS PTFE layer PTFE layer PTFE layer PTFE layer

Flat Flat Flat Flat Capillary Capillary Flat Flat Flat

125 108 125 175 200 400 25 25 90 300 178 178 178 8.5

70 80 75 70 70 70 50 37 55

0.20 0.22 0.45 0.25 0.2 0.2 0.07 0.05 0.1

80 80 80 78

0.2 0.45 1.0 0.2

supported supported supported supported

by by by by

PP PP PP PP

net net net net

Flat Flat Flat Flat



Type

260 Separation, extraction and concentration processes in the concentration of pineapple juice by OD (Nagaraj et al., 2006). This is mainly because of the higher osmotic activity of calcium chloride that results in a higher vapour pressure gradient across the membrane. Aqueous solutions of propylene glycol, glycerol and glycerol–salt mixtures were investigated as an alternative to calcium chloride in order to overcome the problem of corrosion and scaling associated with the use of brines (Celere and Gostoli, 2004). Propylene glycol and glycerol solutions (70–75 wt.%) were less effective than highly concentrated CaCl2 and exhibited a similar extractive power. However, propylene glycol cannot be recommended as an extractant in juice concentration owing to its low penetration pressure through the membrane pores and the not negligible volatility. Ternary mixtures water–glycerol–NaCl are characterized by lower viscosities than glycerol alone, and offer similar fluxes. The transmembrane flux in OD increases with an increase in the flow rate of the osmotic agent (Courel et al., 2000b; Nagaraj et al., 2006). This can be attributed to a stronger shear stress along the condensation side of the membrane leading to a reduction in the hydrodynamic boundary layer thickness and, consequently, to the concentration polarization effect. Ravindra Babu et al. (2008) observed an 8% increase in transmembrane flux when the flow rate of pineapple juice at about 12 °Brix was increased from 25 mL min–1 to 100 mL min–1; the increase in transmembrane flux can be explained by assuming a reduction in the concentration polarization effect on the feed side. The increase in flux, however, was more prominent (about 20%) by increasing the osmotic agent velocity. This phenomenon can be attributed to a lower concentration polarization on the feed side than on the brine side. A minor role of feed velocity on reducing the polarization problem in OD was observed also by Bui and Nguyen (2006) in the concentration of aqueous glucose solutions. They assumed that the heat transfer in OD is only a little involved or not involved at all; on the contrary, feed flow rate is essential to maintain the temperature gradient across the membrane in MD. The evaporation flux in OD is also affected by the feed temperature. Courel et al. (2000b) studied the influence of the temperature under isothermal conditions on the evaporation fluxes of pure water and sugar solutions of 35–65 w/w% sucrose content at 20–35 °C. A calcium chloride solution with an initial concentration of 45 w/w% was used as stripping solution. Evaporation fluxes ranged from 0.5 kg m–2 h–1 for a sugar solution of 65 w/w% (at 20 °C) to 12.8 kg m–2 h–1 for pure water (at 35 °C). The extent of mass transfer increase depended on the solute content: in the range 20–35 °C the evaporation flux increased by 120%, for a 35 w/w% sucrose solution, and only by 32% at 60 w/w% solute content. This phenomenon can be explained by assuming an exponential type relation between the vapour pressure difference across the membrane and the temperature according to Clapeyron’s law. Moreover, an increase in temperature results in a decrease in the feed and brine viscosities and an increase in the solute diffusion


Advances in membrane-based concentration 261 coefficient: the higher the solute content, the stronger the thermal effect. Bui and Nguyen (2006) reported a 200% increase in the evaporation flux for a feed temperature increase of 20 degrees in the concentration of 40 w/w% and 50 w/w% aqueous glucose solutions by means of PVDF hollow fibres. OD applications Because OD can be operated at room temperature and atmospheric pressure, with minimal thermal and mechanical damage of the solutes, it has been successfully applied to the concentration of liquid foods such as milk, fruit and vegetable juices, instant coffee, tea and pharmaceuticals. The low temperature employed avoids the chemical or enzymatic reactions associated with heat treatment. The low operating pressure results in lower energy consumption and capital investment, reduced fouling phenomena and the possibility of using membranes characterized by lower mechanical resistance than those in pressure-driven membrane processes (Courel et al., 2001). OD is particularly suited for fruit juice concentration, producing concentrated juices with a quality and composition very close to fresh ones and higher than conventional products obtained by thermal evaporation (Cassano and Drioli, 2007; Jiao et al., 2004). Plate-and-frame modules with a net-shaped spacer on the extract side and a smooth juice side path were developed for the concentration of whole juice with a high pulp content (Cervellati et al., 1998). This configuration, even if not completely efficient in terms of mass transfer, allows unclarified juices to be processed. Helically wound hollow-fibre modules offer an improvement in the hydrodynamic conditions on the shell-side compared with axial flow modules; consequently, higher concentration of solutes and higher evaporation fluxes can be obtained when viscous feeds are processed (Costello et al., 1997). Rodrigues et al. (2004) evaluated the performance of the OD and RO processes in the concentration of camu-camu juice. RO allowed higher fluxes to be reached (50 kg m–2 h–1) than OD, but lower concentration of soluble solids (25 °Brix). OD allowed the juice to be concentrated up to 63 °Brix with evaporation flux values of 10 kg m–2 h–1. A pre-treatment of the whole juice by UF or MF allows evaporation fluxes to be improved in OD. However, the performance of the OD process is affected by pore diameters of UF membranes. Bailey et al. (2000) observed higher evaporation fluxes when Gordo grape juice was preliminarily ultrafiltered with membranes having pore diameters of 0.1 mm or less. UF membranes with a nominal pore diameter of 0.5 mm did not improve the OD fluxes. UF pretreatment results also in a small increase in juice surface tension with a consequent reduction in the tendency for membrane wet-out to occur. Several kinds of clarified fruit juices (orange, apple, grape, cactus pear, kiwi, passion fruit, and pineapple) were concentrated by OD (Alves and Coelhoso, 2006; Cassano and Drioli, 2007; Cassano et al., 2003, 2004, 2006,


262 Separation, extraction and concentration processes 2007; Durham and Nguyen, 1994; Galaverna et al., 2008; Hongvaleerat et al., 2008; Jiao et al., 1991; Koroknai et al., 2006a, 2006b; Rektor et al., 2006; Rodrigues et al., 2004; Vaillant et al., 2001, 2005). An integrated membrane process was developed by Cisse et al. (2005) to produce concentrated orange juice. The process included a MF pre-treatment followed by OD. The quality of the concentrated juice was similar to the freshly squeezed one. Cassano et al. (2003, 2004, 2006 and 2007) proposed integrated membrane processes including UF, RO and OD to produce concentrated fruit juices such as kiwifruit, orange, lemon and cactus pear juice. Depectinized juices were clarified by UF and then optionally preconcentrated by RO. The OD process was employed as a concentration step to produce concentrated juices with final total soluble solids (TSS) up to 63–65 °Brix. The proposed process employs partial batch recycle on the feed side of the OD step in order to minimize feed viscosity changes through the membrane contactors and continuous counter current recycle plus evaporative reconcentration of the brine strip. The residual fibrous phase coming from the UF treatment (retentate) could be submitted to a stabilizing treatment (pasteurization, ohmic heating, high pressures, electric fields at high voltage) and successively added to the final OD concentrate for the preparation of fibre enriched beverages (Fig. 9.3). The total antioxidant activity (TAA) of the clarified or pre-concentrated juice was kept constant during the OD process independently of the achieved degree of TSS. Both aroma and colour were similar to those of the fresh juice. Ascorbic acid and health-promoting substances were also well preserved. On the contrary, a larger decrease in content of antioxidant compounds was detected in thermally evaporated juices with the same TSS content. In the concentrated orange juice, anthocyanins and hydroxycinnamates (particularly ferulic and p-coumaric acid) underwent a reduction of 36 and 55%, respectively; for the ascorbic acid and flavonones removals were in the order of 30% and 23%, respectively (Galaverna et al., 2008). Cassano and Drioli (2007) also evaluated the quality of kiwifruit juice concentrated by OD and thermal evaporation. The juice concentrated by thermal evaporation at 65 °Brix showed an 87% reduction of the ascorbic acid compared with the clarified juice. The TAA was reduced by about 50% independently of the TSS content achieved. On the contrary, the juice concentrated by OD retained almost all the TAA and the ascorbic acid content of the clarified juice. Blackcurrant juices concentrated by OD, with a final TSS content of 63–72 °Brix, showed a colour intensity, transparent ability and acidic flavour intensity similar to those of the raw juice (Kozák et al., 2008). A hybrid plant consisting of UF and RO pretreatment stages and an OD section (containing two 19.2 m2 Liqui-Cel membrane modules) for the concentration of fruit and vegetables juices was developed by Zenon Environmental (Burlington, Ont.). The plant was designed to produce concentrated juice at 65–70 °Brix at an average flow rate of 50 L h–1. It


Fruit juice 10–11 °Brix

Reconcentrated brine

Feed reservoir Evaporator

Condenser

UF

Condensate

Stripping solution reservoir

OD Pulp

Preconcentrated juice 25–26 °Brix

RO

Water

Pasteurization

Pasteurized pulp

Reconstituted juice Concentrated juice 64–65 °Brix Water

Fig. 9.3 Integrated membrane process for the production of concentrated fruit juices (dashed lines refer to the production of the reconstituted juice).



Clarified juice

264 Separation, extraction and concentration processes was developed primarily for the concentration of grape juices destined for the production of high-quality vintage varietal wines such as Chardonnay, Cabernet Sauvignon and Merlot. Concentrates can be stored for long periods without deterioration and can be used as blending stocks to adjust the sugar content of freshly harvested grapes to minimize variations in alcohol content of the resulting wine between vintages. Additionally, the concentrated juice can be shipped over long distances and used to produce high-priced varietal wines starting from local grapes (Hogan et al., 1998). Evaporation fluxes between 7 and 10 kg m–2 h–1 were obtained by Hongvaleerat et al. (2008) in the concentration of clarified pineapple juice. These values were higher than those obtained with the single strength juice owing to the complete removal of suspended solids in the clarification step. At low TSS the evaporation flux decay during the OD process is more attributable to the dilution of the stripping solution whereas, at higher feed concentrations, the evaporation flux depends mainly on juice viscosity and, consequently, on juice concentration (Cassano and Drioli, 2007; Courel et al., 2000a; Vaillant et al., 2001). Evaporation fluxes in OD can be improved by application of acoustic fields. Narayan et al. (2002) observed an enhancement of the evaporation flux from 0.81 to 0.94 L m–2 h–1 during the OD of sugarcane juice with CaCl2 as stripping solution when ultrasounds were applied to the membrane cell. Concentration and temperature polarizations in OD contribute up to 18% to the flux reduction (Bui et al., 2005). However, concentration polarization gave a larger contribution to the flux reduction than temperature polarization. Furthermore, the flux reduction owing to polarization was smaller on the feed side than on the brine side. Hydrophobic membranes with relatively large pore sizes showed higher organic volatiles retention per unit water removal than those with smaller pores when model aroma aqueous solutions were processed by OD (Barbe et al., 1998). Pores with larger diameter at the membrane surface allow greater intrusion of the feed and brine solutions with a consequent increase in the thickness and resistance of the boundary layer at the pore entrance. Consequently, membranes with large surface pore diameters are preferred for OD applications in which retention of volatile flavour/fragrance components are required. Shaw et al. (2001) investigated the retention of flavours in concentrated orange and passion fruit juices prepared by OD. Losses of volatile components between 32 and 39% were observed in the concentrated products. The transfer of volatiles depends on the nature of compounds and on the operating conditions. A significant reduction in the transfer of juice volatiles was obtained by decreasing the circulation velocity or the temperature (Ali et al., 2002). The management of the diluted brine step is one of the drawbacks associated with the commercial application of the OD in fruit juice processing. Although the regeneration of exhausted brines can be realized by thermal evaporation, this operation is expensive owing to corrosion and scaling phenomena. Solar


Advances in membrane-based concentration 265 ponding, reverse osmosis and pervaporation were proposed as alternatives to re-concentrate the diluted brine solutions (Thompson, 1991). Electrodialysis was also suggested for the regeneration of NaCl brines (Petrotos and Lazarides, 2001). The OD process is also a valid approach for producing low-alcohol beverages because it permits a selective removal of single volatile solutes from aqueous solutions (e.g. ethanol from wine and other ferments) by using water as a stripping solvent. The removal of organic solvents after the extraction of intracellular products (antibiotics, hormones and biologicals) from fermentation broths is a necessity in the drug industry. This operation has to be carried out at low temperature in order to preserve the product from thermal degradation and can be performed by OD in which water is employed as stripping solution (Hogan et al., 1998). The concentration of pharmaceuticals and biological compounds such as vaccines, hormones, recombinant proteins, enzymes, antibiotics, fungicides and nucleic acids is another potential use of OD. These products have to be isolated or recovered in the dry state in order to maintain their activity and shelf stability for a long time; OD can be used as a concentration step, eventually after a pre-concentration unit (by RO or NF), in order to obtain concentrates from which bioactive compounds can be more easily recovered (e.g. by crystallization or extraction). OD can be used also as a pre-concentration step for the lyophilization, reducing the water removal load during freeze drying. 9.4.2 Membrane distillation Process fundamentals Similarly to the OD process, in MD the water vapour transfer is promoted by a vapour pressure difference between two sides of a microporous hydrophobic membrane; however, in MD the physical origin of the vapour pressure difference is a temperature gradient rather than a concentration gradient: the feed is maintained at high temperature while cold water is used as a stripping permeate. Therefore, membrane distillation is a thermaldriven process. The mass transfer in MD can be described as a three-phase sequence: (1) formation of a vapour gap at the warm solution–membrane interface; (2) transport of the vapour phase through membrane pores; (3) its condensation at the cold side membrane–solution interface. Theoretical aspects and potential applications of the MD process have been extensively studied (Bandini et al., 1991; Calabrò et al., 1994; El-Bourawi et al., 2006; Lawson and Lloyd, 1997; Schofield et al., 1987; Tomaszewska, 2000a). The process takes place at atmospheric pressure and at temperatures that may be much lower than the boiling point of the treated solutions. Consequently, it can be used to concentrate solutes sensitive to high


266 Separation, extraction and concentration processes temperatures such as fruit juices and pharmaceuticals. Feed temperatures in MD are typically 60–90 °C, although temperatures as low as 30 °C have been used. Operating pressures are generally of about 0–100 kPa, hence much lower than conventional pressure-driven membrane processes such as RO. Consequently, lower equipment costs and increased process safety can be achieved. Furthermore, the mechanical resistance of the membrane is greatly reduced. Because MD operates on the principles of vapour–liquid equilibrium, another advantage over traditional pressure-driven membrane processes is represented by its high rejection (theoretically 100%) towards ions, macromolecules, colloids, cells and other nonvolatile compounds (Lawson and Lloyd, 1997). Finally, vapour spaces can be reduced compared with conventional distillation processes. As in the OD process, the main drawback of MD is the risk of wetting of the hydrophobic membrane with a consequent reduction in the evaporation flux and separation performance. Therefore, the process solutions must be aqueous and sufficiently dilute. This limits MD to applications such as desalination, removal of trace volatile compounds from wastewater and concentration of nonvolatile aqueous solutions. Membrane distillation (MD) configurations As depicted in Figure 9.4, the MD process can be realized according to four types of configuration. In the direct contact membrane distillation (DCMD), the membrane separates the hot feed from the cold distillate. In this instance, the vapour pressure gradient, which results from the transmembrane temperature difference, is the driving force of the mass transport across the membrane. Volatile molecules evaporate at the hot liquid/vapour interface, cross the membrane pores in the vapour phase, and condense on the cold liquid/vapour interface inside the membrane module (Gryta, 2002; Lawson and Lloyd, 1997; Mengual and Peña, 1997; Tomaszewska, 2000b). DCMD is usually employed for applications in which water is the major fluxing component, such as desalination or concentration of aqueous solutions. In air gap membrane distillation (AGMD), a condensing surface is separated from the membrane by an air gap. Volatile molecules cross both the membrane pores and the air gap and finally condense over a cold surface inside the membrane module. In the sweeping gas membrane distillation (SGMD), a cold inert, gas sweeps the permeate side of the membrane carrying the volatile molecules. In this instance, condensation occurs outside the membrane module (Khayet et al., 2000). In the vacuum membrane distillation (VMD), vacuum is applied on the permeate side of the membrane by means of a vacuum pump and, similarly to SGMD, condensation takes place outside the membrane module (Bandini et al., 1992).


Feed

Retentate

Feed

Retentate Membrane

Membrane

Permeate

Permeate in

Cooling water out

Air gap

Condensing plate

(a)

Feed

(b)

Retentate

Feed

Retentate Membrane

Membrane

Sweep gas in Condenser

Permeate

Sweep gas out

(c)

Cooling water in

Condenser

Permeate

Vacuum

(d)

Fig. 9.4 Membrane distillation configurations: (a) DCMD; (b) AGMD; (c) SGMD; (d) VMD.



Permeate out

268 Separation, extraction and concentration processes Mass transfer and polarization phenomena In a similar way to that of the OD process, classical models of gas diffusion in porous membranes, such as molecular diffusion or Knudsen diffusion, can be used to describe the mass transfer across MD membranes (Courel et al., 2000a; Gryta, 2005). These models suggest a linear relationship between the volume flux per unit surface area of the membrane and the transmembrane water vapour pressure difference at each interface, which depends on composition and temperature of both compartments. The osmotic and thermal contributions can operate either in a synergistic or in an antagonistic way (in which one of them prevails on the other) (Godino et al., 1995). The relationship between the permeate flux (Jw) and the driving force (DP) which causes the mass transfer across the membrane pores is given by:

Jw = Kw DP

[9.18]

where Km is the membrane mass transfer. The driving force in MD is a partial pressure gradient in the vapour phase given by:

DP = Pwf – Pwd

[9.19]

where Pwf and Pwd are water vapour pressures at the feed/membrane and feed/distillate interface, respectively. The mass transfer through the membrane, as in other membrane processes, is caused by the chemical potential difference (Dm) on both sides of the membrane (distillate and feed sides), which, for a ith component is given by:

D mi = D mid – D mif = RT ln

apid a = RT ln id apif ai

[9.20]

where apid and aid are the activity of ith component in the vapour and liquid if of ith component in phase on the distillate side, apif and aif the activity the vapour and liquid phase on the feed side, R the gas constant and T the temperature. The driving force expressed in equation [9.19] requires a knowledge of both temperature and solute composition at the vapour–liquid interfaces. Because the interfacial conditions are not always accessible, the water transport, referred to the bulk conditions, is often described as:

1 1ˆ Ê1 b b J w = K (Pwf – Pwd )=Á + + Ë K f K m K d ˜¯

–1

DP Pwb

[9.21]

where the overall mass transfer coefficient (K) is given by the series of water transport resistances from the bulk of the dilute solution towards the evaporation surface (1/Kf), water vapour transport through the membrane


Advances in membrane-based concentration 269 (1/Km) and from the condensation surface to the bulk of the distillate side (1/Kd). The overall mass transfer coefficient depends on the membrane morphology, module design, solution concentration, temperature and hydrodynamic conditions (Gryta et al., 2005). When the temperatures in the layers near the membrane differ from those measured in the bulk of the feed and permeate side, a temperature polarization phenomenon occurs. However, temperature profiles formed in the DCMD are different from those observed in OD. The temperature gradient results from the evaporation at the feed side and condensation at the permeate side even if the bulk temperatures of two liquids are equal, as in OD. However, the heat conduction from the brine side to the feed side decreases the temperature polarization phenomenon in OD. On the contrary, in the case of DCMD, heat and mass transfer flow through the membrane in the same direction (from the feed to the distillate side) increasing the temperature polarization effect (Gryta, 2005). In all MD variants, as in OD, only water passes through the membrane causing an increase in the solute concentration on the feed side and a decrease in the solute concentration on the distillate side relative to the bulk condition. This phenomenon, called concentration polarization, results in a driving force reduction across the membrane. Several authors assume that the concentration polarization is substantial in OD, whereas the performance of DCMD is affected mainly by the temperature polarization effect, except for the concentration of solutions with a solute content close to the saturated state (Gryta, 2002). Alves and Coelhoso (2006) compared the performance of OD and MD in the concentration of sucrose solution (used as a model fruit juice), in terms of water flux and aroma retention. At the same applied overall driving force, water fluxes in MD were less than half of those observed in OD owing to temperature polarization effects. Moreover a higher retention of aroma compounds was observed for the OD process. MD membranes MD membranes must be porous and hydrophobic with good thermal stability and excellent chemical resistance to feed solutions. They must be characterized by high liquid entry pressure which is defined as the minimum hydrostatic pressure that must be applied to the feed liquid solution before it overcomes the hydrophobic forces and penetrates into the membrane pores. Membranes with high liquid entry pressure can be obtained by using materials with high hydrophobicity and small maximum pore size. The most suitable materials for hydrophobic MD membranes include PVDF, PP, PE and PTFE. These membranes are available in capillary or flatsheet configurations (Ding et al., 2002). PVDF appears of particular interest in MD processes because of its high melting point and good temperature resistance; its resistance to oxidation and gamma radiation, to solvents and to abrasion are also of interest. Hydrophilic membranes, such as cellulose


270 Separation, extraction and concentration processes acetate membranes, can be treated in order to make their surfaces hydrophobic and, consequently, suitable for MD applications (Wu et al., 1992). Composite MD membranes consisting of hydrophobic and hydrophilic layers or a hydrophobic layer sandwiched between two hydrophilic layers have been also realized (Cheng et al., 1982). The size of micropores can range between 100 nm and 1.0 mm. In particular, the pores should be large enough to facilitate the required flux but also small enough in order to prevent liquid penetration through the membrane. For cylindrical pores, the maximum critical pore size at which the liquid penetrates the microporous phase is defined by the Laplace equation:

P = 2g

cos q r

[9.22]

where g is the surface tension of the liquid, q the contact angle between the liquid and the membrane, r the radius of the pore and P the applied pressure. For a given pore size a critical pressure Pc exists. For applied hydrostatic pressures higher than the Pc values, the liquid phase is transported across the membrane. Microporosity can be induced by mechanical stretching or by thermal phase separation technique. The relationship between the transmembrane flux and membrane characteristic parameters is given as:

N µ

ra e dm c

[9.23]

where N is the molar flux, r the mean pore size of the membrane pores, a a factor whose value equals 1 or 2 for Knudsen diffusion and viscous fluxes, respectively, dm the membrane thickness, e the membrane porosity and c the membrane tortuosity. According to equation [9.23], the MD flux increases with the increase in pore size. However, in order to avoid membrane pores wettability, the pore size should be as small as possible. Consequently, an optimum value for the pore size has to be determined for each MD application on the basis of the type of the feed solution to be treated. Schneider et al. (1988) recommended a maximum pore radius of 0.5–0.6 mm in order to avoid membrane wetting owing to fluctuations in process pressure and temperature. As for other membrane processes, the permeate flux in MD is inversely proportional to the membrane thickness: consequently thinner membranes produce higher fluxes. On the contrary, the membrane should be as thick as possible in order to prevent heat loss by conduction through the membrane matrix (Schofield et al., 1987). An optimal membrane thickness, considering the thermal conductivity of commercial membranes, should be within the range of 30–60 mm (Laganà et al., 2000). The porosity is the most influential factor affecting mass transfer rate in MD: membranes with higher porosity produce higher evaporation fluxes (Schneider et al., 1988). Membranes


Advances in membrane-based concentration 271 with high porosity also allows the amount of heat lost by conduction to be reduced, because the heat transfer coefficient of the gas entrapped within the membrane pores is generally an order of magnitude smaller than that of the membrane material. MD membrane modules can be realized with flat-sheet membranes, as plate and frame and spiral-wound configurations, or with capillary membranes in tubular configuration. The design of the MD modules provides a high feed flow rate, to increase turbulence of the feed solution, low pressure drop and high packing density; moreover, because the MD is a nonisothermal process, good heat recovery and thermal stability is also guaranteed. Laboratory-scale modules are usually realized with flat-sheet membranes that are much more versatile than capillary membranes. They can be easily removed from their modules for cleaning, examination or replacement. Consequently, the same membrane module can be used to test different MD membranes. On the other hand, tubular membrane modules are preferred for commercial applications because they do not require a support and allow high membrane surface area/ module volume ratios to be realized. Effect of operating parameters on MD fluxes Evaporation fluxes in MD increase with an increase in the feed temperature. This is because the exponential increase of the vapour pressure of the feed solution with temperature increases the vapour pressure difference across the membrane and, hence, the driving force of the process. In DCMD applications the effect of decreasing the permeate temperature is to increase the evaporation flux (Lawson and Lloyd, 1999). A reduction in the flow rate implies a reduction in the Reynolds number and, consequently, in the transport coefficients. When the mean temperature is kept constant, the permeate flux increases linearly with the temperature difference. On the other hand, when the temperature difference is fixed, the permeate flux increases exponentially with the mean temperature (Mengual and Peña, 1997). When non-volatile solutes are considered, permeate fluxes in all MD configurations decrease by increasing the feed inlet concentration: this phenomenon can be attributed to the reduction of the driving force owing to the decrease in the vapour pressure of the feed solution and to the exponential increase in the viscosity of the feed solution. At high concentration ratios, MD fluxes are higher than those observed in other pressure-driven membrane processes (Cath et al., 2004). Permeate fluxes generally increase by increasing the solute concentration when aqueous solutions containing volatile components (such as alcohols) are processed. This can be explained by assuming that an increase in the volatile compound concentration in the feed side is associated with an increase in its transmembrane partial pressure. The increase in the permeate flow velocity causes an increase in the permeate flux in MD process. The increase in permeate flow velocity increases the heat transfer in the permeate side of the membrane reducing the temperature and concentration polarization phenomena. Consequently, the temperature


272 Separation, extraction and concentration processes at the membrane surface approaches the temperature in the bulk permeate side increasing the driving force and the permeate flux. Finally, a linear increase in the MD flux with the transmembrane vapour pressure difference is typically observed in all MD configurations considered above. MD applications MD configurations previously described can be applied in different areas of interest for the separation of both nonvolatile (ions, colloids, macromolecules) and volatile (benzene, chloroform, trichloroethylene) compounds from water, the extraction of organic compounds such as alcohols from diluted aqueous solutions, the production of distilled water and the concentration of aqueous solutions. Desalination and pure water production from brackish water is the bestknown MD application (Banat and Simandl, 1998; Khayet et al., 2003, 2005). The concentration of radioactive solutions and wastewater treatment in the nuclear industry is another area under investigation (Khayet et al., 2006). In the chemical industry, the separation of azeotropic aqueous mixtures such as alcohol–water mixtures, the removal of volatile organic compounds from water and the concentration of acids, such as sulfuric, hydrochloric and nitric acid, can be achieved by MD (Banat and Simandl, 2000; Duan et al., 2001; Garcia-Payo et al., 2002; Tomaszewska, 2000b; Tomaszewska et al., 1995). Removal of dyes and wastewater treatment in the textile industry have also been reported (Banat et al., 2005; Calabrò et al., 1991). In the pharmaceutical and biomedical areas MD proved attractive for the removal of water from blood and protein solutions and in the wastewater treatment (Ortiz de Zárate et al., 1998; Sakai et al., 1986). MD can be successfully applied in the food industry and in areas where high temperature applications lead to the degradation of process fluids such as concentration of fruit juices and milk processing. A blackcurrant juice concentrate with a high TSS content was produced by MD without degradation of its valuable substances maintaining the juice side temperature at 26 °C and the water side temperature at 11 °C (DT = 15 °C) (Kozák et al., 2009). An increase of a few degrees Celcius in the driving force (DT = 19 °C) influenced significantly the evaporation flux and the operation time of the process. Gunko et al. (2006) used DCMD to investigate the concentration of apple juice. A TSS content of 50 °Brix was obtained when the permeate flux reached about 9 L m–2 h–1. Further concentration to 60–65 °Brix resulted in reduced productivity (up to 3 L m–2 h–1). Highly concentrated apple juices up to 64 °Brix were also produced by using polypropylene hollow fibre DCMD modules with trans-membrane fluxes of 1 kg m–2 h–1 (Curcio et al., 2000; Laganà et al., 2000). Flux rates were dependent essentially upon temperature polarization phenomena located mainly on the feed side, rather than concentration polarization. PVDF membranes used for the concentration of orange juice by MD


Advances in membrane-based concentration 273 showed a very good retention of soluble solids, sugars and organic acids with rejection of sugars and organic acids equal to 100%. The colour and flavour of concentrated juice were satisfactory (Calabrò et al., 1994; Drioli et al., 1992). A protein concentrate containing at least 25% of protein was obtained by Christensen et al. (2006) in the processing of whey protein by DCMD as an alternative to thermal evaporation. The optimal temperature for the whey protein concentrate was 55 °C leading to less protein denaturation than evaporation and therefore a higher quality product. Bandini and Sarti (2002) studied the vacuum membrane distillation (VMD) for the concentration of grape must up to 50 °Brix. The process allowed production of juice concentrates that still retained interesting amounts of the aroma compounds. Bagger-Jørgensen et al. (2004) and Diban et al. (2009) evaluated the potential of VMD to recover aroma compounds from blackcurrant and pear juice, respectively. The highest values of enrichment factor (up to 15) for pear aroma compounds were obtained working at lower temperatures and higher downstream pressures. The highest concentration factors for the blackcurrant aroma compounds (from 21 to 31) were obtained at high feed flow rate (400 L h–1) and low temperatures (10 °C). At 5 vol.% feed volume reduction the recovery of highly volatile compounds was between 68 and 83 vol.% and between 32 and 38 vol.% for the hardly volatile compounds. Also in MD, an improvement in the evaporation flux in fruit juice concentration can be obtained when the juice is preliminarily submitted to a UF treatment. Drioli et al. (1992) found that the UF of the single-strength orange juice resulted in an increase in MD flux that remained almost constant during an approximately two-fold concentration. On the contrary, the MD flux of the unfiltered juice decreases steadily over the same concentration range. The improvement of the MD flux can be attributed to a reduction of juice viscosity as a result of pulp and pectin removal. PVDF membranes showed a very good retention of orange juice components such as total soluble solids, sugars and organic acids; on the other hand, the amount of ascorbic acid decreased by 42%, probably owing to its degradation associated with high temperature and oxidation. By referring to the retention of volatile organic flavour/fragrance components from liquid foods it has been found that membranes having an open fibrous structure rather than discrete pore offer the best volatiles retention for a given amount of removed water. 9.4.3 Coupled operation of osmotic distillation and membrane distillation Table 9.3 summarizes the main advantages and disadvantages of MD and OD processes. Because the water flux in OD is relatively low (usually between 0.07 and 7.2 kg m–2 h–1), the surface area available for flux should be increased. As described above, the thermal effect in OD reduces the driving


274 Separation, extraction and concentration processes Table 9.3 Positive and negative aspects of MD and OD processes Advantages

Disadvantages

Low operating temperature

Low evaporative capacity with a long time of treatment Possibility of wetting of the hydrophobic membrane and consequent loss of flux and separation performance Production costs higher than those of thermal evaporation High cost of membrane replacement

Low operating pressure

No or less degradation of heat-sensitive compounds High TSS concentration in the concentrated products Modularity, easy scale-up Management of diluted brine solutions Possibility to treat solutions with high levels of suspended solids Possibility to concentrate several different products with the same unit No fouling problems Constant permeate flux in time Low investment cost

force of the water transport across the membrane. This phenomenon can be exploited to obtain a coupled process where the brine and feed solutions are thermostatically controlled at different temperatures: the osmotic solution on the cold side and the solution to be concentrated on the warm side. This coupled operation of MD and OD, referred to as membrane osmotic distillation (MOD), allows the water flux across the membrane to be enhanced (Wang et al., 2001). Although the aqueous solution is gently heated during the operation, this coupled method still works under mild conditions, because the temperature difference applied is lower than 15 °C (Bélafi-Bakó and Koroknai, 2006). The effect of varying experimental conditions (solute concentration, stirring rate, mean temperature and bulk temperature difference) on the water flux involved in a coupled process MD/OD was investigated by Godino et al. (1995). Koroknai et al. (2006a and 2006b) obtained concentrated fruit juices (apple, red- and blackcurrant, sour cherry and raspberry) at 60 °Brix in an operation time of 15–20 h, maintaining a temperature difference of 15 °C and using as an osmotic solution CaCl2 6M. In particular, the driving force of the process was greatly enhanced by decreasing the temperature of the osmotic solution, as in the range 15–25 °C only a slight drop in the water vapour pressure, for saturated CaCl2 solution, occurs. Consequently, maintaining the osmotic solution at room temperature, the energy consumption is minimized and the coupled process can be accelerated by increasing the temperature of the solution to be concentrated. Similarly, Bélafi-Bakó and Koroknai (2006) found that the coupled process is more effective than MD © Woodhead Publishing Limited, 2010

Process

Maximum achievable concentration (°Brix)

Product quality

Evaporation rate or flux

Possibility of treating Operating cost different products with the same installation

Thermal evaporation Cryoconcentration Reverse osmosis Direct osmosis Membrane distillation Osmotic distillation

65–75 30–50 25–30 50 60–70 60–70

Poor Very good Very good Good Good Very good

200–300 L h–1 – 5–10 L m–2 h–1 1–5 L m–2 h–1 1–10 L m–2 h–1 1–10 L m–2 h–1

No No No Yes Yes Yes

Moderate High High High High High

Capital investment

Energy consumption

Moderate Very high Moderate Moderate Low Low

Very high Very high High Low Low Low



Table 9.4 Key factors of conventional evaporation and membrane concentration techniques (adapted from Jariel et al., 1996)

276 Separation, extraction and concentration processes or OD alone. The use of short membrane modules in cascade series, with heat exchangers placed between them, was suggested in order to minimize heat losses.

9.5 Conclusions Key factors of conventional evaporation and membrane concentration techniques are summarized in Table 9.4. Several advantages of membranebased processes over conventional evaporation have been successfully demonstrated, including improved product quality, easy scale-up and low energy consumption. Low evaporation fluxes in OD and MD seem to be the main drawbacks of these processes when compared with RO and thermal evaporation. However, when the solution to be concentrated contain solutes sensitive to mechanical or thermal degradation there are serious limitations in using these technologies without significant deterioration in the quality. Currently membrane contactors are emerging technologies in the processing of foods, pharmaceuticals and beverages and will become breakthrough technologies when enhanced effectiveness is attained. Their integration with standard membrane operations is a valid approach for a sustainable industrial growth within the process intensification strategy. The aim of this strategy is to introduce in the productive cycles new technologies characterized by low encumbrance volume, advanced levels of automation capacity, modularity, remote control and reduced energy consumption. In order to improve the competitiveness of membrane contactors towards conventional technologies, efforts should be devoted to the development of new membranes characterized by high selectivity and stability for long-term applications, as well as to improvements in process engineering, including module and process design.

9.6 Nomenclature a a i api C d D h J k B k

water activity activity of the ith component in the liquid phase activity of the ith component in the vapour phase (fugacity) solute molar concentration (mol L–1) diameter (m) diffusion coefficient (m2 s–1) liquid heat transfer coefficient (W m–2 K–1) mass flux (kg m–2 h–1) or volume flux (m3 m–2 s–1) Boltzmann constant (1.3807 ¥ 10–23 J K–1) mass transfer coefficient (m s–1)


Advances in membrane-based concentration 277 K Kn M N P P* Q r R Re Sc Sh T u U Greek g d e q DP Dm l m C r s

mass transfer coefficient (kg m–2 h–1 Pa–1) Knudsen number molecular weight (kg mol–1) molar flux (mol m–2 s–1) water vapour pressure (Pa) pure water vapour pressure (Pa) heat flux (W m–2) pore radius (m) gas constant (8314 J mol–1 K–1) Reynolds number Schmidt number Sherwood number temperature (°C, K) velocity of the fluid (m s–1) overall heat transfer coefficient (W m–2 K–1) symbols liquid surface tension (N m–1) thickness (m) porosity contact angle driving force chemical potential difference mean molecular free path (m) liquid dynamic viscosity (Pa s) tortuosity liquid density (kg m–3) mean collision diameter (m)

Subscripts b bulk d distillate f feed h hydraulic m membrane p pore or permeate s stripping solution or solute w water or vapour Superscripts b bulk location K Knudsen diffusion m membrane location M molecular diffusion


278 Separation, extraction and concentration processes Abbreviations DO direct osmosis MD membrane distillation MF microfiltration OA osmotic agent OD osmotic distillation OMD osmotic membrane distillation PP polypropylene PTFE polytetrafluoroethylene PVDF polyvinylidene fluoride RO reverse osmosis TAA total antioxidant activity TSS total soluble solids UF ultrafiltration

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280 Separation, extraction and concentration processes in osmotic distillation using asymmetric membrane’, J Membrane Sci, 173, 107– 122. Courel M, Tronel-Peyroz E, Rios GM, Dornier M and Reynes M (2001), ‘The problem of membrane characterization for the process of osmotic distillation’, Desalination, 140, 15–25. Curcio E, Barbieri G and Drioli E (2000), ‘Operazioni di distillazione a membrane nella concentrazione dei succhi di frutta’, Industria delle Bevande, XXIX, 113–121. Deblay P (1995), ‘Process for at least partial dehydration of an aqueous composition and devices for implementing the process’, US Patent 5,382,365. Diban N, Voinea OC, Urtiaga A and Ortiz I (2009), ‘Vacuum membrane distillation on the main pear aroma compound: experimental study and mass transfer modelling’, J Membrane Sci, 326, 64–75. Ding Z, Ma R and Fane AG (2002), ‘A new model for mass transfer in direct contact membrane distillation’, Desalination, 151, 217–227. Drioli E, Jiao B and Calabrò V (1992), ‘The preliminary study on the concentration of orange juice by membrane distillation’, Proceedings of VII International Citrus Congress, Acireale (Italy), 3, 1140–1144. Duan SH, Ito A and Ohkawa A (2001), ‘Removal of trichloroethylene from water by aeration, pervaporation and membrane distillation’, J Chem Eng Jpn, 34, 1069–1073. Durham RJ and Nguyen MH (1994), ‘Hydrophobic membrane evaluation and cleaning for osmotic distillation of tomato puree’, J Membrane Sci, 87, 181–189. El-Bourawi MS, Ding Z, Ma R and Khayet M (2006), ‘A framework for better understanding membrane distillation separation process’, J Membrane Sci, 285, 4–29. Gabelman A and Hwang S (1999), ‘Hollow fiber membrane contactors’, J Membrane Sci, 159, 61–106. Gabino F, Belleville MP, Preziosi-Belloy L, Dornier M and Sanchez J (2007), ‘Evaluation of the clearing of a new hydrophobic membrane for osmotic evaporation’, Sep Purif Technol, 55, 191–197. Galaverna G, Di Silvestro G, Cassano A, Sforza S, Dossena A, Drioli E and Marchelli R (2008), ‘A new integrated membrane process for the production of concentrated blood orange juice: effect on bioactive compounds and antioxidant activity’, Food Chem, 106, 1021–1030. Garcia-Payo MC, Rivier CA, Marison IW and Stockar UV (2002), ‘Separation of binary mixtures by thermostatic sweeping gas membrane distillation: II. Experimental results with aqueous formic acid solutions’, J Membrane Sci, 198, 197–210. Geankoplis CJ (1993), ‘Principles of mass transfer’, in Transport processes and unit operation, London, Prentice-Hall, 381–413. Godino MP, Peña L, Ortiz de Zárate JM and Mengual JI (1995), ‘Coupled phenomena membrane distillation and osmotic distillation through a porous hydrophobic membrane’, Sep Sci Technol, 30, 993–1011. Gostoli C (1999), ‘Thermal effects in osmotic distillation’, J Membrane Sci, 163, 75–91. Gryta M (2002), ‘Concentration of NaCl solution by membrane distillation integrated with crystallization’, Sep Sci Technol, 37, 3535–3558. Gryta M (2005), ‘Osmotic MD and other membrane distillation variants’, J Membrane Sci, 246, 145–156. Gunko S, Verbych S, Bryk M and Hilal N (2006), ‘Concentration of apple juice using direct contact membrane distillation’, Desalination, 190, 117–124. Herron JR, Beaudry EG, Jochums CE and Medina LE (1994), ‘Osmotic concentration apparatus and method for direct osmotic concentration of fruit juice’, US Patent 5,281,430. Ho WSW and Sirkar KK (1992), Membrane handbook, Chapman & Hall, New York. Hogan PA, Canning RP, Peterson PA, Johnson RA and Michaels AS (1998), ‘A new option: osmotic distillation’, Chem Eng Prog, 94, 49–61.


