(Functional Foods and Nutraceuticals) John Shi - Functional Food Ingredients and Nutraceuticals Processing Technologies-CRC Press (2006)

2441_title 7/18/06 11:28 AM Page 1
Functional Food
Ingredients and
Nutraceuticals
Processing Technologies
Edited by
John Shi, Ph.D.
Boca Raton London New York
CRC is an imprint of the Taylor & Francis Group,
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© 2007 by Taylor & Francis Group, LLC
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Library of Congress Cataloging‑in‑Publication Data
Functional food ingredients and nutraceuticals : processing technologies / edited
by John Shi.
p. cm. ‑‑ (Functional foods & nutraceuticals series ; 9)
Includes bibliographical references and index.
ISBN 0‑8493‑2441‑6 (alk. paper)
1. Functional foods. I. Shi, John. II. Series.
QP144.F85F85 2006
613.2‑‑dc22
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Foreword
The linkages between diet and health, and their economic implications, continue to be
elucidated and to gain public acceptance as credible science in this fascinating field of
study. Knowledge of the specific contributions that functional foods, bioactive compounds, and nutraceuticals make to our health has grown immensely over the past
decade. So, too, has the desire of consumers to purchase and integrate these materials
into healthful diets, and for mainstream retail chains to give these products lucrative
shelf space increased. To effectively disseminate this knowledge and achieve broad
public benefit from this work, it is essential that emerging factual information in the
scientific literature be distilled, assembled, and consolidated into more concise communications, as presented here, that can be put into action throughout the food continuum.
This ninth volume in the CRC Press Functional Foods and Nutraceuticals Series,
edited by Dr. G. Mazza (AAFC-PARC, Summerland, BC), continues the work of the
earlier volumes and moves farther along the continuum to address the most recent
advances in food engineering and processing. These advances hold the greatest promise
for the immediate future to achieve stable, high-volume production of functional food
and nutraceutical products of defined and reliable composition, thereby fostering the
health and economic benefits that could ensue. Dr. John Shi, an internationally wellknown scientist in this field, and coeditor of an earlier volume of the series, steps out
independently to take the role of editor as well as contributing author on this volume.
The team of contributing authors, representing five continents, reflects the international
distribution of interest and expertise on this topic, and the essential widespread collaboration that is required to generate advances most effectively.
In 16 chapters, this volume provides a sequential study of key factors in the
preparation of functional foods and nutraceuticals, from the selection of sources
through the extraction, purification, decontamination, packaging, and preservation
of a variety of products in these categories. Where the earlier volumes focused on
an array of products and their biochemical constituents, this volume is process
oriented, although it touches on a variety of products as well as their characteristics
and how individual properties influence the suitability of the processes described.
The authors and editor have done a masterful job and are to be congratulated
for their success in selecting the most significant issues and the most promising
opportunities for presentation in this enlightening publication. The dissemination of
the information this volume contains will accelerate the integration of these
approaches and technologies into the production processes of tomorrow.
This book will be an excellent resource for students of food science, life sciences,
as well as food industry professionals interested in functional foods.
Yvon Martel, Ph.D.
A/Assistant Deputy Minister
Agriculture and Agri-Food Canada
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Preface
People from North America and the European community are committing more time
and effort to have greater control over their health by exploring alternative or herbal
medicines and natural health products for disease prevention and better health. There
has been a growing interest in the role of special micronutrients (phytochemicals)
on human health and well-being. The relationships among special food components,
their physiological functionality, and health benefits have been revealed progressively in the recent years. Functional foods contain significant levels of natural
extracts, concentrates, or natural ingredients that are extracted from natural sources.
These can reduce current healthcare costs by health improvement and disease prevention. During the past decade, the trend of functional food consumption was
consumer driven. It also serves the needs of the aging population that wants greater
control over their health and well-being. This trend is expected to continue, and as
a result, scientific information on all aspects of functional foods is vital to the
advancement of this emerging sector. The increase in consumer demand for functional foods has prompted international health organizations and government agencies to develop specific guidelines for their production and use. The scientific
community must, therefore, utilize modern technologies to ensure efficacy and safety
in the manufacturing of functional foods.
Manufacturers are always eager to fulfill the consumer’s desire for functional
food products that could be used to promote health. In order to provide a better
understanding and to disseminate the latest developments in this rapidly expanding
field, this ninth volume of the CRC Press Functional Foods and Nutraceuticals Book
Series, Functional Foods and Nutraceuticals: Processing Technologies, was developed. Sixteen chapters in this book cover a broad spectrum of functional foods from
biological material, applications of engineering techniques in functional food production, process engineering and modeling, functional food bioavailability, to product quality. The emphasis is on (1) applications of engineering techniques such as
high pressure, supercritical fluid, membrane, microencapsulation, and molecular
distillation in the processing of functional foods; (2) stability of bioactive components and antioxidative properties during processing and shelf life; (3) improvement
in bioavailability of bioactive components by physical and chemical methods; and
(4) mechanisms of antioxidant action and clinical and epidemiological evidence of
functionality.
The contributing authors are international experts in their respective fields, and
I am grateful to each and every one of them for their thoughtfulness in contributions
to this book. This book will be of interest to a wide spectrum of professionals from
food scientists and technologists, nutritionists, biochemists, and engineers to entrepreneurs worldwide. It will also serve as a unique reference for food scientists for
the R&D departments of food companies that are working with functional foods
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and ingredients. Additionally, it will serve as a source of basic information for college
and university students majoring in food science and technology, food processing,
and engineering. Readers will obtain sound scientific knowledge of engineering
techniques and the quality of functional foods and nutraceuticals.
John Shi, Ph.D.
Editor
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Series Editor’s Preface
The Functional Foods and Nutraceuticals Book Series, launched in 1998, was developed to provide a timely and comprehensive treatment of the emerging science and
technology of functional foods and nutraceuticals which are shown to play a role in
preventing or delaying the onset of diseases, especially chronic diseases. The first
eight volumes in the series, Functional Foods: Biochemical and Processing Aspects,
Volumes 1 and 2; Herbs, Botanicals and Teas; Methods of Analysis for Functional
Foods and Nutraceuticals; Handbook of Fermented Functional Foods; Handbook of
Functional Dairy Products; Handbook of Functional Lipids; and Dictionary of
Functional Foods and Nutraceuticals, have received broad acceptance by food,
nutrition, and health professionals.
The latest volume, Functional Foods and Nutraceuticals: Processing Technologies, edited by Dr. John Shi, addresses the most recent advances in processing
technologies for functional food ingredients and nutraceuticals. Distinctive features
of this book include in-depth treatments of the peer-reviewed literature on supercritical fluid extraction, pressurized low polarity water extraction, membrane separation, distillation, dehydration, food pasteurization, and sterilization with high pressure, microencapsulation of omega-3 fatty acids, and bioprocessing. Other topics
addressed include microbial modeling for bioreactor design, biochemical reactions
in supercritical fluids, modeling of supercritical fluid extraction of plant material,
stability of lycopene during processing, functional foods packaging, fruits with high
antioxidant activity, and biological antioxidation mechanisms quenching of peroxynitrite. The book contains 16 excellent chapters written by 32 international experts
at the forefront of functional food and nutraceutical science and technology. It is
hoped that the effort will be beneficial to process engineers; food, nutrition, and
health practitioners; and students, researchers, and entrepreneurs in industry, government, and university laboratories.
Earlier volumes in the series addressed a range of topics and include:
Functional Foods: Biochemical and Processing Aspects, Volume 1, the first
volume of the series, edited by G. Mazza, is a bestseller, and is devoted to functional
food products from oats, wheat, rice, flaxseed, mustard, fruits, vegetables, fish, and
dairy products. In Volume 2, edited by Drs. John Shi, G. Mazza, and Marc Le Maguer,
the focus is on the latest developments in the chemistry, biochemistry, pharmacology,
epidemiology, and engineering of tocopherols and tocotrienols from oil and cereal
grain, isoflavones from soybeans and soy foods, flavonoids from berries and grapes,
lycopene from tomatoes, limonene from citrus, phenolic diterpenes from rosemary
and sage, organosulfur constitutes from garlic, phytochemicals from echinacea,
pectin from fruit, and omega-3 fatty acids and docosahexaenoic acid from flaxseed
and fish products.
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The book Herbs, Botanicals and Teas, edited by Drs. G. Mazza and Dave Oomah,
provides an in-depth literature review of the scientific and technical information on
the chemical, pharmacological, epidemiological, and clinical aspects of garlic, ginseng, Echinacea, ginger, fenugreek, St. John’s wort, ginkgo biloba, kava kava,
goldenseal, saw palmetto, valerian, evening primrose, liquorice, bilberries and blueberries, and green and black teas. The book, which is superbly referenced, also
contains chapters on international regulations and quality assurance and control for
the herbal and tea industry.
The volume Methods of Analysis for Functional Foods and Nutraceuticals,
edited by Dr. W. Jeffrey Hurst, presents advanced methods of analysis for carotenoids, phytoestrogens, chlorophylls, anthocyanins, amino acids, fatty acids, flavonoids, water-soluble vitamins, and carbohydrates.
The fifth volume of the series, Handbook of Fermented Functional Foods, edited
by Dr. Edward R. Farnworth, provides a comprehensive, state-of-the-art treatment
of the scientific and technological information on the production of fermented foods,
the microorganisms involved, the changes in composition that occur during fermentation and, most importantly, the effects of these foods and their active ingredients
on human health.
The Handbook of Functional Dairy Products, edited by Drs. Colette Shortt and
John O’Brien, contains outstanding chapters dealing with probiotic lactobacilli and
bifidobacteria, lactose hydrolyzed products, trans-galactooligosaccharides as prebiotics, conjugated linoleic acid (CLA) and its antiatherogenic potential and inhibitory
effects on chemically induced tumors, immuno-enhancing properties of milk components and probiotics, and calcium and iron fortification of dairy products.
On lipids, we have the volume, Handbook of Functional Lipids, edited by
Professor Casimir C. Akoh, which presents up-to-date information on all major
scientific and technological aspects of functional lipids, including isolation, production, and concentration of functional lipids; lipids for food functionality; lipids with
health and nutritional functionality; and the role of biotechnology for functional
lipids.
Finally, the Dictionary of Functional Foods and Nutraceuticals, written by N.
A. Michael Eskin and Snait Tamir, is essentially a mini-encyclopedia that provides
the reader with valuable and up-to-date information on the occurrence, chemistry,
and biological activity/efficacy of 480 functional foods and nutraceuticals. The
information is based solely on peer-reviewed literature, and it is presented alphabetically in a clear and concise manner.
G. Mazza, Ph.D., FCIFST
Series Editor
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Acknowledgments
This book is a product of collaborative efforts, and a number of scientists gave their
valuable time to review the manuscripts. The editor is indebted to their contributions
and support that improved this book significantly. The reviewers include Dr. Ireneo
Kikic (University of Trieste, Italy); Dr. Thomas Gamse (Graz University of Technology, Austria); Drs. Yukio Kakuda, Gauri S. Mittal, and Warrer Stiver (University
of Guelph, Canada); Drs. Selma Guigard and Marleny Saldana (University of
Alberta, Canada); Dr. Paulyn Appah (Food Development Centre, Manitoba, Canada);
and Drs. Giuseppe Mazza, Christopher Young, and Lamin S. Kassama (Agriculture
and Agri-Food Canada).
The editor also would like to express appreciation to Dr. Yvon Martel (A/Assistant Deputy Minister, Agriculture and Agri-Food Canada) for his kind preparation
of the foreword for this book; and to Drs. John Lynch (Science Director) and Maria
Nazarowec-White (Program Coordinator) for their help and support. The editor also
wishes to acknowledge the encouragement and help from Dr. Jerry King (Los Alamos
National Laboratory, Chemistry Division, United States); Dr. Asbjørn Gildberg (Norwegian Institute of Fishery and Aquaculture, Norway); Drs. Amparo Chiralt and
Pedro Fito (Polytechnic University of Valencia, Spain); Drs. Albert Ibarz and Joaquin
Giner Segui (University of Lleida, Spain); Dr. Sam K. C. Chang (North Dakota State
University, United States); Dr. James H. May (University of Hawaii at Manoa, United
States); Dr. Yueming Jiang (South China Institute of Botany, Chinese Academy of
Sciences, China); Dr. Eleanor Riemer, Ms. Susan Lee, and Ms. Patricia Roberson
(CRC/Taylor & Francis) for their support and encouragement in the preparation of
the book proposal and manuscript.
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Editor
Dr. John Shi is a senior research scientist in the Federal Department of Agriculture
and Agri-Food Canada, and is also an adjunct professor of food engineering, at the
School of Engineering, University of Guelph. He is co-editor of two books, Functional Foods II – Biochemical and Processing Aspects, and Asian Functional Foods,
published by CRC Press, now Taylor & Francis. He graduated from Zhejiang University, China, earned a Masters degree in 1985, and Ph.D. in 1994 from Polytechnic
University of Valencia, Spain. Dr. Shi is an international editor of the Journal of
Food Science and Nutrition and Nutraceuticals and Foods, and also a member of
the editorial boards of the Journal of Medicinal Foods and Journal of Agriculture,
Food and Environment.
As a post-doctoral fellow, he conducted research at North Dakota State University, USA; and as visiting professor he conducted international collaborative research
at the Norwegian Institute of Fishery and Aquaculture, Norway, and at Lleida
University, Spain. He was keynote speaker at a number of international conferences
in the United States, Canada, Japan, China, Korea, Thailand, Spain, and Colombia.
He has published more than ninety research papers in international scientific journals
and twenty book chapters. His current research interests focus on processing technologies to separate health-promoting components from natural products and to
develop functional foods.
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Contributors
Mary Ann Augustin, Ph.D.
Science Manager
Food Chemistry and Formulation
Science
Food Science Australia
Victoria, Australia
Caye M. Drapcho, Ph.D.
Associate Professor
Department of Biosystems
Engineering
Clemson University
Clemson, SC, United States
Alberto Bertucco, Ph.D.
Professor
Institute of Chemical Engineering
University of Padova
Padova, Italy
Hideo Etoh, Ph.D.
Professor
Faculty of Agriculture
Shizuoka University
Shizuoka, Japan
Mércia de Fátima M. Bettini, Ph.D.
Technical Director of Flavor
Tec - Aromas de Frutas Ltda
Pindorama, Brazil
Maja Habulin, Ph.D.
Associate Professor
Faculty of Chemistry and Chemical
Engineering
University of Maribor
Maribor, Slovenia
Juan Eduardo Cacace, M.S.
Visiting Research Engineer
Pacific Agri-Food Research Center
Agriculture and Agri-Food Canada
Summerland, BC, Canada
Feng Chen, Ph.D.
Associate Professor
Department of Food Science and
Human Nutrition
Clemson University
Clemson, SC, United States
Louise Deschênes, Ph.D.
Research Scientist
Food Research and Development
Centre
Agriculture and Agri-Food Canada
St. Hyacinthe, QC, Canada
© 2007 by Taylor & Francis Group, LLC
Yueming Jiang, Ph.D.
Professor
South China Institute of Botany
Chinese Academy of Sciences
Guangzhou, China
Yukio Kakuda, Ph.D.
Professor
Department of Food Science
University of Guelph
Guelph, ON, Canada
Lamin S. Kassama, Ph.D.
Post-doctoral Research Fellow
Guelph Food Research Centre
Agriculture and Agri-Food Canada
Guelph, ON, Canada
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ˇ
Zeljko
Knez, Ph.D.
Professor
Faculty of Chemistry and Chemical
Engineering
University of Maribor
Maribor, Slovenia
Aditya Kulkarni, M.S.
Research Assistant
Faculty of Agriculture
Shizuoka University
Shizuoka, Japan
Ashwani Kumar, Ph.D.
Research Scientist
Institute for Chemical Process and
Environmental Technology
National Research Council of Canada
Ottawa, ON, Canada
Chiara G. Laudani, Ph.D.
Associate Professor
Faculty of Chemistry and Chemical
Engineering
University of Maribor
Maribor, Slovenia
Giuseppe Mazza, Ph.D., FCIFST
Principal Research Scientist
Pacific Agri-Food Research Center
Agriculture and Agri-Food Canada
Summerland, BC, Canada
Gauri S. Mittal, Ph.D.
Professor
School of Engineering
University of Guelph
Guelph, ON, Canada
Valérie Orsat, Ph.D.
Research Engineer
Bioresource Engineering Department
Macdonald Campus of
McGill University
Ste-Anne de Bellevue, QC, Canada
© 2007 by Taylor & Francis Group, LLC
ˇ ˇ Ph.D.
Mateja Primozic,
Associate Professor
Faculty of Chemistry and Chemical
Engineering
Laboratory for Separation Processes
University of Maribor
Maribor, Slovenia
G. S. Vijaya Raghavan, Ph.D.
Professor
Bioresource Engineering
Department
Macdonald Campus of
McGill University
Ste-Anne de Bellevue, QC, Canada
Luz Sanguansri, Ph.D.
Research Scientist
Food Science Australia
Victoria, Australia
John Shi, Ph.D.
Research Scientist
Guelph Food Research Centre
Agriculture and Agri-Food Canada
Guelph, ON, Canada
Helena Sovová, Ph.D.
Professor
Institute of Chemical Process
Fundamentals
Academy of Sciences of the Czech
Republic
Prague, Czech Republic
Sara Spilimbergo, Ph.D.
Professor
Department of Materials Engineering
and Industrial Technologies
Faculty of Engineering
University of Trento
Trento, Italy
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Terry H. Walker, Ph.D.
Associate Professor
Department of Biosystems Engineering
Clemson University
Clemson, SC, United States
Shiow Y. Wang, Ph.D.
Plant Physiologist/Biochemist
Fruit Laboratory
ARS-USDA
Beltsville, MD, United States
© 2007 by Taylor & Francis Group, LLC
Xiaoqin Zhou, M.S.
Professional Engineer
Department of Chemical Engineering
University of Waterloo
Waterloo, ON, Canada
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Table of Contents
PART I
Supercritical Fluid Extraction Technology
Chapter 1
Supercritical Fluid Technology for Extraction of Bioactive Components ...............3
John Shi, Lamin S. Kassama, and Yukio Kakuda
Chapter 2
Solubility of Food Components and Product Recovery in the
Supercritical Fluid Separation Process....................................................................45
John Shi and Xiaoqin Zhou
Chapter 3
Modeling of Supercritical Fluid Extraction of Bioactives from
Plant Materials .........................................................................................................75
Helena Sovová
Chapter 4
Biochemical Reactions in Supercritical Fluids .....................................................111
ˇ
ˇ
Zeljko
Knez, Chiara G. Laudani, Maja Habulin, Mateja Primozič
PART II
Pressurized Low Polarity Water Extraction,
Membrane Separation, Distillation, and
Dehydration Technologies
Chapter 5
Pressurized Low Polarity Water Extraction of Biologically Active
Compounds from Plant Products ..........................................................................135
Juan Eduardo Cacace and Giuseppe Mazza
Chapter 6
Purification of Orange Peel Oil and Oil Phase by Vacuum Distillation ..............157
Mércia de Fátima M. Bettini
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Chapter 7
Dehydration Technologies to Retain Bioactive Components ...............................173
Valérie Orsat and G. S. Vijaya Raghavan
Chapter 8
Membrane Separation Technology in Processing Bioactive Components ...........193
Ashwani Kumar
PART III
Bioprocessing Technology
Chapter 9
Bioprocessing Technology for Production of Nutraceutical Compounds ............211
Terry H. Walker, Caye M. Drapcho, and Feng Chen
Chapter 10
Microbial Modeling as Basis for Bioreactor Design for
Nutraceutical Production .......................................................................................237
Caye M. Drapcho
PART IV
Preservation and Packaging Technologies
Chapter 11
Food Pasteurization and Sterilization with High Pressure ...................................269
Alberto Bertucco and Sara Spilimbergo
Chapter 12
Microencapsulation and Delivery of Omega-3 Fatty Acids .................................297
Luz Sanguansri and Mary Ann Augustin
Chapter 13
Packaging Technologies of Functional Foods.......................................................329
Louise Deschênes
PART V
Antioxidant Properties and Material
Chapter 14
Biological Antioxidation Mechanisms: Quenching of Peroxynitrite ...................341
Aditya Kulkarni and Hideo Etoh
© 2007 by Taylor & Francis Group, LLC
Chapter 15
Stability of Lycopene During Food Processing ....................................................353
John Shi, Yukio Kakuda, Yueming Jiang, and Gauri S. Mittal
Chapter 16
Fruits with High Antioxidant Activity as Functional Foods ................................371
Shiow Y. Wang
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1
Supercritical Fluid
Technology for
Extraction of Bioactive
Components
John Shi and Lamin S. Kassama
Agriculture and Agri-Food Canada
Yukio Kakuda
University of Guelph
CONTENTS
1.1
1.2
Introduction ......................................................................................................4
Process Concept Schemes and Systems ..........................................................6
1.2.1 Process Scheme and System................................................................7
1.2.1.1 Single-Stage Extraction Process...........................................7
1.2.1.2 Multistage Extraction Process ..............................................9
1.2.2 Physicochemical Properties of Supercritical (CO2) Fluids ...............10
1.2.2.1 Phase Diagram ....................................................................10
1.2.2.2 Physical Properties..............................................................10
1.3 Applications in the Food Industry .................................................................12
1.3.1 Extraction of Bioactive Compounds..................................................12
1.3.2 Fractionation of Flavors and Fragrances ...........................................18
1.3.3 Cholesterol-Free Food Products ........................................................20
1.3.4 Separation of Spices and Essential Oils............................................21
1.3.5 Decaffeination of Coffee and Tea......................................................23
1.3.6 Fish Oil Concentration.......................................................................26
1.4. Factors Affecting Extraction Yield ................................................................27
1.4.1 Pressure ..............................................................................................27
1.4.2 Temperature........................................................................................28
1.4.3 Moisture Content of Raw Materials ..................................................29
1.4.4 Cosolvent............................................................................................30
3
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4
Functional Food Ingredients and Nutraceuticals
1.4.5 Particle Size........................................................................................32
1.4.6 Flow Rate ...........................................................................................32
1.4.7 Effect of Time on Yield .....................................................................34
1.5 Summary ........................................................................................................36
References................................................................................................................37
1.1 INTRODUCTION
Some chemicals are toxic and if consumed can lead to cancer and other ailments of
public concern. As a result, the conventional solvent extraction methods are viewed
with suspicion with regards to their role in the manufacturing of functional foods.
Extracts from natural sources are key elements in the manufacturing of functional
foods. These are any food fortified with extracts from natural sources or nutraceutical. A new paradigm, supercritical extraction technology, was developed to extract
bioactive components for use as supplements for functional foods. This technology
uses supercritical fluids as the extracting solvent which has a significant attribute
paramount to the food and pharmaceutical industry because it leaves no residue in
the extract and is gaseous at ambient temperature.
Extraction with organic solvents is a well-established technique for selective
separation of specific constituents from plant products. Organic solvents with low
boiling point such as ethyl acetate, methanol, dichloromethane, and so forth, have
been successfully used to isolate bioactive components from hops, spices, oil seeds,
and other plant products. The procedure is used to decaffeinate coffee and tea, and
to remove nicotine from tobacco. The solvents used for this purpose must meet the
legal requirements put in place to ensure food quality and safety, and these regulations vary from country to country. These requirements are: high degree of purity,
chemical stability, inert (no reaction with food constituent), low boiling point, and
no toxic effects. Criteria for these regulations are set by national and international
bodies such as the US Food and Drug Administration (FDA); European Economic
Commission (EEC) Codex committee; the Canadian Food Inspection Agency
(CFIA); and FAO/WHO Codex Alimentarius Commission. Most countries have
regulations stating which extraction solvents are generally regarded as safe (GRAS).
For example, the Canadian food additive regulation (FDAR: B.10.045) for food
chemical codex specification and the EEC directive (88/344/EEC) state the regulations regarding the use of extraction solvents in the production of foodstuffs and
food ingredients within Canada and the EEC. Although only mandatory within
Canada and the EEC, in general they are comparable to those in other countries.
Public health, environmental, and safety issues are the major concerns in the
use of organic solvents in food processing. The possibility of solvent residues
remaining in the final product has been a growing concern to consumers, thus
warranting stringent environmental regulations. The demand for ultrapure and highadded-value products is redirecting the focus of the food and pharmaceutical industries into seeking the development of new and clean technologies for their products.
The supercritical fluid extraction (SFE) technology has provided an excellent alternative to the conventional organic solvent extraction methods. Although the
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Supercritical Fluid Technology for Extraction of Bioactive Components
5
technology was known for more than 100 years, its application in the food and
pharmaceutical industry began only three decades ago.1,2 Since that time, over 100
plants of different capacities have been built globally for extraction of desired solutes
from solid materials.3
Supercritical extraction is a novel separation technique that utilizes the solvent
properties of fluids near their thermodynamic critical points.4 A variety of processes
involving extractions with supercritical fluids (SCF) have been developed and are
regarded as a viable extraction technology that meets the food quality and safety
requirements. The physicochemical properties of supercritical fluids, such as the
density, diffusivity, viscosity, and dielectric constant can be controlled by varying
the operating conditions of pressure and temperature or both in combination.1,3,5
Many supercritical fluids (carbon dioxide, ethane, propane, butane, pentane, ethylene, ammonia, sulfur dioxide, water, chlorodifluoromethane, etc.) are used in supercritical extraction processes. Sihvonen et al.,1 Brunner,3 and Rozzi and Singh6 recommended carbon dioxide (CO2) in their reports because of its favorable properties
and the ease of changing selectivity by the addition of agents such as ethanol and
other polar solvents. Supercritical carbon dioxide (SC-CO2) is the most desirable
supercritical fluid for extracting natural products for foods and medicinal uses. Its
characteristic traits are inert, nonflammable, noncorrosive, inexpensive, availability,
odorless, tasteless, environmentally friendly, and GRAS status.7 Its near-ambient
critical temperature makes it ideal for thermolabile natural products.8,9
Supercritical CO2 has the following advantageous attributes over other solvents:
(a) it has a solvating power similar to organic liquid solvents and higher diffusivities,
lower surface tension, and viscosity; (b) separation can be affected by simply changing the operating pressure or temperature to alter the solvating power of the solvent;
and (c) modifying CO2 with a cosolvent can significantly augment the selective and
separation power and in some cases extend its solvating powers to polar components.
Supercritical carbon dioxide is being given a great deal more attention as an alternative to industrial solvents as a result of (a) increased governmental scrutiny and
new regulations restricting the use of common industrial solvents such as chlorinated
hydrocarbons; (b) its nontoxic and environmentally friendly attributes, given that it
leaves no traces of solvent residue in food; (c) sharp increase in energy cost, which
increased the cost of traditional energy-intensive separation technique, such as distillation; (d) carbon dioxide is cheap, safe to use, recyclable, and has minimum
disposal cost required; (e) stringent pollution-control legislation prompting industries
to seek alternative means of waste treatment and utilization; and (f) increased
performance demands on materials, which traditional processing techniques cannot
meet.
It must, however, be stated that commercial applications of the SFE technology
remain limited to a few high-value products due to high capital investment,10,11 its
novelty, and complex operating system. Adoption of the technology is on the rise
as a result of advances in processing, equipment, and the realization of producing
high-value products with high profitability.1,10 Supercritical fluid extraction is one
of the most promising technologies being adopted in the chemical, food, pharmaceutical, and neutraceutical industries. Analytical techniques are superior to the
conventional organic solvents when applied for qualitative and quantitative extraction
© 2007 by Taylor & Francis Group, LLC
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6
Functional Food Ingredients and Nutraceuticals
of natural thermolabile products.12,13 Research efforts have proven that the SFE
technologies could be successfully employed in the extraction and fractionation of
fats and oils; purification of solid matrix; separation of tocopherols and antioxidants;
removal of pesticide residue from herbs, medicines, and food products; detoxification
of shellfish; and concentration of fermented broth, fruit juices, essential oils, spices,
and coffee; and separation of caffeine, and so forth.3,14–16
1.2 PROCESS CONCEPT SCHEMES AND SYSTEMS
The supercritical fluid extraction technology was conceptualized on the basis of
obtaining pure extract without solvent residues which could be detrimental to consumers of food and pharmaceutical products. Extraction is an analytical process
used to separate and isolate a targeted component from other substances. The success
of the process is dependent on the distribution of the analyte between two phases,
the separation and stationary phases.17 In the phase equilibrium between liquid and
gas, the partition of the liquid phase increases with increasing pressure and decreases
with increasing temperature. If the temperature and pressure are simultaneously
increased, the transport properties of both liquid and gas increases and thus convergence occurs. When used under practical pressure and temperature conditions of 5
to 50 MPa and ambient to 300°C, respectively, the solubility properties of the
supercritical fluid are greatly influenced by its density, diffusivity, and viscosity. The
SC-CO2 is liquid-like and has a higher extraction flux than those obtained with
organic liquids solvents. King et al.17 stated that at high CO2 densities, its solvent
properties were similar to organic solvents like chloroform and acetone, and if
intermediate compression were applied, it behaved like a nonpolar hydrocarbon such
as n-pentane or diethyl ether.
The separation phase occurs during the dynamic extraction period, while the
stationary phase is the sample material loaded as a fixed-bed in the extraction column.
Supercritical extraction (SCE) involves the use of compressed gases at or above their
critical temperature (Tc) and pressure (Pc). It utilizes the ability of these special fluids
to become excellent solvents to solvate certain solutes (bioactive components) from
a solid matrix.6 The solute extraction stream from the sample matrix is directly
proportional to the product of solubility and diffusivity in the supercritical medium.
Hence the solute’s solubility increases with pressure, while its corresponding diffusivity is expected to decrease by two orders of magnitude. The solvent capacity is
mainly the function of density and can be improved with the addition of a cosolvent,
which modifies the density and polarity of the supercritical fluid, thus significantly
increasing the yield.
This technology has been successfully applied in the extraction of bioactive
components (antioxidants, flavonoids, lycopene, essential oils, lectins, carotenoids,
etc.) from a variety of biological materials such as hops, spices, tomato skins, and
other raw or waste agricultural materials. The process requires intimate contact
between the packed bed formed by a ground solid substratum (fixed-bed of extractable material) with a supercritical fluid.18 During the supercritical extraction process,
the solid phase comprised of the solute and the insoluble residuum (matrix) is brought
© 2007 by Taylor & Francis Group, LLC
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Supercritical Fluid Technology for Extraction of Bioactive Components
Back pressure valve
Pressure valve
CO2 outlet
Separator
Extractor column
Heat exchanger
Heat
exchanger
CO2 Pump
Cooling
bath
7
Extracts outlet
CO2 Source
FIGURE 1.1 Schematic diagram of a typical single-stage supercritical fluid extraction system
with CO2.
into contact with the fluid phase which is the solution of the solute in the supercritical
fluid (solvent). The extracted material is then conveyed to a separation unit.
The power of supercritical fluid extraction is linked to the solubility and phase
equilibrium of substances in the compressed gas. The targeted bioactive component
being extracted must be soluble in the supercritical fluid. Controlling the pressure
and temperature of SCF varies the solubility and phase equilibria. The extraction of
pure and high-value extract is accomplished without risk of environmental pollution
or residual solvent contamination in the final product.
1.2.1 PROCESS SCHEME
AND
SYSTEM
The extraction with supercritical fluids is comparable to liquid-liquid solvent extraction even though, with supercritical extraction, compressed gas is used instead of
organic solvents and the applied pressure is crucial. The supercritical fluid extraction
process is governed by four key steps: extraction, expansion, separation, and solvent
conditioning. The steps are accompanied by four generic primary components:
extractor column (high-pressure vessel), pressure control valves, separator column,
and pressure intensifier (pump) (Figure 1.1) for the recyclable solvent.3,5,17,19,20 The
system has other built-in accessories, such as heat exchangers for providing a source
of heating; condensers for condensing supercritical fluids to liquid; storage vessels;
and a supercritical fluid source. Raw materials are usually ground and charged into
a temperature-controlled extractor column forming a fixed bed, which is usually the
case for batch and single-stage mode.
1.2.1.1 Single-Stage Extraction Process
The supercritical fluid is fed at high pressure by means of a pump, which pressurizes
the extraction tank and also circulates the supercritical medium throughout the
system. Figure 1.2 shows an example of a typical single-stage supercritical extraction system. Once the SCF and the feed reach equilibrium in the extraction vessel,
through the manipulation of pressure and temperature to achieve the ideal operating
conditions, the extraction process proceeds. The mobile phase consisting of the SCCO2 fluid and the solubilized components are transferred to the separator where the
© 2007 by Taylor & Francis Group, LLC
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Functional Food Ingredients and Nutraceuticals
Three stage separator columns
CO2 inlet
Extractor column
Pressure
regulator
Dry test
meter
Extracts
8
Ice water trap
CO2 pump
Co-solvent inlet
Mixer
CO-solvent pump
FIGURE 1.2 Schematic diagram of supercritical fluid extraction system used to fractionate
bioactive components from plant matrix using supercritical carbon dioxide.
solvating power of the fluid is decreased by increasing the temperature or decreasing
the pressure of the system. The extract precipitates in the separator while the supercritical fluid is either released or recycled back to the extractor. In the case where
highly volatile components are being extracted, a multistage configuration may have
to be employed as shown in Figure 1.2.
As the solutions leave the extractor and flow to the first separation vessel via
the pressure regulator, the pasty oleoresins settle to the bottom as they separate and
can be collected, while the remaining solution goes to the second-stage separator
where the fractionation of the volatile components occurs. For more sensitive products, the third stage of separation would be required for the complete isolation of
pure volatile components. Saltzman et al.21 presented a design (Figure 1.2) where
the solution flows through a heated valve and precipitates into a preweight U-tube
in an ice-water bath. The glass wool at the U-tube exit traps the entrained solutes
in the gas, while the gas flows through a dry-test meter which monitors the flow
rate. Oszagyan et al.22 used a similar system as illustrated in Figure 1.2 to extract
essential oil from Lavandula intermedia Emeric ex aloisel and herb of Thymus
vulgaris L., and further fractionated volatile components (-Cymene, -Terpinene,
Thymol, and Carvacrol) while Ozcan23 used it to fractionate volatile components
from Turkish herbal tea (Salvia aucheri Bentham Var. canaescen Boiss and Heldr.).
Duquesnoy et al.24 and Boutekedjiret et al.25 extracted and fractionated volatile
compounds from plant materials using SCE with a similar multistage fractionation
method.
The processes described above are semibatch continuous, where the SCF flows
in a continuous mode while the extractable solid feed is charged into the extraction
vessel in batches. In a commercial processing plant, multiple extraction vessels are
sequentially used to enhance process performance and output. Although the system
is interrupted at the end of the extraction period, when the process is switched to a
© 2007 by Taylor & Francis Group, LLC
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Supercritical Fluid Technology for Extraction of Bioactive Components
9
2 Stage separation vessels
Storage tank
3 Stage extraction columns
Co-solvent inlet
CO2 inlet
Pu mps
FIGURE 1.3 Schematic diagram of commercial scale multistage supercritical fluid extraction
system used to fractionate bioactive components.
prepared vessel for extraction, the unloading and/or loading of the spent vessels can
be carried out while extraction is in progress, reducing the downtime and improving
the production efficiency.
1.2.1.2 Multistage Extraction Process
A semicontinuous approach on a commercial scale uses a multiple-stage extraction
process which involves running the system concurrently by harnessing a series of
extraction vessels in tandem as shown in Figure 1.3. In this system the process is
not interrupted at the end of extraction period for each vessel, because the process
is switched to the next prepared vessel by control valves for extraction while unloading or loading the spent vessels; although imperfect, continuity is attained.
The primary extraction stages operate in a similar mode to the ones depicted
in Figure 1.2 and Figure 1.3. The raffinate from the premier stage enters the first
separation vessel while separation and fractionation of different compounds occurs
based on their relative solubility. The options of cosolvent are available to enhance
the solvent power of separation of specific components.
This is effective for cases where more than one targeted component is to be
extracted, giving the flexibility to vary the extraction parameters such as pressure
and temperature to achieve different solubilities for different components being
extracted at each stage of operation. Gamse26 suggested that highly soluble
substances could be extracted at the initial stages at low SCF density, and by increasing the density in the subsequent stages remove the less soluble substances. The
supercritical pressure, temperature, and flow rate at each stage could be controlled
independently.
© 2007 by Taylor & Francis Group, LLC
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10
Functional Food Ingredients and Nutraceuticals
1.2.2 PHYSICOCHEMICAL PROPERTIES
FLUIDS
OF
SUPERCRITICAL (CO2)
The physicochemical properties of the supercritical fluids are crucial to the understanding of the process design calculation and modeling of the extraction process.
Therefore, selectivity of solvents to discriminate solutes is a key property for the
process engineer. Physical characteristics such as density and interfacial tension are
important for separation to proceed; the density of the extract phase must be different
from that of the raffinate phase and the interfacial properties influence coalescence,
a step that must occur if the extract and raffinate phase are to separate.
1.2.2.1 Phase Diagram
Supercritical state of fluid is influenced by temperature and pressure above the critical
point. The critical point is the end of the vapor-liquid coexistence curve as shown
on the pressure-temperature curve in Figure 1.4 where a single gaseous phase is
generated. When pressure and temperature are further increased beyond this critical
point, the fluid enters a supercritical state. At this state no phase transition will occur
regardless of any increase in pressure or temperature nor will it transit to a liquid
phase. Hence, diffusion and mass transfer during supercritical extraction are about
two orders of magnitude greater than in the liquid state.
1.2.2.2 Physical Properties
Substances that have similar polarities will be soluble in each other, but increasing
deviation in polarity will make solubility increasingly difficult. Intermolecular polarities exist as a result of van der Waals forces, and, although solubility behaviors
Pressure (MPa)
Supercritical
region
Liquid phase
PC
Solid phase
Triple
point
Gas phase
Tc = 31.1°C
Pc = 7.38 MPa
TC
Temperature (°C)
FIGURE 1.4 Supercritical pressure-temperature diagram for carbon dioxide.
© 2007 by Taylor & Francis Group, LLC
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Supercritical Fluid Technology for Extraction of Bioactive Components
11
TABLE 1.1
Comparison of Physical Properties of Supercritical CO2 at 20 MPa and
55°C with Some Selected Liquid Solvent at 25°C
Properties
CO2
n-Hexane
Methylene
Chloride
Methanol
Density (g/mL)
Kinematic viscosity (m2/s)
Diffusivity (m2/s)
Cohesive energy density (δ(cal/cm∏))
0.75
1.0
6.0 × 109
10.8
0.66
4.45
4.0 × 109
7.24
1.33
3.09
2.9 × 109
9.93
0.79
6.91
1.8 × 109
14.28
Source: Modified from King et al.17
depend on the degree of intermolecular attraction between molecules, the discrimination between different types of polarities is also important. Substances dissolve
in each other if their intermolecular forces are similar, or if the composite forces
are made up in the same way. Properties such as the density, diffusivity, dielectrical
constant, viscosity, and solubility are paramount to supercritical extraction process
design. The dissolving power of SCF depends on its density and the mass transfer
characteristic, and is superior due to its high diffusivity, low viscosity, and interfacial
tension to liquid solvents.
Although many different types of supercritical fluids are in existence and have
many industrial applications, CO2 is the most desired for SCE of bioactive components. Table 1.1 shows some physical properties of compressed (20 MPa) supercritical CO2 at 55°C compared to condensed liquids commonly used as extraction
solvents at 25°C. It should be noticed that supercritical CO2 exhibited similar density
as those of the liquid solvents, while less viscous and highly diffusive. This fluidlike attribute of CO2 coupled with its ideal transport properties and other quality
attributes outlined above make it a better choice over other solvents.
The specific heat capacity (Cp) of CO2 rapidly increases as the critical point
(31.1°C temperature, 7.37 MPa pressure, and 467.7 g/L flow rate) is approached.
Like enthalpy and entropy, the heat capacity is a function of temperature, pressure,
and density.5 Under constant temperature both the enthalpy and entropy of supercritical CO2 decreases with increased pressure and increases with temperature at
constant pressure. The change in the specific heat as a result of varying the pressure
and temperature is also dependent on density. For example, under constant temperature, specific heat increases with increased density up to a certain critical level.
Above this critical level, any further increase of density decreases the specific heat.
Sample matrix is an important parameter that influences solubility and mass
transfer process during SCE. Properties such as particle shape and size distribution,
porosity and pore size distributions, surface area, and moisture content influence
solubility and mass transfer. The presence of water (moisture content) in the sample
matrix during supercritical extraction also has an effect on the extraction outcome.
© 2007 by Taylor & Francis Group, LLC
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12
Functional Food Ingredients and Nutraceuticals
1.3 APPLICATIONS IN THE FOOD INDUSTRY
One of the most important trends in the food industry today is the demand for allnatural food ingredients that are free of chemical additives. Natural food antioxidants
are derivatives of plant by-products. A quantum leap in supercritical fluid extraction
technology was made by its applications in decaffeinating coffee, tea, and other
bioactive (essential oils from spices) components used as ingredients in foods.
Likewise, SCF extraction is used to extract flavor and fragrance components, highvalue compounds used in the food, pharmaceuticals, and neutraceutical industries.
With this technology, extracts of natural nutrients could be utilized without the fear
of organic solvent residues. A compendium of process parameters used for different
product applications is listed in Table 1.2.
Various processes of extracting bioactive components from agricultural materials
have been used commercially. The major hurdles to overcome in SCE are to obtain
extracted compounds with similar bioactivity as the synthetic compounds and also
to maintain the required flavor, odor, and color components that may be detectable
in the treated food product. Many extraction processes employ the following methods
to extract bioactive components from plants (Labiatae family): solvent (polar and
nonpolar) extraction and aqueous alkaline58; extraction of vegetable oils and
mono/diglycerides59 by steam distillation and molecular distillation.23 These processes are limited by numerous disadvantages, because the solvents used are not
very selective for the bioactive components. Consequently, the resulting extracts are
not as pure as synthetic chemical compounds. The solvents used include hydrocarbons such as hexane, acetone, and methyl chloride which leave unwanted residues
in the food products, which are prohibited in some instances by regulatory bodies
for use in food as discussed earlier. Many researchers applied SCE technology for
the extraction of bioactive components.3,56,60–62
1.3.1 EXTRACTION
OF
BIOACTIVE COMPOUNDS
Some bioactive components are phenolic or polyphenolic compounds of plant origins
which interfere with the formation of free radicals, thus preventing the formation of
hydroperoxides. However, during food processing, especially conventional thermal
processing of food products, these bioactive components are lost. To restore these
components necessitates fortification. Some of the more common bioactive compounds include lycopene, flavonoids, tocopherol, lecithin, ascorbic acid, citric acid,
polyphenols, and so forth.
Most of the separation procedures involve physical and chemical processes such
as centrifugation, filtration, membrane separation, precipitation, chromatography,
solvent extraction, crystallization, evaporation, molecular distillation, and SC-CO2.
To overcome the separation problems encountered when producing soluble materials
via such processing procedures requires conditions that may have detrimental effects
on the nature of the extraction technique or the product stability. In order to achieve
extract labile compounds, a rapid separation process may be needed to avoid any
significant product loss. Most bioactive components used as food additives are in a
concentrated form. Appropriate extraction procedures are consequently required
© 2007 by Taylor & Francis Group, LLC
Product
Extracted
Raw
Material
Sunflower oil
Soybean oil
Sunflower
seed
Soybean
Corm germ oil
Seed oil
Corn germ
Avocado
Raw
Material
Pretreatment
Component
Concentration
(%)
Temp
(°C)
Pressure
(MPa)
Ground
40−50 32–35
Flaked,
20.1
0.38–5.1mm
Dried, milled 23.4
Dried, ground
(< 0.25 mm)
50
7.58–8.27
50
70
55.2
75.8
50
50
70–75
50
Cottonseed
Paprika
Peanuts
Peanut
hearts
Rice bran
Sorghum
bran
Sorghum
germ
Soybean
Wheat bran
Wheat germ
© 2007 by Taylor & Francis Group, LLC
CO2
Flow Rate Time
(L/h)
(h)
MC
(%)
2.5
Recovery
(%)
Cosolvent
Source
Ethanol
36
Cocero &
Calvo27
Friedich et al.28
Ethanol
50
58.2
Ronyai et al.29
Friedich &
Pryde30
900–1080
9.8
1140
24
3.5
3.4
55.2
55.2
68.9
68.9
8
7
8
11
8.6
5
9.0
2.8
30.8
7.2
48.0
42.6
70
78
65.5
66.2
6
11
6.3
6.1
19.2
5.0
70
68.9
7
9.4
16.8
50
50
50
55.2
55.2
55.2
8
5
6
11.4
11.4
10.1
19.4
4.0
7.0
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TABLE 1.2
Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Product
Extracted
(Essential oil)
Limonene
Carvone
Anethole
Eugenol and
Caryophyllene
Yaleranone
Wheat germ oil
Licochalcone A
Licochalcone B
Defatted
mustard
Canola oil
Bergaptene
Raw
Material
Raw
Material
Pretreatment
Component
Concentration
(%)
Sweet
Dried ground
orange
Sieved (1, 0.7,
Caraway
0.4, 0.2, 0.08
seed
mm)
Anise seed
Cloves
Spikenard
Wheat germ Milled,
10.2
powder
(20 µm
diameter)
Licorile
Cut
root
Mustard
Dried, fully
16
seed
pressed
(1mm)
Canola seed Flaking
(0.2–0.5 mm)
cooking
(90°C)
Bergamot
Peel dried
0.15
Citrus
ground
© 2007 by Taylor & Francis Group, LLC
Temp
(°C)
Pressure
(MPa)
CO2
Flow Rate Time
(L/h)
(h)
23–40 9–10
60
35–50 13–41
0.12
2
35
12.7
360
0.5
40
30.4
300–400
3
45–70 41–62
180
3
40–60 8–10
2.6
2
MC
(%)
Recovery
(%)
Cosolvent
Source
6.7
60
Sovava et al.31
4.3–11.5
98.7
Ge et al.32
5% Ethanol 25–30
Ethanol
17–19
80
Taniguchi et al.33
44
Dunford and
Temelli34
85
Poiana et al.35
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TABLE 1.2 (CONTINUED)
Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Defatted &
decholesterol
of muscle
Oil
Bixin pigment
Defatted &
decholesterol
beef
Bixin pigment
Evening
primrose oil
Cardamom oil
Alfalfa
Leaf protein
concentration
Dehydrated Spray dried
beef
powder + air
dried chunk
Antarctic
Freeze-dried 16.2
krill meal
& ground
Annatto
Seed, 3.92
seed
mm
Fresh
19
frozen
Ground
beef
Annatto
Whole seed 1.3
seed
(11.6 oil)
40
Evening
primrose
seed
Cardamom
seeds
Decholesterol
beef tallow
β-carotene
Crude beef
tallow
Sweet
potato
Lanolin
Woolgrease
Dried,
ground,
0.355 mm
Freshly
ground
seeds, mesh
Freeze dried
ground
( 0.25 mm)
© 2007 by Taylor & Francis Group, LLC
30–
70
300–360
90
70
Favati et al.36
1.4–3.2
87
Wehling et al.37
7.83
99
Yamaguchi et
al.38
Chao et al.39
56-lipid
26-cholesterol
Chao et al.40
3
45–55 23–3927.6 300–600
80
24.5
51.4
3–4
50
60
34.5
31
1–2
35
31.
117.2 –
144.8
117.2 –
144.8
50
29.6
1.2
21.9
60
70
64.8
40–60
40
10
0.15–0.2
40
34.5
226.7250
94
48
41.4
840–1080
80
38
396–2880
4–8
Degnan et al.41
Soybean oil
20% v
1.7–5 < 8
97
Favati et al.42
3
85–95
Gopalakrishnan
& Narayanan43
60–70
Chao et al.44
80
Spanos et al.45
4–6
15.5
10
CygnarowiczProvost et al.46
2441_C001.fm Page 15 Thursday, July 20, 2006 9:35 AM
Carotene
lutein
Product
Extracted
Raw
Material
Phospholipid
mixture
Soybean
flakes
Tocopherol
enrichment
Soybean
flakes
Rice bran
Carrot
Carotene
Essential oil
Star anise
fruit
Protein
Spring
mackerel
Black
pepper
Eucalyptus
leaves
Ginger
rhizomes
Palm oil
fiber
Essential oil
Piperine
Essential oil
Essential oils
gingeroles
Triglycerides
carotenoid
Raw
Material
Pretreatment
Component
Concentration
(%)
Ground,
5.134 mg/g
thinly flakes,
(0.1–0.25
mm)
Frozen puree
(0.93mm)
Dried,
ground,
particle size
(0.57 mm)
Freeze-dried,
chopped
Dried ground
Oven dried
© 2007 by Taylor & Francis Group, LLC
Pressure
(MPa)
CO2
Flow Rate Time
(L/h)
(h)
MC
(%)
Source
68.2
43.6–109
80
25
300
500 ppm
55
20.7
90
8–9
26.5
6.5
5.4
1.5
12.7
97
35
34.5
1080
15
1.6
87
40
9–15
2.55
9
18
Perakis et al.49
50
9
1.1
2.5
9.5
2.4
Porta et al.50
138
2
8.8
8.4
Catchpole et al.51
1.45
2.25
5
7
Franca and
Meirele52
1.5
5.7
30
40
45–
55
30
20–30
10.8
Recovery
(%)
Cosolvent
80
Air drying
Dried ground
Temp
(°C)
81
Ethanol
(5% v)
Ethanol
10%
60-Soybean
King et al.47
70-Rice bran
80-Wheat germ
Vega et al.48
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TABLE 1.2 (CONTINUED)
Supercritical Extraction Process Parameters for Some Selected Bioactive Components from Agricultural Material by SC-CO2
Essential oil
vanillin
Essential oil
Capsaicine
alkaloids
Essential oil
Antioxidants
Essential oil
Tomatoes
Peppermint
&
Spearmint
Vanilla pod
Chili
pepper
Dried skin
ground
Cut leaves
65
35
Dried ground
36
36
11
Dried ground
2
40
30
50
15
Coriander
Dried grounds 96
(0.4 mm)
Sweet Thai Dried ground
Tamarinds (mesh
40–70)
Grape seed Wash dry
80
ground
© 2007 by Taylor & Francis Group, LLC
40–80 17–28
30
0.5
24–43 6–18
3.42
4–9
35–80 10–30
35–75 20–47
Cadoni et al.53
Chloroform
14–82
76
Barton et al.54
97
2
3
3
0.3
5
0.75
Catchpole et al.51
8.8
0.61
10
Anitescu et al.55
Ethanol
Luengthanaphol
et al.56
2% Ethanol 77
Lee et al.57
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Lycopene
-Carotene
Essential oil
Menthol
2441_C001.fm Page 18 Thursday, July 20, 2006 9:35 AM
18
Functional Food Ingredients and Nutraceuticals
when preparing them from their original matrices. Some compounds in the concentrated form are thermolabile, volatile, and prone to degradation when subject to
intensive heat.
Supercritical extraction with CO2 is the most viable method for food applications.
Baysal et al.63 extracted lycopene and -carotene from tomato using SC-CO2. The
processing conditions used were extraction pressure of 20, 25, and 30 MPa; temperatures of 35°C, 45°C, 55°C, and 65°C; resident time of 1, 2, and 3 h; and CO2
flow rate of 2, 4, and 8 kg/h. The best conditions for lycopene extraction were 2 h
at a flow rate of 4 kg/h, pressure of 30 MPa, temperature of 55°C, and with the
addition of 5% cosolvent (ethanol). They noted that if too much ethanol is used, it
decreased the homogeneity of the extraction mixture, and reduced the separation
efficiency. Brunner and Peter64 and Vega et al.48 corroborated this finding. Luengthanaphol et al.56 compared the SC-CO2 extraction to other methods and their effects
on bioactivity. Their results compared to Tsuda et al.65 indicated the superiority of
SC-CO2 coupled with cosolvent as shown in Table 1.3.
Both studies show the superiority of antioxidant extraction with SC-CO2 and
modifiers, although some disparity occurred which could have been caused by the
variety used. Macias-Sanchez et al.61 extracted carotenoids and chlorophyll from
Nannochloropsis gaditana and achieved the highest yield at 20 MPa and 60°C, the
optimal pressure and temperature, respectively. Wang et al.66 also reported that the
antioxidant activity of Bupleurum kaoi Liu fractionated with SC-CO2 gave the
highest yield of phenol and the strongest antioxidant capacities.
1.3.2 FRACTIONATION
OF
FLAVORS
AND
FRAGRANCES
The extraction of compounds, for flavor and fragrances, by supercritical CO2 is
paramount in the food industry. Mother Nature is a splendid synthesizer of flavors
and fragrances in natural products. The cleaner and safer attributes make supercritical
TABLE 1.3
Yields and Activities of Antioxidants Extracted by Different Methods from
Sweet Thai Tamarind Seed. SC-CO2 Extraction at 30 MPa and 80°C; Organic
Solvent Extraction at Room Temperature
PV after 24 h
(meq/kg dry weight)
(-) Epicatechin Yield
(mg/100 g dry weight )
Extraction
SC-CO2
SC-CO2 +
Cosolvent
(10%
Ethanol)
Ethanol
α-tocopherol
Luengthanaphol et al.56 Tsuda et al.65
Luengthanaphol et al.56
Tsuda et al.65
0.022
13
0.336
26
–
231
–
≈26
25
32
–
157
≈25
56
65
Source: Modified from Luengthanaphol et al. and Tsuda et al.
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Supercritical Fluid Technology for Extraction of Bioactive Components
19
TABLE 1.4
The Results of Gas Chromatography and Mass Spectrophotometer
Analysis of Flavor Compounds Extracted by the Likens-Nickerson
Extraction and the Supercritical CO2 Extraction Methods of Basmati Rice
Likens-Nickerson Extraction
Peak
No.
Retention
Time
(min)
2
3
5
8
13
14
19
21
22
24
7.51
8.11
9.51
14.52
21.98
22.68
27.71
28.53
30.45
32.14
Hexamethyl disiloxane*
Silicate anion tetramer*
Dibutyl phthalate*
Hexane cyclotrisiloxane*
Pentanal
Butan-2-one-3-Me
Heptanol
2-Heptanone
Octanol
Octanal
28
30
31
34
35
37
35.14
36.12
37.34
40.22
42.77
43.82
2-Octanone
Nonanal
Decanol
2-Decenal
Undecane
Benzoic acid 2,5-bis
(trimethylsiloxy) benzene*
39
46.28
Compound
SC-CO2
Peak
No.
Retention
Time
(min)
Compound
12
13
19
20
21
23
29
30
31
32
34
37
40
22.11
22.56
28.32
29.27
30.92
32.26
35.19
35.44
36.13
37.42
40.31
42.52
45.11
Pentanal
Butan-2-one-3-Me
Heptanol
2-Heptanone
Octanol
Octanal
2-Octenal
2-Octanone
Nonanal
Decanol
2-Decenal
Undecane
Dodecane
44
45.24
Tetradecane
Compounds identified as artifacts. Source: Modified from Bhattacharjee et al.62
technology an ideal candidate for extracting such valuable and heat-sensitive products when compared to toxic organic solvents. The high-value-added natural products
are good for use in soft drinks. An example is ginger extract which gives the
pungency and flavor in ginger ale drinks.
Bhattacharjee et al.62 compared the Likens-Nickerson extraction and the SCCO2 extraction methods on Basmati rice. The results showed that the SCE technique
was superior and extracted the most flavor components that bore the closest resemblance to the original Basmati flavor (Table 1.4). The SC-CO2 technique extracted
more flavor compounds and produced purer (resembles original basmati flavor)
extracts than its counterpart (Liken-Nickerson method).
Desired fragrances are isolated from concentrates extracted from flowers. The
process consists of initial solvent extraction, usually with hexane, which yields an
intermediate product called concrete. This product contains fragrances and other
components like paraffin, fatty acids, fatty acids methyl ester, di-, and tri-terpenic
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20
Functional Food Ingredients and Nutraceuticals
compounds, pigments, and so forth. Postprocessing of the concrete can be done
using SC-CO2 extraction. Reverchon67 used single-step SCE at a pressure of 8 MPa
and temperature of 40°C followed by a two-stage separation procedure at a pressure
of 8 MPa and temperature of –16°C at the first stage and a pressure of 1.5 MPa and
a temperature of 0°C at the second stage. Under these optimum conditions the
extracted volatile rose oil contains 50% 2-phenylethanol. When a cosolvent (ethanol)
is mixed with SC-CO2, a yield of 50% to 60% was observed.68 Jasmine fragrance
extracted at 12 MPa and 40°C gave results superior to those of other solvents. Sastry
and Mukhopadhyay68 experienced increased yield of 45% to 53% with the use of
cosolvents. Similarly, SC-CO2 has been used effectively to extract fragrances from
orange, marigold, sandalwood, vetiver, and so forth.
1.3.3 CHOLESTEROL- FREE FOOD PRODUCTS
Cholesterol is an unavoidable substance we require for the daily maintenance of our
body. Lipoproteins are vehicles that transport cholesterol to various body tissues to
be used, stored, or excreted, but if cholesterol is in excess it can lead to coronary
heart problems (atherosclerosis). Low-density lipoprotein (LDL) termed “bad” cholesterol causes fat buildup in the arteries increasing the risk of heart disease. Highdensity lipoprotein (HDL) termed “good” cholesterol transports cholesterol back to
the liver, where endogenous metabolism prevents cholesterol buildup and reduces
the risk of heart disease. The indiscriminate consumption of saturated fats in our
diet may raise the total LDL (> 100 mg/dL) level and decrease HDL (< 35 mg/dL)
level, thus increasing the risk of heart disease.69 The recommended daily intake of
cholesterol is about 300 mg.70 The correlation between serum cholesterol level and
mortality rate of cardiovascular disease has been reported in many studies.69,71
Pork has cholesterol content of 30 to 450 mg/100 g, poultry 70 mg, fish 35 to
70 mg/100 g, and beef 65 to 331mg. One common source of cholesterol is from the
consumption of fried fast-food products. The fast-food industry uses hydrogenated
fats for their deep-fat frying processes because of its stability and high economic
turnover. The hydrogenated fat is the potential source of trans fatty acids which are
taken up by the fried foods during cooking (French fries, onion rings, chicken
nuggets, etc.) and ultimately ingested by the consumer. Trans fats have been shown
to increase LDL cholesterol levels and reduce HDL cholesterol levels, thus raising
the risk of heart disease. Public health initiatives such as the National Cholesterol
Education programs have raised consumer awareness, resulting in the advocation
for healthy foods with low cholesterol. Thus, the food industry is under tremendous
pressure to address this consumer concern.
Supercritical fluid extraction is an emerging technology with great potential to
revolutionize the oil/fat industry. Many researchers (Dunford and Temelli34; Temelli
et al.34,72) reported the feasibility of supercritical fluid extraction of lipids from food
without compromising their organoleptic quality. Chao et al.44 used a similar extractor configuration (Figure 1.3) as discussed earlier with three stage separations, to
remove cholesterol. By decreasing the pressure at each stage sequentially from 17
to 11 to 4 MPa, they were able to achieve higher selectivity for cholesterol at the
lower pressures. The results also showed that the fraction collected from the third
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Supercritical Fluid Technology for Extraction of Bioactive Components
21
TABLE 1.5
Solubility of Cholesterol in Supercritical CO2 under
Different Operating Conditions
Run
No.
Pressure
(MPa)
Temperature
(°C)
Density of
CO2 (g/L)
Solubility of
Cholesterol (mg/L)
1
2
3
4
5
6
7
8
10
10
10
10
13
13
13
13
40
45
50
55
40
45
50
55
602.4
494.4
408.1
342.3
723.4
655.8
588.5
527.7
146.9
82.6
47.2
28.5
289.2
234.8
182.7
141.1
Source: Modified from Yeh et al.74
separator at 4 MPa contained concentrated cholesterol ranging from 272 to 433
mg/100 g lipid. Chao et al.40 used SC-CO2 at an operating pressure of 10 to 30 MPa
and temperature range of 30°C to 50°C to reduce the cholesterol level in ground
beef. Similarly, Hardardottir and Kinsella73 explored the removal of lipids and
cholesterol from fish muscle with SC-CO2. They removed over 80% to 99% cholesterol using pressures of 14 to 35 MPa and temperatures of 40°C to 50°C. Although
the authors noted limited effect on lipid/cholesterol yield with increased extraction
pressure and temperature, increased extraction time from 3 to 9 h significantly
increased the yield. Yeh et al.74 used eight operating conditions shown in Table 1.5
to optimize their process and observed that at 10.3 MPa and 55°C operating pressure
and temperature, respectively, cholesterol level was reduced from 2867 mg/100 g
to 14.1 mg/100 g. Supercritical-CO2 technology was used to fractionate milk fat,
which is an excellent raw material with specific functionalities used in many products.75 Extracting cholesterol from anhydrous milk fat with SC-CO2 used in conjunction with adsorbents (silica gel) to maximize yield was demonstrated by Huber
et al.76
1.3.4 SEPARATION
OF
SPICES
AND
ESSENTIAL OILS
Spices have strongly flavored or aromatic components that can be used in small
quantities in food as a preservative or flavoring ingredient. Chili (capsicium species),
ginger (Zingiber officinalis), and pepper (piper nigrum L) are classic pungent flavorines while ginger and chili have additional nutraceutical values. These products
have high economic value in their concentrated form. The extraction of spices is
usually carried out in two stages: stage one separates pungent oleoresins and the
second stage the essential oil fractions. Essential oils are typically volatile terpenes
and esters. Essential oils are concentrated pure plant extracts that have long been
revered for their therapeutic applications and are derivatives from flowers, leaves,
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Functional Food Ingredients and Nutraceuticals
stems, berries, rinds, resins, or roots of plants. These are very important ingredients
and food additives of high value.
Catchpole et al.51 performed a detailed study on the extraction of spices and
essential oils using SC-CO2, propane, and dimethyl ether. They reported ginger to
be the easiest of all spices in terms of optimized yield relative to pressure and
temperature, while capsaicin in chili could be extracted at moderate pressure and
temperature, especially with the use of modifiers. Chili oil fraction contains fatty
oil and carotenoids, and is speculated that the fatty oil acts as modifier for the
capsaicins.77 The author extracted black pepper with much duress, because of its
viscous characteristics, thus requiring higher pressure and moderate to high temperatures. The use of supercritical propane for extracting spices was reported by Illes
et al.78 They found propane to adequately extract fatty oils, tocopherols, and carotenoids, but inadequate for capsaicins, while CO2 was adequate for capsaicinoids,
fatty oils, and tocopherols, but not for carotenoids.
Figure 1.5 shows the supercritical extraction of ginger with three extraction
fluids (CO2, propane, and dimethyl ether).51 Propane gave the lowest yields while
dimethyl ether gave the highest yield. They reported dimethyl ether to have mutual
solubility with water. Ginger contains high amounts of volatiles, and CO2 extraction
offers the advantage of dividing the extract into oleoresins and essential oil fractions
by using a two-stage separation procedure with sequential pressure reduction. Similarly, if propane or dimethyl ether is used, considerable heating is required which
ultimately results in thermal degradation, and a larger energy requirement in the
form of cooling, depressurization, and boiling to recover the essential oils. In the
case of ginger, the oxygenated fraction was much greater than the steam distilled
oils and the gingerols in the oleoresin were extracted without decomposition. Oleoresins and piperine from pepper were extracted with insignificant losses although
a longer processing time was required. Similar trends were observed for chili and
pepper (Figure 1.6 and Figure 1.7). The extracts contained carotenoid pigments, and
those obtained with SC-CO2 were bright red with pink residues, while those from
propane and dimethyl ether were dark red. The extract obtained from chili with SCCO2 corroborated the results of other researchers5,79 in being viscous, pastry yellow,
and semisolid, while those extracted with dimethyl ether were yellow/black and
liquid at room temperature with a high quantity of water resulting in the dilution of
the essential oil and piperine content. Nguyen et al.80 described the extraction of
antioxidants from Labiatae herbs (rosemary, sage, oregano, and thyme) with SCCO2 at pressures in the vicinity of 50 MPa and temperatures ranging from 80°C to
100°C. The extracted oleoresin was precipitated into two fractions at various pressures and temperatures. The first fraction consisted of a green-brown, oil-soluble,
heat-stable, resin containing less than 2% essential oil and exhibiting remarkable
antioxidant properties. The second fraction was the essential oil containing more
than 95 mL steam distilled oil/100 g.
The use of SC-CO2 for the production of essential oils or oleoresins from spices
is possible by selecting suitable pressures and temperatures. The oils extracted with
supercritical technology were found to be superior in terms of their chemical composition and higher percentage of sesquiterpene compounds. Supercritical extraction
with CO2 and hydrodistillation extraction methods were used to extract essential oil
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Supercritical Fluid Technology for Extraction of Bioactive Components
23
160
Yield (g/kg)
120
80
40
0
0
5
10
15
Solvent (kg/kg ginger )
CO2, 313 K
Propane, 323 K
20
25
Dimethylether, 313 K
FIGURE 1.5 Supercritical extraction yield of ginger using CO2, propane, and dimethyl ether
(modified from Catchpole et al.51).
160
Yield (g/kg)
120
80
40
0
10
5
0
15
20
25
Solvent (kg/kg chili)
CO2, 313 K
Propane, 323 K
Dimethylether, 313 K
FIGURE 1.6 Supercritical extraction yield of chili using CO2, propane, and dimethyl ether
(modified from Catchpole et al.51).
(Juniperus communis L.)9 (Table 1.6). Oils obtained by SC-CO2 and hydrodistillation
showed significant differences; the former was more selective and particularly efficient for the isolation of -thoujone and limonene. Anitescu et al.55 did a comparative
analysis of coriander oil with supercritical CO2 and stream distillation (Table 1.7).
They concluded that oils obtained by supercritical extraction gave a superior aroma
compared to both the commercial and hydrodistillation extracted oils.
1.3.5 DECAFFEINATION
OF
COFFEE
AND
TEA
Caffeine (1,3,7-trimethylxanthine) is a bioactive plant component commonly found
(since 1820s) in popular beverages such as teas (Camellia sinensis), coffees (Coffee
Arabica, canephora, liberica), and soft drinks.81 Caffeine is a secondary metabolite,
the product of nucleic acid catabolism, and belongs to the group of compounds
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24
Functional Food Ingredients and Nutraceuticals
200
Yiled (g/kg)
150
100
50
0
0
5
10
CO2, 313 K
15
20
25
Solvent (kg/kg pepper)
Propane, 323 K
30
35
Dimethylether, 313 K
FIGURE 1.7 Supercritical extraction yield of pepper using CO2, propane, and dimethyl ether
(modified from Catchpole et al.51).
TABLE 1.6
Essential Oil (Juniperus communis) Oil Extracted by Comparing
Supercritical Extraction and Hydrodistillation (HD) Methods
Pressure
Temperature
Dynamic time
Modifier
No.
Compound
1
α-Thoujone
2
α-Pinene
3
Sabinene
4
Myrcene
5
3-Carene
6
Limonene
7
Terpinolene
(MPa)
(°C)
(min)
(µL)
RI*
928
943
972
987
1007
1020
1071
20
45
20
–
1
25.1
1.3
2.3
2.8
35.0
22.0
2.6
20
45
30
–
2
26.2
1.4
2.1
3.8
36.8
24.2
–
20
55
30
–
3
26.9
1.4
2.3
3.8
37.1
22.9
2.6
35
45
30
–
4
17.0
1.8
2.1
1.3
19.9
9.0
0.8
35
55
30
–
5
13.0
–
1.8
1.2
17.1
8.6
2.1
35.
55
30
80
6
22.0
1.9
1.9
2.4
29.1
15.6
2.7
35
55
30
400
7
4.0
22.4
34.6
4.2
2.6
6.1
2.7
HD
–
24.5
0.4
3.4
39.4
–
3.1
* Retention Index; Source: Modified from Pourmortazavi et al.9
known as purine alkaloids. Excessive ingestion of caffeine may cause certain health
problems such as palpitations, gastrointestinal disturbance, anxiety, tremor, increased
blood pressure, dizziness, and insomnia.82–85 The aroma and flavor coupled with the
stimulant effects comes from caffeine. Coffee beans have about 2% to 3% while tea
leaves have about 5% caffeine,82 depending on the variety and species. Decaffeinated
coffee must contain less than 0.1% caffeine on a dry weight basis, as specified by
EEC regulations. Therefore, decaffeination of coffee and tea poses significant challenges to both the producers and processors. The demand for decaffeinated coffee
is high on the world market. It accounts for more than 20% of all coffee sales in
the USA, with a 50% growing demand among the adult population.82 Research in
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Supercritical Fluid Technology for Extraction of Bioactive Components
25
TABLE 1.7
Results of Comparative Analysis of Essential (Coriander) Oil
between Supercritical CO2 and Steam Distillation Processes
No.
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
α-Thujene
α-Pinene
Camphene
Sabinene
β-Pinene
Ββ-Myrcene
∆3-Carene
Limonene
1,8-Cineole
Linalol
Camphor
Menthol
ρ-Cymen-8-ol
Cis-Hex-3-enyl butyrate
α-Terpineol
β-Citronellol
Neral
Carvone
Anethole
Carvacrol
Neryl acetate
Geranyl acetate
β-Caryophylene
α-Humulene
Eugenyl acetate
β-Caryophylene
Unidentified Compounds
Kovats
RI
Comm
Oil
Steam
Distillation
Oil
SC-CO2
Oil
928
936
951
975
980
990
1006
1030
1033
1103
1147
1174
1184
1186
1192
1226
1241
1245
1287
1299
1363
1382
1428
1463
1526
1594
Trace (Tr)
3.3
0.6
0.1
1.0
1.2
1.1
2.4
Tr
63.8
5.5
Tr
Tr
Tr
1.0
0.1
0.1
0.5
0.7
Tr
0.1
1.0
Tr
Tr
–
Tr
3.8
Tr
2.3
0.4
0.3
0.3
0.8
0.3
2.3
0.1
62.8
5.6
0.1
0.1
0.1
0.9
0.3
0.1
1.0
0.4
0.1
0.1
1.8
2.1
0.3
Tr
Tr
2.9
0.1
2.8
1.5
0.9
0.9
1.0
0.3
2.7
0.1
61.9
5.6
0.1
0.3
0.2
0.6
0.2
0.2
1.0
0.4
0.2
0.2
2.4
0.8
0.2
0.2
0.2
1.7
Source: From Anitescu et al.55
genetics engineering to produce transgenic tea and coffee plants deficient in caffeine
is in progress.82,83,86 However, the consumption of genetically modified products is
still contentious globally, and SCE technology gives the best option in combating
these critical issues. The decaffeination of coffee and tea using SCE technology is
among the first known commercial operation in the food industry.
In the past, methylene chloride was used for decaffeination of coffee with one
cycle of production lasting from 24 to 36 h and the end products usually contained
toxic residues, thus posing more harm than the caffeine. Due to its suspected
carcinogenic effect, the FDA placed regulations against methylene chloride use.
However, decaffeination process with SC-CO2 fluid can be accomplished on green
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26
Functional Food Ingredients and Nutraceuticals
coffee or roasted coffee beans or tea leaves without deleterious effects on the flavor
even after 10 h of processing, and many patents exist for such processes. The process
requires charging the extraction vessel containing the coffee beans with CO2 at a
pressure of 7 to 22 MPa and temperature of 31°C. The caffeine is dissolved in the
SC-CO2 stream, which subsequently enters a washing tower or alternatively activated
carbon scrubbers, distillation, recrystallization, or reverse osmosis are used in some
instances to entrain caffeine. The method can strip coffee of its caffeine content
(0.7% to 3%) by 71% to 97%.87 The caffeine recovered is sold for medicinal purposes
and or for use in soft drinks. Peker et al.88 reported that soaking raw coffee beans
in water prior to processing enhances the rate of decaffeination.
1.3.6 FISH OIL CONCENTRATION
Fish oils are characterized by a high percentage of unsaturated straight-chain fatty
acids ranging from C14 to C22 with one to six double bonds. They contain essential
fatty acids (EFA) and polyunsaturated acids, grouped into omega-6 and -3 EFAs.
The main sources of omega-3 (-3) are flaxseed, walnut, and marine plankton and
fish. This review would focus on -3 oils derived from fish. Eicosapentaenoic acid
(EPA) and decosahexaenoic acid (DHA) are predominant in fish oil, and have been
reported to contribute to the prevention of atherosclerosis, heart attack, depression,
and cancer if consumed in sufficient quantities.89 Fish oil derivatives in the form of
-3 oils are in high demands as food additives. For example, Ocean Nutrition’s ME3TM Omega-3 powder is currently used in several breads. In the US, Wegman’s
Food Markets, Rochester, NY, launched breads fortified with MEG omega-3 fats,
two slices of which offers 80 to 90 mg of omega-3s.90 Encapsulated omega-3 fatty
acids forms are available for fortified bakery products.
Fish oils are processed as fatty acids or as methyl or ethyl esters which are more
stable than the free acids form.91 Fatty acids are highly soluble in CO2 and as a
result SC-CO2 extraction is a preferred method of fractionation. With this technology,
it is possible to separate heat-sensitive compounds (ω-3 fatty acids) and avoid toxic
solvent residues in the final product. The isolation and fractionation of ω-3 PUFA
(polyunsaturated fatty acid) from fish, fish oil, and esters using SC-CO2 have been
studied by several researchers.72,92,93 Eisenbach93 fractionated the ethyl esters from
cold fish oil using SC-CO2 at a pressure of 15 MPa and an extracting temperature
of 50°C. Alkio et al.94 produced EPA and DHA with 50% and 90% purity, respectively, from trans-esterified tuna oil using carbon dioxide. Temelli et al.72 obtained
the highest yield of -3 fatty acids at 35 MPa and 35°C without denaturing the protein
during SCE. They also compared solvent (hexane) extraction to SC-CO2 as shown
in Table 1.8. A higher concentration of -3 was achieved with SC-CO2 (Table 1.8).
At 25 MPa pressure and temperature from 40°C to 80°C, no significant effect on
yield was observed in oil extraction from krill.38 Hardardotti and Kinsella73 did not
see any yield change on the recovery of rainbow trout at operating pressure ranging
from 13 to 35 MPa and temperature range of 40°C to 50°C.
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Supercritical Fluid Technology for Extraction of Bioactive Components
27
TABLE 1.8
Fatty Acid Composition of Fall Atlantic Mackerel Oil Concentrate
Extracted with Hexane and SC-CO2 at Pressure 34.5 MPa and
Temperature of 35°C Fractionated by Gas Chromatograph
Fatty
Acid
GC Retention
Time (min)
Hexane
Extract
SC-CO2
Extract
C16:0
C16:1
C18:0
C18:1
C18:3
C20:0
C20:1
C20:5 (EPA,ω-3)
C22:5
C20:5 (DHA,ω-3)
EPA+DHA (ω-3)
8.07
8.53
12.49
13.09
15.58
18.05
18.14
22.79
30.08
31.43
15.86
6.65
3.53
3.59
0.88
11.22
1.20
6.07
1.34
8.58
14.65
16.66
7.94
3.16
3.16
1.04
7.84
1.20
8.76
1.38
8.97
17.73
Source: Modified from Tamelli et al.72
1.4. FACTORS AFFECTING EXTRACTION YIELD
Several bioactive components were extracted successfully by the SC-CO2 extraction
method as outlined in the preceding sections. Optimization of yield is a function of
various independent parameters. Process parameters such as solvent flow rate, resident time, moisture content, particle sizes, and particle size distribution in conjunction with supercritical pressures and temperatures are key parameters for achieving
optimum results. Most of these parameters can have individual or combined effects
on the extraction rate of a process; for example, the resident time can have an
immense influence on the composition of the extracted compound.
1.4.1 PRESSURE
Figure 1.8 is a typical extraction rate curve, and it is apparent that pressure significantly influences the rate of extraction, and likewise the extraction time. Extrapolating the normalized yield at the point where the yield curve become asymptotic
gives significant different normalized yields of 15, 11, and 4% for pressures of 10,
9, and 8 MPa, respectively.8 Macias-Sanchez et al.61 observed similar trends in the
SC-CO2 extraction of carotenoids and chlorophyll a from Nannochloropsis gaditana,
although as pressure increased beyond a critical point the yields dropped as a result
of increased density. Higher density causes a double effect: increases the solvation
power and reduces the interaction between the fluids and matrix, thus decreasing
the diffusion coefficient. Excessive pressure also increases the compactness of the
sample matrix, thus reducing the pore sizes and apparently reducing the mass
transport, which eventually diminishes the yield.95
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Functional Food Ingredients and Nutraceuticals
16
Yield (%)
12
8
4
0
0
100
200
8 MPa
300
400
Time (min)
9 MPa
500
600
700
10 MPa
FIGURE 1.8 The effect of pressure change on bioactive compound yield during supercritical
extraction of essential oil from Juniperus oxycedrus on extraction rate at flow rate 1.5 kg/h
and temperature 50°C (modified from Marongiu et al.8).
The selectivity of solute extraction is a function of extraction pressure, and if
it increases, different solutes are extracted. D’Andrea et al.96 also found that with a
working pressure of 25 MPa and temperature of 55°C, they attained optimum yield
for the extraction of azadirachtin and 3-tigloylazadirachtol from neem seeds. Similarly, Tonthubthimthong et al.95 reported optimum yield at a pressure of 23 MPa
and a temperature of 55°C.
1.4.2 TEMPERATURE
Temperature is a parameter with significant influence on SFE; therefore manipulating
it could have adverse implication on the process and yield. Figure 1.9 shows the
general trend of increased extraction yield as temperature increases relative to the
pressure. Tonthubthimthong et al.95 reported similar trends for extracting nimbin
from neem seeds at 20 MPa and a CO2 flow rate of 0.62 mL/min, and found 35°C
the optimum temperature for their process. Ge et al.32 indicated a temperature of
35°C gave the highest yield in the first 45 min of extraction, but during prolonged
extraction from 45 to 120 min, the highest temperature condition was shown to
produce the highest yield (Figure 1.9). Although many literature reports correlated
increased temperature to yield,45 many others showed no particular trends as far as
temperature was concerned.97 Some researchers reported yield was inversely proportional to temperature under 15 MPa.
The combined effect of pressure and temperature on cholesterol extraction was
studied by Chao et al.44 At a pressure-temperature setting of 34 MPa and 50°C,
cholesterol yield of 160 mg/100 g was realized compared to 430 mg/100 g when
the temperature drops to 40°C and 2.5 kg of CO2 was used. As the mass of CO2
increased, the yields decreased, but the lowest temperature still maintains the highest
yield as shown in Figure 1.10. Also, under constant temperature, increases were
achieved when pressure decreased. The results demonstrated that higher selectivity
is possible at lower pressures and higher temperatures. Froning et al.98 corroborated
this fact based on their experiment with lipid and cholesterol extraction from
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Yield (mg/100 g)
2600
2200
1800
1400
10
15
20
25
Pressure (MPa)
35°C
40°C
30
45°C
35
40
50°C
FIGURE 1.9 The effect of temperature change on bioactive compound yield during supercritical extraction of wheat germ on extraction, time 120 min, rate at flow rate 2.0 mL/min
and sample size 5g (modified from Ge et al.32).
Cholesterol (mg/100 g)
500
400
300
200
100
0
0
5
10
15
20
25
Carbon dioxide (kg)
34.5 MPa/50°C
34.5 MPa/40°C
13.8 MPa/50°C
24.1 MPa/40°C
FIGURE 1.10 Supercritical carbon dioxide extraction of cholesterol in beef tallow, different
pressures and temperatures (modified from Chao et al.44).
dehydrated chicken meat. The combination of pressure and temperature of 38.6 MPa
and 55°C, respectively, yielded lipid (89%) and cholesterol (90%), while a pressure
and temperature combination of 30.3 MPa and 45°C, respectively, produced much
lower yield.
1.4.3 MOISTURE CONTENT
OF
RAW MATERIALS
Moisture content is a factor that influences extraction yield of bioactive compounds
as shown in Table 1.9. A maxima yield of 1678 mg/100 g was achieved at 5%
moisture content, and any further increase or decrease in moisture reduced that yield.
Therefore, it is important to establish this magic number in order to maximize yield
in the SCE process. The effect of sample pretreatment is crucial in attaining this
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Functional Food Ingredients and Nutraceuticals
TABLE 1.9
The Effect of Moisture
Variation on Bioactive
Compound Yield during
Supercritical CO2 Extraction
of Wheat Germ
Water Content
(% wet basis)
Yield
(mg/100 g)
4
5
8
12
1470
1678
1352
1290
Source: Modified from Ge et al.32
objective. Water removal in most cases frees the internal pores and thus increases
the mass transport intensity, because water in the sample matrix would inhibit the
flow of SCF by changing surface tension and contact angles as a result of phase
interaction between the three components (water, sample matrix, and SCF). For
example, the higher the moisture content, the higher the probability for the formation
of a thin film of water between the sample matrix and the SCF phase. Water has a
small but finite solubility in SC-CO2, and as a result it can also be extracted with
the targeted components, and its separation can then only be done at the end of the
process.
1.4.4 COSOLVENT
The use of cosolvent (entrainers) during SCF extraction is key to enhancing the
extraction efficiency and cost effectiveness of the processes. Joslin et al.99 indicated
two significant attributes of cosolvents: the interaction between the cosolvent and
the solute (direct effect) and the cosolvent-solvent interactions (indirect effect).
Cosolvents used in small doses (1% to 5% mol) in SCF can change the overall
characteristics of the extraction fluid such as polarity, solvent strength, and specific
interactions. These changes in turn can significantly alter the density and compressibility of the original SCF.5 Additionally, they can improve selectivity for desired
components and facilitate selective fractional separations. Table 1.10 summarizes
the results of different cosolvents (ethanol, methylene chloride, and methanol).
Ethanol was found to have the greatest enhancement factor while methanol had the
lowest.100 The solubility enhancement is the result of complex interaction between
the -carotene, SC-CO2 , and cosolvent as indicated by Joslin et al.99 Temelli et al.101
observed an enhancement factor of 64, 63, and 29 by using ethanol as cosolvent to
extract palmitic acid, stearic and behenic fatty acids, respectively. Baysal et al.63
used ethanol at different concentrations (5%, 10%, and 15%) to recover -carotene
and lycopene from tomato paste. Although they observed that, with a high ethanol
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31
TABLE 1.10
The Effect of Fractionation of
β-Carotene in Cosolvent in
Supercritical Carbon Dioxide
Mixtures at the Temperature of 70°C.
Enhancement Factors were
Determined Based on Density
(17 mol–1)
Pressure
(MPa)
21.2
24.9
28.7
32.8
35.8
40.0
43.9
CO2 Density
(mol–1)
β-Carotene CO2
15.71
16.73
17.65
18.43
18.91
19.49
19.95
Yields
(× 107)
1.95
3.33
6.23
10.00
12.50
19.10
25.4
β-Carotene CO2 + 1 wt % Ethanol
(Enhancement factor = 4.7)
22.3
15.92
9.6
24.9
16.73
19.5
31.6
18.22
25.2
37.4
19.14
37.5
β-Carotene CO2 + 1 wt % methylene chloride
(Enhancement factor = 3.5)
23.4
16.28
12.8
24.7
16.67
13.3
31.2
18.16
21.7
37.0
19.08
27.7
β-Carotene CO2 + 1 wt % Methanol
(Enhancement factor = 2.1)
18.0
13.92
3.98
26.8
17.26
9.62
33.0
18.47
15.60
37.3
19.12
30.60
Source: Modified from Cygnarowicz et al.100
concentration, the extraction was hindered due to a decrease in the homogeneity of
the extraction mixture, no statistically significant differences were found between
the 10% and 15% concentrations. Quancheng et al.102 also investigated the effect of
cosolvents on the extraction of tocopherols. They extracted tocopherols from rapeseed deodorizer distillate at 2%, 4%, and 6% with methanol, ethanol, and mixed
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Functional Food Ingredients and Nutraceuticals
ethanol as cosolvents, under operating conditions of 12 MPa and 60°C. The use of
4% cosolvent gave the best tocopherol yield followed by 2% and 6%, and the 2%
and 6% methanol concentration gave the best yield of the three and the mixed ethanol
the worst.
1.4.5 PARTICLE SIZE
Particle size may have a significant impact on the flow behavior of SCF in the sample
matrix. The mechanism of sample pretreatment, for example the methods of drying
(air, oven, vacuum, or freeze drying), would influence particle sizes when subjected
to attrition or size reduction. The sizes of particles, shapes, and their random layout
(size distribution) would determine what goes through the medium and how fast.
The layout would influence the type of pore, either open or blind pores, or their
degree of interconnectedness. Process parameters such as pressure influence particle
size distributions. Pressure tends to create compactedness,95 thus decreases the intergranular porosity resulting in increased solid density. The smaller the particle sizes,
the larger is the surface area, and as a result bioactive components are released easily.
However, Coelho et al.103 observed no significant effect of particle sizes on the
extraction yield as a function of extraction time at a fixed flow rate. The oxygenated
compounds increased from 81% to 85% as particle size decreased, as shown in Table
1.11. However, the findings of Ge et al.32 were contrary to Coelho et al.103 in their
study on the effect of particle sizes on wheat germ (Table 1.12). Papamichail et al.104
extracted essential oil from celery with SC-CO2. They experienced increased yield
(more oil released) as the particle sizes of the seed decreased and attributed that to
the pretreatment milling and sieving.
Maximum yield of 1838 mg/100 g was obtained with optimum particle size of
0.505 mm. Also, they observed that very fine and big particle sizes have low
extraction yield probably due to higher resistances to mass transfer because of the
compact tendency reflecting reduced pore sizes in finer particle sizes while less
interactions with the supercritical fluids in the case for the latter. Likewise larger
particles contain undamaged cell walls rendering then impervious,60 although some
believed that higher SC-CO2 flow rate is capable of degrading protein structure to
release the targeted bioactive components.105 Nelson106 revealed the need to break
the lipoprotein matrix in order to release the embedded lipids in fish during SCE.
Papamichail et al.104 reported that it was possible to extract more essential oil per
kg of CO2 at a lower flow rate due to the intraparticle diffusion resistance. Therefore,
a threshold level for each product has to be experimentally determined in pursuance
to pilot plant or industrial processing.
1.4.6 FLOW RATE
Coelho et al.103 presented three scenarios of 2.3, 1.5, and 0.85 kg/h flow rates as a
function of extraction time and concluded the highest flow rate of 2.3 kg/h gave the
higher rate of extraction of Foeniculum vulgare volatile oil with SC-CO2 at 9 MPa
and 40°C. A similar trend was observed by Ge et al.32 who reported a flow rate of
3 mL/min yielded 1927 mg/100 g of bioactive component from wheat germ. Some
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Supercritical Fluid Technology for Extraction of Bioactive Components
TABLE 1.11
Comparative Supercritical CO2 and Hydrodistillation
Extraction of Volatile Components from Fennel
(Foeniculum vulgare) Fruits of Different Particle Sizes
SCE CO2
(%)
HD
(%)
Volatile Components
Canfene
Sebinene
Myrcene
-Phellandrene
Limonene
-Terpinene
Terpinolene
Fenchone
Estragol
(E)-Anethole
Piperitenone oxide
Unknowns
Waxes
Stalks
Fruits
(0.5 mm)
Fruits
(0.55 mm)
Fruits
(0.35 mm)
0.2
0.1
1.2
2.0
2.1
Tr
0.5
15.8
18.9
42.5
0.2
4.8
0.2
0.2
1.4
2.2
3.6
0.1
0.6
16.8
20.9
42.2
0.2
3.4
0.2
0.2
1.4
2.2
3.5
Tr
0.6
16.2
21.0
42.5
0.3
5.5
0.6
0.1
0.2
1.3
1.9
3.1
Tr
0.6
17.1
21.9
44.6
0.3
0.3
Tr (Trace < 0.05)). Source: Modified from Coelho et al.103
TABLE 1.12
The Effect of Particle Sizes on
Bioactive Compound Yield during
Supercritical CO2 Extraction of
Wheat Germ
Sieving
(mesh)
Particle Size
(mm)
Yield
(mg/100 g)
No grinding
20
30
40
60
80
100
2.1
0.86
0.51
0.40
0.22
0.18
0.13
1610
1710
1838
1550
1070
890
742
Source: Modified from Ge et al.32
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Functional Food Ingredients and Nutraceuticals
Yield (kg/kg feed)
0.18
0.12
0.06
0
0
50
100
150
Time (min)
1.1 kg/h
200
250
3 kg/h
FIGURE 1.11 The effect of flow rate on supercritical extraction of essential oil from celery
at pressure 15 MPa and temperature 45°C (modified from Baysal et al.63).
of these facts were corroborated by Peker et al.88 in their experimental study on
extraction rates of coffee beans with SC-CO2. They indicated the need for long
extraction time in conditions where low flow rate is used. Figure 1.11 shows an
apparent yield at high flow rate for extracting celery oil104 and Juniperus oxycedrus
essential oil.8 Summarized in Table 1.13 are the results of Baysal et al.63 where a
flow rate of 4 kg/h was identified as the optimum condition for attaining the highest
yield. Similar trend was observed by Ferreira et al.18 for extracting essential oil from
black pepper. They observed larger yield at 30 MPa using the upper-level flow rate
(10.54 kg/s). When maximum solubility is attained, the highest CO2 flow rate would
offer the highest recovery in extracting lipids from fish.72
1.4.7 EFFECT
OF
TIME
ON
YIELD
Several factors have direct or indirect implication on yield during SCE. Resident
time is an important factor that influences yield and the economic viability of the
process. Other factors such as temperature and pressure could have individual or
TABLE 1.13
Effect of Flow Rate on the Supercritical
Extraction of Lycopene and -Carotene
from Tomato Paste
Flow Rate
(kg/h)
Extraction
Time
(h)
Lycopene
(%)
β-carotene
(%)
2
4
8
4
2
1
14
22
20
30
43
34
Source: Baysal et al.63
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35
60
Content (%)
50
40
30
20
10
0
0
50
100
HM
150
200
Time (min)
OM
250
HS
300
350
OS
Cumulative quantity (g)
(a)
1.5
1
0.5
0
0
50
100
150
200
250
300
350
Time (min)
HM
OM
HS
(b)
OS
Overall
FIGURE 1.12 Families of flavor compounds extracted from Santolina Insularis at different
supercritical extraction times. HM: hydrocarbon monoterpenes; OM: Oxygenated monoterpenes; HS: Hydrocarbon sesquiterpenes; and OS: Oxygenated sesquiterpenes (modified from
Cherchi et al.107).
combined effects. Cherchi et al.107 performed detailed analysis of flavor compounds
in essential oil extracted from Santolina insularis by SC-CO2 extraction. They
reported the change in concentration exhibited by the monoterpenes from 50% in
the fraction collected after 30 min to 10% in the fraction collected after 240 min
(Figure 1.12a) under optimum conditions of 9 MPa and 50°C and a two-stage
separation. The first-stage separation was accomplished under 9 MPa and −12°C,
while the final stage used 2 MPa and 15°C. The yield becomes asymptotic at 1.75%
with increased extraction time while the rate decreases (Figure 1.12b). Hawthorne
et al.108 studied the extraction rate of basil conducted at 30 MPa and 45°C for 10
min and identified 1,8-cineole, estragole, eugenol, and selinene. Yield in most cases
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Functional Food Ingredients and Nutraceuticals
was dependent on time. Tamelli et al.72 considered 3 to 4 h sufficient time to extract
all extractable lipids from freeze-dried krill and 6 h for rainbow trout regardless of
the conditions.
1.5 SUMMARY
Supercritical carbon dioxide extraction has been shown to be a viable alternative to
the conventional solvent extraction technique to extract bioactive components from
agricultural materials. It offers a unique advantage of adding value to agricultural
waste by extracting antioxidants and flavonoids (lycopene from tomato skin, essential oils, and flavor and fragrances, etc.), which are then used for the fortification of
foods and other applications. Its drawbacks are the difficulties in extracting polar
compounds and its susceptibility of extracting compounds from a complex matrix
where the phase interaction with the intrinsic properties of the product inhibits its
effectiveness. Many of these drawbacks can be ameliorated by using cosolvents.
However, much investigation is required to understand the solvation effects on
targeted bioactive components being extracted.
Supercritical fluid extraction technology can be utilized to provide healthy snack
foods, a problem that faces the snack food industry, with regard to fat/oil contents,
which is becoming a greater public health concern. A lot of cost is incurred reformulating products such as French fries, onion rings, and other snack foods to
eliminate trans fats and high cholesterol content. Defatting and decholesterol with
SCF has been demonstrated as applicable to food products. Although most of the
tests were conducted on dehydrated products, research has shown successful application of SC-CO2 on high moisture products where extraction could be accomplished
without compromising the organoleptic characteristics.
The SCF technology is available in the form of single-stage batch and could
be upgraded to multistage semicontinuous batch coupled with a multiseparation
process. Although much work was accomplished, it would not warrant complacency,
since batch modes render the SCF technology cumbersome for certain industrial
applications, and has been the drawback for broader commercial adaptation. However, the needs to improve the design into continuous modes are growing. One
possibility is to integrate membrane technology into a supercritical process. Study
on membrane and SC-CO2 has been attempted and needs an aggressive pursuance.
This concept would make feasible the extraction of bioactive components from
aqueous, less viscous, and pumpable substances in a continuous mode.
Supercritical carbon dioxide extraction could only be cost effective under largescale production, which made it ideal for decaffeination of coffee, tea, and hops.
Although it is expensive, it is much more economical when compared to conventional
solvent extraction at high production scales. With improved processing conditions
and reduced cost, SCE will become even more economical at low throughput.
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2
Solubility of Food
Components and
Product Recovery in the
Supercritical Fluid
Separation Process
John Shi1 and Xiaoqin Zhou2
1
2
Agriculture and Agri-Food Canada
University of Waterloo
CONTENTS
2.1
2.2
2.3
Introduction ....................................................................................................45
Solubility of Food Components in SFE ........................................................46
Factors Affecting Solubility in SFE-CO2 ......................................................48
2.3.1 Solvent Selectivity .............................................................................51
2.3.2 Polar and Nonpolar Solvents .............................................................52
2.3.3 Cosolvents-Modification of SFE Fluid Phase Behavior ...................53
2.3.4 Pressure Effects..................................................................................57
2.3.5 Effect of Water Content in Material ..................................................58
2.4 Solubility Prediction ......................................................................................60
2.4.1 Equations of State (EOS)...................................................................65
2.5 Summary ........................................................................................................67
References................................................................................................................68
2.1 INTRODUCTION
Supercritical fluid extraction (SFE) has been used in the food industry since 1979,1
and has accrued 23 years of experience in the extraction of oils from plant materials.
Initially, it was mainly used in the decaffeination of coffee and tea and the extraction
of spices and hops. Those had to be large-scale processing operations in order to
make them economically feasible and cost effective. However, with the development
45
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Functional Food Ingredients and Nutraceuticals
and advancements in processing conditions and equipments, the cost effectiveness
improved allowing the small-scale productions of ultrahigh quality or high margin
product using SFE for commercial distribution. The hottest area for supercritical
extraction application is with nutraceuticals, as supercritical fluid extraction can be
operated at mild conditions and will retain the bioactivity of components.
Solubility is the amount of a substance dissolved by the solvent at thermodynamic equilibrium. For a multicomponent system (different components in the analyzed matrix and a mixture of solvents), the solubility of various components determines the selectivity and gives the opportunity of solving a separation problem.
To design a supercritical fluid extraction process, the solubility of solutes in the
supercritical fluid is fundamental. It influences the ideal operating conditions of the
extractor and recovery unit. Also, it controls the minimum amount of supercritical
fluid required to complete the extraction. In addition, the primary characteristics of
a supercritical fluid which make it so attractive is the continuously changing solvating
power. Since the solvating power is sensitive to temperature and pressure in the
supercritical region, it can be finely adjusted by varying temperature and pressure.
The goal of optimizing temperature and pressure of an SFE system is to maximize
the solubility of solutes and increase their upper limit of yield.
In past years, study has focused on the experimental solubility data in the
supercritical region, solubility prediction, and improvement (modifying solvent). As
the molecular structures of the food component in nutraceuticals become more
complex, the interaction between solute, solvent, and the solubility of biocomponents
in SCF become more complex. In the following sections, recent developments of
the research of solubility in SFE related with food science are reviewed.
2.2 SOLUBILITY OF FOOD COMPONENTS IN SFE
Because solubility is important to the process design of SFE, a lot of experimental
work has been done on the measurements of solubilities of food component in SCF.
Typical solubility behavior of a solid solute in SCF solvent is shown at Figure 2.1.
Two convergence points are shown at PL and PU, and one minima (Pmin). At low
pressure region (< Pmin), the solubility decreases as pressure increases, while in the
region of pressure Pmin< P < PU, the solubility increases sharply with the increase
of pressure. This region is usually observed in the near-critical and high compressed
region of an SCF solvent. It indicates that in the supercritical fluid extraction, the
solubility can be controlled by pressure. In other words, it is related to the state of
the supercritical fluid. Hence, the prediction of solubility is usually based on the
equation of state (EOS) of the solvent.
The crossover points PL and PU are attracting some interest as a method of
separating components with small difference in selectivity, such as isomers. For a
multicomponent system, the crossover point of each component may not overlap,
so there is a “crossover region” where most of the crossover points are located. At
this point or in this region, the solubility of components are similar. Johnston et al.2
pointed out that the crossover point in fact is the turning point of the isotherm line
of solubility, where
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Solubility of Food Components and Product Recovery
T3
47
T2
T1
Mole fraction
T3 > T2 > T1
Pmin
PL
Pressure
PU
FIGURE 2.1 Solubility behavior of a solid solute in an SCF solvent.
∂ ( ln y2 )
=0
∂T
where y2 is the mole fraction of solute in gas phase and T is temperature. For one
component, a slight increase in temperature at PU will cause the solubility to increase
above the crossover pressure and decrease below the crossover pressure. The results
are reversed at PL. Under these conditions a retrograde region is formed. For multicomponent systems, at the crossover point of one component, its solubility will
not change with temperature, while others do, so the selectivity of those components
increases. Through several cyclings of retrograde crystallization or solvation, the
components are separated. The operation becomes similar to distillation and requires
a temperature gradient. Chimowitz and Pennisi,3 and Foster et al.4 gave a detailed
description of the operation in the crossover pressure region. Because fewer data
are available in the critical region, this “distillation” operation runs more often in
the upper crossover pressure than in lower crossover pressure.
The other interesting observation is the sharp change of solubility with pressure.
In view of the macroscopic thermodynamics, the influence of the pressure to the
solubility can be explained by partial molar volume V2 directly
ln φ2 =
1
RT
∫
P
V2 dp
0
where V2s is the saturate volume, f2 is the fugacity coefficient, and R is the gas
constant. The partial molar volume is a differential quantity that describes the
solution behavior at a particular pressure. Here, fugacity coefficient f2 is the pressure
integral of partial molar volume (Equation 2.3). Using the equation above, the
solubility behavior vs. pressure can be easily explained.
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Functional Food Ingredients and Nutraceuticals
When the pressure is much lower than the critical point, the partial molar volume
is not a function of composition, so the equation is simplified to
∂ ( ln y2 ) V2s − V2
=
∂P
RT
At low pressure V2s << V2 hence the solubility decreases with pressure, and as
the pressure increases V2s decreases more slowly than V2 . When the partial molar
volume equals the saturate volume, the solubility reaches its minimum at a certain
pressure. At high pressures, that are significantly above the critical pressure, V2
increases slowly; where V2 exceeds V2s, because of the repulsive force, the solubility
will reach a maximum and decrease slowly with the increase of pressure. In this
region, the solubility does not change much. The quickest increase of solubility
occurs at a pressure corresponding to the minimum V2 . This theory explains the
solubility behavior in the supercritical region (Figure 2.1), and is supported by some
experimental data.5,6,7,8 Unfortunately, due to the lack of adequate experimental data
for biosubstances, the practical application of the partial molar volume model is
limited. So under these circumstances, the factors that influence solubility are usually
analyzed phenomenologically.
Biocompounds are usually present as a mixture in natural tissues and are often
not used in their pure form, thus they are regarded as one pseudocomponent in
research. Some researchers will measure the pure substance and others just measure
the “oil solubility.” Staby and Mollerup9 published a list for fish oil related component from1970s. Bartle et al.10 compiled solubility data from 1980s, which included
solubility in supercritical carbon dioxide (SC-CO2 ) of not only bioactive components
but also other chemical components. Guclu-Ustundag and Temelli11 collected data
on pure lipids in SC-CO2 from 1970 to 1999. Foster et al.4 collected some solubility
data of various chemical substances in various supercritical fluids from 1968 to
1990. Recent solubility information for bioactive components from different sources
is listed as Table 2.1.
2.3 FACTORS AFFECTING SOLUBILITY IN SFE-CO2
The most common fluid for SFE is supercritical carbon dioxide, because it is safe,
cheap, nonflammable, and the critical state is easy to obtain (31°C, 7.8 MPa). This
critical temperature is close to the room temperature and it preserves the bioactivity
of biocompounds. In food processing and nutraceutical fields, it is approved as safe
without declaration.
In the liquid and supercritical states, CO2 behaves very similarly to hydrocarbon
solvents with very low polarizability.12 In general, the extractability of compounds
with supercritical CO2 depends on the occurrence of the individual functional groups
in these compounds, their molecular weights, and polarity. The nonpolar, lipophilic
solutes have the greatest solubility in SC-CO2, whereas the introduction of polar
function groups will decrease the solubility. Extensive experiments have revealed
the following13:
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Solubility of Food Components and Product Recovery
49
TABLE 2.1
Publications on Solubility of Bioactive Components in Supercritical CO2
Substance
Temperature
(°C)
Pressure
(bar)
Resources
40, 50, 60
40–60
40–60
70–190
Singh et al.59
100–250 Yun et al.60
100–500 Murga et al.61
1993
1991
2004
Vanillic acid
Echium, Borage, and Lunaria
Seed Oils
Essential oil from black pepper
Fish oil
Hazelnut oil
Hippophae Rhamnoides L. seed
oils
Palm kernal oil
Tomato seeds oil
Lipids &
Fatty acid (C6, C12, C16) and
Fatty acids Triglyceride (C24, C36, C48)
Lipids
Triglycerides
Triglycerides with acryl chain,
Phospholipid and
monoglyceride
Fatty acids Lauric acid
Lauric acid and oleic acid
40–60
10–55
100–500 Murga et al.61
60–300
Gaspar et al.62
2004
2003
Lauric, linoleic, myristic, oleic,
palmitic, and stearic acid
Methyl oleate, oleic acid, oleyl
glycerols, oleyl Glycerol
mixture, mono-, di, and
trioleylglycerol
Myristic acid (tetradecanoic
acid), palmitic acid
(hexadecanoic acid), and cetyl
alcohol
Perilla oil ( poly-unsaturated
fatty acid)
2-ethyl-1-hexanol, 2ethylhexanoic acid
2-ethyl-1-hexanol+ 2ethylhexanoic acid
Ethyl esters of C18:1, C20:3
and C22:6
Fish oil fatty acid ethyl esters
Sterols
Phenolic
compounds
Fatty acid
esters
Cholesterol
Cholesterol
Syringic acid
© 2007 by Taylor & Francis Group, LLC
16–20
60–350
Ferreira et al.63
20, 40, 80, 120 60–650
Botch-Jensen et al.64
40–60
150–600 Ozkal et al.51
30–45
150–300 Yin et al.65
1993
1997
2005
2002
40–80
40–70
40–80
207–483 Hassan et al.66
108–245 Roy et al.49
25–300
Bharath et al.67
2000
1996
1993
40–80
40
100–300 Tilly et al.68
150–350 Hammam15
1990
1992
40
80
345–483 Norulaini et al.69
2004
276–483 Nik Norulaini et al.70 2004
30–60
130–400
Maheshwari et al.71
1992
50, 60
172–309
Nilsson et al.72
1991
35
81–218
Iwai et al.73
1991
40
200–400
Ikawa et al.74
1991
40, 50
68–180
Ghaziaskar et al.75
2003
40–100
138
Ghaziaskar et al.75
2003
40–100
90–250
Liong et al.76
1992
10, 40, 70
20–220
Staby et al.77
1993
2441_C002.fm Page 50 Tuesday, July 18, 2006 8:12 AM
50
Functional Food Ingredients and Nutraceuticals
TABLE 2.1 (CONTINUED)
Publications on Solubility of Bioactive Components in Supercritical CO2
Substance
Temperature
(°C)
•
•
•
•
•
•
•
Resources
Artemisinin
37–65
100–270
Xing et al.78
2003
α-Carotene in SC-CO2 or
Halocarbon
β-Carotene
40–70
60–340
Hansen et al.79
2001
35–50
96–300
Sakaki80
1992
γ-Carotene
40–70
200–300
Cygnarowicz et al.38
1990
Caffeine, uracil, and
erythromycin
Capsaicin in SC-CO2 or
Halocarbon
Fat-soluble vitamins A, D, E,
and K
Limonene + linalool 40%
limonene or 60% limonene
Limonene + linalool
40–60
Burgos-Solorzano
et al.81
Hansen et al.79
2004
35–70
Around
300
60–332
40, 60, 80
200–350
1997
41,42
70–100
Johannsen and
Brunner82
Chafer et al.83
45, 55
69–111
Berna et al.84
2000
Nitroaromatics and adamantane 50–100
•
Pressure
(bar)
100–450
2001
2001
Poly(α-hydroxybutyrate)
35–75
p-t-butylcalix[n] arenes
40–80
Lewin-Kretzschmar
et al.85
122–355 Khosravi-Darani
et al.86
150–300 Graham et al.87
Pure limonene and linalool
40, 50
69–111
Berna et al.84
Triacylglycerols
40, 60
172–310
Nilsson and Kudson88 1993
2002
2003
1998
2000
Nonpolar and slightly polar components with low molecular weight such
as hydrocarbons and some typical lipophilic compounds can be extracted
at lower pressure.
Solvent power of SC-CO2 for low molecular weight compounds (<250)
is high and decreases with increase of molecular weight. Organics with
molecular weights greater than 400 are almost insoluble.
SC-CO2 has high affinity with oxygenated organic compounds of medium
molecular weight.
Free fatty acids and their glycerides exhibit low solubilities.
Pigments are even less soluble.
Water has a low solubility (<0.5% w/w) at temperatures below 100°C.
Compounds with strongly polar functional group such as –OH, COOH,
are more difficult to extract. For instance, phenols with one carboxyl and
three or more hydroxyl groups cannot be extracted by SC-CO2.
When pressure increases, less volatile, higher molecular weight, and more
polar component may be dissolved.
© 2007 by Taylor & Francis Group, LLC
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Solubility of Food Components and Product Recovery
•
51
The highly polar substances such as sugar and amino-acid need pressure
up to 40 MPa to be extracted. Proteins, polysaccharides, and mineral salts
are insoluble.
2.3.1 SOLVENT SELECTIVITY
An important feature of an SCF solvent is that it can selectively dissolve more of a
certain compound from a mixture of compounds having similar volatility but different chemical structures. Strictly speaking, the selectivity of a solvent is the ratio
of partition coefficient (K2, K2) of two components (Equation 2.5). For a solid-fluid
system, it is often approximated as the ratio of binary solubility (y2 , y2) in SC-CO2
(Equation 2.6).
S=
y2
y2′
Two key factors that influence the selectivity are the volatilities of the solids
and the strength of solute-solvent interactions in the supercritical phase. They are
described in the following sections. Generally, for many high molecular weight
compounds, the selectivity increases with pressure. For low molecular weight compounds, at low pressure, the selectivity attains a maximum, and then decreases with
pressure, and a high level of selectivity is usually reached at higher temperatures.
As a nonpolar solvent, SC-CO2 has high selectivity on lipids, due to strong solute-solvent interactions.
A cosolvent may increase the selectivity of polar components, as it increases
the solubility of components in the mixture at different folds. Usually the quantity
of cosolvent is small, so the effect on selectivity is mainly due to the change of
interaction between solute and solvent. This effect is limited, but if the partial
pressure of the solute is changed by the cosolvent, the effect on selectivity is greater.
Wong et al.14 showed that the selectivity of Terpinen-4-ol and other components
increased at least twofold with water (Figure 2.2).
A general rule is that the solubility increases with increasing pressure and
decreasing molecular weight. Molecular weight mainly influences the vapor pressure
of the components. High molecular weight substances are usually not volatile.
Hence, increased molecular weight leads to decreased solubility of a homologous
series in SC-CO2. Materials with molecular weights above 500 daltons have limited
solubility. Low-to-medium molecular weight compounds such as aldehydes, ketones,
esters, alcohols, and ethers are very soluble. Low molecular weight, nonpolar,
aliphatic hydrocarbons with up to 20 carbons and small aromatic hydrocarbons are
soluble. If the molecular weight and structure of two components are similar, a great
deal more comprehensive analysis is required to determine selectivity. Hammam15
investigated different molecular weights of lipids and found that above 20 MPa,
dilaurin was less soluble than trilaurin, reflecting its more polar characteristic,
whereas below 20 MPa, the effect is opposite due to the lower molecular weight of
dilaurin.
© 2007 by Taylor & Francis Group, LLC
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52
Functional Food Ingredients and Nutraceuticals
60.0
% Composition
50.0
40.0
a-Pinene
a-Terpinene
30.0
g-Terpine
Terpinen-4-ol
20.0
10.0
0.0
Whole dried leaves
Rehydrated whole dried leaves
FIGURE 2.2 Recovery of target analytes from tea leaves. Comparison of whole dried leaves
and rehydrated whole dried leaves (modified from Wong et al., 200114).
2.3.2 POLAR
AND
NONPOLAR SOLVENTS
Besides vapor pressure, solubility is strongly affected by the interaction of solvent
and solute. A biocomponent usually has an aromatic group which has high polarity
or other functional groups which have specific force such as electron donor-acceptor
properties. Obviously, these compounds will have high solubility in polar solvent.
Primary supercritical solvents are listed in Table 2.2. Organic solvents are usually
used in polymer devaporization and petrol purification procedures.
TABLE 2.2
Critical Conditions for Some Primary Supercritical Solvents
Substance
Nonpolar
Nonpolar
Nonpolar
Polar
Polar
Polar
Polar
Polar (weak)
Polar (weak)
Polar (weak)
Polar (weak)
Polar (weak)
Carbon dioxide
Ethylene
Xenon
Acetone
Ammonia
Chlorotrifluoromethane
Water
Diethyl ether
n-Pentane
Propane
Ethane
Propylene
© 2007 by Taylor & Francis Group, LLC
Critical Temperature
(K)
304.2
282.4
289.7
508.1
405.6
302
647.3
467.7
469.6
369.8
305.4
365
Critical Pressure
(MPa)
7.38
5.03
5.84
4.7
11.3
3.92
22
3.64
3.37
4.24
4.88
4.62
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Solubility of Food Components and Product Recovery
53
The polarizability of CO2 is 2.65 × 10–24 cm3, less than that of all the hydrocarbons other than methane.16 Under this condition the solubility of biocomponents
(mostly hydrocarbons) in SC-CO2 will decrease several orders of magnitude below
the ideal solubility of a solid in a liquid. Xenon possesses a polarizability of 4.01
× 10–24 cm3,17 which is slightly greater than CO2 and close to the polarizability of
lower alkanes, such as ethane and propane which are used widely in the petroleum
field. It is suitable for solvating some polymers whose molecular weight are approximately 100,000.18 Because of its convenient critical properties (Tc = 289.7K, Pc =
5.8 MPa), safety, and larger polarity, it may be well suited for extraction of bioactive
components. However, xenon is much more expensive than CO2, so its application
is limited.
Halocarbons (CFCs) are very good solvents in SFE due to their high density,
which is a good indication of strong solvating power. Their polarities are similar to
diethyl ether,19 a solvent that is able to dissolve many solutes. Because of their effect
on the ozonosphere, the use of chloro-fluoro-hydrocarbons is restricted by the Montreal Protocol. Recently, some modified halocarbons with zero ozone-depletion
potential, such as difluoromethane (R-32), 1,1,1,2-tetra-fluoroethane (R-134a), and
1,1,1-trifluoroethane (R-143a) have been used for extracting biocomponents.19,20 The
solubility of those polar biocomponents was increased by 10 to 20 times when
compared to their solubility in SC-CO2 under the same conditions. However, the
critical temperatures of these halocarbons range from 100°C to 70°C. The extraction
of biocomponents has to proceed near or under supercritical conditions to avoid
destroying the bioactivity.
Polar solvents, such as acetone, have strong solvating powers, but due to the
safety problems in food, they are not used. Generally, the common safe polar solvents
are water, ethanol, and, sometimes, oil. They have high critical pressure and temperature, and are often used as a cosolvent.
2.3.3 COSOLVENTS-MODIFICATION OF SFE FLUID PHASE BEHAVIOR
Adding a cosolvent (entrainer or modifier) will dramatically increase solubility.
Usually, the amount of additive is 1–5 mol %. Adding 3.5 mol % methanol in SCCO2 will improve the solubility of cholesterol sevenfold compared to pure CO2 at
15 MPa21 and increase the solubility of 2-aminobenzoic acid sixfold. Chandra and
Nair22 observed much higher extraction yields of carotene from carrots with the
addition of hexane or chloroform to the SC-CO2, and this higher yield results from
a combined effect of increased solubility and increased availability of the carotene
by competing with sorption sites within the matrix. Table 2.3 lists some material
systems that show the effect of cosolvent. Generally, the role of a cosolvent in SFE
is to increase the polarity and solvent strength of the pure SC-CO2 while retaining
its solubility sensitivity to temperature and pressure. A suitable cosolvent is one that
as a liquid can normally dissolve the solutes.
When considering safety, ethanol, ethyl acetate, and water are the best natural
cosolvents for food-grade products and they are “Generally Recognized As Safe”
(GRAS). Recently, some new compounds have come into use as entrainers, but they
are cosolutes. They act as surfactants in SC-CO2 because of their ability to increase
© 2007 by Taylor & Francis Group, LLC
Cosolvents
Results
Cholesterol
(1.75–6.0 mol %) of acetone and hexane
Cotton seed
Ethanol or IPA
Fatty acids (palmitic acid and stearic
acid and higher alcohols (cetyl
alcohol and stearyl alcohol)
1–10 mol % ethanol and octane at 308.2K,
9.9–19.7 MPa
Glucose
Ethanol and water
Grape seeds
Maize
Ethanol 5–30 wt %
Methanol 500 µL
o- and m-Hydroxybenzoic acid
3.5 mol % acetone- or 3.5 mol %
methanol-
Soybean lecithin
5 wt % ethanol or 10 wt % acetone
Besides SC-CO2, also check other primary
supercritical systems and ethane-acetone
produced the best solvate power
Gossypol can be dissolved; Phosphorus
content doubled
Stearic acid: 4 mol % ethanol 10–15-fold; 4
mol % octane 3-fold
Palmitic acid: 5 mol % ethanol 12–13-fold, 5
mol% octane 8–11-fold
Stearyl alcohol: 5 mol % ethanol 4-fold,
Mole % octane, 4–6-fold
Cetyl alcohol: 3 mol % ethanol, 2–3-fold
3 mol % octane, 3-fold
Glucose dissolved and ternary and quaternary
phase diagram was obtained
phenolic compounds, 25%, highest solubility
Final yield doubled and comparable with
organic solvent extraction
Solubility was increased significantly; and
exhibited a retrograde vaporization
phenomenon and a common upper crossover
pressure for each ternary system
Made deoiling process possible at 170 bar
© 2007 by Taylor & Francis Group, LLC
Resources
Foster et al., 199389
Kuk and Hron, 199490
Koga et al., 199691
Dohrn et al., 199392
Chafer et al., 200493
Ambrosino et al., 200494
Gurdial et al., 199395
Teberikler et al., 200126
2441_C002.fm Page 54 Tuesday, July 18, 2006 8:12 AM
TABLE 2.3
Solubility Data and Cosolvent Effects
3.5 mol % cosolvent: acetone, ethanol, or
methanol
Sunflower seed oil
Ethanol, up to 20%, 150 to 350 bars and
42 to 80°C
Xanthines and Cocoa Butter
Ethanol, 20–25wt %
Various binary, ternary, and quaternary
systems
3.5 mol % cosolvent
© 2007 by Taylor & Francis Group, LLC
Cholesterol-CO2-Acetone (T = 35°C)
increase 6–7-fold
Cholesterol-CO2-Methanol (T = 35°C) 8–9
Stigmasterol-CO2-Ethanol (T=35°C) 6–9
Oil solubility in SC-CO2 greatly increases
over the whole range of pressure and
temperature conditions. Some phospholipids
are coextracted at levels directly proportional
to the added ethanol. A large amount of them
was recovered in the ethanolic phase. Acidity
of the extracted oil was lower than that
without alcohol. Part of the free fatty acids
was found in the ethanolic phase
Targeted component got higher solubility (2fold), and selectivity; caffeine no change,
Increased 3–7.2
Wong and Johnston, 198621
Cocero et al., 199696
Li and Hartland, 199697
Dobbs et al., 198716
2441_C002.fm Page 55 Tuesday, July 18, 2006 8:12 AM
Sterols
2441_C002.fm Page 56 Tuesday, July 18, 2006 8:12 AM
56
Functional Food Ingredients and Nutraceuticals
solubility. These include some modified sugars (peracylated sugars), which have
hydroxyl groups, will help those philize hydroxylated compounds come into CO2.23
The effect of cosolvents is based on the interaction between solute-solvent or
solvent-cosolvent. For the SFE process, there exists an extraction stage and a regeneration stage (recycle of the solvent). In the cosolvent-solute-solvent system, the
interaction of solute and cosolvent is key in the extraction stage while the solventcosolvent interaction becomes important in the regeneration stage. In the extraction
stage, the solubility of the solute in the cosolvent can be used to make the initial
selection. In the regenerating stage, raising temperature at a constant pressure is
preferred, so the solvent-cosolvent binary should have a wide two phase region at
that temperature and pressure.24
The quantity of cosolvent is usually small, since large quantity may cause the
critical points to move and may form multiphases.25 This is not expected in the
operation. Due to lack of multicomponent phase equilibrium data, the mixture phase
behavior at different ratios cannot be predicted. It is usually determined experimentally. Empirically, the quantities of cosolvent are controlled at about 5% of the
weight. That requires a flexible cosolvent system to obtain the operation flexibility
and efficiency. In addition, large quantities of cosolvent may coextract other components as byproducts, which is not desired. Teberikler et al.26 observed that 5% of
the weight ethanol made the deoiling process for soybean lecithin possible at 17
MPa, whereas no oil was extracted when pure SC-CO2 was used at the same pressure.
On the other hand, 5% of the weight ethanol did not resulted in any coextraction of
phospholipids. However, when the ethanol was increased to 10% of the weight to
accelerate the deoiling process, the phospholipids were coextracted.
Recently, Yuan et al.27 pointed out that the Lennard-Jones energy parameter and
dipole moment of cosolvent influenced the solute chemical potential significantly,
which is related to solubility directly. However, that research was focused on qualitative analysis. If solubility is increased significantly by the addition of small
amounts of cosolvent, the economics of existing or proposed processes through
reduction in the pressure and/or recycle ratio could be improved.16 Furthermore,
cosolvents could lead to more selective extraction of components which have similar
volatility but different types of chemical forces, and for compounds which have
extremely limited solubility in pure fluids, for example, biomolecules. Some detailed
studies have shown that the cosolvent may work differently on different solutes. For
example, Wong and Johnston21 found that in a sterol mixture, 3.5 mol % acetone
will improve the solubility of cholesterol 5.7-fold, stigmaterol 1.8-fold, and almost
no effect on ergosterol at 35°C and 12.3 MPa. Obviously the selectivity of cholesterol
increases. Dobbs and Johnston28 observed the selectivity of benzoic acid-hexamethylbenzene increased from 1.9 to 2.5-fold with the addition of 3.5 mol % methanol,
and the yield also increased. However, Brunner29 found that the selectivity always
decreased with an increase in yield for hexadecanol-octadecane-CO2 system using
acetone as cosolvent. Generally, if a pair of nonpolar solutes were chosen, a nonpolar
cosolvent would increase each solubility about the same, thus causing little change
in the selectivity.30 Similarly, a nonpolar cosolvent will not change the selectivity of
a pair consisting of either a nonpolar and a polar solute or two polar solutes.
© 2007 by Taylor & Francis Group, LLC
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Solubility of Food Components and Product Recovery
57
3.0
Tr = T/Tc = 0.8
Reduced density
0.9
1.0
2.0
1.1
1.2
1.0
CP
1.55
0.0
0.1
1.0
Reduced pressure Pr = P/Pc
10.0
FIGURE 2.3 Variation of the reduced density (ρr) of a pure component near its critical point.
2.3.4 PRESSURE EFFECTS
Cosolvents change the interaction of solute and solvent, and this phenomenon is
often called the “chemical effect.” Another effect that significantly affects the solubility is the “physical effect” of temperature and pressure. In SFE, the solubilities
are often extremely sensitive to pressure and temperature. That results in an important
feature of SFE that allows the yield and the selectivity of the solvent to be finely
tuned by temperature and pressure above the critical temperature. Usually, pressure
is preferred as a control factor. The advantage of pressure control is that the solubility
can be changed on a nearly instantaneous basis, whereas the temperature control
suffers from thermal lag and the temperature gradients that limit the maximum useful
speed of temperature increase, because the gradient elution requires the time for
complete mixing of two liquid streams.31 Therefore, in most cases, the physical
effect is usually referred to as the “pressure effect.” This extreme pressure sensitivity
of solubility can be explained by its compressibility, which is expressed as a gradient
of density over pressure. As the solubility parameters are proportional to the density
of the solvent-SC-CO2, the sharp variance of density near the critical region (Figure
2.3) shows why solubility changes greatly at different temperatures and pressures.
At the critical point, the slope of density-pressure is almost vertical, which means
that the rate of change is close to infinite and results in extreme pressure sensitivity.
To further understand the impact of pressure on the solubility, an enhancement
factor E was derived
E = y2
© 2007 by Taylor & Francis Group, LLC
P
P2 sat
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58
Functional Food Ingredients and Nutraceuticals
6.5
6
Acridine
Antracence
Hexamethylbenzene
log E
5.5
5
4.5
4
16
17
18
19
20
21
22
Density (mol/L)
FIGURE 2.4 Enhancement factors at various SC-CO2 densities, modified from Dobbs and
Johnston (1987).28
where P2sat is the solute saturated pressure. This equation removes the effect of
pressure and focuses on the solvent-solute interaction in the SCF phase. For the
sterols in SC-CO2, at 35°C and 20 MPa, the enhancement factors have almost the
same value. In fact, the enhancement factor varies very little for many organic solids.
Figure 2.4 shows the E values vary within a range of only 1.5 orders of magnitude
for substances with a variety of polar functional groups, whose actual solubilities
may vary by many orders of magnitude.28 These results indicate that in SFE, pressure
is primarily responsible for the solubility, not the solute-solvent interaction.
Furthermore, vapor pressures of the components in the mixture have significant
impact on the selectivity. Vapor pressure measures the volatility of solutes. If the
components in the mixture have similar chemical structures, their selectivity is
mainly determined by their vapor pressure. A typical example is the components in
a sterol mixture that contains three sterols: cholesterol, stigmaterol, and ergosterol.
The CO2 is surprisingly more selective for cholesterol than ergosterol, despite the
fact that both of them have very similar chemical structures. The solubilities of these
two components differ by two orders of magnitude. The reason is that their vapor
pressures are different. Although their vapor pressure is as low as 10-11 MPa, which
is negligibly small, the selectivity follows the ratio of pressure.
It should be mentioned that if an inert component is added to a mixture which
does not strongly interact with the extracted component but is also volatile, that
volatile component will decrease the solubility of the desired component as its partial
pressure is decreased by the added volatile component. Water behaves in this manner
with many biocomponents.
2.3.5 EFFECT
OF
WATER CONTENT
IN
MATERIAL
Many natural products contain water, and its solubility is close to oil, at normal
temperature (30°C) and pressure (10 MPa or so). Wiebe and Gaddy32 measured the
© 2007 by Taylor & Francis Group, LLC
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Solubility of Food Components and Product Recovery
59
0.008
Solubility (g.water/L. CO2 S.T.P)
0.007
0.006
0.005
0.004
0.003
0.002
25°C
31.04°C(Tc)
50°C
0.001
0
0
100
200
300
400
500
Total pressure, atm
600
700
800
FIGURE 2.5 Composition of phase rich in carbon dioxide, modified from Wiebe and Gaddy
(1941).32
vapor phase composition of carbon dioxide-water mixtures at various temperatures
and pressures up to 70 MPa. Their measurements revealed the similar solubility
behavior of the water-SC-CO2 system with the oil-SC-CO2 system (Figure 2.5).
There was a minimum solubility at a certain pressure. Near this pressure, water
solubility changes greatly, and at high pressures and above the supercritical region,
water solubility increases slowly. King et al.33 obtained more mutual solubility data
on water-SC-CO2 at lower temperatures (down to 15°C) and at lower pressures (a
little over 20 MPa), which is the liquid and supercritical region, and their data agreed
well with data from Wiebe32 in the overlapped region. According to their data, over
the temperature range 15°C to 40°C and pressure range 5.1 to 20.3 MPa, the
solubility of water in liquid and SC-CO2 varies from 2.2 × 10–3 to 5.8 × 10–3 mole
fraction. It is close to the oil solubility and that may be the reason why water is
extracted along with the oil. Further, as discussed above, if water comes into the
SCF phase in large quantities, the partial pressure of oil in the SCF phase will
decrease. That will influence the solubility of oil in SCF. The binary phase diagram
is shown in Figure 2.6.33 The turning point D on Figure 2.6 corresponds to the
minimum solubility. Dunford et al.34 observed that the solubility of Atlantic mackerel
oil decreases significantly at moisture levels > 40.5%.
However, for some proteins or other substances with hydroxyl groups, their
water binding potential may help them dissolve in the SC-CO2, like a cosolvent.
Due to its dual effects, the moisture content needs to be optimized. Dunford et al.35
reported an optimum moisture content of 10.2% for the extraction of oil and residual
proteins from Atlantic mackerel. Figure 2.7 shows the typical effect of water on
© 2007 by Taylor & Francis Group, LLC
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60
Functional Food Ingredients and Nutraceuticals
Pressure
Pressure
D
0
Water rich
CO2
1
0
1
Mole fraction of CO2
Mole fraction of CO2
(a)
(b)
FIGURE 2.6 Pressure-composition diagram for CO2/water system. a) Temperature below
critical point of CO2. b) Temperature above critical point of CO2.
supercritical fluid extraction of oil. Figure 2.7 also shows that for the optimized
moisture level, from C1 to C2 (for Atlantic mackerel, 0% to 26% of moisture), the
solubility does not decrease significantly. Figure 2.7 proves again that the effect of
pressure is much larger than that of solute-solvent interaction. If the extracted
component has no or weak interaction with water, before the partial pressure of
water changed significantly, water has almost no effect on the solubility and extract
yield of oil. Snyder et al.36 observed that moisture level between 3% and 12% had
little effect on the extraction of soybean oil at pressure of 1.7 MPa and a temperature
of 50°C.
In terms of an industrial process, the low sensitivity to water content has some
advantages. The fact that in some regions the solubility is not sensitive to water
content will reduce the need for a stronger drying process. Samples that do not
require extensive drying will have lower drying cost, because it is usually an energy
intensive process.
2.4 SOLUBILITY PREDICTION
Obtaining solubility data requires a lot of experimental work, so the ability to predict
solubility is important for design. The solubility can be predicted through properties
of pure substances. Its foundation is built on the thermodynamics of solvent equilibrium state, which is described by phase equilibrium and chemical potential equilibrium. In the view of phase equilibrium, the fugacity of species i in the supercritical
phase is equal to the fugacity of the pure solid. The compressed gas model37 is used
to describe this equilibrium on the solid-gas interface
© 2007 by Taylor & Francis Group, LLC
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Solubility of Food Components and Product Recovery
61
Oil solubility
ymax
C1
C2
C3
Water content
FIGURE 2.7 Oil solubility vs. water concentration; plotted according to data of Dunford et
al. (1997,1998).34,35
(
 Vi P − Pi sat
Pi sat exp 
RT