Advances in membrane-based concentration 281 Hongvaleerat C, Cabral LMC, Dornier M, Reynes M and Nigsanond S (2008), ‘Concentration of pineapple juice by osmotic evaporation’, J Food Eng, 88, 548–552. Jariel O, Reynes M, Courel M, Durand N, Dornier M and Deblay P (1996), ‘Comparison of some fruit juice concentration techniques’, Fruits, 51, 437–450. Jiao B, Cassano A and Drioli E (2004), ‘Recent advances on membrane processes for the concentration of fruit juices: a review’, J Food Eng, 63, 303–324. Jiao B, Molinari R, Calabrò V and Drioli E (1991), ‘Application of membrane operations in concentrated citru juice processing’, Agro-Ind Hi-Tech, 19–27. Karode SK, Kulkarni SS and Ghorapade MS (2000), ‘Osmotic dehydration coupled reverse osmosis concentration: steady-state model and assessment’, J Membrane Sci, 164, 277–288. Khayet M, Godino MP and Mengual JI (2000), ‘Theory and experiments on sweeping gas membrane distillation’, J Membrane Sci, 165, 261–272. Khayet M, Mengual JI and Matsura T (2005), ‘Porous hydrophobic/hydrophilic composite membranes. Application in desalination using direct contact membrane distillation’, J Membrane Sci, 252, 101–113. Khayet M, Mengual JI and Zakrewska-Trznadel G (2003), ‘Theoretical and experimental studies on desalination using the sweeping gas membrane distillation’, Desalination, 157, 297–305. Khayet M, Mengual JI and Zakrewska-Trznadel G (2006), ‘Direct contact membrane distillation for nuclear desalination. Part II. Experiments with radioactive solutions’, Int J Nuclear Desalination, 56, 56–73. Koroknai B, Gubicza L and Bélafi-Bakó K (2006a), ‘Coupled membrane process applied to fruit juice concentration’, Chem Pap, 60, 399–403. Koroknai B, Kiss K, Gubicza L and Bélafi-Bakó K (2006b), ‘Coupled operation of membrane distillation and osmotic evaporation in fruit juice concentration’, Desalination, 200, 526–527. Kozák A, Bánvölgyi S, Vincze I, Kiss I, Békássy-Molnár E and Vatai G (2008), ‘Comparison of integrated large scale and laboratory scale membrane processes for the production of black currant juice concentrate’, Chem Eng Proc, 47, 1171–1177. Kozák A, Békássy-Molnár E and Vatai G (2009), ‘Production of black-currant juice concentrate by using membrane distillation’, Desalination, 241, 309–314. Laganà F, Barbieri G and Drioli E (2000), ‘Direct contact membrane distillation: modelling and concentration experiments’, J Membrane Sci, 166, 1–11. Lawson KW and Lloyd DR (1997), ‘Membrane distillation’, J Membrane Sci, 124, 1–25. Lebfevre MSM (1988), ‘Method of performing osmotic distillation’, US Patent 4,781,837. Luh BS, Feinberg B, Chung JI and Woodroof JG (1986), ‘Freezing fruits’ in Woodroof JG and Luh BS, Commercial fruit processing, Westport, AVI Publishing Co., 263–351. Maccarone E, Campisi S, Cataldi Lupo MC, Fallico B and Nicolosi Asmundo C (1996), ‘Thermal treatments effects on the red orange juice constituents’, Ind Bevande, 25, 335–341. Mansouri J and Fane AG (1999), ‘Osmotic distillation of oily feeds’, J Membrane Sci, 153, 103–120. Mengual JI, Ortiz de Zárate JM, Peña L and Velázquez A (1993), ‘Osmotic distillation through macroporous hydrophobic membranes’, J Membrane Sci, 82, 129–140. Mengual JI and Peña L (1997), ‘Membrane distillation’, Colloid Interf Sci, 1, 17–29. Michaels AS (1998), ‘Methods and apparatus for osmotic distillation’, US Patent 5,824,223. Michaels AS (1999), ‘Osmotic distillation process using a membrane laminate’, US Patent 5,938,928. Milleville H (1990), ‘Direct osmotic concentrates juices at low temperature’, Food Proc, 51, 70–71. © Woodhead Publishing Limited, 2010

282 Separation, extraction and concentration processes Mulder M (1998), Basic principles of membrane technology, 2nd edn. London, Kluwer Academic Publishers, 280–303. Nagaraj N, Patil G, Babu BR, Hebbar UH, Raghavarao KSMS and Nene S (2006), ‘Mass transfer in osmotic membrane distillation’, J Membrane Sci, 268, 48–56. Narayan AV, Nagaraj N, Hebbar HU, Chakkaravarthi A and Raghavarao KSMS (2002), ‘Acoustic field-assisted osmotic membrane distillation’, Desalination, 147, 149–156. Ortiz de Zárate JM, Rincón C and Mengual JI (1998), ‘Concentration of bovine serum albumin aqueous solutions by membrane distillation’, Sep Sci Technol, 33, 283–296. Petrotos KB, Quantick PC and Petropakis H (1998), ‘A study of the direct osmotic concentration of tomato juice in tubular membrane module configuration. I. The effect of certain basic process parameters on the process performance’, J Membrane Sci, 150, 99–110. Petrotos KB, Quantick PC and Petropakis H (1999), ‘Direct osmotic concentration of tomato juice in tubular membrane module configuration. II. The effect of using clarified tomato juice on the process performance’, J Membrane Sci, 160, 171–177. Petrotos KB and Lazarides HN (2001), ‘Osmotic concentration of liquid foods’, J Food Eng, 49, 201–206. Ravindra Babu B, Rastogi NK and Raghavarao KSMS (2006), ‘Mass transfer in osmotic membrane distillation of phycocyanin colorant and sweet-lime juice’, J Membrane Sci, 272, 58–69. Ravindra Babu B, Rastogi NK and Raghavarao KSMS (2008), ‘Concentration and temperature polarization effects during osmotic distillation’, J Membrane Sci, 322, 146–153. Rektor A, Vatai G and Békássy-Molnár E (2006), ‘Multi-step membrane processes for the concentration of grape juice’, Desalination, 191, 446–453. Rodrigues RB, Menezes HC, Cabral LMC, Dornier M, Rios GM and Reynes M (2004), ‘Evaluation of reverse osmosis and osmotic evaporation to concentrate camu-camu juice (Myrciaria dubia)’, J Food Eng, 63, 97–102. Romero J, Draga H, Belleville MP, Sanchez J, Come-James C, Dornier M and Rios GM (2006), ‘New hydrophobic membranes for contactor processes – applications to isothermal concentration of solutions’, Desalination, 193, 280–285. Sakai K, Muroi T, Ozawa K, Takesawa S, Tamura M and Nakaue T (1986), ‘Extraction of solute-free water from blood by membrane distillation’, Trans Am Soc Artif Intern Organs, 32, 397–400. Schneider K, Holz W and Wollbeck R (1988), ‘Membranes and modules for transmembrane distillation’, J Membrane Sci, 39, 25–42. Schofield RW, Fane AG and Fell CJD (1987), ‘Heat and mass transfer in membrane distillation’, J Membrane Sci, 33, 299–313. Shaw PE, Lebrun M, Dornier M, Ducamp MN, Courel M and Reynes M (2001), ‘Evaluation of concentrated orange and passion fruit juices prepared by osmotic evaporation’, Lebensm Wiss Technol, 34, 60–65. Sheng J, Johnson RA and Lefebvre MS (1991), ‘Mass and heat transfer mechanism in the osmotic distillation process’, Desalination, 80, 113–121. Sirkar KK (1997), ‘Membrane separation technologies: current developments’, Chem Eng Commun, 157, 145–184. Thompson D (1991), ‘The application of osmotic distillation for the wine industry’, Aust Grapegrower Winemaker, 11, 11–14. Tomaszewska M (2000a), ‘Concentration and purification of fluosilicic acid by membrane distillation’, Ind Eng Chem Res, 39, 3028–3041. Tomaszewska M (2000b), ‘Membrane distillation – examples of applications in technology and environmental protection’, Pol J Environ Stud, 9, 27–36. Tomaszewska M, Gryta M and Morawski AW (1995), ‘Study on the concentration of acids by membrane distillation’, J Membrane Sci, 78, 277–282. © Woodhead Publishing Limited, 2010

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10 Separation of value-added bioproducts by colloidal gas aphrons (CGA) flotation and applications in the recovery of value-added food products P. Jauregi and M. Dermiki, The University of Reading, UK Abstract: This chapter describes the application of a flotation method based on the formation of colloidal gas aphrons (CGA), which are surfactant-based microbubbles, to the recovery of value-added food products from fermentation broths and waste streams of food and agricultural industries. Their main properties are described and contrasted with those of conventional foams. The fundamentals of the separation are described and illustrated by several case studies on CGA applications to the recovery of whey proteins, polyphenols and carotenoids. Finally, a critical evaluation of the feasibility of CGA for industrial applications is carried out based on the astaxanthin case study and future applications of CGA are discussed. Key words: colloidal gas aphrons, flotation, bioproducts, surfactants, food ingredients, recovery.

10.1 Introduction Many studies have been conducted in recent years into additional biological functionalities of several food components and ingredients such as the dairy proteins, peptides, polyphenols or carotenoids so that they could be produced as ingredients in functional foods. The development of functional foods poses new challenges to the food industry as new processes and technologies are required. The food industry must adapt to these changing times and to the revolution caused by newly emerging functional foods. In addition, in the last few years reducing the environmental impact of industrial wastes has been a subject of increasing concern. In industrial wastewaters, these compounds considerably increase biochemical and oxygen demands, with detrimental effects on the flora and fauna of discharge zones, whereas in solid residues © Woodhead Publishing Limited, 2010

Separation of value-added bioproducts by colloidal gas aphrons 285 used as fertiliser they may inhibit germination. Furthermore sustainability is given high priority by governments and industries are encouraged to develop practices for the usage and recycling of wastes. Food processing waste streams and agricultural wastes can be exploited as cheap sources of high-value products. For example, proteins and peptides can be extracted from whey which is a by-product in cheese production and polyphenols can be extracted from winemaking waste, such as grape skin and marc. Separation and purification of these products is carried out by a number of steps including liquid–liquid extraction, membrane techniques and/or chromatographic methods. Alternative separations such as flotation may be used for the recovery stage although further processing will be necessary if high purity products are required. Foam fractionation has been applied for the recovery of proteins. Flotation, which is the foam separation of insoluble compounds, has been applied mainly to wastewater treatment and to the recovery of minerals. Separation of biomass has also been successfully carried out by flotation. This chapter deals mainly with a particular type of flotation using microbubbles which are also called colloidal gas aphrons (CGA). CGA were first described by Sebba (1972) as surfactant-stabilised microbubbles (10–100 mm) generated by intense stirring (>8000 rpm) of a surfactant solution. In this chapter, the application of CGA to bioseparations is reviewed particularly in relation to the recovery of value-added food products. First, the structural and dispersion characteristics of CGA are described. Then, the separation fundamentals are described and illustrated with the application of CGA to the separation of proteins. In addition, the feasibility of CGA for industrial applications is considered, focusing on: (i) scalability using a flotation column and (ii) removal and recycling of surfactant. All these aspects are illustrated in several case studies of CGA separations for the recovery of bioactives from plant extracts (norbixin and polyphenols) and recovery of astaxanthin produced by microbial fermentation.

10.2 Colloidal gas aphrons (CGA) properties 10.2.1 Structure of CGA Colloidal gas aphrons (CGA) are surfactant-stabilised microbubbles (10– 100 mm) generated by intense stirring of a surfactant solution at high speeds (>8000 rpm). Sebba (1987) postulated that they are composed of multilayers of surfactant molecules; as depicted in Fig. 10.1 surfactant molecules adsorb at the interface with hydrophilic heads towards the aqueous phase and the hydrophobic tails towards the gas phase. Sebba’s hypothesis on the structure was based on several experimental observations such as delayed coalescence and hence higher stability of CGA than conventional foams. Jauregi et al. (2000) investigated the drainage kinetics of CGA and compared measured drainage rates with those obtained by applying predictive models for foams with and without modifications. The main modifications were in relation to © Woodhead Publishing Limited, 2010

286 Separation, extraction and concentration processes Inner surface of shell

Air core

Viscous water Shell

Electrical double layer

Outer surface of shell

Fig. 10.1 Proposed structure of CGA by Sebba (1987).

structural differences between foams and CGA upon drainage, such as aphrons being surrounded by a liquid film so that they do not adopt a dodecahedral shape. Interestingly, the model giving the best prediction was the one including differences in structural features which further supports Sebbas’s theory. Jauregi and coworkers used for the first time small angle x-ray diffraction in an attempt to determine the thickness of the surfactant film and the number of surfactant layers of CGA generated by the anionic surfactant sodium bis(2-ethyl hexyl) sulfosuccinate (AOT). The analysis of the data proved to be difficult as for the same sample different film thickness values could be obtained. However, an interesting finding was that samples containing aphrons gave similar scattering regardless of the surfactant concentration and these corresponded to 5.4 nm (which is equivalent to seven layers of surfactant assuming the full length of the surfactant molecule arranged in layers and vertically at the interface). Moreover, the same surfactant solutions with no aphrons gave a different scattering signal and, in this instance, differences between different concentrations of surfactant solutions were found; for example, at concentrations around the cmc (critical micellar concentration), the scattering corresponded to a bilayer, which could correspond to micelles whereas, above the cmc, the scattering corresponded to a multilayer of 3–5 molecules, confirming the hypothesis that AOT forms lamellar structures (Jauregi et al., 2000). In a number of research studies predictive models for liquid drainage in CGA were developed based on measurements of liquid drainage rate (Table 10.1). Most of the studies highlight the difference between CGA and conventional foams. Bhatia et al. (2005) studied the effect of stirring speed and stirring time on the stability of CGA generated using a range of


Table 10.1 Summary of studies for the characterisation of CGA (c, cationic; a, anionic; n-i, nonionic) (adapted from Dermiki, 2009) Surfactant

Parameters studied

Main findings


Zhao et al., SDS (a) 2009; Larmignat CTAB (c) et al., 2008 Tween 20 (n-i)

Surfactant type, Csurf, pressure CGA rheology is not affected by pipe shape or hydraulic diameter, CGA drop, T, volumetric and mass can be treated as shear thinning fluid for all surfactant solutions. Increase flow rate, pipe diameters and in Csurf leads to increased shear stress for a given shear rate shape

Feng et al., 2009 Tergitol (n-i) Ramnolipid (a)

Csurf, pH, Csalt

Stability increases with Csurf and decreases with increasing pH, Csalt. Three distinct phases in the liquid drainage

Moshkelani and SDS (a) Amiri, 2008

Electrical conductivity of surfactant solution

Three clear stages of drainage of CGA can be identified, compared with two stages for conventional foams

Tseng et al., 2006

Pressure drop, T, mass and volumetric flow rate of CGA

CGA can be considered shear thinning fluid. Heat transfer coefficients for CGA made of water and surfactant are smaller than that of water, and they are constant and independent of mass flow and heat flux

tstir, rpm, Csurf, mixing two oppositely charged CGA

Development of an empirical correlation for variation of gas hold-up with tstir, rpm Ê t ˆ – 4.025ˆ a = (0.004 + 44.017) Á 0.2407 ln ÊÁ –2.00 ˜¯ ˜¯ Ë2 Ë

Bhatia et al., 2005

SLS (a) CTAB (c)

The mixing of the two oppositely charged CGA showed no effect on drainage Yan et al., 2005 SDS (a) CTAB (c) Tween 80 (n-i)

Type of surfactant, Csurf, T

There was no effect of the type of surfactant on the stability. Mathematical rn model which describes drainage of CGA: Vt = Vmax n . Two distinct K + rn stages of drainage are determined by two independent mechanisms

Jarudilokkul et al., 2004

Tween 20 (n-i)

tstir, pH, rpm, CNaCl, Csurf, protein separation

Increasing Csurf, tstir increases the stability Increase of Csurf leads to decrease of protein separation

Dai and Deng, 2003

HTAC (c)

pH, concentration of silica sol for stabilisation

CGA were stable for up to 12 h at concentration of silica sol 0.15–0.25 mol dm–3 and pH 7–10

Separation of value-added bioproducts by colloidal gas aphrons 287

Reference


Reference

Surfactant

Parameters studied

Main findings


AOT (a)

Csurf, pH, Csalt, T, tstir

X-ray diffraction for the characterisation of CGA showed evidence of the existence of surfactant multilayers


AOT (a)

Csurf, pH, salt, T, tstir

Increase of Csurf increases the stability, whereas addition of salt leads to the opposite effect Significant interactive effects: (i) Csurf · salt, (ii) pH · Csurf, (iii)T · tstir, (iv) Csurf · T Gas hold-up depends on tstir, Csurf, and salt

Bredwell et al., 1995

SDS (a) Csurf, salt CPC (c) Triton X-100 (n-i)

Save and DTAC, CTAC, Pangarkar, 1994 CPC, DMDSAC (c) SDBS, SLS (a) Chaphalkar et al., 1993

pH, tstir, Csurf, Csalt, viscosity, additives, impeller clearance

SDBS (a), CTAB Type and concentration of (c), surfactant, ionic strength Tergitol (n-i)

Amiri and TTAB (c) Woodburn, 1990

Csurf, pH

Increasing Csurf leads to decrease of formation time of CGA and increase in CGA stability. No effect of salt. No effect of surfactant type and surfactant concentration on the gas hold-up pH and impeller clearance had no effect on stability. Type of surfactant, viscosity, addition of enzymes, polymers, nonionic surfactants, solvents and salts affect gas hold-up and stability MDtergitol<MDCTAB<MDDDBS. Increase of Csurf leads to decrease of MD. Addition of salts reduces the MD of ionic surfactants There is agreement between the predicted and observed rise velocities Stability of CGA depends on the Csurf and pH

MD, mean diameter; tstir, stirring time; Csurf, concentration of surfactant; Csalt, concentration of salt; T, temperature; rpm, stirring speed. SDS, sodium dodecyl sulfate; SDBS, sodium dodecylbenzenesulfonate; CTAB, cetyltrimethyl ammonium bromide; SLS, sodium lauryl sulfate; AOT, sodium bis (2-ethyl hexyl) sulfosuccinate. HTAC, hecadecyl trimethyl ammonium chloride; CPC, cetyl piridinium chloride; DTAC, dodecyltrimethylammonium chloride; CTAC, cetyltrimethylammonium chloride; DMDSAC, dimethyl distearyl ammonium chloride; TTAB, tetradecyl trimethyl ammonium bromide.



Separation of value-added bioproducts by colloidal gas aphrons 289 surfactants and they developed a first order kinetics model for the drainage. Interestingly, drainage kinetics did not change when they used a mixture of an anionic and a cationic surfactant as opposed to conventional foams (Bhatia et al,. 2005). Yan et al (2005) developed a mathematical kinetic model by studying CGA generated using a range of concentrations of surfactants at varying temperatures. According to this model there were two distinct stages of CGA drainage (Yan et al., 2005). On the other hand, in a more recent study Moshkelani and Amiri (2008) measured the electrical conductivity of CGA dispersions and found that there are three separate stages in the liquid drainage of aphrons as opposed to the two stages in conventional foams (Moshkelani and Amiri, 2008). This was further supported in a more recent study by Feng et al. (2009) where the volume of drained liquid was measured and also photographs of the foams at different stages were taken. At the first stage they showed how drainage increases with time owing to a combination of upflow migration of bubbles and downward liquid drainage under gravity. Then, in the following stage, the dispersion behaves like conventional foams and, at the final stage, the foam becomes water deficient and begins to behave like dry foam. At this stage, the foam drainage is very slow owing to slow liquid release from films under capillary suction , i.e.: liquid is released from the film owing to lower pressure in the capillaries between the bubbles (Feng et al., 2009). Overall these studies support further the hypothesis that there are structural differences between CGA and conventional foams. 10.2.2 Characteristics of CGA Owing to their unique structure CGA possess the following important properties: ∑

∑

Higher stability than conventional foams. Owing to the multilayer structure, CGA exhibit high stability. When two aphrons collide, the momentum may not be enough to break the barrier of six surfactantstabilised interfaces, as opposed to the two surfactant-interfaces when two bubbles of conventional foams collide (Sebba, 1987). Stability is measured in terms of the liquid drainage rate, which is generally considered to follow first order kinetics (Bhatia et al., 2005) consequently, half-life (t) is defined as the time required for half the original volume of liquid to drain. CGA are also characterised in terms of gas hold-up (e), which is defined as the ratio between the gas volume (Vg) and the dispersion final volume after stopping stirring and at time = 0 of drainage (Va0): e = Vg/Va0

[10.1]

The buoyancy of the encapsulated gas leads to easy separation of the aphron phase from the bulk liquid phase. Therefore, no centrifuges are needed to separate the two phases. On the other hand, creaming can be


290 Separation, extraction and concentration processes avoided when necessary by stirring CGA at such a rate that the lateral movement conveyed to the bubbles is greater than the upward buoyancy owing to gravity. ∑ Adherence of particles and molecules to the encapsulating shell. It is possible to modify the surface properties of CGA by using different types of surfactant. Depending on the surfactant used to generate the CGA, the outer surface of the bubble can be negatively (anionic surfactant), positively (cationic surfactant) or non-charged (nonionic surfactant). Consequently, oppositely charged molecules adsorb. Thus, the selectivity of adsorption can be modified by changing the type of surfactant. This is illustrated in experiments carried out in our laboratory with two oppositely charged dyes, the cationic methylene blue, and the anionic methyl orange and CGA generated by the cationic surfactant CTAB (see Plate I, between pages 292 and 293). CGA are contacted with the aqueous solution containing both dyes and then the dispersion is allowed to settle so it separates into two phases (Plate Ic). The top CGA phase contains mainly methyl orange (orange colour top phase) and the liquid phase contains mainly methylene blue (blue colour liquid phase). Basu and Malpani (2001) also reported the effective separation of these dyes using CGA generated by CTAB. ∑ Low viscosity of the system. Flow properties are similar to those of water, as long as the gas hold-up does not exceed 65% (Roy et al., 1995), regardless of the type of the surfactant used to generate them. Consequently, CGA can be pumped easily from the generation point to the point where they are going to be used, whereas, for conventional foams, their characteristics change during pumping owing to the elastic nature of the bubble. This is advantageous when applied to a flotation column for fractionation and flotation in the removal and/or recovery of products. Several researchers have investigated the rheology of CGA, as shown in Table 10.1 (Larmignat et al., 2008; Tseng et al., 2006; Zhao et al., 2009). An interesting application which takes advantage of CGA rheological properties is described in a patent by Brookey (2004) where CGA are used to produce fluids with improved shear thinning properties, and this has led to a successful industrial application for well-drilling fluids by MASI technologies LLC. ∑ Small size of bubbles, resulting in larger interfacial area per unit volume, and thus a large capacity for adsorption of molecules and fine particles. Bubble size depends on the concentration and type of surfactant, ionic strength and the presence of other molecules or particles. Jauregi (1997) found that bubble size of AOT-generated CGA increased with surfactant concentration, ionic strength and stirring time up to 10 min. Size distribution of CGA generated by selected surfactants can be seen in Table 10.2.


Separation of value-added bioproducts by colloidal gas aphrons 291 Table 10.2 Characteristics of CGA generated by ionic and nonionic surfactants (values found at optimum conditions) Surfactant

cmc (mM)

Gas hold-up

Half life (s)

CTAB (c)

0.9 (in H2O)

0.70 1 mm) and natural astaxanthin ( 1.7) leads to a further advantage in terms of cheese yield. A distinction must be made between retentates concentrated to an intermediate level (1.7 < VRF < 5) in which some serum proteins are retained and syneresis still takes place, and concentration to the final composition of the cheese (VRF ~5-7), also called pre-cheeses, where the protein content is similar to the protein content of the final cheese and very little whey drainage is observed (Fig. 12.5). Intermediate concentrated retentates have been applied to numerous cheese varieties, ranging from soft to hard. Feta cheese manufacture, which is the main industrial application of this operation, yield increases of 14% while maintaining good quality products. The pre-cheeses concept was first applied to camembert cheese but many applications have been developed successfully for the manufacture of other cheese varieties, mainly fresh unripened cheeses such as quarg, ricotta and cream cheese, and soft and semi-hard cheeses including mozarella, Saint Paulin and feta, the manufacture of which being unquestionably the greatest success worldwide of the MMV process. The advantages of the MMV process are numerous. The overall cheese yield is about 10–30% higher than in the traditional process owing mainly to the retention of serum proteins and enzyme usage is generally reduced. The MMV process eliminates the need for the large storage tanks traditionally used for heating and cooking the curds, resulting in a saving in both capital investment and energy costs. In addition, it is possible to use the MMV process to convert much of the cheese production in a continuous operation, leading to significant advantages in terms of overall capital costs and operational efficiency. However, during UF, the concentration of serum proteins, with higher water-binding capacities than caseins, and of minerals associated with the casein micelles, that are lost in the whey during coagulation in a conventional process, led to the inherent properties of UF milk cheeses. Thus, rather than duplicating traditional cheese varieties, which requires numerous adaptations (Mistry and Maubois, 2004), UF technology leads to the development of new types of cheeses, well-accepted by consumers. For fifteen years, manufacturing technologies including UF have also emerged and have become important for the production of milk protein concentrates, presenting interesting new technical possibilities in cheesemaking. These concentrates contain both major milk protein groups in proportions similar to milk and are often dried. Because there are no specific standards


354 Separation, extraction and concentration processes of identity for such concentrates, they cover a wide range of compositional and functional characteristics and are manufactured mainly using UF and diafiltration, for the reduction of the lactose content. Milk MF has also recently emerged for the production of protein concentrates with various casein–serum proteins ratio (12.5.2).

12.5 Isolation of whole casein in the dairy industry The characteristics of the two groups of milk proteins (casein and serum proteins) differ significantly, permitting their ready separation from each other by at least three techniques. 12.5.1 Isoelectric precipitation and rennet coagulation There are two principal ways of manufacturing casein on industrial scale: isoelectric precipitation and rennet coagulation, both based on the precipitation/ aggregation of casein. In isoelectric precipitation, destabilization of the casein micelles can be accomplished at pH around 4.6: when milk is acidified, the net charge of the micelles decreases, the calcium and phosphate are removed and the micelles become less and less stable until caseins precipitate. Acidification of the milk may be carried out by one of the following processes: – Inoculation of milk with a mixed or multiple defined strain starters, such as lactic acid-producing bacteria, which degrade some of the lactose to lactic acid during the period of incubation (about 14–18 h). – Direct addition of dilute mineral acid to the milk. Adding acid (HCl or H2SO4) to milk heated at 50 °C results in a casein curd (‘acid casein’) that can be separated by straining and washing several times. The coproduct, which is a mixture of acid whey and part of the washing water, has a high ash and chloride content (where HCl is used), which poses some difficulties during spray-drying. Acid destabilization of casein is exploited in the manufacture of cottage cheese, quarg and fermented milks. – Indirect acidification of the milk. A number of alternative processes have been proposed and patented to improve the quality of the acid whey. The acidification can be performed by contact of all or part of the milk with cation-exchange resins (strong hydrogen ion exchange resin), electrodialysis (Noël, 1992) or using carbon dioxide (Hofland et al., 2003). It is believed that these processes have relatively low commercial significance (Maubois and Ollivier, 1997). Regardless of the processes used, isoelectric caseins may be dried and used as such but they are insoluble in water, which limits their applications. Therefore caseins are usually converted to a soluble form, namely caseinate, © Woodhead Publishing Limited, 2010

Separation technologies in dairy and egg processing 355 by neutralization with alkali, usually NaOH (producing sodium caseinate) and less frequently KOH, NH4OH or CaOH2. Soluble caseinates may then be sprayed or roller dried. Destabilization of casein can also be caused by the proteolysis of the micelle stabilizing protein, namely k-casein. The chymosin, the principal enzyme present in calf rennet, releases the glycomacropeptide (C-terminal segments of glycosylated k-casein), and renders the casein micelles susceptible to precipitation with Ca2+ at the natural concentration in milk. This principle is used in the manufacture of rennet casein and most cheese varieties. 12.5.2 Microfiltration for fractionation of casein micelles and serum proteins The most promising technology for the selective separation of casein micelles is undoubtedly membrane MF using a membrane with a pore size of 0.1–0.2 mm. For the past 15 years, this operation has enjoyed rapid industrial development and is still expected to increase significantly over the next few years owing to the high quality and properties of the two fractions produced: – The retentate, enriched specifically in native casein micelles, improves the rennet coagulability in the cheesemaking process. Such casein enrichment reduces rennet coagulation time, accelerates curd firmness kinetics and increases final curd firmness compared with milk or caseinate. Consequently, casein and fat retentions into the cheese curd are significantly improved (less fines and fat in the drained whey) and that leads to an increase in cheese yield. The fact that a smaller amount of serum proteins end up in the cheese manufacturing process is also advantageous, because there are then fewer flavour and texture defects attributed to serum proteins and also fewer detrimental effects of heat treatment on rennet milk coagulability. The decrease in b-lactoglobulin (b-LG) content evidently lowers the extent of the formation of the complex k-casein/b-LG during heat treatment and, consequently, renneted micelles aggregate well. This property was used to develop a new high-heat milk powder, with a cheesemaking ability similar to that of raw milk (Fig. 12.6, Quiblier et al., 1991). – The permeate, with a composition close to that of a whey, contains soluble proteins in their native state, is sterile, and free of phage particles, cellular debris, fat and glycomacropeptide. It therefore becomes a useful fluid for preparing whey protein concentrates (WPC) and isolates (WPI) (protein ratio up to 97%) with very high functional and nutritional properties, in particular compared with WPC obtained from cheese whey (Foegeding et al., 2009). Despite these advantages, the microfiltrate is sometimes used in the cheesemaking process by reincorporation in the caseinenriched fraction after denaturation under moderate concentration and heat treatment (Fig. 12.6).


356 Separation, extraction and concentration processes Skimmed milk MF 0.1 mm VRF = 3 Microfiltrate

Retentate Cream

UF 10–20 kg mol–1 VRF = 6

Cheese

Permeate

Concentration spray drying HT (95 °C, few minutes) Retentate WPC

Milk powder b-Lactoglobulin High cheese-making ability

Fig. 12.6 Primin process (Quiblier et al., 1991). VRF, volume reduction factor; WPC, whey protein concentrates; HT, heat treatment.

Finally, purified casein micelles obtained by diafiltration of retentate against water, and WPI obtained after UF of milk microfiltrate, are excellent starting fluids for further fractionation of individual caseins or serum proteins. From an engineering point of view, this separation is classically conducted using ceramic membrane either with the UTP system or membranes with linear hydraulic resistance gradient, as is done for bacterial removal (12.3.2). Separation is usually performed at 50–55 °C, at a crossflow velocity of 7 m s–1, TMP ~ 50 kPa and VRF of 2–4. At VRF = 3, serum proteins transmission is 65–80% depending on the milk heat treatment (pasteurization, thermalization or raw skimmed milk). Permeation flux is ª75 L h–1 m–2 for 10 h according to the critical stability criterion, which allows the industry to perform long runs with very moderate fouling and high selectivity (Gésan-Guiziou et al., 1999). Owing to the high running costs and investment required by the tubular ceramic equipment, the industry is starting to operate MF with spiral-wound polymer membranes. These membranes, which can not be used with the UTP system, are operated at low temperature (90 °C and pH 90% w/w) in the desired phosphopeptides. The glycomacropeptide, the C-terminal part of the k-casein released in whey by the action of chymosin was shown to be separated from sodium caseinate using UF membrane or centrifugation. This peptide has numerous uses, such as action on satiety and inhibition of Escherichia coli cells adhesion to intestinal walls and, in particular, it contains no phenylalanine, which makes it suitable for use as a nutritional protein supplement for patients suffering from phenylketonuria, who cannot digest protein containing phenylalanine owing to their lack of the appropriate degrading enzyme. A number of dairy starter cultures, as well as proteolytic enzymes isolated from lactic acid bacteria also lead to the formation of bioactive peptides from milk proteins, in particular during the manufacture of fermented dairy products. For example, several studies have demonstrated that Lactobacillus helveticus strains are capable of releasing antihypertensive peptides. A UHT milk fermented by the GG strain of Lb. rhamnosus and subsequently digested with pepsin and trypsin produced hydrolysate fractions that were immunosuppressive. The occurrence of many bioactive peptides in bovine milk is now well established (Korhonen, 2009), but currently the industrial-scale production of such peptides is limited by a lack of suitable separation technologies. Among them, membrane techniques, such as NF or UF, are used industrially to produce ingredients that contain bioactive peptides based on casein or whey protein hydrolysates (Table 12.7) and seem to be the best technology available for the enrichment of bioactive peptides.