yi =
Pφ i
) 

where the subscript i means the component i, and Vi is the volume of pure substance.
The fugacity coefficient φ is calculated by equation of state (EOS). The most widely
used EOS are the Peng-Robinson (P-R) equation and Redlich-Kwong (R-K) equation. They will be described in the next section.
Cygnarowicz et al.38 used this method to estimate the solubility of β-carotene
in supercritical carbon dioxide at temperature ranging 40°C to 70°C, and pressure
ranging from 20 to 45 MPa, and the result agreed well with experimental data.
Soave39 applied R-K EOS and Huron-Vidal mixing rules to calculate fugacity coefficient. This method contained two adjustable parameters and agreed well with the
isothermal experimental data.
In the view of chemical potential equilibrium, if the chemical potential of
component i is the same in each phase, then the following equation is obtained to
calculate the solubility
(
 Vi P − Pi vap
 − ∆µ i 

Pi vap exp 
exp
 kT 
kT

yi =
ρkT
© 2007 by Taylor & Francis Group, LLC
) 

2441_C002.fm Page 62 Tuesday, July 18, 2006 8:12 AM
62
Functional Food Ingredients and Nutraceuticals
where ∆µ is excess chemical potential, ρ is density, and k is the Boltzmann constant.
Calculating the excess chemical potential ∆µ is a great challenge. Goldman et al.40
developed a method based on the Redlich-Kwong equation of state and LennardJones intermolecular potential (Equation 2.11):
 σ ij  12  σ ij  6 
µ ij = 4 εij 
 −  r  
 r 

where εij is the potential well depth for the interactions of component i, j and σij
measure their range. They obtained an expression for chemical potential directly in
terms of the intermolecular potentials at infinite dilution of components for ternary
system
∆µ 2
= − log (1 − bρ) − γz ( x1 , x3 ) log (1 + bρ) + y ( x1 , x3 )
kT
 bρ

bρ  
+ γ  log (1 + bρ) −


1 + bρ  

 1 − bρ
where
γ = 7.45 ( ε kT )
3/ 2
y ( x1 , x3 ) = 2 x1
σ12 3
σ 3
+ 2 x3 233 − 1
3
σ
σ
z ( x1 , x3 ) = 2 x1
σ12 3
σ 3
+ 2 x3 233 − 1
3
σ
σ
and ε and σ are the counterpart parameters of mixture, b is an adjustable parameter,
and x is the mole fraction of each component. This model passed a simulation test
and agreed well with the theory. The obvious advantage is that it can be used to
directly demonstrate the cosolvent effect through solute chemical potential which is
strongly dependent on the molecular interaction. In a view of solute property, Politzer
et al.41 built relationships of molecular structure and solubility in SC-CO2 through
surface potential. The logarithm solubility shows a linear relationship with the square
of surface potential. Their work encompassed 22 solutes in CO2 at 308K and two
pressures: 14 and 20 MPa.
The methods above based on the thermodynamic equilibrium have a strong
theoretical background. However, the inherent weakness is that they need extensive
chemical physical data to characterize the solvent and solute. Therefore they are
usually applied to simple structures where properties have been a fully investigated
substance. That limits their application in the bioproducts, as those substances
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Solubility of Food Components and Product Recovery
63
usually have complicated structures and only a limited amount of physical data
available for calculation.
Since solubility does not directly influence the mass transfer, sometimes a
qualitative analysis is used. The solubility parameter concept is a good tool for
representing the solubility level. It can be easily obtained from thermodynamic
properties. Even if there is a lack of thermodynamic data, it still can be approximated
by the UNIFAC model, which is based on summation of all the functional group
contributions. The obvious advantage of this method is a simpler analysis that
estimates the solubility based on the molecular structure.
Giddings et al.42 obtained the correlation of solubility parameters d by extending
the theory of the solubility power and using it to rapidly estimate a solubility level
in the condensed solvent
χ = V1 ( δ1 − δ 2 ) RT + χ s
2
δ = 1.25 Pc 0.5 ( ρr ,SF ρr .L )
where the subscript r stands for the reduced property and 1 is the solvent, 2 is the
solute, χ is the total interaction parameter, and χs is the entropic interaction parameter.
Fedors43 correlated chemical structure and solubility parameter. They collected many
thermodynamic properties of typical atoms and function as group contributions. With
this knowledge, the solubility parameter and the molar volume for both solvent and
solute were estimated. In addition, a very simple correlation was obtained for
temperature dependence of solubility parameter
V 
δ 22 = δ12  1 
 V2 
2.27
+
R
V2
  V1  1.27

T1   − T2 
  V2 

It was suitable for both polar and nonpolar liquids. When |T1 - T2| < 150°C and both
are at or below the normal boiling point of the liquid, this equation can be simplified
as
V 
δ 2 = δ1  1 
 V2 
1.13
ρ 
= δ1  2 
 ρ1 
1.13
It is built on the relationship between the solubility temperature dependency and the
temperature dependency of density. When |T1 - T2| < 50°C, it can be further simplified
as
V2 = V1 1 + α (T2 − T1 ) 
δ 2 = δ1 1 + 1.13α (T1 − T2 ) 
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Functional Food Ingredients and Nutraceuticals
where α was the cubic coefficient of expansion, and the prediction accuracy was
around 2%.
To further utilize the advantage of solubility parameters, Prausnitz et al.7 built
the relationship between solubility parameters and solubility, where ∆Hf is the
change of enthalpy of fluid. It is valid for a nonpolar solute in a nonpolar solvent.
ln y2 =
∆H f  1 1  ν2 φ12 ( δ1 − δ 2 )
−
−
R  T f T 
RT
2
Later, the reduced solubility parameter concept was developed by King and
Friedrich44
∆=
δ1
δ2
where δ1 and δ2 are the solubility parameters of solvent and solute, respectively. It
described solubility parameters in a more general way, and even the relationship
with partition coefficient can be built through the ∆ term. When plotting the data of
various extraction systems, it showed apparent correlation between partition coefficient and reduced solubility parameter, which was confirmed in one equation, that
was formalized by Giddings et al.42
(
) (
)
RT V2 δ 22 ln K normal K = 2 − ∆ ∆
where subscript “normal” means low-pressure reference state. This implied that the
partition coefficients can be predicted by other well-known solutes under similar
conditions if their reduced solubility parameters were calculated.
All of the above correlations were based on thermodynamics and equation of
state, so they have complicated forms. To simplify the calculations, the empirical
models are still accepted by researchers. The empirical models of Chrastil45and Mitra
and Wilson46 are based on the phenomenological method, which builds the correlation of solubility with their effect factors directly. One of most widely used empirical
models was made by Chrastil.45 This is a simple empirical correlation.
ln C2 = k ln ρ1 + a1 / T + a0
where
a1 = ∆H R
a0 = ln ( M 2 + kM 1 ) + q − k ln M 1
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Solubility of Food Components and Product Recovery
65
where Mi is the molecular weight of component i, and k is an adjustable parameter.
This correlation describes the fact that the solubility is proportional to the solvent
density. Because it relates solubility with CO2 density directly and has high accuracy
in prediction (4% error), it is used widely in the SC-CO2 extraction field.11,47 Some
modification of this model was made by Del Valle and Aguilera48 (Equation 2.23),
where C is the concentration of solute, but it did not improve the accuracy. However,
it did successfully predict the solubility of vegetable oil (Equation 2.24) in the
temperature range of 293K to 353K and pressure range of 20 to 80 MPa. It also
showed that the solubility behavior of some bioactive components such as lycopene
was very similar to vegetable oil49, and that their solubilities can be predicted by
this equation
C = exp( 40.361 − 18708 / T + 2186840 / T 2 ) × ρ10.724 ± 2.7
Adachi and Lu50 developed a five-adjustable-parameter equation (ki and ai are all
adjustable parameters), however, the accuracy did not improve much. The determinant coefficient of regression for the solubility for hazelnut oil increased to 0.995
from 0.985 based on the prediction from the Chrastil equation.51
(
)
ln C2 = k1 + k2 ρ1 + k3ρ12 ln ρ1 + a0 + a1 / T
As for the solubility of cosolvent, Walsh et al.52 gave an associated-perturbedanisotropic-chain theory, and obtained a complicated phase equilibrium calculation
method. Usually, researchers choose suitable mixing rules for EOS to simplify
calculation.
2.4.1 EQUATIONS
OF
STATE (EOS)
Equations of state are used to describe the thermodynamic states through the relationship of state parameters such as temperature, pressure, and volume. In SFE
processing, EOS describes the behavior of solute being dissolved in supercritical
fluids. The Peng-Robinson equation53 is usually used to describe a compressed gas.
A general form of the Peng-Robinson equation is
P=
RT
aα
−
V − b V (V + b ) + b (V − b )
where
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Functional Food Ingredients and Nutraceuticals
a = 0.45724
( RTc )2
Pc
(
)
α = 1 + m 1 − Tr 


2
m = 0.37464 + 1.54226ω − 0.26992ω 2
b = 0.07780
RTc
Pc
(
)
ω = −1.000 − log P s Pc
Tr =0.7
For mixtures, the following mixing rule is used to calculate the fugacity coefficient.
a=
n
n
i =1
j =1
∑∑ y y a
i
j ij
aij = ai a j 1 − kij + ( kij − k ji ) yi 
Redlich-Kwong (R-K)54 is simplified to the following forms
a = 0.42747
( RTc )2
Pc
(
)
α = 1 + m 1 − Tr 


2
m = 0.48 + 1.57ω − 0.176ω 2
b = 0.08664
RTc
Pc
(
ω = −1.000 − log P s Pc
)
Tr=0.7
Both P-R and R-K predict the state well in the compressed gas region, but P-R
shows better prediction in the saturated liquid field. Although P-R and R-K are
widely accepted by researchers, Schmitt and Reid55 pointed out that these kinds of
equations of state are not appropriate for a solid solute system, as the essential
assumptions are applicable for a liquid vapor system, which is considered the main
reason that the prediction of P-R is not perfect over a wide pressure range even with
optimized parameters. They gave a modified P-R equation accounting for the solid
solute and obtained better prediction over a wide pressure range (from 5 to 45 MPa).
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Solubility of Food Components and Product Recovery
67
Hartono et al.56 pointed out that a two-parameter equation Mohsen-Nia-ModdaressMansoori (MMM) was as accurate as the modified P-R equation for a biocomponent
in a supercritical region. An alternative choice is the Virial equation of state. The
Virial equation of state57 directly describes the multicomponent fluid mixture with
molecular interactions. There is no adjustable parameter, and the solubility is
obtained directly by the intermolecular potentials of the interaction of solvent, solute,
and cosolvent. This method performs under SFE operation conditions with fourbody interaction level.
Although the EOS above have good prediction performance, they require a lot
of critical physical properties of solutes, which are not easily available and only few
are available in the published literatures. Hence some empirical expressions are still
widely used by researchers. For example, Saeki58 expressed an empirical equation
based on power law and Maxwell thermodynamic relation. It has good agreement
with some fluids such as neon, hydrogen, deuterium, and carbon monoxide in the
supercritical state.
Equations of state have a central role in supercritical fluids because they not
only predict the solubility, but also give the qualitative phase behavior. Hence, more
complicated theories and more multiparameter empirical methods continue to be
developed to formulate the state more accurately. Although the present model accuracy is limited by its character, they can be used to build most of the known binary
phase diagrams, which are the fundamentals of supercritical fluid extraction.
2.5 SUMMARY
As discussed above, solubility determines the capacity of SFE, and measuring the
solubility of targeted components in SC-CO2 will build a solid base for prediction
and further process design. The measurement and prediction of solubility includes
not only solubility in SC-CO2, but also the phase equilibrium data in multisolvent
systems, as the modification of SC-CO2 will play a leading role in the future
development which is based on the reality that a lot of bioactive components have
low solubility in SC-CO2, and a small quantity of cosolvent can change the solubility
dramatically. Cosolvents will decrease the pressures applied and further decrease
the operation cost, but how much cosolvent is suitable, and how much the solubility
will change, needs more attention. So far, many researchers studying cosolvents are
at the qualitative stage and require many more experiments to determine the optimal
conditions. The difficulty of prediction is that cosolvents will change the polarity
and phase state of the SC-CO2 system. Few phase equilibrium data in or near the
critical region are available, which makes the theory difficult to validate. So, developing an accurate and convenient EOS which can deal with multicomponents will
be important. Through EOS, the solubility phase diagram can be determined, which
is a design base of SFE. Furthermore, a lot of bioactive components are extracted
together with byproducts. In order to improve the purity or decrease the untargeted
component, the selectivity of SFE systems should be improved. Crossover pressure
operation is a choice, which in fact is similar to distillation at the supercritical region.
Its limitation is that usually there is not enough phase equilibrium data in the
supercritical region, and temperature control is not as convenient as pressure control.
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Functional Food Ingredients and Nutraceuticals
Complex cosolvents can be an alternative choice. Some researchers have observed
that at different operational stages, the extraction speeds of various components are
different, so that the components in the extracted mixture will change. If the batch
operation can be changed to a semibatch operation, the cosolvent can be added to
the system at a suitable time to keep the selectivity at a high level. Optimizing the
water content is also important. There are opportunities to decrease the pretreatment
cost without change or even to increase the efficiency of SFE. To decrease the
operation cost, pretreating tissues, breaking down the cell wall and releasing the oil,
needs to be improved. Flaking and grinding are widely used methods because the
cell breakage percentage is high. However, those are lab-scale methods. If applied
to industry, decreasing the operation costs and increasing the percentage of cell
breakage should be considered. In a word, to applied SFE technology, the solubility
prediction and phase diagrams must be accurate to give a solid base for design, and
the operation costs have to be decreased further.
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71. Maheshwari, P., Nikolov, Z.L., White, T.M., and Hartel, R., Solubility of fatty acids
in supercritical carbon dioxide, Journal of the American Oil Chemists’ Society, 69,
11, 1069–1076, 1992.
72. Nilsson, W.B., Gauglitz, E.J., and Hudson, J.K., Solubilities of methyl oleate, oleic
acid, oleyl glycerols, and oleyl glycerol mixtures in supercritical carbon dioxide,
Journal of the American Oil Chemists’ Society, 68, 2, 87–91, 1991.
73. Iwai, Y., Fukuda, T., Koga, Y., and Arai, Y., Solubilities of myristic acid, palmitic
acid, and cetyl alcohol in supercritical carbon dioxide at 35°C, Journal of Chemical
and Engineering Data, 36, 4, 430–432, 1991.
74. Ikawa, N., Nagase, Y., Imanishi, N., and Fukuzato, R., Separation of polyunsaturated
fatty acid by supercritical fluid extraction and chromatography, KOBELCO Technology Review, 12, 40–43, 1991.
75. Ghaziaskar, H.S., Eskandari, H., and Daneshfar, A., Solubility of 2-Ethyl-1-hexanol,
2-Ethylhexanoic acid, and their mixtures in supercritical carbon dioxide, Journal of
Chemical and Engineering Data, 48, 2, 236–240, 2003.
76. Liong, K., Keat, F., Neil R., and Ting, S.S.T., Solubility of fatty acid esters in
supercritical carbon dioxide, Industrial and Engineering Chemistry Research, 31, 1,
400–404, 1992.
77. Staby, A., Forskov, T., and Mollerup, J., Phase equilibria of fish oil fatty acid ethyl
esters and sub- and supercritical CO2, Fluid Phase Equilibria, 87, 2, 309–340, 1993.
78. Xing, H.B., Yang, Y.W., Su, B.G., Huang, M., and Ren, Q.L., Solubility of artemisinin
in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 48, 2,
330–332, 2003.
79. Hansen, B.N., Harvey, A.H., Coelho, J.A.P., Palavra, A.M.F., and Bruno, T.J., Solubility of capsaicin and β-carotene in supercritical carbon dioxide and in halocarbons,
Journal of Chemical and Engineering Data, 46, 5, 1054–1058, 2001.
80. Sakaki, K., Solubility of β-carotene in dense carbon dioxide and nitrous oxide from
308 to 323K and from 9.6 to 30 MPa, Journal of Chemical and Engineering Data,
37, 2, 249–251, 1992.
81. Burgos-Solorzano, G.I., Brennecke, J.F., and Stadtherr, M.A., Solubility measurements and modeling of molecules of biological and pharmaceutical interest with
supercritical CO2, Fluid Phase Equilibria, 220, 1, 57–69, 2004.
82. Johannsen, M. and Brunner, G., Solubilities of the fat-soluble vitamins A, D, E, and
K in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 42,
1, 106–111, 1997.
83. Chafer, A., Berna, A., Monton, J.B., and Mulet, A., High pressure solubility data of
the system limonene + linalool + CO2, Journal of Chemical and Engineering Data,
46, 5, 1145–1148, 2001.
84. Berna, A., Chafer, A., and Monton, J.B., Solubilities of essential oil components of
orange in supercritical carbon dioxide, Journal of Chemical and Engineering Data,
45, 5, 724–727, 2000.
85. Lewin-Kretzschmar, U., Blasberg, L., and Harting, P., Solubility of nitroaromatics
and adamantane in pure and surfactant-modified compressed gases, Tenside, Surfactants, Detergents, 39, 1, 29–35, 2002.
86. Khosravi-Darani, K., Vasheghani-Farahani, E., Yamini, Y., and Bahramifar, N., Solubility of poly (α-hydroxybutyrate) in supercritical carbon dioxide, Journal of Chemical and Engineering Data, 48, 4, 860–863, 2003.
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87. Graham, B.F., Lagalante, A.F., Bruno, T.J., Harrowfield, J.M., and Trengove, R.D.,
Solubility of p-t-butylcalixarenes in supercritical carbon dioxide: a comparison of
static and dynamic measurement techniques, Fluid Phase Equilibria, 150, 151,
829–838, 1998.
88. Nilsson, W.B. and Hudson, J.K., Solubility of simple and mixed triacylglycerols in
supercritical CO2, Journal of American Oil Chemists’ Society, 70, 8, 749–754, 1993.
89. Foster, N.R., Singh, H., Yun, S.L.J., Tomasko, D.L., and Macnaughton, S.J., Polar
and nonpolar cosolvent effects on the solubility of cholesterol in supercritical fluids,
Industrial and Engineering Chemistry Research, 32, 11, 2849–2853, 1093.
90. Kuk, M.S. and Sr. Hron, R.J., Supercritical carbon dioxide extraction of cottonseed
with co-solvents, Journal of the American Oil Chemists’ Society, 71, 12, 1353–1356,
1994.
91. Koga, Y., Iwai, Y., Hata, Y., Yamamoto, M., and Arai, Y., Influence of cosolvent on
solubilities of fatty acids and higher alcohols in supercritical carbon dioxide, Fluid
Phase Equilibria, 125, 1–2, 115–128, 1996.
92. Dohrn, R., Bunz, A.P., Devlieghere, F., and Thelen, D., Experimental measurements
of phase equilibria for ternary and quaternary systems of glucose, water, CO2 and
ethanol with a novel apparatus, Fluid Phase Equilibria, 83, pt 2, 149–158, 1993.
93. Chafer, A., Fornari, T., Berna, A., and Stateva, R.P., Solubility of quercetin in supercritical CO2 + ethanol as a modifier: Measurements and thermodynamic modelling,
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94. Ambrosino, P., Galvano, F., Fogliano, V., Logrieco, A., Fresa, R., and Ritieni, A.,
Supercritical fluid extraction of Beauvericin from maize, Talanta, 62, 3, 523–530,
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95. Gurdial, G.S., Macnaughton, S.J., Tomasko, D.L., and Foster, N.R., Influence of
chemical modifiers on the solubility of o- and m-hydroxybenzoic acid in supercritical
CO2, Industrial and Engineering Chemistry Research, 32, 7, 1488–1497, 1993.
96. Cocero, M.J. and Calvo, L., Supercritical fluid extraction of sunflower seed oil with
CO2-ethanol mixtures, JAOCS, 73, 11, 1573–1578, 1996.
97. Li, S. and Hartland, S., New industrial process for extracting cocoa butter and
xanthines with supercritical carbon dioxide, JAOCS, 73, 4, 423–429, 1996.
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3
Modeling of
Supercritical Fluid
Extraction of Bioactives
from Plant Materials
Helena Sovová
Academy of Sciences of the Czech Republic
CONTENTS
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Supercritical Fluid Extraction from Plant Materials .....................................76
Extraction Bed and the Concept of Broken and Intact Cells .......................78
Mass Balance and Model Equations for Plug Flow .....................................79
3.3.1 Mass Balance for Solvent and Matrix...............................................79
3.3.2 Mass Balance for Solute ....................................................................80
3.3.3 Time Constants and Dimensionless Model Equations......................82
SFE of Oils from Seeds .................................................................................85
3.4.1 Oil Solubility in SC-CO2 ...................................................................85
3.4.2 Model for Vegetable Oil Extraction................................................... 87
3.4.3 Equilibrium-Controlled Period ..........................................................89
3.4.4 Diffusion-Controlled Period...............................................................90
3.4.5 Approximate Solution for Both Periods............................................90
SFE of Essential Oils.....................................................................................92
3.5.1 Solute-Matrix Interaction...................................................................92
3.5.2 Simulation of Essential Oil Extraction..............................................92
3.5.3 Two Extraction Periods......................................................................92
SFE for Combined Equilibrium.....................................................................94
3.6.1 Equilibrium According to Perrut et al. ..............................................94
3.6.2 Simulation of SFE with Combined Equilibrium...............................96
3.6.3 Two Extraction Periods......................................................................96
Flow Patterns..................................................................................................96
3.7.1 Axial Dispersion ................................................................................96
3.7.2 Models for Differential Extraction Bed (Ideal Mixer)......................98
3.7.3 Natural Convection ............................................................................98
3.7.4 Channeling and Scale Up of Extractor............................................100
75
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Functional Food Ingredients and Nutraceuticals
3.8
Preliminary Evaluation of Extraction Curves..............................................101
3.8.1 Splitting Extraction Curve into Two Sections.................................101
3.8.2 Equilibrium-Controlled Period ........................................................103
3.8.3 Diffusion-Controlled Period.............................................................103
3.9 Conclusions ..................................................................................................106
Acknowledgment ...................................................................................................106
References..............................................................................................................107
3.1 SUPERCRITICAL FLUID EXTRACTION FROM
PLANT MATERIALS
A substance is in a supercritical state when both its pressure and temperature are
higher than its critical pressure and temperature. Supercritical fluids, due to their
liquid-like density and gas-like viscosity, are very well suited for extraction of
valuable substances from plant materials. The most frequently used supercritical
solvent is supercritical carbon dioxide (SC-CO2) because of the following characteristic attributes:
Strong dependence of solvent properties on pressure and temperature,
enabling wide control of solvent power in a broad range of operating
conditions
Easy separation of extract from the solvent by depressurization provides a
distinct separation of solute from solvent since CO2 is gas at ambient
conditions
High diffusivity of solutes in supercritical fluids and, consequently, high mass
transfer rates
No traces of organic solvents in extracts
Thermo-labile components are not exposed to high temperatures (critical
point temperature and pressure of CO2 are 31.1C and 74 bars, respectively)
No degradation of residual botanic material that can be used as foodstuff,
feedstuff, or as a source of substances insoluble in supercritical solvent
The main drawback of supercritical fluid extraction (SFE) is higher capital cost of
the necessary high-pressure extraction equipment, compared to conventional extraction. Supercritical CO2 is the solvent of choice in the SFE of bioactive components
from plant materials. Since CO2 is an environmentally friendly compound, it presents an alternative to hazardous traditional solvents. Though its solvent power for
nonpolar compounds is lower than that of many organic solvents and it is even
lower for the polar compounds (this disadvantage can be partially overcome by the
addition of a polar modifier to the solvent), benefits of SC-CO2 prevail. Besides the
advantages mentioned above it is also inflammable, nonexplosive, cheap, and easily
accessible. About 100 industrial plant materials using SC-CO2 are onstream worldwide, most of them processing natural products for the food industry, nutraceuticals,
or phytopharmaceuticals.
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In a typical extraction procedure, botanic material is pretreated by drying and
grinding to form small particles. The extractor is filled with the particles, heated to
the desired temperature, and pressurized with solvent. The solvent is then passed
through an extraction bed at constant flow rate until the material is depleted or until
the extraction rate drops below an economically acceptable value. Thus, the extraction can be carried out in a semicontinuous mode. The CO2 is recycled between the
extractor and separator that is operated at a lower pressure corresponding to lower
solubility where the solute precipitates, while gaseous CO2 passes to a condenser.
When pressure is increased by a high-pressure pump, the supercritical solvent enters
the extractor again. Temperature control is necessary in all parts of the high-pressure
equipment.
The capacity of industrial extractors ranges from hundreds of liters to several
cubic meters. The extraction unit usually consists of two or more extractors; while
one extractor is depressurized and refilled with fresh material, the solvent is passed
through the other extractor under pressure and thus a continuous flow of extract to
the separator is achieved. Extraction units are usually equipped with a series of
separators operating at different pressures and temperatures, which enables partial
fractionation of extract according to different solubility of its components. As the
aim of industrial-scale SFE is to isolate a desired compound from a matrix at
minimum cost and with minimum ballast substances coextracted, a careful optimization of extraction pressure, temperature, time, and solvent flow rate is necessary
(besides optimization of conditions in separators). The solvent is usually fully saturated with solute in the initial extraction period.
Analytical SFE, on the other hand, operates extractors of small capacity (a few
cm3) and aims to extract soluble substances from matrix quantitatively as fast as
possible. The extract is usually collected in a trap containing a liquid solvent, and
then injected in a chromatograph or other analytical equipment. Different liquid
modifiers are added to SC-CO2, and relatively high solvent flow rate, pressure, and
temperature are set up in order to accelerate the extraction process. Fluid phase
concentration usually does not reach saturation values because of the high solvent
flow rates. As total solvent consumption is very low due to the small size of the
extracted sample, the solvent is not recycled.
The size of laboratory equipment ideal for SFE studies usually ranges from tens
of cm3 to several liters. It is used to study the relationship between operating
conditions (pressure, temperature, solvent flow rate, and extraction time, and eventually modifier concentration in the solvent) and material pretreatment on one side
and extraction yield and extract composition on the other side, and also to optimize
SFE. To scale up the process, detailed knowledge is required. The knowledge based
on laboratory experiments is condensed in mathematical models, which are again
tested by comparison with a wide range of experimental data. Thus, the knowledge
of the process is gradually improved as a result of interaction between experimental
work and theory.
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Functional Food Ingredients and Nutraceuticals