12.8 Treatment of effluents and technical fluids in the dairy industry In many countries, the dairy industry is considered to be one of the largest generators of food-processing wastes. Owing to large water volumes for factory cleaning and disinfection, the dairy industry produces 0.2–11.0 L effluents/L of processed milk. Cleaning-in-place (CIP) operations significantly contribute to water consumption and are responsible for 50 to 95% of the overall volume of waste streams (Daufin et al., 2000, Alvarez et al., 2007). © Woodhead Publishing Limited, 2010

Separation technologies in dairy and egg processing 367 The polluting load of these effluents ranges from 0.2 to 5.0 g L–1 of chemical oxygen demand (COD), and is mainly caused by loss of raw material (0.5 to 2% of milk). CIP systems are also mainly responsible for the high pH value (9–11) of the end-of-pipe wastewaters. Although a high portion of dairy wastewaters is still land spread, the treatment of the effluents mainly takes place in biological treatment plants at most dairies. For a factory of average capacity (106 L of milk a day), the sludge produced is usually used for land spreading (1 to 3 tonne of dry matter) and purified water drained to rivers (0.3 ¥ 106 to 3 ¥ 106 L). Current European regulations relating to landfill management, land spreading and purified water quality along with social pressure, has recently forced the dairy industry to significantly reduce its production of sludge and to improve purified water quality. Over the past few years, the dairy industry has been attempting to find new concentration and separation processes to reduce its effluents production. Several types of effluents are currently treated in the industry, benefiting mainly from the potential of membrane technology: washing waters of rennet casein precipitate were treated, respectively, using NF and dead-end filtration; white (flushing) and pre-rinsing waters, corresponding to the first step of CIP, were treated using UF, NF or RO (Blanchard, 1991; Delbecke, 1981). The outcome is a highly significant improvement of water quality after treatment [suspended solids (SS) < 2 mg L–1; chemical oxygen demand (COD) 80% recovery at a 200 cm h–1 flow rate. Billakanti and Fee (2009) characterized a cryogel monolith chromatography for extraction of minor proteins (LF) by cation-exchange chromatography from whole milk feeds at 550 cm h–1 in a single-step. Yield and purity of LF extracted using this process were >85 and 90%, respectively. Wu and Xu (2009) reported a purification process for isolation of LF and IgG from bovine colostrum using a serial cation–anion exchange chromatography system with 95 and 97% final purities, respectively. Lu et al. (2007) designed a productionscale technology for extraction of LF from bovine colostrum with final yields of 83% and purities of 94% using an ultrafiltration system coupled with a fast-flow strong cation-exchange chromatography column. Fee and Chand (2006) have investigated and optimized a process to isolate LF and LP from


Table 16.6 Author and year

Purification examples for the basic whey proteins Configuration/material

Tu et al. 2002 Anti-LF immunoglobulins (IgYLF) immobilized on Sepharose 4B support Ye et al. 2000 Carboxymethyl cation-exchange chromatography

Target protein


LF

Methods for purification of dairy nutraceuticals 465


Colostral whey Final products have purification folds of 103–136 of initial concentration and recoveries of 82–99% Bovine They performed the adsorption process using 50 mM LFa and LFb colostrum phosphate buffer at pH 7.7 LFa was eluted in 0.38 M NaCl and LFb at 0.43 M NaCl buffer solutions. Also investigated variation in the LF concentrations of lactation time and species Li-Chan et al. Immobilized yolk antibody on a LF Milk and Binding capacity was a function of ligand density: at 1998 monoaldehyde-activated support cheese whey 9.2 mg mL–1 of IgYLF, binding capacity was 20% (mol %). This could be increased to 80% by using low ionic strength buffers Camperi et al. Red HE-3B dye affinity chromatography LF Rennet whey This process was achieved with 82% recovery and 98% 2000 purity Jyh-Ping and Microfiltration affinity purification LF and IgG Cheese whey Maximum binding capacity on heparin-Sepharose gel Cheng-Hsin was 124 mg mL–1. LF recovered in this process has 92% activity and 95% purity whereas IgG has 86% activity 1991 and 90% purity Korhonen Review Igs Bovine The latest developments and progress in separation 2004 colostrum, technologies for isolation of Igs in both small and largewhey and milk scale applications Stec et al. Isolation of polyclonal Igs by HPLC Igs Bovine serum Ammonium sulfate precipitation was performed as 2004 a first step of Igs isolation followed by HPLC. This method is applicable for preparative-scale applications DeSilva et al. Conference paper. Novel approaches Whey Dairy whey Recent developments in isolation and purification 2003 to meet the challenges in processing proteins and technologies of various proteins and peptides functional dairy components peptides

Author and year

Continued


Target protein


Xu et al. 2000 Separation of IgG and glycomacropeptides using a polystyrene anion exchanger IRA93 and Amicon YM100 membrane

IgG

Dairy whey

Akita and LiChan 1998

IgG1 and IgG2

Kim and LiChan 1998

Configuration/material

Specific antibody-immobilized immunoaffinity chromatography for isolation of bovine immunoglobulin G subclasses Single-step process for isolation of IgG using avidin-biotinylated IgY chromatography

Konecny et al. Purification of monospecific polyclonal 1994 antibodies from hyper immune bovine whey using immunoaffinity chromatography Labrou and Review Clonis 1994

IgG

Igs

Igs

Successful for concentration of IgG while removing all major whey protein contaminates. A continuous process using IRA 93 resin and 100 kD membranes, designed to produce enriched IgG GMP and WPI ingredients from rennet dairy whey Bovine milk, Gave >98% purity of IgG subclasses. Binding capacities colostrum and of the column were 27 and 38% (molar masses) for whey IgG1 and IgG2, respectively. Antibody columns were stable for more than a year with minimal antibody loss Cheddar Binding capacities achieved in this investigation were cheese whey 50–55% (w/w of IgG and IgY ligands). Bound IgG was eluted at pH 2.8 using glycine–HCl buffer with 99% purity Cheese whey

Various modes of affinity adsorption technologies used for isolation of Igs both in laboratory- and industrialscale applications


Table 16.6

Methods for purification of dairy nutraceuticals 467 whole milk at milking pH and temperatures using SP Sepharose Big Beads as a packed chromatographic resin. In their investigation, they successfully captured both LP and LF (total dynamic capacity of 49 mg mL–1 resin) from raw milk at 300 cm h–1 flow rate with minor leakage (500 cm h–1). Fee and Chand (2005) reported on-farm capture of high-value minor milk proteins (LF and LP) from the raw milk of individual animals using a fully automated protein fractionation robotic system. This method was rapid, used a single step and was designed to minimize microbial contamination, while offering full product traceability to individual animals. Claycomb (2004) holds a patent for on-farm fractionation of high value milk proteins directly from raw milk using an automated milking system (AMS). Dionysius (1991) investigated a CM-Sephadex resin in a stirred tank system, mixing the resin with whey (cheese, rennet and acid) for 60 min at pH 7.0, and captured more than 80% of LP and 90% of LF, although the resin capacity changed with nature of whey. The bound LF was subsequently eluted by NaCl gradient elution. Heeboll-Nielsen et al. (2004) developed a superparamagnetic cation-


468 Separation, extraction and concentration processes exchange chromatographic process for fractionation of bovine whey proteins including LF and LP with 28-fold purification over the starting material. The resin could be recovered magnetically in a high-gradient magnetic fishing system, allowing the resin size to be minimized for fast adsorption, while being recovered without the need for fine particle filtration, settling or centrifugation. Ulber et al. (2001) described a two-step membrane processing method for separation of LF from sweet whey, the first membrane removing lipids and other solids and the permeate then passing through a cation exchange membrane adsorber. Plate et al. (2006) used 2-m2 membrane modules for preparative-scale isolation of LF and LP from sweet whey with >90% recovery yields and purities. Chiu and Etzel (1997) described a method for extraction of LF and LP from cheese whey using microporous (3–5 mm) cationic membranes and achieved 50 and 70% recoveries, respectively. Mitchell et al. (1994) used cation-exchange membranes operated in either dead end or cross-flow configuration to isolate LF and LP from cheese whey. Affinity chromatography Affinity chromatography is the second most popular chromatography method for isolation of minor milk proteins from complex protein mixtures on a laboratory scale. Wolman et al. (2007) used Red HE-3B dye coupled with hollow fibers as a membrane affinity chromatography matrix for isolation of LF in a single step, achieving a higher adsorption capacity (111.0 mg mL–1) than the same ligand immobilized on agarose beads (9.3 mg mL–1 resins). They could achieve >94% purity and 91% recovery in the final product using whey and colostrum as feed materials. Chen et al. (2007) evaluated micrometre-sized monodisperse superparamagnetic polyglycidyl methacrylate (PGMA) particles coupled with heparin (PGMA-heparin) as an affinity method for isolation of LF from bovine whey in a single step. This magnetic affinity method resulted in a very high binding capacity of 164 mg g –1 resin and may hold promise as a fast process tool for industrial-scale purification of high-purity LF, although one must always consider the potential for leaching of toxic compounds from affinity ligands. Noppe et al. (2006; 2007) described immobilized affinity peptides as a new platform for rapid development of alternative affinity chromatography ligands for isolation of target proteins from crude feeds. In their method development, they used selected bacteriophages immobilized on a microporous monolith column for extraction of LF from complex fluids such as milk and blood serum. Bound LF was eluted with 1 M NaCl and fractions were recovered with >95% purity. Kawakami et al. (1987) used an immobilized LF-monoclonal antibody resin for a single-step isolation of LF from both human and bovine skim milk and colostrum, with very high purities and recoveries of >97%. Unlike LF, isolation of LP has attracted limited attention because of its low concentration and low commercial value. Shin et al. (2001) used


Methods for purification of dairy nutraceuticals 469 immunoaffinity chromatography for selective isolation of LP from human whey but such processes have not been extensively studied for large-scale applications. Other chromatographic methods Size-exclusion (gel-filtration) chromatography is not commonly used as an early step in large-volume protein purification because linear velocities are low, but it is often used as a final purification step after other chromatography steps to enhance protein purity. For example, Al-Mashiki and Nakai (1987) applied size-exclusion chromatography for isolation of LF and immunoglobulins from different whey streams. Tomita et al. (2002) reported the possibility of LF and LP isolation from cheese whey or skim milk using a semi-large scale hydrophobic interaction chromatography method. Yoshida (1989) used hydrophobic interaction chromatography followed by DEAE ion exchange chromatography to isolate LF and LP from acid whey. Non-chromatographic methods Noh et al. (2005) selectively purified LF using a cationic surfactant as a micelle foaming agent, manipulating the protein behaviour by changing pH and salt concentration in the aqueous phase and surfactant concentration in the organic phase. LF was partitioned into the aqueous phase whereas all other proteins were solubilized into the organic phase. Fuda (2004) obtained approximately 25-fold enrichment of LF and LP from sweet whey using colloidal aphrons (CGAs), which are surfactant microbubbles generated by intense stirring of the anionic surfactant sodium bis-2ethylhexyl sulfosuccinate (AOT). Noel et al. (2001) investigated a low-cost foam fractionation process as the first step in separating LF from milk and achieved approximately 40% mass recovery at pH 10. Similarly, Saleh and Hossain (2001) developed a semi-batch foaming process to separate LF from a multicomponent mixture of BSA, a-Lac and LF by utilizing protein surface active properties. 16.5.2 Lysozyme purification Owing to its high isoelectric point (pI>11), lysozyme typically co-elutes with other basic proteins (LF and LP), but there have been few studies of the isolation of this protein. However, lysozyme has been successfully purified from milk or acid whey by a combination of affinity chromatography on heparin-Sepharose, followed by gel filtration on Sepharose 4B (Wang and Kloer 1984) or Sephadex-G50 (Boesman-Finkelstein and Finkelstein 1982). Duhaiman (1988) used heparin–Sepharose 4B, Sephadex G-75 and hydroxyapatite for chromatographic extraction of lysozyme from camel milk. Recently, using recombinant technology, human lysozyme was expressed at high concentrations (0.5% dry weight) in transgenic rice seed (Huang et


470 Separation, extraction and concentration processes al. 2002). Wilken and Nikolov (2006) found cation-exchange purification of lysozyme to be optimal at pH 4.5.

16.6 Immunoglobulins in the dairy nutraceutical industry IgG is an important component of bovine milk and comprises the major protein in bovine colostrum. Consumer acceptance of value-added whey protein products that involve Igs began in Asia and migrated to Europe and the US, targeting sports nutrition, infant formulae, dietary supplements and physiologically functional foods (Gapper et al. 2007). Immunoglobulins are g-globulin proteins that are found in blood and other body fluids of human, bovine and all other lactating species. These are used in the immune system to identify and neutralize bacteria, viruses and other antigens. Human immunoglobulins are broadly classified into five different classes: IgA, IgD, IgE, IgG, and IgM, whereas bovine milk and colostrum mainly contain immunoglobulins IgG, IgM and IgA. All immunoglobulin classes share a basic structural unit that comprises four polypeptide chains with two identical heavy (H) chains and two identical light (L) chains, held together with disulfide bonds. Molecular masses of the heavy- and light-chain peptides are approximately 50–70 kDa and 25 kDa, respectively. 16.6.1 Immunoglobulin G Immunoglobulin G is the most abundant class of antibody in the colostrum and milk of several mammalian species, comprising 80–90% of total antibodies. The primary structure of IgG contains two heavy (each of 450–550 amino acids) and two light chains (each of 211–217 amino acids) with a total molecular mass of approximately 150–160 kDa and a pI of 5.5–8.3. Commonly, IgG presents in two-sub classes such as IgG1 (MW 160 kDa, pI 5.5–6.8) being the richest class, at 15–180 g L–1, and IgG2 (MW 150 kDa, pI 7.5–8.3), at 1–3 g L–1. 16.6.2 Immunoglobulin A Similar to other immunoglobulins, the monomeric structure of IgA also contains two heavy and two light chains but it occurs as a monomer or a dimer, the latter joined by a J-chain and a secretory component. This complex structure is called secretory IgA (sIgA) and has a molecular weight of 380–417 kDa. IgA is present in blood, milk and several mucosal surfaces of lungs and gastrointestinal tracts. This antibody acts as a primary defence system against several pathogens. The IgA concentration in bovine colostrum ranges from 1 to 5 g L–1, whereas bovine milk contains only 0.05 to 1 g L–1.


Methods for purification of dairy nutraceuticals 471 16.6.3 Immunoglobulin M The monomeric structure of IgM is similar to IgG but IgM consist of five subunits, linked together to form a pentameric structure in a circular mode by disulfide bonds and a J-chain. The molecular mass of IgM is approximately 900–1000 kDa. In bovine milk, the IgM concentration ranges from 0.04 to 1.0 g L–1, whereas bovine colostrum contains 5 to 10 times greater amounts.

16.7 Purification technologies for immunoglobulins in the dairy nutraceutical industry Immunoglobulins from bovine milk have applications as supplements in infant formulae, hyperimmune foods, functional foods, nutraceutical and pharmaceutical products. Although there are many processing methods available for separation of immunoglobulins from various sources, including colostrum and milk, appropriate technologies for large-scale productions are still lacking. Traditionally, isolation of Igs from colostral whey has been achieved by precipitation with either ammonium sulfate or ethanol, followed by chromatography. Although such methods yield rather pure IgG fractions, most are feasible only in small-scale applications. In particular, they are not appropriate for large-scale production of Igs from bovine colostrum or milk (El-Loly 2007). Because milk is a complex fluid and contains high amounts of fat, it is difficult to isolate Igs using conventional methods. However, rapid developments in separation technologies and particularly in the application of membrane separation methods have made industrial-scale isolation of immunoglobulins from various streams, including whey, colostrum and recombinant cell culture supernatants a possibility. 16.7.1 Ion exchange chromatography Use of ion-exchange chromatography to isolate immunoglobulins from dairy feeds is limited but it is used as an intermediate or final purification step in combination with other chromatographic or membrane processing methods. For example, Wu and Xu (2009) applied cation- and anion-exchange chromatography in series for isolation of IgG and LF from bovine colostral whey. Similarly, Pessela et al. (2006) investigated a simple method for isolation of Igs from whey protein concentrate with >80% recovery and reasonable purity using a combination of DEAE–agarose anion-exchange chromatography followed by low-substitution aminated adsorbents. In the first step, they could remove the main contaminant, BSA, and during the second step, Igs were selectively bound on the aminated resin with minor contamination by a-Lac and b-Lac. Noel (2007) developed a second-generation, robust EBA method for


472 Separation, extraction and concentration processes isolation of Igs from cheese whey, which involved a mixed-mode chemical ligand that has selectivity towards IgG over other whey proteins. The feed whey solution was heated to 50 °C to maintain pasteurization, reduce the viscosity of the solution and maximize flow rate. They could thus load the column at greater than 3000 cm h–1 flow rate without reducing its adsorption capacity. Using a 880-L EBA column, they could produce 13 kg of IgG per day with >90% of final product yields. The eluent from this column was passed through a 160 L anion exchange column for further purification. 16.7.2 Membrane chromatography Since membrane technology was introduced to the dairy industry in the 1970s, many membrane processes have been developed for isolation of Igs from whey and milk using either a single-step process or a combination of several individual steps. For example, UF has been employed either alone or in combination with ion-exchange or gel-filtration chromatography for largescale fractionation of Igs from cheese or colostral whey streams (Abraham 1988; Korhonen 2004; Syväoja et al. 1994). Al-Mashikhi et al. (1988) successfully isolated IgG from acid and cheddar whey using UF, combined with immobilized metal chelate affinity chromatography, obtaining purities of 77 and 53%, respectively. Korhonen (1998) used a multi-step process with UF, microfiltration and reverse osmosis, followed by a cation-exchange resin to achieve 40–75% enrichment of Igs from colostral whey. 16.7.3 Affinity chromatography Affinity adsorption methods have been widely used for isolation of Igs from colostral or cheese whey. Protein-A and Protein-G affinity media are well characterized and used for fractionation of Igs, particularly IgG. Many pilot- and industrial-scale methods using membrane technologies and affinity chromatography have been developed for extraction of Igs from whey and colostrum either alone or in combination with other chromatography techniques (Kochan et al. 1996; Mukkur and Froese 1971; Schmerr et al. 1985). Labrou and Clonis (1994) reviewed various affinity-based purification technologies for Igs. Human and secretory IgA molecules have been isolated by precipitation, ion-exchange and affinity chromatography from human serum (Kondoh et al. 1987; Monteiro et al. 1985; Roque-Barreira et al. 1986) and human milk (Khayam-Bashi et al. 1977). Hutchens et al. (1990) developed a salt-promoted thiophilic adsorption method for simultaneous isolation of all bovine Igs from colostral whey. Mukker and Froese (1971) described isolation of bovine IgM from colostral whey using a combination of molecular sieve and DEAE–cellulose chromatography. Su and Chiang (2003) described a simple non-chromatographic method for isolation of Igs from other whey proteins. They used reverse micelles


Methods for purification of dairy nutraceuticals 473 to extract more than 90% of IgG into an aqueous phase, with 90% purity, whereas all other whey proteins were partitioned into the reverse micelle phase. 16.7.4 Protein-A mimetic (PAM) affinity peptides Protein A and protein G affinity chromatography can achieve capacities of up to 30–40 mg IgG per mL of resin under optimal conditions. However, these affinity ligands suffer from several inherent drawbacks such as high cost, their biological origin, possible contaminants, low stability towards sanitizing agents and the fact that their selectivity is limited to IgG. Recently, with the help of combinatorial chemistry and modern analytical tools, researchers synthesized protein A mimetic (PAM) peptide ligands (D’Agostino et al. 2008; Fassina 1994; Newcombe et al. 2005; Ulbricht and Yang 2005; Yang et al. 2005) for isolation of human immunoglobulins from mammalian cell cultures. Compared with protein A or G, these synthetic peptide ligands have several benefits (D’Agostino et al. 2008), including high stability towards chemical and biological reagents, low production cost and the absence of biological contaminants. Moreover, these synthetic affinity ligands may also have selectivity towards other classes of immunoglobulins (IgG, IgE, IgM and IgA) (Fassina et al. 1998; Yang et al. 2005), which are not recognized by conventional protein A or G ligands. Several synthetic peptides have been proposed as potential replacements for protein A in affinity chromatography (Ulbricht and Yang 2005). However, the lack of selectivity for antibodies from various classes has limited their widespread use and some of these ligands are not compatible with the retention of antibody activities in proposed buffer solutions. However, more recently, Yang et al. (2005) synthesized a hexamer peptide that has a wide-ranging selectivity towards various classes of immunoglobulins at near-neutral pH. By using a gradient elution with 0.2 M sodium acetate buffer, they were able to isolate various classes of immunoglobulins from human serum (Yang et al. 2005). They have also reported the specificity of this ligand towards immunoglobulins of human, bovine and several other species. Although most of this work has been carried out on human immunoglobulins, there has been recent interest in applying this PAM peptide affinity chromatography for isolation of bovine immunoglobulins from early colostral milk, hyperimmunized cows and transgenic animals.

16.8 Future trends In the past few decades, the dairy industry has moved from being solely a commodity food-based industry producing significant waste (whey) volumes to one that has increasingly sought added value and the minimization of waste. The nature of dairy products has expanded from simple nutritional © Woodhead Publishing Limited, 2010

474 Separation, extraction and concentration processes foods to separate fractions sold as individual ingredients or as blends to meet the specific functional and nutritional requirements of secondary producers. This trend will continue, as milk volumes increase to meet the new demand for dairy products from the developing world and greater bulk production efficiencies drive companies to find new products to maintain their competitive advantages. Nutraceutical products, with implicit claims of health benefits, are now commonplace and there are likely to be more such products in the future, with some fractions in the future perhaps even meeting the more stringent proof of efficacy requirements of pharmaceuticals for highly targeted applications, for example around neonatal gut development, anticancer agents or immune system enhancers. The commercial recovery of very minor fractions with useful bioactivities, such as oligosaccharides (including those incorporating sialic acid), the myriad proteins that make up the milk fat globule membrane, and the complex lipids therein (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and glycolipids such as gangliosides) will be a challenge. Recovery of such fractions may require specific affinity techniques and the challenge will be to use techniques to extract minor proteins economically on a large scale, while maintaining the safety and integrity of the residual milk or whey for further processing i.e. without contaminating the flow-through stream with leachates. On-farm extraction of minor products from milk may offer advantages in the production of proteins and other bioactives that are heat-labile and are currently largely lost during the pasteurization required inside the factory gate. On-farm extraction may also offer direct benefit to farmers who select for certain genetic traits or apply unique feeding regimes to enhance the production of specific high-value products in their herds. In summary, higher volumes of milk production and increased global competition in the dairy industry will probably drive further growth in niche bioactive products over the next few decades. High-volume, economically efficient separation technologies with high specificity and compatibility with food production will be required to support this growth.

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17 Methods of concentration and purification of omega-3 fatty acids S. P. J. Namal Senanayake, Danisco USA, Inc., USA

Abstract: An overview is presented of the various methodologies used for producing highly purified omega-3 fatty acids from natural source materials. Omega-3 fatty acids derived from fish, krill and microalgae, consisting of eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3), have beneficial effects in the prevention and management of cardiovascular disease and other chronic disorders. Production of high-purity omega-3 fatty acids is increasingly important in both the nutraceutical and pharmaceutical industries. The physical, chemical and enzymatic methods used include urea adduction, chromatography, low-temperature fractional crystallization, supercritical fluid extraction and distillation. Key words: omega-3 fatty acids, urea adduction, chromatography, crystallization, supercritical fluid extraction, distillation, enzymatic methods.

17.1 Introduction Omega-3 fatty acids are essential fatty acids that have diverse biological effects in human health and disease. They are essential to human health but cannot be manufactured by the body. For this reason, omega-3 fatty acids must be obtained from food. Omega-3 polyunsaturated fatty acids (PUFAs) can be found in fish, such as salmon, tuna, and halibut, other marine organisms such as algae and krill, certain plants, and nut oils. The heart-health benefits of the omega-3 fatty acids are well known. Omega-3 fatty acids are considered to have beneficial effects in the prevention of cardiovascular disease, inflammation, hypertension as well as other chronic disorders (Kris-Etherton et al., 2001; Lands, 2003; Senanayake, 2000; Senanayake and Shahidi, 2000b; Shahidi and Senanayake, 2006; Yokoyama et al. 2007). In 2004, the US Food and © Woodhead Publishing Limited, 2010

484 Separation, extraction and concentration processes Drug Administration gave ‘qualified health claim’ status to eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) found in fish, stating that ‘supportive but not conclusive research shows that consumption of EPA and DHA omega-3 fatty acids may reduce the risk of coronary heart disease’ (United States Food and Drug Administration, 2004). For clinical and nutritional applications, the natural sources of omega-3 fatty acids, as such, may not provide the required amounts of these fatty acids and hence concentration and purification of omega-3 fatty acids may be necessary. A concentrated source of these fatty acids is desired to achieve sustainable benefits. Highly purified omega-3 fatty acids may be produced in the free fatty acid, simple alkyl ester and triacylglycerol forms. To achieve this, physical, chemical and enzymatic techniques may be employed. Methods traditionally employed for the concentration and purification of omega-3 fatty acids include urea adduction, chromatographic methods, low-temperature fractional crystallization, supercritical fluid extraction, distillation, and enzymatic and integrated methods.

17.2 Urea adduction in the concentration and purification of omega-3 fatty acids Urea adduction is one of the most efficient and simplest techniques for concentration and purification of omega-3 fatty acids from natural sources (Senanayake, 2000). The formation of complexes between urea and straightchain saturated fatty acids is a well established and potentially valuable separation technique for fractionation of free fatty acids or esters (Shahidi and Senanayake, 2006). Initially, the triacylglycerols (TAGs) of oil are hydrolyzed into their constituent fatty acids via alkaline hydrolysis using alcoholic KOH or NaOH. The resultant free fatty acids (FFAs) are then mixed with an ethanolic solution of urea for complex formation. Urea molecules readily form solid-phase complexes with saturated and monounsaturated fatty acids and crystallize out on cooling and may be removed by filtration. The liquid fraction is highly enriched with omega-3 fatty acids. In general, the crystallization temperature can be ranged from ambient to –20 °C. Since the process involves relatively mild operating conditions and chemicals (free fatty acids, urea, ethanol, water) that are generally recognized as safe (GRAS) by the US Food and Drug Administration, it can be labeled as eco-friendly. This process can be used as a preliminary fractionation step in conjunction with other purification methods, such as low-temperature fractional crystallization, enzyme-catalyzed methods, and molecular distillation, resulting in a highpurity free-fatty-acid omega-3 product. Urea-based fractionation of fatty acids has been extensively used for enriching omega-3 fatty acids in marine oils (Hayes et al., 2000). Urea adduction of fatty acid ethyl esters of squid (Illex argentinus) visceral oil,



using cyclohexane and methanol, can increase the EPA content from 11.8 to 28.2% and DHA content from 14.9 to 35.6% (Hwang and Liang, 2001). Haagsma et al. (1982) described a urea adduction method for enriching the EPA and DHA levels of cod liver oil from 12 to 28 and 11 to 45%, respectively. Wanasundara and Shahidi (1999) used an experimental design to optimize the conditions on a laboratory scale that led to the maximum concentration of EPA and DHA from seal blubber oil, using the urea-based fractionation. The authors obtained 88.2% of total omega-3 fatty acids at an urea/fatty acid ratio of 4.5, a crystallization time of 24 h and a crystallization temperature of –10 °C. Senanayake and Shahidi (2000a) concentrated and purified DHA from the oil extracted from the microalgae Crypthecodinium cohnii and reported a DHA enrichment from 47.4 to 97.1% with a process yield of 32.5% of the mass of the original algal oil. The fatty acid composition of algal oil and its DHA concentrate obtained by urea adduction are reported in Table 17.1 (Senanayake and Shahidi, 2000a). In another study, mackerel processing waste comprising skins, viscera, and muscle tissue was evaluated by Zuta et al. (2003) for concentrating omega-3 fatty acids by urea adduction. Fish oil was extracted using either chloroform/methanol (2:1 vol/vol) or hexane/isopropanol (3:2 vol/vol). The oil yield and iodine value (which measures the degree of unsaturation in fats and oils) were determined for fresh fish oil extracts. Omega-3 fatty acid concentrates were prepared from saponified fish oil via urea adduction. The mean oil yields were 9.18, 9.2, and 38.1% for viscera, muscle, and skin, respectively. The mean baseline iodine value was 134, which increased to 296 after urea adduction. Hence, it was possible to concentrate omega-3 fatty acids from mackerel processing waste. The type of tissue used did not affect the amount of omega-3 fatty acids concentrated. Mackerel skin was most desirable because of its high oil content. Hayes (2006) investigated the effect of cooling rate on the degree of removal of saturated acyl groups from FFAs derived from canola oil and Table 17.1 Fatty acid composition of algal oil and its DHA concentrate after urea adduction (adapted from Senanayake and Shahidi, 2000a) Fatty acids 10:0 12:0 14:0 16:0 16:1 18:1w-9 18:2 w-6 22:5 w-3 22:6 w-3 Iodine value, calculated

Algal oil (%) 0.6 1.1 15.0 9.0 2.2 19.0 1.0 0.5 47.4 234

DHA concentrate (%) 0.5 0.5 0.1 0.0 0.3 0.2 0.7 0.4 97.1 437


486 Separation, extraction and concentration processes the isolation of di- and polyunsaturated acyl groups from FFAs derived from vegetable and fish oil during urea adduction. Traditionally, slow cooling has been used (–1 °C min–1). A more rapid cooling rate (–47 °C min–1) produced urea inclusion compounds (crystals) of similar morphology and thermodynamic properties, but of a size an order of magnitude smaller than the urea inclusion compounds formed during slow cooling. Fractionations used only renewable materials (urea, FFAs, and 95% ethanol as solvent) and benign operating conditions (ambient pressure, 25–75 °C, and neutral pH). When the recovery of FFAs was relatively high (>60%), the selectivity of urea adduction toward the inclusion of saturated fatty acids and against PUFAs was not affected by the cooling rate. In contrast, when the FFAs recovery was low, representing those instances where an increase in the purity of the PUFAs is a more important economic goal, a slower cooling rate resulted in a significantly greater discrimination against PUFA groups, hence to a FFA product with a measurably greater purity.

17.3 Chromatographic methods for the concentration and purification of omega-3 fatty acids Another method for concentration and purification of omega-3 fatty acids is the use of chromatography. High-performance liquid chromatography, silver resin chromatography and supercritical-fluid chromatography have been used for concentration of omega-3 fatty acids from natural sources. Hayashi and Kishimura (1993) isolated 63–74% pure DHA from skipjack tuna eye orbital oil by stepwise elution with hexane, diethyl ether/hexane and diethyl ether on a silicic acid column. Teshima et al. (1978) employed a silver nitrate-impregnated silica gel column to separate DHA and EPA from squid-liver oil after forming methyl esters. They were able to isolate 95–98% DHA and 85–96% EPA with yields of 48 and 39%, respectively. Guil-Guerrero and Belarbi (2001) purified EPA and DHA from cod liver oil using a silver nitrate-impregnated silica gel column. The oil was saponified and treated with urea, and the non-complexed fatty acids were then converted to methyl esters before chromatography. The column was washed with a sequence of mobile phases. A 64% recovery of DHA with 100% purity was obtained. The recovery of EPA was 29.6%, with a final purity of 90.6%. Perretti et al. (2007) investigated the fractionation of fish oil fatty acid ethyl esters with the aim of obtaining a lipid fraction enriched in omega-3 fatty acids and with a suitable EPA/DHA ratio. They reported the possibility of modifying the original fatty acid ethyl ester concentrations by optimizing the extraction conditions in terms of pressure, temperature, and supercritical CO2 flow rate: 2-h runs, pressures of 100, 140, 150, and 300 bar, and liquid CO2 flow rates of 2.5, 3.5, 5, and 10 kg h–1, at 40, 50, and 60 °C, in the three sections of the column starting from the bottom, respectively. They



stated that supercritical fluid fractionation appears to be a useful processing technique for changing the composition of lipids in order to obtain high added-value functional products. A preparative reversed-phase high-performance liquid chromatography method with gradient elution using acetonitrile–chloroform and evaporative light-scattering detection was used by Mansour (2005) to purify milligram quantities of microalgal PUFAs, separated as methyl esters. PUFA-methyl esters purified included methyl esters of DHA, EPA and the unusual very-longchain highly unsaturated fatty acid, octacosaoctaenoic acid (28:8n-3) from the marine dinoflagellate, Scrippsiella sp. Other fatty acids purified from various microalgae using this method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-methyl esters, and resolution from closely eluting PUFA-methyl esters, which determined the maximum loading. The purity of these fatty acids was determined by electron-impact gas chromatography–mass spectrometry (EI GC–MS) and the chain length and location of double bonds were determined by EI GC– MS of 4,4-dimethyloxazoline derivatives formed using a low-temperature method. The advantages over silver-ion HPLC for purifying PUFA-methyl esters are that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-methyl esters with the same degree of unsaturation but different chain length. In addition, PUFA-methyl esters were not strongly adsorbed, but eluted earlier than their more saturated corresponding PUFA-methyl esters of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. Centrifugal partition chromatography (CPC) is a separation method based on the liquid partition of compounds. Owing to the high centrifugal field force, one phase stays in the rotor (the liquid stationary phase), the other one is the mobile phase as in classical liquid chromatography. CPC has been used for isolation and purification of PUFAs from various natural sources. Several important advantages of centrifugal partition chromatography are reported in Table 17.2. A CPC method to purify DHA from microalgal oil on a laboratory scale, has been developed by Wanasundara and Fedec (2002). The starting microalgal oil contained 39.7% DHA and 15.2% DPA (n-6), along with several other fatty acids. The mobile phase was hexane–methanol–water in a normal phase-ascending mode. A good separation was achieved and recoveries of 84.6% DHA and 84.9% DPA were obtained. Bousquet and Goffic (1995) have examined CPC separation of microalgal oil-based DHA and EPA and were able to isolate pure DHA and EPA from this oil with excellent yields. The first separation used heptane as the stationary phase and aqueous 3% acetonitrile as the mobile phase. The minor fatty acids were eluted, leaving a mixture of four major PUFAs. This mixture was subjected to a second separation, using heptane as a stationary phase and aqueous methanol as the mobile phase. Under these conditions, it was possible to isolate pure DHA and EPA with high yields. High-speed countercurrent chromatography was used by Du et


488 Separation, extraction and concentration processes Table 17.2 Advantages of centrifugal partition chromatography Advantages Cost

Solid supports, which in many cases are costly, are not required but instead a solvent system is used Recovery Elimination of solid supports avoids problems associated with irreversible retention of highly retentive sample components. Almost 100% recovery of the compounds is guaranteed Quantity Compared with conventional liquid chromatography, the volume ratio of the stationary phase to the total column (rotor) volume is greater. Hence, large quantities of materials can be retained in the stationary phase Speed The stationary phase is retained by centrifugal force, enabling the mobile phase to be pumped at high speeds through the apparatus resulting in reduced separation times Stability Mild operating conditions are used. Therefore, decomposition and oxidation of PUFAs are virtually nonexistent under these conditions Versatility Any two-phase solvent system can be used, including many prepared from nontoxic and commonly available solvents

al. (1996) to separate DHA and EPA ethyl esters in a mixture containing 39.3 and 56.4%, respectively, with hexane–dichloromethane–acetonitrile (5:1:4 v/v/v) as mobile phase. However, the separation was not successful with this solvent system because of the low partition coefficients of the esters under the experimental conditions used. Murayama et al. (1988) separated a mixture of stearic, oleic, linoleic and a-linolenic (18:3n-3) acid ethyl esters because their partition coefficients were distributed over a wide range in the mobile phase comprising hexane–acetonitrile (1:1 v/v). The ethyl esters of linoleic acid and a-linolenic acids were successfully separated during the first normal ascending elution whereas the ethyl ester of oleic acid was isolated by switching the elution mode to descending. Despite the developments in chromatographic techniques to concentrate and purify omega-3 oils, the use of very large volumes of solvents, loss of column resolution after repeated use, and potential product solvent residues are likely to hinder scale-up to production scale volumes.

17.4 Low-temperature fractional crystallization for the concentration and purification of omega-3 fatty acids Low-temperature fractional crystallization is one of the simplest methods employed for production of purified omega-3 fatty acids. This process takes advantage of the existing differences in the melting points of different fatty acids as neat oils or in different solvent systems. The more saturated fatty acids have higher melting points and crystallize out of the mixtures



leaving behind the more unsaturated fatty acids (Senanayake, 2000). The melting points of fatty acids are dependent on their degree of unsaturation. For example, EPA and DHA melt at –54 and –44.5 °C compared with 13.4 and 69.6 °C for 18:1 and 18:0, respectively (Merck Index, 1983). As the temperature of a mixture of saturated and unsaturated fatty acids decreases, the saturated fatty acids, having a higher melting point, start to crystallize out first and the liquid phase becomes enriched in the unsaturated fatty acids (Shahidi and Senanayake, 2006). However, as the number and type of fatty acid components in the mixture increases, the crystallization process becomes more complex and repeated crystallization and separation of fractions must be carried out to obtain purified fractions. For fish oils, not only is there a very wide spectrum of fatty acids but the fatty acids exist, not in the FFA form, but esterified in TAGs. However, the principle of low-temperature fractional crystallization can still be applied to marine oils to partially concentrate TAGs rich in omega-3 PUFAs (Shahidi and Wanasundara, 1998). Seal blubber oil in the TAG and FFA forms were subjected to low-temperature fractional crystallization using solvents such as hexane and acetone in order to obtain omega-3 fatty acid concentrates. Preparation of omega-3 fatty acid concentrates from seal blubber oil by low-temperature crystallization was also reported by Wanasundara (1996), who subjected seal blubber oil in the TAG or FFA form to solvent-based fractionation, using hexane and acetone as solvents, at different temperatures. The content of omega-3 fatty acids in the non-crystalline fraction was increased with decreasing the crystallization temperature. Under all temperature conditions, acetone gave rise to the highest concentration of total omega-3 fatty acids. Low-temperature solvent crystallization of seal blubber oil, in the FFA form, at –60 and –70 °C in hexane, resulted in total omega-3 fatty acid content of up to 58.3 and 66.7%, respectively. However, the content of total omega-3 fatty acids in acetone increased up to 56.7 and 46.8%, respectively. In another study, Han et al. (1987) found that alkali salts of less unsaturated fatty acids crystallize more rapidly than those of highly unsaturated fatty acids containing four or more double bonds, when the saponified solution is cooled. They also compared the cooling temperature and the rate of cooling on the enrichment of omega-3 fatty acids of alkali salts of sardine oil fatty acids. The DHA and EPA from sardine oil were concentrated more than 2.3fold with minimum yields of 91 and 87%, respectively. Fatty acid profiles of the prepared concentrates showed that the cooling rate and temperature had little effect on the yield and contents of DHA and EPA. Chen and Ju (2001) utilized a modified low-temperature solvent crystallization process for the enrichment of PUFAs in borage and linseed oil fatty acids. The effects of solvent, operation temperature, and solvent-to-FFA ratio on the concentration of PUFAs were investigated. The best results were achieved when a mixture of 30% acetonitrile and 70% acetone was used as the solvent. With an operating temperature of −80 °C and a solvent-to-FFA ratio of 30 mL g –1, g-linolenic acid (GLA; 18:3n-6) in FFA of saponified borage oil was raised


490 Separation, extraction and concentration processes from 23.4 to 88.9% with a yield of 62.0%. At a yield of 24.9%, a-linolenic acid (ALA; 18:3n-3) in linseed oil was increased from 55.0 to 85.7%.

17.5 Supercritical-fluid extraction for the concentration and purification of omega-3 fatty acids Supercritical-fluid extraction is a relatively novel technique which has found use in food and pharmaceutical applications (Senanayake, 2000). The properties of a supercritical fluid are used to selectively extract a specific compound or to fractionate mixtures by changing the temperature and pressure without any phase change. The most important property of these fluids is the high solvation capacity in their supercritical region (Shahidi and Senanayake, 2006; Shahidi and Wanasundara, 1998). This method is mild and, because it uses CO2, minimizes autoxidation (Senanayake, 2000). A number of gases are known to have good solvent properties at pressures above their critical values. For food applications, CO2 is the solvent of choice because it is inert, inexpensive, non-flammable, environmentally acceptable, non-toxic, relatively safe, completely recoverable, and readily available and has a moderate critical temperature (31.1 °C) and pressure (1070 psig). The fatty acids were most effectively separated on the basis of chain length; hence the method works best for oils with low levels of long-chain fatty acids. Because this method is derived from separation of compounds based on their molecular weight and not their degree of unsaturation, prior concentration steps, such as urea adduction or low-temperature crystallization, may be necessary in order to purify the omega-3 fatty acids. The use of supercritical fluids for extraction and purification of omega-3 fatty acids from fish and krill oils has been reported (Mishira et al., 1993; Yamagouchi, et al., 1986). Fish oils in the form of free fatty acids and fatty acid esters have been extracted with supercritical CO2 to yield concentrates of EPA and DHA. The use of high pressures and high capital costs might limit the widespread use of this technique in large-scale applications (Shahidi and Senanayake, 2006). Alkio et al. (2000) evaluated the technical and economic feasibility of producing ethyl ester concentrates of DHA and EPA from transesterified tuna oil using supercritical fluid chromatography. A systematic experimental procedure was used to find the optimal values for process parameters and the maximal production rate. DHA ester concentrates up to 95 wt% purity were obtained in one chromatographic step with supercritical fluid chromatography, using CO2 as the mobile phase at 65 °C and 145 bar and octadecylsilane-type reversed-phase silica as the stationary phase. DHA ester, 0.85 g/(kg stationary phase/h) and 0.23 g EPA ester/(kg stationary phase/h) can be simultaneously produced at the respective purities of 90 and 50 wt%. The process for producing 1000 kg of DHA concentrate and 410 kg of EPA concentrate per year requires 160 kg of stationary phase



and 2.6 t h–1 of CO2 as recycling mobile phase. The operating cost of the supercritical fluid chromatography was US$550 kg–1 for DHA and EPA ethyl ester concentrate. The US Patent 4,675,132 (Stout and Spinelli, 1987) demonstrated that fish oil esters could be fractionated by supercritical fluid extraction to produce an oil with a DHA content of 60–65%. However, the recovery of omega-3 fatty acids was low during this process. Jachmanián et al. (2007) studied the solubility of various ethyl esters derived from hake liver oil in supercritical CO2. A selectivity factor was used to determine optimal conditions to fractionate the ethyl ester mixture. A strong influence of solvent pressure and temperature was observed at 8.63–18.04 MPa and 40–70 °C. The lowest total solubility of the ethyl ester mixture was obtained when using supercritical CO2 at the lowest density (the lowest pressure and the highest temperature value tested). The highest discrimination against longchain PUFAs (e.g. EPA and DHA) was also obtained under these conditions. Conversely, higher solubility and lower selectivity were obtained when solvent density increased. Considering this inverse correlation between selectivity and solubility, a single-step batch-fractionation process was designed to increase the DHA ethyl ester content from an initial value of 17.5% in the starting material to 55% in the final extract. Davarnejad et al. (2008) examined the solubility of fish oil in supercritical CO2 at temperatures of 40, 50, 60, and 70 °C and pressures of 13.6, 20.4, and 27.2 MPa. The fractionated fish oil samples collected were then esterified using methanol with sodium methoxide catalyst. The samples were analyzed by GC to determine the amount of four fatty acid methyl ester (FAME) components extracted, namely, methyl palmitate, methyl oleate, methyl EPA (5,8,11,14,17-eicosapentanoate), and methyl DHA (4,7,10,13,16,19docosahexenoate). The results showed that the highest solubility of the fish oil (0.921 g of oil in 100 g of CO2) was obtained at optimum conditions of 40 °C and 27.2 MPa. The solubility of fish oil in supercritical CO2 was found to be higher at lower temperature and at lower fractionation time. Furthermore, the average yield obtained for the combined total of the four FAME components was 66%, with methyl palmitate having the highest at 30.5% under extraction conditions of 50 °C and 13.6 MPa whereas methyl EPA has the lowest at 3.24%. Létisse et al. (2006), and Létisse and Comeau (2008) evaluated the enrichment of EPA and DHA from sardine heads, a waste product from the fish canning industry, via supercritical fluid extraction. These studies were done on a laboratory scale. Various parameters, such as pressure, temperature, CO2 rate and time were optimized in order to obtain the highest yield of extracted oil with the highest amount of EPA and DHA in the extraction product. In the first approach, the oil yield was measured. Then, a quadratic model with three variables was employed to maximize the EPA and the DHA concentrations. A multicriteria optimization, using the desirability function, was performed to determine the best level for each parameter. Pressure, temperature and CO2 rate were, respectively, set at 300 bar, 75 °C and © Woodhead Publishing Limited, 2010

492 Separation, extraction and concentration processes 2.5 ml min–1 during the 45 min extraction. A yield of 10.36% of extracted oil (compared with the dry material) was achieved with an amount of 10.9% of EPA and 13.0% of DHA (compared with all fatty acids of the extract). These yields were lower than with a traditional solvent extraction. However, the advantages of supercritical fluid extraction were shorter extraction time, prevention of heating, and better organoleptic properties by excluding the use of toxic organic solvents. Catchpole et al. (2000) reported the countercurrent extraction and fractionation of a range of crude fish oils using supercritical CO2 and CO2– ethanol mixtures. Vitamin A palmitate was extracted from model mixtures of cod liver oil and vitamins using pure CO2. The separation factor was low, owing to similar solubilities of the vitamin ester and the oil. Vitamin A was also recovered from cod liver oil ethyl esters–vitamin A mixtures. The separation factor was substantially improved over the non-esterified oil, owing to large differences in the solubilities of the esters and vitamin A in supercritical CO2. Solubilities of fish oils and squalene are reported using CO2–ethanol mixtures at 333 K, ethanol concentrations of 0 to 12% by mass, and pressures of 200–300 bar. Solubilities of all oils and squalene increased exponentially with linear increases in the ethanol concentration. The solubility of polar components increased more rapidly than non-polar components. Pilot-scale removal of fatty acids from Orange Roughy oil and squalene from deep sea shark liver oil was carried out using CO2–ethanol mixture as the solvent. The extent of fatty acid removal from Orange Roughy oil was higher than with pure CO2, whereas the degree of separation of squalene from shark liver oil was lower. However, throughput was substantially increased relative to pure CO2 in both cases. Temelli et al. (1995) optimized the supercritical CO2 extraction temperature and pressure for oil removal from freeze-dried, fall Atlantic mackerel. The effect of extraction conditions on pH and water-binding potential of the protein residue was evaluated. For the temperature range (35–55 °C) and pressure interval (20.7–34.5 MPa), a combination of 34.5 MPa and 35 °C gave the highest oil yield and concentration of omega-3 fatty acids.

17.6 Distillation methods for the concentration and purification of omega-3 fatty acids Fractional distillation is another simple process for separation of mixtures of omega-3 fatty acids under ultralow pressure. This method takes advantage of differences in the boiling point and molecular weight of fatty acids under the conditions of high temperature (180–250 °C) and reduced pressure (0.1 to 1.00 mm Hg). Fatty acid enrichment is achieved by exploiting the differences in vapour pressure through countercurrent contacting of vapour



and liquid phases in stages using plates or continuously using random or structured packing. Molecular distillation, often also called ‘short path distillation’, may use moderately lower temperatures and short heating intervals. Molecular distillation provides high selectivity, but as a result of its relatively high operating temperatures and reduced pressures, it leads to high operating costs, possible thermal degradation of omega-3 fatty acids and safety concerns. Several advantages and disadvantages of molecular distillation are reported in Table 17.3. Molecular distillation can be used for the concentration of omega-3 fatty acids from fish oils. The content of omega-3 fatty acids in fish oils mainly depends on the fish species itself, their habitat and the season. A typical base material for commercial production of omega-3 fatty acid concentrates is anchovy or sardine oil with a content of approximately 18% EPA and 12% DHA. Tuna oil is also used for the large-scale production of DHA concentrates, as the crude oil contains up to 25% DHA. In general, the concentration of omega-3 fatty acids in the crude oil should not be less than 30% in total. The state-of-the-art method for the concentration of omega-3 fatty acids from fish oil by molecular distillation is by the fractionation of their ethyl esters (Fig. 17.1). The latter can be obtained by interesterification of fish oil with ethyl alcohol. The equipment used in this process consists of two stills (two-stage molecular distillation) with a degasser. The product distillation requires a minimum of three passes through the system. The first, degassing pass, removes any moisture left after interesterification. The residue from this pass is then used as the feed for the second, ‘lights’ removal, pass. The second pass concentrates the DHA and EPA by separating and removing the ‘lights’ from the feed. The second pass removes 20–50% of C10–C18 fatty acid esters. The residue from this pass is then used as the feed for the third, ‘product distillation’, pass. The third pass further concentrates the DHA and EPA (up to 40–80%) by separating the heavier fraction from the feedstock. The heavy fraction (5–10%) consists of longer chain fatty acids, including fatty acids chain length >C24 as residue. Operating pressures and temperatures required are 0.005–0.01 torr and 170–190 °C, respectively. The recoveries can be up to 70% at DHA and EPA concentrations of 55–65 wt%. Table 17.3 Advantages and disadvantages of molecular distillation Advantages

Disadvantages

Stability: the vacuum allows oils to be processed at minimal temperatures, reducing the risk of thermal decomposition and oxidation of PUFAs

Cost: cost is relatively high

Purity: separating the oil components by weight allows contaminants to be reduced far below industry specifications

Natural form: the starting natural triglyceride form is lost in the process

Concentration: weight grouping allows the processor to concentrate specific fatty acids


494 Separation, extraction and concentration processes Crude fish oil

Refining, bleaching and deodorization

Refined fish oil

Interesterification

Fish oil ethyl esters

Molecular distillation

Purified omega-3 fatty acid esters

Fig. 17.1 Purification of crude fish oil into highly concentrated omega-3 fish oil esters.

The DHA to EPA ratio primarily depends on their content in the base material and on the degree of concentration, as EPA ethyl ester, the more volatile component, also accumulates in the mid-chain-length fatty acid ester fraction. Reprocessing of the latter fraction by molecular distillation increases the recovery of omega-3 fatty acids. Stout et al. (1990) pointed out the practical difficulty of purifying omega-3 fatty acids from menhaden oil via molecular distillation in the natural TAG form. The distillation of menhaden oil, in its natural TAG form, concentrated only EPA from an initial value of 16.0% to 19.5%. Hence, this study did not result in a significant improvement in the concentration of omega-3 fatty acids, as they are more or less uniformly distributed in the TAGs. Furthermore, distillation of triglycerides require high temperatures, risking thermal decomposition of unsaturated fatty acids, and a very low operating pressure, which results in an inefficiently low specific feed rate. However, molecular distillation of its ethyl esters increased the EPA content from 15.9% to 28.4% (Stout et al., 1990). The degree of concentration of DHA was even more remarkable. Whereas DHA doubled from 8.4% to 17.3% in the TAG form, in the alkyl ester form it increased from 9.0% to 43.9% (Stout et al. 1990). Although the greater volatility of the fatty acid esters allows the use of lower temperatures (compared with process temperatures required for FFA fractionation), temperatures are still moderately high, and exposure to distillation conditions over a prolonged period of time can be © Woodhead Publishing Limited, 2010


detrimental to the omega-3 fatty acid constituents, causing polymerization, hydrolysis, isomerization and thermal degradation. Methyl esters of Atlantic herring oil containing 62% monoethylenic fatty acids were subjected to batch fractional distillation, under vacuum on a pilot-plant scale, to study the feasibility of fractionating fatty acid esters of marine oils of low iodine value into monounsaturated fractions with increased commercial value for industrial chemical uses (Ackman et al., 1973). A total of 64 methyl ester fractions were collected and recoveries of the major saturated and monounsaturated acids were close to 100%, and some fractions contained over 90% of the desired 22:1 long chain monounsaturated acids. The short-chain polyunsaturated acids were recovered in high yields, but the long-chain highly unsaturated acids were recovered in yields of 60% or less owing to oxidative and thermal decomposition in the particular apparatus employed. If small amounts of unsaturated acids are acceptable, fractional distillation of low iodine value marine oils could inexpensively supply fractions with high concentrations of methyl esters of longer chain (C20 and C22) monounsaturated and shorter chain (C14) saturated acid or (C16) saturated–monounsaturated acid mixture. Liang and Hwang (2000) employed short-path distillation to fractionate EPA and DHA ethyl esters from squid visceral oil. The elimination temperatures of squid visceral oil ethyl esters (SVOEE) ranged from 50 to 140 °C, increasing with the carbon number of ethyl esters. The elimination temperature of cholesterol was higher than those of SVOEE. When SVOEE originally containing 9.0% EPA, 14.7% DHA, and 11.21 mg g–1 of cholesterol was distilled from 50 to 150 °C, the 130 °C distillate contained 15.5% EPA and 34.7% DHA with 0.99 mg g–1 of cholesterol, and the yield was 21.8%. The 150 °C distillate had 43.1% DHA with 4.96 mg g–1 of cholesterol. Furthermore, the distillates collected from 110 to 150 °C contained 24.4 to 50.2% of EPA plus DHA, and their total yield was 58.3%.

17.7 Enzymatic methods for the concentration and purification of omega-3 fatty acids For the concentration of omega-3 fatty acids on a large scale, each of the above physical and chemical separation methods have some drawbacks either in terms of low yield, a requirement for large volumes of solvent or sophisticated equipment, a risk of structural changes in the fatty acid products, or high operational costs (Senanayake, 2000). Lipases work under mild conditions of temperature and pH (Gandhi, 1997), a factor which favors their potential use for the enrichment of omega-3 fatty acids in oils. Lipases are enzymes that catalyze the hydrolysis, esterification, interesterification, acidolysis and alcoholysis reactions (Senanayake and Shahidi, 2000b; Shahidi and Senanayake, 2006; Shimada et al., 2006; Weete et al., 2008). The common


496 Separation, extraction and concentration processes feature among lipases is that they are activated by an interface. Lipases have been used for many years to modify the structure and composition of foods. Lipases which act on neutral lipids generally hydrolyze the esters of PUFAs at a slower rate than those of more saturated fatty acids (Villeneuve and Foglia, 1997). Use has been made of this relative substrate specificity to increase the concentration of omega-3 PUFAs in seal blubber and menhaden oils by subjecting them to hydrolysis by a number of microbial lipases (Wanasundara and Shahidi, 1998). Concentration of omega-3 fatty acids by enzyme-assisted reactions involves benign reaction conditions and provides an alternative to the traditional concentration methods such as distillation and chromatographic separation (Shahidi and Senanayake, 2006). Furthermore, concentration via enzymatic means may also produce omega-3 fatty acids in the acylglycerol form, which is nutritionally preferred. Studies over the past two decades have used microbial lipases to produce purified omega-3 fatty acids via hydrolysis, esterification or transesterification of marine oils. Tanaka et al. (1992) used six types of microbial lipases (lipases derived from Aspergillus niger, Candida cylindracea, Chromobacterium viscosum, Rhizopus javanicus, Rhizopus delemer and Pseudomonas sp.) to hydrolyze tuna oil and found that the lipase from Candida cylindracea was the most effective one in increasing the DHA content in the non-hydrolyzed fraction of the oil. The DHA content in the non-hydrolyzed fraction was increased three-fold compared with the original oil; however, other lipases were not very effective. Various microbial lipases were evaluated for the enrichment of omega-3 fatty acids from cod liver and sardine oils by selective hydrolysis (Hoshino et al., 1990). The best hydrolysis results were obtained for the lipases from Candida cylindracea and Aspergillus niger, but none of the lipases were able to increase the EPA content of the oil considerably. However, over 50% of the total omega-3 fatty acids were produced when these two lipases were used. A Japanese patent (Noguchi and Hibino, 1984) describes a method based on the discrimination of lipases on DHA and EPA for preparation of omega-3 fatty acids. Ethyl esters from sardine and mackerel were hydrolyzed with several lipases derived from Candida cylindracea, Mucor miehei and Aspergillus niger. Selective hydrolysis afforded ethyl ester concentrates of up to 17% DHA and 25% EPA after separation of the hydrolyzed fatty acids. Transesterification of various fish oil TAGs with a stoichiometric amount of ethanol catalyzed by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation of EPA and DHA (Haraldsson and Kristinsson, 1998). When FFAs from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. When tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained. The residual glyceride mixture contained 49% DHA and 6% EPA, with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil FFAs were directly esterified with ethanol, 68% conversion was obtained.



The residual FFAs comprised 74% DHA and only 3% EPA. The recovery of both DHA into the residual FFA fraction and EPA into the ethyl ester product was reported to be very high at 83 and 87%, respectively (Haraldsson and Kristinsson, 1998). Schmitt-Rozieres et al. (2000) studied the enrichment of PUFAs from sardine cannery effluents via enzymatic selective esterification. The sardine canning industry produces vast quantities of effluents that need expensive reprocessing. Their oily component contains EPA and DHA up to 10% each. The author’s goal was to develop a process allowing the recovery of these fatty acids. After the removal of solid particles, proteins, and peptides from the crude effluent, the resultant oil was hydrolyzed and EPA and DHA were enriched by selective enzymatic esterification. Using Lipozyme™, DHA was enriched up to 80%, but no enrichment was observed for EPA. By immobilizing Candida rugosa lipase on Amberlite IRC50 cation-exchange resin, a 30% EPA enrichment was obtained. Shimada et al. (2001) also attempted to purify PUFAs by taking advantage of the enzyme-catalyzed reactions. When FFAs originating from PUFA-containing oil were selectively esterified with lauryl alcohol (LauOH) using a lipase acting on the desired PUFAs very weakly, the PUFA was efficiently enriched in the FFA fraction. In addition, when selective alcoholysis of ethyl esters originating from PUFA-containing oil with LauOH was carried out, the PUFA ethyl ester (EtPUFAs) was enriched to a desired purity in the unreacted ethyl ester fraction. These reaction mixtures contain LauOH, PUFA (EtPUFAs), and lauryl esters, and their molecular weights are different from one another. Hence, PUFA or EtPUFAs can be easily separated by conventional distillation. Selective esterification increased the purity of DHA, GLA, and arachidonic acid (ARA; 20:4n-6) to 91, 98, and 96 wt%, respectively. Selective alcoholysis was also effective for increasing the purity of ethyl docosahexaenoate to 90 wt%. Kojima et al. (2006) studied the enzymatic acidolysis and acylglycerol synthesis using PUFAs with lipases from Pseudomonas fluorescens HU380 (HU-lipase), P. fluorescens AK102 (AK-lipase), and Candida rugosa (CR-lipase). The acidolysis of triolein with EPA or DHA in n-hexane was evaluated with lipases immobilized on Celite 545. HU-lipase showed the highest incorporation rate at a low temperature (10 °C) with either EPA or DHA as the acyl donor, and the rate decreased with increasing reaction temperature. At 45 °C, the rates for EPA and DHA were 7.1 and 0.5 relative to those at 10 °C, respectively. The EPA incorporation rate was even higher at a low temperature (10 °C), and the DHA incorporation rate increased with decreasing temperature. Although AK-lipase showed the reverse tendency for incorporation rate, the DHA incorporation rate increased with increasing reaction temperature with both PUFAs. HU-lipase reacted well with PUFAs such as DHA, EPA, ARA, mead acid (MA), and dihomo-g-linolenic acid (DGLA) on acidolysis and glyceride synthesis. The reactivities of AK-lipase toward these PUFAs except for DGLA, i.e. MA, ARA, EPA, and DHA, were low for both reactions.


498 Separation, extraction and concentration processes Halldorsson et al. (2003) investigated the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their fatty acid selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40 °C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the fatty acids, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. When FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained. The residual FFA contained 78% DHA and only 3% EPA, with 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration of EPA and DHA.

17.8 Integrated methods for the concentration and purification of omega-3 fatty acids Most of the separation methods described above, when used alone, can only concentrate and purify omega-3 fatty acids to a limited extent. Therefore, two or more procedures are often required to produce highly purified omega-3 fatty acids. A simple and inexpensive method involving saponification of wet biomass, followed by transmethylation, winterization and urea adduction in a sequential manner has been recently developed for concentration of DHA from Crypthecodinium cohnii biomass (Mendes et al., 2007) (Fig. 17.2). The algal biomass grown in shake flasks is concentrated by centrifugation and the wet concentrate is kept frozen until needed. Fatty acids are extracted by direct saponification of wet biomass with KOH-ethanol. Before extracting unsaponifiable matter with hexane, water is added to shift the equilibrium distribution of unsaponifiable matter to the hexane phase. The hydroalcoholic phase, containing the soaps, is acidified with HCl and free fatty acids are extracted with hexane. The organic phase, containing FFAs, is dried with anhydrous sodium sulfate and the solvent is evaporated to recover FFAs. The FFAs are then methylated using transmethylation reagent and sulfuric acid. The mixture containing methyl esters are then winterized at –18 °C. Subsequently, the liquid fraction is separated from the winterized crystals. The liquid fraction is added to a urea-methanol saturated solution and the urea-fatty acids adducts are crystallized; the crystals are separated by centrifugation and the filtrate (non-urea complexing fraction), which contains DHA, is finally recovered. The highest DHA recovery (49.9%) was obtained at 24 °C at a urea/fatty acid ratio of 4.0, corresponding to 89.4% of DHA of the total fatty acids (Mendes et al., 2007).


Methods of concentration and purification of omega-3 fatty acids 499 Microalgal wet biomass Saponification Free fatty acids Transmethylation Fatty acid methyl esters (FAMEs) Winterization DHA methyl esters Urea adduction Highly purified DHA methyl esters

Fig. 17.2 Scheme for the DHA concentration and purification of microalgal biomass from Crypthecodinium cohnii.

Medina et al. (1995) described a two-step process, urea adduction followed by preparative high-performance liquid chromatography, for concentration of omega-3 fatty acids from the marine microalga, Isochrysis galbana. By the urea adduction method, a mixture that contained 94% (w/w) stearidonic (SA), EPA, and DHA acids (4:1 urea/fatty acid ratio and 4 °C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse-phase C18 columns. These isolations were scaled up to a semi-preparative column. A PUFA concentrate was isolated from I. galbana with methanol/water (80:20, w/w) or ethanol/ water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively. Guil-Guerrero and Belarbi (2001) also used an integrated method to purify DHA and EPA from cod liver oil. The process consisted of four main steps: (i) saponification of the oil, (ii) use of urea inclusion adducts method to obtain PUFAs, (iii) PUFA methylation, and (iv) silica gel column chromatography of the methylated PUFAs. Silica gel chromatography yielded highly pure DHA in the process (100% purity, 64% yield). For EPA, the recovery in the combined process was 29.6%, and the final purity was 90.6%, owing to the simultaneous elution of other PUFA esters. The recovery in the urea adduction method was strongly enhanced by application of orbital agitation during the crystallization process, in which EPA yield increased from 60–70% without agitation to 90–97% at 800 rpm; stearidonic acid (18:4n−3) yield ranged from 60–75% without agitation to 87–95% at 800 rpm, and DHA yield varied from 53–73% without agitation to 85–99% at 800 rpm. Chakraborty and Paulraj (2008) used enzymatic hydrolysis followed © Woodhead Publishing Limited, 2010

500 Separation, extraction and concentration processes by urea adduction to purify EPA and ARA from sardine oil. The enzyme used for the hydrolysis of sardine oil was the lipase derived from Bacillus licheniformis MTCC 6824. The enzyme exhibited more hydrolytic resistance toward the ester bonds of EPA and ARA than those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 °C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued. Lipasecatalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided FFAs containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea adduction proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil. Yuzo et al. (2006) employed enzymatic hydrolysis, using lipase from Pseudomonas fluorescens strain HU380, and urea adduction to concentrate EPA and DHA from refined cod oil. The starting oil had 12.2% EPA and 6.9% DHA. Lipase-catalyzed hydrolysis followed by urea adduction provided FFAs with 43.1% EPA and 7% DHA. The resulting yield of concentrated total fatty acids comprised 2.6% of the fatty acids from the cod oil. Thus, EPA was particularly concentrated in the fatty acids derived from refined cod oil as a result of enzymatic hydrolysis followed by urea adduction. On the other hand, hydrolysis of cuttlefish oil with AK-lipase (lipase from Pseudomonas fluorescens strain AK102), followed by urea adduction increased the EPA content from 14.2 to 16.8%, and DHA content from 16.3 to 44.6%. The yield of purified total fatty acids by urea concentrate was 9.4% of the fatty acids from the cuttlefish oil. Thus, DHA was particularly concentrated in this study. A US patent (Zaks and Gross, 1999) disclosed an enzymatic process for preparing an oil-based product significantly enriched in omega-3 fatty acids. The process is a two-step procedure involving lipase-catalyzed transesterification of TAGs followed by low-temperature crystallization. The process yields a mixture of highly pure monoglycerides, at least 60% of which contain omega-3 fatty acids. The process can also be used to produce omega-3 enriched TAG products. Enzymatic methods of enrichment of omega-3 fatty acids have also been investigated under supercritical conditions. Lin et al. (2006) examined the enrichment of omega-3 fatty acids in TAGs of menhaden oil by lipasecatalyzed trans-esterification under supercritical CO2. Before the reaction, menhaden oil was treated by urea adduction to produce 80.1% of omega-3 PUFAs containing 29.4% EPA and 41.8% DHA. Using the sn-1,3-specific lipase from Mucor miehei, the effect of various operating parameters on the reaction was studied, including co-solvent concentration, reaction temperature,



time, pressure, and substrate ratio (free omega-3 fatty acids: TAGs). Water and ethanol were examined as co-solvents, and both fluids exhibited a maximum for omega-3 fatty acid content in TAGs as a function of concentration. Pressure up to 103.4 bar had a significant positive effect on the conversion of omega-3 PUFA onto TAGs. By further increasing pressure, the conversion rate decreased owing to the transformation of the spatial structure of lipase that leads to deactivation. The enzyme exhibited a good performance and stability in the region of 323 K. The optimal substrate molar ratio of TAG to omega-3 PUFA is about 1:4 taking into consideration the TAG inhibition. The conversion in supercritical CO2 appeared 40% higher than in n-hexane at ambient pressure after 5h. In another study, Lin and Chen (2008) used esterification of free omega-3 fatty acids with enriched omega-6 TAGs to produce a desired structured lipid with an omega-3/omega-6 ratio of 4 by using lipases under supercritical CO2. Comparing four different types of lipases, sn-1,3-specific lipase from Mucor miehei had the highest degree of incorporation under 10 wt% loading amount of total substrates. The optimal operating parameters under 10.2 MPa and 323.15 K supercritical CO2 could attain the desired omega-3/ omega-6 ratio in 6 h. Because of the negative effect on the enzyme activity by the enriched omega-6 TAGs, the optimal substrate ratio of the enriched omega-6 TAGs and the omega-3 fatty acids was chosen as 1/4. To enhance the solubility of omega-3 fatty acids in supercritical CO2, ethanol was applied as a co-solvent and reached an optimal input at 10% of total substrates. The enzyme maintained 81% of its initial activity because of moisture removal from the surface of the enzyme after seven cyclic pressurization/ depressurizations.

17.9 Conclusions Production of purified omega-3 fatty acids from natural source materials may be achieved via a number of techniques, namely urea adduction, chromatography, low-temperature crystallization, supercritical fluid extraction, enzymatic splitting, molecular distillation, as well as a combination of any of the above methods. Purified omega-3 fatty acid products thus produced may be in the form of free fatty acids, alkyl esters or acylglycerols. Each method had its own benefits and drawbacks. Of the methods described above, molecular distillation is highly energy consuming and results in a significant distraction of labile highly unsaturated fatty acids. Other methods, which require the use of organic solvents, include low-temperature crystallization, chromatography and supercritical CO2 extraction, among others. These processes have a number of drawbacks. For example, low-temperature crystallization of acylglycerols typically results in only a small omega-3 enrichment of the product, and chromatography and supercritical fluid extraction are expensive and difficult to scale up. Owing to the potential benefits of having the concentrates in the © Woodhead Publishing Limited, 2010

502 Separation, extraction and concentration processes acylglycerol form, enzymatic procedures have become popular. However, there are several limitations to the enzymatic processes as well. These include the necessity for a complex separation of the product from free saturated fatty acids, use of complex multi-enzymatic systems and the low efficiency that results in an insufficient degree of enrichment. Urea adduction has been very successful in enriching omega-3 fatty acids on an industrial scale and the products thus formed are in the free fatty acid or simple ester forms. Efficient and cost-effective methods of enriching the level of omega-3 fatty acids will continue to be needed in order to reduce the cost and to meet the future demand for highly purified omega-3 products.

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18 Extraction of natural antioxidants from plant foods E. Conde, A. Moure, H. Domínguez and J. C. Parajó, University of Vigo, Spain

Abstract: An overview of the most studied vegetal sources of compounds with antioxidant properties and their biological action is firstly presented. The conventional and alternative extraction processes are discussed, special emphasis is given to those using biorenewable, environmentally friendly solvents. Selected examples of integrated processes for extraction, concentration and purification of extracts are included. Key words: antioxidants, plants, extraction, purification.

18.1 Introduction The utilization of phytochemicals in the food and cosmetic industries has attracted public and scientific interest, because of their perceived efficiency, low cost and lack of toxicity. The number of recent publications and patents on antioxidants has increased considerably. The demand for natural antioxidants, which are presumed to be safer, has risen owing to concerns about the long-term safety and negative consumer perception of some synthetic antioxidants. Many natural compounds show antioxidant activity and may act as flavorings, colorants, preservatives and reinforcers of endogenous antioxidant systems (Astley, 2003; Valenzuela et al., 2003; Halliwell, 2002; Halliwell and Gutteridge, 1999). There is epidemiological and clinical evidence relating antioxidant-rich diets with a decreased risk of degenerative diseases, reduced morbidity and mortality. However, the safety limits of many natural antioxidants (which can consist of a mixture of several active substances) are unknown, and their lack of toxicity should be confirmed (Galati and


Extraction of natural antioxidants from plant foods 507 O´Brien, 2004; Pokorny, 2007). The biological activity and bioavailability of natural antioxidants are the subject of much research. Edible vegetals and agroindustrial residues are considered to be abundant and promising sources of natural antioxidants. The extraction method, of crucial importance for both technical and economic reasons, ideally should be non-destructive, time efficient and suitable for producing high quantities of extracts, which should be processed by selective techniques to yield concentrates of enhanced antioxidant capacity. This chapter presents an overview of the most studied vegetal sources of antioxidants and extraction processes (in particular those using biorenewable, environmentally friendly solvents). Selected examples of integrated processes for extraction, concentration and purification of extracts are also included.

18.2 Antioxidant activity in food systems According to a classic definition, an antioxidant is any substance that, when present at low concentrations compared with those of an oxidizable substrate (such as lipids, proteins, DNA or carbohydrates), significantly delays or prevents oxidation of that substrate (Halliwell, 2002; Halliwell and Gutteridge, 1999). In food systems, the term antioxidant is used to designate the inhibitors of lipid peroxidation, whereas in biological systems it usually refers to protection of lipids, proteins and DNA against oxidative damage by processes or reactions involving reactive oxygen and nitrogen species (ROS and RNS, respectively). Antioxidant activity and antioxidant capacity, although often used interchangeably, have different meanings. Activity refers to the rate constant of the reaction between the antioxidant and the oxidant species, and capacity refers to the amount (in moles) of a given free radical scavenged by a sample. The basic mechanism of lipid peroxidation (Frankel, 2005; Laguerre et al., 2007; Pokorny, 1991) is presented in Fig. 18.1. Lipid peroxidation is induced by oxygen in the presence of initiators such as heat, free radicals, light, photosensitizing pigments and metal ions. Once these free radicals are formed, lipid peroxidation progresses at a high rate by a radical chain reaction, and oxidation ends by producing secondary non-radical compounds. In this scheme, antioxidants can act at different levels: ∑ ∑ ∑ ∑ ∑

scavenging species responsible for oxidation initiation or preventive antioxidants; interruptors of the propagation of the autoxidation chain reaction or chain-breaking antioxidants; singlet oxygen quenchers; synergists or compounds increasing the activity of chain-breaking antioxidants in a mixture; reducing agents;


508 Separation, extraction and concentration processes Initiation Formation of radicals: peroxyl (RO2•), alkoxy (RO•) or alkyl (R•) Propagation R• + O2 Æ RO2• RO2• + RH Æ ROOH + R• RO• + RH Æ ROH + R•

ROOH Æ RO• + •OH 2 ROOH Æ RO2• + RO• + H2O Termination 2 R• R• + RO2• Æ Stable products 2 RO2•

Fig. 18.1 Mechanism of lipid oxidation.

∑ ∑

metal chelators, which stabilize metal pro-oxidants (iron or copper cations); inhibitors of specific oxidative enzymes (especially lipoxygenases).

Antioxidants often act by several mixed and co-operative mechanisms. An antioxidant can behave as a pro-oxidant depending on the structure, chemical environment and operational conditions: for example, typical antioxidants such as flavonoids, a-tocopherol or ascorbic acid can act as pro-oxidants in the presence of transition metal ions, whereas carotenoids show pro-oxidant activity at high oxygen pressure (Galati and O’Brien, 2004). Table 18.1 lists representative in vitro tests employed in the assessment of antioxidant properties. The need for developing simple and reliable in vitro analytical antioxidant tests is widely acknowledged, and the convenience of a common standard has been claimed. A mixture of caffeic acid, catechin and epigallocatechin3-gallate, hesperetin, and morin was proposed for this purpose (Luthria and Vinyard, 2008). The problems associated with the determination of antioxidant activity were summarized in several critical reviews (Andrade et al., 2008; Becker et al., 2004; Decker et al., 2005; Fernandez-Panchón et al., 2008; Frankel and Finley, 2008; Gordon, 2001; Halliwell, 2002; Karadag et al., 2009; Kiokias et al., 2008; Magalhães et al., 2008; Moon and Shibamoto, 2009; McDonald-Wicks et al., 2006; Prior et al., 2005; Roginsky and Lissi, 2005; Laguerre et al., 2007; Sánchez-Moreno, 2002; Singh and Singh, 2008; Strube et al., 1997; Verhagen et al., 2003). The antioxidant activity depends on a number of factors, including type of substrate, medium, and initiators; oxidation conditions, partitioning properties of the antioxidant between lipid and aqueous phases, and the experimental method used (Antolovich et al., 2002; Decker et al., 2005; Frankel and Meyer, 2000; Gordon, 2001; Yanishlieva, 2001). Pitfalls of in vitro antioxidant research include the low stability of antioxidants, which can lead to formation and accumulation of oxidation products (Haenen et al., 2006; Knasmüller et al., 2008). © Woodhead Publishing Limited, 2010

Table 18.1 In vitro methods usually employed for testing the antioxidant activity of pure compounds and extracts. The activities considered include antiradical, anti-lipoperoxidation, metal chelation and reducing capacity Antioxidant activity assay

Substrate/initiator



bC (b-carotene bleaching)

ROO˙


CAT (conjugated autoxidizable triene)

ROO˙

Chain-breaking

Crocin bleaching

ROO˙


CUPRAC (cupric ion reducing antioxidant activity)

Cu2+

Reducing capacity 2+

Deoxyguanosine oxidation

HO˙, H2O2, Fe

Deoxyribose oxidation

HO˙, H2O2, Fe2+

Scavenging, chain-breaking, chelating Scavenging, chain-breaking, chelating

DMDP

DMDP˙+ (N,N-dimethyl-pphenylenediamine)


DPPH

DPPH˙ (a,a-diphenyl-bpicrylhydrazyl)


FIC (ferrous ion-chelating assay)

Fe2+, Cu2+

Chelating

FRAP (ferric-reducing antioxidant power)

Fe3+-TPTZ

Reducing capacity, indirect competition method of chain lipid peroxidation (chain-breaking)

FTC (antioxidant activity in the linoleic acid system with ROO˙ ferrothiocyanate reagent)


HOSC (hydroxyl radical scavenging capacity)


HO˙

HPS (hydrogen peroxide scavenging)

H2O 2


Hypochlorous acid scavenging

HOCl



ABTS or TEAC (Trolox equivalent antioxidant capacity) ABTS·+ [2, 2¢-azinobis(3ethylbenzothiozoline-6sulfonate)]

Mechanism

Antioxidant activity assay

Substrate/initiator

Mechanism

Liposome oxidation

ROO˙, Fe2+

Scavenging, chain-breaking, chelating

CD (conjugated dienes)

LDL

Oxidation by copper, aldehydes formation


In vitro LDL oxidizability

LDL

Oxidation by copper, conjugated dienes determination

Iodometric assay of lipid peroxides

LDL

Iodide oxidation by lipid peroxides

TBARS (thiobarbituric acid-reacting substances)

LDL, ROO˙

Adduct malondialdehyde–thiobarbituric acid (MDA– TBA) formation, scavenging, chain-breaking

ORAC (oxygen-radical absorbance capacity)

Oxygen radical, ROO˙


Peroxyl radical scavenging

ROO˙


Peroxynitrite scavenging

ONOO–


2 –

PHOTOCHEM (photochemiluminescence)

O˙


PM (phosphomolybdenum method)

Mo6+

Reducing capacity

Reduction of the Fremy’s radical

Fremy’s (potassium nitrosodisulfonate)


RP (reducing power)

Fe3+

Reducing capacity

SRS (superoxide radical scavenging)

O 2˙ –


TAR (total antioxidant reactivity)

ROO˙


TOSC (total oxidant scavenging capacity)

ROO˙, O2˙–, HO˙, HOCl, LO(O) , ONOO–, 1O2


TRAA (a-tocopheroxyl radical attenuating ability)

a-Tocopheroxyl

Scavenging

TRAP (total radical-trapping antioxidant parameter)

ROO˙


Trichloromethyl peroxyl scavenging

CCl3O2˙

Scavenging



Extraction of natural antioxidants from plant foods 511 The need to use a variety of antioxidant activity assays is generally agreed, as the results obtained in individual tests can be contradictory. Some recommendations on the in vitro determination of antioxidant activity include: identification and determination of active compounds; evaluation of protection against oxidation in foods or physiological model systems under conditions resembling the real chemical, physical, and environmental conditions in the systems to be protected; detailed understanding of the oxidation mechanisms; measurement of both initial and secondary products, and utilization of reagent concentrations that are physiologically relevant. Low oxidation levels should also be determined, and possible mechanism modification under accelerated oxidation conditions should be considered (Antolovich et al., 2002; Choe and Ming, 2006; Collins, 2005; Decker et al., 2005; Frankel, 1993; Frankel and Meyer, 2000; Frankel and Finley, 2008; Gordon, 2001; Prior et al. 2005; Huang et al., 2005; Schlesier et al., 2002). Several classifications of antioxidants assays have been proposed, based on mechanisms such as the ability to quench free radicals by hydrogen donation, the ability for electron transfer, or a combination of both (Huang et al., 2005; Prior et al. 2005; MacDonald-Wicks et al., 2006). Results from simple in vitro experiments are difficult to extrapolate to the heterogeneous conditions of multifaceted in vivo systems (Haenen et al., 2006), where the possible action on the target tissues is affected by absorption, distribution, metabolism and excretion. The animal model used and a battery of well-validated tests to assess the broad diversity of oxidative damage and antioxidative defence parameters, are crucial for antioxidant research in vivo. Measurement of total antioxidant capacity was proposed as an integrated parameter to consider the balanced action of all antioxidants and their synergistic interactions in plasma and body fluids (Aruoma, 2003; Ghiselli et al., 2000; Halliwell, 2009; Sies, 2007). However, pitfalls and serious limits of applicability of this concept have been manifested (Frankel and Finley, 2008; Sies, 2007) as well as the strong dependency on the analytical method used (Fernández-Panchón et al., 2008).