y = y (H, t) Q
h=H
V
h=0
y=0

Q
FIGURE 3.1 Schematic drawing of extractor, extraction bed of volume V and height H with
·
solvent flow rate Q and fluid phase concentration y.
3.2 EXTRACTION BED AND THE CONCEPT OF
BROKEN AND INTACT CELLS
The extractor is a high-pressure cylindrical vessel usually vertically positioned, as
shown schematically in Figure 3.1. The solvent flows through a fixed bed formed
by solid particles and is gradually saturated with extracted substances. Let us assume
for the sake of simplicity that only one component (or pseudocomponent consisting
of more substances that behave similarly) is extracted. Solid phase thus consists of
the solute and insoluble residuum called matrix, and fluid phase is a solution of the
solute in the solvent.
Different models for SFE based on solute mass balance for solid and fluid phase
are available in literature. The mass balance is in the form of partial or ordinary
differential equations and contains a term for mass flux from solid to fluid phase,
which again is a function of equilibrium relationship between the solid and fluid
phase. Solution to the equations yields time-dependent concentration profiles, and
the resulting fluid phase concentration at extractor outlet serves for calculation of
extraction yield. Different models for SFE from plant materials are based on different
assumptions on phase equilibrium, mass transfer in solid phase, and flow pattern.
The existence of different models is justified by differences in many aspects of
extraction process like the type of plant tissue and its permeability, solute location
in the tissue, extent of solute-matrix interaction, flow distribution, and flow direction
in the extraction bed. The underlying practical problem is how to select the proper
model to describe the concrete extraction process as closely as possible.
In this chapter we discuss the main aspects of SFE from plant materials utilizing
a generalized model1 enabling us to refer to different types of phase equilibrium,
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79
flow pattern, and mass transfer. The model belongs to a group of SFE models based
on the concept of broken and intact cells of disintegrated plant tissue.2–5 The concept
results from a well-known fact that SFE from plant materials can be split into two
periods. The faster extraction in the first period is controlled mainly by phase
equilibrium, while the slower extraction in the second period is controlled mainly
by intraparticle diffusion.
During grinding of the botanic material, outer layers of the formed particles are
mechanically damaged. Reverchon and coworkers3–5 published scanning electron
microscope images of disintegrated plant materials, showing that the particle surface
consists solely of broken cells. (The term “cell” in SFE models is not used in the
same context as in botany; it refers to a cavity containing solute.) According to the
model, the solute in the broken cells becomes easily accessible for extraction. Its
diffusion to the bulk of fluid phase is fast due to favorable transport properties of
supercritical solvents. Walls of intact cells in the particle core, on the other hand,
are not easily permeable and the solute diffusion from the core is therefore orders
of magnitude slower than the diffusion from broken cells. Thus, the first extraction
period corresponds to the extraction from broken cells, and the solute from intact
cells is extracted in the second period.
Reverchon and co-workers assumed that broken cells may form a monolayer on
a particle surface and by surface image they were able to estimate the total volume
of broken cells in particles. They also assumed that the broken cells, in contrast to
the cells forming the particle core, were initially completely filled with solute. Such
assumptions correspond well to the extraction from materials where the solute is
not evenly distributed. For example, in the leaves of some plant materials essential
oil is concentrated on the leaf surface in the glandular trichomes.6 Ground seeds or
roots, however, have more or less homogeneous solute distribution in particles. In
order to simulate sufficiently large amounts of easily accessible solute in homogeneous particles, a deeper region of broken cells consisting of many layers must be
assumed in the model.
The model with homogeneous initial solute distribution in a particle,1 where the
fraction of particle volume filled with broken cells, r, equals the fraction of solute
located initially in broken cells, is discussed below. For r = 0, all solute is initially
closed in intact cells and the model is identical with conventional SFE models that
do not distinguish between broken and intact cells; for r = 1, all solute is easily
accessible and the model is identical to the models for extraction from a particle
surface.
3.3 MASS BALANCE AND MODEL EQUATIONS FOR
PLUG FLOW
3.3.1 MASS BALANCE
FOR
SOLVENT
AND
MATRIX
The mass of solvent, M, and the mass of matrix, N, in the extraction bed are
N = V (1 − ε ) ρs
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Functional Food Ingredients and Nutraceuticals
where V is the extraction bed volume, ε is the void fraction, ρs is the solid phase
density, and ρf is the solvent density. The matrix consists of broken cells of mass
rN and intact cells of mass (1 - r)N. Solvent-to-matrix mass ratio in the extraction
bed is
γ=
ερ f
M
=
N (1 − ε ) ρs
(3.2)
Let us assume that the solvent flow through the extraction bed is uniform, with no
axial mixing (plug flow). The solvent residence time in the extraction bed, the time
of contact with the botanic material, is
tr =
M γ H
= =
Q
q u
(3.3)
·
where u is the interstitial velocity, Q is the mass flow rate, q· is the specific mass
flow rate, and H is the bed height.
3.3.2 MASS BALANCE
FOR
SOLUTE
It is convenient to express solute concentrations as solute-to-matrix and solute-tosolvent mass ratios x and y, respectively. In particles we further distinguish the mean
concentration in a particle core consisting of intact cells, xc, and the concentration
in broken cells, xb. Solute mass balance is
Nxu =
N (1 − r )
H
H
∫
xc ( h, t ) dh +
0
Nr
H
H
∫
xb ( h, t ) dh +
0
M
H
H
∫ y ( h, t ) dh +
0
(3.4)
t
∫
+ Q y ( h = H , t )dt
0
where xu is the concentration in untreated botanic material, the first term on the
right-hand side stands for mass of solute in the intact cells, the second term for mass
of solute in broken cells, the third term is for solute in fluid phase inside the extraction
bed, and the fourth term is the mass of extract, E
t
∫
E = eN = Q y ( h = H , t ) dt
0
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81
Solvent starts flowing out from the extractor at t = 0. To examine the relationship
between concentrations and mass transfer rate, the mass balance is written in its
differential form for unit bed volume as
ρs (1 − ε ) (1 − r )
∂xc
∂x
∂y 
 ∂y
+ ρs (1 − ε ) r b + ρ f ε  + u  = 0

∂h 
∂t
∂t
∂t
(3.6)
together with mass balance for broken cells and a particle core that are
ρs (1 − ε ) r
∂xb
= jc − j f
∂t
ρs (1 − ε ) (1 − r )
∂xc
= − jc
∂t
(3.7)
(3.8)
where jf is the mass flux from broken cells to fluid phase and jc is the mass flux
from intact cells to broken cells, per unit volume of extraction bed. Mass flux jf is
driven by difference between concentrations in the bulk fluid phase and at a particle
surface, y+
(
j f = k f a0 ρ f y + − y
)
(3.9)
where kfa0 is the volumetric fluid phase mass transfer coefficient. As mass transfer
resistance in broken cells is neglected in the model, solid phase concentration in the
whole region of broken cells is assumed to be in equilibrium with y+
y + = y + ( xb )
(3.10)
Specific surface area a0 is the surface area per unit volume of the extraction bed. It
is expressed with help of l, the characteristic particle dimension defined as particle
volume divided by particle surface
a0 = (1 − ε )
particle surface
1− ε
=
particle volume
l
(3.11)
Mass flux jc is driven by concentration difference between intact and broken cells:
jc = kc ac ρs ( xc − xb )
(3.12)
The specific surface area of particle cores, ac, is calculated according to the following
relationship:
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Functional Food Ingredients and Nutraceuticals
ac = (1 − ε )
=
core surface
corre volume
= (1 − ε )
particle volume
particle volume
core surface
=
core voolume
(1 − ε ) (1 − r )
lc
(3.13)
where lc is the characteristic particle core dimension defined as the core volume
divided by core surface.
As the mass transfer resistance in fluid phase is very low, part of the solute is
transferred from the broken cells to the solvent during extractor pressurization.
Therefore, at t = 0 the concentration in the broken cells, xb0, is lower than xu, while
the fluid phase concentration, y0, is higher than zero. As these concentrations were
established in the period of “static extraction” without solvent flow, they are independent of axial coordinate h. Due to the low mass transfer resistance we further
assume that equilibrium between the broken cells and the fluid phase is established
at t = 0. On the other hand, the period of pressurization is too short for the concentration in the intact cells to change significantly. Thus, the initial and boundary
conditions are assumed as follows
xu − xb 0 =
γ
y0
r
y0 = y + ( xb 0 )
xc 0 = xu
y ( h = 0, t ) = 0
(3.14)
Equation 3.6 through Equation 3.10 and Equation 3.12 and Equation 3.14 represent
a complete mathematical model for concentration profiles in the extraction bed with
solvent plug flow. The equations are integrated numerically and extraction yield, e,
is calculated from the outlet fluid phase concentration y(h = H, t) according to
Equation 3.5. To fit the model to the experimental data, it is not necessary to rely
solely on the numerical solution; a preliminary analysis of the extraction data based
on time constants is possible.
3.3.3 TIME CONSTANTS
AND
DIMENSIONLESS MODEL EQUATIONS
Each individual process component has its own time constant, independent of the
·
others. Thus, the solvent flow is characterized by a mean residence time tr = M/Q
(see Equation 3.3). The external mass transfer time is
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tf =
ε
ε l
=
k f a0 1 − ε k f
83
(3.15)
and the intraparticle diffusion time is
tc =
(1 − ε ) (1 − r ) =
kc ac
lc
kc
(3.16)
Finally, the equilibrium extraction time te, which is the time necessary to extract all
solute that was initially in the broken cells when the external mass transfer resistance
is negligible, is represented by
te =
rxu
y0 q
(3.17)
If the mean residence time tr is regarded as a characteristic time, then the extraction
time t and the time constants can be expressed as the dimensionless time τ and
dimensionless model parameters, respectively, as follows
τ=
Θf =
t
tr
tf
lqε
=
t r k f γ (1 − ε )
Θc =
t c lc q
=
t r kc γ
Γ=
t r γy0
=
t e rxu
(3.18)
Dimensionless parameters Θf and Θc are the external and intraparticle mass transfer
resistances, respectively, and Γ compares the amount of solute in fluid phase at
equilibrium at t = 0 with the amount of solute present originally in the broken cells.
Introducing these parameters and dimensionless variables Xb = xb/xu, Xc = xc/xu, Y =
y/y0, z = h/H, the following dimensionless model equations are constituted after
rearrangement
τ
∫
e xu = r Γ Y ( z = 1, τ ) d τ
0
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Functional Food Ingredients and Nutraceuticals
∂Y ∂Y
+
= Jf
∂τ ∂z
(3.6a)
∂Xb 1 − r
=
J c − ΓJ f
∂τ
r
(3.7a)
∂Xc
= − Jc
∂τ
(3.8a)
Jf =
(
1
Y+ −Y
Θf
)
Y + = Y + ( Xb )
Jc =
1
( X c − Xb )
Θc
(3.9a)
(3.10a)
(3.12a)
Xb0 = 1 − Γ ,
Y0 = 1
Xc0 = 1
Y ( z = 0, τ ) = 0
(3.14a)
The dimensionless extraction yield, e/xu, is in the range 0 to 1.
Different algorithms for solution of partial differential equations Equation 3.6a
through Equation 3.8a are available. We have chosen to transform the partial differential equations to 3n ordinary differential equations written for a series of n identical
ideal mixers of dimensionless height 1/n, followed by their integration using the
Runge-Kutta method. The complete set of model equations for n mixers is
τ
∫
e xu = r Γ Yn d τ
(3.5b)
0
dY j
+ n (Y j − Y j −1 ) = J fj
dτ
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85
dXbj 1 − r
J cj − J fj
=
dτ
r
(3.7b)
dXcj
= − J cj
dτ
(3.8b)
J fj =
(
1
Y j+ − Y j
Θf
)
Y j+ = Y j+ ( Xbj )
J cj =
1
( Xcj − Xbj )
Θc
(3.9b)
(3.10b)
(3.12b)
Xb 0 j = Xb 0 = 1 − Γ
Y0 j = Y0 = 1
Xc 0 j = Xc 0 = 1
Y j−1 ( τ ) = 0
for
j = 1,2,...,n
(3.14b)
The higher n is, the closer the model simulates plug flow. The applications to SFE
of different substances are discussed below.
3.4 SFE OF OILS FROM SEEDS
3.4.1 OIL SOLUBILITY
IN
SC-CO2
Vegetable oils in seeds serve as a store for energy for future plant use, and their
mass fractions in seeds are therefore high, typically 10 to 50% wt. Common vegetable oils are composed of triglycerides of unsaturated and saturated fatty acids. In
the extraction of C16-C18 vegetable oils, the oil can be regarded as a pseudocomponent, where its solubility in SC-CO2 is predicted according to the equation
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86
Functional Food Ingredients and Nutraceuticals
(
c = ρ10f .724 exp − 18708 T + 40.361 + 2186840 T 2
)
± 2.7
(3.19)
developed by del Valle and Aguilera.7 The symbols are c (kg/m3) for the solubility,
ρf (g/cm3) for the density of pure CO2, and T (K) for the temperature. The equation,
based on solubility data for soybean oil, sunflower oil, cottonseed oil, and corn oil,
was validated for temperatures ranging from 20°C to 80°C, the pressures between
15 and 89 MPa, and solubility below 100 kg/m3. Several solubility isotherms calculated according to the equation are depicted in Figure 3.2 in terms of solute-tosolvent mass fraction ys = c/ρf. According to Equation 3.19, the solubility increases
with increasing solvent density, and, at constant solvent density, also with increasing
temperature. A crossover region is obvious at lowest pressures where the solubility
values decrease at constant pressure with increasing temperature. It is a result of a
rapid drop in solvent density with increasing temperature near the critical point.
Equation 3.19 has been successfully applied to many vegetable oils where C18 fatty
acids are dominant. Only the solubility of vegetable oils rich in free fatty acids is
underestimated because the free fatty acids are more soluble in CO2 than their
triglycerides.
Lee et al.9 examined the equilibrium relationship y+ = y(xb) for oil extracted with
CO2 from flaked canola seeds at 55C and 36 MPa. They prepared samples of partially
extracted flakes with oil concentration ranging from 0.2–0.7 g/g oil-free seed and
measured the concentration of oil in the solvent phase in equilibrium with seeds for
each sample. The concentration was 0.011 ± 0.001 g/g CO2, which is close to
ys = 0.012 g/g CO2 calculated according to Equation 3.19, for all samples. Thus,
0.1
20°C
0.08
40°C
ys , kg/kg
60°C
0.06
80°C
0.04
0.02
0
15
25
35
45
55
Pressure, MPa
65
75
85
FIGURE 3.2 Solubility of C18 vegetable oils in dense CO2 in dependence on pressure and
temperature according to del Valle and Aguilera7; CO2 density calculated with Altunin and
Gadetskii equation.8
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
87
TABLE 3.1
Calculation of Dimensionless Model Parameters for Extraction of a
Solute from Spherical Particles; Distribution Coefficient K Is Valid in
the Case of Solute-Matrix Interaction
Physico-Chemical
Parameters
xu
d, mm
ε
γ
q,· kg/kg min
r
K
kf, m/s
kc, m/s
Dimensionless Model
Parameters
0.025
0.60
0.40
0.50
0.125
0.60
0.24
1.11 · 10–3
2.56 · 10–5
r
0.60
Γ
0.1667
Θf
0.25
Θc
12.0
–
K
1.20
—
—
Initial concentrations
xb0
0.02083
y0
0.005
Equations
—
Equation
Equation
Equation
Equation
—
—
Equation
Equation
3.18
3.11 and Equation 3.18
3.13 and Equation 3.18
3.28
3.14 and Equation 3.27
3.37
over the range of seed oil concentrations used, the seed tissue has no physical or
chemical affinity to oil and acts merely as an inert substrate. The equilibrium
concentration of oil in the solvent phase is therefore assumed to be constant as long
as any oil is present in the broken cells
y+ = ys for xb > 0, y+ = 0 for xb = 0
(3.20)
We shall further use the term “constant solubility” for equilibrium described by
Equation 3.20. Dimensionless form of the equation for j-th mixer is
Y j+ = 1 for Xbj > 0 , Y j+ = 0 for Xbj = 0
3.4.2 MODEL
FOR
(3.21)
VEGETABLE OIL EXTRACTION
To illustrate the dependence of concentration profiles and the extraction curve on
the type of phase equilibrium, we have chosen a basic set of model parameters, r =
0.6, Γ = 0.167, Θf = 0.25, and Θc = 12 (see Table 3.1). To simulate SFE of vegetable
oils with plug flow, model Equation 3.5b through Equation 3.9b plus Equation 3.12b
and Equation 3.14b were numerically integrated together with Equation 3.21 using
the basic set of parameters and n = 40; the results are shown in Figure 3.3. The
depicted solid phase concentration is calculated as mean concentration in particle X
= rXb + (1 - r)Xc. The concentration profiles in both phases gradually shift to the
right-hand side of the figure, in the direction of solvent flow. As long as the fluid
phase concentration at solvent outlet remains constant, the extraction curve e/xu
versus τ is a straight line. When the extraction of solute from broken cells is complete,
mass transfer from particles continues much slower and concentration profiles flatten; the slope of the extraction curve decreases proportionally with the decrease in
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Functional Food Ingredients and Nutraceuticals
1
0.8
Y
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
0.6
0.8
1
z
1
0.8
X
0.6
0.4
0.2
0
0
0.2
0.4
z
1
0.8
e/xu
0.6
0.4
0.2
0
0
10
20
t
30
40
FIGURE 3.3 Simulated concentration profiles and extraction curve for plug flow and constant
solubility. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, n = 40. Fluid phase
concentration profiles Y(z) and solid phase concentration profiles X(z) shifting to the right and
decreasing: (_______) solution of complete model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40.
Extraction yield e/xu: (_______) solution of complete model equations; (+ + + +) approximate
sections of extraction curve according to Equation 3.24 and Equation 3.25.
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
89
outlet fluid phase concentration. The transition from equilibrium-controlled period
to diffusion-controlled period occurs at extraction yield higher than the yield of
easily accessible solute rxu . The reason for the increase is that a certain amount of
solute from the intact cells is transferred into the broken cells and becomes easily
accessible while the solute deposited initially in broken cells is transferred into the
fluid phase.
3.4.3 EQUILIBRIUM-CONTROLLED PERIOD
Several researchers assume constant solubility and plug flow in order to model the
first period of oil extraction with SC-CO2. Lee et al.9 solved the mass balance
equations for oil extraction from rape seed flakes numerically and adjusted external
mass transfer coefficient kfa0 to experimental data with the result that kfa0 was
proportional to u0.54.
Brunner10 pointed out that oil extraction is a steady-state process as long as any
easily accessible oil remains at the solvent inlet, where it is depleted first. (It can
be shown that the steady-state extraction lasts until τ = τ1 = Θf(1 – Γ)/Γ. Thus, in
our example depicted in Figure 3.3, τ1 = 1.25.) During the steady-state extraction,
both fluid phase concentration profile and extraction rate are constant and the mean
driving force in the extraction bed ys – ymean is related to measurable quantities ys
and y(h = H) according to
ys − ymean =
y (h = H )
ys
ln
ys − y ( h = H )
(3.22)
Taking into account that in a steady-state period ∂y/∂t = 0, volumetric mass transfer
·
coefficient kfa0 can be determined from mass flux Vkfa0ρf (ys – ymean) = Qy(h = H).
Ferreira et al.11 applied this procedure to evaluate the external mass transfer coefficient with experimental data for black pepper with SC-CO2 at 15–30 MPa and 30C
–50C. The botanic material was ground to a mean size of approximately 0.1 mm,
and the interstitial solvent velocity, u, was in the range of 0.2–0.9 mm/s. Mass
transfer coefficient kf , increasing with increasing interstitial velocity, was found to
be independent of pressure and temperature and its value was between 3 × 10–4 and
9 × 10–4 m/s.
In terms of the present model, Equation 3.22 corresponds to a steady-state fluid
phase concentration profile
 z 
Y = 1 − exp  −
 Θ f 
(3.23)
(depicted in Figure 3.3a) for τ = 1, and the dimensionless extraction yield is based
on the following relationship
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Functional Food Ingredients and Nutraceuticals

 1
e
= Γr τ 1 − exp  −
xu
 Θf


 

(3.24)
The exponential term for Θf < 0.22 is lower than 0.01. For these values of Θf , the
solvent flowing out from the extraction bed is practically saturated with solute. After
exhaustion of the easily accessible solute from particles at the solvent inlet, that is,
when xb(z = 0) becomes zero, the fluid phase concentration profile starts shifting
toward the solvent outlet and the overall extraction rate gradually slows down.
3.4.4 DIFFUSION-CONTROLLED PERIOD
In the second part of the extraction curve the fluid phase concentration is much
lower than the solute solubility in the solvent. If the assumption on equilibrium at
a particle surface and constant solubility holds, the solute that arrives from the
particle core to its surface dissolves immediately in the solvent and the concentration
in broken cells becomes practically zero. The driving force for internal diffusion
according to Equation 3.12 is then equal to xc and the extraction yield in the second
period is approximated by the following equation
e
 τ − τc 
= 1 − (1 − r ) exp  −

xu
Θ c 
(3.25)
where the shift in time, τc > 0, is related to the fact that the driving force for solute
transfer from the intact to broken cells was decreased in the first extraction period
by the nonzero concentration in the broken cells. As shown in Figure 3.3, the
extraction yield calculated using the complete model equations and the yield according to Equation 3.25 with τc adjusted to 3.75 overlap in the second extraction period.
3.4.5 APPROXIMATE SOLUTION
FOR
BOTH PERIODS
An approximate model with plug flow based on the concept of broken and intact
cells was derived by Sovová2 as an extension of Lack’s model. The model simulates
both concentration profiles and extraction yield, which is given as

 1
e
= Γr τ 1 − exp  −
xu
 Θf


Θf
  for τ ≤ τ m = Γ

(
)



exp  τ − τ m Θc  − 1 + r
e

= r Γτ − Θ f exp  r ΓΘc ln
− 1

xu
r




for
© 2007 by Taylor & Francis Group, LLC


Θ f 

 
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
τ < τn =
91

Θf
 1 
+ Θ c ln  1 − r + r exp 
,
 r ΓΘ c  
Γ


e
 1  
 τ − τ m  

− 1 expp  −
= 1 − r ΓΘ c ln 1 + (1 − r )  exp 


 r ΓΘ c  

Θ c  
xu


for
τ ≥ τn
(3.26)
The steady-state extraction period according to Equation 3.26 finishes when τ = τm.
Then the fluid phase concentration profile starts shifting through the extraction bed.
At time τ = τn the last easily accessible solute is transferred from the broken cells
to fluid phase and the extraction is further controlled solely by intraparticle diffusion.
A model2 was derived for γ 0 where the solute accumulation in the fluid phase
was not taken into account. Therefore a discrepancy exists between extraction curves
calculated according to Equation 3.26 and those obtained by numerical solution to
the complete model equations, as illustrated in Figure 3.4 for the basic set of
parameters r = 0.6, Γ = 0.167, Θf = 0.25, and Θc = 12. To simulate precisely the
extraction curve calculated with the complete model, the approximate parameters
would have to be adjusted to r = 0.697, Γ = 0.211, Θf = 0.917, and Θc = 11.6. Thus,
the approximate model is useful to smooth the experimental data and obtain the first
estimation of model parameters, which, however, should be refined by further calculations. As the approximate model does not involve solute-matrix interaction, it
should not be applied for the system where solute interacts with matrix.
1
0.8
e/xu
0.6
0.4
0.2
0
0
10
20
τ
30
40
FIGURE 3.4 Comparison of extraction curves calculated for constant solubility, plug flow,
and model parameters r = 0.6, Γ = 0.167, Θf = 0.25, Θc= 12, and n = 40. (_______) solution of
complete model equations; (+ + + +) approximate model2 with analytical solution.
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Functional Food Ingredients and Nutraceuticals
3.5 SFE OF ESSENTIAL OILS
3.5.1 SOLUTE-MATRIX INTERACTION
Essential oils as mixtures of volatile and predominantly low-polar compounds are
more soluble in SC-CO2 than vegetable oils. Their main components, terpenes and
oxygenated terpenes, are fully miscible with SC-CO2 at temperatures of 40°C –50°C
and pressures of 10–20 MPa, as Reverchon states in a comprehensive review on
SFE of essential oils.12 Nevertheless, when essential oils are extracted with SC-CO2
from leaves or other botanic materials where their concentrations are only a few
percent or less, their equilibrium fluid phase concentration is much lower than that
measured in absence of tissue matrix. For example, Goto et al.13 observed that the
equilibrium fluid phase concentration of menthol, a major component in peppermint
essential oil, during SFE from peppermint leaves, was two orders of magnitude lower
than the solubility of pure menthol in SC-CO2. To explain how the matrix affects
the essential oil equilibrium, the authors assumed that the essential oil is adsorbed
on the lipids present in the leaves and thus the experimental equilibrium concentration is represented by an adsorption isotherm. To describe the solute-matrix interaction they applied the simplest and the most frequently used relationship for SFE,
linear equilibrium with partition coefficient K
y + = Kxb
(3.27)
Its dimensionless form for j-th mixer is
Y j+ = KXbj , K = 1 (1 − Γ )
3.5.2 SIMULATION
OF
(3.28)
ESSENTIAL OIL EXTRACTION
Model Equation 3.5b through Equation 3.9b plus Equation 3.12b and Equation 3.14b
together with Equation 3.28 were numerically integrated for the basic set of model
parameters and n = 40. Due to the linear equilibrium, completely different concentration profiles from vegetable oil extraction were obtained, as shown in Figure 3.5.
As the solid phase concentration gradually decreases during the extraction process,
the equilibrium concentration in fluid phase at a particle surface also decreases
likewise the driving force y+ - y. As a result, the concentration profiles corresponding
to solute-matrix interaction are flatter and the extraction becomes slower than in the
case without solute-matrix interaction.
3.5.3 TWO EXTRACTION PERIODS
The first and second extraction periods cannot be completely separated as it was in
the case without solute-matrix interaction. On one hand, the solute diffusing from
the intact to broken cells during the first period increases the concentration in broken
cells and therefore also the equilibrium fluid phase concentration. On the other hand,
as the concentration in the broken cells is directly proportional to the fluid phase
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93
1
0.8
Y
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
0.6
0.8
1
z
1
0.8
X
0.6
0.4
0.2
0
0
0.2
0.4
z
1
0.8
e/xu
0.6
0.4
0.2
0
0
10
20
τ
30
40
FIGURE 3.5 Simulated concentration profiles and extraction curve for linear equilibrium and
plug flow. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, and n = 40. a) Decreasing
fluid phase concentration profiles Y(z) and solid phase concentration profiles X(z): (_______)
solution of complete model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40. Extraction yield e/xu:
(_______) solution of complete model equations; (+ + + +) approximate sections of extraction
curve according to Equation 3.24 and Equation 3.29.
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Functional Food Ingredients and Nutraceuticals
concentration at a particle surface, it is neither zero nor constant in the second
extraction period. Nevertheless, at least the initial slope of the extraction curve can
be estimated according to Equation 3.24 as in the case of constant solubility (Figure
3.5). The extraction in the second extraction period is slower than that derived for
constant solubility with Equation 3.25, because the nonzero concentration xb reduces
the driving force. An acceptable estimate for extraction yield in the second period
is given in the following equation1

τ − τc
e
= 1 − (1 − r ) exp  −
xu
 Θ c 1 + (1 − r ) K ΓΘ c r

(
)


 

(3.29)
The agreement of Equation 3.29 with the results obtained by solution of complete
model equations for the basic set of model parameters is shown in Figure 3.5.
3.6 SFE FOR COMBINED EQUILIBRIUM
3.6.1 EQUILIBRIUM ACCORDING
TO
PERRUT
ET AL.
A versatile SFE model would combine the constant solubility and linear equilibrium
in one expression. Two papers independently solving this task were published almost
simultaneously. Goto et al.14 suggested the BET adsorption isotherm which is often
used to describe adsorption equilibria. The BET isotherm simulates a smooth
increase in equilibrium fluid phase concentration with increasing solid phase concentration, from y+ determined solely by solute-matrix interaction at lowest solid
phase concentrations to y+ asymptotically approaching ys at high solid phase concentrations. The other equation, proposed by Perrut et al.15 as a simplified description
of phase equilibrium during SFE from plant materials, defines the equilibrium fluid
phase concentration as a discontinuous function of solid phase concentration as
depicted in Figure 3.6
y
ys
b
a
c
KXf
d
Xf
X1
FIGURE 3.6 Combined equilibrium relationship with constant solubility for high solid phase
concentrations and with linear equilibrium for low solid phase concentrations according to
Perrut et al.15
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
y + = ys for xb > xt
y + = Kxb for xb ≤ xt ,
95
(3.30)
where ys > Kxb. The sudden fall in equilibrium concentration y+ at xb = xt can be
explained as follows. Matrix has certain capacity of interacting with solute, as
expressed by transition concentration xt. As long as the solid phase concentration is
less than or equal to xt, all solute in solid phase interacts with matrix and the
equilibrium fluid phase concentration is determined by the distribution coefficient
K. When the solid phase concentration is higher, part of solute remains free, unbound
to the matrix, and its equilibrium fluid phase concentration equals solute solubility.
According to this simplified model, when SFE starts at xb0 > xt, solid phase
concentration gradually decreases to xt and when it reaches this value, the equilibrium
fluid phase concentration at the particle surface falls from ys to Kxt, below the fluid
phase concentration in the diffusion layer close to the surface. Mass transfer from
the broken cells to fluid phase is interrupted until the fluid phase concentration drops
below y = Kxt. In the meantime, the solute is only washed out
(
j f = k f a0 ρ f y + − y
)
for xb ≠ xt or xb = xt , y < Kxt , j f = 0 otherwise.
(3.31)
Equation 3.30 includes both equilibrium relationships mentioned above: for xt = 0
it is identical with Equation 3.20 for constant solubility, and for xt > xb0 it describes
linear equilibrium according to Equation 3.27. It seems that any solute can occur
both in free form and bound to the matrix, depending on its solid phase concentration
and on matrix and solvent properties. Indeed, Perrut et al.15 observed the combined
equilibrium during SFE of vegetable oil from seed, where constant equilibrium
would be expected.
When the extraction with plug flow and combined equilibrium (Equation 3.30)
starts in section “a” of the equilibrium curve (Figure 3.6), the whole period of solute
extraction from the broken cells is governed by constant solubility and the solutematrix-interaction does not influence the course of the extraction. When the extraction starts in section “b,” the outlet concentration firstly approaches the solubility,
then the equilibrium value at transition concentration xt, and finally the intraparticle
diffusion-controlled extraction follows. When the extraction starts in section “c,” the
amount of free solute present in the extraction bed at t = 0 is not sufficient to saturate
the solvent and thus the initial outlet concentration is lower than solubility; as soon
as the initial solution is washed out of the extractor, only the solute interacting with
matrix is extracted. Finally, when the extraction starts in section “d,” all solute
interacts with matrix, as described in Section 3.5.
In the dimensionless model for a series of mixers, Equation 3.30 reads as follows
Y j+ = 1 for Xbj > Xt , Y j+ = KXbj for Xbj ≤ Xt
© 2007 by Taylor & Francis Group, LLC
(3.32)
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Functional Food Ingredients and Nutraceuticals
–
–
where Xt = xt/xu and K = Kxu/y0. Section “a” is characterized by 1 – Γ ≥ 1/K, section
“b” by Xt < 1 – Γ < 1/ K , section “c” by Xt = 1 – Γ and K < 1 (1 − Γ ) , and section
“d” by Xt ≥ 1 – Γ and K = 1 (1 − Γ ) .
3.6.2 SIMULATION
OF
SFE
WITH
COMBINED EQUILIBRIUM
Model Equation 3.5b through Equation 3.9b plus Equation 3.12b and Equation
3.14b, together with Equation 3.32 were numerically integrated with the basic set
of the same parameter values and number of mixers as in the case of constant
equilibrium (r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, and n = 40), using two more
–
equilibrium parameters Xt = 0.48 and K = 0.5. Thus, the extraction starts in section
“b.” The results shown in Figure 3.7 apparently indicate an extraction with constant
equilibrium where the second, diffusion-controlled period starts as soon as the
dimensionless solid phase concentration in the broken cells drops to Xt. Further
analysis, however, will show that the shape of the extraction curve starting in section
“b” is different from that of constant equilibrium.
3.6.3 TWO EXTRACTION PERIODS
The first extraction period consists of two sections of different slopes. If the external
mass transfer resistance is neglected for the sake of simplicity, the shape of the
extraction curve in the first extraction period is calculated as1
e
1 − Γ − Xt
= Γr τ for τ ≤ τ1 = 1 +
xu
Γ 1 − KXt
(
e
= Γr  τ1 + KXt ( τ − τ1 )  for τ >τ1
xu
)
(3.33)
In the second, diffusion-controlled period, the solute interacting with matrix in
broken cells is practically in equilibrium with the solute in fluid phase, and the
extraction curve is approximately described by Equation 3.29 (unless Xt = 0, when
Equation 3.25 should be applied). As Figure 3.7 shows, the agreement of estimated
extraction curve sections with complete extraction curve is good.
3.7 FLOW PATTERNS
3.7.1 AXIAL DISPERSION
In the previous sections we have shown how phase equilibrium and diffusion resistance affect the shapes of concentration profiles and the extraction curve. Another
aspect that should be taken into account and which is often omitted is the flow
pattern in the extraction bed. To express deviations from plug flow, Goodarznia and
Eikani16 inserted an axial diffusion term into the differential mass balance equation
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97
1
0.8
Y
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
0.6
0.8
1
z
1
0.8
X
0.6
0.4
0.2
0
0
0.2
0.4
z
1
0.8
e/xu
0.6
0.4
0.2
0
0
20
40
τ
60
80
FIGURE 3.7 Simulated concentration profiles and extraction curve for plug flow and com–
bined equilibrium. Model parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, Xt = 0.48, K =
_______
0.5 concentration profiles X(z) shifting to the right and decreasing: (
) solution of complete
model equations for τ = 1, 2, 4, 6, 8, 10, 20, 40. Extraction yield e/xu: (_______) solution of
complete model equations; (- - - -) approximate equilibrium-controlled section according to
Equation 3.33; (+ + + +) approximate diffusion-controlled section according to Equation 3.29.
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Functional Food Ingredients and Nutraceuticals
ρs (1 − ε ) (1 − r )
 ∂y
∂xc
∂x
∂2 y 
∂y
− Dax 2  = 0
+ ρs (1 − ε ) r b + ρ f ε  + u
∂h
∂t
∂t
∂h 
 ∂t
(3.34)
where Dax is the axial dispersion coefficient. Reis-Vasco et al.6 applied Equation
3.34 on SC-CO2 extraction of mint oil from leaves and found out that Equation 3.34
yields a better representation of experimental extraction curves than the plug flow
model. The axial dispersion values adjusted to fit experimental data were decreasing
with increasing flow rate and were about one order of magnitude larger than those
obtained from the correlations found in literature. Nevertheless, the authors regarded
the introduction of an additional adjustable parameter into the model as too high a
cost for slightly better accuracy and therefore selected the plug flow model as the
most useful one.
Another possibility to express axial dispersion in extraction bed is decreasing
the number of mixers in the model Equation 3.5b through Equation 3.14b. Though
the changes in the degree of axial dispersion are not continuous as according to
Equation 3.34, it is possible to cover a wide range of flow patterns from plug flow
for n ∞ up to ideal mixer for n = 1, which is discussed in the next section.
3.7.2 MODELS
FOR
DIFFERENTIAL EXTRACTION BED (IDEAL MIXER)
The models published for SFE from plant materials by Goto et al.13,14 consider a
differential extraction bed where no concentration gradient is assumed. Thus, the
differential extractor is described by ordinary differential equations for an ideal mixer
(Equation 3.6b through Equation 3.8b for n = 1). Such models are simpler than the
plug flow models and some of them have analytical solutions. They would be ideal
for SFE application in short extraction beds or when strong axial dispersion exists
in fluid phase.
Compared to plug flow, the extraction in an ideal mixer is slower in the first
extraction period due to the lower driving force (Figure 3.8). The following equation
y=
y+
1+ Θf
and
Γr τ
e
=
xu 1 + Θ f
(3.35)
holds for the initial steady-state period. The effect of external mass transfer resistance
on outlet concentration and the extraction rate is more pronounced than in the case
of plug flow (Equation 3.24).
3.7.3 NATURAL CONVECTION
The best extractor performance is achieved with a plug flow. Unfortunately, supercritical fluids are extremely prone to natural convection, due to their low kinetic
viscosity. Such convection can be induced by solvent density differences caused by
the differences in local solute concentrations. The occurrence of natural convection
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99
1
0.8
e/xu
0.6
y + = ys
0.4
0.2
0
0
10
20
τ
30
40
1
0.8
e/xu
0.6
y+ = Kxb
0.4
0.2
0
0
10
20
30
τ
40
50
60
1
0.8
e/xu
0.6
y+ = ys for xb > xt
y+ = Kxb for xb ≤ xt
0.4
0.2
0
0
20
40
τ
60
80
FIGURE 3.8 Effect of flow pattern on extraction curves for three types of equilibrium. Model
parameters: r = 0.6, Γ = 0.167, Θf = 0.25, Θc = 12, n = 40, and for combined equilibrium Xt
–
= 0.48 and K = 0.5. (_______) plug flow; (– – – –) ideal mixer; (- - - - -) two parallel flows
with unc = 0.75u.
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Functional Food Ingredients and Nutraceuticals
depends also on the direction of solvent flow through the extraction bed, as Beutler
et al.17,18 discovered. They observed this phenomenon in SC-CO2 extraction of
different solutes, such as black pepper oleoresin or fat, from botanical matrix. When
the flow was from the top to the bottom, the fat extraction yield was twice as high
as for the opposite flow direction. The authors explained this phenomenon by higher
density of solute-laden CO2 than that of pure solvent; the solute-laden solvent can
be more easily withdrawn from the bottom of the extractor than from its top. The
better withdrawal of solute leads to higher concentration gradients in the solid phase
and thus to a higher mass transfer driving force. Barton et al.19 observed the effect
of flow direction when they extracted peppermint oil with dense CO2. Though the
extraction yields by downflow CO2 reached 90 to 98% of those extracted by steam
distillation, the yield of peppermint oil in the CO2 upflow mode was only 76%, even
at higher solvent treatments and extraction times.
Also, Sovová et al.20 who extracted grape seed oil with SC-CO2 observed retardation in extraction when the flow direction was switched to the upflow mode; the
effect was more pronounced for lower interstitial velocities. Equation 3.26 was used
to simulate experimental data. While a simple plug flow model was adequate for
SFE in downflow mode, the natural convection in upflow mode was simulated
assuming parallel flows of different velocities in the extraction bed. The simplest
application of this approach is splitting the bed into halves with interstitial velocities
u + unc and u – unc. The resulting extraction curves calculated for the basic set of
model parameters with unc = 0.75u are compared with the results of the simple plug
flow in Figure 3.8; the effect of different velocities is most pronounced for constant
solubility.
The existence of natural convection in a fixed bed where supercritical solvent
flows slowly in the direction to the top was confirmed by Dams21 and Stüber et al.22,
who dissolved in dense CO2 solid substances from porous particles. Dams observed
the natural convection indicated by flattening of solid phase concentration profiles.
Stüber et al. measured extraction curves for a wide range of extraction conditions;
to simulate the results they applied the model for a differential bed and included the
effects of gravity-opposing flow (upflow) and gravity-assisting flow (downflow) on
extraction rate into correlations for external mass transfer coefficient.
3.7.4 CHANNELING
AND
SCALE UP
OF
EXTRACTOR
Another unfavorable type of flow pattern occurring in extraction beds is channeling.
When the extraction bed is inhomogeneous, the solvent flows preferably through
the paths of minimum hydraulic resistance and extracts the solute from their vicinity.
Most of the solute in the extraction bed has very limited contact with the flowing
solvent and as a result the extraction may even be retarded by orders of magnitude.
With an increase in the cross section of the extractor the probability of inhomogeneous particle distribution and the tendency of channeling increase. This tendency
depends also on the properties of extracted material; it is large for example when
extracted particles are sticky or when they are too small and the extraction bed has
therefore large hydraulic resistance. Berna et al.23 published experimental results on
SC-CO2 extraction of orange peel essential oil from different cultivars and on
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
101
different scales, showing a large extraction retardation on production scale compared
to the pilot scale. When a bed of inert particles of diatomaceous earth was placed
in the extractor as solvent flow distributor, the difference between extraction yields
measured in a small and a large extractor was significantly reduced.
We have shown1 that the model for a differential extraction bed can simulate
SFE in channeling extractor, when the external volumetric mass transfer coefficient
kfa0 is adjusted to unrealistically low values, corresponding to the difficult solute
approach from distant particles to flow channels. Prediction of scale-up effects is,
however, difficult because there is no rule for predicting the extent of channeling in
a larger-scale extraction bed. On the other hand, if the channeling is suppressed by
proper measures, and flow pattern in the larger extractor is similar to that in the
smaller extractor, the model with equilibrium and mass transfer parameters adjusted
to the data measured in the smaller extractor is able to predict well the large-scale
extraction.
3.8 PRELIMINARY EVALUATION OF EXTRACTION
CURVES
3.8.1 SPLITTING EXTRACTION CURVE
INTO
TWO SECTIONS
Before the SFE is simulated using the complete model equations, model parameters
should be roughly estimated from the shapes of extraction curves. The first condition
for measurement of “readable” extraction curves is to suppress deviations from plug
flow and particularly the channeling. This can be achieved relatively easily in a
small-scale extractor, especially when the gravity-assisting solvent flow direction is
chosen. In the case without solute-matrix interaction, the equilibrium- and diffusioncontrolled sections of extraction curves are obvious at first sight (Figure 3.3).
In the case of solute-matrix interaction the differentiation is more difficult but
possible especially when at least two extraction curves measured at identical
conditions except for residence times are available. The extraction of easily accessible solute should be close to equilibrium extraction, which is possible due to
excellent transport properties of supercritical solvents. Let us assume that the
second experiment will be performed at identical solvent flow rate and with twofold
solid charge in the extractor. (It is better to maintain the flow rate constant, as its
change would affect not only the residence time but also the external mass transfer
coefficient.) In our example, extraction yields e were calculated first for model
parameters given in Table 3.1 and then for the same parameters except for a
doubled residence time. The obtained extraction curves e(q) and e(t) are compared
in Figure 3.9. The slopes of e(q) are in the initial, equilibrium-controlled extraction
period practically equal to the equilibrium fluid phase concentration until about
11 g/(kg matrix) is extracted. The curves stop overlapping in the following,
transition, period controlled by both equilibrium and intraparticle diffusion.
Finally, when approximately 21 g/(kg matrix) has been extracted and the extraction
is controlled almost solely by intraparticle diffusion, the shapes of both curves
e(t) are almost identical, as can be proved by shifting the first extraction curve by
a proper time interval as indicated in Figure 3.9. After identification of equilibrium-
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Functional Food Ingredients and Nutraceuticals
30
e, g/kg
20
10
0
0
10
20
q, kg/kg
30
40
30
e, g/kg
20
10
0
0
50
100
150
t, min
200
250
300
FIGURE 3.9 Estimation of model parameters from extraction curves e(q) and e(t) measured
close to plug flow and with almost saturated solvent in the first extraction period. Solute
matrix interaction (linear equilibrium) is assumed. (_______) simulated extraction curve for
residence time tr = 4 min, model parameters according to Table 3.1; (- - - -) the same curve
shifted by 36 min to the right; (– – – –) simulated extraction curve for residence time tr = 8
min (q· = 0.0625 min-1) and other physico-chemical parameters unchanged; (+ + + +) equilibrium model Equation 3.36.
and diffusion-controlled extraction curve sections, each section can be evaluated
independently according to different relationships.
It should be taken into account that the models simulate the extraction of one
compound or one pseudocompound. When two or more solutes with completely
different solubilities and comparable contents in botanic material are extracted and
when the only experimental data available is the extraction curve measured for total
extract, the curve should be simulated as a superposition of extraction curves for
individual components and not as an extraction curve for a single compound.
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
103
3.8.2 EQUILIBRIUM-CONTROLLED PERIOD
The equilibrium parameters are estimated by fitting approximate model equations
to the first, equilibrium-controlled part, of the extraction curve. The equations given
in Sections 3.4.3 and 3.6.3 as functions of dimensionless parameters can be rearranged for Θf → 0 to
e = qys = ys qt
(3.36)
for the case without solute-matrix interaction, and to
, y0 =
e = qy0 = y0 qt
Kxu
1+ Kγ r
(3.37)
for solute-matrix interaction described by linear equilibrium. For combined equilibrium according to Equation 3.30, depicted in Figure 3.6, the shape of the first section
of extraction curve depends on where on the equilibrium curve the extraction starts,
as we discussed in Section 3.6.1. Equation 3.36 should be applied for the extraction
starting in section “a,” Equation 3.37 for the extraction starting in “d,” and the
following equation
for t ≤ t1 =
e = qys = ys qt
r ( xu − xt ) − γKxt
,
q ( ys − Kxt )
e = q  ys t1 + Kxt ( t − t1 )  for t > t1.
(3.38)
for the extraction starting in section “b.” From the slopes of the curve we can read
equilibrium characteristics ys and Kxt, and from t1 we can estimate xt, after r is
estimated from the diffusion-controlled part of the extraction curve. The equation
for the extraction starting in section “c” is similar to Equation 3.38 but its initial
slope e/q is lower than ys. More details on modeling the extraction with combined
equilibrium are given elsewhere.1
3.8.3 DIFFUSION-CONTROLLED PERIOD
The intraparticle diffusion time is much larger than the time constants for other
phenomena affecting SFE and thus the diffusion characteristics can be obtained
easily from the second extraction period data. Extraction yield in this period is
according to Equation 3.25 and Equation 3.29 approximated by

1− r
 t 
for y + = Kxb1 .
e = xu 1 − C1 exp  −   , C2 = t c for y + = ys , C2 = t c +


C
Kq
2 

(3.39)
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Functional Food Ingredients and Nutraceuticals
The content of extractable solute in botanic material, xu, and constants C1, C2 are
determined by fitting Equation 3.37 to the experimental extraction curve.
The fact that the shape of the extraction curve in the second period is known
has practical impact in analytical SFE as it enables the prediction of analyte content
in extracted sample, Nxu, from several (at least three) measurements in the second
period. If the amount of extract is determined in this period at three time points with
the same time intervals, the initial amount of solute in the extraction bed is calculated
after rearrangement of Equation 3.39 as
E∞ = Nxu = m1 +

( e2 − e1 )2 
m22
= N  e1 +

2 e2 − e1 − e3 
m2 − m 3

(3.40)
where m1 = E(t1) is the amount of extract in the first measurement, m2 = E(t2) - E(t1)
is the increase in extract in the first time interval and m3 = E(t3) - E(t2) is the increase
in extract in the second interval. Nowadays, with computers, a direct fitting of
Equation 3.39 to more than three experimental points is recommended instead of
the three-point extrapolation, which is extremely sensitive to experimental error.
To express the relation between intraparticle diffusion time (tc), effective intraparticle diffusivity (De), and particle size and shape, Reverchon24 used the results of
Villermaux25 who examined approximate relationships between diffusion times and
particles of different shapes and found the following relationship
tc = µ
l2
De
(3.41)
where µ is equal to 3/5, 1/2, and 1/3 for spheres, cylinders, and slabs, respectively,
and l is the characteristic particle dimension. Though the model applied by Reverchon and Villermaux does not take into account the existence of broken cells and
easily accessible solute in particles, it can be easily applied to the second extraction
period when the characteristic particle core dimension, lc, is substituted for l:
tc = µ
lc2
De
(3.42)
As Equation 3.16 holds simultaneously, the solid phase mass transfer coefficient is
approximately
kc =
© 2007 by Taylor & Francis Group, LLC
De
µlc
(3.43)
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105
Particles are usually assumed to be of spherical shape. Characteristic dimension for
spherical particles of diameter d is equal to d/6 and their core characteristic dimension is
d (1 − r )
6
13
lc =
(3.44)
Inserting Equation 3.44 in Equation 3.42 we obtain the intraparticle diffusion time
for spherical particles
tc =
d 2 (1 − r )
60 De
2 3
(3.45)
Equation 3.45 written for r = 0 and modified for simultaneous external mass transfer
resistance was applied in SFE from plant materials several times, for example, by
del Valle et al.26 Reverchon24 demonstrates with experimental data for essential oil
extraction from ground leaves of different particle sizes the importance of correct
modeling of particle shape. The internal diffusion time was less dependent on particle
size than according to Equation 3.45 derived for spherical particles. When it was
taken into account that the extracted particles had a form of slabs whose characteristic
dimension is less dependent on particle size and more on the slab thickness, which
is not affected by grinding, a good agreement of model calculations with experimental extraction curves was achieved.
Other published models are based on the assumption that the intraparticle mass
transfer coefficient is related to solute diffusion in particle pores; mass balance
equations then include the rate of solute transfer from solid phase to the pores or
solid-fluid equilibrium at pore walls, and the diffusion in pores. For example, Goto
et al.13 assumed adsorption equilibrium according to Equation 3.30 on the pore
surface. Diffusion in the pores filled with solvent is also involved in shrinking core
models (Roy et al.27 and Akgün et al.28). These models are based on the assumption
that as the solute in spherical particle is depleted, its concentration in solid phase
remains unchanged but the volume of the solid phase containing the solute, which
is regarded as a particle core, is diminishing; the intraparticle mass transfer resistance
increases as the solute has to diffuse through increasing length of pores between the
shrinking core and particle surface. Shrinking core models do not require any
knowledge of equilibrium equation y+ = y+(x) as the equilibrium solid phase concentration at a particle core surface remains constant.
Whatever models are used to simulate intraparticle diffusion, all of them, after
some simplification, lead to the equation for extraction yield in the form of Equation
3.39.
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Functional Food Ingredients and Nutraceuticals
3.9 CONCLUSIONS
Supercritical extraction of botanic materials is characterized, in contrast to other
extraction processes, by the combination of special properties of supercritical fluids
(liquid-like density, very low kinematic viscosity, and good transport processes) and
extracted plant materials (low permeability of intact dry plant tissue in contrast to
the easily accessible solute in broken cells close to the particle surface). The process
of supercritical fluid extraction can be split into two periods, the first one governed
by phase equilibrium where solute is extracted from broken cells, and the second
one controlled by intraparticle diffusion where the solute is extracted from intact
plant tissue in the particle core. Using mathematical model with broken and intact
cells, we have analyzed the effects of three phenomena — phase equilibrium, mass
transfer, and flow pattern — on the extraction process. A simple experimental design
enables us to distinguish the extraction periods and estimate phase equilibrium and
mass transfer parameters separately. Solvent flow pattern significantly affects extraction yields. Scale up of the extraction process is usually connected with unfavorable
changes in flow pattern, accompanied by a decrease in extractor performance. These
changes are related to the inhomogeneous structure of the extraction bed formed by
solid particles. Though the changes in flow pattern are not fully predictable, they
can be largely suppressed when appropriate measures including the selection of
solvent flow direction are taken.
ACKNOWLEDGMENT
Financial support of project AS CR No. K4040110 is gratefully acknowledged.
GLOSSARY
a0
c
d
Dax
De
e
E
h
H
j
k
K
l
M
n
N
q
q
Specific particle surface, m2/m3
Fluid phase concentration, kg/m3
Particle size, m
Axial dispersion coefficient, m2/s
Effective diffusivity in particle core, m2/s
(=E/N) extraction yield, kg/(kg matrix)
Extract, kg
Axial coordinate, m
Extraction bed height, m
Flux across interface, kg/m3 s
Mass transfer coefficient, m/s
Distribution coefficient (equilibrium parameter) defined by Equation 3.27
Characteristic particle dimension, m
Solvent in extraction bed, kg
Number of mixers
Insoluble solid phase (matrix), kg
(=Q/N) specific solvent amount, kg/kg
(= Q /N) specific solvent flow rate, kg/kg s
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Modeling of Supercritical Fluid Extraction of Bioactives from Plant Materials
Q
Q
r
t
te
tr
T
u
V
xb
xc
xt
107
Solvent passed through extraction bed, kg
Solvent flow rate, kg/s
Grinding efficiency (fraction of broken cells in plant tissue)
Extraction time, s
(=tr/Γ) equilibrium extraction time, s
Mean residence time, s
Temperature, K
Interstitial velocity, m/s
Extraction bed volume, m3
Concentration in broken cells, kg/(kg matrix)
Mean concentration in intact cells (particle core), kg/(kg matrix)
Transition concentration (equilibrium parameter) defined by Equation
3.30, kg/(kg matrix)
Solute in untreated material, kg/(kg matrix)
(=x/xu) dimensionless solid phase concentration
Fluid phase concentration, kg/(kg solvent)
Solubility (equilibrium parameter), kg/(kg solvent)
Equilibrium fluid phase concentration, kg/(kg solvent)
(y/y0) dimensionless fluid phase concentration
Dimensionless axial coordinate
xu
X
y
ys
y+
Y
z
Greek letters
γ
Γ
ε
Θ
ρ
τ
Solvent-to-matrix ratio in extraction bed, kg/kg
(=γy0/(rxu)) initial free solute ratio, kg/kg
Void fraction
Dimensionless mass transfer resistance defined by Equation 3.18
Density, (kg solvent or matrix)/(m3 fluid or solid phase)
(= t/tr) dimensionless time
Subscripts
0
c
f
s
Initial conditions
Particle core
Fluid phase
Solid phase
REFERENCES
1. Sovová, H., Mathematical model for supercritical fluid extraction of natural products
and extraction curve evaluation, J. Supercrit. Fluids, 33, 35, 2005.
2. Sovová, H., Rate of the vegetable oil extraction with supercritical CO2: I. modelling
of extraction curves, Chem. Eng. Sci., 49, 409, 1994.
3. Marrone, C. et al., Almond oil extraction by supercritical CO2: experiments and
modelling, Chem. Eng. Sci., 53, 3711, 1998.
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Functional Food Ingredients and Nutraceuticals
4. Reverchon, E. et al., Supercritical fractional extraction of fennel seed oil and essential
oil: experiments and mathematical modeling, Ind. Eng. Chem. Res., 38, 3069, 1999.
5. Reverchon, E. and Marrone, C., Modeling and simulation of the supercritical CO2
extraction of vegetable oils, J. Supercrit. Fluids, 19, 161, 2001.
6. Reis-Vasco, E.M.C. et al., Mathematical modelling and simulation of pennyroyal
essential oil supercritical extraction, Chem. Eng. Sci., 55, 2917, 2000.
7. del Valle, J.M. and Aguilera, J.M., An improved equation for predicting the solubility
of vegetable oils in supercritical CO2, Ind. Eng. Chem. Res., 27, 1551, 1988.
8. Angus, S., Armstrong, B., and de Reuck, K.M., International Thermodynamic Tables
of Fluid State, CO2, Pergamon Press, Oxford, 1976, p. 37.
9. Lee, A.K.K., Bulley, N.R., and Meisen, A., Modelling of supercritical CO2 extraction
of canola oilseed in fixed beds, J. Am. Oil Chem. Soc., 63, 921, 1986.
10. Brunner, G., Mass transfer from solid material in gas extraction, Ber. Bunsenges.
Phys. Chem., 88, 887, 1984.
11. Ferreira, S.R.S. et al., Supercritical fluid extraction of black pepper (Piper nigrum
L.) essential oil, J. Supercrit. Fluids, 14, 235, 1999.
12. Reverchon, E., Supercritical fluid extraction and fractionation of essential oils and
related products, J. Supercrit. Fluids, 10, 1, 1997.
13. Goto, M., Sato, M., and Hirose, T., Extraction of peppermint oil by supercritical
carbon dioxide, J. Chem. Eng. Japan, 26, 401, 1993.
14. Goto, M. et al., Modeling supercritical fluid extraction process involving solute-solid
interaction, J. Chem. Eng. Japan, 31, 171, 1998.
15. Perrut, M., Clavier, J.Y., Poletto, M., and Reverchon, E., Mathematical Modeling of
sunflower seed extraction by supercritical CO2, Ind. Eng. Chem. Res., 36, 430, 1997.
16. Goodarznia, I. and Eikani, M.H., Supercritical carbon dioxide extraction of essential
oils: modeling and simulation, Chem. Eng. Sci., 53, 1387, 1998.
17. Beutler, H.J. et al., Einflu der Lösungsmittelführung auf den Hochdruck-ExtraktionsProze, Chem. Ing. Tech., 60, 773, 1988.
18. Beutler, H.J., Lenhard, U., and Lürken, F., Erfahrungen mit der CO2-Hochdruckextraktion auf dem Gebiet der Fettextraktion, Fat Sci. Technol., 90, 550, 1988.
19. Barton, P., Hughes, R.E., Jr., Hussein, M.M., Supercritical carbon dioxide extraction
of peppermint and spearmint, J. Supercrit. Fluids, 5, 157, 1992.
20. Sovová, H., Kuera, J., and Jeû, J., Rate of vegetable oil extraction with supercritical
CO2 : II. extraction of grape oil, Chem. Eng. Sci., 49, 415, 1994.
21. Dams, A., Stoffübergang bei der überkritischen Extraktion im Festbett, Chem. Ing.
Tech., 61, 712, 1989.
22. Stüber, F. et al., Supercritical fluid extraction of packed beds: external mass transfer
in upflow and downflow operation, Ind. Eng. Chem. Res., 35, 3618, 1996.
23. Berna, A. et al., Supercritical CO2 extraction of essential oil from orange peel: effect
of the height of the bed, J. Supercrit. Fluids, 18, 224, 2000.
24. Reverchon E., Mathematical modeling of supercritical extraction of sage oil, AICHE
J., 42, 1765, 1996.
25. Villermaux, J., Chemical engineering approach to dynamic modelling of linear chromatography, J. Chromatogr., 406, 11, 1987.
26. del Valle, J.M., Napolitano, P., and Fuentes, N., Estimation of relevant mass transfer
parameters for the extraction of packed substrate beds using supercritical fluids, Ind.
Eng. Chem. Res., 39, 4720, 2000.
27. Roy, B.C., Goto, M., and Hirose, T., Extraction of ginger oil with supercritical carbon
dioxide: experiments and modeling, Ind. Eng. Chem. Res., 35, 607, 1996.
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28. Akgün, M., Akgün, N.A., and Dincer, S., Extraction and modeling of lavender flower
essential oil using supercritical carbon dioxide, Ind. Eng. Chem. Res., 39, 473, 2000.
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4
Biochemical Reactions in
Supercritical Fluids
ˇ
Zeljko
Knez, Chiara G. Laudani, Maja Habulin,
ˇ
Mateja Primozič
University of Maribor
CONTENTS
4.1
4.2
4.3
Introduction ..................................................................................................111
Enzyme Catalysis in Nonconventional Media ............................................114
Enzyme Catalysis in Supercritical Fluids (SCFs) .......................................117
4.3.1 Enzyme Stability in Supercritical Fluids.........................................118
4.3.2 Effect of Pressure.............................................................................119
4.3.3 Number of Pressurization-Depressurization Steps ..........................121
4.3.4 Effect of Temperature ......................................................................122
4.3.5 Effect of Water Activity ...................................................................122
4.3.6 Enzyme Reactors..............................................................................124
4.3.6.1 Process Schemes and Downstream
Processing Schemes..........................................................124
4.3.7 Processing Costs ..............................................................................126
4.4 Conclusion....................................................................................................126
References..............................................................................................................127
4.1 INTRODUCTION
Enzymatic catalysis has gained considerable attention in recent years as an efficient
tool for synthesis of natural products, pharmaceuticals, fine chemicals, and food
ingredients.
The production of fine chemicals results in the generation of considerable volumes of waste, as the syntheses generally include a number of steps. The yield of
each of these steps is usually 60% to 90%, but 10% is not unusual. Based on these
data we can conclude that typically 1 kg of end product leads to the generation of
15 kg of wastes or more. Most generated wastes are solvents and by-products from
solvents and intermediates. Therefore, ideally several reactions should be performed
in water or in supercritical fluids.
111
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Functional Food Ingredients and Nutraceuticals
The high selectivity and mild reaction conditions of enzymatic transformations
make them an alternative to the synthesis of complex bioactive compounds, which
are often difficult to obtain by standard chemical routes. However, the majority of
organic compounds are not highly soluble in water, which was traditionally perceived
as the only suitable reaction medium for the application of biocatalysts. The realization that most enzymes can function perfectly well under nearly anhydrous conditions and, in addition, display a number of useful properties, for example, highly
enhanced stability and different selectivity, has dramatically widened the scope of
their application to organic synthesis. Another great attraction of using organic
solvents rather than water as a reaction solvent is the ability to perform synthetic
transformations with relatively inexpensive hydrolytic enzymes. Generally, in vivo,
the synthetic and hydrolytic pathways are catalyzed by different enzymes. However,
elimination of water from the reaction mixture enables the “reversal” of hydrolytic
enzymes and thus avoids the use of the expensive cofactors or activated substrates
that are required for their synthetic counterparts. Water is the most common solvent
for biochemical reactions but in a biotechnological perspective, there are a lot of
advantages of conducting enzymatic conversions in monophasic organic solvents as
opposed to water.1 Some are listed below:
•
•
•
•
High solubility of most organic (nonpolar) compounds in nonaqueous
media
Ability to carry out new reactions impossible in water because of kinetic
or thermodynamic restrictions
Reduction of water-dependent side reactions
Insolubility of enzymes in organic media, which allows their easy recovery
and reuse
However, the use of organic solvents can be problematic because of their toxicity
and flammability, and also there are increasing environmental concerns. As a result,
supercritical fluids (SCFs) have attracted much attention in recent years as an
alternative to organic solvents for enzymatic reactions.
The present research activity will be focused on the development of selective
methods for the production of polyfunctional molecules by enzymatic reactions in
supercritical carbon dioxide (SC-CO2). Among all the possible SCFs, carbon dioxide
is largely used. The use of SC-CO2 instead of organic solvents in biocatalysis presents
several additional advantages. It performs mainly as a lipophilic solvent, nontoxic,
nonflammable, and cheap. Supercritical CO2 has been most frequently used as a
medium for biotrasformations: its critical pressure (73.8 MPa) is “acceptable” and
temperature (31.1°C) is consistent with the use of enzymes and labile solutes. It has
the GRAS (Generally Regarded As Safe) status. In addition, its “naturalness” is
greatly appreciated by the food and healthcare related industries. The use of SCFs
as solvents for enzymatic transformations is a relatively new area of research, which
is expected to expand in the future.
Close to the critical point, small changes in temperature or pressure can produce
large changes in density and solvation ability of SCFs. This attribute of SCFs can
be fruitfully exploited and integrated in biotransformation and downstream
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processing steps in a single bioreactor. Beyond the critical point, both phases are
indistinguishable, and the fluid is monophasic and occupies all the vessel volume.
It can be described as a dense gas or an expanded liquid. Generally, SCFs exhibit
liquid-like density and therefore have good solvating power, but they retain gas-like
compressibility. Consequently, it is possible to control their solvating power by
changing the pressure and/or temperature, with a continuous transition from a good
to poor solvent. Moreover, low viscosity and high diffusion coefficients of these
fluids enhance mass transport and reaction kinetics. These unique properties of SCFs
enable one to design efficient integrated processes by coupling an enzymatic reaction
with subsequent fractionation and product recovery steps. Molecules in the supercritical (SC) phase are not uniformly distributed in space, but the solvent molecules
aggregate around the solute through solvent-solute intermolecular interactions forming clusters, where the aggregated molecules are in dynamic equilibrium with free
solvent molecules. Thus, the solvation depends strongly on the density of the SCFs
and differs from that in the liquid solution or gas phase.2 When catalytic reactions
are performed in SCFs, the outcome of the reactions can be affected in a number
of ways. In general, replacement of conventional liquid solvents by SCFs can
increase the rate and tune the selectivity of reactions for the following reasons:3
•
•
•
Rapid diffusion of solutes or weakening of the solvation around reacting
species facilitates the reactions and sometimes changes the reaction pathway
Local clustering of solutes or solvents resulting in an appreciable increase
in the local concentration of substrate (and catalyst) causes acceleration
of the reaction
Reduction or increase in the cage effect often affects the reaction performance of rapid chemical transformations such as radical reactions
In addition to these benefits, SCF chemistry, as a reaction media it poses economical,
technical, environmental, and health advantages. The high volatility of CO2 allows
it to be completely and easily removed from the product, resulting in an overall
“solvent-free” reaction. By using SC-CO2 , an integrated production process can be
performed, because it can act as solvent for the reaction and also as a separation
medium. The variable solvating power of SC-CO2 and other SCFs facilitates the
integration of biocatalytic and downstream processing steps in a single robust bioreactor. The main drawback of SC-CO2 is that it has limited solvating power with
respect to polar compounds. This is a serious limitation for biotechnological applications where most natural molecules of interest (e.g., alkaloids, carotenoids, phenols, proteins, and sugars) are only sparingly soluble in SC-CO2. In this case, a polar
cosolvent or so-called entrainer, such as acetone, ethanol, methanol, or water, is
added in order to increase the polarity of the medium and to solubilize the target
solute via the formation of hydrogen bonds. Typically, cosolvents are added to the
SCF at moderate concentrations of less than 10 mol %.4 In a batch reactor, the
cosolvent can be added directly into the reactor prior to pressurization, whereas in
a continuous process, the addition should be made to the CO2 inflow via a liquid
pump to deliver a constant flow rate at the operating pressure. However, the use of
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another component in the system further increases the complexity and may also
complicate downstream processing. Moreover, the solubility enhancement effect of
a cosolvent is usually limited in the case of very polar compounds. Two alternative
methods have been developed for some specific cases. To solubilize polyols (e.g.,
glycerol and sugars) has been proposed5 to form a hydrophobic complex between
the polyol and phenylboronic acid (PBAC), which is much more soluble in the SC
phase. This method was used to perform the esterification of glycerol and sugar with
oleic acid in SC-CO2. Another method involves the adsorption of polar substrates
onto a solid hydrophilic support such as silica gel. Compared with the former, this
approach is more general because it is not necessary to have two vicinal hydroxyl
groups in the substrate molecule.5 In addition, recent advances in the understanding
of the chemical properties of materials that are soluble in CO2 have permitted the
development of novel surfactants that allow dissolution of both hydrophilic and
hydrophobic materials in CO2.
The use of SCFs decreases the mass transfer limitations because of the high
diffusivity of reactants in the SC medium, the low surface tension, and because of
the relatively low viscosity of the mixture. The Schmidt number, Sc = η/ρ⋅D (where
η is dynamic viscosity, D is diffusivity, and ρ is density), for CO2 at 200 bars is 45
times lower than for water at 1 bar and 20°C. High diffusivity of SCF and low
surface tension lead to reduced internal mass transfer limitations for heterogeneous
chemical or biochemical catalysis.
One of the main advantages of the use of dense gases as a solvent for enzymecatalyzed reactions is the simple downstream processing. The physico-chemical
properties of dense gases are determined by their pressure and temperature, and are
especially sensitive near their critical point. By reducing the solvent-power of a
dense gas in several stages, fractionation of the product and unreacted reactants is
possible. Fractionation is also possible by extracting the mixture, usually with the
same dense gas as used in the reaction, but under different process conditions. In
all downstream processing schemes, various particle formation techniques or chromatographic techniques can be integrated.
4.2 ENZYME CATALYSIS IN NONCONVENTIONAL
MEDIA
The breakthrough of biocatalysis in nonaqueous media started in the early 1980s.6–10
Nowadays, it is well established that many enzymes, such as lipases, can remain
active and stable in pure organic solvents. Changing the hydrophobicity of predominantly aqueous media through addition of organic solvents causes the hydrophobic
effects, which keep the hydrophobic residues buried in the core of the proteins and
folded in an aqueous environment where enzymes are kinetically trapped in their
native structure in organic solvents.11–14 In addition, in the media of low water
content, enzyme inactivation, caused by hydrolysis of peptide bonds and deamidation
of asparagine and glutamine residues, is reduced.11 Indeed, many times enzymes are
more stable in organic solvents than in water. For instance, increased thermal stability
in dry organic solvents with substantial increase in enzyme half-life at 100°C
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TABLE 4.1
Advantages of Biocatalysis in Nonaqueous
Media
Increased substrate solubility
Simplified recovery of biocatalyst
Shift to synthetic reactions
Mild reaction conditions and minimization of side-reactions
Environmentally benign catalyst
High selectivity
Simplified work-up of products
Avoids microbial contaminations
compared to water has been observed.9,15 There are a number of advantages of using
enzymes in organic solvents. These are listed in Table 4.1.
Many substrates that are insoluble in water can be dissolved by organic solvents.
Enzymes are often insoluble, an advantage that simplifies their recovery and reuse
many times and is economically very important. A change from an aqueous environment can favor a shift in the equilibrium, enabling synthetic reactions to be
achieved with hydrolytic enzymes. Moreover, the unique selectivity and activity of
enzymatic reactions is achieved under mild reaction conditions. The enzyme instability in harsher reaction conditions is common in industrial processes, which can,
of course, be a disadvantage. The search for enzymes from various extremophilic
organisms may, however, result in biocatalysts that can withstand more extreme
conditions.16,17 Furthermore, enzymes are environmentally benign and are, unlike
metal catalysts, completely degraded in nature. Although some enzymes do display
perfect selectivity, many others can receive a broad range of unnatural substrates
still with high chemo-, regio- or enantioselectivity. Additionally, the use of an organic
solvent often simplifies work-up procedures and avoids microbial contamination of
the reaction medium.11,18
The conventional notion that enzymes are only active in aqueous media has long
been discarded, thanks to the numerous studies documenting enzyme activities in
nonaqueous media, including pure organic solvents and supercritical fluids (SCFs).
Enzymatic reactions in nonaqueous solvents offer new possibilities for producing
useful chemicals (emulsifiers, surfactants, wax esters, chiral drug molecules,
biopolymers, peptides and proteins, modified fats and oils, structured lipids, and
flavor esters). According to conventional notion, enzymes are active only in water.
Historically, enzymatic catalysis has been carried out primarily in aqueous systems.
Although water is a poor solvent for preparative organic chemistry, it is the unique
specificity of enzymes that drew the interest of chemists who were seeking highly
selective catalytic agents. Experiments to place enzymes in systems other than
aqueous media date back to the end of the nineteenth century.19–23 Initial studies
considered the addition of small quantities of water-miscible organic solvents like
ethanol or acetone to aqueous enzyme solutions ensuring high availability of water
to retain the catalytic activity of enzymes. Then, the biphasic mixtures (aqueous
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enzyme solution emulsified in a water-immiscible solvent such as isooctane or
heptane) were used, in which the substrates from the organic phase diffuse into the
aqueous phase, that undergoes enzymatic reaction and the products diffuse back.
The size of water droplets may be reduced to facilitate mass transfer resulting in
the formation of microemulsions or reverse micelles, whose stabilization is achieved
by adding surfactants.24,25
Developments in using enzymes in nearly nonaqueous solvents containing traces
of water have stimulated research in achieving various kinds of enzymatic transformations.26–34 Enzymatic reactions in nonaqueous solvents offer numerous possibilities for the biotechnological production of useful chemicals using reactions that are
not feasible in aqueous media. These reactions include chiral synthesis or resolution;35–38 production of high-value pharmaceutical substances;39–43 modification of
fats and oils;44 synthesis of flavor esters and food additives;45–51 production of
biodegradable polymers,52 peptides, proteins, and sugar-based polymers.53 In nonaqueous solvents, hydrolytic enzymes could undergo synthetic reactions while they
also exhibit altered selectivities,11 pH memory,10,15,54 increased activity and stability
at elevated temperatures,9,55 regio-, enantio- and stereoselectivity,44,56 and may also
be affected by their water activity.57 Currently, there is a considerable interest in the
use of enzymes (particularly lipases, esterases, and proteinases) as catalysts in
organic synthesis.18,44,56,58–62
Five major technological advances are believed to have significantly influenced
the industry for adopting enzymatic biotransformations:63 (1) the development of
large-scale downstream processing techniques for the release of intracellular
enzymes from microorganisms; (2) improved screening methods for novel biocatalysts;64–68 (3) the development of immobilized enzymes; (4) biocatalysis in organic
media; and most recently, (5) recombinant-DNA (r-DNA) technology to produce
enzymes at a reasonable cost. There seems to be no agreement as to why the
biocatalysis in organic media could not have taken off earlier.69–71 Perhaps the
traditional belief that most enzymes are incompatible with most organic syntheses
in nonaqueous media, which also poses a psychological hurdle. Also, until recently,
there was no demand for enantiopure compounds, and hence, no enzymes needed
to be used. The establishment of industrial processes,72,73 and the realization that
most enzymes can function well in organic solvents,9,10,74,75 have heightened interest
in the use of enzymes. Also, the need for enantiomerically pure drugs is driving the
demand for enzymatic processes. This combined with the discovery of strikingly
new properties of enzymes in organic solvents has led to the establishment of organic
phase enzyme processes in industry.56,60
Enzymes occupy a unique position in synthetic chemistry due to their high
selectivities and rapid catalysis under ambient reaction conditions. Nevertheless,
synthetic chemists have been reluctant to employ enzymes as catalysts, because most
organic compounds are water insoluble, and the removal of water is tedious and
expensive. The fact that enzymes are stable, and in some cases, improve their high
specificity in near anhydrous media, has dramatically changed the prospects of
employing enzymes in synthetic organic chemistry. The problems that arise for most
biotransformations are low solubility of reactants and products, and limited stability
of biocatalysts. Carrying out reactions in an aqueous-organic two-phase system
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Biochemical Reactions in Supercritical Fluids
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would be a solution to overcome the first problem. This is not always possible due
to the limited stability of enzymes at liquid-liquid interface or in organic solvents.
Hence, other approaches are necessary. These include addition of complexing agents
such as dimethylated cyclodextrins or adsorbing materials like Amberlite™ XAD7 resins (Eli Lilly, Indianapolis, IN, USA), use of membrane-stabilized interface
(Sepracor™, Marlborough, MA, USA) and continuous extraction of reaction products (Forshungs-zentrum-Julich, Julich, Germany). The catalyst’s stability can be
increased using a variety of methods including the addition of antioxidants (e.g.,
dithiothreitol), immobilization, cross-linking, separation from deactivating reagents,
variation of reaction conditions, and by genetic engineering.76
Solvent systems used as the reaction media for enzymatic catalysis may be
categorized as: (1) aqueous; (2) water: water-miscible (monophasic aqueous-organic
system); (3) water: water-immiscible (biphasic aqueous-organic system); (4) nonaqueous (monophasic organic system); (5) anhydrous; (6) supercritical fluids; (7)
reversed micelles; (8) solvent-free systems; (9) gas phase; and (10) ionic liquids.
4.3 ENZYME CATALYSIS IN SUPERCRITICAL FLUIDS
(SCFs)
During the last decade, the tremendous potential of enzymes as a practical catalyst
for chemical processes in nonaqueous environments has been well recognized. The
use of biocatalysts in organic solvents offers many advantages over using pure water.
Among these media, SCFs, such as carbon dioxide (SC-CO2), exhibit properties
similar to organic solvents, but with the additional capacity of enhancing transport
phenomena (due to their high diffusivities) and facilitate reaction products separation
by tuning solvent power, which makes them more attractive to be used as “greendesigner” solvents.77,78 The interest of using biocatalysts in SC-CO2 has been growing
rapidly in recent years, mainly in industrial and pilot plant applications.79
The growing interest in SCF technology results from the attractive possibilities
offered by this technique: processing at moderate, usually ambient temperatures, use
of nontoxic, nonflammable, and environmentally acceptable solvents (usually pressurized CO2) and easy-to-change solvent power (not possible with conventional
organics). For example, SCFs at temperatures and pressures slightly above the critical
points (e.g., 31°C and 73.8 MPa for CO2), exhibit unique combined properties:
liquid-like density (and hence solvent power) and high compressibility, very low
viscosity, and high diffusivity. The first two properties make the solvent power
controllable by changing pressure and/or temperature, while low viscosity and high
diffusivity markedly enhance mass transport phenomena and hence kinetics of a
process. However, enzymes are not soluble in SCFs, therefore enzymatic catalysis
in SCFs would always be heterogeneous.
The use of enzymes as catalysts in nonaqueous media has been described in
scientific literature since the middle of the 1980s.10 Organic media offer certain
advantages over aqueous media: stabilization of enzymes, dissolution of hydrophobic
compounds, and the feasibility of shifting thermodynamic equilibrium toward the
synthesis of esters and amides (e.g., in the case of hydrolytic enzymes). Certain
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SCFs, such as CO2, may prove an interesting alternative inasmuch as they exhibit
properties similar to organic solvents and their solvent power, which is dependent
on the specific density (and hence easily controllable by regulation of pressure and
temperature), may be advantageously used in the recovery of products. This opens
the way for an integrated production of product recovery processes.
The use of SCFs as media for enzymatic reaction was first proposed in the
middle of the 1980s by Randolph et al.,80 Hammond et al.,81 and Nakamura et al.82
The aim of the initial studies was to demonstrate that enzymes are active in SCCO2. Randolph et al.80 showed that alkaline phosphatase and cholesterol oxidase are
active in SC-CO2; Hammond et al.81 demonstrated the same for polyphenyl oxidase;
and Nakamura et al.82 carried out successful transesterification reactions using lipase.
Successively, Randolph et al.83 examined the effect of aggregation of cholesterol on
cholesterol oxidase activity and found that the addition of cosolvents (entrainers)
promotes aggregation and thus resulted in an increase in reaction rate proportional
to the degree of aggregation. Recently, the benefit of using SCFs for enzymatic
reactions has been demonstrated by Mori and Okahata84 and Kamat et al.85,86 and
Chaudhary et al.,87 for example, for improved reaction rates, control of selectivities
by pressure, and so forth. Some examples of enzymatic reactions are shown in Figure
4.1 and Figure 4.2.
Most of the research published to date dealt with two problems: (1) conformation
and stability in the supercritical environment (mainly CO2) and the effect of pressure
on reaction rate; (2) the effect of water/moisture content on the activity of enzymes.
4.3.1 ENZYME STABILITY
IN
SUPERCRITICAL FLUIDS
The use of enzymes in supercritical fluids is full of potential problems. For example,
the number of parameters that influences the stability of the enzymes increases
dramatically when using supercritical fluid. This is the reason why, up to now, no
prediction could be made on whether enzymes are stable under supercritical conditions or if the equivalent of even higher activity and selectivity is available compared
OH O
.
OH O
OH O
OEt
Pseudomonas SP
OH
OEt +
SC CO2
FIGURE 4.1 Reaction scheme of the enantioselective hydrolysis of HPAE in SC-CO2.88
R
R1
+
H
O
O
OH
C
CH3 C
O
C
CH3
Pseudomonas SP
SC CO 2
+
C
R
1
H
OH
R
OH
R
C
R
1
+ CH3
O
C
OH
H
FIGURE 4.2 Reaction scheme of the enzymatic esterification of secondary alcohols in
SC-CO2.89
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Biochemical Reactions in Supercritical Fluids
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with the reactions in organic solvents. In the following sections the influence of
parameters on the enzyme stability will be discussed.
Early investigations80–82 demonstrated that certain enzymes are active in SCCO2. Randolph et al.83 first studied the conformation of several spin-labeled variants
of cholesterol oxidase in SC-CO2 and concluded that these proteins were not influenced by the SC-CO2 environment; a similar result has recently been obtained for
lipase.90 In contrast, Kasche et al.91 reported that α-chymotrypsin, trypsin, and
penicillin amidase were partially denaturated by SC-CO2 and suggested that the
decompression process led to their denaturation, but no in situ measurements were
conducted to substantiate this suggestion. Most recently Zagrobelny and Bright92
carried out a more detailed examination of the same problem. The conformation of
trypsin in SC-CO2 was studied at the pressure range of 5 to 25 MPa and monitored
the conformational changes of trypsin in situ as a function of pressure. Their results
clearly demonstrate that: (1) significant changes in protein conformation can be
induced by SC solvents; (2) most of the conformational changes occur during
compression; (3) the native trypsin conformation is only slightly more stable than
the unfolded form.
Performance characteristics, specific rates of conversion, and yield factors are
essential for rating any technological process. On the whole, enzymatic reactions in
SC-CO2 proceed at rates similar to those of organic solvents such as n-hexane93,94
and cyclohexane.90 As well as ensuring similar rates of processing and enzyme
stability, the SC technology offers important advantages over organic solvent technology, such as ecological friendliness and product fractionation, which can easily
be linked with direct micronization and crystallization from SC-CO2 by fluid expansion. In addition, CO2 does not usually oxidize substrates and products, allowing
the process to be operated at a temperature of 40°C. Although many enzymes are
stable in SCFs, one should pay considerable attention to finding the correct reaction
conditions for each substrate/enzyme/SCF system. Although successful reactions
have been reported with Subtilisin Carlsberg protease and Candida lipases in SCCO2, there is also evidence for their instability85,95,96 or the existence of a narrow
pressure range of activity.97,98 These enzymes are fairly stable in other SCFs such
as fluoroform, ethylene, ethane, propane, and sulphur hexafluoride.85 Immobilized
Mucor miehei lipase appears to be very stable in SC-CO2; it is a monomeric enzyme
with three stabilizing disulfide bonds,99 which may play a role in maintaining its
activity in SC-CO2. Nevertheless, one of the most important advantages of using the
SCFs as reaction media is that upstream processing after reaction can be greatly
simplified as the technique is easily combined with other unit operations.
4.3.2 EFFECT
OF
PRESSURE
The influence of the system pressure on the stability of enzymes is not so significant
up to 30 MPa. This is of great advantage because it means that the solvent-power
of the supercritical fluid can be adjusted for reaction performance. On the one hand,
the solubility of substances increases with higher pressures because of a higher fluid
density and this is essential to bring the initial products in the reactor and remove
the end products from the reactor. On the other hand, higher pressure normally
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results in higher reaction rates. Therefore a pressure increase is in most cases positive
for enzymatic reactions.
An isothermal change of pressure of SCFs may change the reaction rate by
changing either solubility or the reaction rate constant. A pressure increase improves
solubility, thereby increasing production rates, and this effect is most pronounced
in the near-critical region. Certain enzymes show considerable apparent pressure
activation.100 Pressure can modify the catalytic behavior of enzymes by changing,
for example, the rate-limiting step101 or modulating the selectivity of the enzyme.102
If an enzyme is stable in a supercritical fluid, its stability is usually not influenced
by pressure ranges of up to 30 MPa. Conversely, reaction rates may be influenced
by pressure. In most cases a pressure increase acts positively for enzymatic reactions
or there are no changes in the reaction rates. Pressure-induced deactivation of
enzymes takes place mostly at pressure exceeding 150 MPa. Reversible pressure
denaturation mostly occurs at pressures below 300 MPa, and higher pressures are
required to cause irreversible denaturation.103
The effect of pressure on the reaction rate constant has not yet been determined,
but the effect on the overall production rate has been examined in several papers.
Erickson et al.104 carried out transesterification of triglycerides, using lipase from
Rhizopus arrhizius. The reactants were trilaurin and palmitic acid and the pressure
ranged from 10 to 30 MPa. A strong negative effect of pressure increased the rate
of palmitic acid incorporation into triglyceride was detected, especially in the nearcritical region. The interesterification of trilaurin and myristic acid, catalyzed by
lipase, was investigated by Miller et al.90 in the pressure range of 6 to 11 MPa. The
interesterification rate and the overall rate (based on total trilaurin conversion)
increased with increase in pressure; however, the interesterification rate increased
much more rapidly than the overall rate, indicating that the selectivity of the reaction
for interesterification over hydrolysis improved at higher pressures. The operational
stability of enzymes in SC-CO2 is of crucial importance from the point of view of
application. Miller et al.90 measured the interesterification rate over 80 h of continuous operation and observed no loss of activity of lipase. Cholesterol oxidase is
stable at 10 MPa and 35°C for at least 50 h.83 Pressure has also been found to have
little effect on the stability of lipase from Mucor miehei in the range from 13 to 18
MPa, causing only 10% loss of activity93 after six days at 40°C, unlike temperature,
which contributed to a 20% loss at 60°C. Additionally, in some cases a negative
effect of pressure on the catalytic activity of biocatalysts in compressed gases may
be observed. The catalytic efficiency of subtilisin Carlsberg suspended in compressed
propane, near-critical ethane, near-critical carbon dioxide, and tert-amyl alcohol at
constant temperature and pressure up to 30 MPa and fixed enzyme hydration was
lowered.105 In near-critical fluids an increase in pressure of only 20 MPa caused a
sixfold decrease in the catalytic efficiency of subtilisin in CO2.
In SC-CO2 the formation of carbamates are responsible for lower enzymatic
activity in this medium. Carbamates are the product of the reaction between basic
free amino groups in the enzymes and acidic SC-CO2.95 On the other hand, lysozyme
lipase unfolded and partially oligomerized in moist SC-CO2 at 80°C and its denaturation was not caused by interaction with SC-CO2 but by heating the protein in
the presence of water, as found by Weder.106
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One of the advantages of using SCFs as enzymatic reaction media is separation
of products from the reaction mixture by changing the pressure of the supercritical
fluid. With respect to the facts mentioned previously, the solvent power of the SCF
can be adjusted for running reactions, and products can be easily removed from the
reactor. When the lipid-coated lipase was employed in SC-CHF3 the enzyme
activity107 could be switched on and off by adjusting the pressure or temperature of
the SC-CHF3.
The effect of pressure on the extent of conversion and product composition of
the enzyme-catalyzed hydrolysis of canola oil in SC-CO2 was investigated using
lipase from Mucor miehei immobilized on macroporous anionic resin.108 A conversion of 63% to 67% (triglyceride disappearance) was obtained at 24 to 38 MPa.
Monoglyceride production was favored at 24 MPa. The amount of product obtained
was higher at 24 to 38 MPa due to its enhanced solubility in SC-CO2.
4.3.3 NUMBER
OF
PRESSURIZATION-DEPRESSURIZATION STEPS
The influence of pressurization-depressurization steps in batch reactors on the
enzyme activity is of importance to many researchers.109 Pressurizing an enzyme
does not play an important role, but depressurization is usually the step that influences residual enzyme activity. The supercritical fluid permeates through the
enzymes by diffusion, a process that is relatively slow. After a certain time the
enzymes are saturated with the SCFs. If the expansion is too fast and the fluid
diffuses out of the enzyme slowly, this causes a higher fluid pressure in the enzyme
than in the system. The pressure difference results in cell-cracking, where the cell
membranes are broken by the resulting pressure inside the cells. This causes the
unfolding of the enzymes and the loss in tertiary structure, which is required for
their activity and selectivity. Experiments have shown that depressurization from
supercritical CO2 conditions is much smoother than entering the two-phase region
and expanding the gas of the fluid.110 This is caused by a change in density which
is continuous in supercritical conditions. The expanding liquid CO2 causes evaporation of the fluid accompanied by a large change in density, and this volume
expansion causes the unfolding of the enzyme.
Depressurization is important when using the benefit of SCFs for simple downstream processing. In this case, by operating a cascade of depressurizations (with a
possible change in temperature), product fractionation can be achieved.111 In order
to have successful industrial applications, enzymes as biocatalysts must retain their
activity for a considerable period of time. The activity of the lipase from Candida
antarctica for the production of isoamyl acetate in SC-CO2 was studied by Romero
et al.111 in a tubular reactor. The yield of isoamyl acetate was 100% for 30 days,
and then slowly decreased. Habulin et al.112 found similar results with immobilized
Rhizomucor miehie lipase, reporting a 4% decrease in conversion after one month
of treatment.
Cholesterol oxidase from different sources can exhibit different stabilities in SCCO2.83 The cholesterol oxidase from Gloecysticum retained its activity for three days
and the one from Streptomyces sp. for only one hour. In some cases the half-life of
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the biocatalysts can be increased by adjusting pressure.113 The half-life of α-chymotrypsin increased when the pressure increased from 8 to 15 MPa.
4.3.4 EFFECT
OF
TEMPERATURE
The effect of temperature is much more significant than the pressure effect. For
enzyme stability, a temperature increase above certain levels, depending on the
enzyme sources, results in the deactivation of the enzyme. This limitation places the
temperature limits on the extraction process. Normally, at higher pressure levels an
increase in temperature results in higher solubilities of substances in SCFs because
the increase in the vapor pressure of the compounds to be dissolved overcomes the
reduction in density.
Reaction rate also increases at higher temperatures, although enzyme deactivation at higher temperatures may occur. At the moment no correlation between
temperature and the stability of the different types of enzymes is available.
Lipase from Aspergillus niger was incubated in SC-CO2 at 30 MPa and different
temperatures.114 Its residual activity was optimal at 323K. At higher temperatures a
rapid decrease in activity was observed. This thermal deactivation was connected to
changes in the water distribution in the system.
In microaqueous media, including SCFs, thermal stability of biocatalysts can
also be improved. Reaction rate for subtilisin protease-catalyzed reactions increased
by 80% in SC-CO2.115 The optimal temperature for esterification between n-butyric
acid and isoamyl alcohol, catalyzed by porcine pancreas lipase, increased from 313K
at atmospheric pressure to 323K in near-critical propane.116 The improved stability
of the lipase in the low-water-content environment is a consequence of a well-known
fact that reactions, which may be responsible for the denaturation of enzymes, are
hydrolytic and therefore require water.117
Thermal activation and deactivation (energy of activation and deactivation
enthalpy, respectively) may be determined from the Arrhenius diagram. The ratio
between the amounts of inactive and active forms of the enzyme at a temperature,
where the greatest initial reaction rate is observed, is expressed with a deactivation
constant. If the activation and deactivation enthalpy values are high this indicates
that enzyme activity is very dependent on temperature.
4.3.5 EFFECT
OF
WATER ACTIVITY
Water concentration in the reaction system is one of the most important factors that
influence activity of an enzyme. Water is crucial to enzymes and affects enzyme
action in various ways: by influencing enzyme structure via noncovalent binding
and disruption of the hydrogen bonds; facilitating reagent diffusion; and influencing
the reaction equilibrium.118
The subtilisin Carlsberg catalyzed transesterification of N-acetyl phenylalanine
methyl ester,119 N-acetyl phenylalanine ethyl ester,120 N-trifluoroacetyl phenylalanine
methyl ester,121 and N-trifluoroacetyl phenylalanine ethyl ester122 was studied in
SC-CO2. The water content of the reaction affected the reactivity of the system; for
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Biochemical Reactions in Supercritical Fluids
123
the transesterification of the methyl esters with ethanol the optimum concentration
of water was determined to be about 0.74 M, while during the transesterification of
the ethyl esters123 with methanol it was about 1.3 M.
Use of an enzyme in pure SC-CO2 may lead to removal of the water, which is
included or bonded to the enzyme. The quantity of the removed water is temperature
and pressure depended and if too high may lead to enzyme denaturation and loss
of activity.
The solubility of water in SC-CO2 can be calculated by Chrastil equation124
a