18.3 Natural compounds with antioxidant activity and major sources 18.3.1 Natural compounds Natural antioxidants are synthesized by plants, micro-organisms, fungi, and animals. The most important groups of natural antioxidants are listed below. Phenolics refer to monomeric, oligomeric or polymeric compounds with an aromatic ring bearing one or more hydroxyl substituents and functional derivatives, such as esters, methyl ethers and glycosides. Phenolics (including


512 Separation, extraction and concentration processes simple phenols, coumarins, flavonoids, stilbenes, lignans, hydrolyzable and condensed tannins, and phlorotannins), have powerful antioxidant activities in vitro (von Gadow et al., 1997; Miller and Ruiz-Larrea, 2002; Tabart et al., 2009) and play a role in signals between plants, plant defense against predation (by micro-organisms, insects and herbivores) or in response to environmental stress (such as air pollution, heavy metal ions and UV-B radiation). Simple phenolics include hydroxybenzoic and hydroxycinnamic acids. The main subclasses of flavonoids are anthocyanins, flavanols, flavanones, flavonols, flavones and isoflavones, and anthocyanidins and their glycosides. Tannins are polyphenolic compounds with varying molecular masses. Plant tannins include hydrolyzable tannins (gallotannins, which yield glucose and gallic acid upon hydrolysis, or ellagitannins, which produce ellagic acid), and condensed tannins (proanthocyanidins). Algae contain phlorotannins, formed by the polymerization of phloroglucinol (Singh and Bharate, 2006). Table 18.2 summarizes the phenolic composition of some plants. Terpenes are a large and diverse class of lipophilic secondary plant metabolites made up of isoprene units, and are classified into hemi-, mono(C10), sesqui- (C15), di- (C20), sester- (C25), tri- (C30), and tetraterpenoids (carotenoids), having eight isoprenoid C 5 residues. Monoterpenes, sesquiterpenes and diterpenes are the main components of essential oils, which also contain oxygenated derivatives and other compounds (including aldehydes, ketones, phenolic, acetates and oxides). Antioxidant potential has been reported for terpenes (Escuder et al., 2002; Grassmann, 2005; Grassmann et al., 2002) as well as synergistic effects with phenolics (Milde et al., 2004). Carotenoids (carotenes and xanthophylls) have received attention because of their provitamin and antioxidant roles (Bohm et al., 2002). Vitamin E includes a family of tocopherols and tocotrienols and some of their ester derivatives. Tissues and seeds of plants are the major sources of vitamin E, which is associated with membrane lipids or appears in lipid storage stuctures. Vitamin E is the most important natural antioxidant in vegetable oil-derived foods from rice bran, palm, and wheat germ (Weber and Rimbach, 2002). The richest source is a by-product of soybean processing (‘oil deodorizer distillate’). Vitamin E effectively inhibits the peroxidation of lipids by peroxyl radical scavenging, and shows relevant activities related to the regulation of enzymes and gene expression (Brigelius-Flohé, 2009). Small proteins and peptides isolated from various protein hydrolyzates show antioxidant activity (Chen et al., 2006; Kitts, 2005; Kitts and Weiler, 2003). Their low-molecular-weight (low-MW) and simple structure, easy absorption, stability under different conditions and the lack of immunoreaction have been cited as advantages (Chen et al., 1996). Some peptides from hydrolyzed food proteins exert antioxidant activities against enzymatic and non-enzymatic peroxidation of lipids and essential fatty acids (Chen et al., 2006; Hook et al., 2001). Certain hydrophobic amino acids and their derivatives present high activity, and show synergistic effects with non-peptidic antioxidants


Extraction of natural antioxidants from plant foods 513 Table 18.2 Phenolic contents (mg g–1 dry weight) of some plants Plant (Latin name)


Achyrocline F satureioides Anethum graveolens F Averrhoa carambola L B Capparis spinosa L

CA, 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA pAA, Ap, BA, CA, C, EC, CGA, pCouA, PCys, GA, GeA, K, Lt, Myr, Q, SiA CGA, GA, Q CGA, GA CA, pCouA, FeA, Q, R, VA

TPC/TF

145/48** 78.3 30.1 15.9

Cassine orientalis

L

Castanea vulgaris

L

C, EC, EGC, PCyB2, GA, K, 24/24 Myr, Q Ap, FeA, GeA, Ng, Q, R 11.9

Casuarina equisetifolia Cinnamomun zeylanicum Coffea macrocarpa

B L B

CA, FeA, GA, Q K, Q, R CA, FeA, GA, Q

72.1 57.7 48.9

L

24/18

Diospyros neraudii

L

Eugenia tinifolia

L

Ficus microcarpa

B

Geranium purpureum Ilex paraguariensis

L

Monimiastrum acutisepalum Moringa oliefera

L L

C, EC, ECG, EGC, EGCG, PCyB1, PCyB2, GA, K, Q C, EC, ECG, EGCG, PCyB1, K, Q C, EC, EGC,EGCG, PCyB1, PCyB2, GA, Q, K Cat, Cou, pPGu, pVGu, OlA, pPPh, PCA, PR, Syr, Sy, V, iVA HBA, CA, C, EC, pCouA, GeA, Q, R, SyA, VA CA, 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA, 3FQA, 4FQA, 5FQA C, EC, ECG, EGC, PCyB1, GA,Q, K CGA, EA, FeA, GA, Q

Myonima obovata

L

Phytolacca americana Pimpinella anisum

L

L

S

C, ECG, EGC, PCyB1, PCyB2, K, Q HBA, CA, pCouA, FeA, R, VA 4CQA, 5CQA, 3,4dCQA, 3,5dCQA, 4,5dCQA, 3FQA, 4FQA, 5FQA

75/19 23a/17a 237/6.3*

Reference Marques and Farah, 2009 Shyu et al., 2009 Prakash et al., 2007b Proestos et al., 2006 Soobrattee et al., 2008 Proestos et al., 2006 Prakash et al., 2007b Prakash et al., 2007b Soobrattee et al., 2008 Soobrattee et al., 2008 Neergheen et al., 2006 Ao et al., 2008

28.2

Proestos et al., 2006 Marques and Farah, 2009

10a/9a

Neergheen et al., 2006 Prakash et al., 2007b Neergheen et al., 2006 Proestos et al., 2006 Marques and Farah, 2009

32.9 31a/32a 9.2


514 Separation, extraction and concentration processes Table 18.2 Continued Plant (Latin name)


Ricinus communis

L

EC, EA, GA, GeA, Q, R

Rubus ulmifolius Ruta graveolens

L

Salix aegyptiaca

C

CA, 4CQA, 5CQA, CGA, FeA, KGu, KCouGPy, KCGPy, QGu HBA, CA, C, GeA, FeA, Q, R CA, C, EGCG, pCouA, GA, R, V CA, C, EGCG, pCouA, GA, Myr, V CA, C, EGCG, pCouA, GA, Myr, Q, R, V CA, pCouA, GeA, Lt, Q, VA

L B Spartium junceum

F

Styrax officinalis

L

Syzygium glomeratum Juglans regia

L

Achillea millefolium

TPC/TF

L H L ST

Reference Singh et al., 2009 Dall’Acqua et al., 2008

4.3

Proestos et al., 2006 107/351** Enayat and Banerjee, 2009 64/165** 212/479** 4.8

HBA, CA, C, EC, CouA, GA, 18.4 GeA, FeA, Ng, Q, VA C, EC, ECG, EGCG, EGC, 84a/39a PCyB1, PCyB2, GA, Q, K 5CQA, 3CouQA, 4CouQA, CouA, QA, QGal, QP-d, QR, QX Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi Ap, Apg, CGA, Lt, Ltdg, Ltg, R, Vi

Proestos et al., 2006 Proestos et al., 2006 Neergheen et al., 2006 Pereira et al., 2007 Benetis et al., 2008

B, bark; C, catkins; F, flowers; H, herbs; L, leaves; S, seeds; ST, stems. a Composition of phenols (mg g–1 fresh weight) of some plants. TF, total flavonoid expressed as mg quercetin equivalent g–1 on a dry weight basis. * Total flavonoid expressed as mg rutin equivalent g–1 on a dry weight basis. ** Total flavonoid expressed as mg catechin equivalent g–1 on a dry weight basis. TPC: total phenolic content expressed as mg gallic acid equivalent g –1 on a dry weight basis. 3,4dCQA, 3,4-dicaffeoylquinic acid; 3,5dCQA, 3,5-dicaffeoylquinic acid; 3CouQA, 3-coumaroylquinic acid; 3FQA, 3-feruloylquinic acid; 4,5dCQA, 4,5-dicaffeoylquinic acid; 4CouQA, 4-coumaroylquinic acid; 4CQA, 4-caffeoylquinic acid; 4FQA, 4-feruloylquinic acid; 5CQA, 5-caffeoylquinic acid; 5FQA, 5-feruloylquinic acid; Ap, apigenin; Apg, apigenin-7-O-glycoside; BA, benzoic acid; C, catechin; CA, caffeic acid; Cat, catechol; CGA, chlorogenic acid; Cou, coumaran; EA, ellagic acid; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate; FeA, ferulic acid; GA, gallic acid; GeA, gentisic acid; HBA, p-hydroxybenzoic acid; iVA, isovanillic acid; K, kaempferol; KCouGPy, kaempferol-3-O-(6≤-p-coumaroyl)-b-d-glucopyranoside; KCGPy, kaempferol-3-O-(6≤-caffeoyl)-b-d-glucopyranoside; KGu, kaempferol-3-O-glucuronide; Lt, luteolin; Ltdg, luteolin-3¢,7-di-O-glycoside; Ltg, luteolin-7-O-glycoside; Myr, myricetin; Ng, naringenin; OlA, oleanolic acid; pAA, p-anisic acid; PCA, protocatechuic acid; pCouA, p-coumaric acid; PCyB1, procyanidin B1; PCyB2, procyanidin B2; PCys, proanthocyanidins; pPGu, p-propylguaiacol; pPPh, p-propylphenol; PR, 4-n-propylresorcinol; pVGu, p-vinylguaiacol; Q, quercetin; QA, quercetin 3-arabinoside; QGal, quercetin 3-galactoside; QGu, quercetin-3-O-glucuronide; QP-d, quercetin-3pentoside derivate; QR, quercetin 3-rhamnoside; QX, quercetin 3-xyloside; R, rutin; SiA, sinapic acid; Sy, syringol; SyA, syringic acid; Syr, syringaldehyde; V, vanillin; VA, vanillic acid; Vi, vicenin-2.


Extraction of natural antioxidants from plant foods 515 such as phenolic compounds (Erdmann et al., 2008). Antioxidant peptides have been obtained from soybean (Pyo and Lee, 2007), soy-fermented foods (Gibbs et al., 2004), soy protein fractions (Moure et al., 2006), barley hordein (Chiue et al., 1997), and potato (Wang and Xiong, 2005). Different hydrolysis conditions result in peptide mixtures with different properties (De Mejia and De Lumen, 2006). Food-derived bioactive peptides from animal or plant proteins have regulatory functions in humans after being released in vitro or in vivo in the gastrointestinal tract (De Mejia and De Lumen, 2006). A number of beneficial health effects have been claimed for food-derived products containing bioactive peptides, including blood pressure-lowering effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption and/or bioavailability, and cyto- or immunomodulatory effects. Maillard reaction products (MRP) such as Schiff bases, premelanoidins and melanoidins are formed by reactions involving the condensation of the carbonyl group of reducing sugars with the amino group of amino acids and proteins. Polyphenols, ascorbic acid and other carbonyl compounds can participate in the Maillard reaction. MRP are naturally formed during food processing and storage. Their characteristics and MW depend on both the type of reagents and the processing conditions (temperature, time, pH, water activity) (Jing and Kitts, 2004). The antioxidant properties of MRP are positively correlated with color when the mechanisms responsible for the formation of antioxidants and color follow the same pathway (Manzocco et al., 2001). Formation of potentially harmful substances (acrylamide and hydroxymethylfurfural) increases rapidly with temperature and time (Morales et al., 2009). The antioxidant capacity of MRP formed from model compounds has been reported (Borrelli et al., 2003; Chen and Kitts, 2008; Dittrich et al., 2003; Kim and Lee, 2008; Morales and Babbel, 2002). Maillardderived antioxidants increase the plasma tocopherol, decrease the plasma thiobarbituric acid reactive substances (TBARS) concentration (Somoza, 2005), prevent low density lipoprotein (LDL) oxidation (Mesa et al., 2008), show reducing activity (Yilmaz and Akgun, 2008) and radical scavenging capacity (Michalska et al., 2008), and may be employed as multifunctional ingredients (Lindenmeier et al., 2002). Carbohydrates including polysaccharides, such as dextran, pullulan, mannan, and lipopolysaccharide, exhibit higher free radical scavenging activity than their constituent sugars, although lower than Trolox™ (Tsiapali et al., 2001). Although glucans are weak free radical scavengers in solution, they stimulate free radical activity in a murine macrophage cell line and modestly augment the generation of free radicals. Arabinoglucogalactan from Panax noto ginseng roots exhibited a, a-diphenyl-b-picrylhydrazyl (DPPH) free radical scavenging activity (Wu and Wang, 2008), whereas porphyran, a sulfated polysaccharide isolated from Porphyra, delayed the aging process in mice by enhancing the amount of antioxidative enzymes and reducing the risk of lipid peroxidation (Zhang et al., 2003). Fucoidans,


516 Separation, extraction and concentration processes sulfated algal polysaccharides made up of fucose as the major component, exhibit antioxidant and biological properties (anticoagulant, anti-inflamatory, antiviral, or antitumoral activities) (Zhao et al., 2008). 18.3.2 Sources The number of potential sources of antioxidant compounds is increasing. Table 18.3 summarizes the composition of some examples of the major vegetal sources of bioactive compounds. Food by-products have the additional advantages of being renewable, widely distributed, largely available and inexpensive. Waste streams from the processing of agricultural or industrial feedstocks are particularly attractive as sources of antioxidants, particularly when they are concentrated and create environmental problems (Balasundram et al., 2006; Moure et al., 2001; Obied et al., 2005). Typical wastes employed for antioxidant production and composition of some representative cases are summarized in Tables 18.4 and 18.5. Table 18.3 Phenolic composition of fruits, vegetables and cereals (% oven-dried substrate) Vegetal source Fruits Acerola (Malpighia emarginata DC.) Andean blackberry (Rubus glaucus Berth.) Blueberry (Vaccinium species) Brazilian mango (Mangifera indica L.) Capulí cherry (Prunus serotina Ehrh. var. Capulí). Grape (Vitis vinifera L.)

Phenolic compounds

Reference

CGA, EC, EGCG, PCyB1, R

Mezadri et al., 2008 pCouA-d, CyG, EA-d, EC, Et, GA, Gs, Vasco et al., PCYs, PeG, Q-d 2009 pHBA, C, CA, CGA, pCouA, Cy, De, Taruscio et al., EC, FeA, Myr, Ma, Peo, Pet, Q 2004 KG,Ma, iMa, Q, QAp, QAf, QG, QGal, Ribeiro et al., QR, QX 2008 C, CGA, CyG, EC, K-d, PCYs, Q-d Vasco et al., 2009

C, CA, HCA, oCouA, pCouA, GA, R, Q Kedage et al., 2007 Lemon (Melissa officinalis CA, mCouA, Er7G, He, Het, Na, Ng, Dastmalchi L.) RA et al., 2008 Plum (Prunus salicina CA-d, nCGA, pCouA-d, Cyg, EC, K-d, Vasco et al., Lindl.) (var. ‘Santa Rosa’ PCYs, Q-d 2009 and ‘Beauty’) Portuguese pear (Pyrus Ar, monC, terC, CQA, pCouMA, Ferreira et al., communis L. var. S. monEC, terEC, extEC 2002 Bartolomeu) Strawberry (Fragaria × CyG, EA, K, K-CouG, Pe, PeG, PeR, Zhang et al., ananassa Duch.) HPhAC, Q 2008 CGA, nCGA, pCouQA, CyG, EC, PeR, Usenik et al., Sweet cherry (Prunus avium L.) PeoR, R 2008 © Woodhead Publishing Limited, 2010

Extraction of natural antioxidants from plant foods 517 Table 18.3 Continued Vegetal source

Phenolic compounds

Reference

Vegetables Broccoli (Brassica oleracea L.) Escarole (Cichorium indivia var. latifolium L.) Lettuce (Lactuca sativa var. capitata L.) Onion (Allium cepa)

nCGA, FeGe, PCA, dSiGe, SiFeGe, SiGe, dSiFeGe, SidFeGe dCQA, CGA, CGA-d, CtA, ChiA, KGl, KMG dCQA,CGA, CGA-d, CtA, ChiA, Q-d, QMG FeA, GA, K, PCA, Q

Vallejo et al., 2003 Degl’innoocenti et al., 2008 Degl’innoocenti et al., 2008 Prakash et al., 2007 Reddivari et al., 2007 Degl’innoocenti et al., 2008 Aehle et al., 2004

Potato (Solanum Axa, C, CA, CGA, Cxa, GA, Lu, Vxa tuberosum L.) Rocket salad (Eruca sativa HCAs-d, K-d Mill.) Spinach (Spinacia Pa, Sp oleracea) Cereals Barley (Hordeum vulgare C, CA, pCouA, EC, FeA, GA, PCA, L.) SyA, VA Corn (Zea mays L.) CyG, aCyG, HCAs, pCouA, PCA, He-d, PeG, aPeG, PeoG, aPeoG, Q-d, VA Millets

pHBA, C, CA, CinA, pCouA, FeA, GA,GeA, L, Or, iOr, PCA, SaA, Sap, SiA, Tr, VA, Vi, Vt, iVt Rice (Oryza sativa L.) HBA, CA, CGA, pCouA, FeA, FS, PCA, SiA, SiS, SyA, VA Rye (Secale cereale L.) ALRs, pHBA, CA, FeA, SiA, SyA, VA, VeA Sorghum (Sorghum Apf, Ap, Api, ApiG, MApi, MApiG, bicolor L.) 5MApi, PApi, pHBA, CA, EC, pCouA, CinA, PCyB1, PDe, Er, Er5G, FeA, GA, PCA, GeA, KRG, Lut, Ltn, LtnG, PLtn, Lt, MLtn, MLtnG, 7MLtn, Ng, Ta, TaG, SaA, SiA, SyA, VA Wheat (Triticum aestivum CA, oCouA, pCouA, FeA, SyA, VA L.)

Zhao et al., 2006 Pedreschi and CisnerosZevallos, 2007 Dykes and Rooney, 2006 Tian et al., 2004 Heiniö et al., 2008 Dykes and Rooney, 2006

Mpofu et al., 2006

5MApi, 5-methoxyapigeninidin; 7-MApiG, 7-methoxyapigeninidin-5-glucoside; 7MLtn, 7-methoxyluteolinidin; aCyG, acylated cyanidin-3-glucoside; ALRs, alkylresorcinols; Ap, apigenin; aPeG, acylated pelargonidin-3-glucoside; aPeoG, acylated peonidin 3-glucoside; Apf, apiforol; Api, apigeninidin; ApiG, apigeninidin-5-glu; Ar, arbutin; Axa, antheraxanthin; C, catechin; CA, caffeic acid; CA-d, caffeic acid derivatives; CGA, chlorogenic acid; CGA-d, chlorogenic acid derivates; ChiA, chicoric acid; CinA, cinnamic acid; CQA, caffeoylquinic acid; CtA, caffeoyltartaric acid; Cxa, canthaxanthin; Cy, cyanidin; Cyg, cyanidin glycosides; CyG, cyanidin-3-glucoside; dCQA, dicaffeoylquinic acid; De, delphinidin; dSiFeGe, 1,2¢-disinapoyl-2-feruloylgentiobiose; dSiGe, 1,2-disinapoylgentiobiose; EA, ellagic acid; EA-d, ellagic acid derivatives; EC, epicatechin; EGCG, epigallocatechin gallate; Er, eriodictyol; Er5G, eriodictyol 5-glucoside; Er7G, eriodictyol-


518 Separation, extraction and concentration processes Table 18.3 Continued 7-O-glucoside; Et, ellagitannins; extEC, extension epicatechin; FeA, ferulic acid; FeGe, 1,2diferuloylgentiobiose; FS, 6¢-O-feruloylsucrose; GA, gallic acid; GeA, gentisic acid; Gs, galloyls; HBA, hydroxybenzoic acid; HCA, hydrocaffeic acid; HCAs, hydroxycinnamic acids; HCAs-d, hydroxycinnamic derivates; He, hesperidin; He-d, hesperidin derivates; Het, hesperetin; HPhAC, 3,4,5trihydroxyphenyl-acrylic acid; iMa, isomangiferin; iOr, isoorientin; iVt, isovitexin; K, kaempferol; K-CouG, kaempferol-3-(6¢-coumaroyl)glucoside; K-d, kaempferol derivatives; KG, kaempferol 3-O-glucoside; KGl, kaempferol-3-O-glucuronide; KMG, kaempferol-3-O-(6-O-malonylglucoside); KRG, kaempferol 3-rutinoside-7-glucuronide; L, lucenin-1; Lt, luteolin; Ltn, luteolinidin; LtnG, luteolinidin-5-glucoside; Lu, lutein; Lut, luteoforol; Ma, mangiferin; MApi, 7-methoxyapigeninidin; mCouA, m-coumaric acid; MLtn, 5-methoxyluteolinidin; MLtnG, 5-methoxyluteolinidin-7-glucoside; monC, monomeric catechin; monEC, monomeric epicatechin; Myr, myricetin; Na, naringin; nCGA, neochlorogenic acid; Ng, naringenin; oCouA, o-coumaric acid; Or, orientin; Pa, patuletin; PApi, proapigeninidin; PCA, protocatechuic acid; pCouA, p-coumaric acid; pCouA-d, p-coumaric acid derivatives; pCouMA, p-coumarylmalic acid; pCouQA, p-coumaroylquinic acid; PCyB1, procyanidin B1; PCYs proanthocyanidins; PDe, prodelphinidin; Pe, pelargonidin; PeG, pelargonidin-3-glucoside; Peo, peonidin; PeoG, peonidin 3-glucoside; PeoR, peonidin 3-rutinoside; PeR, pelargonidin-3rutinoside; Pet, petunidin; pHBA, p-hydroxybenzoic acid; PLtn, proluteolinidin; Q, quercetin; QAf, quercetin 3-O-arabinofuranoside; QAp, quercetin 3-O-arabinopyranoside; Q-d, quercetin derivatives; QG, quercetin 3-O-glucoside; QGal, quercetin 3-O-galactoside; QMG, quercetin-3-O(6-O-malonylglucoside); QR, quercetin 3-O-rhamnoside; QX, quercetin 3-O-xyloside; R, rutin; RA, rosmarinic acid; SaA, salicylic acid; Sap, saponarin; SiA, sinapic acid; SidFeGe, 1-sinapoyl-2,2¢diferuloylgentiobiose; SiFeGe, 1-sinapoyl-2-feruloylgentiobiose; SiGe, 1,2,2¢-trisinapoylgentiobiose; SiS, 6¢-O-sinapoylsucrose; Sp, spinacetin; SyA, syringic acid; Ta, taxifolin; TaG, taxifolin-7-glucoside; terC, terminal catechin; terEC, terminal epicatechin; Tr, tricin; VA, vanillic acid; VeA, veratric acid; Vi, violanthin; Vt, vitexin; Vxa, violaxanthin.

Table 18.4 Residual sources proposed for antioxidant manufacture Feedstock

References

Residues from grape and wine

Cruz et al., 2004; Corrales et al., 2008; Lafka et al., 2007; Makris et al., 2007; Pinelo et al., 2006; Arvanitoyannis et al., 2006; Louli et al., 2004

Apple pomace

Schieber et al., 2003

Olive

Obied et al., 2005; McDonald et al., 2001

Husks and hulls

Moure et al., 2000; Conde et al., 2008; Goli et al., 2005; Takeoka and Dao, 2003; Oliveira et al., 2008; DeliormanOrhan et al., 2009; Rubilar et al., 2007

Woods

Pérez-Bonilla et al., 2006; Castro et al., 2008; Moure et al., 2005

Leaves

Ferreira et al., 2007; Ozsoy et al., 2008

Seeds

Liu and Yao, 2007; Siddhuraju and Becker, 2007

Peels

Berardini et al., 2005; Li et al., 2006

Fruits and vegetables and their processed products and by-products have been extensively investigated. Their activity has been ascribed to the presence of phenolic compounds (Cieślik et al., 2006; Heinonen and Meyer, 2002; Macheix et al., 1990; Peschel et al., 2006; Proteggente et al., 2003; Robards et al., 1999; Sakakibara et al., 2003). Abundant research on fruits © Woodhead Publishing Limited, 2010

Extraction of natural antioxidants from plant foods 519 Table 18.5 Chemical composition of solid agro-industrial by-products (% oven-dried substrate) Solid agro-industrial Phenolic compounds by-product Almond hulls

References

CGA, cCGA, nCGA

Takeoka and Dao, 2003 Almond shells HCinAs, pCouA, VA, SyA Moure et al., 2007 Apple, peach and CA, p-CouA, FeA Gorinstein et al., pear peels 2002 Apple peels CA, C, EC, CGA, FeA, Q, QA, QG, QGal He and Liu, 2008 Schieber et al., 2003 Apple pomace C, EC, CGA, pCouA, pCouQA, PCyB2, FeA, Pht, Phx, Phz, Q, QA, QG, QGal, QR, QRu, QX Banana peel/pulp C, EC, CG Someya et al., 2002 Barley husks dHB, pCouA, GA, SyA, VA, V Conde et al., 2008 Quettier-Deleu Buckwheat hulls/ B2D, B2G, EC, ECG, QGal, QRu, Q et al., 2000 flour Chestnut wood 4HB, Con, EA, FeA, GA, Sin, Sc, SyA, Canas et al., 1999 Syra, V, VA, Um Coconut husks 4HBA, FeA Dey et al., 2003 Corn cobs AV, HMCin, iEu, MEu, Gu, Gua, EGu, Garrote et al., 2007a VGu, VPh, Sy, ASy, Syra, V Citrus peel and seed CA, p-CouA, Er, nEr, FeA, He, nHe, Na, Bocco et al., 1998 Nr, SiA Eucalypt wood 3HB, HMCin, Co, dHEu, MEu, Gu, VGu, Garrote et al., 2007b iEu, V, iVA, HVA Grape skin and seeds C, EC, CA, oCouA, pCouA, CyGl, GA, Lafka et al., 2007 PCA, SyA, Ty, HTy, VA Olive tree leaves ApG, C, CA, Di, DiG, Lt, LtG, Ol, R, Ty, Benavente-García HTy, V, VA, Ve et al., 2000 Olive tree pruning dHB, HMCin, Ol, Sy, Syra, Ty, HTy, V, Conde et al., 2009 HV, VA Potato peel GA, CGA, CA Kanatt et al., 2005 Rice husks AV, 4HB, HMCin, VPh, Gu, Gua, VGu, Garrote et al., 2007 Eu, iEu, MEu, Sy, ASy, Syra, V, HVA Sweetpotato leaves CA, CGA, 3,4dCQA, 3,5dCQA, Islam et al., 2002 4,5dCQA, tCQA Winemaking waste dHB, 3HBA, CinA, pCouA, GA, PCA, Cruz et al., 2004 solids SyA, VA, iVA Wood of olive tree De, iEu, MEu, Gu, Sy, Syra, HSyA, Ty, Castro et al., 2008 branches HTy, Ol, V, HV 3,4dCQA, 3,4-di-O-caffeoylquinic acid; 3,5dCQA, 3,5-di-O-caffeoylquinic acid; 3HB, 3-hydroxybenzaldehyde; 3HBA, 3-hydroxybenzoic acid; 4,5dCQA, 4,5-di-O-caffeoylquinic acid; 4-HB, 4-hydroxybenzaldehyde; 4HBA, 4-hydroxybenzoic acid; ApG, apigenin-7-glucoside; ASy, acetosyringone; AV, acetovanillone; B2D, proanthocyanidin B2; B2G, B2-3´-O-gallate; C, catechin; CA, caffeic acid; cCGA, cryptochlorogenic acid; CG, catechin gallate; CGA, chlorogenic acid; CinA,


520 Separation, extraction and concentration processes Table 18.5 Continued cinnamic acid; Co, coniferol; Con, coniferaldehyde; CYGl, cyanidin glycosides; De, desaspidinol; dHB, 3,4-dihydroxybenzaldehyde; dHEu, dihydroeugenol; Di, diosmetin; DiG, diosmetin-7-glucoside; EA, ellagic acid, EC, epicatechin; ECG, epicatechin gallate; EGu, 4-ethylguaiacol; ER, eriocitrin; Eu, 4-eugenol; FeA, ferulic acid; GA, gallic acid; Gu, guaiacol; Gua, guaiacylacetone; HCinAs, hydroxycinnamic acids; He, hesperidin; HMCin, 4-hydroxy-2-methoxycinnamaldehyde; HSyA, homosyringic acid; HTy, hydroxytyrosol; HV, homovanillyl alcohol; HVA, homovanillic acid; iEu, isoeugenol; iVA, isovanillic acid; Lt, luteolin; LtG, luteolin-7-glucoside; MEu, methoxyeugenol; Na, naringin; nCGA, neochlorogenic acid; nEr, neoeriocitrin; nHe, neohesperidin; Nr, narirutin; oCouA, o-coumaric acid; OL, oleuropein; PCA, protocatechuic acid; pCouA, p-coumaric acid; pCouQA, p-coumaroylquinic acid; PCY-B2, procyanidin B2; Pht, phloretin; Phx, phloretin xyloglucoside; Phz, phloridzin; Q, quercetin; QA, quercetin-3-arabinoside; QG, quercetin-3-glucoside; QGal, quercetin3-galactoside; QR, quercetin-3-rhamnoside; QRu, quercetin-3-rutinoside; QX, quercetin-3-xyloside; R, rutin; Sc, scopoletin; SiA, sinapic acid; Sin, sinapaldehyde; Sy, syringol; SyA, syringic acid; Syra, syringaldehyde; tCQA, 3,4,5-tri-O-caffeoylquinic acid; Ty, tyrosol; Um, umbelliferone; V, vanillin; VA, vanillic acid; Ve, verbascoside;VGu, 4-vinylguaiacol; VPh, 4-vinylphenol.

is available, including citrus (Dastmalchi et al., 2008; Di Majo et al., 2005; Jayaprakasha and Patil, 2007; Tripoli et al., 2007), berry fruits (Szajdek and Borowska, 2008; Vasco et al., 2009; Taruscio et al., 2004; Zhang et al., 2008) and cherries (Piccolella et al., 2008). Results have also been reported for juices (Gardner et al., 2000), teas (Gupta et al., 2008; von Gadow et al., 1997; Wiseman et al., 1997) coffee (Nardini et al., 2002), wines (Burns et al., 2001; Fogliano et al., 1999; Minussi et al., 2003; Sanchez-Moreno et al., 2003; Villaño et al., 2005), and several beverages (Arts et al., 2000; Richelle et al., 2001; Tabart et al., 2009). The potential of aromatic herbs, spices and medicinal plants was first assessed in the 1950s (Chipault et al., 1952). Their major antioxidant components are vitamins, phenolic acids, flavonoids and flavonoid derivatives, terpenoids, carotenoids, phytoestrogens and minerals. Specific compounds from plants include carnosic acid, carnosol, rosmarinic acid, rosmanol, thymol, carvacrol, gingerol-related compounds, curcumins, capsaicin, capsaicinol, and ascorbic acid (Suhaj, 2006; Yanishlieva et al., 2006). Their effectiveness in different substrates and other biological activities have been reviewed (Srinivasan, 2005; Yanishlieva et al., 2006; Zheng and Wang, 2001). The in vitro antioxidant capacity of cereals is significantly correlated with their polyphenol content, except for maize (Decker et al., 2002). During digestion, the antioxidant capacity of cereals is increased, providing a favorable antioxidative environment for the epithelium in the large intestine (Fardet et al., 2008). However, the in vitro antioxidant capacity of cereals is only an approximate reflection of their in vivo antioxidant effect, owing to differences in solubility/bioavailability within the digestive tract and to the metabolism/conjugation with compounds such as polyphenols. About 6000 seaweed species have been identified and they are grouped into green (chlorophytes), brown (pheophytes) and red (rhodophytes) algae.


Extraction of natural antioxidants from plant foods 521 Seaweeds are traditionally exploited mainly for soluble polysaccharides and direct utilization as foods. The soluble fraction of red seaweeds is mostly composed of sulfated galactans (agar, carrageenans), whereas the brown seaweeds (alginates, fucans, and laminarans) correspond mostly to reserve b-glucans. The recovery of valuable biomolecules, including vitamins, minerals, lipids, polyphenols, proteins, sterols, pigments, tocopherol derivatives and related isoprenoids, is a topic of growing interest (Herrero et al., 2006; Kumar et al., 2008). The most potent antioxidants of algae are phlorotannins and sulfated polysaccharides (Rupérez et al., 2002; Wang et al., 2008; Xue et al., 2001; Ye et al., 2008). A recent work has reported the irradiation-dependent production of butylhydroxytoluene (BHT) by green algae and cyanobacteria (Babu and Wu, 2008). Mushrooms are also a source of phenolic antioxidants (Cheung et al., 2003).

18.4 Biological activities of natural antioxidants Reactive oxygen species (ROS) play a dual role: a certain physiological level of ROS is crucial for the regulation of cell functions (Wang and Yi, 2008), but ROS and other radicals are also involved in diabetes, cancer, liver disease, aging, arthritis, AIDS, macular degeneration, and autoimmune, inflammatory, cardiovascular and neurodegenerative diseases. Antioxidants prevent ROS concentrations from reaching a damaging level and have been considered as a promising therapy for the prevention and treatment of these diseases (Seifried et al., 2007). The results from many intervention trials with antioxidants failed to demonstrate benefits in humans, probably because the reactive species may not be important, the pathologies were too advanced, the doses used may be wrong or the administered antioxidants do not decrease the oxidative damage (Halliwell, 2009). Antioxidant properties alone are not sufficient to explain their biological properties, some of which are indicated in Table 18.6. Although an antioxidant is a redox agent that in the presence of metal ions could act as a pro-oxidant, in vivo most transition metal ions are protein-conjugated and not available to catalyze free radical reactions (Hadi et al., 2007). The most studied compounds for controlling various disorders (including cardiovascular, neurological and neoplastic diseases) are plant-derived polyphenols. The use of the cardioprotective properties of dietary antioxidants against cardiovascular diseases has been studied and some conflicting findings have been found (Seifried et al., 2007). Natural polyphenolic compounds possess antioxidant, vasorelaxant and antihypertensive properties (Depeint et al., 2002; Erlund, 2004; Ghosh, 2005; Ullah and Khan, 2008). Some effects of polyphenols (such as anti-atherogenesis) can be attributed to their antioxidant properties (German and Walzem, 2000). Selected polyphenols



Biological activities

Plant or extract

Active compounds

Reference

Antiallergic Antiarthritic

Coronopus didymus Cleome gynandra

Antiasthmatic Antibacterial Anticancer Anticoagulant Antidiabetic Antiglycative Antihyperglycemic

Laurencia undulata Cyperus rotundus Prunus serrulata Cirsium japonicum Psidium guajava L. Helichrysum plicatum Psidium guajava L. Punica granatum

Mantena et al., 2005 Narendhirakannan et al., 2005 Jung et al., 2009 Kilani et al., 2008 Lee et al., 2007 Yin et al., 2008 Hsieh et al., 2007 Aslan et al., 2007 Hsieh et al., 2007 Bagri et al., 2009

Antihyperlipidemic

Coronopus didymus Helichrysum plicatum Punica granatum

Flavonoids, tannins Polyphenolic compounds, tannins, anthroquinones, flavonoids Phlorotannins Tannins, flavonoids, coumarins, sterols Phenolic glucosides, flavonoids, anthocyanins Phenolic acids, flavonoids Phenolic compounds Phenolic acids, flavonoids Phenolic compounds Alkaloids, flavonoids, anthocyanins, hydrolyzable tannins Flavonoids, tannins Phenolic acids, flavonoids Alkaloids, flavonoids, anthocyanins, hydrolyzable tannins Flavonoids and phenolics acids Polyphenols, tannins Bioflavonoids, catechins, procyanidins, phenolic acids Monoterpene glycosides Eugenol Curcuminoids Polyphenols Oligomeric procyanidins, catechins, condensed tannins

Souza et al., 2007 Kim et al., 2007 Hasegawa et al., 2008 Panich et al., 2009 Panich et al., 2009 Ahn et al., 2008 Esquenazi et al., 2002

Anti-inflammatory

Antimelanogenic

Antimicrobial

Geranium robertianum L. Uncaria tomentosa Myracrodruon urundeuva Pycnogenol® Eucalyptus globulus Almina galanga Curcuma aromatic Sasa quelpaertensis Nakai Cocos nucifera L.

Mantena et al., 2005 Aslan et al., 2007 Bagri et al., 2009 Amaral et al., 2009


Table 18.6 Biological activities reported for antioxidant extracts from some plant foods

Pycnogenol® and Ginkgo biloba Hibiscus tiliaceus L.

Antiparkinson Antipyretic Antitumor Antiulcer Antiviral Apoptotic activities Cytotoxic Hepatoprotective

Mucuna pruriens Coronopus didymus Senna alata Myracrodruon urundeuva Cocos nucifera L. Cyperus rotundus Cyperus rotundus Coronopus didymus Commiphora berryi

Polyphenols, procyanidins Polyphenols, carotenoids, tocopherols, flavonoids, anthocyanins, phytosterols Polyphenols Flavonoids, tannins Flavonoids, tannins, polyphenols Polyphenols, tannins Oligomeric procyanidins, catechins, condensed tannins Tannins, flavonoids, coumarins, sterols Tannins, flavonoids, coumarins, sterols Flavonoids, tannins Phenolic compounds, tannins

Decalepis hamiltonii

Phenolic compounds

Ecklonia cava

Polyphenols

Dhanasekaran et al., 2008 Mantena et al., 2005 Pieme et al., 2008 Souza et al., 2007 Esquenazi et al., 2002 Kilani et al., 2008 Kilani et al., 2008 Mantena et al., 2005 Gowri Shankar et al., 2008 Srivastava and Shivanandappa, 2009 Ahn et al., 2008

Morus alba L.