c = ρk ⋅ exp  + b 
T

where c is the solubility (g/L), ρ is the CO2 density (g/L), and T the temperature
(K). The calculated water parameters are k = 1.549, a = −2826.4, and b = –0.807.
Water is important in the supercritical fluid because water-saturated CO2 causes
the inhibition of enzymes and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Enzymes require a
specific amount of water to maintain their active conformation. Enzyme stability
generally decreases with increasing water concentration, whereas their activities
require some water to be present. Therefore, the water content has to be optimized
to find the best balance between enzyme life and activity. If water acts as a substrate
for an enzymatic reaction, the optimal parameters for continuous reaction require
among other things enough moisture to compensate for complete reaction and
sufficient enzyme moisture for losses due to water solubility in SC-CO2.125,126 However, if the water concentration in the supercritical medium is too high or if it is a
product of the reaction, the increase in moisture may cause enzyme deactivation.
Not surprisingly, all studies published so far have pointed out the strong influence
of moisture on enzymatic activity and reaction rates. Optimum water content in the
support was estimated at 10 wt %, irrespective of the operating conditions,94 but this
value may be contested in view of other reports.127 To prevent dehydration of the
enzyme, the fluid in contact with the protein must contain water. The most hydrophilic hydrocarbons (e.g., hexane) dissolve 0.01% water, but SC-CO2 may dissolve
as much as 0.3% to 0.35% water. However, it is not the solubility of water itself but
the partition of water between the enzymatic support and the solvent (SC-CO2),
which matters. Marty et al.94 carried out an extensive analysis of the partition of
water between the enzymatic support and SC-CO2 as a function of pressure and
temperature. They found that increasing temperature and pressure had a negative
effect on the adsorption of water by the support. This is opposite to the results
obtained by gas adsorption, which suggests that the solvation effects predominate
over the vapor pressure effects. The same authors also extensively studied the
influence of ethanol (entrainer) content in SC-CO2 and found that ethanol has a
strong “drying” effect on the enzyme support; indeed, the more hydrophilic the fluid,
the more pronounced is the dehydration of the enzymes. Increasing water content,
above the optimum level, adversely affects the overall performance. This appears to
be related to hydrophilic hindrance of the hydrophobic substrate on its way to the
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Functional Food Ingredients and Nutraceuticals
active sites on the enzyme, and eventually makes the thermodynamic equilibrium
less favorable.128 Chulalaksananukul et al.129 measured the residual activity of lipase
from Mucor miehei after a day in SC-CO2 at a temperature range of 40°C to 100°C
and at various water concentrations. As the temperature increased, the enzyme
molecule at first unfolded reversibly and then underwent one or more of the following
reactions: formation of incorrect or scrambled structures, cleavage of disulfide bonds,
deamination of trypsine residues, and hydrolysis of peptide bonds. Each process
requires water and is therefore accelerated with increasing water concentration.
4.3.6 ENZYME REACTORS
4.3.6.1 Process Schemes and Downstream Processing Schemes
Batch-stirred tank, extractive semibatch, recirculating batch, semicontinuous flow,
continuous packed-bed, and continuous-membrane reactors have used dense gases
as solvents.
4.3.6.1.1 Batch-Stirred-Tank Reactor
Batch-stirred-tank reactors are usually used for screening enzymatic reactions in
dense gases.130,131 The design of the system is shown in Figure 4.3. Initially, the
substrates are pumped into the reactor and then the enzyme-preparation is added.
Finally, dry gas is pumped into the reactor, up to the desired pressure. The initial
concentration of the reactant must never exceed its solubility limit in the gas.
T = const.
PI
P
2
Gas
1
FIGURE 4.3 Design of experimental batch-stirred-tank apparatus for synthesis under high
pressure. 1 magnetic stirrer and heater; 2 reactor; P high-pressure pump; PI pressure indicator.
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Biochemical Reactions in Supercritical Fluids
125
PI
PI
HPP
PI
TIR
TIR
H
Gas
3
FI
HPP
HPP
4
1
2
FIGURE 4.4 Design of experimental continuous packed-bed apparatus for synthesis under
supercritical conditions. 1, 2 substrates; 3 reactor; 4 separation column; HPP high-pressure
pump; PI pressure indicator; TIR temperature indicator and regulator; H heat exchanger; FI
flow indicator.
4.3.6.1.2 Continuous Packed-Bed Reactor
A continuous packed-bed reactor is presented in Figure 4.4. The pump delivers a
high-pressure gas into the system. The substrates are pumped into the system, using
a liquid pump. The initial concentration of the reactant must never exceed its
solubility limit in the gas. Supercritical CO2 is depressurized through the expansion
valves into separator column 4, where the product and the unreacted substrates are
recovered. The gas phase is finally vented into the atmosphere after measuring the
flow rate with a rotameter. The gas can be condensed and recycled when used on a
pilot or industrial scale.
4.3.6.1.3 Continuous High-Pressure Enzyme Membrane
Reactor
In a continuous high-pressure enzyme membrane reactor (Figure 4.5), a 35-mmdiameter membrane is placed between two sintered plates and fitted in the reactor.
A measured amount of the catalyst (hydrated enzyme preparation) is put in the
reactor, which is electrically heated, with a heating jacket, to constant temperature.
The substrates and the gas are pumped into the membrane reactor with the highpressure pump. The products and unreacted reactants are collected in the separator.
The catalyst remains in the reactor (behind the membrane).
Continuous processes in particular are favored in the industry because they are
cost efficient and the reactors can be kept relatively smaller in size.132,133 This
reduction in size reduces both costs and safety problems of the high-pressure equipment needed for SC reactions.
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Functional Food Ingredients and Nutraceuticals
TIR
PI
P
PI
H
4
FI
TIR
Gas
P
P
3
2
1
1
FIGURE 4.5 High-pressure continuous stirred-tank membrane reactor. 1 substrates; 2 magnetic stirrer and heater; 3 reactor with membrane; 4 separation column; P high-pressure pump;
TIR temperature indicator and regulator; PI pressure indicator; FI flow indicator.
4.3.7 PROCESSING COSTS
Economic evaluations were made for the enzyme-catalyzed production of oleyl
oleate in a high-pressure batch-stirred-tank reactor (HP BSTR) and in a high-pressure
continuously operated reactor (HP COR). It was assumed that the reaction mixture
was completely precipitated from CO2. The production costs are strongly dependent
on the solubility of the substrates in dense gas, the enzyme lifetime, and the productivity of the biocatalyst.
The study shows that the return on investment would take less than one year.109
4.4 CONCLUSION
The application of SCFs as reaction media for enzymatic synthesis has several
advantages, such as the higher initial reaction rates, higher conversion, possible
separation of products from unreacted substrates, over solvent-free, or solvent systems (where water or organic solvents are used). Owing to the lower mass transfer
limitations and mild (temperature) reaction conditions, at first the reactions which
were performed in nonaqueous systems will be transposed to supercritical media.
An additional benefit of using SCFs as reaction media is that they give simple and
ecologically safe (no heat and solvent pollution) recovery of products. However, for
some specific reactions, solvent-free systems are preferred because of their higher
yields. The main area of development should be related to the hydrolysis of glycerides, transesterification, esterification, and interesterification reactions. As lipases
have high and stable activity in SC-CO2 (even at high temperatures) even greater
and more intensive developments are expected in the future. Only a few papers on
the separation and synthesis of chiral compounds have been published so far. Because
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Biochemical Reactions in Supercritical Fluids
127
enzymes have high selectivity, and owing to the great importance of enantioselective
synthesis or enantiomeric resolution in the pharmaceutical industry, this area of
research is expected to have substantial growth.
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ˇ ˇ M., and Knez, Ž., Stability of proteinase form Carica papaya
110. Habulin, M., Primozic,
latex in dense gases, J. Supercrit. Fluid, 33, 27, 2005.
111. Romero, M.D. et al., Enzymatic synthesis of isoamyl acetate with immobilized
Candida antarctica lipase in supercritical carbon dioxide, J. Supercrit. Fluid, 33, 77,
2005.
112. Habulin, M., Krmelj, V., and Knez, Ž., Supercritical carbon dioxide as a medium for
enzymatically catalyzed reaction, in Proc. High Pressure Chemical Engineering, von
Rohr, R. and Trepp, Ch., Eds., Elsevier, Amsterdam, 1996, 85.
113. Lozano, P. et al., Stability of immobilized alpha-chymotrypsin in supercritical carbon
dioxide, Biotechnol. Lett., 18, 1345, 1996.
ˇ ˇ M., Hydrolases in supercritical CO2 and their
114. Knez, Ž., Habulin, M., and Primozic,
use in a high-pressure membrane reactor, Bioprocess. Biosyst. Eng., 25, 279, 2003.
115. Pasta, P. et al., Subtilisin-catalyzed trans-esterification in supercritical carbon-dioxide,
Biotechnol. Lett., l1, 643, 1989.
116. Habulin, M. and Knez, Ž., Activity and stability of lipases from different sources in
supercritical carbon dioxide and near-critical propane, J. Chem. Technol. Biotechnol.
76, 1260, 2001.
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Functional Food Ingredients and Nutraceuticals
117. Mattiasson, B. and Aldercreutz, P., Tailoring the microenvironment of enzymes in
water-poor systems, Trends in Biotechnol., 9, 394, 1991.
118. Krishna, H.S., Developments and trends in enzyme catalysis in nonconventional
media, Biotechnol. Adv., 20, 239, 2002.
119. Aaltonen, O., Enzymatic catalysis, in Chemical Synthesis using Supercritical Fluids
Jessop, P.G. and Leiner, W., Eds., Wiley-VCH, Weinheim, 1999.
120. Turner, C. et al., Lipase-catalyzed reactions in organic and supercritical solvents:
application to fat-soluble vitamin determination in milk powder and infant formula,
Enzyme Microb. Tech., 29, 111, 2001.
121. Rezaei, K. and Temelli, F., On-line extraction-reaction of canola oil using immobilized lipase in supercritical CO2, J. Supercrit. Fluid, 19, 263, 2001.
122. Schmitt-Rozieres, M., Deyris, V., and Comeau, L.C., Enrichment of polyunsaturated
fatty acids from sardine cannery effluents by enzymatic selective esterification, J.
Am. Oil. Chem. Soc., 77, 329, 2000.
123. Smallridge, A.J., Trewhella, M.A., and Wang, Z., The enzyme-catalysed stereoselective transesterification of phenylalanine derivatives in supercritical carbon dioxide,
Aust. J. Chem., 55, 259, 2002.
124. Chrastil, J., Solubility of solids and liquids in supercritical gases, J. Phys. Chem., 86,
3016, 1982.
125. Hampson, J.W. and Foglia, T.A., Effect of moisture content on immobilized lipasecatalyzed triacylglycerol hydrolysis under supercritical carbon dioxide flow in a
tubular fixed-bed reactor, J. Am. Oil. Chem. Soc., 76, 777, 1999.
126. Martinez, J.L., Rezaei, K., and Temelli, F., Effect of water on canola oil hydrolysis
in an online extraction-reaction system using supercritical CO2, Ind. Eng. Chem. Res.,
41, 6475, 2002.
127. Leitgeb, M. and Knez, Ž., The influence of water on the synthesis of n-butyl oleate
by immobized Mucor miehei lipase, J. Am. Oil. Chem. Soc., 67, 775, 1990.
128. Basheer, S., Mogi, K., and Nakajima, M., Surfactant-modified lipase for the catalysis
of the interesterification of triglycerides and fatty acids, Biotechnol. Bioeng., 45, 187,
1995.
129. Chulalaksananukul W., Condoret J.S., and Combes D., Geranyl acetate synthesis by
lipase-catalyzed transesterification in supercritical carbon dioxide, Enzyme Microb.
Technol., 15, 691, 1993.
130. Knez, Ž., Habulin, M., and Krmelj, V., Enzyme catalyzed reactions in dense gases,
J. Supercrit. Fluid, 14, 17, 1998.
131. Knez, Ž. and Habulin, M., Compressed gases as alternative enzymatic-reaction solvents: a short review, J. Supercrit. Fluid, 23, 29, 2002.
132. Hitzler, M.G. et al., Selective catalytic hydrogenation of organic compounds in
supercritical fluids as a continuous process, Organic Process Res. Dev., 2, 137, 1998.
133. Tundo, P., Continuous Flow Methods in Organic Synthesis, Ellis Horwood, Chichester, 1991.
© 2007 by Taylor & Francis Group, LLC
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5
Pressurized Low Polarity
Water Extraction of
Biologically Active
Compounds from Plant
Products
Juan Eduardo Cacace and Giuseppe Mazza
Agriculture and Agri-Food Canada
CONTENTS
5.1
5.2
Introduction ..................................................................................................135
Pressurized Low Polarity Water Extraction Process ...................................136
5.2.1 Basic Concepts.................................................................................136
5.2.2 Equipment ........................................................................................138
5.3 Applications of PLPW Extraction ...............................................................142
5.3.1 Effect of the Extraction Temperature and Pressure ........................142
5.3.2 Fractionation of Compounds of Different Polarity .........................147
5.4 Modeling of PLPW Extraction of Bioactives from Plant Materials...........150
5.5 Conclusions ..................................................................................................152
References..............................................................................................................152
5.1 INTRODUCTION
Plants synthesize many classes of organic chemical compounds ranging from simple
structures to complex molecules as part of their normal metabolic processes. These
compounds are broadly characterized as: (1) primary metabolites which encompass
substances such as nucleic acids, proteins, lipids, and polysaccharides that are the
fundamental biologically active chemical units of living plant cells, and (2) secondary metabolites which typically have larger, more complex chemical architectures
that incorporate one or more primary metabolites into their structures. Various types
135
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136
Functional Food Ingredients and Nutraceuticals
of secondary metabolites synthesized by plants are commonly referred to as phytochemicals and include carotenoids, phenolics, alkaloids, terpenes, sterols, saponins,
nitrogen-containing compounds, and organosulfur compounds.1 The most studied of
the phytochemicals are the phenolics and carotenoids. Phenolics are compounds
possessing one or more aromatic rings with one or more hydroxyl groups and
generally are categorized as phenolic acids, flavonoids, stilbenes, coumarins, tannins,
isoflavones and lignans. The flavonoids are further classified as flavonols, flavones,
flavanols (catechins), flavanones, and anthocyanins.
It is known that many phytochemicals can significantly affect human metabolism
and health, and therefore, there is considerable interest in extraction of these compounds for their incorporation into functional foods, nutraceuticals, and pharmaceutical products. Also, certain classes of phytochemicals (terpenes and pyrazines) are
useful for the production of flavor and fragrances and for incorporation into topical
preparations.
Phytochemicals typically are not soluble in water under ambient conditions due
to their organic nature and the preponderance of nonionic bonds in their architectures.
However, they are readily soluble in various organic solvents such as aliphatic
alcohols, hexanes, dioxanes, acids, ethers, methylene chloride, trichloroethylene,
and acetonitrile. Numerous methods are known for extracting phytochemicals from
plant materials; most are based on sequential extraction processes incorporating one
or more organic solvents in combination with washing steps.2–7 Some methods teach
the use of alkali or alkaline solvents in combination with organic solvents for
increased extraction efficiency.8–10 Starting plant materials are usually physically
disrupted by means of grinding, shredding, chopping, pulverizing, compressing, or
macerating in order to improve extraction efficiency. Phytochemical extracts have
to be further processed to remove all trace of the organic solvents, and/or to separate
and purify individual phytochemicals. More complex techniques such as hydrodistillation, pressurized solvent extraction, microwave-assisted extraction, supercritical
CO2 extraction, and pressurized low polarity water (PLPW) extraction have been
used in an attempt to reduce the use of organic solvents and increase the efficiency
of the extraction process.2,5,11–13
5.2 PRESSURIZED LOW POLARITY WATER
EXTRACTION PROCESS
5.2.1 BASIC CONCEPTS
Pressurized low polarity water (PLPW) extraction, also known as subcritical water
extraction, is a technology that modifies the properties of water by increasing the
temperature up to 374ºC and keeping the pressure high enough to maintain the water
in the liquid state to improve its extraction ability. It is known that the physical and
chemical properties of water within sealed systems can be manipulated by concurrently controlling the temperature and pressure, whereby the water remains in the
liquid state even though its temperature may be significantly increased above its
atmospheric boiling point of 100ºC. In this condition, pressurized low polarity water
can be maintained in the liquid form up to a temperature of 374ºC and a pressure
© 2007 by Taylor & Francis Group, LLC
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Pressurized Low Polarity Water Extraction
1000
°C
4.5
70
3
0
50
3
m
kg/
400
m
/
kg
1.5
60
0
kg
/
3.5
3.0
4.0
0
kg
m3
3
300
3
/m
8
kg
/m
kg
0
90
2.5
kg /
m3
2.0
95
0
/m
00
°C
20
0°C
T
3
500
1000
2000
40
0°C
0
=0
°
20 C
°
40 C
°
60 C
°
80 C
1 0 °C
0°C
10000
137
50
10.0
360°C
340°C
320°C
300°C
280°C
260°C
1.0
Pressure (bar)
100
Density =
1000 kg/m 3
Entropy = 0.5 kj/kg K
200
240°C
220°C
200°C
180°C
160°C
140°C
= 90%
80%
70%
60%
50%
40%
30%
T = 100°C
Quality
Qu
alit
y
1.0
20%
=1
0%
120°C
80°C
60°C
0.1
0
1000
Enthalpy (kj/kg)
2000
FIGURE 5.1 Pressure-enthalpy chart of water (adapted from Haar et al.14).
of 22.1 MPa (221 bars) after which it becomes supercritical water. The low polarity
water can be considered in a region from normal atmospheric pressure and room
temperature to 22 MPa and 374ºC (Figure 5.1). The polarity, viscosity, surface
tension, and dissociation constant of subcritical water are significantly lower compared to water at ambient temperature and pressure conditions, thereby significantly
altering its chemical properties to approximate those of organic solvents. Thus,
increases of water temperature from 25ºC to 200ºC, for instance, would decrease its
dielectric constant from 79 to 35, reaching values similar to those for ethanol (24)
or methanol (33). A pressure of 5 MPa (50 bars) would be high enough to keep the
© 2007 by Taylor & Francis Group, LLC
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138
Functional Food Ingredients and Nutraceuticals
water in the liquid phase during this increase of temperature (Figure 5.1). A higher
extraction pressure would be detrimental for the process because of a slight increase
in the dielectric constant and a considerable increase in the cost of the equipment.
At 25ºC, the dielectric constant of water increases from about 79 to 93 when the
pressure increases from 10 to 600 MPa (100 to 6000 bars).14 The polarity of pure
water would be comparable to water-methanol or water-acetonitrile mixtures at
25ºC.15 Consequently, pressurized low polarity water can easily solubilize organic
compounds such as phytochemicals, which are normally insoluble in ambient
water.16
Extraction using PLPW has been compared with and reported to be superior to
conventional extraction techniques including hydrodistillation,17–20 solid-liquid extraction,13,18,21 and supercritical CO2 extraction.22,23 Some of the benefits that have been
mentioned include higher selectivity, cleanliness, speed, and cost savings of both raw
material and energy. The heat advantage of PLPW extraction over hydrodistillation
has been reported at 20 times per kg of water, although 10 kg more water would be
used.24 While methods based on sequential extraction processes with organic solvents
are useful for extraction and purification of small quantities of phytochemicals for
research purposes, they are difficult to scale up to commercial volumes because of
problems associated with cost and safe removal and recovery of the organic solvent
from the extracts and spent plant materials. Furthermore, the types and concentrations
of organic solvents must be carefully selected in order to avoid structural changes to
the target phytochemicals during extraction that may adversely affect one or more of
their desirable physical, chemical, or biological properties.
5.2.2 EQUIPMENT
The most commonly used equipment in PLPW systems (Figure 5.2) consists of
storage tank (T) for pure water connected to a high-pressure pump (P), which is
connected to a valve (V1) and to a heating coil (HC) housed within a temperaturecontrolled chamber (O). The pressure in the line is displayed by a pressure gauge
installed outside the oven. The heating coil is connected to an extraction vessel (EV),
which is also mounted inside the temperature-controlled chamber. The chamber is
equipped with programmable temperature controller and recorder (TC). The extraction vessel is connected to a cooling coil (CC), which in turn is connected to the
inlet of a pressure regulation valve or backpressure regulator (BPR). The pressure
regulation valve is connected to a collection vessel (CV).
The collection vessel can be changed periodically to provide a plurality of
collection volumes, thereby separating and individually collecting multiple eluant
fractions. A source plant material is loaded into the extraction vessel and it is
maintained inside the vessel during extraction by fitting adequate frits in the inlet
and outlet of the vessel. Water is pumped from the storage tank into the extraction
vessel until a desired in-line pressure is achieved, usually in the range of about 4 to
7 MPa. Liquid chromatography (LC) pumps and gas chromatography (GC) ovens
have been used most frequently for pumping and heating the water (Table 5.1).
The pressurized water within the extraction vessel is then heated by raising the
temperature within the extraction chamber while in-line pressure is maintained at a
© 2007 by Taylor & Francis Group, LLC
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Pressurized Low Polarity Water Extraction
139
V1
TC
P
CC
T
T
BPR
HC
O
EV
CV
FIGURE 5.2 Diagram of pressurized low polarity water extractor: T, water tank; P, water
pump; HC, heating coil; TC, temperature controller; EV, extraction vessel; O, oven; CC,
cooling coil; BPR, backpressure regulator; CV, collection vial.
desired level by the pressure regulation valve. Stainless steel tubing coils of various
lengths have been used as heating and cooling elements. Varied pressure regulators
have been used from very simple home tubing restrictors to automatic backpressure
regulators (Table 5.1). Self-contained equipments have been also used for pressurized
liquid or accelerated liquid extraction. Flow-control devices taking action on the
pump achieve precise flow rates of subcritical water through the extraction vessel.
The temperature within the chamber is set and maintained by an automatic controller
during an extraction procedure. Components of the system between the pump and
the pressure regulation valve must be connected by tubing resistant to high pressure
(8 to 10 MPa) required for PLPW extraction.
Extraction of bioactives using PLPW has been usually performed using pure
water. Water may be further processed by distillation or filtration, and optionally,
could be purged with nitrogen to remove all dissolved oxygen prior to its use. Purified
water typically has a pH in the range of 5.9 to 6.2. However, if so desired, the pH
of purified water can be adjusted into a range of 3.5 to 9.5 with acids or bases, prior
to its use, to enhance solubilization and extraction of various phytochemicals.
Extraction of bioactives from flaxmeal was optimized by modifying the pH of
subcritical water. Thus, buffered water at either high or low pH was used to improve
extraction of bioactives.25 Yields of proteins and the lignan secoisolariciresinol
diglucoside (SDG) were maximized at alkaline pH 9. The pH was the factor that
defined the equilibrium yields of SDG irrespective of the temperature. At pHs 4 and
9, extraction of SDG from flaxmeal at 160ºC and 190ºC reached the same equilibrium
yield (Figure 5.3). It is known that alkaline pH helps to solubilize protein.26,27 At
the same time, pH could have helped to extract lignans by breaking phenolic-protein
interaction and thus releasing the lignans from the plant matrix. Alkaline pH may
© 2007 by Taylor & Francis Group, LLC
Targeted
phytochemicals
Essential oils
Essential oils
Essential oils
Oxygenates with AO4
activity
Fragrance and flavor
compounds
Anthocyanins
Phenolics from
whole flaxseed and
flaxseed meal
1
Cell
Pump
80 × 3 ID2 mm
Frit 2 µm
150 × 11 ID mm (14 mL)
Frit 2 µm
(10.4 mL) Frit 0.5 µm
LC3 Pump
(0.5–3.0 mL/min)
LC pump
(2.0 mL/min)
LC pump
(1–3 mL/min)
Isco 100 D
(1 mL/min)
LC pump
50 × 9.4 ID mm (3.47 mL)
0.5 µm and 2 µm frits
(10.4 mL)
14.8 ID mm (55 mL)
100
100
100
200
×
×
×
×
7 0 ID mm
9.4 ID mm
19.3ID mm
7.7 ID mm
(24 mL/min)
LC pump
(0.3–4 mL/min)
Heater
Coil/oven
coil/oven
3 m × 0.76 mm
ID coil/oven
3 m coil/oven
1 m coil/oven
Coil/oven
3 m × 0.76 mm
ID coil/oven
Backpressure regulator; 2 Inside diameter; 3 Liquid chromatoraphy; 4 antioxidant
© 2007 by Taylor & Francis Group, LLC
Cooler
BPR1
Coil/cooling recirculation
bath (25°C)
1 m coil/cooling
recirculation bath (25°C)
1 m coil/ice water bath
Home variable restrictor
(2–20 MPa)
40 cm coil/water bath
(25°C)
Coil/cooling water bath
1 m × 0.76 mm ID
coil/water bath (room
temperature)
Reference
13
2,17,18,21
Pressure control valve
(2–9 MPa)
BPR (6–7 MPa)
11 cm to 100 µm
restrictor
Micro metering valve
(4 MPa)
BPR (5.2 MPa cartridge)
43,46
23,31
24,22
48
40,25
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TABLE 5.1
Components of PLPW Extraction Systems Used for Extraction of Plant Bioactives
2441_C005.fm Page 141 Tuesday, July 18, 2006 8:32 AM
141
25
119
20
95
15
71
Yield (%)
SDG (mg/g meal)
Pressurized Low Polarity Water Extraction
48
10
160°C pH 4
160°C pH 9
190°C pH 4
190°C pH 9
5
24
0
0
0
100
200
300
400
500
Time or volume (min or mL)
FIGURE 5.3 Pressurized low polarity water extraction of SDG from flaxmeal at 160ºC and
190ºC and pH 4 and 9.
have also hydrolyze d complex polymeric phenolics, reducing them to more available
and easily extracted compounds. Whatever mechanisms pH affected, high pH raised
SDG and protein yields. Thus, pH effect overcame the increase of solubility and
yield obtained by temperature.
Carbohydrate extraction from flaxmeal was improved using PLPW at pH 4.25
Also, high-temperature pressurized liquid extraction (80°C to 100°C) using acidified
water was as effective as acidified 60% methanol in extracting anthocyanins from
grape skins.28
Another modification of water to improve its extraction efficiency has been the
addition of sulfur dioxide. Sulfured PLPW (containing 1400 g/mL sodium metabisulfite) extractions of grape skin phenolics over a temperature range of 100ºC to
160°C were compared with conventional hot water or aqueous 60% (v/v) methanol
extractions (50°C, 1 h). The PLPW extracts from modified sulfured water had higher
levels of total anthocyanins and total phenolics than extracts that used pure water.
Furthermore, subcritical water and subcritical sulfured water extracts had comparable or higher levels of anthocyanins than extracts obtained using conventional hot
water or 60% methanol.29
A modification of the heating system in the subcritical water extraction could
be the replacement of the oven with a water heater interconnected between the pump
and the extraction vessel. In such a system the extraction vessel would be provided
with a jacket or a surface heater, thereby maintaining the temperature of pressurized
low polarity water flowing through the extraction vessel. The jacket may be integral
to the extraction vessel, or alternatively, be mountable onto the exterior surface of
the vessel.30
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Functional Food Ingredients and Nutraceuticals
5.3 APPLICATIONS OF PLPW EXTRACTION
PLPW has been applied to extract a variety of biochemicals from a wide range of
plant species (Table 5.2). It has been used to extract antioxidants from rosemary31
and Taiwan yams;32 anthocyanins from red grape skin;28,29 ginsenosides from American ginseng,33 catechins and epicatechin from tea leaves and grape seeds,34 oil from
cedarwood;35 essential oil from oregano,17 anthraquinones (antibacterial, antiviral,
and anticancer compounds) from roots of Morinda citrifolia,36 tanshinone I and IIA
from Salvia miltiorrhiza used in Chinese medicine;37 flavones, anilines, and phenols
from orange peels;38 kava lactones from kava roots;39 lignans from whole flaxseed40
and flaxseed meal,25 and saponins from cow cockle (Saponaria vaccaria) seed.41
Plant material for the extraction can include homogenous samples; or mixtures
of whole plant parts such as seeds, flowers, leaves, stems, and roots; and also, with
source plant materials disrupted and processed by methods including one or more
of grinding, shredding, chopping, pulverizing, compressing, and macerating. The
biomass can be fresh hydrated plant materials or plant materials may be dehydrated
prior to extraction, or alternatively, processing by one or more of the methods
described above prior to extraction. The plant materials may be packed into an
extraction vessel either alone or in combination with inert physical substrates such
as glass wool, glass beads, resin beads, silica sand, stainless steel wire cloth, and
other like substrates whereby the inert substrates maintain spacing and distribution
of the source plant materials throughout the vessel during the course of the extraction
procedure thereby facilitating mass transfer while preventing migration and packing
of the plant material against the outlet frits.30 Recently, Ho et al.25 showed that
extraction of lignans, proteins, and carbohydrates with PLPW was positively affected
by the addition of glass beads to flaxseed meal for packing the cell. The overall
lignan yield increased from 10% to 50% at all temperatures tested with copacking
the meal with glass beads.
5.3.1 EFFECT
OF THE
EXTRACTION TEMPERATURE
AND
PRESSURE
The extraction of bioactives from plant material with the PLPW process is clearly
affected by the temperature. Thus, SDG, the major lignan in flaxseeds, along with
two other phenolic compounds, p-coumaric acid glucoside and ferulic acid glucoside,
were extracted with varied success at different temperatures in a PLPW system.
Extraction yields of lignans and other phenolic compounds from flaxseed increased
from 10 % at 100ºC up to approximately 90% at 140ºC to 160ºC.40 Temperature
also affected PLPW extraction of lignan SDG from flaxmeal. Yield of flaxmeal SDG
increased from 30% at 130ºC to a maximum of almost 100% at 190ºC.25 In PLPW
extraction of phenolic compounds from grape seeds at 150ºC, catechin increased by
32% and epicatechin increased by 99% over the recovery at 50ºC extraction.42
Saponin yields of PLPW extracts increased with temperature and time. While only
33.2% (w/w) of total saponins was extracted at 125°C in 3 h, 60.2% (w/w) was
recovered at 175°C in the first 15 min extraction from cow cockle ground seeds.41
The temperature has such a remarkable effect on the extraction and composition of
the final extract that a study of temperature effect is a prerequisite in any new PLPW
© 2007 by Taylor & Francis Group, LLC
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Pressurized Low Polarity Water Extraction
143
extraction research. The effect of temperature on extraction of bioactives using
PLPW has also been reported in extractions of lactones from kava root,39 phenolics
from grape skin,28 and essential oils from peppermint and savory,23 fennel,21 rosemary,24,31 thyme;43 clove,44 Origanum micranthum,45 Rosa canina,46 and Origanum
onites.47 It is clear from these and others studies (Table 5.2) that temperature has to
reach a minimum value for the water to gain the properties required to extract a
given bioactive. Below the required temperature the extraction does not occur or
results in a very low yield.
A very good example of the effect of temperature on compound yield is the
extraction of volatile compounds from peppermint (Figure 5.4). Four groups of
compounds were extracted: the first included carvone, pulegone, and piperitone;
the second eucalyptol, menthone, neomenthol, and menthol; the third menthyl
acetate; and the fourth β-caryophyllene.23 Only one line per group has been plotted
in Figure 5.4 to simplify the plot. As can be observed, extraction yields increased
with temperature from values as low as zero to as high as 100%, depending on
the group of compounds evaluated. At temperatures below 50ºC, only compounds
of the first two groups were extracted, and yields varied depending on the extraction
time. Menthyl acetate and β-caryophyllene were not extracted or their yields were
very low because their solubility in water at those temperatures was likely very
low. In order to begin extraction of menthyl acetate and β-caryophyllene, temperatures above 50ºC and 150ºC had to be used. Increasing the extraction time did
not considerably increase the yield of these compounds when the extraction temperature was kept below 50ºC and 150ºC, respectively. Yield points represented
in Figure 5.4 are yields of 15 min extraction at each temperature. Thus 80% yield
at 175ºC was calculated by adding yields of five extractions of 15 min each.
However at 50ºC, menthyl acetate yield after 15 min extraction was 1% or 2%;
if the extraction time were increased five times, the yield would be at most 5% to
10%. Effect of temperature has been attributed to an increase on the solubility of
the compounds.24 It is also possible that the temperature affects the extraction by
breaking interactions between the analytes and the plant matrix.42 Because the
extraction yields increase with temperature and also temperature has to reach a
minimum value for the bioactives to be extracted with water, the extraction temperature must be selected for the targeted compounds.
There is an optimal temperature at which the yield is maximized for every
bioactive. Ideally this temperature would be related to the modified properties of
the water required for the extraction in the PLPW system, which in turn are linked
to the properties of the bioactive that has to be extracted. Thus, the most efficient
extraction of the lignan SDG and other phenolics from flaxseed occurred in the
temperature range from 140ºC to 160ºC40 and from flaxmeal at 190ºC.25 Recommended temperatures for PLPW extraction of other phytochemicals are: 150ºC for
phenolics compounds from grape seeds;42 120ºC for anthocyanins from berries;48
100ºC for volatile flavor and fragrance compounds from Rosa canina,46 and 150ºC
for essential oils from Achillea monocephala.49
Most of these extraction temperatures are relatively high, and may be detrimental
when heat sensitive bioactives are present in the raw material. Thus, at 160ºC the
total SDG measured in flaxseed extracts, solvent wash, and extracted seed residues
© 2007 by Taylor & Francis Group, LLC
Target Phytochemicals
Plant Material
Temperature/Pressure
Major Compounds
Eucalyptol, linalool, terpinen-4-ol, αterpineol, geraniol, ETMCb
Essential oils
Laurel
50ºC to 200ºC (150ºC)/5 MPa
1,8-cineole, 2 unidentified peaks
Essential oils
Clove
125ºC, 250ºC/2.4, 5, 10, 17 MPa
Eugenol, eugenyl acetate
Essential oils
Thyme
100ºC, 125ºC, 150ºC, 175ºC (150ºC)/2, 6, 9 MPa α-pinene, p-cymene, γ-terpinene,
limonene, E-3-caren-2-ol, thymol,
carvacrol, caryophyllene
Essential oils
Oregano
100ºC–175ºC (150ºC)
Thymol
Essential oils
Achillea monocephala 100ºC, 125ºC, 150ºC, 175ºC/6 MPa
1,8-cineole,camphor, α-campholenal,
borneol, terpinen-4-ol and manymore
Essential oils
Origanum micranthum 100ºC, 125ºC, 150ºC, 175ºC/4 to 8 MPa
α-terpineol, linalool, borneol, terpinen-4ol, and many more
Essential oils
Fennel
50ºC to 200ºC (150ºC)/2 MPa
α-pinene, mircene, limonene, camphor,
phelandrene, anethol
Phenolic antioxidants
Sage
70ºC, 100ºC or 150ºC/10 MPa
Rosmarinic acid, carnosol, carnosic acid,
methyl carnosate
Antioxidant compounds Rosemary leaves
25°C, 100°C, 150°C, 200°C and 100°C, 150°C, Scutellarein,rosmanol, genkwanin,
200°Cc/6 MPa
carnosol, carnosic acid, NI 1
Fragrance and flavor
Peppermint
150°C, 175°C and 50°C, 100°C, 125°C, 150°C, Carvone,pulegone, eucalyptol, menthone,
compounds
175°C, 200°Cc/6 MPa
neomenthol, menthol, menthyl acetate
Fragrance and flavor
Savory
100°C, 150°C, 175°C and 50°C, 100°C, 125°C, p-cymene, thymol, carvacrol, linalool,
compounds
150°C, 175ºC, 200ºCc/6 MPa
borneol, thymoquinone
Fragrance and flavors
Rosemary
125ºC, 150ºC, 175ºC
α-pinene, limonene, camphene, camphor,
1,8-cineole, borneol
Essential oils
Marjoram
© 2007 by Taylor & Francis Group, LLC
100ºC – 175ºC (150ºC)a/2–20 MPa
Reference
13
18
44, 22
43
17
49
45
21
50
31
23
23
24
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TABLE 5.2
Applications of PLPW Technology to the Extraction of Plant Bioactives
Rosa canina
50ºC, 100ºC, 150°C/2.5, 5, 7.5 MPa
Eldelberry, raspberry,
bilberry, chokeberry
110°C–160°C (100°C–120°C)
Anthocyanins and
phenolics
Phenolics/lignans
Grape skin
100°C to 160°C by 10°C
Whole flaxseed
100°C, 120°C, 140°C, 160°C/5 MPa
Lignans, proteins
Flaxseed meal
130°C, 160°C, 190°C/5 MPa
Saponins
Kava lactones
Cow cockle
Kava root
125°C, 175°C/5 MPa
25°C, 50°C, 100°C to 200°C by 25°C/6–7 MPa
a
Benzaldehyde, benzyl alcohol, phenylethyl 46
alcohol, eicosane, and more
Phenolic acids, ellagic acid, catechin,
48
epicatechin, resveratrol, quercetin,
kaempferol, anthocyanins
Monoglucoside and acylated anthocyanins 29
SDG d, p-coumaric acid glucoside, ferulic
acid glucoside
SDGd
proteins, carbohydrates
Saponins, cyclopeptides
Dihydrokavain, kavain,
desmethoxyyangonin, yangonin,
dihydromethysticin
40
25
41
39
Selected temperature in brackets; b ethenyl-α,α-4trimethyl-3-(1-methylethenyl)-cyclohexanemethanol; c sequential equal time extractions at listed temperatures of one sample load; d SDG Secoisolariciresinol diglucoside.
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Fragrance and flavor
compounds
Anthocyanins
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Functional Food Ingredients and Nutraceuticals
100
Yield (%)
80
60
40
Carvone
Menthol
Menthyl acetate
β-caryophyllene
20
0
50
100
150
Temperature (ºC)
200
FIGURE 5.4 Effect of temperature on pressurized low polarity water extraction of volatile
compounds from peppermint (adapted from Kubátová et al.23).
was lower than the amount achieved at 140ºC; and there was a lower SDG percentage
in the seed residue at 160ºC, which may be attributed to degradation of phenolics.
At temperatures higher than 160ºC degradation of SDG also occurred in PLPW
extractions of bioactives from flaxmeal. However, the detrimental effect of temperature could be alleviated when a high rate of extraction removes most of the bioactives in a relatively short time. Thus, SDG yields of 96% to 98% were reached at
temperature as high as 190ºC when the rate of the extraction was increased by the
increase of pH to 9.25 Equilibrium time and maximum recovery were reached in less
than 100 min in extractions avoiding degradation of SDG.
Elevated temperature increases the extraction rate, which in turn reduces the
volume of water required for the extraction and the time to reach maximum recovery.
The increase of the extraction temperature increased the extraction rate of lignans
from flaxmeal, thus the volume of water required for the extraction and the time to
reach maximum recovery were reduced. A raise of temperature from 160ºC to 190ºC
reduced the extraction time by half.25 Increased extraction rates produced by temperature raises have been reported for the extraction of kava lactones from kava
roots39 and flavor and fragrance compounds from savory and peppermint,23 rosemary,24 and clove.44 However, for some plant material and targeted compounds the
raise in temperature cannot be unlimited.
The combination of high temperatures and long time of extraction must be
avoided when temperature sensitive bioactives are being extracted. Ju and Howard28
reported that extraction temperatures higher than 100°C resulted in anthocyanin
degradation in pressurized liquid extraction, which was especially marked at 140°C.
Reductions from 24% to 40% of the total anthocyanins content have been reported
with pure and modified PLPW in the range of temperatures from 110ºC to 160°C.29
It has been demonstrated that most phenolic compounds react easily at high
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147
temperature (65ºC) when they are in contact with air. However, when higher temperatures are applied under nitrogen atmosphere, there are no degradations, since
the degradation process for phenolic compounds is an oxidative process requiring
the presence of oxygen.42 At high temperatures, dark brown PLPW extracts with
strong burning smell and increased viscosity have been found in extractions from
parsley,30 flaxmeal,25 Origanum micranthum,45 oregano,17 and leaves and flowers of
Achillea monocephala.49 This has been attributed to the presence of browning reaction products such as furfural, acetylfuran, and 5-methylfurfural in the extracts of
flowers of Achillea monocephala at 175ºC.49 However, the degradation in PLPW
extractions is lower than the effects measured at atmospheric pressure extractions
even at lower temperatures. Thus, PLPW allows for the use of temperatures higher
than those used in conventional extraction techniques, probably because PLPW
requires shorter extraction times and an oxygen-reduced environment.
The effect of pressure in PLPW extraction of bioactives from diverse plant
material has also been reported. Rovio et al.44 studied the effect of four pressures at
two temperatures in PLPW extraction of flavor and fragrance from clove. No significant differences were found for the recoveries of eugenol and eugenyl acetate
from clove at 25, 50, 100, and 175 kg/cm2 (2.4, 4.9, 9.8, and 17.2 MPa). Similar
responses have been reported on PLPW extractions from Rosa canina,46 oregano,17
Origanum micranthum,45 and marjoram.13 These results are in agreement with the
change of water dielectric constant with an increase of pressure. As it has been
mentioned above, an increase of pressure of 590 MPa (from 10 to 600 MPa at 25°C)
results in a small increase of dielectric constant from 79 to 93.14
5.3.2 FRACTIONATION
OF
COMPOUNDS
OF
DIFFERENT POLARITY
The extraction performance of a compound in a PLPW system is related to the
polarity of the compound. Extraction yields of major phenolics present in flaxseed
were not markedly different, indicating that they have a similar polarity. This suggests that fractionation of the major phenolic compounds in flaxseed using a PLPW
system may not be viable. However, fractionation of phenolic compounds has been
achieved in black currants and parsley.30 A sequential-temperature extraction of
frozen black currant particles was performed in a PLPW system at temperatures
from 80ºC to 240ºC. The total phenolic concentration of the extracts decreased from
80ºC to 120ºC and the yield of the extraction increased until it reached a plateau.
However, when the temperature was further increased up to 240ºC, the concentration
and the yield increased continuously with the temperature (Figure 5.5). The concentration of anthocyanins decreased continuously to zero and the yield increased
to reach equilibrium and remained at those values even after further temperature
increase up to 240ºC. HPLC chromatograms of samples collected at 80ºC, 120ºC,
and 200ºC (Figure 5.6) indicate that high polarity compounds were extracted at the
initial lower temperatures, and their content in the extraction cell decreased with
further extraction. Although the identification of the compounds being extracted was
not pursued, the increase in yield at the highest temperatures could be attributed to
the extraction of newly generated high polarity compounds by hydrolysis of low
polarity polymeric compounds or to the extraction of different molecules that
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Functional Food Ingredients and Nutraceuticals
1000
5000
Concentration (mg/L)
4000
TPhen
Anthoc
Yield (mg/100 g)
600
3000
TPhen
Anthoc
400
2000
200
1000
0
Yield (mg/100 g)
Concentration (mg/L)
800
0
50
100
150
200
Temperature (ºC)
300
250
FIGURE 5.5 Total phenolic and anthocyanic yield and extract concentration in pressurized
low polarity water sequential-temperature extractions from frozen black currant at 80ºC to
240ºC.
(C) 200°C
(B) 120°C
(A) 80°C
0
10
20
30
40
50
Retention time (min)
60
70
80
FIGURE 5.6 HPLC chromatograms at 280 nm of frozen black currant extracts collected at
80ºC, 120ºC, and 200ºC in pressurized low polarity water sequential-temperature extractions.
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149
otherwise would not be extracted. Extractions of those compounds are indicated by
the appearance of new peaks at the beginning of the 280 nm chromatograms of the
extracts collected at 200ºC and 240ºC (Figure 5.6). On the other hand, anthocyanins
were extracted continuously and completely with extractions at 80ºC, 120ºC, and
200ºC and no new compounds that absorb at 525 nm were extracted with further
increase of temperature. These observations clearly suggest that by suitably controlling the extraction temperature and the collection of the extracted fractions, PLPW
extraction can be used for the recovery or fractionation of compounds of different
polarity.
Extractions of phenolics from grape seeds using PLPW42 and antioxidant compounds from rosemary produced similar results.31 In these studies, depending upon
the temperature used, there were large differences in both the identity and recovery
rate of the phenolic compounds extracted. In grape seed extraction at 150ºC, catechin
increased by 32% and epicatechin increased by 99% over the recovery from the
50ºC extraction. There were also some compounds that were not detected in the
extracts produced at 50ºC and 100ºC but which were detected in the extracts obtained
at 150ºC. It was considered that the use of higher extraction temperature helped in
the breaking of bonds between the analytes and the matrix, increasing yields of some
compounds or extracting compounds that otherwise would not be extracted. In
rosemary extractions, when water was heated to 200ºC, the dielectric constant of
water was reduced and the solubilities of less polar compounds increased by several
orders of magnitude, changing the composition of the extracts.31 Similarly, Palma
et al.42 reported that the behavior of the analytes depended on the matrix, and the
analytes in grape seeds were more strongly bonded to the matrix than in grape skin.
Therefore, for grape skins shorter extraction times and lower temperatures would
result in significant increase of yield and fractionation of phenolics.42 The temperature of the water affects the extraction in two ways: first by changing the dielectric
constant of the water and thus the solubility of targeted compounds, and second by
breaking the interactions between the analytes and the matrix.
Production of essential oils richer in oxygenated fragrance compounds of more
value and with less contaminant monoterpenes and sesquiterpenes from peppermint
can be produced by adequate selection of extraction temperature (Figure 5.4). Oxygenated compounds extract at substantially lower water temperatures than nonoxygenated compounds.23 Extraction at 50ºC to 100ºC will result in extracts richer on
valuable oxygenated compounds with a low fraction (< 5%) of terpenes such as pcymene or β-caryophyllene. Similarly, desirable carnosol-rich rosemary extracts can
be produced by PLPW extraction of rosemary leaves at 100ºC. Furthermore, by
using such a procedure it is possible to obtain enriched extracts with very high
antioxidant activity.31 Also, continuous PLPW extraction of oil from marjoram
resulted in extracts richer in odoriferous and hence more valuable oxygenated compounds than hydrodistilled oils, which contained 11 to 22 times larger amounts of
monoterpenes.13 Therefore, extracts produced by PLPW extractions can give a
higher-quality and higher-value product with more intense characteristic natural
aroma.
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Functional Food Ingredients and Nutraceuticals
5.4 MODELING OF PLPW EXTRACTION OF
BIOACTIVES FROM PLANT MATERIALS
Two simple models have been applied to describe the extraction profiles obtained
with PLPW extraction.51 The first of these models is based on the thermodynamic
distribution coefficient (KD), which assumes that analyte release from the matrix is
rapid compared to elution; and the second model is a two-site kinetic model which
assumes that the extraction rate is limited by the analyte release rate from the matrix,
and is not limited by the thermodynamic (KD) partition that occurs during elution.
The two models are defined by the following equations51
Thermodynamic model:
Sa
(1– )
Sb
Sa
0
S
=
+
S0  KDm
 S0
 (Vb − Va )d + 1


(5.1)
ST
= 1 − [ Fe− k 1t ] − [(1 − F )e− k 2 t ]
S0
(5.2)
Kinetic model:
Sa: cumulative mass of the analyte extracted after volume Va (mL)
Sb: cumulative mass of the analyte extracted after volume Vb
S0: initial total mass of analyte in the matrix.
Sb /S0 and Sa /S0: cumulative fraction of the analyte extracted by the fluid of the volume
Vb and Va
ST: mass of the analyte removed by the extraction fluid after time t
KD: distribution coefficient; concentration in matrix/concentration in fluid
F: fraction of the analyte released quickly
k1 and k2: first order rate constant (mL–1) for the quickly and slowly released fractions
d: density of extraction fluid at given condition (g/mL)
m: mass of the extracted sample (g)
The kinetic model does not include solvent volume, but relies solely on extraction
time. Therefore, doubling the extractant flow rate should have little effect on the
extraction efficiency per unit time if the extraction efficiency is controlled by the
kinetics of the initial desorption step (assuming the other extraction parameters
remain constant). On the contrary, the thermodynamic model is only dependent on
volume of extractant used. Therefore, the mechanism of thermodynamic elution and
desorption kinetic can be compared simply by changing the flow rate in PLPW
extraction. If the concentration of bioactive compounds increases proportionally with
increase in flow rate at certain extraction time, the extraction mechanism can be
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Pressurized Low Polarity Water Extraction
151
explained by the thermodynamic model. However, if the increase of flow rate has
no significant effect on the extraction of the bioactive compounds, with the other
extraction parameters kept constant, the extraction mechanism can be modeled by
kinetic diffusion.40,51 In this case, plots for several flow rates of the extracted amount
or yield of the analyte as a function of the extraction time must lie on the same line
indicating no effect of the flow rate. The mechanism of control and therefore the
model valid for PLPW extraction may be different depending on the raw material,
the targeted analyte, and extraction conditions. Thus, phenolic extractions from
flaxseed performed at flow rates from 1 to 4 mL/min were affected by the flow rate,
which indicates that the mass transfer of the solute from the surface of the solid into
the bulk of the water regulated most of the extraction process in a similar way to
the thermodynamic model. However, there was no difference in the extraction rate
among phenolics extractions performed at 1, 0.5, and 0.3 mL/min, which were not
affected by the flow rate. Thus, extractions at low flow rates would have been
controlled by the diffusion in the seeds, as the two-site kinetic model establishes
above. It has been suggested that at low flow rates (<1 mL/min), Biot number may
have increased to values higher than 50 to 100; in this condition the effect of the
boundary layer is negligible: the extraction would be controlled by the diffusion
inside the seeds and not show any effect of the flow rate. Also, no effect of the flow
rate has been found in extractions of SDG, proteins, and carbohydrates from
flaxmeal.40
The data from PLPW extraction for savory, presented in Figure 5.7, clearly show
that the extraction rates of essential oils had an increase proportional to the water
100
Yield (%)
80
60
40
0.25 mL/min
0.5 mL/min
1 mL/min
2 mL/min
4 mL/min
20
0
0
10
20
30
Time (min)
40
50
60
FIGURE 5.7 Effect of flow rate on thymol yields in pressurized low polarity water extractions
from savory at 100ºC (adapted from Kubátová et al.51).
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Functional Food Ingredients and Nutraceuticals
flow rate, and thus the thermodynamic elution of analytes from the matrix is the
prevailing mechanism as evidenced by the fact that extraction rates increased proportionally with the water flow rate. This is also confirmed by the fact that simple
removal calculations based on a single KD (for each essential oil compound) give
good fits to experimental data for flow rates from 0.25 to 4 mL /min.51 The increased
recovery rates directly proportional to extraction flow rates show that high water
flow rates can be used to shorten PLPW extraction times without increasing the
amounts of fluid needed to achieve a high recovery.51
5.5 CONCLUSIONS
Pressurized low polarity water (PLPW) extraction, also known as subcritical water
extraction, is a highly promising “green” technology for the extraction and fractionation of biologically active compounds for use as functional food ingredients and
nutraceuticals. PLPW has been applied to extract a variety of phytochemicals from
a wide range of plant species including antioxidants from rosemary and yams,
anthocyanins from berries and red grape skin, ginsenosides from American ginseng,
catechins and epicatechin from tea leaves and grape seeds, essential oil from oregano,
kava lactones from kava roots, lignans from flaxseed and flaxseed meal, and saponins
from cow cockle (Saponaria vaccaria) seed. PLPW extraction has been compared
and reported to be superior to conventional extraction techniques including solidliquid extraction,18,21 hydrodistillation, and supercritical CO2 extraction. Key benefits
include higher selectivity, cleanliness, speed, and cost savings of both raw material
and energy.
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Pressurized Low Polarity Water Extraction
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44. Rovio, S., Hartonen, K., Holm, Y., Hiltunen, R., and Riekkola, M.L., Extraction of
clove using pressurized hot water, Flav. Frag. J., 14(6), 399–404, 1999.
45. Gogus, F., Ozel, M.Z., and Lewis, A.C., Superheated water extraction of essential
oils of Origanum micranthum, J. Chromatog. Sci., 43(2), 87–91, 2005.
46. Ozel, M.Z. and Clifford, A.A., Superheated water extraction of fragrance compounds
from Rosa canina, Flav. Frag. J., 19(4), 354–359, 2004.
47. Ozel, M.Z. and Kaymaz, H., Superheated water extraction, steam distillation and
Soxhlet extraction of essential oils of Origanum onites, Analyt. Bioanalyt. Chem.,
379(7–8), 1127–1133, 2004.
48. King, J.W., Grabiel, R.D., and Wightman, J.D., Subcritical water extraction of anthocyanins from fruit berry substrates, Proc. 6th Intl. Sym. Supercritical Fl., tome 1,
409–418, 2003.
49. Gogus, F., Ozel, M.Z., and Lewis, A.C., Extraction of essential oils of leaves and
flowers of Achillea monocephala by superheated water, Flav. Frag. J., In press, 2006.
50. Ollanketo, M., Peltoketo, A., Hartonen, K., Hiltunen, R., and Riekkola, M.L., Extraction of sage (Salvia officinalis L.) by pressurized hot water and conventional methods:
antioxidant activity of the extracts, Eur. Food Res. Tech. 215(2), 158–163, 2002.
51. Kubátová, A., Jansen, B., Vaudoisot, J.F., and Hawthorne, S.B., Thermodynamic and
kinetic models for the extraction of essential oil from savory and polycyclic aromatic
hydrocarbons from soil with hot (subcritical) water and supercritical CO2, J. Chromatog. A., 975(1), 175–188, 2002.
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6
Purification of Orange
Peel Oil and Oil Phase by
Vacuum Distillation
Mércia de Fátima M. Bettini
Flavor Tec – Aromas de Frutas Ltd, Brazil
CONTENTS
6.1
Introduction.................................................................................................157
6.1.1 Steam Distillation.............................................................................158
6.1.2 Simple Distillation and Fractional Distillation................................158
6.1.3 Vacuum Fractional Distillation ........................................................159
6.1.3.1 Significant Reduction of Thermal Hazard .......................159
6.1.3.2 Greater Fractionation Efficiency ......................................160
6.1.3.3 Enhanced Purity of Distillate ...........................................160
6.2 Industrial Scale Purification of Orange Peel Oil.........................................160
6.2.1 Citrus Industry..................................................................................160
6.2.2 Sweet Orange Oil.............................................................................161
6.2.2.1 Purification of Orange Peel Oil........................................161
6.2.2.2 Method of Deterpenation — Vacuum
Fractional Distillation .......................................................162
6.2.2.3 Folded Oils........................................................................162
6.2.2.4 Uses and Applications ......................................................163
6.2.2.5 Experimental Results ........................................................163
6.3 Conclusions ..................................................................................................170
References..............................................................................................................172
6.1
INTRODUCTION
Distillation is a process of heating a substance until the most volatile constituents
change into the vapor phase, and then cooling the vapors to recover the constituents
in liquid form by condensation. The main purpose of distillation is to separate a
mixture into individual components by taking advantage of their different level of
volatilities. Distillation is one of the main methods of extracting essential oils from
plants. The percentage of each constituent in the vapor phase usually depends on
157
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its vapor pressure at a certain temperature. The principle of vacuum distillation may
be applied to substances such as oils that would be damaged by overheating by the
conventional method [1]. There are different methods of distillation, depending on
the desired product; some of these processes are described below.
6.1.1 STEAM DISTILLATION
Steam distillation is one of the common methods of distillation. Although steam
distillation appears to be the best method for extracting essential oils, supercritical
CO2 extraction is becoming quite popular. Most aromatherapists believe that essential
oils are only effective when extracted by steam distillation and all other essential
oils should be labeled with their extraction methods, that is, cold pressed oils,
concretes, absolutes, and so forth. These preparations should not be confused with
“true” essential oils.
In the steam distillation process the material to be extracted is carefully gathered
and placed into a copper or stainless steel vat, known as the distillation chamber.
Steam is then generated by heating (preferably low heat) to build pressure within
the distillation chamber causing the glands of the plant to rupture and release its
essence which is then carried by the steam vapor. The steamy vapor is cooled and
condensed in a coil, which is usually submerged in refrigerated cold water. The
water and essential oil separate and the essential oils usually stay on top of the water
due to their lower densities. The water and essential oil are sent to a collection
chamber where the oil is easily skimmed off the top of the distillated water. The
distillated water may contain many low water soluble components of the plant. These
by-products of the distillation process are known as “hydrosol” and can be used for
skin care, usually for children and the elderly.
When subjecting a plant material to heat or steam as in the distillation process,
the physicochemical properties of the material may be altered. The thermolabil
components are degraded and here the information given by some authors with
regard to the essential oils may be flawed. To achieve effective aromatherapeutic
benefits, it is important to recognize that the extracted essential oils are not always
in the same form when they were in the plant. Therefore, when subjecting plant
materials to heat or steam as in the distillation process, the chemical structures within
the essential oil after distillation are different than those found in the original plant
material. For instance, chamomile is converted to the chemical azulene, which is
not found in the plant itself but is produced during distillation [2].
When designing equipment for high vacuum process, parameters such as pressures, pipe sizes, and vessel sizes, must be adequate to avoid excessive vapor speed
and pressure drops. The entire system must be free of leaks [3], and compensation
for the excess pressure drop (2 or 3 mm Hg) across the entire system must be
incorporated into the design process.
6.1.2 SIMPLE DISTILLATION
AND
FRACTIONAL DISTILLATION
If one desires to get a pure compound, the distillation process has to be repeated
many times and this will make it a cumbersome process. In practice, the vapors are
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condensed and revaporized in fractionating columns connected sequentially in series.
Equilibrium between liquid and vapor presumably occur on each plate. However, it
is not likely that equilibrium is reached on each plate, therefore, it is imperative to
calculate the plate efficiency, an important design factor. The column is packed with
an inert material to promote contact between the liquid and the vapor. One of the
most important design factors in packed columns is to prevent channeling — a
condition where the vapors and liquid flow in opposite directions without coming
into contact.
The number of theoretical plates is determined under a specific set of operating
conditions during the construction of the column. The efficiency of design is referred
to as the Height Equivalent per Theoretical Plate (HETP).
When all the vapors that go up through the column condense and flow back
down, the returning liquid is called the reflux. The total reflux consists of the total
product received from the outlet when no distillate is produced. As soon as the
product is drawn from the condensed vapors, the amount of liquid returning through
the column is reduced and produces changes to the operating conditions of the
column. If all the condensed vapors are drawn off, then there will be no downflow
to wash the upcoming vapors and in effect the column would stop functioning
altogether and become just a long pipe. However, most columns would retain some
amount of reflux and, for practical considerations, there would be some heat losses
through radiation and convection along the column [3].
6.1.3 VACUUM FRACTIONAL DISTILLATION
Vacuum distillation is a method of distilling substances at temperatures below their
normal boiling point (standard atmospheric pressure). By reducing the pressure, a
much lower distillation temperature will be required.
High vacuum distillation may be used for certain classes of chemical compounds
that decompose, polymerize, react, or are destroyed by conventional distillation
methods. Low cost per pound and high throughput may be obtained on certain groups
of compounds such as vitamins, epoxy resins, highly concentrated pure fatty acids,
plasticizers, fatty acid nitrogen compounds, and a host of other heat-sensitive materials which may require only deodorizing and decolorizing. The pharmaceutical
industry, in their continuing search for new products with high and intermediate
molecular weights, finds that it is still an invaluable commercial practice to separate
products by molecular weight from excess reactants and catalysts.
6.1.3.1 Significant Reduction of Thermal Hazard
High vacuum distillation is a safe process to separate mixtures of organic or silicon
compounds, most of which cannot withstand prolonged heating above 250ºC without
excessive structural change or decomposition. As opposed to conventional atmospheric and pressure packed distillation towers, the high vacuum process utilizes the
heat of condensation as a source of radiant heat emission to heat the surface film
on the evaporator. With short residence time and lower distilling temperatures,
thermal hazard to the organic material is greatly reduced.
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6.1.3.2 Greater Fractionation Efficiency
The best approach to a mechanical method of creating a uniform film thickness with
a fast-moving liquid is to feed the liquid to the center of a heated spinning disc. By
centrifugal action, the material spreads as a film across the heated disc that intimately
faces a large condensing surface. The lighter compound evaporates and condenses
in a fraction of a second. The heavier residues that are not evaporated slide off the
outer edge of the disc into a concentric residue collector and are discharged. The
degree of separation is a function of the molecular weight differences of the distilled
mixture. The greater the difference in the molecular weights, the purer the distillate.
The closer the molecular weights of the mixture are, the less efficient is the fractionation process, resulting in the need for successive runs of the distillate.
6.1.3.3 Enhanced Purity of Distillate
Purity of the distillate also depends on the film thickness. Controlling positive
pressure and supply to the heated evaporator surface will usually provide a uniform
film throughout the distillation.
The absence of air molecules in the high vacuum distillation column permits
most of the distilling molecules to reach the condenser with relatively few molecules
returning to the liquid film surface in the evaporator. Experimental results show a
relationship between the molecular weight and distillation temperature for a broad
range of different materials. There is no predictable temperature at which distillation
may occur; condensation begins whenever a sufficient temperature difference occurs
between the evaporator and condenser. There is an infinite number of sets of operating conditions for every material that is fed through the system. Since pressure is
constant, the only variables are flow rate (governing film thickness) and evaporator
temperature [4].
6.2 INDUSTRIAL SCALE PURIFICATION OF ORANGE
PEEL OIL
The orange peel oil and essences are produced by the orange juice industry, as will
be explained in Sections 6.2.1, citrus industry and 6.2.2.1, purification of orange
peel oil.
With the objective of preparing products rich in aromatic compounds, compounds that are very important for flavor, the deterpenation process on those oils
and essences will be discussed in Sections 6.2.2.2 and 6.2.2.3.
6.2.1 CITRUS INDUSTRY
Citrus juice processing generates a large amount of wastes with almost 50% of the
fruit weight ending up as waste in the form of peel, segment, membranes, rags, and
seeds. Besides that, during juice concentration, volatile constituents contained in the
vapor phase are condensed by the aroma recovery systems. The depletion of these
flavoring components results in decreased juice quality. Unless these compounds
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are reincorporated back into the juice, the nutritional value will be diminished. The
aroma recovery system consists of two separate phases: essence oil (oil phase) and
aqueous essences (water phase). The average yield of these products is very low.
All these products are rich in aldehydes, esters, and other special volatile compounds.
As the storage and transportation of these products are expensive, the evaluation of
the concentration processes is of great interest [5]. It is very important that the
special light fractions and the concentration of flavoring volatile compounds from
the oil phase and peel oil be obtained by vacuum fractional distillation.
6.2.2 SWEET ORANGE OIL
Expressed sweet orange oil is primarily used for flavoring beverages, soft drinks,
ice cream, sweets, pharmaceutical preparations, and also perfumes. It contains
limonene, minor amounts of other monoterpene hydrocarbons, linalol terpineol,
aliphatic alcohols, and aldehydes such as octanal, nonanal, decanal, traces of the
sesquiterpenic aldehydes and alpha and beta sinensal, methyl anthranilate, coumarins, and waxes. For flavoring foods and beverages, the so-called concentrated
terpeneless oils or folded oils are preferred and are commercially available.
6.2.2.1 Purification of Orange Peel Oil
Sweet orange oil is expressed from the fresh peels of ripe citrus fruitsaurantium
L.var.dulcis (Citrus sinensis Os beck) (fam. Rutaceae). The tree probably originated
from the Far East between the Himalayas and Southwest China. It is now cultivated
in the United States (California, Texas, and Florida), throughout the Mediterranean
region (southern Italy, Sicily, Spain, Algeria, Tunisia, Morocco, Israel, and Cyprus),
South Africa, Brazil, and the West Indies.
The main producers of the equipment that expresses oil are the United States
and Cyprus, whereas the hand-pressed equipment is produced in Guinea [6]. Many
investigators have pointed out that the quality of citrus oils is dependent upon many
factors such as soil type, weather, method of extraction, and maturity of the fruit.
Citrus oils are located in the oval, balloon-shaped oil sacs or vesicles located in
the outer ring or flavedo of the fruit. Winton and Winton [1] described the exact
location of these oil sacs in their discussion of the microscopic structure of the
flavedo of the orange.
To extract the oil from the peel of citrus fruits, the oil sacs must be ruptured by
either pressure or rasping. The methods used in Florida were investigated by von
Loesecke and Pulley [6] and they revealed that extraction method had an effect on
the quality of the oil [7].
Cold-pressed citrus oils may be further processed to remove all or part of the
terpenes and sesquiterpenes. The resulting products are known as terpenefree, terpeneless, or sesquiterpeneless oils. For products with low sesquiterpene concentrations, the term terpeneless is generaly used. Citrus oils from which terpenes have
been removed are also called folded oils as the remaining flavorful oxygenated
compounds are more concentrated. The degree of concentration is often calculated
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Functional Food Ingredients and Nutraceuticals
from the ratio of the principal constituent in the concentrated oil to that in the prime
oil from which it was made.
6.2.2.2 Method of Deterpenation — Vacuum Fractional
Distillation
The bulk of terpeneless oils are produced by fractional distillation under high vacuum
(1 to 2 mm Hg) so that the oil boils at a relatively low temperature to ensure minimum
damage to the heat sensitive constituents during the long distillation period. At these
pressures the boiling points of the constituents may be too low for efficient separation, but as the terpenes are rarely separated individually, this is usually a design
problem, and reflux balance can be readily solved if necessary.
The aromatic vapors rise through a fractionating column that enables fractions
at defined boiling ranges to be separated and collected.
Depending on the nature of the starting material and the desired character of
the end product, the various fractions may be reblended as necessary and any
unwanted fractions rejected [8].
6.2.2.3 Folded Oils
Folded orange oil is the concentrated product obtained from the winterized oil of
fresh oranges. The process used will remove unneeded materials while retaining the
highly aromatic components. Folded oils are several times more potent than the
original oil and maintain the best fragrance. The two primary commercial varieties
of folded orange oil are 5-fold and 10-fold. The higher the fold (distillation) the
more concentrated is the finished product. Stable emulsions are more easily formed
with folded oils since they required less oil to produce the desired flavor and
fragrance levels [9]. The oil is primarily used as a flavoring agent for both foods,
beverages, and pharmaceutical products, and is 10 times stronger than the usual
orange oil.
Concentrated fractions with high contents of fragrant compounds such as ethylbutyrate, valencene, aldehyde C 10, and others, can be obtained. Their smell and
taste, that come from the original esters and aldehydes of the raw materials, are very
good.
Cold-pressed orange and grapefruit oils contain about 90% d-limonene.
D-limonene is a hydrocarbon and has the following structure
CH3
H2C
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D-limonene boils at 178ºC, and it is generally thought that d-limonene contributes very little to citrus flavor. However the oil flavor and taste can be varied by
adjusting the d-limonene concentration. In general, the term five-fold oil is used
when starting with 5 lb of cold-pressed orange oil and distilling 4 lb; the remaining
1 lb is referred to as five-fold oil.
The folding of oils is surrounded by secrecy and mysticism and various flavor
houses have developed their own methods. Some use solvent extraction, usually
about 70% ethanol followed by chilling to separate the extracted oil.
Since d-limonene boils at about 27ºC and 2 mm Hg, and reliable mechanical
vacuum pumps are available, it is possible to fractionate d-limonene at 5 to 10 mm
Hg absolute pressure. This is probably the most common method used today. There
are some low boiling point esters and aldehydes that are distilled with d-limonene,
and their separation and recovery is presently the subject of research.
6.2.2.4 Uses and Applications
Folded orange oils are used as a food ingredient and also as a flavor enhancer in
beverages. They can be incorporated into new and existing formulations to enhance
fragance and color. A wide array of industrial applications from general purpose
cleaning to air fresheners have been enhanced by the addition of folded orange oils
[10].
Original citrus oils are characterized by the presence of large percentages of
terpenes (C10/H16) and smaller amounts of sesquiterpenes (C15/H24).
Both of them, original and folded oils, carry oxygenated compounds comprising
alcohols, aldehydes, ketones, acids, and esters, which are responsible for the characteristic odor and flavor profiles. The terpenoid composition of various original
citrus oils is similar, and their principal component is d-limonene. The terpenes
possess little intrinsic odor or flavor value but it would be incorrect to say that they
have no flavoring effect. An original citrus oil from which the terpenes have been
removed is significantly flatter and lacks the characteristic freshness associated with
a complete peel oil [8]. When part of its terpenes is removed, folded oils will be
obtained.
6.2.2.5 Experimental Results
Figure 6.1 illustrates the industrial production of essences, as oil and water phases.
In one of the stages of the evaporator, there is a column where the volatile compounds, the oil and water phase, are evaporated and recovered in a tank.
The Essence Recovery System is connected to TASTE evaporators (Thermally
Short Time Evaporator) which have the first juice stage in the first evaporator effect
(that is, the first stage is the hottest effect or boiler steam effect). The essence-bearing
vapors are “boiled-off” with the water vapors in the first stage. The vapors are
condensed in the shell side of the second stage (second effect). The essence-bearing
vapors rise to the top of the shell side of the second stage and through a series of
baffles in the tube nest to remove as much condensable water vapor as possible. The
noncondensable portion of these vapors is removed through the vent line at the top
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Back pres regulator
& strainer
Product &
noncondensable cooler
Ammonia surge
drum
Noncondensable
Water out
Dual pressure
Relief valve
Overhead
condenser
Reflux
chamber
Sporian UMC-DA-5
Liquid level control
Matering
valve
Barometric isolation valve
Off
Atm steam
Water out
valve
Column vacuum
gauge
On
Hot water to hot well
No 2 T/N vacuum
Essence vacuum
gauge
Bottom dump
NH3 liquid out
2 T/N
NH3 liquid in
Scrubber
Stripping cal
& reboiler
Condenser
Essence recovery
column assy
Flow meter
500 Gal
cold wat
tank
Ammonia surge
drum
Essence storage tank
Water
To hot well
Botton seal
tank
Essence valve
Water to
baro cond
Hot well
Steam header
To essence vacuum gauge
FIGURE 6.1 Recovery system of water phase plus oil phase in the orange juice industry.
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Barometric
condenser
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of the tube, which is connected to the barometric condenser with a bypass for the
essence system. This is also repeated in the next tube.
When the essence system is “on” the noncondensable vapors are routed to the
essence system through remote-controlled air-operated valves, and the vent line to
the barometric condenser is shut off.
The essence vapors enter the essence system at the vent condensers. The vent
condenser condenses approximately 90% to 95% of the essence product. Cold water
is used as a condensing medium.
The essence product (condensed) flows downward through a single line to a dirt
trap.There are two lines running up from the trap: one line is the essence product
line running to the product chillers, the second line is the reflux line, which is used
to control the strength of the essence. The amount of reflux flow is dependent on
the desired strength of the water phase essence. The reflux rate is controlled only
by the amount of product taken from the system. There is a constant amount of
essence product generated by the overhead condenser, the essence product that does
not continue through the system flows back to the tube nest as reflux.
The essence product flows from the dirt trap to the double-circuit chiller condenser. This unit uses flooded NH3 refrigeration on the shell side. One circuit of the
chiller-condenser is used to chill the essence product, and the other circuit is used
to further condense the noncondensable vapors that rise from the vent condenser.
The second circuit (noncondensable) of the chiller condenser is connected to
the bottom of the refrigerated condenser. This condenser is a vertical shell and tube
heat exchanger using flooded NH3 on the shell side. The noncondensable vapors
flow through the tubes in a final condensing stage. The chilled product flows to the
decanter assembly. The remaining noncondensable vapors flow upward and into the
evaporator barometric condenser.
The product lines from the bottom of the scrubber and the final condenser run
to the decanter assembly located in the cold wall storage tank. The product line from
the bottom of the scrubber to the decanter assembly has a flow meter located in the
line. This flow meter measures product flow into the decanter assembly.
The essence system product flow is created by vacuum generated by the evaporator barometric condenser. There is a continuous vacuum path from the evaporator
tube to the evaporator barometric condenser, through each unit. The vacuum in the
essence system is controlled by remote controlled valves [3].
The purification process of orange peel oil and orange essence oil at Flavor Tec
is described by Figure 6.2. Peel oil, oil phase, and water phase, are by-products of
the citrus processing plants. These products can be concentrated to obtain folded
oils and folded essences (in the case of the oil phase and water phase), besides the
special light fractions and terpenes. Figure 6.2 shows the flow diagram for the
processing of raw materials at Flavor Tec. As shown, the light fractions and folded
oils or essences, are processed by vacuum fractional distillation.
As shown in the first step of Figure 6.2, the volatile compounds that will be the
raw materials for the FTNF (From The Named Fruit) aromas, are obtained from the
natural fruit in the citrus processing plant, as follow: the peel oil comes from the
peel in a special process of washing, cleaning, centrifugation, and dewaxing. The
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Functional Food Ingredients and Nutraceuticals
Raw material (fruit)
Volatile compounds - peel oil, oil phase,
water phase
Distillation/fractional process
Terpenes
Folded oils/folded
essences
Light fractions
Selling
FTNF aromas
Analytical evaluations
Shelf-life study
Juice addition
Shelf-life study
FTNF = From the named fruit
FIGURE 6.2 Citrus oils concentration at Flavor Tec by distillation.
oil phase and water phase are also recovered by the citrus juice process, as shown
in Figure 6.1.
In the Flavor Tec process these raw materials feed the distillation equipment to
produce the folded oils or folded essences, plus terpenes and light fractions by high
vacuum fractional distillation (second and third steps in Figure 6.2).
By blending the different percentages of the folded oils, folded essences, and
light fractions, the FTNF aromas are produced at Flavor Tec. The percentage of each
of the compounds, will change according to the needs of the final flavor, for example,
freshness, sweetness, ripeness, and so forth. All the distilled products are evaluated
by physicochemical and sensory analytical methods.
After the analytical evaluations of all the obtained products are completed, their
shelf life at room temperature and cold store will be studied. The shelf life of the
FTNF aromas used in citrus juices will also be evaluated.
Based on these evaluations it will be possible to guarantee the quality of the
aromas, as well as their addition to the final feed. A typical industrial fractional
vacuum distillation plant is shown in Figure 6.3. In order to increase the yield and
quality of the final products at Flavor Tec, the distillator is continuously fed with
raw materials 24 hours a day.
Figure 6.4 shows the chromatographic profile of a 10-fold orange oil. Figure
6.5 shows the profile of the orange terpene obtained during the distillation process.
The average yield of the terpenes on the process of a 10-fold oil is about 90% and
about 80% on the process of a 5-fold oil. The purity grade of d-Limonene is a
minimum of 96% by high-resolution gas chromatography (HRGC). The different
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FIGURE 6.3 Vacuum fractional distillation at Flavor Tec.
concentration of each component of single, 5-fold, 10-fold oils and terpene can be
seen in Table 6.1.
The values of the components as area percentage (area %) were analyzed by
HRGC, using the following analytical conditions:
Gas Chromatograph Shimadzu, 14BPFSC, 115/220 V AC (50/60 Hz) and 2
KVA max. capacity. Detector: Flame Ionization; T = 220ºC. Injector: Split / splitless
(Split = 1:100);T = 200°C. Column: Capillary – Shimadzu, Type bonded phase.
Phase: CBP1 (nonpolar). Size: 30m × 0.25 mm. Film thickness: 0.25 micron. Material: Fused silica. Temperature program = 40ºC to 190ºC (4ºC/min).
The area percentage (HRGC) of each identified component of the orange essence
oils, single, 5-fold, and 10-fold products, is listed in Table 6.2. There are more
aromatic or fragrant components in the essence oil as compared to peel oil, as can
be seen in Tables 6.1 and 6.2. It is therefore advisable to remember that the peel oil
is obtained from the orange peel while the essence oil is from the concentration of
the orange juice. The latter has fresher compounds than the former . Special attention
might be given to the valencene content in the folded oil phases. This component
is very important for the fruit taste characteristic. During the concentration process
of the essence oil, special light fractions can be obtained, if processed under ideal
conditions. Table 6.3 shows the concentration of one of these fractions, obtained
from the essence oil (oil phase). It is a very light fraction because it is rich with top
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3
7
5
9
11
10
12
8
2
1
4
1
2
3
4
5
6
7
8
9
10
11
12
6
α - Pinene
Sabinene
Mircene + Octanal
Phelandrene
D – Limonene
Nonanal
Linalol
Citronelal
Decanal
Neral
Geranial
Valencene
FIGURE 6.4 Chromatographic profile of 10-fold orange oil (HRGC).
note components, for example, ethyl-butyrate and octanal, as can be seen in Figure
6.6.
Special attention might also be given to the high level of the light components,
for example, hexanal and ethyl-butyrate, which are very important for the top notes
of a flavor. The FTNF aromas are produced by blending the folded oils and oil
phases plus the special light fractions, as show in Figure 6.2.
At Flavor Tec, this blending is done with different fractions, as show in Figure
6.2. The FTNF aromas are produced by blending the folded oils and folded oil
phases. It depends on the desired characteristics of the final flavor, for example,
fruity, fresh, green, and so forth. As an example, Table 6.4 shows the concentration
of some components of two different FTNF aromas called A and B. They are products
of Flavor Tec, after concentration of the raw materials and blending with light
fractions.
The main differences between the aromas A and B are: A is fresher than B, as
demonstrated by its ethyl-butyrate content, and B has more juice note, as shown in
its valencene content.
These special characteristics of each component will give unique flavor notes
when added to the final food product.
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Purification of Orange Peel Oil and Oil Phase by Vacuum Distillation
3
5
1
2
4
1
2
3
4
5
6
7
8
6
7
8
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-limonene
Nonanal
Linalol
Citronelal
FIGURE 6.5 Chromatographic profile of orange terpene (d-limonene) (HRGC).
TABLE 6.1
Area Percentage by HRGC of the Identified Components of
the Orange Peel Oils in Several Concentrations
Evaluated Products (area %)
Pick No.
Components
1
2
3
4
5
6
7
8
9
10
11
12
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-limonene
Nonanal
Linalol
Citronelal
Decanal
Neral
Geranial
Valencene
© 2007 by Taylor & Francis Group, LLC
Single Oil 5-Fold Oil 10-Fold Oil Terpene
0.49
0.32
2.19
0.15
95.36
0.03
0.38
0.04
0.24
0.06
0.10
0.04
0.31
0.20
1.54
0.10
91.92
0.11
1.01
0.13
1.15
0.29
0.50
0.14
0.13
0.13
1.08
0.05
81.71
0.10
2.08
0.35
3.07
0.92
1.51
0.58
0.53
0.43
2.35
0.16
96.30
0.02
0.13
0.01
–
–
–
–
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Functional Food Ingredients and Nutraceuticals
TABLE 6.2
Area Percentage by HRGC of Some Components of the Orange
Essences or Orange Oil Phases in Several Concentrations
Evaluated Products (area %)
Pick No.
Components
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Hexanal
Ethyl-butyrate
T-2-hexanal
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-limonene
Nonanal
Linalol
Citronelal
Decanal
Neral
Geranial
Valencene
Single Oil Phase 5-Fold Oil Phase 10-Fold Oil Phase
0.01
0.03
0.01
0.38
0.34
2.17
0.08
94.57
0.10
0.70
0.01
0.30
0.11
0.10
0.23
0.29
0.14
0.02
0.93
0.34
1.46
0.09
85.75
0.09
2.13
0.13
1.21
0.54
0.49
2.31
0.71
0.33
0.06
2.10
0.67
2.30
0.12
68.20
0.14
2.78
0.57
3.70
2.04
1.00
4.08
TABLE 6.3
Area Percentage by HRGC of the Main Components
of the Light Fraction of the Orange Oil Phase
Pick No.
Components
Oil Phase Light Fraction (area %)
1
2
3
4
5
6
7
8
Hexanal
Ethyl-butyrate
T-2-Hexanal
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-limonene
5.38
2.54
0.41
13.84
4.34
12.76
0.64
57.51
6.3 CONCLUSIONS
Fractional distillation has been shown to be an efficient process. At high vacuum, it
is a safer process from an operational point of view, but also much gentler on the
raw materials, because of the short residence time and low distillation temperature.
This results in better fragrant characteristics in the final products. To obtain topquality products, the raw materials, orange peel oil and oil phase, need to be well
preserved from harvest until their use in the process.
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Purification of Orange Peel Oil and Oil Phase by Vacuum Distillation
1
4
2
3
171
6
5
8
7
1
2
3
4
5
6
7
8
Hexanal
Ethyl-butyrate
T-2-hexanal
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-Limonene
FIGURE 6.6 Chromatographic profile of special light fraction of orange oil phase (HRGC).
TABLE 6.4
FTNF Orange Aromas (A and B) HRGC Data
Pick No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Components
Sample A (area %)
Acetaldehyde
Hexanal
Ethyl-butyrate
T-2-hexanal
α-Pinene
Sabinene
Mircene + Octanal
Phelandrene
D-limonene
Nonanal
Linalol
Citronelal
Decanal
Neral
Geranial
Valencene
TR
0.27
0.18
0.10
1.23
0.60
2.98
0.17
87.36
0.05
1.02
0.12
1.38
0.40
0.52
0.53
© 2007 by Taylor & Francis Group, LLC
Sample B (area %)
TR
0.10
0.05
0.05
0.74
0.28
1.91
0.09
86.08
0.10
1.70
0.17
1.80
0.54
0.80
1.17
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Functional Food Ingredients and Nutraceuticals
It is well known that single citrus oils and essences can come in large volumes
from the industry. The storage and transportation costs of these products are high,
which is another reason why more interest is been shown in the concentrated
products. Research results have shown limonene to be a good candidate for human
clinical chemoprevention trials. Figure 6.4, Figure 6.5, and Figure 6.6 with chromatograms and Table 6.1, Table 6.2, and Table 6.3 of the main components that
were obtained from this work at Flavor Tec, show that the folded oils, essences, and
the aromas, besides their fragrant components, also maintain good levels of dlimonene concentration when added to juice and other beverages. Although considerable differences have been observed with these preparations, wide acceptance by
sensory panels has been achieved. It is very important to note that these folded oils
and aromas are useful in both aspects: (1) as functional foods, because of the
limonene content, and (2) the taste and fragrance, because of their fragrant compounds such as esters, aldehydes, and so forth. It is very important to bear in mind
that the aroma is not a curative but will in general enhance food products.
REFERENCES
1. PE Chemical Engineering Review by Wagar Ahmad, PhD. Binary distillation weight
and mole fractions, www.reviewpe.com/pe/samples/b6_7htm, (retrieved October 5,
2004).
2. Steam distillation. Infuse your life with excellence, www.serene-aromateraphy.de/
steamdistillation.html, (retrieved October 28, 2004).
3. Redd, J.B., Hendrix, D.L., and Hendrix, C.M.Jr., Process procedures, Quality Control
Manual For Citrus Processing Plants, Ag Science, Inc., Auburndale, Florida vol II,
section I, division B, 1992.
4. Myers Vacuum, Molecular distillation: the unique principle behind a unique process,
www.myers-vacuum.com/mv1.html, (retrieved November 3, 2004).
5. Bettini, M.F.M., Orange concentrated essences by fractional distillation, presented at
25th IFU – Symposium, Stuttgart, Germany, May , 2004.
6. Formacek, V. and K.-H. Kubeczka, Orange oil, sweet, Essential Oils Analysis by
Capillary Chromatography and Carbon-13 NMR Spectroscopy, John Wiley & Sons,
New York, 185, 1982.
7. Kesterson, J.W., Hendrickson, R. and Braddock, R.J., Introduction, review of literature, Florida Citrus Oils, technical bulletin 749, Gainesville, FL, 3,1971.
8. Heath, H. B, London, England and Reineccius G., Dept. of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota, Flavor Chemistry and Technology,
England, 1986.
9. Florida Chemical Company, Inc., Folded orange oil, www.d-limonene.com/
datasheets/foldedorangeoil.html, (retrieved October 6, 2004).
© 2007 by Taylor & Francis Group, LLC
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7
Dehydration
Technologies to Retain
Bioactive Components
Valérie Orsat and G. S. Vijaya Raghavan
Macdonald Campus of McGill University
CONTENTS
7.1 Introduction ..................................................................................................173
7.2 Drying of Biological Materials....................................................................174
7.3 Heating of Biological Materials ..................................................................175
7.4 Quality Changes in Biological Materials ....................................................176
7.5 Artificial Drying...........................................................................................179
7.6 Convective Drying .......................................................................................180
7.7 Drum Drying ................................................................................................180
7.8 Spray Drying ................................................................................................181
7.9 Fluidized and Spouted Bed Drying .............................................................181
7.10 Freeze Drying...............................................................................................182
7.11 Vacuum Drying ............................................................................................183
7.12 Refractance Window Drying........................................................................183
7.13 Solar Energy in Drying................................................................................184
7.14 Combination Drying ....................................................................................184
7.15 Microwave-Vacuum Drying .........................................................................186
7.16 Heat Pump Hybrid Dryers ...........................................................................186
7.17 Conclusion....................................................................................................187
Summary ................................................................................................................187
Acknowledgment ...................................................................................................188
References..............................................................................................................188
7.1 INTRODUCTION
The phenomenal growth in the use of phytochemicals in functional and nutraceutical
foods forces the food industry to control the quality of those ingredients in terms
of efficacy and safety of the ingredients claiming to be health promoting.1 The
173
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industry must ensure that there are governing guidelines during processing to ensure
the nutritional and functional activity in the finished products.
The choice of drying system depends upon the annual volume produced, the
marketing pattern, the material to be dried, and the capacity and nature of existing
facilities. Presently, most commercially available dryers are based on heated air
(convection dryers); however, there has been substantial research aimed at adapting
other technologies to agroproducts drying. Two general areas are involved: (1)
improvement of heated air dryers to improve energy efficiency and eliminate overdrying (e.g., fluidized and spouted beds concepts), and (2) alternate modes of heat
and mass transfer. Among the alternative modes are conduction drying, infrared
drying, microwave drying, and heat pump drying. Although improving fuel efficiency
is one of the main motivations for continued research, several other advantages may
be sought. Among them are faster drying, better quality of dried products, and use
of renewable energies; however, advances in one direction often require a trade-off
somewhere else.
7.2 DRYING OF BIOLOGICAL MATERIALS
The removal of moisture prevents the growth and reproduction of microorganisms
which cause decay and minimizes many of the moisture driven deterioration reactions. Differences in relative humidity in the environment in which the food is kept
can make differences in the rate of microorganism multiplication. Water activity is
related to the relative humidity. Relative humidity, RH, is defined as the ratio of the
partial pressure of water vapor in the air to the vapor pressure of pure water at the
same temperature. Water activity, aw , is a property of solutions and is the ratio of
vapor pressure of the solution compared with the vapor pressure of pure water at
the same temperature. Under equilibrium conditions water activity equals RH/100.
At the usual temperatures permitting microbial growth, most bacteria require a water
activity in the range of about 0.90 to 1.00. Qualitatively, water activity is a measure
of unbound, free water in a system available to support biological and chemical
reactions. Water activity, not absolute water content, is what bacteria, enzymes, and
chemical reactants encounter and is affected by at the microenvironmental level in
food materials. Dehydration targets to lower the product’s aw to reach an equilibrium
between product quality and product shelf life.
The quality of the dried product and its cost are greatly influenced by the drying
operation. The drying time, temperature, and water activity influence the quality of
the final product. Low temperatures generally have a positive influence on the quality
of biological materials, but require longer processing times, which have a detrimental
effect on quality and a higher cost of operation. Low water activity retards or
eliminates the growth of microorganisms, but results in higher lipid oxidation.2
Storage stability of a food product increases as the water activity decreases, and
lipid-containing foods are susceptible to lipid oxidation at low water activities and
must be stored in oxygen impermeable packages. In certain circumstances, there is
poor color retention when the food is dried too rapidly. Other food materials
have different drying and handling problems to which targeted solutions must be
developed.2
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Drying is a process of simultaneous heat and mass transfer for moisture removal.
External heat evaporates the surface moisture, while the internal moisture can be
forced to the surface and then evaporated or it can be evaporated internally and then
transported to the surface. The transfer of heat depends on the air temperature, air
humidity, air flow rate, pressure, surface area, physical nature of the material, its
composition, and the process by which the heat is transferred to the material whether
it be by conduction, convection, or radiation.
To speed up the heat and mass transfer in the fruits, vegetables, or plant materials
to be dehydrated, they are cut into small pieces or thin layers. Increased surface area
provides more contact with the heating medium and more surface from which
moisture can escape. The greater the temperature gradient between the heating
medium and the food, the greater will be the rate of heat transfer into the food,
which provides the driving force for moisture removal. The hotter the air, the more
moisture it will be able to carry away from the drying product.
In convective drying, the drying rate is generally high at the beginning of the
process since convective drying depends largely on the concentration of moisture at
the surface. Afterward, the drying rate falls rapidly with gradual reduction of moisture. With thermal treatment under convection, the limitation resides in the fact that
in order for the center of the material to reach the desired temperature, the surface
may have to be overheated, leading to losses in product quality. This is where
dielectric heating can benefit with its volumetric heating where selective absorption
of energy by the water molecules takes place inside the product.
7.3 HEATING OF BIOLOGICAL MATERIALS
In convection heating, the heat transfer occurs between the surface of a material and
its surrounding fluid. The rate of transfer then depends on the properties of the fluid
and the fluid flow. The characteristics of the fluid such as density, specific heat, and
so forth, are expressed in the convective heat transfer coefficient which also takes
into account the geometrical shape of the object undergoing heating.
In conduction heating, the heat transfer mode involves energy transfer from
regions of higher to lower temperatures by contact between matter. The rate of heat
transfer is then proportional to the temperature gradient from the hotter region to
the colder region and the capacity of the material to conduct the heat.
Electroheat is defined as the branch of science and technology dealing with the
transformation of electrical energy into thermal energy for a given purpose.3 Electrotechnology applications range over the entire electromagnetic spectrum, with
ultraviolet radiation and x-rays in the high end (>109 MHz); infrared radiation (107
MHz); microwaves (915 to 22,125 MHz); radio frequency (0.5 to 900 MHz) and
induction and resistance heating in the lower frequency range. Research interest in
agrifood applications lies mostly in high-frequency applications with dielectric,
induction, and resistance heating.
High-frequency electromagnetic heat transfer, in the microwave and radio frequency range, can be considered as heat arising from the oscillation of molecular
dipoles (microwave) and the movement of ionic constituents (radio frequency) in
response to an alternating electric field. Unlike conventional energy, this energy is
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absorbed throughout the volume of the wet material. The evaporation of moisture
within the material results in an increase of the internal pressure which can rapidly
drive out the moisture from the interior of the solid.
Microwave cooking/heating is the most rapidly expanding meal preparation
technology in the world. Its effect on the functional activity of phytochemicals is
not well defined and more research is needed in this area. Processing as a whole
affects the content, activity, and availability of biological compounds, hence there
is a need to better understand the mechanisms affecting the nutraceutical stability
of functional foods.
The degree to which a given material responds to an electromagnetic field
depends on the frequency and intensity of the field and on the characteristics of the
material itself. The electrical basis of interaction is described in terms of the dielectric
properties: dielectric constant and dielectric loss factor. These, respectively, represent
the proportion of impinging energy that can penetrate the material, and the amount
of energy that can be absorbed. The dielectric properties of materials are of major
importance in the consideration of high-frequency or microwave applications with
biological materials. Fundamental studies aimed at characterizing the dielectric
responses of complex biological materials over a wide range of frequencies, temperatures, and moisture contents are still required to be undertaken.
Resistance or ohmic heating is the term used to describe direct electrical resistance of food products. The process allows the rapid, uniform heating for highquality processing of liquid food products. Direct resistance heating uses the fact
that metallic materials, insulators, and foodstuffs are highly conductive at main
frequencies so that the source energy is dissipated ohmically. In such applications,
the material comes in direct contact with the electrodes.
Induction heating on the other hand has not had many applications with biological materials but has rather been used in the metal industry. The majority of
applicators for induction are based on coils wrapped around the workpiece. The
current flowing in the coil establishes an axial magnetic flux which in turn sets up
a circumferential current in the material, the depth of which depends on the operating
frequency. The flowing current is responsible for imparting electrical energy to the
material.4
7.4 QUALITY CHANGES IN BIOLOGICAL MATERIALS
Conditions in drying and thermal processing influence greatly the quality of processed biological materials. Browning reactions occur in the presence of high heat
with lower moisture causing decrease in nutritional value, textural changes, and
overall quality. Color loss, shrinkage, denaturation, loss of aroma and flavor, vitamin
loss, and textural changes are important quality changes affected by thermal processing. Degradation of nutrients, color, texture, and so forth during dehydration
can be described by a first order model of the form: X = Xo exp (-kt), for constant
processing conditions.5 Thermal processing has often been demonstrated to have a
negative effect on the nutrient content. As the temperature of the product increases,
the reaction rate constant is increased. As such, a low-temperature drying process
results in lower nutrient degradation. Hence, a longer constant-rate drying period
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177
should be favored for quality retention. The drying process of choice must target to
minimize chemical degradation reactions since loss of nutrient can be viewed as the
decomposition of a particular chemical compound.
Importance has to be given to the appearance and functional properties of the
dried products.6 In a study conducted by Nindo et al.,7 it was demonstrated that the
drying method significantly affects the antioxidant activity of dried asparagus. The
drying methods studied were tray drying, spouted bed drying, microwave spouted
bed, refractance window, and freeze drying; freeze drying and refractance window
obtained the highest retention of total antioxidant activity.
In the case of folate, its stability in dried food is mediated by the content of
ascorbic acid. During thermal processing, levels of folate and ascorbic acids may
both decrease depending on the intensity of the process. In a study conducted by
Stralsjo et al.,8 folate retention during drying of rosehip berries was determined.
Folate degradation was shown to be influenced by drying time and drying temperature. The duration of drying appears most important at higher water activity (greater
than 0.75).
The major chemical components of chamomile were studied after the flowers
were dried by different drying methods, namely, sun drying, shade drying, and oven
drying at 40°C.9 The drying methods studied had no effect on the different components present; however, the drying methods had a significant effect on the proportion
of the various components and their retention. Shade drying yielded the highest oil
yield, whereas oven drying was better suited for concentrating particular components
such as isobutyl and isobutyrate since oven drying vaporized some of the more
volatile components.
In order to optimize practices for thermal processing and drying of biological
materials, extensive research has been carried out around the world on various drying
methods, such as fluidized bed drying, spouted bed drying, particulate medium
conduction drying, flash drying, freeze drying, spray drying, drum drying, tunnel
convective drying, tray drying, microwave drying, radio frequency drying, infrared
drying, electric field drying, superheated steam drying, osmotic dehydration drying,
conveyor dryers, impingement drying, solar drying, rotary drying, and so forth.10
All drying methods have been developed to optimize the drying of target materials,
where not all drying processes are suitable for all types of biological materials.11
The quality of biological materials is so specific that drying of each product should
be studied on an individual basis. In the case of biomaterials, their phytochemicals’
content and quality must be maintained during drying as research is demonstrating
their ability to provide health benefits.1
Phytochemicals are very reactive and their content varies depending on plant
cultivar, ripeness at harvest, storage conditions, and level of processing. Production
of vegetal ingredients must ensure the preservation of their inherent quality attributes.
In general, the concentration of bioactive compounds will steadily decrease during
processing. The development of processing methods to minimize the loss of bioactive
components is imperative.12
When processing plant materials, a variety of changes will occur to the antioxidants present. Changes in the functionality of the antioxidants depend on the
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Functional Food Ingredients and Nutraceuticals
energy transfer, oxygen availability, temperature, food composition, time, and light
exposure.13
Retention of chlorophyll and ascorbic acid from cabbage samples air dried (60°C
to 85°C) followed by microwave vacuum drying is higher than in samples air dried
only. It is recognized that rapid drying can retain more chlorophyll and nutrients in
the dried product.14
In drying with hot air, the processing time and temperature are typically long
and high. The material’s natural protective layer is destroyed, exposing the antioxidants to the free access of air with rapid lipid oxidation which in turn destroys the
functionality of antioxidants. Lipid oxidation and antioxidant losses can be reduced
by short drying times, low temperatures, and low pressures.13
A study was conducted by Piga et al.15 on the influence of drying air temperature
on the phenolic compounds and antioxidant activity of dried plums. High temperature drying destroyed anthocyanins and significantly reduced flavonoids and ascorbic
acids. The analysis is, however, more complex when studying the functional properties of the dried foods since during processing the polyphenols in their intermediate
stage of oxidation may have a greater antioxidant power than initially, and high
temperature treatments may cause the formation of new compounds, or degradation
components that have a higher antioxidant activity.
In the case of thermodegradable vitamin C in wild-rose fruits (Cinebasti fructus),
Jinescu et al.16 confirmed that the presence of a large amount of polyhydroxilic acids
such as malic and citric acid improves the stability of the vitamin C during drying.
Furthermore, it was determined that by allowing a greater maximum allowable
moisture content of the dried product, lower process temperatures and longer drying
times ensured the maximum retention of vitamin C. Similar results were obtained
by da Silva et al.17 for hot air drying of camu-camu slices (Myrciaria dubia). The
tree fruit berries were dried in a vertical tray dryer. The highest ascorbic acid retention
of 78% was obtained with the lowest drying temperature of 50°C and highest final
moisture content of 10% (wet basis).
A predictive model was developed by Ramallo and Mascheroni18 for predicting
vitamin C retention during hot air drying of pineapple slices. Excellent predictive
agreement was obtained with maximal retention of ascorbic acid when the drying
temperature was kept at the lowest temperature tested of 45°C.
In a study on processing of tea leaves, it was clearly demonstrated that type and
intensity of processing causes a significant effect on their total polyphenolic content
and their antimutagenic activity. In the case of tea leaves, low intensity processing
is recommended.19
There is no doubt that food processing, and specifically heating, affects the
properties of processed products. However, it does not always have a detrimental
effect. In the case of lycopene in the processing of tomatoes, the lycopene degradation occurs through isomerization and oxidation. Heat induced isomerization converts the all-trans lycopene to its cis form which has lower stability. However,
lycopene absorption as a health promoting nutrient, is higher in the cis-isomeric
form than in the unprocessed trans form.20 When considering a processing method,
a good balance must be maintained between the stability and quality of the product
and its functionality as a nutritious and health-promoting food ingredient.
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Dehydration Technologies to Retain Bioactive Components
179
Some other interesting plant components are also favored by a thermal treatment.
This is the case of taxol, a plant-based cancer chemotherapeutic agent used in the
treatment of certain cancers. In a thin layer drying study of taxus clippings, it was
demonstrated that the lowest yields of the plant component were obtained when
drying took place at the lowest temperature setting of 30°C, while the highest yield
was obtained with the highest temperature setting studied of 60°C.21
7.5 ARTIFICIAL DRYING
Several types of dryers and drying methods, each better suited for a particular
situation, are commercially used to remove moisture from a wide variety of food
products and biomaterials. While sun drying of crops is still practiced for certain
fruits such as plums, apricots, grapes, and dates, atmospheric dehydration processes
are used for apples, plums, herbs, and several vegetables; continuous processes such
as tunnel, belt through, fluidized bed, and foam-mat drying are mainly used for
vegetables.
Spray drying is suitable for fruit juice concentrates, and vacuum dehydration
processes are useful for low moisture-high sugar fruits like peaches, pears, and
apricots.
The selection of a particular dryer or drying method depends on the type of raw
material and its properties (Table 7.1), the desired characteristics of dried product,
and the restrictions on the operating conditions and costs.
TABLE 7.1
Common Dryer Types for Liquid and
Solid Foods
Dryer Type
Usual Food Type
Air convection dryers
Kiln
Cabinet, tray or pan
Tunnel
Continuous conveyor belt
Belt trough
Air lift
Fluidized bed
Spray
Drum or roller dryers
Atmospheric
Vacuum
Vacuum shelf
Vacuum belt
Freeze dryers
Pieces
Pieces
Pieces, slurries, liquids
Pieces
Pieces, slurries, liquids
Pieces
Small pieces, granules
Small pieces, granules
Liquid, slurries
Pieces and slurries
Pieces, slurries, liquids
Pieces, slurries, liquids
Pieces, slurries, liquids
Slurries, liquids
Pieces, liquids
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There are three basic types of drying process: mainly, sun and solar drying;
atmospheric drying, including batch or continuous (kiln, tower, and cabinet dryers,
tunnel, belt, belt trough, fluidized bed, explosion puff, foam mat, spray, drum and
microwave); and subatmospheric dehydration (vacuum shelf/belt/drum and freeze
dryers).
7.6 CONVECTIVE DRYING
Convective drying consists of passing heated air through layers of product. It can
be conducted with tray or cabinet dryers, where perforated trays hold thin layers of
materials. Tunnel dryers are similar to cabinet dryers, where trays of material are
stacked and moved through a tunnel. For a continuous process, conveyor belts are
used.
A study conducted by Mendez et al.22 investigated the effect of convective hot
air drying under constant conditions on garlic drying kinetics and quality. Garlic
slices were dried in a conventional hot air tunnel, under constant conditions. Three
air temperatures were compared (40°C, 50°C, and 60°C), in combination with two
air velocities (1 and 2 m/s). Allicin and pyruvic acid contents were measured as an
indicator of the effect of the process on garlic quality. The allicin content decreased
with rising drying air temperature, and moderate temperatures (40°C and 50°C)
allowed better allicin retention than did the higher temperature (60°C). In the case
of air-dried bay leaves, no significant loss of quality was experienced when drying
the leaves at 60°C.23 The level of thermal sensitivity is product dependent.
The effect of drying temperature on the quality of dried Echinacea purpurea
was determined in a study by Stuart and Wills.24 An increase in process temperature
from 40°C to 70°C had a significant effect on the concentration of cichoric acid,
whereas temperature had no effect on the concentration of alkylamides.
Larrauri et al.25 studied the effect of drying temperatures (60°C, 100°C, and
140°C) on the polyphenol content and antioxidant activity of dried red grape pomace.
When drying at higher temperatures, a significant reduction in total extractable
polyphenols and condensed tannins was observed along with a reduction in total
antioxidant activity. Drying at the lower temperature of 60°C did not significantly
affect the functionality of the dried grape pomace.
7.7 DRUM DRYING
In drum drying, heat is transferred to the material by conduction with heated revolving drums (Figure 7.1). The dried product is flaked off the drum with a scraper.
Drum dryers have high drying rates and high energy efficiency. Typically, the
temperature of the process is approaching the boiling point of water and the process
time is in the order of seconds. This process is adequate for viscous or pureed foods
that can withstand high temperatures for short time.26
A study was conducted by Hsu et al.27 to determine the antioxidant activity of
yams dried by various methods, including drum drying. In their study, freeze drying,
hot air drying (60°C) and drum drying (95°C) all reduced the antioxidant activity