Flavonol glycosides

Katsube et al., 2006

Castanea sativa

Polyphenols

Almeida et al., 2008

Immunomodulatory activity Inhibitor LDL oxidation Skin protector

Križková et al., 2008 Rosa et al., 2006



Antimutagenic

524 Separation, extraction and concentration processes (extracts from grape and wine, resveratrol, genistein, dihydrodaidzeins) relax endothelial function (Shen et al., 2006). Flavonoids can have favorable effects on atherosclerosis. Lipid-lowering activity has been reported for tea flavonoids (Li et al., 2005), a-tocopherol, and b-carotene, which inhibit the oxidation of LDL and the atherosclerotic process (Odeh and Cornish, 1995). Tocopherols show enhanced immune response and regulation of platelet aggregation (Weber and Rimbach, 2002). Protection against coronary heart disease was reported for resveratrol (Szewczuk and Penning, 2004), anthocyanidins, EGCG, and vitamin E (Violi et al., 2006). Biologically active peptides promote diverse activities (antithrombotic, hypocholesterolemic and antihypertensive actions) relevant to cardiovascular health (Erdmann et al., 2008). Inhibitors of angiotensin-converting enzyme (ACE) play an important role in the regulation of blood pressure and fluid and salt balance in mammals. Immunomodulatory activity has also been reported for peptides (Erdmann et al., 2008). Antiallergenic activity has been reported for flavonoids (Das and Rosazza, 2006; Ghosh, 2005; Hodek et al., 2002; McKay and Blumberg, 2006), which may also preserve T-cell-mediated immunity (Horváthová et al., 2001; Strickland, 2001). Bioactive compounds such as flavonoids (Horváthová et al., 2001) and vitamin E protects against cataracts. Vitamin E decreases oxidative stress and the levels of erythrocyte osmotic fragility in patients on dialysis (Uzum et al., 2006) and had beneficial effects on diabetic patients (Levy and Blum, 2007). Epidemiological studies suggested a positive association between a diet rich in fruit and vegetables and a lower incidence of cancer (Ames et al., 1993; Pan et al., 2008a; Ren et al., 2003; Tanaka and Sugie, 2008). The antioxidant properties may only partly explain the antitumor effects of dietary phenolics, which may exert modulatory actions in cells, interfering in the steps leading to the development of malignant tumors (Kanakis et al., 2007). Supplemental antioxidants may help to prevent cancer only in dietdeficient populations or individuals, and antioxidant anticancer agents may affect patients differently according to their health (Vickers, 2007). Although dietary antioxidants have attracted great interest and are considered safe, they have to pass controlled clinical trials for therapeutic or chemopreventive use, and potential toxic flavonoid–drug interactions should be considered (Galati and O’Brien, 2004). Many natural agents have shown potential in bioassays and animal models, and some of them have been selected for ongoing phase I–III clinical trials (Bonfili et al., 2008). An ideal, effective and acceptable agent should be non-toxic in normal and healthy cells, low cost and efficient against multiple cancers, act according to a known mechanism, deserve public acceptance, and be suitable for oral consumption. Potential cancer preventive agents suppress carcinogenesis by several major mechanisms: inhibiting phase I enzymes or blocking carcinogen formation, inducing phase II (detoxification) enzymes, scavenging DNA reactive agents, suppressing the over-expression of pro-oxidant enzymes, modulating hormone homeostasis,


Extraction of natural antioxidants from plant foods 525 suppressing hyper-cell proliferation induced by carcinogens, inducing apoptosis, counteracting angiogenesis, and/or inhibiting certain phenotypic expressions of preneoplastic and neoplastic cells (D’Archivio et al., 2008; Shklar, 1998; Ren et al., 2003; Pan et al., 2008a). Various dietary antioxidants are under investigation for their anticancer properties, including curcumin (Tsvetkov et al., 2005; Maheshwari et al., 2006), oleuropein (Bonfili et al., 2008), and tangeretin, nobiletin and resveratrol (Narayanan, 2006). Green tea inhibits tumor incidence and multiplicity in various organs, and recent phase I and II clinical trials have been conducted to explore its anticancer effects (Dou, 2008). Flavonoids in human diet may reduce the risk of various cancers (Kanadaswami et al., 2005), including hormone-dependent breast and prostate cancers (Hodek et al., 2002), intestinal neoplasia (Hoensch and Kirch, 2005) and skin cancer (Singh and Agarwal, 2002). Lycopene shows both in vitro and in vivo anticancer activities, possibly through ROS scavenging, up-regulation of detoxification systems, interference with cell proliferation, induction of gap-junctional communication, inhibition of cell cycle progression and modulation of signal transduction pathways (Bhuvaneswari and Nagini, 2005). A beneficial role of vitamin C on cancer has been claimed, although further clinical trials are needed (Verrax and Calderón, 2008). Vitamin E and vitamin E-based compounds are potent antioxidants regulating peroxidation, some forms display apoptotic activity against cancer cells and restore multidrug resistant tumor cells sensitivity to chemotherapeutic agents (Kline et al., 2007; Sylvester, 2007). Among soy peptides, lunasin suppresses transformation of cells induced by carcinogens and viral oncogenes (De Mejia and De Lumen, 2006), and the Kunitz trypsin inhibitor suppresses ovarian cancer cell invasion by blocking urokinase up-regulation (Xiao et al., 2005). Combinations of antioxidant nutrients act synergistically, and their use is proposed to offer a better quality of life (Shklar, 1998), and to present a co-operative action with chemotherapeutic drugs and radiation therapy (Bonfili et al., 2008; Prasad et al., 2002). The nervous system, rich in fatty acids and iron, is vulnerable to free radical generation (Ullah and Khan, 2008). Reactive species are constantly produced in the brain, for example by leakage of electrons from the mitochondrial electron transport chain to generate superoxide radical (O2–). Increased levels of oxidative damage may occur early in the progression of many neurodegenerative diseases (Philips et al., 1993), as oxidative damage may aggravate the accumulation and precipitation of proteins. Epidemiological studies indicate that dietary antioxidants can limit the incidence of neurodegenerative disorders (Morris, 2002). Polyphenols are active agents in neuroprotection owing to their ability to influence and modulate several cellular processes such as signaling, proliferation, apoptosis, redox balance, and differentiation. Their role in neurological disorders has been studied (Dajas et al., 2002; Horváthová et al., 2001; Singh and Singh, 2008). Promising compounds for neuroprotection include polyphenols such as epigallocatechin gallate (EGCG), curcumin, naringenin, extracts of


526 Separation, extraction and concentration processes blueberries and Scutellaria (Ullah and Khan, 2008), and extracts from green tea (Bastianetto et al., 2006). Flavonoids and their derivatives are reported to inhibit the growth and development of HIV by suppressing acute infection and inhibiting protease, integrase and reverse transcriptase (Critchfield et al., 1996), whereas scutellarin showed anti-HIV activity (Zhang et al., 2005). The overall role of reactive species in chronic inflammatory diseases is not clear. Reactive species may help explain why such diseases increase cancer risk, but ironically these species can sometimes be anti-inflammatory. Hence the use of antioxidants to treat chronic inflammatory diseases may not be as simple as it originally sounded (Halliwell, 2009). Natural antioxidants with anti-inflamatory activity include phenolics (Lomnitski et al., 2000), resveratrol, flavonoids (Ghosh, 2005; Das and Rosazza, 2006; Hodek et al., 2002; Kim et al., 2004), terpenoids (De las Heras et al., 2003), and glucans (Tsiapali et al., 2001). Therapeutic potential for combating bronchial asthma has been reported for polyphenolic extracts from Laurencia undulata, an edible red alga (Jung et al., 2009). Tyrosinase inhibitors can ameliorate cutaneous hyperpigmentary disorders. Their use is becoming increasingly important in the cosmetic industry owing to their skin whitening action and preventive effects, and there is a growing interest in their medicinal and cosmetic applications (Parvez et al., 2007). Some carotenoids (a- and b-carotene and b-cryptoxanthin) are precursors of vitamin A and protect against chemical oxidative damage and against several kinds of cancer, age-related macular degeneration and UV-induced erythema.

18.5 Extraction of natural antioxidants from plant foods and residues Solid–liquid extraction is a heterogeneous operation involving transfer of solutes from a solid to a fluid. Extractable compounds of vegetal origin are a complex mixture of solutes that can be extracted at different rates depending on their location (outer surface, pores and vacuoles). Extraction involves the following sequential steps: (i) transport of solvent from the bulk solution to the external surface of the particle, (ii) solvent penetration and diffusion in the solid matrix, (iii) solubilization of the components, (iv) transport of the solute(s) through the solid matrix, and (v) transport of the solute(s) from the external surface of the solid to the bulk solution. 18.5.1 Conventional solvent extraction The type of solvent is one of the most influential variables on both extraction yield and type of extracted solutes. Methanol, ethanol, and water are widely


Extraction of natural antioxidants from plant foods 527 employed for extracting phenols. Methanol exhibits the highest capacity to extract phenolics (Pinelo et al., 2005), but shows toxicity, whereas hot water may extract polyphenols without safety concerns. Ethanol–water mixtures are suited to penetrate the hydrophobic areas of the vegetable matrix and help to precipitate soluble proteins, facilitating further processing. Representative examples of extraction processes involving these solvents are summarized in Table 18.7. Temperature is one of the most influential variables affecting the release of phenols. Increased temperatures favor extraction by enhancing both solubility and diffusion. However, temperature cannot be increased indefinitely, because instability of phenolic compounds and denaturation of membranes may take place at temperatures above 50 °C (Cacace and Mazza, 2003). Partially oxidized polyphenols can exhibit higher antioxidant activity than non-oxidized phenols. Thermal treatment of crude extracts can be performed to modify their composition by oxidation, hydrolysis and isomerization, and can result in the formation of compounds with new antioxidant properties. Therefore, heat-induced antioxidants balance the thermal loss of antioxidants, or the overall antioxidant properties can be increased upon heating (Delgado-Andrade and Morales, 2005). This behavior was reported for catechin, resveratrol, grape extract (Pinelo et al., 2005), and citrus peel (Xu et al., 2007). Steam-processing of broccoli may release more bound phenolic acids from the breakdown of cellular constituents (Roy et al., 2009). Heating vegetal products such as sweet corn increased the total antioxidant activity and the phenolic content (Dewanto et al., 2002). Heat treatment of huyou (Citrus paradisi Changshanhuyou) peel increased the free fraction of phenolic acids and decreased the ester, glycoside, and ester-bound fractions (Xu et al., 2007), whereas the antioxidant capacity of citrus peel [measured by the ABTS and ferric-reducing antioxidant power (FRAP) assays] increased with heating time and temperature owing to the increase of lower MW phenolics (benzoic acids and cinnamic acids). High solvent-to-solid ratios favor phenol yields, but a balance between high costs (derived from solvent usage) and absence of saturation effects has to be found (Pinelo et al., 2005). However, operational variables such as temperature, solvent-to-solid ratio, and type of solvent used only affect solutes weakly linked to the cell wall structure and those contained in vacuoles. Extraction of compounds forming part of the cell walls may require other strategies, such as cell wall breaking or enzymatic treatments. Even though conventional extraction with organic solvents is widely used, it has low efficiency and requires large volumes of solvent, long extraction times and high temperature. This could lead to loss of biological activities and decreased selectivity, as well as to problems derived from organic solvent cost and disposal (Robards 2003). Additionally, organic solvents must be completely removed from the exhausted solids. Recently, there has been an increasing interest in using environmentally friendly extraction technologies for producing extracts of high quality and activity. Some of these techniques



Raw material

Solvent

Solubles yield or Antioxidant activity TEP* (% dry weight)

Reference


W

8

DPPH(–)*1; bC(–)*1

Sharififar et al., 2009


E

2.84

TEAC(–)*2; DPPH(–)*2,3; RP(–)*2,3; FIC(–)*4; bC(–)*2

EA

8.28

Hordeum vulgare (seeds) Chamaecyparis obtusa (bark)

E (70%)

4.70

TEAC(–) ; DPPH(–) ; RP(–) ; FIC(–) ; bC(–) RP(–)*1,3; DPPH(–)*1,3; FTC(+)*1(=)*3

E

9.07 (GA)

DPPH(–)*2; TEAC(–)*5; RP(–)*5; bC(–)*5

EA

10.28 (GA)

Cirsium japonicum (leaves) Chukrasia tabularis (leaves)

W

24.13

DPPH(–) ; TEAC(–) ; RP(–) ; bC(–) DPPH(–)*3,6; RP(–)*6; HOSC(–)*6

EA W E (95%)

98.35 (GA) 60.88 (GA) 9.68 (PC)

DPPH(–)*7; RP(–)*7; HOSC(+)*7 DPPH(–)*7; RP(–)*7; HOSC(–)*7 DPPH(+)*1; HOSC(+)*1; RP(+)*1; TA(+)*1; LPPO(+)*1

Kaur et al., 2008

E (96%)

17.74

DPPH(–)*1,8; bC(–)*1,8

Moure et al., 2001

W

17.36

EA E W W

1.09 (GA) 1.94 (GA)

DPPH(–)*1,8; bC(–)*1,8 DPPH(–)*1,(+)*6; FIC(–)*3; RP(–)*1 DPPH(–)*1,(+)*6; FIC(–)*3; RP(–)*1; FTC(+)*1 DPPH(–)*1,6; RP(–)*1 DPPH(–)*2,3; RP(–)*2,3; TEAC(–)*2

Dimocarpus longan Lour. (peel) Gevuina avellana (seeds) Kappaphycus alvarezii

Litchi chinenesis (flowers)

9.92

*2

*2

*2,3

*5

*2,3

*5

*4

Shyu et al., 2009

*2

Liu and Yao, 2007 Marimuthu et al., 2008

*5

Yin et al., 2008

Pan et al., 2008b

Kumar et al., 2008

Liu et al., 2009


Table 18.7 Examples of extraction of plant food antioxidants using conventional solvent extraction. Symbols (+), (–) and (=) indicate higher/ lower/equal activity compared with the reference standard compound

W

1.60

DPPH(–)*1,8; bC(–)*1,8

W

30.96

FTC(+)*6; bX(+)*7(–)*8; DPPH(–)*7,8; SRS(–)*7; HOSC (–)*7,8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4

E

34.46

FTC(+)*6; bC(+)*7(–)*8; DPPH(–)*7,8; HOSC(+)*7(–)*8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4

EA

10.61

E

37 (GA)

FTC(+)*6, bC(+)*7(–)*8, DPPH(–)*7,8; HOSC(+)*7(–) *8; HPS(–)*7,8; RP(–)*7,8; FIC(–)*4 DPPH(–)*2,3,5,7,12(+)*1; SRS(+)*2,12; HOSC(–)*1,3,5 (+)*2,12

E

34 (GA)

DPPH(–)*1,2,3,5,7,12; SRS(–)*2,12; HOSC(–)*1,2,3,5,12

E

27 (GA)

DPPH(–)*1,3,7(+)*2,5,12; SRS(–)*2,12; HOSC(–)

W

–

FTC(+)*6; RP(+)*6; SRS(+)*1,6,8; DPPH(–)*5(+)*8; FIC(+)*1,6,8; HPS(–)*1,6,8

W E (50%)

8, 5.4 (GA) 12, 16.8 (GA)

Citrus sinensis

E EA

10, 10.4 (GA) 9.12, 6.43 (C)

Citrus grandis

EA

3.41, 9.72 (C)

Randia echinocarpa W (pulp) Rosa rubiginosa (seeds) E (96%) Smilax excelsa (leaves)

Syzygium cumini (kernel) Syzygium cumini (pulp) Syzygium cumini (seed coat) Urtica dioica (aerial parts)

9.46

*1,2,3,5,12

Ozsoy et al., 2008

Benherlal and Arumughan, 2007 Benherlal and Arumughan, 2007 Benherlal and Arumughan, 2007 Gülçin et al., 2004



1.97

DPPH(–)*3,6, 8,9,10; TBARS(–)*6,8; SRS(–)*11; HOSC(–)*8 Ningappa et al., 2007 DPPH(+)*2,3,8,9,10; TBARS(+)*6,8; SRS(+)*11; HOSC(+)*8; RP(+)*8; FIC(–)*4 DPPH(–)*3,6, 8,9(+)*10; TBARS(+)*6,8; SRS(–)*11; HOSC(–)*8 DPPH(–)*3; ORAC(–)*12; ABTS(–)*3; RP(–)*2; PM(+)*13 Jayaprakasha et al., 2008 *3 *12 *3 *2 *13 Jayaprakasha et al., DPPH(–) ; ORAC(–) ; ABTS(–) ; RP(–) ; PM(+) 2008 *1 Santos–Cervantes bC(–) et al., 2007 *1,8 *1,8 Moure et al., 2001 DPPH(–) ; bC(–)

Murraya koenigii (leaves)


*Total extractable polyphenols. E, ethanol; EA, ethyl acetate; W, water. C, catechin; GA, gallic acid; PC, pyrocatechol. *1 , butylhydroxytoluene (BHT); *2, (+)-catechin; *3, ascorbic acid; *4, ethylene diamine tetra-acetic acid (EDTA); *5, quercetin;*6, a-tocopherol; *7, gallic acid; *8, butylhydroxyanisol (BHA); *9, curcumin; *10, b-carotene;*11, superoxide dismutases (SOD); *12, trolox; *13, propyl gallate. bC, b-carotene bleaching method; DPPH, a,a-diphenyl-b-picrylhydrazyl radical scavenging assay; FIC, ferrous ion-chelating assay; FTC, antioxidant activity in the linoleic acid system with ferrothiocyanate reagent; HOSC, hydroxyl radical scavenging assay; HPS, hydrogen peroxide scavenging assay; LPPO, lipid peroxidation in peanut oil; ORAC, oxygen-radical absorbance capacity; PM, phosphomolybdenum method; RP, reducing power; SRS, superoxide radical scavenging assay; TA, total antioxidant capacity assay; TBARS, thiobarbituric acid-reacting substances; TEAC, Trolox equivalent antioxidant capacity assay.



Extraction of natural antioxidants from plant foods 531 (enzyme-assisted aqueous extraction, some novel methods including subcritical water, pressurized liquid extraction, supercritical fluid extraction (SFE) with CO2, and ultrasound- and microwave-assisted processes) are considered in the following subsections. 18.5.2 Enzyme-aided extraction Enzyme processing is used as a pretreatment for conventional and alternative solvent extraction technologies, owing to the ability of enzymes to disrupt cell walls. Enzyme-aided extraction is considered more environmentally friendly than chemical treatments, and the further utilization of both residue and extract is possible (Mandalari et al., 2006). Enzyme treatments have been considered for a variety of materials, including algae, fruits, herbs and cereals. Edible and other abundant and underutilized seaweeds have been studied for extracting water-soluble compounds with antioxidant activity. Most antioxidant compounds are soluble in polar solvents, but the large amounts of highly viscous polysaccharides act as a physical barrier. Enzymes can break down the cell walls and storage materials to release both free compounds and polysaccharides attached to polyphenols (Heo et al., 2005). Representative studies of antioxidant extraction from algae biomass using hydrolytic enzymes are summarized in Table 18.8. Synergistic effects between different enzyme activities (including endopeptidases, carbohydrases and proteases) enhance the release of antioxidants (Siriwardhana et al., 2008; Heo et al., 2005). The variables affecting the extraction yield and activity include: type of raw material and pre-conditioning stages, temperature, type and concentration of solvent, and type and concentration of enzyme(s). This latter effect is shown in Fig. 18.2. Incubation results in increased extraction efficacy (Siriwardhana et al., 2008). Thermal degradation of high-MW polysaccharides enables higher extraction of target antioxidants. Some enzymatic hydrolysates are stable at 100 °C (Athukorala et al., 2006; Heo et al., 2005) and the DPPH radical scavenging activity increases with heating time (Siriwardhana et al., 2008). Heating followed by enzymatic hydrolysis enhances the access of endopeptidases and b-glucanase to laminarin and proteins. Increased antioxidant activity at alkaline pH was observed in Hizikia fusiformis, owing to hydrolysis, ion exchange, desulfation and prevention of the formation of the protein–polyphenol complex (Siriwardhana et al., 2008). A simple procedure to reduce the viscosity of porphyrans of (sulfated polysaccharide of Porphyra) various MW, consisting of treatments with hydroxyl radical, has been reported (Zhang et al., 2003). Algal enzymatic extracts show a variety of other biological activities. The enzymatic extract of Ecklonia cava inhibited proliferation of cancer cells strongly and selectively (Athukorala et al., 2006), and showed in vivo anti-inflammatory, inmunomodulatory and antiproliferative activity (Ahn et al., 2008). Highly sulfated fucose and galactose polysaccharides from


Table 18.8 Examples of enzyme-aided extraction of plant food antioxidants

Algae Ecklonia cava Hizikia fusiformis © Woodhead Publishing Limited, 2010

Ishige okamurae Sargassum fullvelum Sargassum horneri Sargassum coreanum Sargassum thunbergii Scytosipon lomentaria Undaria pinnatifida

Terrestrial plants Buckwheat (Fagopyrum esculentum) Caroleo (Olea europaea)

Enzymes



Reference

Viscozyme Alcalase 2.4L FG Alcalase and Ultraflo

1352 mg GAE g–1 1095 mg GAE g–1

DPPH; HO˙; O2˙; H2O2

Heo et al., 2005

DPPH; H2O2

Ultraflo L Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Viscozyme Protamex Viscozyme Alcalase 2.4L FG Ultraflo L Alcalase 2.4L FG Alcalase, Flavourzyme, Promozyme, Viscozyme, Celluclast

275 mg GAE g–1 420 mg GAE g–1 313 mg GAE g–1 366 mg GAE g–1 384 mg GAE g–1 533 mg GAE g–1 1123 mg GAE g–1 996 mg GAE g–1 386 mg GAE g–1 416 mg GAE g–1 149 mg GAE g–1 207 mg GAE g–1


Siriwardhana et al., 2008 Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005


Heo et al., 2005

DPPH; HO–; O2–

Je et al., 2009

Alcalase

0.371 mg R g–1

DPPH; reducing power; inhibition of LA peroxidation

Tang et al., 2009

Bioliva

234.1 mg CA kg–1

Oxidative stability; peroxide value Ranalli et al., 2003b



Leccino (Olea europaea) Leccino (Olea europaea) Lemon peel (Citrus limon cv. Meyer) Lemon peel (Citrus limon cv. Yen Ben) Mandarin peel (Citrus reticulara cv. Ellendale)

Bioliva

362.2 mg CA kg–1


Rapidase adex D

128 mg CA kg–1


Cytolase

170 mg CA kg–1


Cytolase

79 mg CA kg–1


Rapidase adex D

92 mg CA kg–1


Cellulase

220 mg F g–1

Huang et al., 2009

Pectinases and cellulases

89 mg CA g–1

HO˙; O2–; inhibition of lipid peroxidation; reducing power DPPH; oxidative stability

Cellulase MX Cellulase CL Kleerase AFP Bioliva

1.52 mg GAE g–1 1.30 mg GAE g–1 1.25 mg GAE g–1 121.2 mg CA kg–1

FRAP

Li et al., 2006

Cytolase

121 mg CA kg–1

Oxidative stability

Ranalli et al., 1999

Cellulase MX Cellulase CL Kleerase AFP Cellulase MX Cellulase CL Kleerase AFP Cellulase MX Cellulase CL Kleerase AFP

0.4429 mg GAE g–1 0.4895 mg GAE g–1 0.4375 mg GAE g–1 1.131 mg GAE g–1 0.99 mg GAE g–1 1.108 mg GAE g–1 1.39 mg GAE g–1 1.15 mg GAE g–1 1.19 mg GAE g–1

FRAP

Li et al., 2006

FRAP

Li et al., 2006

FRAP

Li et al., 2006

Ramadan et al., 2008




Coratina (Olea europaea) Coratina (Olea europaea) Coratina (Olea europaea) Dritta (Olea europaea) Dritta (Olea europaea) Eucommia leaf (Folium eucommiae) Goldenberry (Physalis peruviana) Grapefruit peel (Citrus x paradisi)



Enzymes



Moringa concanensis seeds

1.283 mg T kg–1 1.236 mg T kg–1 1.176 mg T kg–1

Oxidative stability; peroxide value; Latif and Anwar, conjugated diene/triene 2009

Sunflower (Helianthus annuus)

Kemzyme Natuzyme Feedzyme Protex 7L Kemzyme Alcalase 2.4L Viscozyme L Natuzyme

Sweet orange peel (Citrus sinensis cv. Navel)

Cellulase MX Cellulase CL Kleerase AFP

0.80 mg GAE g–1 0.79 mg GAE g–1 0.90 mg GAE g–1

FRAP

Li et al., 2006

Macer8 Fj (M) Grindamyl pectinase (G) (M) + (G) Olivex and Celluclast

358 mg GAE L–1 237 mg GAE L–1 383 mg GAE L–1 5.2 g C kg–1

Conjugated diene

Landbo and Meyer, 2001

DPPH

Soto et al., 2008

Grindamyl pectinase (G) Celluclast (C) (G) + (C) Ultrazym 100G and Cellubrix Ferulic acid esterase

3072 mg GAE L–1 2773 mg GAE L–1 3017 mg GAE L–1 0.25 g C g–1

Conjugated diene

Meyer et al., 1998

DPPH

Collao et al., 2007

6937 mg SAE g–1

DPPH; conjugated diene; hydroperoxide; hexanal; liposome model system

Vuorela et al., 2004

Residues Blackcurrant pomace (Ribes nigrum) Borage seeds (Borago officinalis) Grape seeds pomace (Vitis vinifera) Evening primrose seeds (Oenothera biennis) Rapeseed (Brassica rapa)

13 14 13 15 13

mg mg mg mg mg

GAE GAE GAE GAE GAE

g–1 g–1 g–1 g–1 g–1

Reference

DPPH; inhibition of LA; oxidation Latif and Anwar, 2009

C, catechin; CA, caffeic acid; F, flavonoid; GAE, gallic acid equivalents; R, rutin; SAE, sinapic acid equivalents; T, tocopherol.



Extraction of natural antioxidants from plant foods 535 Extractable compounds (mg/100 g)

4.5

1

4.0 3.5

2

3

4

3.0 2.5 5

2.0 1.5

7

1.0

9

0.5 0

6

8

10 0

2

4 6 Enzyme concentration (%)

8

10

Fig. 18.2 Effect of enzyme concentration on the yield of extractable compounds. 1: Bilberry (Biopectinase CCN) (Puupponen-Piniä et al., 2008). 2: Bilberry (Pectinex BE 3L) (Puupponen-Piniä et al., 2008). 3: Blackcurrant (Biopectinase CCN) (PuupponenPiniä et al., 2008). 4: Blackcurrant (Pectinex BE 3L) (Puupponen-Piniä et al., 2008). 5: Pigeonpea (Fu et al., 2008); lutein (mg/100 g) ¥ 10–1; enzyme concentration ¥ 10–2. 6: Grape pomace (Maier et al., 2008); enzyme concentration ¥ 10–1; 7: Apple pomace (Pinelo et al., 2008). 8: Pigeonpea (Fu et al., 2008); apigenin ¥ 10–1; enzyme concentration ¥ 10–2. 9: Meyer lemon (Li et al., 2006). 10: Yen Ben lemon (Li et al., 2006).

Ecklonia cava showed antiproliferative action (Athukorala et al., 2006). The phlorotannin derivatives of this alga exhibited inhibitory effect on human immunodeficiency virus type 1 reverse transcriptase and protease (Ahn et al., 2004). Enzymatic extracts of Stellaria dichotoma inhibited the in vitro hydroxyl radical-induced DNA damage (Lim et al., 2008). A peptide with ACE-inhibitory activity was isolated from the pepsin hydrolyzate of Chlorella vulgaris waste (Sheih et al., 2009), whereas some glucans (hexaose, schizophyllan, and laminarin) showed inhibition against AAPH (Tsiapali et al., 2001). The major antioxidants of fruits and juices are phenolic compounds. Phenols may be found in cell walls, bound to polysaccharides by hydrophobic interactions and hydrogen bonds, linked to the protein matrix of vacuolar inclusions, confined inside the cellular vacuoles, and near or associated with the cell nucleus (Pinelo et al., 2006). Plant cell walls are a complex network of cellulose, hemicelluloses (mainly of xyloglucans, mannans, xylans, and arabinogalactans), pectins and lignin (a hydrophobic polymer derived from p-coumaryl, coniferyl, and sinapyl alcohols). Degradation of the cell-wall polysaccharide structure is a fundamental step in the release of phenols linked to the cell wall or associated with cell vacuoles. Ferulic and p-coumaric acids, the major lignin monomers that link hemicellulose sugars and lignin, are potent in vitro antioxidants (Meyer et al., 1998; Nardini et al., 1995). Enzymatic degradation of cell-wall polysaccharides is assumed to increase © Woodhead Publishing Limited, 2010

536 Separation, extraction and concentration processes the overall substrate porosity, facilitating solvent penetration and extraction efficiency, but lignin and tannins may block the enzyme action. The use of enzymes that degrade plant cell walls to enhance the extraction of phenolic antioxidant compounds has been reported for food products and by-products. Although no interactions between proteases and pectinases was confirmed for blackcurrant juice production, degradation of the protein network enhanced both substrate porosity and phenol recovery (Landbo and Meyer, 2001). In grape skins, cellulases, hemicellulases, pectinases, and other hydrolytic enzymes can be used (Pinelo et al., 2006). Despite the high cellulose content of the grape cell wall, cellulolytic activity did not influence the extraction yield of phenols from pomace (Meyer et al., 1998), but pectinases, cellulases and proteases increased the extraction yields from apple skin (Pinelo et al., 2008). Preferential enzymecatalyzed solubilization of pectin and cellulose from bergamot peel caused the selective release of glucose and galacturonic acid (Mandalari et al., 2006). Glucanases and pectinases were used for processing citrus peels (Li et al., 2006). Neutral protease, papain and alkaline protease have been assayed on the fruit of Physalis alkekengi (Ge et al., 2009), whereas culture broths of Aspergillus niger enriched in cinnamoyl esterases were used to release hydroxytyrosol from olive oil by-products (Bouzid et al., 2005). The combined effect of the major operational variables (including particle size, time, temperature, and enzyme to substrate ratio) is evaluated using experimental designs (Meyer et al., 1998). The particle size is relevant for degrading plant cell wall carbohydrates, as well as for solvent penetration and solute recovery. A favorable effect of the reduction of particle size on the extraction of phenolics was reported for spent coffee grounds (Pinelo et al., 2007), blackcurrant pomace (Landbo and Meyer, 2001), apple skin (Pinelo et al., 2008), and citrus peels (Li et al., 2006). Reduced particle size favored the degradation of polysaccharides by pectinase without affecting the recovery of phenols (Meyer et al., 1998). Although a beneficial effect of high enzyme concentration (5%) was reported for citrus peel, the optimal value for this material was 1–3% (Li et al., 2006). Increased enzyme dosage enabled higher juice yields and phenol concentrations from blackcurrant mash. However, the non-enzyme-treated juices exhibited higher antioxidant activity than the enzyme-treated ones, presumably because of differences in their phenolic profiles (Bagger-Jørgensen and Meyer, 2004). The effects of enzymatic treatments were different for various citrus fruits (lemon, mandarin, grapefruit and orange) (Li et al., 2006), and various effects of temperature, particle size reduction and ethanol concentration were reported for the extraction of individual compounds from apple skins (Pinelo et al., 2008). Optimal temperatures of 50–60 °C were reported for citrus peel (Li et al., 2006), apple skins (Pinelo et al., 2008), and elderberry (Landbo et al., 2007). Prolonged incubation time (24 h) with pectinolytic and cellulolytic enzymes was necessary to solubilize carbohydrates and flavonoids from bergamot peel (Mandalari et al., 2006), but a negative effect of long reaction


Extraction of natural antioxidants from plant foods 537 times was observed for grape owing to degradation of phenolic compounds (Meyer et al., 1998). Enzyme-assisted aqueous of citrus peels required an incubation period of 3 h, compared with 6 h for water extraction without enzymes (Li et al., 2006). The amount of quercetin in enzyme-treated juices increased with incubation time, but the quercetin content did not involve a higher antioxidant activity (Sun et al., 2005). The yields of soluble carbohydrates were higher in enzyme-treated samples than in untreated ones. A positive linear correlation between extent of cell wall degradation and phenol extraction was observed for grape pomace (Meyer et al., 1998), elderberry (Bagger-Jørgensen and Meyer, 2004), and blackcurrants (Bagger-Jørgensen and Meyer, 2004; Landbo and Meyer, 2001), but no significant differences were found in carbohydrate degradation, distribution of phenolics and release of anthocyanins and total phenols (Landbo et al., 2007). Similarly, pectinolysis affected both juice yield and contents of phenolic acids, flavonoids and anthocyanins (Kaack et al., 2008). Even though the amount of phenols extracted from the pomace is enhanced, specific compounds may decrease: for example, enzyme-mediated anthocyanin degradation may occur owing to enzymatic hydrolysis of glycosylated anthocyanins by polyphenol oxidases. Pectinase preparations produced by Aspergillus niger, containing b-glucosidase, b-galactosidase, and a-l-arabinosidase, affect anthocyanin pigments and color quality (Landbo and Meyer, 2001). Despite b-galactosidase and b-glucosidase causing color degradation, benefits for extraction of potent antioxidants from grape pomace were reported (Meyer et al., 1998). Enzymatic release of esterified hydroxycinnamic acids can be achieved by synergistic action of cell-wall-degrading enzymes (esterases and xylanases). Commercial pectinases facilitate the release of ferulic acid and other phenolic acids from ground rye grain (Andreasen et al., 1999). A cold pressing process was used for defatting borago before extracting phenolics with conventional solvents. Enzymatic pretreatment of seeds enhanced the extraction of phenolic compounds with DPPH radical scavenging capacity from the meal; the tocopherol content of the resulting oil was maintained by cold pressing (Soto et al., 2008). Treatments with cell-wall-degrading enzymes were proposed for releasing ferulic acid from agro-industrial byproducts such as sugar beet pulp or maize bran (Bouzid et al., 2005). Other less studied materials include microbial sources. Saccharomyces cerevisiae b-glucan, isolated by hot water and enzyme treatment, exhibited antioxidant activity (Jaehrig et al., 2007). Protease treatments can be included within the enzyme-assisted extraction methods. Relatively short bioactive peptides (2–9 amino acids) can be obtained from sources such as wheat, corn, soybean and mushrooms (Dziuba et al., 2004). Commercial proteases have been used to process a number of substrates (Tang et al., 2009; Wang et al., 2007). Hydrolysis increased ACE inhibition and the radical-scavenging activity of protein hydrolyzates from potato isolates and by-products, possibly related to peptides and /or


538 Separation, extraction and concentration processes free amino acids liberated during digestion (Pihlanto et al., 2008). Rapeseed protein hydrolyzates present DPPH radical-scavenging activity, provide protection against the peroxidation of lipids and/or fatty acids (Amarowicz and Shahidi, 1997; Peña-Ramos and Xiong, 2002; Sakanaka et al., 2004) and show reducing power and Fe2+ chelating ability (Moure et al., 2006; Tang et al., 2009). Esterified phenolic acids (p-coumaric acid, ferulic acid) present in sugarcane bagasse were released by alkaline hydrolysis (Ou et al., 2009). Ferulic acid (FA), a scavenger of free radicals approved in certain countries as a food additive to prevent lipid peroxidation (Srinivasan et al., 2007), can be extracted using enzymatic, alkaline or acidic extractions. 18.5.3 Pressurized-liquid extraction Pressurized-liquid extraction (PLE) is an efficient, innovative and environmentally clean technique, which is performed at high temperature and pressure to maintain the solvent in the liquid state. Compared with traditional extraction techniques, it is faster and requires lower amounts of solvent. Alternatively, it is known as accelerated solvent extraction (ASE), or high-pressure liquid extraction (HPE) (Adil et al., 2008). The structure, activity and properties of the extracts are usually unaffected in an oxygenfree and light-free environment, and micro-organisms and enzymes may be inactivated (Qadir et al., 2009). The influence of HPE on the antioxidant capacity of food products was recently reviewed by Oey et al. (2008). PLE was proposed for the extraction of a variety of natural products and wastes, including the production of anthocyanins from grape skins (Ju and Howard 2003), flavonoids from spinach (Howard and Pandjaitan, 2008) and processing of berry substrates (King et al., 2003). The use of non-toxic solvents (water and ethanol) offers environmental advantages. Ethanol–water mixtures gave high extraction yields from Phormidium species (Rodríguez-Meizoso et al., 2008), sour cherry pomace (Adil et al., 2008), and dried spinach (Howard and Pandjaitan, 2008); whereas methanol was used for extracting catechin and epicatechin from tea leaves and grape seeds (García-Marino et al., 2006; Piñeiro et al., 2004), and flavonoids from apple extracts (Alonso-Salces et al., 2001). The compounds recovered and their extraction efficiency differed with the solvent, providing different properties to the extracts, including color and odor compounds (Howard and Pandjaitan, 2008). The amount of carotenoids extracted from Dunaliella salina with hexane was more than seven times higher than the value extracted with ethanol, but the antioxidant activity was only twice as high owing to structural differences (Herrero et al., 2006). Combinations of pressure, temperature, solid/solvent ratio and extraction time can be addressed by experimental designs (Adil et al., 2008; Herrero et al., 2006). Temperature, vapor pressure of the most volatile compounds and improved mass transfer have been reported as the major variables involved in


Extraction of natural antioxidants from plant foods 539 PLE regardless the solvent used (Rodríguez-Meizoso et al., 2008). Temperature was the most influential factor in the extraction of antioxidant compounds from Dunaliella salina (Herrero et al., 2006). The optimal temperature for water and ethanolic extraction of Spinacia oleracea flavonoids was 50–130 °C for water, and 50–150 °C for ethanol (Howard and Pandjaitan, 2008), compared with 100–130 °C for PLE processing of grape by-products (Ju and Howard 2003; Piñeiro et al., 2004) and berries (King et al., 2003). The optimal temperature depends on the solvent considered. Optimal antioxidant activity of Phormidium species in hexane extracts was obtained operating at 50 °C, whereas in ethanol and water extracts it was observed at 150 and 200 °C, respectively (Rodríguez-Meizoso et al., 2008). Thermal degradation of apple polyphenols was observed at extraction temperatures higher than 60 °C in methanol, whereas activity decreased at 110–190 °C in water and ethanol (Alonso-Salces et al., 2001). Thermal degradation of flavonoids was reported for extraction temperatures >130 °C (Howard and Pandjaitan, 2008). The reduction in antioxidant activity observed at prolonged extraction times during PLE of Dunaliella salina with hexane and ethanol suggested some kind of carotenoid degradation (Herrero et al., 2006). HPE extraction can give a moderately high extraction efficiency of bioactive compounds in a short time, but treatments for prolonged periods could result in degradation (Qadir et al., 2009). The development of brown, highly polar, odoriferous compounds with high antioxidant capacity at high extraction temperatures suggested the participation of Maillard reactions in ethanolic (Howard and Pandjaitan, 2008) and in water extracts (Rodríguez-Meizoso et al., 2008). Extracts of propolis obtained by high hydrostatic pressure extraction in 1 min presented similar properties to the ethanolic extracts obtained by leaching at room temperature for a few days (Jun, 2006). No degradation of compounds present in ethanolic extracts occurred after exposure to light and air at room temperature for 2 days (Herrero et al., 2006). Other reported biological activities include cytotoxic effects on the growth of human promyelocytic leukemia cells (HL-60) of sweet potato extracts (Rabah et al., 2005), toxicity on preformed monolayers of Vero cells (African green monkey kidney cell line) of Phormidium extracts from PLE (Rodríguez-Meizoso et al., 2008). Antimicrobial activity of Phormidium sp. extracts has been ascribed to terpenes (i.e., b-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) (Rodríguez-Meizoso et al., 2008). Subcritical-water extraction (SWE) is an environmentally friendly technology for the selective extraction of bioactive compounds from plant materials with advantages derived from its simplicity, reduced extraction time, high quality of the extract, low cost of the extracting solvent and possibility of scaling-up (Rodríguez-Meizoso et al., 2006). Extract with water under pressure (between 100 °C and the critical temperature of 374 °C) is also known as superheated water extraction, subcritical water extraction, high-temperature water extraction, pressurized hot water extraction, or hot


540 Separation, extraction and concentration processes water extraction. Most studies have been performed in batch mode on a small scale, but continuous flow equipment has also been described (Budrat and Shotipruk, 2008). The feedstocks studied include fruits or vegetables and their processing wastes, seeds, herbs and algae (Table 18.9). SWE was more efficient than conventional solvents for extracting flavanols from grape seeds (García-Marino et al., 2006) or antioxidant phenolics from bitter melon (Budrat and Shotipruk, 2008), and was sucessfully employed to concentrate and isolate antioxidant compounds from oregano leaves (Rodríguez-Meizoso et al., 2006). Temperature has a marked effect on yield and selectivity. The dielectric constant of water decreases with temperature, enabling the extraction of compounds having different polarities at different temperatures. High temperature also enhances diffusivity, facilitating the transport of solutes from the solid matrix, and a compromise to avoid thermal degradation must be reached. The release of hydroxycinnamates from cell walls is favored by temperature, but anthocyanins undergo degradation reactions. Lignin decomposes in sub- and supercritical water to give phenol, which are degraded at high temperatures (Budrat and Shotipruk, 2008; Garrote et al., 2003). Sequential extraction with stepwise pressure increase was proposed for processing black tea leaves (Chambers et al., 1984) as well as to recover quercetin glycosides from onion waste (Turner et al., 2006), and catechins and proanthocyanidins from winery by-products (García-Marino et al., 2006). The major variable is temperature, because at 150 °C or above, substrates are hydrolyzed, and the solvent polarity decreases. Optimization of operational conditions has to be carried out for each material. The yield of total phenolics from sweet potato and bitter melon increased with temperature (Budrat and Shotipruk, 2008; Rabah et al., 2005). Although higher temperatures resulted in higher amounts of extracted phenolics, their antioxidant activity was higher operating at lower temperatures (Budrat and Shotipruk, 2008). Carnosic acid, the most potent antioxidant in rosemary, was preferentially extracted at 200 °C (Rodríguez-Meizoso et al., 2006). Extraction yields of oregano leaves increased with temperature: at the lowest temperature considered, the more polar compounds (flavanones and dihydroflavonol structures such as dihydroquercetin, eriodictyol and dihydrokaempferol) were preferentially extracted, whereas less polar compounds could also be extracted at 200 °C (Rodríguez-Meizoso et al., 2006). The phenolic content was a maximum at 25 °C and a minimum at 200 °C, but the highest antioxidant activities were achieved at 150–200 °C. Boiling ground coffee beans in water at 110 °C under elevated pressure gave an efficient extraction of antioxidants (Budryn and Nebesny, 2008). The total phenolic contents and the antioxidant activities of HPE extracts from longan fruit were higher than those obtained with conventional solvent extract (Prasad et al., 2009), whereas enhanced extraction of onion flavonols was observed at 100–400 MPa (Roldán-Marín et al., 2009). The conditions of SWE influence the degree of polymerization and structure


Table 18.9 Examples of extraction of plant food antioxidants using high pressure systems Raw material


Elderberry leaves (Sambucus nigra L.) Elderberry berries (Sambucus nigra L.) Elderberry flowers (Sambucus nigra L.) Phormidium species

Red grape skin (Vitis vinifera)


0.32 mg GAE g–1

DPPH

Bonoli et al., 2004

Fiber: 14.99 g GAE kg–1 Flour: 20.93 g GAE kg–1

DPPH

Papagiannopoulos et al., 2004

28.91 mg GAE g–1

DPPH ABTS FRAP DPPH bC bleaching


DPPH bC bleaching DPPH bC bleaching TEAC Antimicrobial activity

Dawidowicz et al., 2006 Dawidowicz et al., 2006 Rodríguez-Meizoso et al., 2008

ORAC

Ju and Howard, 2003

1 g; ECV(22); 17 (g F/100 g ¥ 10–2) SE: E:W (80:20); -; 10 min; 60 bar; 200 ºC; PLE: FV( 60% ECV); P(120) 20.18 (g F/100 g ¥ 10–2) 214.2 (g F/100 g ¥ 10–2) 1 g sample H: 20 min; 200 ºC; E: 20 min; 200 ºC; W: 20 min; 200 ºC; 0.2 g + 30 g sea sand; ECV(22); SE: acidified W; 3 cycles; 5 min; 10.1 MPa; 50 ºC PLE; FV (50% ECV); P(90)

6.54% 40.91% 22.81% 179.9 mg GAE g–1

Dawidowicz et al., 2006



Pressurized liquid extraction (PLE) Barley flour 2 g + 4 g Hydromatrix; ECV(33); (Hordeum vulgare L.) SE: E:W (4:1); 2 cycles; 5 min, 20 MPa 60 °C PLE: FV (60% ECV); P(60) Carob fruits 1 g + 2 g diatomaceous earth; (Ceratonia siliqua L.) SE: A:W (1:1); 2 cycles; 5 min; -; 60 °C; PLE: 50% FV; Cow Cockle seeds 2 g; (Saponaria vaccaria) W; 3 h; 125–175 °C; F(2)


Raw material


Spinach (Spinacia oleracea)

0.5 g + 30 g Ottawa sand 42 mmol GAE kg–1 SE: E:W (7:3); 3 cycles; 5 min; 13.8 MPa; -; PLE: R (70% ECV) 2.5 g; ECV(11) PE; 9 min; 170 ºC; 77.0 mg extract H; 15 min; 170 ºC; 95.5 mg extract E; 9 min; 170 ºC; 270.1 mg extract W; 15 min; 170 ºC; 290.0 mg extract 2.5 g; ECV(11) PE; 9 min; 170 ºC; 2.94% H; 9 min; 170 ºC; 4.28% E; 9 min; 170 ºC; 19.70%

Spirulina plantensis © Woodhead Publishing Limited, 2010

Spirulina plantensis


Antioxidant activity Reference ORAC

DPPH

DPPH

Howard and Pandjaitan, 2008 Herrero et al., 2004

Jaime et al., 2005

High pressure extraction (HPE) Korean barberry bark (Berberis koreana)

1 g mL–1; 15 min; 500 MPa; RT

50% E; 0.02 g mL–1; 30 min; 500 MPa; 50 ºC E; 0.06–0.07 g mL–1; 25 min; 176–193 MPa; 60 °C High pressurized fluid extraction (HPFE) Brazilian propolis RSL 1:10; 20% E; 40 g surfactant; 50 psig; 393 K Longan fruit pericarp (Arillus longan) Sour cherry pomace

11.04% (317.35 mg GAE g–1)

DPPH Inhibition of xanthine oxidase

Qadir et al., 2009

30% (23 mg GAE g–1)

Prasad et al., 2009

3.80 mg GAE g–1

DPPH O2– DPPH

Adil et al., 2008

25.2%

DPPH

Chen et al., 2007



Taiwan Red Pine (Pinus taiwanensis)

70% E; 180 min; 689 kPa; 343 K; F (10)

26.3 mg g–1

Taiwan White Pine (Pinus morrisonicola)

70% E; 180 min; 689 kPa; 343 K; F (10)

39.8 mg g–1

Propolis crude

RSL 1:35, E 75%; 1 min; 500 MPa; RT

Accelerated solvent extraction (ASE) Spirulina plantesis 2.5 g; ECV (11); 15 min; -; 170 °C

–

DPPH

Rieger et al., 2008

–

DPPH

Rieger et al., 2008

317 mmol GAE g–1 dm

ABTS


–

DPPH

Rieger et al., 2008

TPA: 0.20, TF: 7.10, TOD: DPPH ORAC 63.5 mg GAE g–1

Súarez et al., 2009

290 mg GAE g–1

bC bleaching DPPH

Xi, 2006

6.43 g GAE g–1

bC bleaching DPPH

Xi and Shouqin, 2007

H: 4.3%; PE: 4.01% E: 17.14%; W: 10.12%

DPPH

Herrero et al., 2005



High hydrostatic pressure (HPP) Bilberry ECV(11); (Vaccinium myrtillus) SE: 3 cycle, 7 min, 60 bar, 80 ºC; PLE: FV (100%), P (100) Elderberry ECV (11); (Sambucus nigra) SE: 3 cycle; 5 min; 68.9 bar; 60 °C; PLE: FV (100%), P (60) Grape skins RSL 1:4.5; E:W (1:1); 1 h; 600 MPa; (Dornfelder V. vinifera) 70° C; Heather ECV (11); (Calluna vulgaris) SE: 3 cycle, 5 min; 60 bar; 60 ºC PLE: FV (100%), P (60) Olive cake 30 g OC; ECV (100) E:W (80:20), FV (60% ECV), 2 cycles; 5 min Propolis extract RSL 1:35, E 75%; 1 min; 500 MPa; RT

DPPH, Fe Lin et al., 2009 chelating, reducing ability, O2–, NO DPPH, Fe Lin et al., 2009 chelating, reducing ability, O2– NO

Raw material





Hot water extraction (HWE) Canola meal RSL 1:16.7; W; 30 min; -; 80 °C (Brassica napus)

0.15 g g–1; 7.83 g SA g–1

Citrus peels

RSL 1:20; bddW; 30 min; -; 100 ºC

~32.5 mg GAE g–1 dw

Formosa koa leaves (Acacia confusa)

bdd H2O; 4 h; -; -

7.3 g

Green tea

RSL 1:100; dW; 5–30 min; -; 80 ºC

Herbal tea (Anagallis arvensis) Japanese horse chestnut (Aesculus turbinata ) Kaffir lime fruit peel (Citrus hystrix) Lettuce extract (Lactuca sativa) Mallotus japonicus leaves

RSL 1:166.7; bW; 5 min;- ; -

TP: 2050 mg GAE L–1 TF: 1700 mg GAE L–1 1.47 mmol Trolox g–1

RSL 1:100; bW; 2 h

1560 mg (280 mg EE)

DPPH TEAC Reducing power bC bleaching DPPH FRAP DPPH NBT Reducing power FRAP ABTS CUPRAC ABTS DPPH

0.2 g; ECV (10.8); 15 min; 200 ºC

23.7 mg GAE mL–1

DPPH

RSL 1:2; W; 80 ºC; 10 min

–

RSL 1:50 mL; bdW; 20 min

7.5 mg GAE mL–1

Morinda citrifolia roots

0.5g, ECV(10); 4 MPa; 2 h; 170 °C F (5)

92.55%

Peroxidation of liposomes DPPH O2– HO· DPPH

Hassas-Roudsari et al., 2009 Xu et al., 2008 Tung et al., 2009 Rusak et al., 2008 Apak et al., 2006 Ogawa et al., 2008 Khuwijitjaru et al., 2008 Altunkaya et al., 2008 Tabata et al., 2009 Pongnaravane et al., 2006



Samor thai fruits (Terminalia chebula Retz.) Sasa palmata leaf

15.96 mg TP g–1 dw

ABTS

Rangsriwong et al., 2009

SE: 50 g; steam; 0.5–20 min; 180–260 °C (1.0–4.9 MPa) HWE: RSL 1:100; dW; 2 h; 98 °C RSL 1:100; dW; 2 h; 98 ºC

128.24 mg GAE g–1

DPPH

Kurosumi et al., 2007

12.08 mg GAE g–1

DPPH

Kurosumi et al., 2007 Spigno and De Faveri, 2009 Rusak et al., 2008

Tea

RSL 1:100; bW; 30–210 s

White tea

RSL 1:100; dW; 5–30 min; 80 ºC

Hot pressurized water extraction (HPWE) Boldo RSL 1:10; W; 3 h; 110 ºC (Peumus boldus M.) Sage 0.15 g + sea sand; 60 min; 100 kg cm–2; 100 ºC; F(1) (Salvia officinalis) Yam (Dioscorea alata)

LSR: 20 kg kg–1; ECV(1000); W; 3 h; 1.34 MPa; 120 ºC; F(10)

Subcritical water extraction (SWE) Bitter melon 1 g; ECV (10); (Momordica charantia) 3 mL min–1; 60 min, 200 °C, 10 MPa Canola meal 30 min; 6.89 MPa; 160 °C; F(1)

ABTS TP: 1300 mg GAE L–1 TF: 900 mg GAE L–1

FRAP ABTS

51.4% 5.2 mg B kg–1

ABTS DPPH

Del Valle et al., 2005 Ollanketo et al., 2002

2.143 g kg–1

DPPH

Chen et al., 2004

53 mg GAE g–1

ABTS

0.45 g g–1 15.42 g SA g–1

TEAC bC bleaching DPPH Reducing power

Budrat and Shotipruk, 2008 Hassas-Roudsari et al., 2009



RSL 1:150; 100 °C; 120 min


Raw material




Eucalyptus leaves (Eucalyptus globulus) Licorice roots (Glycyrrhiza glabra) Oregano (Origanum vulgare)

60 g; ECV (300) 200 mL W; 30 min; 4.91 MPa; 200 ºC 0.1 g; ECV (18.57) 10 mL W; 60 min; 0.002–5 MPa; 300 °C 0.75 g; ECV (11) SE:W; 15–30 min; 1500 psi; 25–200 °C; SWE: R (70% ECV) 0.40 g + 30 g sea sand; ECV (22) SE: 1 cycle; 40 s; – 110 ºC; SWE: R (70% ECV), P (90) 1.0 g; ECV (10) 37.5 min; 4 MPa; 180 °C; F (4) RSL 1:50; 10–60 min; 0.47–4.76 bar; 80–150 °C

4.8 mg g–1

Peroxynitrite

1521.3 ppm GAE 54% (0.149 g GAE g–1)

Radical scavenging Baek et al., 2008 Reducing power DPPH Rodriguez-Meizoso et al., 2006

52.3 mg TP g–1

ORAC

Ju and Howard, 2005

21.43 mg TP g–1

ABTS

0.73 mg TC g–1 dm 5.1 mg TF g–1 dm

ABTS

Rangsriwong et al., 2009 Prommuak et al., 2008

Red grape skin Samor thai fruits (Terminalia chebula) Thai silk waste *

Kulkarni et al., 2008

Total extractable polyphenols. B, boldine; bdW, boiled distilled water; bddW, boiled double distilled water; bW, boiled water; bC, b-carotene bleaching; dm, dry matter; dW, distilled water; E, ethanol; ECV, extraction cell volume; EE, epicatechin equivalent; F, flux (mL/min); FV, flush volume; GAE, gallic acid equivalent; H, hexane; LSR, liquid–solid ratio; P, purge (s); PE, petroleum ether; PLE, pressurized liquid extraction; pm, plant material; R, rising; RT, room temperature; SE, static extraction; TC, total carotenoids; TF, total flavonoids; TP, total phenolics; W, water.



Extraction of natural antioxidants from plant foods 547 of molecules, as reported for procyanidin extraction (García-Marino et al., 2006). Enhanced recoveries of flavanol dimers and trimers, and gallic acid was obtained in greater quantities by a single extraction at 150 °C, in which gallic acid accounted for 61% of total phenolics. Higher temperatures favored the extraction of oligomeric fractions. When the galloylated moieties were situated in the terminal sub-unit, sequential extraction (50–100 °C) was preferable, whereas a single extraction at 150 °C was better for the rest of the galloylated derivatives (García-Marino et al., 2006). Treatment of lignocellulosic substrates with water or steam at 160–240 °C (autohydrolysis processing) results in depolymerization of hemicelluloses and in breakage of lignin-carbohydrate bonds, leading to solubilization of lignin fragments of low MW. The aqueous treatments of lignocellulosics for both the hydrolytic degradation of hemicelluloses and the solubilization of antioxidant compounds have been reviewed (Garrote et al., 2004; Meireles, 2009). Other related technologies, based on the utilization of chemicals (for example, solvents, oxygen or acids) added to water, were proposed to provide higher extraction yields. Steam treatments were efficient in releasing hydroxytyrosol and 3,4-dihydroxyphenylglycol (DHPG), from the semisolid waste of olive oil extraction systems (alperujo) (Fernández-Bolaños et al., 2002; Rodríguez et al., 2009). The amount of solubilized hydroxytyrosol increased with temperature and time, reaching 1.4−1.7 g/100 g of dry alperujo (FernándezBolaños et al., 2002). 18.5.4 Supercritical-fluid extraction Supercritical-fluid extraction (SFE), based on the utilization of a fluid under supercritical conditions, is suitable for the extraction and purification of natural compounds with bioactive and antioxidant properties. The interest in SFE is promoted by increasingly restrictive environmental, toxicological, and health regulations. Carbon dioxide shows favorable properties, including environmental safety, high availability at low cost, high purity, and suitability for extracting heat-labile, natural compounds with low volatility and polarity. The physicochemical properties of supercritical CO2 (high diffusivity, low viscosity and low surface tension compared with conventional solvents) facilitate mass transfer, enabling an environmentally friendly operation. The properties of the final product are improved by high selectivity, low processing temperatures, absence of light and oxygen, and absence of solvent in extracts. The extracts are regarded as natural, and do not require additional sterilization. The major disadvantages of supercritical CO2 are the high critical pressure (requiring expensive equipment), and the poor solvent power derived from its low polarity. The use of cosolvents enhances the solubility of some compounds and may increase the extraction selectivity, allowing operation at a lower pressure. Representative antioxidant compounds extracted by SFE are summarized


548 Separation, extraction and concentration processes in Table 18.10. SFE successfully recovered phenolic compounds from liquid solutions (Gamse, 2004), and countercurrent contact has been employed for processing propolis (Wang et al., 2004), B. kaoi (Wang et al., 2005) and orange juice (Señoráns et al., 2001). Studies on the SFE of bioactive compounds with antioxidant activity are available (Del Valle et al., 2005; Díaz-Reinoso et al., 2006; Herrero et al., 2006; Meireles, 2003; Mukhopadhyay, 2000; Reverchon, 1997). The most influential variables in SFE of compounds with antioxidant properties from natural sources are summarized here. The drying technique (Mechanical and thermal conditioning) affects both the structure of the solid matrix and the thermal stability of the active compounds. Mass transfer is facilitated by a higher degree of grinding, but the performance of fixed beds during leaching could be limited by too fine particles. Pressure and temperature are the variables that determine the solubility equilibrium, an understanding of which is essential for process design. Results are mostly available for a single solute in the supercritical solvent, but the presence of other solutes may affect the equilibrium, and the natural products are a complex mixture of compounds. Studies dealing with the solubility of solid mixtures of phenolic compounds in supercritical CO2 have been reported (Del Valle et al., 1998; Díaz-Reinoso et al., 2006; Fornari et al., 2005; Lucien and Foster, 2000; Reverchon, 1997). Solvent density increases with pressure, but beyond a certain threshold, the increased solvent viscosity reduces the diffusion coefficients. When the target compounds are lipophilic, the operational conditions may be milder than those required for phenolic antioxidants. Highly labile compounds require mild temperatures (below 50 °C) to avoid alteration. A stepwise increase in the extraction pressure allows selective extraction of different compounds. The presence of a modifier increases the solvent density resulting in a higher interaction of solutes with the solvent, causing alterations and swelling of the vegetal matrix. A suitable cosolvent may improve the extraction yield and selectivity. However, cosolvents of hazardous nature or resulting in decreased selectivity should be avoided. The common modifiers used in SFE are alcohols, which induce dipole/dipole interactions and hydrogen bonding with polar functional groups. Ethanol has been employed to increase the solubility of ginsenoids (Wang et al., 2001), antioxidants from tamarind seed coat (Luengthanaphol et al., 2004), Eucalyptus (El-Ghorab et al., 2003) and olive leaves (Tabera et al., 2004), lipophilic compounds from marigold (Baumann et al., 2004), and flavonoids and terpenoids from Ginko biloba (Yang et al., 2002). Methanol was used for the extraction of magnolol (Dean et al., 1998), soy isoflavones (Rostagno et al., 2002), phenolics from chamomille (Scalia et al., 1999), pistachio hulls (Goli et al., 2005) or aloe extracts (Hu et al., 2005). Isopropanol was used for the extraction of ginger oleoresin (Zancan et al., 2002). Water is the cosolvent employed in the industrial extraction of vanilla. The extracts resulting from the unselective extraction of compounds at high pressure can be fractionated in multiple-stage separators. The pressure and


Table 18.10 Examples of extraction of plant food antioxidants using SFE Extraction yield (total extract, TE and active compounds)


Reference

Aloe vera leaf skin (Aloe barbadensis) Baical skullcap root (Scutellaria baicalensis) Boldo (Peumus boldus) Boldo (P. boldus) Coriander seeds (Coriandrum sativum) Eucalyptus leaf oil (Eucalyptus camaldulensis) Eucalyptus leaves (E. camaldulensis)

TF = 1.50 TF = 0.27

DPPH DPPH

TE = 2.9; B (0.0031)e TE = 4.9; B (7.4)e TE = 1.92 MTp H (0.242); STp (0.0825) pCl (1.16); oHC (0.17); MEP (0.34); Thy (0.98)a TE = 16.6

TEAC ABTS DPPH Inhibition of LA oxidation Inhibition of LA oxidation

Hu et al., 2005 Bimbato and Tamanini, 2003 Del Valle et al., 2005 Del Valle et al., 2005 Yépez et al., 2002 Fadel et al., 1999

Ginger (Zingiber officinale)

Monoterpenes, sesquiterpenes and gingerols

b-C, LA

Leal et al., 2003

Ginger (Z. officinale)

TE = 2.31

b-C, LA

Zancan et al., 2002

Helichrysum dried flower heads (Helichrysum italicum) Hop (Humulus lupulus) Lemon balm (Melissa officinalis subs. officinalis) (M. officinalis subs. inodora) Marjoram (Origanum vulgare) Hungarian marjoram (O. majorana) Egyptian marjoram Nigella seeds (Nigella sativa)

Flavonoids; TE = 4.5–4.9

O2–;

Marongiu et al., 2003

TP = 0.52–3.79/TF = 0.2–0.92 TE = 1.9 TE = 0.7

LA oxidation LA oxidation

Lermusieau et al., 2001 Marongiu et al., 2004

TE = 3.2 TE = 3.76 TE = 5.39 T.O. (1.14)b; Tq (0.49); p-Cy (0.23)

Peroxide value Inhibition of oil oxidation

Uy et al., 1991 Vági et al., 2005

b-C, LA

Machmudah et al., 2005

DPPH; b-C, LA

El-Ghorab et al., 2003






Extraction yield (total extract, TE and active compounds)


Reference

Black pepper oleoresin (Piper nigrum)

RER = 5.43 (Pp: 39.4)

Tipsrisukond et al., 1998

Rosemary (Rosmarinus officinalis) Rosemary (R. officinalis)

TE = 5.2 TE = 7.15

Hexanal formation; TBARS Peroxide value

Rosemary (R. officinalis) Rosemary (R. officinalis)

Carnosic acid solubility TE = 4.5; Cm (39.6); Vbn (20.3); t-C (10.3)a


Sage (Salvia officinalis) Sage (S. officinalis)

F1: Rsl (3.6); Gk (11.9); Cr (5.6); CrA (66.0); MCr(1.6)a F2: Rsl (8.0); Gk (0.13); Cr (6.4); CrA (60.7); MCr(4.6)a TE = 1.6; Cm (0.025 ); Cn (0.001); CrA (0.236); RA(0.065)c TE = 5.7 TE = 5.02

Sage (S. officinalis)

TE = 46.26; F1 (024)/F2 (27.50)/F3 (18.52)


Black sesame seed (Sesamum indicum) Black sesame seed (S. indicum) Summer savory (Satureja hortensis)

TE = 51.83 Lignans (sesamin, sesamolin) S1: Crv (32); Lnl (24); Myc (22); Myl (21)c S2: Crv (44); Lnl (12); Myl (11); g-Tpnn (20)c S3: Crv (55); g-Tpnn (38)c Tamarind seed coat (Tamarindus indica) 0.13 mg EC g–1

b-C, LA Peroxide value; TBARS

Uy et al., 1991 Dapkevicius et al., 1998 Ramírez et al., 2005 Leal et al., 2003

b-C, LA DPPH

Cavero et al., 2005

b-C, LA

Carvalho et al., 2005

Peroxide value

Uy et al., 1991 Dapkevicius et al., 1998

AAb-c PF

Daukšas et al., 2001

DPPH; Lipid oxidation DPPH; lipid oxidation Inhibition oil oxidation

Hu et al., 2004 Xu et al., 2005a Esquível et al., 1999

Peroxide value

Luengthanaphol et al., 2004



Tamarind seed coat (T. indica)

Thyme leaves (T. vulgaris) Thyme (T. vulgaris)

TE = 4.92 TE = 0.7; Thy (0.17)d

Turmeric oil (Curcuma longa) Turmeric (C. longa)

TE = 5.5 (STp H)

Green tea (Camelia sinensis)


1

Zg A (40.1); Eg A (18.2); arT (15.7); Zg (3.1)

Peroxide value

Tsuda et al., 1995

Lipid oxidation

Chang et al., 2000

IPDEBTA Peroxide value

Ko et al., 2002 Uy et al., 1991 Dapkevicius et al., 1998

b-C, LA Rancimat Rancimat AOP

a

b-C, LA

Simándi et al., 2001 Zeković et al., 2001 and 2003 Gopalan et al., 2000 Leal et al., 2003

Antioxidant activity reported in the referenced work or, if not indicated, other works referencing antioxidant activity of the plant extracts. Relative percentage of the normalized area detected by LC–MS or GC. b Total oil (essential oil). c Relative weight content in the extract; d mg (100 g)–1. e mg g–1. TE, total extraction yield (%); TF, total flavonoids extraction yield (%); TP, total phenolics extraction yield (%); RER, relative extraction rate. AAb-c = antioxidant activity during bleaching of b-carotene–linoleate solution, scale (0:Low–5:High). AOP = antioxidant potency with linoleic acid in an iron/ascorbate system. b-C, LA = absorbance of the b-carotene–linoleic acid system. Hexanal = percentage of reduction in the formation of hexanal respect to control. IP DEBTA = inhibition of linoleic acid peroxidation by the DEBTA (diethyl-2-thiobarbituric acid) method. PF = protection factor calculated as the ratio between the induction period of the sample with additive and the control sample during vegetable oil oxidation. TBARS = percentage of reduction in the formation of TBARS with respect to control. TEAC = Trolox equivalent antioxidant capacity. O 2– = superoxide radical scavenger capacity of superoxide dismutase. Simple phenolics, derivatives and flavonoids: B, boldine; Crv, carvacrol; DPA, 3,4-dihydroxyphenyl acetate; EgA, E-g-atlantone; EC, epicatechin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate; GA, gallic acid; Gk, genkwanin; HDA, 2-hydroxy-3¢,4¢-dihydroxyacetophenone; MDB, methyl 3,4-dihydroxybenzoate; MEP, 2-methyl-6-ethylphenol; RA, rosmarinic acid; Thy, thymol. Terpenoids: t-C, trans-caryophylene; Cm, camphor; Cn, cineole; Cr, carnosol; CrA, carnosic acid; p-Cy, p-cymene; MCr, methyl carnosate; Lnl, linalool; MTp H, monoterpenes hydrocarbons; Myc, myrcene; Myl, myrtenol; Rsl, rosmanol; STp, sesquiterpenes; STp H, sesquiterpene hydrocarbons; ar-T, ar-turmerone; g-Tpnn, g-terpinene; Vbn, verbenone; ZgA, Z-g-atlantone; Zg, zingiberene. Other compounds: pCl, p-cymen-7-ol; oHC, O-hydroxycumine; Pp, piperine; Tq, thymoquinone. a


Terminalia catappa leaves, seeds Thyme (Thymus vulgaris) Thyme (T. vulgaris)

TE = 0.29; HDA (0.043); MDB (0.068); DPA (0.885); EC (0.164)d EGC (290); EGCG (510); ECG (105); EC (90); GA (7)e Tannin, flavonoid glycosides TE = 2.0 TE = 5.46

552 Separation, extraction and concentration processes temperature in the separators influence the fractionation of extracts. Series of two or three separation stages are used to recover the desirable components. Although the extraction yields are increased under harsh conditions, the selectivity is reduced, leading to extracts with poor antioxidant activity. These instances require optimization of the extraction stage (Gelmez et al., 2009) and/or separation steps. The yield can be similar to the use of a stepwise increase in the extraction pressure, but with a lower consumption of solvent. 18.5.5 Other novel extraction technologies The fundamentals and application of alternative techniques using solvents at low pressure for the extraction of bioactive compounds have been comprehensively reviewed (Meireles, 2009). Some of these techniques have been developed for analytical purposes, but they could be scaled up. Studies dealing with the antioxidant activity of extracts obtained with ultrasonic- and microwave-assisted technologies are summarized in Table 18.11. Ultrasound-assisted extraction (UAE) is a cheap, scalable technique allowing reduced extraction times, decreased extraction temperature, and increased extraction yields. Its effects are mainly attributed to cavitation forces upon the propagation of the acoustic waves. Ultrasounds result in physical, chemical, and mechanical effects promoting the release of soluble compounds from the plant body, enhancing mass transfer and facilitating the access of solvent to cell contents (Knorr et al., 2002; Ma et al., 2008). UAE was used to extract valuable compounds such as phenolic acids and flavanone glycosides (Ma et al., 2008). In UAE, both treatment time and (particularly) temperature have significant effects on the properties of extracts. However, UAE must be applied carefully to avoid degradation of susceptible solutes (Ma et al., 2008). Degradation of phenolics depends on their substitution pattern. High comparative stability has been reported for sinapic and vanillic acids, which have methoxyl-type substituents in their aromatic rings, whereas benzoic acids are more stable than cinnamic acids. In previously dried samples, ultrasound accelerated rehydration and swelling, without significant chemical degradation (Qadir et al., 2009). The ultrasonic power had a positive effect on the contents of phenolic acids from mandarin peels (Ma et al., 2008), tannins from myrobalan nuts (Sivakumar et al., 2008), and anthraquinones from Morinda citrifolia roots (Hemwimol et al., 2006). Differences in the effects of acoustic treatments can be ascribed to the utilization of different equipment, effects of the nature of the solid matrix (hardness, compactness, and solute distribution), and cavitation. Some of the general trends outlined above were confirmed in the extraction of active components from Hypericum perforatum L. (Smelcerovic et al., 2006). Other technologies enhancing mass transfer in plant tissues and permeability of cytoplasmatic membranes include microwave-assisted extraction (MAE), pulsed electric fields (PEF) and high hydrostatic pressure (HHP) (Corrales


Table 18.11 Other extraction technologies applied to the recovery of antioxidants from food plants Operational conditions

Extraction yield


Reference

Ultrasound Almond shells

LSR 1:10; 5% NaOH; RT; 10 min

TEP (%)10.1

DPPH

Bearberry leaves

LSR 1:25; W:E; 60 kHz; 25 ºC; 40 min

2.15 %

Cow cockle seeds (Saponaria vaccaria)

LSR 1:50; (W, 50% E, ACN); RT; 1 h

W: 0.78 g GAE g–1 dw 50%E: 1.58 g GAE g–1 dw ACN: 0.20 g GAE g–1 dw

Dornfelder skins (Vitis vinífera) Ginseng (Panax ginseng) Ginseng (P. ginseng) Indian mulberry (Morinda citrifolia) Indian mulberry roots (M. citrifolia) Mandarin peels (Citrus unshiu)

35 kHz; 70 °C; 1 h

360 mmol GAE g–1 dw

Inhibition of oil oxidation DPPH ABTS FRAP ABTS

Ebringerová et al., 2008 Gribova et al., 2008


LSR 1:50; 70% E; 60 ºC; 40 min; 250 W; 42 kHz LSR 1:50; 70% E; 70 °C; 2 h; 42 kHz; 284 W RLS 100; E; 60 °C; 60 min; 15.7 W

TS: 3.89 %

DPPH

Chen et al., 2010

G: 13.7 mg g–1 TP: 7.1 mg g–1 62.23%

DPPH

Kim et al., 2007

DPPH

79.62%

DPPH –

Pistachio (Pistachia vera) Plum (Prunus domestica)

LSR 1:8; W; M; 45 min

TCE:1935.12 mg g–1 dw FG: 1374.35 mg g–1 dw W: 34.2 mg TAE g–1 dw M: 32.8 mg TAE g–1 dw TP:1.74–3.75 mg GAE g–1 TP:1.18–2.37 mg CE g–1

Hemwimol et al., 2006 Pongnaravane et al., 2006 Ma et al., 2008

Peroxide value

Goli et al., 2005

ABTS

Kim et al., 2003

LSR 1:10; E; 60 ºC; 2 h; 38.5 kHz; 270 W LSR 1:80; 80% M; 30 °C; 10 min; 8 W

LSR 1:10; 80% M; -; 20 min




Raw material


Raw material


Extraction yield


Reference

Sage (Salvia officinalis) Sea-buckthorn (Hippophae rhamnoides) Sempervivum (Sempervivun marmoreum) Wheat bran (Triticum aestivum) Microwave

LSR 1:4; M; RT, 20 min

–

DPPH

LSR 1:10; E; 30 °C; 60 min

15.6 (0.39) mg GAE g–1

DPPH TEAC

Ollanketo et al., 2002 Sharma et al., 2008

LSR 1:10; dW; 25 °C; 60 min; 40 kHz

2.5 mg g–1

Hydroxyl DPPH

Stojičević et al., 2008

LSR 1:10; 5%NaOH; 60 °C; 60 min; 100 W

24.3% polysaccharides

DPPH

Hromádková et al., 2008

Sappan wood (Caesalpinia sappan)

540 W; 20 min

0.747 g

DPPH Nitric oxide

Shrishailappa et al., 2007

Cranberry (Vaccinium macrocarpon)

LSR 1:5.7; 10 min; 125 °C W E A LSR 1:50; 70% M; 50 W; 10 min

Ginseng (Panax ginseng) Ginseng (Panax ginseng) Indian mulberry (Morinda citrifolia)

LSR 1:50; 70% E; 250 W; 10 min 2450 MHz LSR 1:100; E; 720 W; 30 min; 60 °C

17.6% (9.42 mg Q g–1) 2% (1272 mg Q g–1) 3.25% (960 mg Q g–1) G: 9.8 mg TAE g–1 dw TP: 5.8 mg TAE g–1 dw TS: 3.30% 65.88%

Inhibition of lipid Raghavan and oxidation Richards, 2007 TBARS DPPH

Kim et al., 2007

DPPH

Chen et al., 2010

DPPH

Hemwimol et al., 2006



141 W; 83 s; 9.8 mL; 4 cycles

5.94 mg A mg–1

DPPH

Zhao et al., 2009

LSR 1:10; E 95%; 30 min; 500 W; 2450 MHz, 80 °C

96.78 mg TP g–1

Pan et al., 2008b

30 min; 800 W

0.12% oil

LSR 1:3; I (a) T gradually increased for 5 min, 800 W, and (b) 15 min; 500 W; 40–120 ºC LSR 1:10; E; 20 min; 150 W; 60 ºC

0.15 g oil g–1 125.3 mg Vit E g–1

DPPH Hydroxyl, reducing power DPPH, ABTS, FRAP, lipid peroxidation DPPH

DPPH TEAC Scavenging capability

Sharma et al., 2008

Sea-buckthorn (Hippophae rhamnoides) Wheat bran -

23.5 mg GAE g–1 489.5 mg CE

Phutdhawong et al., 2007 Zigoneanu et al., 2008

Oufnac et al., 2007

ACN, acetonitrile; E, ethanol; LSR, liquid:solid ratio; M, methanol; RT, room temperature; W, water; dW, distilled water. A, astaxanthin; CE, catechin equivalent; FG, flavanones glycosides; G, ginsenosides; GAE, gallic acid equivalent; Q, quercetin; TAE, tannic acid equivalent; TCE, total content of extract; TP, total phenolics; TEP, total extractable phenolics; TS, total saponins.



Green algae (Haematococcus pluvialis) Longan peel (Dimocarpus longan Lour.) Teja (Cinnamomum iners Reinw.) Rice bran (Oryza sativa)

556 Separation, extraction and concentration processes et al., 2008). PEF enhances mass transfer by improving softness and texture (Eshtiaghi and Knorr, 2002), and increasing the contents of some solutes, including total phenolics. However, the antioxidant activity varies significantly with the treatment used (Corrales et al., 2008). PEF inactivates degrading enzymes. HHP increases the extraction yields by deprotonation of charged groups, disruption of salt bridges and hydrophobic bonds in cell membranes and decreasing the dielectric constant of water (Corrales et al., 2008). Combinations of the different extraction methods are used. High-pressure extraction combined with sonication was used to extract compounds from Berberis koreana bark, improving the extraction of bioactive compounds compared with the high-pressure process (Qadir et al., 2009).

18.6 Integration of extraction processes and purification Several technologies with different degrees of sophistication (including solvents, resins and membranes) were used to obtain purified compounds or fractions with biological activity from crude extracts. Some examples of their individual and combined application to the concentration and purification of natural antioxidants from several sources are included here. Combinations of conventional and emerging extraction technologies were proposed to extract and/or to purify natural compounds with antioxidant and biological activity. The extraction of oil and/or lipophylic fractions with supercritical fluids at low pressures and the subsequent solvent extraction of the solid residue was proposed to recover essential oils from aniseed (Rodrigues et al., 2003), Melissa officinalis (Marongiu et al., 2004), rosemary, sage (Nakatsu and Yamasaki, 2000), summer savory (Esquível et al., 1999), and oils from borage seeds (Soto et al., 2008). A sequence of SFE and hydrothermal extraction has been proposed to obtain antioxidants from bamboo (Quitain et al., 2004). Alternatively, a first extraction of the raw material with conventional solvents and further purification of the crude extracts by SFE offer operational advantages, including reduction in CO2 consumption, increased extraction yields, and processing of less substrate owing to its enhanced content of bioactive compounds. Two options are used to purify the bioactive compounds from the crude extract: relatively mild SFE to extract non-polar compounds (desirable when antioxidant compounds of a phenolic nature have to be recovered from the residue) (Hadolin et al., 2004), and SFE under harsh conditions to recover polar compounds in the extract (Díaz-Reinoso et al., 2006). A simple purification process based on the sequential extraction of the raw materials or extracts with solvents of different polarity was applied to the extraction of antioxidant compounds from plant foods. Cheung et al. (2003) reported on the processing of edible mushrooms, leading to similar yields as those obtained with methanol and water on large and small scales. The


Extraction of natural antioxidants from plant foods 557 chemical components of the methanol extract were fractionated by solvents of increasing polarity (dichloromethane, ethyl acetate, butanol and water) (Cheung and Cheung, 2005). The dichloromethane subfraction of the methanol extract of Volvariella volvacea had a high antioxidant activity against lipid peroxidation of rat brain homogenate, whereas the ethyl acetate subfraction of the methanol extract of V. volvacea has antioxidant activity against the oxidation of human low density lipoprotein (LDL) similar to that of caffeic acid. Combined extraction involving pressurized hot water extraction as a first stage has been reported for a variety of purposes (Chambers et al., 1984; Wang and Weller, 2006). The advantages of membrane processing include a low energy requirement, no additives, mild operating conditions, separation efficiency and easy scaling up. The utilization of membrane technologies for concentrating and purifying bioactive phenolic compounds from aqueous streams is a topic of growing interest. For example, fractionation of phenolics into low- and high- MW compounds was reported for conventional solvent crude extracts from edible mushrooms (Cheung and Cheung, 2005), grape (Nawaz et al., 2006), grape pomace (Díaz-Reinoso et al., 2009), partially purified extracts from persimmon pulp (Ga et al., 2008), and mulberry root cortices (Yu et al., 2007). Ultrafiltration (UF) membranes are easy and fast to use in the separation of phenolics according to their MW. At the analytical scale, almond skin phenolics were fractionated to separate low MW compounds in permeate and proanthocyanidin oligomers (up to decamers) in retentate. Permeates from a 50 kDa membrane contained proanthocyanidin pentamers (Prodanov et al., 2008). UF membranes can be used for tailoring grape anthocyanins (containing about 60% monomers, 20% polymers, and 20% other forms) from grapes, having benzoic acids, hydroxycinnamates, anthocyanins, flavan-3-ols, and flavonols as major phenolic groups. The membrane (83% of total egg white proteins. The major egg yolk proteins take the form of lipoprotein complexes, which are divided into the plasma and granule fractions comprising lipovitellin, phosvitin, livetin and lipovitellenin. A number of egg proteins and some of their peptide sequences that have biological activities have been identified. For example, lysozyme is the egg protein that has probably attracted the most attention because of its potential use as an antimicrobial agent in foods. Chicken lysozyme contains a peptide sequence that is potently antimicrobial against both Gram-positive and Gram-negative bacteria. Biologically active proteins and peptides occur at relatively low concentrations in foods and pre-concentration of purification © Woodhead Publishing Limited, 2010

Fractionation of egg proteins and peptides 597 is needed to obtain a dose level to produce beneficial effects in situ. It was therefore necessary to develop technologies for fractionation and purification of bioactive molecules of interest for nutraceutical applications. This chapter mainly focuses on the chemistry and biological activities of proteins and peptides derived from egg components and the various technologies applied for fractionation/purification of proteins/peptides.

19.2 Composition and physicochemical characteristics of egg proteins and applications in the nutraceutical industry 19.2.1 Egg white The physicochemical characteristics of some egg white proteins are listed in Table 19.3. Egg white proteins are predominantly globular proteins having an acidic isoelectric point (pI), the exceptions being lysozyme and avidin. Except for lysozyme, egg white proteins are glycoproteins that are rich in sulfur amino acids and very sensitive to heat denaturation. Ovalbumin Ovalbumin has a molecular mass of 45 kDa and is composed of 385 amino acids. This protein belongs to the serpins family and possesses two genetic variants which differ at residue 289 and 311 by a substitution of Glu and Asn, respectively, by Gln and Asp (Ishihara et al., 1981; Wiseman et al., 1972). The primary sequence reveals one disulfide bond between Cys74 and Cys121. Table 19.3 Physicochemical characteristics of egg white proteins [adapted from LiChan and Nakai (1989)]

Amino acid residues

MW (kDa)

pI

T d*

Number of cystein residues [disulfide bridge]

Ovalbumin Ovotransferrin Ovomucoid Ovomucin Lysozyme G2 globulin G3 globulin Ovoinhibitor Ovoglycoprotein Ovoflavoprotein Ovomacroglobulin Avidin Cystatin

385 686 186 8300 129 – – – – 219 – 128 –

45 77.7 28 5500–8300 14.3 30–45 – 49 24.4 32 760–900 68.3 12.7

4.5 6.0 4.1 4.5–5.0 10.7 5.5 4.8 5.1 3.9 4.0 4.5 10 5.1

84 61 79 – 75 92.5 – – – – – 85 –

6 [1] 30 [15] 18 [9] – 8 [4] – – – – 18 [9] – 2 [1] 4 [2]

(–) Data not available. * Denaturation temperature.


598 Separation, extraction and concentration processes Approximately one half of ovalbumin amino acids residues are hydrophobic and one third are acidic, which confer a pI of 4.5 (Li-Chan and Nakai, 1989). The secondary structure of ovalbumin consists of 30% a-helix and 32% b-sheet structure using high resolution x-ray diffraction (Stein et al., 1990, 1991). Three components A1, A2 and A3 have two, one and no phosphate, respectively, and are found in purified ovalbumin (Perlman, 1952). In solution, ovalbumin can be denatured and aggregated by thermal denaturation or by exposure to the air–water surface (Mine et al., 1990). Partial thermal denaturation of ovalbumin occurs at 78 to 86 °C. The protein adopts a more stable form, termed S-ovalbumin to denote its increased stability. At a heating rate of 10 °C min–1 at pH 9, the denaturation temperature of ovalbumin is 84.5 °C compared with 92.5 °C for S-ovalbumin (Donovan and Mapes, 1976). The greater stability, compactness and hydrophobicity of the S-form contrast with that of ovalbumin (Nakamura and Ishimaru, 1981). Ovotransferrin Ovotransferrin, also called conalbumin, belongs to the transferrin family. Ovotransferrin, with a molecular mass of 77.7 kDa and 686 amino acids, contains no phosphorus or free sulfhydryl group. Ovotransferrin has a pI of 6.0 and has the capacity to bind two Fe3+ ions per molecule with two CO2–3 or HCO3– ions (Mine, 2007). Ovotransferrin is folded into two lobes which show similar structural elements (Williams et al., 1982). Each lobe contains a site for iron binding and is further divided into two distinct domains. Ovotransferrin is the most heat-sensitive protein of egg white. The protein easily binds metallic ions, such as Fe3+, Al3+, Cu2+ or Zn2+ forming heatstable complexes. Ovomucoid Ovomucoid is a glycoprotein with a molecular weight of 28 kDa and a pI of 4.1. Its amino acid sequence is composed of 186 residues and it possesses no tryptophan residues. Ovomucoid has nine disulfide bridges and no sulfhydryl groups. Ovomucoid has three domains defined by the amino acid sequences of 1–68, 69–130 and 131–186, each domain is cross linked by three disulfide bridges (Kato et al., 1987). Its secondary structure is composed of 26% a-helix, 46% b-structure, 10% b-turn and 18% random coil structure (Watanabe et al., 1981). Ovomucoid is highly resistant to heat owing to its high cystine content and, consequently, to the high number of disulfide linkages. Under acidic conditions ovomucoid can resist to heat treatments up to 100 °C, but it is rapidly (89 °C) denaturated in alkaline solutions (pH 9) (Matsuda et al., 1982). Ovomucin Ovomucin is a highly glycosylated protein displaying a molecular weight in the range of 5500–8300 kDa depending on the method of isolation and environmental conditions (Li-Chan and Nakai, 1989). Ovomucin differs from


Fractionation of egg proteins and peptides 599 other egg white proteins because it is extremely large. Its structure comprises sulfate esters, large amounts of cystine and 50% of the total sialic acid contents of egg white (Stadelman and Coterril, 1973). Ovomucin is composed of two subunits: a- and b-ovomucin, which differ by a carbohydrate-poor fraction (15% of dry matter) and a carbohydrate-rich fraction (50% of dry matter), respectively, and amino acids composition. Watanabe et al. (2004) estimated the molecular weight of a-ovomucin at 254 kDa whereas that of b-ovomucin has been estimated, using SDS-PAGE, to be between 400 and 720 kDa (Itoh et al., 1987). Ovomucin is heat stable over the pH range between 7.1 and 9.4. Cunningham and Lineweaver (1965) showed that the viscosity and absorbance of ovomucin solutions did not change upon heat treatment at 90 °C for 2 h. Lysozyme Lysozyme is a small enzyme molecule of 14.3 kDa capable of hydrolyzing specific polysaccharides that cleaves the b 1-4 linkages between N-acetylneuraminic and N-acetylglucosamine in bacteria cell. The single polypeptide chain consisting of 129 amino acid residues is cross-linked by four disulfide bridges. Because of its strong basic character (pI = 10.7), lysozyme binds to ovomucin, ovotransferrin and ovalbumin, via electrostatic interactions between positively charged lysozyme and negatively charged residues of sialic acid in these glycoproteins. Young et al. (1994) analyzed the three-dimensional structure of hen egg white lysozyme. The lysozyme molecule has two domains: the N-terminal domain (residues 40 to 88) with a hydrophobic core is composed of antiparallel b-sheets whereas the second hydrophilic domain, residues 1 to 39 and 89 to 129, is made up of a-helix. The enzyme is much more heat sensitive in egg albumen than when present alone. Minor egg white proteins The remaining proteins listed in Table 19.2 and 19.3 (G2 and G3 globulin, ovoinhibitor, ovoglycoprotein, ovoflavoprotein, ovomacroglobulin, cystatin, avidin) account for 400 mM) but exerted a potent vasorelaxant and antihypertensive effect upon oral administration to SHR rats at a dosage of 10 mg kg–1 (Matoba et al., 1999). Non-hydrolyzed whole egg white did not have ACE-inhibitory property whereas its digestion with pepsin resulted in high ACE-inhibitory activity with IC50 = 55.3 mg mL–1 after hydrolysis for 3 h. Treatment with trypsin and chymotrypsin induced very poor ACE-inhibitory activity values, indicating the importance of the enzyme specificity in producing peptides with ACEinhibitory activities (Miguel et al., 2004). Furthermore, these workers showed that the active peptides could be enriched from the hydrolysate using ultrafiltration with a 3 kDa molecularweight-cut-off (MWCO) membrane. The permeate generated contained peptides with molecular mass 90%) was observed under optimized conditions and moderately pure lysozyme (80–90%) could be obtained by carrying out a diafiltration step (Ghosh et al., 2000). Production of ACE-inhibitory peptides from egg white proteins with various proteolytic enzymes was carried out using a membrane reactor. Egg white proteins hydrolyzed with thermolysin produced the highest ACE-inhibitory potential (IC50 = 54.1 mg mL–1). Thermolysin hydrolysate was fractionated using UF membrane of MWCO of 10, 3 and 1 kDa. A lower IC50 (17.2 mg mL–1) was obtained in the UF permeate with 1 kDa MWCO (Chiang et al., 2008). By a modification of polymeric forms of lysozyme under optimal conditions of UF, a preparation comprising 53.3% of lysozyme polymeric forms, i.e. 33.2% of dimer and 20.1% of trimer, was obtained. Modified lysozyme preparation by UF showed the highest bacteriostatic activity against Gramnegative bacteria selected (Lesnierowski et al., 2009). A water-soluble plasma protein from egg yolk granules was obtained with various simple water dilutions, followed by centrifugation or filtration. Two factors were critical, pH and egg yolk dilution, for IgY recovery. Optimum recovery of IgY (93–96%) was obtained by a six-fold water dilution at pH 5.0–5.2 with incubation for 6 h at 4 °C. A 100 kDa MWCO extracted IgY from water-soluble plasma upon the separation of crude IgY by ammonium sulfate precipitation. A maximal recovery of IgY (> 98%) was obtained and this value was similar to the degree of purity obtained by gel filtration (Akita and Nakai, 1992). A serial filtration approach, involving dilutions, paper filtration and delipidation using hydrophobic filters or using different UF membranes, was developed by Kim and Nakai (1996; 1998). The delipidated water-soluble fraction was thereafter purified using 100 kDa UF membranes and both high recoveries (72–89%) and purity (74–99%) were obtained. Although the fractionation of proteins through a membrane depends


612 Separation, extraction and concentration processes on physicochemical parameters such as pH and salt concentration (Kim and Nakai, 1996; 1998), the surface properties of the membrane are also important for the successful protein fractionation. Hernandez-Campos et al. (2010) purified IgY from egg yolk water-soluble protein by UF–diafiltration with different membranes. The best selectivity and purification factors were obtained without salt at pH 5.7 and 6.7 using polyethersulfone and modified polyethersulfone of 100 MWCO membrane, respectively. Compared with chromatographic methods, membrane separation techniques offer the advantage of lower cost and ease of scale-up for commercial production. Membrane-based separation techniques are powerful tools for fractionation/purification of proteins and bioactive peptides. UF membranes can be used as preliminary step for the removal of enzymes and non-hydrolyzed proteins, and to further fractionate the peptide mixture. Various combinations of precipitation and UF using selective membranes can be applied for the fractionation of food proteins. In addition to selective membranes, chromatographic methods can be employed successfully to isolate specific bioactive compounds from a complex mixture. UF membranes were successfully used to enrich peptide fractions. Membranes consisting of negatively charged materials such as NF were used to desalt or to fractionate acidic peptides from hydrolysate mixture (Chay Pak Ting et al., 2007; Wijers et al., 1998). 19.4.4 Other separation methods A new isolation method was described for IgY. Use of food-grade products such as sodium alginate or l-carrageenan was investigated for precipitation of yolk lipoproteins from the water-soluble fraction to purify IgY. Polysaccharides exhibit satisfactory yields of IgY (Hatta et al., 1988; 1990). In another study, the optimal separation of IgY was achieved with 0.15% pectin and at pH 5.0, implying that the interactions between polysaccharides and lipoproteins were mainly ionic bonds, hydrophobic interactions and hydrogen bonds (Chang et al., 2000).

19.5 Conclusion and perspectives Considering that the discovery of novel bioactive peptides and their possible functions and health benefits are constantly increasing, there is a growing need to develop new technologies for the production of specific bioactive peptides from hydrolysate mixtures. Membrane-based separation techniques have traditionally been used to separate molecules of different sizes and lowmolecular-weight components from proteins. UF, alone and in combination with other techniques such as NF and chromatography has shown its potential to separate peptide mixtures on a large scale. However, the further research into membrane areas (such as membrane materials and chemistry, © Woodhead Publishing Limited, 2010

Fractionation of egg proteins and peptides 613 module configurations) for the complete separation/purification of proteins/ peptides is still needed. The addition of affinity ligands to chelate proteins/ peptides could be useful in obtaining maximal selectivity by combining with membrane processes.

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20 Supercritical-fluid extraction of lycopene from tomatoes J. Shi and S. Jun Xue, Agriculture and Agri-Food Canada, Canada, Y. Jiang, The Chinese Academy of Sciences, China and X. Ye, Zhejiang University, China

Abstract: Several process parameters for supercritical CO2 fluid extraction – such as pressure, temperature, flow rates, co-solvent or modifier concentrations, resident time, moisture content, particle sizes, and particle size distribution – have individual or combined effects on the recovery of lycopene from tomatoes. The solubility and bioactivity of lycopene, and the composition of the extract and extraction yield can be affected. Improved processing conditions and reduced cost are required if the extraction of lycopene from tomato materials using the supercritical CO 2 fluid extraction process is to become more economical at low throughputs. Key words: bioactivity, lycopene, supercritical fluid extraction, tomato.

20.1 Introduction Lycopene is a phytochemical responsible for the red pigments found in plants. It is a non-provitamin A carotenoid that plays an important role in the biosynthesis of many carotenoids. Recently, the extraction of lycopene and other carotenoids has attracted attention owing to their biological and physiochemical properties, particularly those which possess natural antioxidant activities. Antioxidants have been associated with disease prevention and reduction (Negre-Salvayre et al., 2006; Papas, 1999; Wu, et al., 1999). Structurally, lycopene is an acyclic, open-chain, C40 polyisoprenoid unsaturated carotenoid having 13 double bonds, of which 11 are conjugated, arranged in a linear array, and it has a molecular formula of C40H56 (Fig. 20.1). Because of this unique molecular structure, lycopene has high antioxidant activity and singlet oxygen quenching ability that is thought to be beneficial



Fig. 20.1 Structure of lycopene.

to human health. The antioxidant protective effect of lycopene and tomatoes has been shown in vitro as well as in vivo (Breinholt et al., 2000; Hadley et al., 2003; Rao and Agarwal, 1998; Suganuma and Inakuma, 1999; Willcox et al., 2003). Therefore, foods containing lycopene are of considerable interest. The singlet oxygen quenching ability of lycopene has been found to be three times greater than that of b-carotene and a-tocopherol (Stahl and Sies, 1992, 1996). Epidemiological studies and investigations have shown important roles of lycopene and other carotenoids in free radical inactivation and fat peroxidation inhibition (Krinsky and Rock, 1998). The health benefits of lycopene-rich diets and its effect on minimizing the risk of cardiovascular ailments and various forms of cancers have been reported (Palozza, 1998; Olson, 1986). More recent studies have demonstrated the important functions of lycopene and other carotenoids in the pathophysiology of chronic diseases (Rao and Agarwal, 1999; Rao and Rao, 2007). Increasingly, in vivo and in vitro clinical studies have shown its protective effect against the growth of tumor cells and its ability to protect against cardiovascular (Arab and Steck, 2000) and coronary heart diseases, and cancer (Clinton, 1998). With its 11 conjugated and two non-conjugated double bonds, lycopene is a more efficient antioxidant (singlet oxygen quencher) than b-carotene, a-carotene, and a-tocopherol (Mascio et al., 1989; Shi, 2002; Shi and Le Maguer, 2000). As the most abundant carotenoid in tomatoes, lycopene is the dominant pigment responsible for the color of tomatoes. Lycopene occupies the largest portion, about 80–90%, of the carotenoids in ripe tomatoes, followed by b-carotene, phytoene, and the other minor carotenoids. Concentrations of lycopene in tomatoes approach 50–100 mg kg–1, and it has a higher colour intensity than b-carotene (Shi and Le Maguer, 2000). Other sources of lycopene include pink grapefruit, guava, watermelon, autumn olive, and apricots. Ripe tomato skins contain approximately five times more lycopene than the pulp. The industrial processing of tomato products produces wastes such as seeds and skins. However, reuse of those wastes is limited to animal feed. Even though ripe tomatoes are the most abundant source of lycopene, over 90% is located in the skin, which constitutes the greater part of the waste, and is a potential natural source for lycopene extraction (Shi and Le Maguer, 2000). Lycopene content in plant materials is dependent on the species and the temperature at which growth and maturation occurs. Factors such as climate trends significantly alter the amount of lycopene in the material, thus affecting the yield. Lycopene is the last pigment to appear during maturation and its


Supercritical-fluid extraction of lycopene from tomatoes 621 development is inhibited by temperatures higher than 30–32 °C, whereas other carotenoids degrade at these temperatures. As a result, the pigment concentrations in plant materials may vary between crop cycles (Shi et al., 1999b). The concentration of lycopene also changes during ripening and storage. Liu et al. (2009) reported that the accumulated lycopene contents of tomatoes did not change significantly during the first four days of storage. However, between days 4 and 21, the lycopene content increased. A growing interest in and demand for healthy, environmentally safe, and cost-efficient products has driven the research and application of new technologies in the food, pharmaceutical and cosmetic industries. Extraction processes are commonly used to enrich and detoxify food through the removal of targeted components from natural products. Organic solvents are used in conventional methods for the extraction of bio-compounds such as lycopene from plant materials. However, these solvents not only generate environmentally hazardous problems and requests for expensive disposal procedures for the chemical extraction solvents, but also chemical residues remaining in the final products become a major safety concern. With increasing government restrictions reflecting consumer concerns on food safety, alternative and reliable extraction techniques are of great interest. Extraction processes are needed for ‘additive-free natural’ products. It is clear that the current concern for safety in food products has increased interest in ‘green’ extraction techniques, instead of the conventional organic solvent extraction processes. The determination of lycopene in food products and development of a safe ‘green’ extraction process to complement the fortification of functional foods is of great public interest. One possible environmentally friendly alternative is supercritical fluid extraction (SFE), in particular using supercritical CO2 ‘green technology’ because it is physiologically harmless, environmentally safe, non-explosive, exhibits high selectivity as a result of low viscosity, high diffusivity, and liquid-like density, as well as being readily available and easily removed from products (Simandi et al., 2002). Because of the CO2 supercritical state (31 °C and 7.38 MPa), procedures should allow supercritical operation of thermally labile compounds that would be easily degraded at high temperatures. SFE has attracted growing interest for the recovery of natural compounds for large-scale industrial production over recent decades (O’Day and Rosenau, 1982). SFE is successfully and widely used for the extraction of lycopene from ripe tomatoes (Cadoni et al., 2000) and tomato processing wastes (Baysal et al., 2000; Kassama et al. 2008; Ollanketo et al., 2001; Rozzi et al., 2002; Sabio et al., 2003; Topal et al., 2006; Vasapollo et al., 2004). One significant thermodynamic advantage of using supercritical fluid is its ease of separation from the extracted solutes by simply modifying the operation conditions, either pressure or temperature. Supercritical fluids have liquidlike densities that give superior mass transfer characteristics compared with organic solvents, and are characterized as low-viscosity and high-diffusivity fluids. In addition, supercritical fluids have low surface tension leading to


622 Separation, extraction and concentration processes easy penetration into the porous biological matrix of plant material while releasing the targeted components.

20.2 Supercritical-fluid extraction (SFE) of lycopene Supercritical fluids are, by definition, at a temperature and pressure greater than or equal to the critical temperature and pressure of the fluid. They actually have physical properties somewhere between those of a liquid and a gas. Supercritical fluids are able to spread out along a surface more easily than a true liquid because they have lower surface tensions than liquids. The supercritical CO2 is fed at high pressure by means of a pump, which pressurizes the extraction tank and also circulates the supercritical fluid throughout the system. Figure 20.2 demonstrates an example of a typical single-stage supercritical CO2 extraction system. Once the supercritical CO2 reaches the equilibrium state in the extraction vessel, the extraction process proceeds with the manipulation of pressure and temperature to achieve the ideal operating conditions. The mobile phase, consisting of the supercritical CO2 and the solubilized lycopene component, are transferred to the separator where the solvating power of the fluid is decreased by increasing the temperature and or decreasing the pressure of the system. The lycopene extract precipitates in the separator whereas the supercritical CO2 is either released or recycled back to the extractor. As the solution containing the extracts of lycopene compounds leave the extractor and flow to the first separation vessel via the pressure regulator, a paste of lycopene oleoresins settles to the bottom as they separate and this is collected, whereas the remaining solution goes to the second-stage separator where the fractionation of the lycopene components occurs. A third stage of separation might be required for the complete isolation of pure lycopene

Back pressure valve

Pressure valve

Separator

Cooling bath

Extract column

CO2 outlet Heat exchanger

Heat exchanger

CO2 pump

Extracts outlet

CO2 source

Fig. 20.2 Schematic diagram of a typical single-stage supercritical fluid extraction system with CO2 (modified from Shi et al., 2006).


Supercritical-fluid extraction of lycopene from tomatoes 623 components. In commercial-scale processing, multiple extraction vessels are normally used to enhance operation efficiency and throughput (Fig. 20.3). The process requires an intermittent batch system because it is interrupted at the end of the extraction period, when the pressure must be released so the extraction vessel can be switched out of the extractor loop. By having two or more interchangeable extraction vessels, the unloading and reloading of a vessel can occur while extraction of a previously charged vessel is in progress. This reduces the downtime and improves the overall production efficiency.

20.3 Factors affecting lycopene yield Many studies have successfully extracted lycopene from tomato materials by SFE. The technology has been continuously developed and users have established environmentally sound and complimentary safe techniques for extracting food-grade, bioactive components used as natural ingredients from agricultural materials. There have been numerous studies on the effects of various independent variables – such as pressure, temperature, flow rates, and co-solvent or modifier concentrations – on lycopene yield using SFE (Baysal et al., 2000; Ollanketo et al., 2001; Ciurlia et al., 2009; Kassama et al., 2008; Yi et al., 2009, Shi et al., 2009a; 2009b; Vaughn et al., 2008). Two-stage separation vessels

Storage tank

Three-stage extraction columns

CO2 inlet

Co-solvent inlet Pumps

Pressure valve Heat exchanger

Fig. 20.3 Schematic diagram of commercial scale multi-stage supercritical fluid extraction system used to fractionate bioactive components.


624 Separation, extraction and concentration processes The optimization of pressure, temperature, and flow rate is necessary to obtain a high recovery of lycopene. Baysal et al. (2000) observed the highest recovery (54%) of lycopene obtained at 55 °C, 30 MPa, and 4 kg h–1 with 5% ethanol as modifier. Ollanketo et al. (2001) recovered 94% of lycopene in 15 min at 40 MPa and 110 °C. Rozzi et al. (2002) obtained a maximum recovery (61%) at 80 °C, 35 MPa, and 2.5 mL min–1. Yi et al. (2009) studied the effects of SFE parameters on the recovery of lycopene and antioxidant activity. In this study, the maximum total lycopene content of 31.25 mg g–1 of raw tomato was extracted at the highest temperature of 100 °C, 40 MPa and 1.5 mL min–1. Process parameters such as solvent flow rate, residence 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, bioactivity, and composition of the extract. For example, the residence time can have an important influence on the composition of the extracted compound. 20.3.1 Effects of temperature, pressure and flow rate on recovery of lycopene Effect of pressure Plate IIa (between pages 292 and 293) shows that pressure significantly influences the rate of extraction. Higher density causes a double effect that increases the solvation power and reduces the interaction between the fluids and matrix, resulting in a decrease in 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 (Tonthubthimthong et al., 2001). It is commonly considered that an increase in pressure results in an increase in CO2 density, increasing the solvating power of the supercritical fluid. Thus, higher pressure is responsible for quantitative recoveries and stronger interactions between the fluid and the matrix (Shi et al., 2009a; Topal et al., 2006). Effect of temperature Temperature is a parameter with significant influence on SFE yield; thus manipulating it could have an adverse implication on the process and yield. Plate IIb shows the general trend of increased extraction yield as temperature increases relative to the pressure. The extraction yields increased both with temperature and pressure, whereas the effect of temperature is more significant than that of pressure. Vági et al. (2007) maximized the recovery of lycopene at 46 MPa and 80 °C by SFE. At 40–100 °C, maximum yields were obtained for extraction of lycopene. The highest temperature gave the maximum lycopene content (Rozzi et al., 2002). Cadoni et al. (2000) reported that at 85 °C a maximum of 65% of lycopene was recovered from the pulp


Supercritical-fluid extraction of lycopene from tomatoes 625 and skins of ripe tomatoes. However, with further elevation of temperature, degradation and isomerization of lycopene was often encountered (Shi et al., 1999a, 2009b, 2002; Shi and Le Maguer, 2000; Yi et al., 2009). It is well-known that increasing the temperature reduces the solvent density and consequently decreases the recovery of lycopene at constant pressure. At the same time, higher temperature promotes the solubility of lycopene and increases the recovery by increasing mass transfer of lycopene in the matrix, and from the matrix to the CO2 extraction medium (Marsili and Callahan, 1993; Shi et al., 2007d). Therefore, the increase in temperature could have either a positive or a negative effect, as a result of the balance between CO2 density (r) and solute vapour pressure. The increase in the recovery of lycopene by SFE depends more on the solute’s vapour effect. Although lycopene is not stable during long heating times and was approximately 53.5% degraded after 60 min at 100 °C through isomerization or auto-oxidation (Boskovic, 1979; Mayeaux et al., 2006; Shi and Le Maguer, 2000; Shi et al., 1999a). However, in the thermal processing of tomato products, the total lycopene contents still increased in the products owing to the elevated release of lycopene from the tomato tissue matrix (Dewanto et al., 2002; Seybold et al., 2004; Toor and Savage, 2006). For SFE, the enhanced release of lycopene from the insoluble fibre portion of tomato skin may also contribute to the increased recovery. Effect of CO2 flow rate In an extraction, the flow rate of supercritical CO2 has an influence on the extraction efficiency. The flow rate controls the amount of solvent (e.g. CO2) to be used and the extraction time. Extraction is a diffusion-based process, with the solvent required to diffuse into the matrix, and the extracted component to diffuse out of the matrix into the solvent at equilibrium state. The flow rate also greatly affects solubility of extracted components in fluid and equilibrium of diffusion between solvent and extracted components during extraction. If the concentration of extracted components is much higher than the solvent, the solubility of extracted components in the solvent could be limited and slow down the extraction process. Therefore, optimization of flow rate is important for extraction time and cost efficiency. For example, to maximize the rate of extraction, the flow rate should be high enough for the extraction to be completely diffusion limited, but this is very wasteful of solvent. In contrast, the lower flow rate would minimize the amount of solvent used, but the extraction should be completely solubility limited, and thus would take a very long time. Therefore, the optimum flow rate is probably somewhere in the region where both solubility and diffusion are significant factors. At optimum temperature and pressure, the extraction process is performed under conditions in which the CO2 flow rate is variable. The linear correlation is observed for the flow rates and the extraction rates. The extraction rate increased with increasing flow rate. When the intra-particle diffusion


626 Separation, extraction and concentration processes resistance is considered as a factor in the material matrix, more CO2 is required to make contact with the targeted components. Thus, the extraction rate, is a function of CO2 consumed: the greater the amount of supercritical gas used, the higher the yield obtained (Friedrich and Pryde, 1984). When the temperature, pressure, and overall extraction time are kept constant, the effect of various solvent flow rates on the carotenoid yield is minor. Plate IIc shows the effect of flow rate on total lycopene yield at constant pressure (30 MPa) or temperature (70 °C). An increase in flow rate from 1.0 to 2.0 mL min–1 did not show a significant change in the yield of lycopene (Yi et al., 2009). Results indicated that the higher flow rate could produce faster extractions and higher recoveries since the higher flow rate could overcome the interface resistance for lycopene transport from the tomato skin tissue to the CO2 fluid (Hawthorne et al., 1995). However, excessively high flow rates would produce undesirable results owing to a decrease in contact times between the solvent and the solute (Topal et al., 2006). This may force the solvent to leave the system without reaching its solubility limit (Anitescu et al., 1997). High flow rates may also cause the sample to compact and restrict the fluid flow, thus reducing the amount of solvent that comes in contact with the solute (Franca and Meireles, 2000). Increasing the CO2 flow rate would increase fluid diffusivity and improve the heat and mass transfer within the system, because the resulting fluid temperature approaches the wall temperature more quickly (Shi et al., 2007a). The effect of solvent flow rate on mass transfer rate and total yield in the constant extraction rate period is significant. Effect of operating time Residence time is an important factor that influences yield and the economic viability of the process. When optimizing extraction conditions, the extraction time of the process is an important parameter. For the extraction of lycopene by SFE Baysal et al. (2000) found that the highest carotene yield was obtained at an extraction time of 2 h, as opposed to 1 or 3 h. A 1-h extraction may not suffice for the maximum amount of carotenoids to be dissolved in the solvent, whereas over 3 h there is an increase in the occurrence of degradation. The duration and intensity of thermal processing are directly correlated to the degree of isomerization and degradation of the targeted compounds (Baysal et al., 2000). Therefore, to minimize the degradation effect, it is advisable to reduce the extraction time as much as possible. In addition, optimizing extraction conditions towards the shortest extraction time possible with the maximum extraction recovery is ideal from the point of view of the extract quality and processing cost. Effect of moisture content of raw materials Water represents approximately 80–90% of the total weight of fresh plant materials and interferes with the effectiveness of the SFE (Hopper and King, 1991). Thus, the initial moisture content may improve or impede the


Supercritical-fluid extraction of lycopene from tomatoes 627 yield results depending on the polarity of the compound. The non-polar compounds such as lycopene produce smaller yields with higher moisture content (Anitescu et al, 1997; Chao et al., 1993). Water has a small but finite solubility in supercritical CO2, therefore, it can also be extracted with the targeted components. The presence of a little water (less than 10%) aided in the extraction of lycopene, particularly at the beginning of the process (Shi et al., 2006). A moisture content of 7–18% had negligible effect on extractability of oil. However, above an 18% moisture content, the presence of water affected the extraction and the efficiency decreased (Nagy and Simándi, 2008). The high water content in the matrix inhibited the flow of SCF by changing surface tension and contact angles as a result of phase interaction. Therefore, water removal in most cases frees the internal pores and thus increases the mass transport intensity. Higher moisture contents cause a higher probability of formation of a thin film of water between the matrix and the SCF phase. Possible effects of water include an increase in distance that carotenoids must travel to reach the solvent and swelling of the cell matrix, which have negative and positive effects on the process, respectively. Effect of particle size Particle size is a factor that influences the extraction recovery, having a significant impact on the flow behaviour of SCF in the sample matrix. The extraction process and diffusivity of the extraction medium are hindered by large particle sizes (>550 mm) because of the distance which the lycopene travels from the inside of the particle to the surface (Shi et al., 2007a). By reducing the size of the particles, components such as carotenoids have less distance to travel. Thus, the collective amount of surface area with which the solvent is in contact is increased, an important factor during the initial stages of extraction (Ollanketo et al., 2001). The particle size is affected by sample pre-treatment. For example, the methods of drying (air, oven, vacuum, or freeze drying), milling, and other mechanical, ultrasonic, high-pulsed electronic field, and non-mechanical treatments can change the particle size by attrition or size reduction. Numerous experimental results have indicated that smaller particle sizes are ideal for extraction processes, and the manner in which the solid material is crushed and powdered also affect the results (Ferreira and Meireles, 2002; Spanos et al., 1993). The degree to which the material is crushed determines the amount of cells that are broken, and this has a positive correlation with recovery. The smaller the particle size, the larger is the surface area, and the bioactive components are released more easily. However, if the particle size is exceedingly small that can favour channelling which commonly results in a decrease in recovery (Ziémons et al., 2005). Only 40% lycopene recovery was obtained from powdered tomato skins crushed with a household blender, relative to the amount achieved when sea sand was used to assist the grinding procedure (Ollanketo et al., 2001). Therefore, optimizing particle


628 Separation, extraction and concentration processes size significantly improves the yield of extraction. Sun and Temelli (2006) noted that the optimal range of particle size was 0.25–0.5 mm for lutein, b-carotene, and a-carotene. However, for certain processes, it is important to be cautious of the degree of crushing. Gouveia et al. (2006) studied the effects of slight, moderate and complete crushing, which gave 20%, 40%, and 65% carotenoid recovery, respectively, and also found that samples that have been extremely crushed may not be affected by the addition of entrainers.

20.4 Effects of pressure and temperature on the antioxidant activity of lycopene Recovery is increased with increasing temperature owing to increasing solubility of the targeted compounds; therefore, for economic reasons, food industries prefer to increase the temperature to achieve higher yields. However, the bioactivity of the lycopene may be compromised by heat because of the instability of lycopene at higher temperatures (Mayer-Miebach et al., 2005; Shi et al., 2002b, 2004b, 2008, 2009b; Vega et al., 1996; Yi et al., 2009). Yi et al. (2009) reported that the recovery and bioactivity of lycopene increased when the temperature increased from 20 to 60 °C. The recovery of lycopene increased slowly and its antioxidant activity declined above 80 °C. Pol et al. (2004) made a remarkable observation that lycopene recovery was increased with increasing temperature, but the bioactivity of the extracted lycopene did not follow the trend of the recovery of lycopene extracts at high temperature. No significant effects of pressure or flow rate on the antioxidant activity were observed (Yi et al., 2009). Cadoni et al. (2000) and Shi and Zhou (2006) reported that the results obtained from SFE of lycopene from tomatoes are poorly reproducible because of the decomposition and isomerization of lycopene during the extraction process. Yi et al. (2009) investigated the effects of the two major supercritical fluid extraction parameters (temperature and pressure) on the antioxidant activities of the extracts as shown in Fig. 20.4. The recovery increased with the increase in pressure, whereas antioxidant activity decreased slightly. The recovery of total lycopene increased with temperature, but the antioxidant activity of the lycopene-rich extract decreased with increasing temperature (Hackett et al., 2004; Yi et al., 2009). At 25 and 50 °C, lycopene in an oleoresin is degraded predominately through oxidation, whereas isomerization increases at 75 and 100 °C. If lycopene is isomerized, the proportion of lycopene isomers is changed, that then further influences the antioxidant activity. In general, the change in antioxidant activity in lycopene-rich extracts is mainly influenced by their lycopene content, proportions of the isomers of lycopene, and the synergistic effects of carotenoids in the extract (Shi et al., 2004a,b, 2004c, 2007c; Shixian et al., 2005). The total antioxidant activity of


0.30

30

0.25

25

0.15

15

0.10

10 5 40


0.20

20

50

60 70 80 Temperature (°C) (a)

90

0.05 100

35

0.30

30

0.25

25

0.20

20 0.15

15

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10 5 20

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30 Pressure (MPa) (b)

35


35




0.05 40

Fig. 20.4 Changes of lycopene yield (mg –1g raw material) (d) and antioxidant activity (mM mg–1 Torlox) of the extract (m) (a) at 40 to 100 °C, 30 MPa and flow rate of 1.5 mL min–1, and (b) at 20–40 MPa, 70 °C and flow rate of 1.5 mL min–1 (modified from Yi et al., 2009).

the extract, that is the antioxidant activity of each unit of lycopene multiplied by the total lycopene amount in the extract, firstly significantly increases as the temperature increases, and then slightly decreases as temperature is kept at a high temperature. The antioxidant activity of extracts first increased with increasing lycopene contents in the extracts between 40 and 80 °C, and then started to decline thereafter. The slight decrease above 90 °C might be affected by the degradation of other carotenoids (Cocero et al., 2003). Dewanto et al. (2002) found that thermal processing increased the total antioxidant activity of tomato from 4.13±0.36 to 6.70±0.25 mmol g–1 of vitamin C equivalents after 30 min of heating at 88 °C. The total antioxidant activity probably increases because larger amounts of lycopene are released from the matrix during thermal processing. HPLC chromatograms of the extracts


630 Separation, extraction and concentration processes obtained at 50 and 90 °C are shown in Fig. 20.5. All-trans lycopene is the major configuration eluting with a retention time of 15.87 min under both conditions. Three major cis-isomer peaks are detected around 6.8, 8.9, and 16.1 min of elution time in extracts prepared at 50 °C. When the extraction temperature is elevated to 90 °C, total lycopene increased 1.18 fold, whereas all-trans lycopene contents increased only 1.03 fold and total cis-isomers increased more than 2.13 fold, respectively. Moreover, more than three major peaks of cis-isomers are detected (Fig. 20.5) because of new cis-isomer generations. The higher increase in cis-isomers than total and all-trans lycopene indicates the transformation of all-trans to cis-isomers. As extraction vessel temperature increased from 50 to 90 °C, the ratio of all-trans to total cislycopene isomers changed from 1.67 to 1.40 (Table 20.1). Further increases in the operating temperature resulted in dramatic isomerization. The lower ratio of trans- to cis-isomers obtained at high temperature indicated higher lycopene bioavailability owing to the cis-isomers generated (Boileau et al., 1999). The isomerization of lycopene in tomato samples during thermal processing above 75 °C, and the decrease in the trans- to cis-isomer ratios from 9:1 to 2:3 after heating at 140 °C, were demonstrated (Cocero et al.,

4 cis isomers mAU

3

trans-lycopene

2 5-cis

1 0

0

5

10

15 20 Time (min) (a)

25

30

25

30

trans-lycopene

4 mAU

cis isomers 3 2

5-cis

1 0

0

5

10

15 20 Time (min) (b)

Fig. 20.5 Chromatograph of SFE extract obtained at (a) 50 °C and (b) 90 °C at 30 MPa and flow rate of 1.5 mL min–1 (modified from Yi et al., 2009). mAu, milli absorbance unit. © Woodhead Publishing Limited, 2010

Supercritical-fluid extraction of lycopene from tomatoes 631 Table 20.1 All-trans and cis-isomers contents and ratio in the SFE extract obtained at 40–100 °C, 30 MPa and a flow rate of 1.5 mL min–1 (modified from Yi et al., 2009) Temperature Total content All-trans (mg g–1) content (mg g–1) 100 90 80 70 60 50 40

29.46 26.12 22.69 14.66 13.85 12.00 10.35

16.75 15.26 13.76 9.04 8.74 7.50 6.52

Total cis Ratio Ratio Ratio* content (trans:total) (cis:total) (trans:cis) (mg g–1) 12.71 10.86 8.93 5.62 5.11 4.50 3.83

0.57 0.58 0.61 0.62 0.63 0.63 0.63

0.43 0.42 0.39 0.38 0.37 0.37 0.37

1.32c 1.40c 1.54bc 1.62ab 1.71a 1.67a 1.70a

*The different letters shows the values that are significantly different (P

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