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Liquid feed deposited
between heated rotating
drums
Knife
Dry
product
FIGURE 7.1 Schematic of drum drying process.
of the dried yam when compared to fresh yam. Drum drying, with its high temperature, had the highest reduction of total antioxidant activity.
7.8 SPRAY DRYING
Spray drying is the transformation of a fluid into a dry particulate by spraying in a
hot medium. Hot air spray drying takes place at temperatures between 150°C and
200°C. The dried product may take the shape of a powder, granules, or agglomerates
depending on the physical and chemical properties of the fluid and the operating
conditions of the spray drying process.
Andrade and Flores28 studied the spray drying of roselle extract to exploit its
importance as a source of pectin, anthocyanin, and ascorbic acid. Operating factors
of importance are the feed rate, the inlet drying air temperature, and the pressure of
the compressed air at the nozzle. Not all materials can be spray dried. In some cases
the material requires a carrier, such as maltodextrine, to favor agglomeration and
provide stability for the components the drying process wants to retain. In spray
drying, as with other drying processes, the degradation of the phytocomponents
follows a first order kinetic.29
7.9 FLUIDIZED AND SPOUTED BED DRYING
Fluidized bed drying involves the fluidizing of particles in a flowing gas stream,
typically heated air. With the fluidized motion of the particles in heated air, drying
occurs rapidly. Fluidized bed drying offers a tight control of process and product
temperatures necessary for processing heat sensitive plant and food products.30 An
alternative to spray or fluidized bed drying for fluids has been investigated by Souza
and Oliveira31 with the spouting of a bed of inert material with warm air and spraying
the fluid to coat the material with a thin film which fragments into a powder as the
material collides in the spouted bed (Figure 7.2). The advantage of this process is
its simplicity and low cost while with the proper operating parameters, it can provide
high quality products with low degradation. Souza et al.32 demonstrated that spouted
bed drying produced end products with high concentration of active substances such
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Functional Food Ingredients and Nutraceuticals
07
06
150 mm
08
Caption
01 - Blower
02 - Valves
03 - Air heater
04 - Flow meter
05 - Spouted bed
06 - Atomizer
07 - Pressure taps
08 - Thermocouples
05
08
01
02
03
07
04
FIGURE 7.2 Schematic diagram of spouted bed dryer as tested by Souza et al.32
as total flavonoids and total tannins. An increase in the drying temperature increases
the degradation of the flavonoid compounds.33
7.10 FREEZE DRYING
Freeze drying operates at low temperatures and low pressures which can help reduce
quality degradation. Under vacuum, frozen water changes directly into vapor, a
process known as sublimation, yielding particular dried product characteristics. The
food pieces are frozen and vacuum is applied in the airtight drying chamber. Thermal
energy is then applied to the food pieces (by heated plates or microwaves) to promote
the sublimation process. In freeze drying, the food remains at a low temperature
yielding high retention of nutrients. Batungwanayo et al.34 studied freeze drying as
a drying method to preserve and concentrate allicin in garlic. Cloves of fresh garlic
were freeze dried either whole, or cut into halves and slices. Both freeze drying
temperature and size of garlic samples had an important effect on drying kinetics.
The allicin content of whole garlic cloves decreased with an increase of freezedrying temperature. However, for garlic slices the effect of the temperature was not
significant. Excellent retention of allicin was experienced with whole garlic samples
freeze dried at a temperature of 20°C.
A freeze drying study of medicinal herbs demonstrated that the quality of freeze
dried product was slightly lower than fresh material, but the quality remained
considerably higher than for oven-dried samples at 35°C to 40°C.35
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To preserve higher levels of alkylamides from Echinacea purpurea, freeze drying
was found to be the best drying method when compared with vacuum microwave
drying, which was in turn a better drying method than air drying at 70°C for drying
roots. In the case of leaves drying, air drying at 50°C was the best process for optimal
retention of alkylamides.36
7.11 VACUUM DRYING
In vacuum drying, the product is heated by steam, conduction (hot oil, heated
elements, etc.), or radiation, while under low pressure. This drying process may
offer product specific advantages such as low-temperature drying and low oxidation.
Kutovoy et al.37 studied the vacuum drying of grape by-products. When dried under
vacuum (500 mm Hg) at a temperature below 50°C, up to 95% of nutritious ingredients, vitamins, ferments, and other biologically active components were preserved.
7.12 REFRACTANCE WINDOW DRYING
Refractance Window™ drying is a novel drying method patented by MCD Technologies Inc. (http://www.mcdtechnologiesinc.com). The process uses circulating
water at 95°C to 97°C to carry heat to dehydrating materials which circulate in a
thin layer on a conveyor belt (Figure 7.3). The drying process is similar to drum
drying, except that the product temperature is much lower, between 70°C and 85°C
compared to 120°C to 150°C with drum drying. Abonyi et al.38 conducted a study
to determine the carotene losses in carrots dried using Refractance Window, freeze
drying, and drum drying. Carotene losses were slightly, but not significantly, higher
for Refractance Window than for freeze drying, whereas drum dried products experienced a considerable carotene loss caused by the more intense heating treatment.
Product
removal
Exhaust
air
Moist air extraction
Cooling
water
Blower
Air
Product
application
T
Cooling water out
Cooling water in
T
Water
heater
Mylar conveyor belt
Hot water pump
T - Temperature sensor
FIGURE 7.3 Schematic diagram of Refractance Window™ System.
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7.13 SOLAR ENERGY IN DRYING
In most tropical countries, a commercial dryer is too expensive to consider its
purchase. In countries where harvesting time occurs at the beginning of the dry
season, the most popular method of drying is exposure to the sun. Crops are often
left to dry in the field before harvesting. In some countries various crops are dried
on scaffolds or inverted latticework cones. At the village level, probably the most
common practice is to spread the harvested product on the ground on a specially
prepared area (e.g., matting, sacking, or concrete) exposed to the sun.
An alternative that is being encouraged in hot, dry countries of Asia and Africa
is solar drying. Solar heat is trapped with a solar collector constructed from an
aluminum sheet painted black.39 The collector may be fixed to the drying bin in such
a way that an air space exists between it and the bin wall. Energy absorbed by the
collector heats the ventilating air by a few degrees as it is forced through the air
space. Various solar energy dryers have been designed around the world. AbdelRehim and Fahmy40 developed a photovoltaic dryer with dual packed beds for drying
medicinal herbs. Their dryer is equipped with electrical heaters powered by a photovoltaic system.
Flat-type solar collector dryers have been designed in Poland with corrugated
absorbers equipped with a backup coal-fired air heater. The solar dryer is equipped
with variable speed fans to modulate the air flow according to transient changes in
solar irradiance and heat output.41
With solar energy, like any other drying process, the temperature and duration
of exposure to light have to be monitored to retain the functional properties specific
to the products being dried.
7.14 COMBINATION DRYING
Rapid drying of plant materials has always been problematic due to the high sensitivity of such products to heat. Indeed thermal treatments may have detrimental
effects on quality characteristics such as color, flavor, texture, and nutritional value.
For this reason, traditional methods of drying require modification and improvement.
Combination of processes can favor complementary process benefits. The traditional
methods of drying high-quality thermosensitive materials are sun drying (inexpensive, slow, and unpredictable), low temperature drying (slow), and freeze drying
(expensive, high-quality products). The potential of microwaves to reduce drying
time while preserving quality has been studied and demonstrated for a number of
agrifood commodities.42,43 The combination of osmotic dehydration for preconcentration, with microwave drying, implemented with convective air has been investigated for a variety of fruits.43–46 The osmotic preconcentration can remove 50% of
the initial weight of the fruits with dry sugar or concentrated syrup. Since the cost
of drying depends on the amount of moisture removed, predehydration can significantly reduce the cost of dried material production. It has been demonstrated by Pan
et al.47 that osmotic pretreatments can, in certain cases, preserve the quality of
subsequently dried products with respect to nutrient retention, compared to direct
drying. Microwaves further offer a great potential for energy savings (in comparison
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to freeze drying) due to the speed of drying and the direct coupling of the energy
with the material.
In a study conducted by Marcotte et al.,46 blueberries and cranberries were dried
using a hybrid technology consisting of pretreatment, osmotic dehydration, and
convective drying. The performance of this hybrid technology was evaluated in terms
of quality attributes such as color, taste, rehydration ratio, and water activity as well
as sugar and anthocyanin content. The most significant loss of anthocyanins took
place during osmotic dehydration for both berries. This was due to intensive mass
exchange between berries and the osmotic syrup. Convective drying at low temperatures gave dried berries of high quality.
Nindo et al.7 studied the combined microwave and spouted bed drying of asparagus and compared the quality of the dried product with refractance window-dried,
tray-dried, freeze-dried, and spouted-bed-dried products. Total antioxidant activity
of the dried asparagus after refractance window and freeze drying was significantly
higher than after tray drying, spouted-bed-drying, and combined microwave and
spouted bed drying. When comparing only methods using heated air, it is the
microwave spouted-bed-drying asparagus which retained the highest total antioxidant activity. Refractance window drying retained the highest level of ascorbic acid.
Hybrid drying of strawberries was studied by Venkatachalapathy and Raghavan.44 Berries were dried to 15% moisture content (wet basis) using a procedure
involving sample preparation, osmotic dehydration, and microwave-assisted drying
leading to a dried berry that was comparable in quality to a freeze-dried berry in a
much shorter time. Convection drying took 9 to 10 hours (Figure 7.4), compared to
2 to 3 hours for microwave-convection dried berries.44
Convection with treat
0.2 W/g
Convection no treat
Moisture, % (dry basis)
1200
1000
0.1 W/g
0.3 W/g
800
600
400
200
0
0
90
180
270 360 450
Time, min
540
630
720
FIGURE 7.4 Drying strawberries by convection alone and by microwave-convective drying
at three power levels.44
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7.15 MICROWAVE-VACUUM DRYING
Further drying improvements can be obtained by using subatmospheric pressures.
Water evaporation takes place at lower temperatures under vacuum, and hence the
product processing temperature can be significantly lower, offering higher product
quality. Many comparisons have been made between microwave-vacuum drying and
other systems, mainly focusing on hot air and freeze drying.
In a comparative study conducted by Popovich et al.,48 a detailed analysis of
ginsenosides was conducted on ginseng dried with different methods. Different
drying methods influence the retention of specific ginsenosides which all have
distinct bioactivity. Freeze drying of ginseng proved to have greater Rg1 and Re
retention, whereas, Rb1 and Rd ginsenosides were found in greater amounts in
microwave-vacuum-dried ginseng.
In a comparative study conducted by Sunjka et al.,43 microwave-vacuum drying
of cranberries exhibited enhanced characteristics when compared to microwaveconvective drying. Drying performance results (defined as mass of evaporated water
per unit of supplied energy) showed that microwave-vacuum drying is more energy
efficient than microwave convective.
Microwave-vacuum drying was compared with freeze drying, air drying, and a
combination drying method of air drying and microwave-vacuum drying for preserving the antioxidant functional properties of Saskatoon berries.49 Drying yielded
a considerable loss of anthocyanin. The best retention was obtained with freeze
drying followed by microwave-vacuum drying, combination drying, and air drying
(75°C).
7.16 HEAT PUMP HYBRID DRYERS
Heat pump dryers have many advantages compared to the conventional air drying
systems, such as improved product quality, reduced energy consumption. and
reduced environmental incidence. An electrically driven heat pump shares the advantage of low temperature drying and manifests the same desirable characteristics of
resistance heat, yet is considerably more energy efficient.50,51 The simplest drying
method is to blow heated air over the moist material and to vent the moist air to
atmosphere. All the latent heat carried in the water vapor is lost to the atmosphere.
In the case of a heat pump, the moisture in the exhaust is condensed at the evaporator
and the latent heat is recovered to the circulating airstream via the condenser.52 The
heat pump, in a closed cycle, reclaims heat and uses it at a cost equal to that of
operating the compressor motor.
In a study conducted by Sosle et al.,53 various agriproducts such as apple, banana,
onion, and potato rings were heat pump dried from 85% wet basis to 10% to 12%
wet basis. The products obtained by the process were of exceptionally high quality.
The color degradation was negligible and the flavor was free from any processinduced aberrations. The low temperature drying achieved in the equipment was less
destructive toward the cell walls and led to a gradual, gentle collapse of the cells,
which recovered their turgor remarkably upon rehydration.
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Novel applications of heat pump drying now include radio frequency54 or microwave energy.55 The ability of microwaves to selectively heat areas with higher
dielectric loss factors is very important in this application. Unlike other conventional
systems, dielectric drying has an inherent ability to “moisture level” a given product;
that is, more power is applied to regions with higher moisture content than to regions
with lower moisture content. The use of dielectric heating provides an efficient
supply of energy required to speed up the process of moisture rejection from the
material undergoing drying. This component overcomes some of the heat and mass
transfer drawbacks of convective air drying.
7.17 CONCLUSION
There exist a great number of processing choices for the dehydration of biomaterials
and foods. The impact of each drying system is product specific in terms of end
product quality and functionality of its components. The choice in processing method
will depend on the intended end use of the product. Any given process can be
detrimental to a certain product, with a significant reduction in the retention of its
bioactive ingredients or their activity level. On the other hand, the exact same process
can be suited to yield higher levels of health promoting bioactive compounds during
the processing of a different plant material. The mechanisms of product degradation
and bioactivity are intimately related to the processing conditions, principally time
and temperature, and their control.
SUMMARY
Drying is one of the oldest methods of food conservation practiced worldwide. The
removal of moisture prevents the growth and reproduction of decay-causing microorganisms and minimizes many of the moisture driven deterioration reactions. Drying offers a substantial reduction in weight and volume thus minimizing storage and
transportation costs and enabling storage of the product at ambient temperature for
extended periods of time. The sharp rise in energy costs has favored a growing
interest in the development of new or improved drying technologies.
Traditionally, dehydration involves the application of heat to vaporize water
combined with some means of removing the water vapor at the surface of the fruit
or vegetable products. Drying is a combined heat and mass transfer operation for
which energy is supplied. Several types of dryers and drying methods, each better
suited for a particular situation, are commercially used to remove moisture from a
wide variety of plant products including fruit and vegetables. This chapter presents
them along with the most recent developments in drying research as they apply to
product quality deterioration during dehydration. The appearance and functional
properties of dried products are important in the choice of a drying process. The
remaining antioxidant and functional activities of dried products promote their
nutraceutical marketability.
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ACKNOWLEDGMENT
The authors acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada and Le Fonds Québéçois de la Recherche sur
la Nature et les Technologies, for the research carried out in their laboratory.
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drying of strawberries, Dry. Technol., 17(4–5), 837–853, 1999.
45. Beaudry, C., Raghavan, G.S.V., and Rennie, T.J., Microwave finish drying of osmotically dehydrated cranberries, Dry. Technol., 21(9), 1797–1810, 2003.
46. Marcotte, M., Grabowski, S., Quan, D., Taherian, A.R., Zareifard, M.R., Poirier, M.,
and Kudra, T., Quality aspects of canadian blueberries and cranberries dried by osmoconvective method, in Proc. 3rd Inter-Am. Dry. Conf., V. Orsat, G.S.V. Raghavan,
and T. Kudra, Eds., McGill University, Montreal, Paper IV-2, 2005.
47. Pan, Y.K., Zhao, L.J., Zhang, Y., Chen, G., and Mujumdar, A.S., Osmotic dehydration
pretreatment in drying of fruits and vegetables, Dry. Technol., 21(6), 1101–1114,
2003.
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Dehydration Technologies to Retain Bioactive Components
191
48. Popovich, D.G., Hu, C., Durance, T.D., and Kitts, D.D., Retention of ginsenosides
in dried ginseng root: comparison of drying methods, J. Food Sci., 70(6), S355–S358,
2005.
49. Kwok, B.H.L., Hu, C., Durance, T., and Kitts, D.D., Dehydration techniques affect
phytochemical contents and free radical scavenging activities of Saskatoon berries
(Amelanchier alnifolia Nutt), J. Food Sci., 69(3), S122–S126, 2004.
50. Strommen, I. and Jonassen, O., Performance tests of a new 2-stage counter-current
heat pump fluidized bed dryer, Drying '96: Proc., Poland, Vol. A., pp. 563–568, 1996.
51. Strommen, I. and Kramer, K., New applications of heat pumps in drying processes,
Dry. Technol., 12(4), 889–901, 1994.
52. Heap, R.D., Heat Pumps, E. & F.N. Spon., London, 1979.
53. Sosle, V., Raghavan, G.S.V., and Kittler, R., Low-temperature drying using a versatile
heat pump dehumidifier, Dry., 21(3), 539–554, 2003.
54. Marshall, M.G. and Metaxas, A.C., Modelling the radio-frequency electric field
strength developed during the radio frequency assisted heat pump drying of particulates, J. Microwave Power Electromag. Energy, 33(3), 167–177, 1998.
55. Jia, X., Clement, S., and Jolly, P., Study of heat pump assisted microwave drying,
Dry. Technol., 11(7), 1583–1616, 1993.
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8
Membrane Separation
Technology in
Processing Bioactive
Components
Ashwani Kumar
National Research Council of Canada
CONTENTS
8.1
8.2
8.3
Introduction ..................................................................................................193
Membrane Basics.........................................................................................194
Applications .................................................................................................195
8.3.1 Preconcentration...............................................................................196
8.3.2 Fractionation.....................................................................................197
8.3.3 Hybrid Processes..............................................................................199
8.3.4 New Membrane Processes ...............................................................201
8.3.5 Cleaning Chemical and Solvent Recycling .....................................203
8.4 Summary ......................................................................................................204
Acknowledgments..................................................................................................205
References..............................................................................................................205
8.1 INTRODUCTION
In recent years there has been a lot of interest in functional foods and nutraceuticals,
particularly their impact on human health and prevention of certain diseases. Manufacturers have responded to this sudden demand by releasing a large number of
products. However, there is a lot of scope of research and development in making
safe, reliable, and effective products from complex matrices of natural products. In
addition to growing good-quality natural products, there are challenges in harvesting,
postharvest treatment, extraction, refining, analysis of active ingredients, and safety
before an effective and reliable product is made. Although all of the above activities
193
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play an important role in making a good product, special attention needs to be
focused on separation and purification mainly due to the fact that 40% to 80% of
the cost of production is assigned to separation and purification unit operations in
making functional foods and nutraceuticals. Therefore, a lot of research effort has
gone into developing efficient extraction and separation schemes. This chapter will
mainly focus on refining and purification of bioactive compounds from natural
products using membrane-based separation schemes.
Membrane-based processes are usually more energy efficient than distillation,
adsorption, and chromatography. Furthermore, membranes have the advantage of
compatibility with a wide range of solvents and chemical products, ability to process
thermally sensitive compounds, easy amenability to automation, smaller footprint,
and seamless scale up. These advantages open up several possibilities of membrane
application in the production of bioactive compounds in the areas of energy efficient
preconcentration of dilute solutions, fractionation of diverse classes of compounds
from a complex mixture, recovery of intermediates, and recycling of solvents.
However, it is also important to be aware of the challenges posed by membranebased separation schemes, such as limited selectivity, fouling leading to performance
decline, cleaning, and higher capital costs in certain installations. This chapter is
divided in to three sections dealing with membrane basics, applications, and final
remarks.
8.2 MEMBRANE BASICS
Membranes could be described as barriers that provide selective and controlled
passage of one component over the other from a multicomponent mixture. Membranes have been classified by pore sizes, materials, driving forces and devices, even
specific applications. The basic definitions and principles of operation have been
described in textbooks.1,2,3 The membranes are usually made of polymeric, ceramic,
and metallic materials and are semipermeable to mixture components. These can be
in flat sheet, spirals, hollow fibers, and tubular form and can be housed in specially
designed housing or modules for applications. Membranes are often used in
cross-flow filtration mode and permeation or rejection of a particular component
mainly depends on the pore size, morphology, and interactions of feed components
with membrane materials. A membrane’s ability to reject a solute is often expressed
in molecular weight cutoff, which is determined by sieving experiments with standard solutes of varying molecular weights. A brief summary of the main types of
membranes and their characteristics is given in Table 8.1 for those processes that
are most relevant for functional foods and nutraceutical applications.
In processing complex matrices of natural products, the extracts usually contain
many dissolved and suspended species with varying physical and chemical properties. The separation and purification steps are complex. Considering that a variety
of membrane-based processes that operate on different principles are available (Table
8.1), one can envision that these processes could have an effective role in separation
schemes. Based on the knowledge of the characteristics of extracts and species of
interest, one can select a suitable membrane process. The process development
generally involves screening of commercially available membranes under different
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TABLE 8.1
Membrane Processes and Their Characteristics
Membrane
Process
Driving
Force
Molecular
Pore Sizes, Weight Cutoff,
nm
Dalton
Pervaporation
Vacuum
<1
—
Reverse
Osmosis
Nanofiltration
Pressure
<1
Low
Pressure
1–10
100–1000
Ultrafiltration
Pressure
10–100
1000–100,000
Microfiltration Pressure
Nonvolatile or
affinity basis
Ions, large
molecules
Multivalent ions,
large molecules
Species
Permeated
Volatile or
affinity basis
Water, alcohol
—
—
Non-ionic solutes
Nonvolatile solute
Monovalent
ions, water,
alcohol
Ions, water,
alcohol, small
molecules
Some macromolecules,
ions, water,
alcohol
Ionic solutes
Water
—
Nonextractable
species
Extractable
species
100–10,000 —
Electrodialysis Electric current —
Osmotic
Partial pressure —
distillation
difference
Membrane
Concentration —
solvent
gradient
extraction
Species
Retained
Macromolecules,
pathogens
Large
macromolecules,
some pathogens
operating variables such as cross-flow velocities, permeate rate, operating pressure,
and temperature. Longer-term process evaluation is also used to establish membrane
cleaning schedules for restoring performance. This basic laboratory data is utilized
to run pilot-scale experiments to ascertain the overall viability of the process for a
particular application. These laboratory experiments and analysis of collected data
are well described, particularly for food applications.1,3
8.3 APPLICATIONS
It is obvious from the description given in Section 8.2 that separation characteristics
of membranes are dependent not only on the physicochemical properties of the
membrane but also on the feed characteristics and operating variables. This observation also provides a broad basis for developing applications of membranes in
separation and purification to suit particular requirements of bioactive compounds
from a complex mixture. The following sections present a summary of membranebased separation processes.
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8.3.1 PRECONCENTRATION
Most of the process streams in the functional food and nutraceutical industries have
very small amounts of dissolved solids of interest. Irrespective of the extracting
solvent, a preconcentration step is essential prior to drying and making the final
products. In traditional processes this step is usually thermal in nature, which is
accomplished by thermal evaporation or distillation. Energy consumption for removing 1 kg of water from dilute solutions is listed in Table 8.2. It is obvious that a
membrane-based preconcentration is very energy efficient. Furthermore, membranebased processing is suitable for thermally sensitive nutraceuticals and functional
foods and helps in maintaining the natural taste of the final product by avoiding the
heat-induced taste profile changes.
In a U.S. Department of Energy study, it was clearly pointed out that current
usage of membrane-based separation schemes in the food processing industry will
expand and have a significant impact on reducing energy consumption.4 Concentration of several food products, such as fruit juices, maple sap, seafood cooker water,
and corn light steep water, using reverse osmosis membranes have been reported.5,6,7
However, it should be noted that these applications are limited by the osmotic
pressure of the dissolved species and fouling of membranes. Concentration applications are implemented more effectively using membrane-based separation as part
of the overall process. Similar applications in nutraceuticals and functional foods
are possible; however, published reports are scant. Ekanayake et al.8 patented a
process describing the concentration of green tea extract with nanofiltration. These
inventors claimed superior quality of the membrane-concentrated product in masking
the taste of artificial sweeteners in beverages. Gugger and Grabiel9 utilized temperature-dependent solubilities of soy isoflavones for concentration by ultrafiltration
membranes. The target compound in the feed stream was first adsorbed on a resin.
The eluted isoflavone fractions were treated with either reverse osmosis or ultrafiltration to remove solvent and achieve a higher isoflavone concentration in the
retentate, which was spray dried to make the final product. Recently, a similar
approach for concentration of isoflavones from soybean process waste and extract
of red clover flowers has been reported by Xu et al.10,11 In these studies concentration
by membranes played an important role in making the final product.
TABLE 8.2
Energy Consumption in Preconcentration of Dilute
Solutions by Different Unit Operations (Kumar et
al. 1999)
Unit Operation
Energy Consumption,
kJ/kg Water Removed
Thermal evaporation
Mechanical vapor recompression (MVR)
Membranes
2100
350
90
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8.3.2 FRACTIONATION
Considering that most nutraceuticals and functional foods are produced from complex matrices such as grains, botanicals, and fermentation broths, isolation and
purification of the desired active components is very important for product efficacy.
A membrane-based separation scheme has vast potential to make an impact in this
area, particularly where compounds of interest are of comparable molecular weight,
have similar chemical makeup, and impurities are significantly different from the
target compounds. Several studies utilizing membranes in fractionation of food
products from grains, meat, and poultry have been described.3,4
Recently the separation of individual whey proteins (alpha-lactalbumin and betalactoglobulin) for use as nutraceuticals was reported. This laboratory study utilized
both cellulosic and polyethersulfone membranes and reported the optimal filtration
conditions, showing that a diafiltration process yielded beta-lactoglobulin as the
retentate product with a purification factor of 100 and recovery of 90%. Alphalactoalbumin was recovered in the filtrate with a purification factor of more than 10
and nearly 99% yield.12 A product made from oat extract in water/alcohol mixture
and fractionated by membranes to get the active ingredient avenanthramides in
reasonably high concentration for skin-care products was patented by Redmond and
Fielder.13 The reported process claimed the use of ultrafiltration membranes for
separating the active ingredients from crude extract and reverse osmosis membranes
for concentration before making the final product. Singh14 has reported a process
for making high soy protein concentrate (> 70%) with reasonably high concentration
of isoflavones and saponins while the polysaccharide concentration was reduced.
Fractionation of the protein was accomplished by adjusting the pH and using a 10
to 30 kDa ultrafiltration membrane.
Yamaguchi et al.15 reported the use of ultrafiltration for isolating nicotianamine
from water extracts of soybeans. Chou16 has patented a process to make antioxidant-rich compositions prepared from sugar cane and beet juices. A relatively high
concentration of polyphenolics and flavonoids present in the juices were fractionated
by various methods including ultrafiltration to get a retentate that showed very high
oxygen radical absorbance capacity (ORAC) even at 50% recovery of thin juice.
Mulligan and Gibbs17 reported using ultrafiltration for purifying glycolipids; however, only 77% of the product was retained. Buchholz et al.18 improved this process
by adjusting the pH of the feed to retain 99% of the product. They have optimized
the operation by using ultrafiltration in regular as well as diafiltration mode. Frangi
et al.19 reported a procedure to extract procynidol from crude vegetable extract of
Vitis vinifera using ultrafiltration membranes. Though the reported product was free
from monomers, the procedure required longer processing time and chlorinated
solvents, which are not desirable due to environmental and health concerns. NafisiMovaghar et al.20 patented a process that used only blends of polar solvent and water
for extraction and employed ultrafiltration for separating proanthocynadins from the
rest of the large molecular weight compounds while final extract was concentrated
by reverse osmosis. Their flow sheet also included steps to recycle solvents. Nwuha21
screened a series of ultrafiltration membranes for separating green tea catechins and
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identified the alcohol stable membranes. However, rejection value for catechins and
caffeine were not sufficiently different to make a caffeine-free catechin product.
There has been concern among a section of consumer about the use of aggressive
chemicals and solvents in processing nutraceuticals and functional foods. In keeping
this in mind, process development for refining compounds of interest from a complex
matrix has been attempted using only water extraction. Considering that membranebased processes largely involve inert material, these could be a natural choice for
fractionation of such extracts. However, it is also important to note that the choice
of extraction procedure plays an important role in downstream processing with
membranes. One interesting example is development of a membrane-based process
for refining natural sweeteners from stevia leaves extract.22,23 Extraction of glycoside
sweeteners was accomplished in lightly packed column at an optimized pH, temperature, and flow rate to extract most of the sweetener and least amount of phenolics
and lipids that might cause fouling problem during subsequent processing by membranes. Microfiltration of the extract removed suspended and colloidal impurities
while sweeteners and dissolved compounds permeated through the membrane. Permeate from microfiltration was ultrafiltered with a membrane of 2 to 3 kDa molecular
weight cutoff , followed by diafiltration in a batch mode until all sweeteners were
washed out. Values of sweetener concentration in permeate at different diafiltration
volumes of the original feed and permeate flux are listed in Table 8.3. It is obvious
from the table that most of the sweetners are recovered at a diafiltration volume of
3 to 4. The permeate from ultrafiltration was processed at higher than ambient
temperatures by a reverse osmosis membrane to concentrate the glycosides and
remove lower molecular weight components that are perhaps responsible for bitter
aftertaste of the product. It was also found that by raising the processing temperature
from 50°C to 800°C, the amount of impurities removedwas increased from 55% to
89%. A conceptual process flow diagram is given in Figure 8.1. Though the final
product from this process had a very good taste profile, the overall yield was lower
than conventional processes based on solvent extraction. In a later study,24 a sequential extraction by solvents such as alcohols and hexane was followed with membrane
processing of the extract. This process gave higher yields and good-quality product.
TABLE 8.3
Stevioside Concentration and Permeation Rates for
Different Diafiltration Volumes in Ultrafiltration Step
Diafiltration Volume
Sweetener Concentration,
g/l
Permeation Rates,
l.m–2h–1
0
2.0
3.0
4.0
1.5
0.75
0.25
ND
35
47
64
65
ND - not detected
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HEX
DF
MF Perm
DF
UF
DF
NF
Sweetener
concentrate
Water recycle
FIGURE 8.1 Conceptual process flow diagram for producing stevia sweetener concentrate;
HEX is heat exchanger; DF, UF, and NF are diafiltration, ultrafiltration, and nanofiltration.
One very interesting finding of this study was that prior solvent treatment of stevia
leaves was helpful in removing some of the natural products such as lipids, phenolics,
and chlorophyll, which are known to foul membranes. Obviously, membrane processing of this extract was relatively easy and the overall yield of the sweeteners
was higher than the previous water-based process.23
8.3.3 HYBRID PROCESSES
Efficiency of a separation process to some extent depends on the type of material
to be separated. In this regard membrane-based separations are no exception. Often,
a membrane-based scheme could achieve good results; however, there are theoretical
(e.g., osmotic pressure of the dissolved species) and material limitations (e.g., surface
characteristics leading to fouling). Considering that functional food and nutraceuticals are present in complex matrices containing a large number of chemically
different species, their separation and purification pose additional challenges. In
order to develop these challenging applications, so-called hybrid processes are often
very effective due to additional controls over the range of operating variables.
Basically, a hybrid process combines more than one unit of operations to create
overall synergy in the separation scheme. Hybrid processes have been reported1
utilizing pervaporation and distillation; reverse osmosis and multistage flash distillation; pressure swing adsorption and membrane permeation; condensation and vapor
permeation; and reverse osmosis and freeze drying. Often the output stream from
one process becomes the input stream for the next process. It appears that this hybrid
process approach has not been often utilized in separation and purification of functional foods and nutraceuticals. Selected examples from literature are described
below:
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Functional Food Ingredients and Nutraceuticals
In a process reported by Cheryan,25 extraction of two major proteins present in
corn meal was done in aqueous alkaline medium and the proteins were separated
by centrifugation. The individual proteins zein and glutelin were separated by using
a combination of ultrafiltration and ethanol extraction. The permeate of ultrafiltration
was also recycled using a reverse osmosis membrane. Seafood process cooker water
usually contains suspended solids, protein, amino acids, and flavoring components.
The suspended solids are removed by centrifugation or sieving, and proteins are
concentrated by several folds with protein recoveries up to 90%.7,26 Nafisi-Movaghar
et al.20 patented a process for extraction and refining of proanthocynadins from plant
material. Their approach was different from earlier processes19 using a number of
solvents, which allowed ultrafiltration only in the batch process. They used heat and
pressure to extract proanthocynadins from plant matter, followed by ultrafiltration
and chromatographic separation. Ethanol eluted proanthocynadins mixture was concentrated by reverse osmosis before drying the concentrate to powder. Borneman
et al.27 reported a process for removing polyphenols from apple juice using a specially made ultrafiltration membrane that utilized adsorptive capabilities of polyvinyl
pyrrolidine.
A new process for making defatted soymilk with higher isoflavone concentration
was reported by Xu et al.28 This process used wet grinding and centrifugation at a
higher ratio of bean to water and separated fat by centrifugation. However, the protein
concentration in the soymilk so produced was lower, which was brought up to 3%
by ultrafiltration. The permeate from a 30 kDa ultrafiltration membrane was also
rich in isoflavones. This stream was used to develop another process for recovering
soy isoflavones (Xu et al.11) by ultrafiltering the permeate from milk production by
a 1 kDa membrane. This lower molecular weight membrane was selected to reject
phytates. The amount of isoflavones in ultrafiltration permeate at varying processing
conditions is shown in Figure 8.2. It is obvious from this figure that permeate from
the high-temperature ultrafiltration of cooked milk was richest in isoflavones. It is
mainly due to the dissociation of isoflavones from proteins at higher processing
temperatures. The permeate was concentrated with a commercial reverse osmosis
membrane by 90%. The concentrate was further dried to make a powder that
contained 11 mg/g isoflavones, which represented 8.4% mass yield of dried beans.
Soy-based isoflavones are commonly used as nutraceuticals; however, these have
been associated with estrogenic activity in a recent publication.29 Red clover flowerbased products have high amounts of isoflavones and do not show any estrogenic
activity. Although there are compositional differences in isoflavones extracted from
soy beans (aglicones and glycosides) and red clover flowers (bichanin A and formononetin), their absorption in humans is similar.30 A process for extraction and
refining isoflavones from red clover flowers was developed31 that involved alkaline
extraction, adsorption on polyvinylpolypyrrolidone powder, elution by ethanol, concentration, and finally freeze drying. The final product contained 20% isoflavones,
which accounted for more tham 50% of the isoflavone content of dried red clover
flowers. Although the recovery and the concentration of isoflavones in the final
product were reasonably high, this process had numerous steps. A simpler process
involving extraction by ethanol-water blends followed by processing with ultrafiltration and reverse osmosis membranes was recently developed.32 The process
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50
Permeate
45
Soymilk
Isoflavone yield in permeate (%)
40
35
30
25
20
15
10
5
0
No cooking, UF at 21°C
With cooking, UF at 21°C
With cooking, UF at 50°C
FIGURE 8.2 Yield of isoflavones in UF permeate as percentage of total amount in soybeans;
results are presented as total amount of isoflavones in soybeans; error bars are based on
duplicate runs.
utilized the solvation properties of ethanol-water blends and formation of micelles
by partial removal of solvents. In this process the overall recovery of isoflavones
from dried red clover flowers was 35% while the concentration of isoflavones in the
final product was 9%, which is lower than the earlier process but was suitable for
making nutraceutical supplements. Process flow diagrams of these two processes
are shown in Figure 8.3. It is obvious that a membrane-based process has a reduced
number of process steps with little penalty to overall recovery. Furthermore, this
process offers the possibility of recycling the solvents. Obviously, in selecting a
proper process, an overall assessment needs to be done.
8.3.4 NEW MEMBRANE PROCESSES
In previous sections, membrane-based processes that are more frequently used in
well-established food process industry are described with examples of applications
in the nutraceutical and functional food industry. However, there are several new
membrane-based processes that are at various stages of development and perhaps
have potential for application in the industry. Selected examples of these developing
membrane processes are given below.
A review of the applications of pervaporation in food processing has been
published by Karlsson and Trägårdh.33 Pervaporation of flavoring compounds from
grape juice34,35 and ester-based flavoring compounds36 have been reported. It was
observed that in the presence of increasing amounts of ethanol vapors the recovered
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Functional Food Ingredients and Nutraceuticals
Ground red clover flowers Ethanol solution
Dried and powdered
red clover blossom
Batch extraction
in water at constant
temperature and pH
Filter on a 50–200
mesh sieve
Dried isoflavone
concentrate
Extraction
Freeze drying
Ethanol extract
Liquid isoflavone
concentrate
Ultrafiltration
Washing
Alkali
Rejects
Condensate
Vacuum
evaporation
PVP adsorbent
for reuse
Adsorbent PVP
with isoflavones
Water washing
of adsorbent
Ethanol-eluted
isoflavones
Ethanol elution of
isoflavones
Retentate
Permeate
Reverse osmosis
Filtrate – + PVP
and adjust pH
Centrifuge
Spent residue
Permeate
Retentate
Ethanol evaporation
Aqueous solution
Supernatant
liquid
Wash water
Refrigeration
Supernatant
Micelles
Drying
Isoflavone-enriched product
(a)
(b)
FIGURE 8.3 (a) Extraction and polymeric adsorption-based process flow diagram, (b) extraction and membrane-based process flow diagram.
aroma profile was changed significantly. A very comprehensive study on the screening of numerous commercial membranes for recovering peach flavors was also
reported.37 A similar approach could be used to recover volatile components such
as essential oils present in plant materials.
A membrane-based solvent extraction process could overcome many of the
problems encountered in a traditional solvent extraction process involving dispersion, coalescence, emulsification, and loading limits. Membrane hollow fibers made
with hydrophobic material could be used to provide significantly higher contacting
area that would provide very high extraction rates for selective solvent extraction of
the desired species present in the aqueous phase across the membrane barrier. Prasad
and Sirkar38,39 have reported the use of this techniques for purifying mevinolinic
acid and thiazoles using different solvents. The purity of the products was very high
and the membranes did not show any deterioration after long-term use. Dahuron
and Cussler40 have reported extraction of proteins using polyethyleneglycol containing potassium phosphate. Recently Kertész et al.41 have reported a simultaneous
solvent extraction and stripping process for recovering phenlanaline in n-alkanes
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containing a phosphoric acid derivative. These authors have presented a comprehensive study of mass transfer and kinetics of this system.
Reviews of potential food application of electrofiltration have been published
by Vradis and Floros42 and Bazinet et al.43 They have provided the theoretical basis
as well as the potential of this technique to overcome some of the fouling problem
in regular pressure driven membranes processes, particularly for the charged species
in the feed. Pressure filtration of bioactive compounds is dependent on the ionic
environment of the feed as it influences the hydrodynamic radius of species. Hofmann and Posten44 have given a sound basis for enhanced flux in separation of
biopolymers and proteins under the influence of electric field. Recently, Labbé et
al.45 have reported feasibility of separating green tea polyphenols by electrofiltration.
These authors reported that up to 50% concentration of epagallocatechin and epagallocatechin gallate could be achieved without significant migration of caffeine.
However, a similar study to recover polyphenols from tobacco extracts encountered
serious membrane fouling.46 A feasibility study of separating bioactive peptide from
casein isolates has been reported by Bargeman et al.47 These authors reported that
active peptide was concentrated from 7.5% to 26%. Obviously, additional studies
to develop these techniques for commercial applications are required.
Osmotic distillation process uses the vapor pressure difference as the driving
force while the feed stream and concentrated osmotic agent stream are separated by
a hydrophobic porous membrane. This technique is particularly suitable for heat-sensitive compounds and has largely been used for concentration of fruit juices.48,49
Recently Babu et al.50 reported concentration of lime juice and phycocynin colorant
by osmotic membrane distillation. They have studied the effects of various parameters on flux and developed models to predict flux using resistance models.
Affinity membrane separation combines affinity chromatography and traditional
pressure-driven membrane separation processes. This technique is suitable if the
feed stream contains several compounds of comparable molecular weight and characteristics to the compound of interest. A ligand or binding agent that has affinity
to the compound of interest is added to the feed solution and separated from other
compounds by membranes. The retentate from the process is treated with a proper
eluent to dissociate the ligand and the compound of interest is separated by a second
membrane filtration step. Matthiasson and Ling51 successfully separated biologically
active concavalin A from jack beans using S cerevisia cells as ligands and D-glucose
as eluent. Considering that micelles are larger than the dissolved organic solutes,
surface active agents also have been used to make micelles with the key component
of the feed for easy separation.52 Ruckenstein and Zeng53 reported the development
of chitin-silica membranes for separating lysozyme from ovalbumin. These membranes were an order of magnitude superior to the chitin beads for this separation
and could be used for larger-scale separation.
8.3.5 CLEANING CHEMICAL
AND
SOLVENT RECYCLING
In a membrane-based separation process, various cleaning chemicals are periodically
used for restoring membrane performance as well as cleaning and sterilizing the
ancillary equipment. These procedures have added importance for the nutraceutical
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and functional food industry for maintaining a higher level of purity and safety of
products for human consumption. The cleaning fluids are composed of caustic
solution, surfactant solution, enzymes, and chemicals, or a combination of these
ingredients. With repeated use, these cleaning fluids are contaminated with impurities
and need to be replaced. This adds to the cost of operation in replacement chemicals
as well as disposal fees. A membrane-based recycling of these cleaning fluids is
attractive as properly selected membranes could remove suspended, colloidal, and
dissolved species while water and active cleaning agent could be recycled. Caustic
recycling in orange juice and dairy plants is in use and has replaced more
energy-extensive evaporation systems.54 By using a multistage membrane separation
scheme, other cleaning agents could also be recovered.
As discussed in Section 8.3.3, organic solvents are used in extraction and elution
of nutraceuticals extensively. A membrane-based approach can recover and concentrate the compound of interest and allow the recycling of solvent. For example, a
typical reverse osmosis membrane will permeate water and ethanol while retaining
compounds of interest. There are some commercial membranes that are suitable for
alcohol and hexane recycling; however, there are very few membranes that are
suitable for other solvents such as ketones and esters that are often used in the
extraction of functional food and nutraceuticals. There have been several reports of
new solvent-resistant nanofiltration membranes55,56,57,58 that might be suitable for
recovery of compounds in the range of 300 to 900 Da molecular weights, which
includes a large number of nutraceuticals.
8.4 SUMMARY
In recent years there has been great interest in using plant-derived functional food
and nutraceuticals. These compounds are usually present in low concentrations in a
complex matrix; therefore, extraction, separation, and purification is generally very
expensive. Opportunities offered by membrane-based processes in concentration,
fractionation, and purification of these compounds have been identified in this work.
Several examples of processes utilizing a membrane unit operation in combination
with other unit operations such as extraction, adsorption, and thermal evaporation
are presented. Multistage membrane processes for refining desired compounds are
also discussed. Several new membrane processes are briefly described that need to
be developed further for use in functional food and nutraceutical production. Membrane-based processes for recycling chemicals and solvents used in the functional
foods are also highlighted as potential applications. Finally, the challenges for
researchers in developing newer products and processes relevant for the functional
food and nutraceutical industry are identified.
Use of membranes in water purification and wastewater treatment alone or in
combination with other unit operations is fairly well established. Membrane-based
separation schemes are now making inroads into many other chemical processes,
food, and dairy industries. Advantages of the membrane-based approach in nonthermal processing, energy efficiency, easy amenability to automation, modular
nature, and generally smaller footprint have been clearly exploited. Potential of a
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hybrid-process approach needs to be exploited further even with the presently available technology.
Many of the new membrane processes have potential applications in functional
food processing; however, these need to be researched further with particular focus
on material and process development. Membrane researchers have many challenges
to overcome in the areas of membrane materials, morphologies, and surface characteristics that have an impact on compatibility of membrane with feed stream,
separation performance, and resistance to fouling. Although many of the specifically
targeted compounds are extracted from complex matrices using aggressive solvents,
often at higher than ambient temperature and extremes of pH, there are very few
membranes in the market to process such streams economically and effectively.
Therefore, there is a need for developing membranes that are suitable for treating
such feeds.
ACKNOWLEDGMENTS
The author is thankful for the support of his colleagues in the Advanced Separation
Technology Project.
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9
Bioprocessing
Technology for
Production of
Nutraceutical
Compounds
Terry H. Walker, Caye M. Drapcho, and
Feng Chen
Clemson University
CONTENTS
9.1
9.2
9.3
Introduction ..................................................................................................212
Commodity Biochemicals and Nutraceutical Bioproduction......................214
Lipid-Based Nutraceuticals..........................................................................215
9.3.1 Polar Lipids ......................................................................................217
9.4 Long-Chain Polyunsaturated Fatty Acids....................................................219
9.5 Large-Molecule Nutraceuticals....................................................................221
9.5.1 Polysaccharide Compounds .............................................................221
9.5.2 Proteins and Nucleotides .................................................................224
9.5.3 Small Molecular Nutraceuticals.......................................................225
9.6 Bioprocess Design........................................................................................226
9.7 Process Analysis...........................................................................................227
9.8 Process Economics.......................................................................................227
9.9 Conclusions ..................................................................................................229
Acknowledgments..................................................................................................230
References..............................................................................................................230
211
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9.1 INTRODUCTION
Nutraceuticals are natural compounds used to supplement the diet as a means to
increase uptake of important nutrients in the diet of humans and other animals.
Nutraceutical compounds are becoming an important commodity in the United States
and globally as market demand increases for both purified and supplemented forms.
In the 1990s, nutraceuticals from natural sources were most widely explored in
“plants, marine organisms, and microorganisms, in particular actinomycetes, and
fungi.”1 Development of biopharmaceuticals primarily from animal tissue culture in
serum media has recently enabled obtaining these products in other eukaryotic
systems such as plants, algae, and fungi. Many nutraceuticals are extracted and
purified from plants (e.g., antioxidants) and animals (e.g., fish oil rich in essential
fatty acids), which are an important component of the total market. However, there
are vast sources of microorganisms capable of production of nutraceutical components and the database is rapidly increasing in recent years. Production of nutraceuticals in microorganisms is usually conducted in highly controlled closed systems
capable of meeting U.S. Food and Drug Administration (FDA) approval criteria.
Obvious expansion of this market requires innovation for new products, and the use
of bioprocessing techniques commonly applied to the food and pharmaceutical
industry may provide a means to enhance products through novel high-density cell
culture techniques as well as new fractionation and bioseparation techniques.
Researchers in biological and chemical engineering are finding ways to effectively
utilize byproducts from the food, seafood, and agricultural industries for first fractionating valuable nutraceutical components and then further processing of coproduct
to serve as feedstock for bioenenergy compounds like ethanol, biodiesel, methane,
and hydrogen.
The combined global market of nutraceuticals and functional foods surpassed
$90 billon in 2004, up approximately 10% per year since 1993 where nearly half
of the market comprised dietary supplements. The combined value of nutraceuticals
and functional foods is expected to reach $130 billion by the year of 2007.2,3 One
example is the Martek Biosciences process for production of nutraceutical oil from
algal and fungal sources containing a high degree of omega-3 and omega-6 essential
fatty acids primarily used as a supplement in baby formulas.
Development of advanced biological conversion processes (enzymatic, microbial, and physicochemical processes) is an important part of the bioprocessing
research agenda. Biological processes are the preferred paths for converting agriculture-based resources into industrial products. Bioprocesses tend to have higher
reaction specificity, milder reaction conditions, and produce fewer toxic byproducts.
These characteristics are very consistent with the goal of developing industrial
processes and systems that are environmentally friendly. Innovative research is
currently underway that links physicochemical processes, such as gasification, with
microbial conversion processes. These novel reaction systems are evolving in
response to the heterogeneity of most biomass resources and the realization that
there are important autotropic microorganisms that are effective in converting common bioorganic compounds into more useful industrial or biomedical compounds.
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Raw materials, byproducts,
agricultural “wastes”
Bioprocessing,
bioconversion,
bioreactor design and
control, “biorefineries”
Bulk
chemicals,
biofuels
Biomaterials
Specialty
biochemicals
FIGURE 9.1 Biorefinery concept for conversion of by-products to bulk chemicals, biomaterials, and specialty biochemicals with emphasis on integrating the 3 Es (environment, education, and economics).
Land grant institutions across the United States represent a great repository of
scientific and engineering knowledge to catalyze the transition of society from a
fossil fuel to a biobased economy. With the biotechnology revolution currently in
place, application of new technologies is sought to address the challenging arenas
of specialty biochemical production (e.g., biopharmaceuticals and nutraceuticals),
bioenergy compounds (e.g., ethanol), and biomaterials (e.g., polylactate, PLA).
Figure 9.1 illustrates the basic flow of raw materials to end products proposed for
a biorefinery concept4 in an initiative established by the U.S. Department of Agriculture (USDA).5 Integrated approaches to bioprocessing show the greatest promise
to complete the cycle driven by the 3 Es including economics, environment, and
educational aspects of the bioindustry. Bottom-line industry economics balanced
with environmentally friendly technologies that contribute to carbon sequestration
within the biological system require educating the increasing masses of people on
the planet of this balance and creating a workforce to tackle the highly innovative
challenges that lie ahead in a widely evolving biotechnological world. Environmental
concerns encourage “green” technologies, economics requires feasibility of the
proposed technologies, and education addresses the importance of all levels of
learning including grade school, undergraduate studies, and graduate research opportunities as a mainstay to achieving the goals to establish a bio-based economy.
Current breakthroughs are not limited to only molecular biology and genetics,
but over the past 15 years, biological and chemical engineers have made notable
advances in the fractionation and bioprocessing of biobased resources of raw materials required to obtain the principal building blocks for the synthesis of new bioproducts. For example, the critical first step in bioconversion of ligno-cellulosic
biomass is the fractionation and modification of constituent biopolymers to facilitate
either enzymatic or microbial conversion resulting in the efficient production of
fermentable sugars. Several promising pretreatment technologies, such as ammonia
fiber explosion (AFEX) and supercritical fluid explosion pretreatment have resulted
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Functional Food Ingredients and Nutraceuticals
in improved yields and reactivity.6 In addition, engineering advances in bioseparation
technologies are improving capabilities to identify and separate secondary metabolites and other bioactive compounds from plant, animal, and microbial materials,
such as simulated moving bed (SMB) chromatography and supercritical fluid fractionation (SFF). Advances in nanobiotechnology such as the fabrication of microfluidic channels in silicon wafers take advantage of differences in physical and
chemical properties such as diffusion, electrophoretic mobility, or chemical affinity
to produce rapid, efficient separations of proteins, secondary metabolites, and other
organic compounds primarily for rapid identification and synthesis. In analytical
biotechnology, chemical and biological techniques are combined and integrated into
engineered devices or sensors for the detection and quantification of secondary
metabolites and other organic compounds in a variety of bioprocesses.
9.2 COMMODITY BIOCHEMICALS AND
NUTRACEUTICAL BIOPRODUCTION
The biorefinery concept utilizes the bioconversion of natural raw materials or industry by-products to purified specialty biochemicals and bulk fuels (see Figure 9.1).
Research to develop bioconversion processes to produce specialty chemicals and
bioenergy consists of several phases: (1) screening for wild-type strains and metabolic engineering of cells to produce desired products; (2) characterization and
pretreatment (hydrolysis) of feedstock prior to bioconversion; (3) determination of
microbial growth, decay, and product formation kinetics of the target microorganism
(filamentous fungi, bacteria, yeast, algae, etc.); (4) design/optimization of reactor
systems to culture cells and produce desired products based on simulations using
mass-balanced-based models and microbial kinetic parameters determined in phase
3; (5) downstream processing to separate, concentrate, and purify products and (6)
recycle waste energy and materials to bio-refinery using sustainable technologies.
The bioprocess involves chemical treatment of feedstocks to hydrolyze the
cellulose and hemicellulose compounds to fermentable sugars, which consist of
mostly glucose and xylose. The fermentable sugars are then fermented to industrial
products (e.g., ethanol, lactic acid, or succinic acid) and nutraceuticals (e.g., amino
acids, omega-3 fatty acids, or high-value biopharmaceuticals). Biopharma compounds range from smaller molecules such as traditional antibiotic products like
penicillin or insulin from mammalian cell culture or modified E. coli (Eli Lilly
process) to larger molecules that encompass bioactive proteins, lipoproteins and
glycoproteins ranging from 1000 to 500,000 daltons with complex three-dimensional structures often dictated by their specific levels of glycosilation and chiral
chemistry. Generally, the purity of substrate is required for increasing product value.
For example the expense of serum media may exceed the value of most of the
products shown in the table. Therefore, effort to utilize substrates from conventional
agricultural byproducts for production of biopharmaceuticals with mammalian culture is of great interest to the biotechnology industry. Carotenoids including βcarotene astaxanthins, and more recently increasing markets for lutein and lycopene,
make up a world market nearing $1 billion with a growth rate of about 3%/yr. Most
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215
carotenoids are produced synthetically by companies like BASF and DSM, but recent
biotechnological advances for carotenoid metabolism have shown promise in fungi
like Blakeslea trispora7 and have been extracted for the nutraceutical market (Nature
Source®) from the algae Dunaliella salina.8–12
There is a great interest in biological chemicals, functional food ingredients and
nutraceuticals regarding the rapid development in biochemistry, genetic engineering,
genomics, biological engineering, food chemistry, and medical research fields. However, bioseparation and downstream processing equipment account for nearly half
of the total cost of production of biological products and nutraceuticals.13,14 Therefore, the competitive nature of the biotechnology, pharmaceutical, and food industries for cost-effective manufacturing has provided much impetus for the development and use of new bioseparation techniques in large scale, but in lower cost.
Except for the conventional bioseparation techniques, such as crystallization, precipitation, distillation, liquid-liquid extraction, and so forth, that have been well
established and commercialized,15 a number of newer bioseparation techniques have
been implemented at the commercial scale, including chromatography, supercritical
fluid extraction, and membrane-based separation. These techniques have been implemented for purification of proteins and nucleotides, characterization of aromas, whey
protein removal from dairy products, extraction of health-benefiting fish oil, and
clarification of beverages including beer, fruit juices, and wine. The following section
provides some examples of analytical- and process-scale bioseparations of biological
products and nutraceuticals based on their polarity (lipid-based nutraceuticals) and
molecular size (large and small molecular nutraceuticals). Table 9.1 lists several
potential products from pretreated feedstocks to form high-value products.16
9.3 LIPID-BASED NUTRACEUTICALS
Lipids include many broad classes of neutral and partially polar compounds with
differing physiological function. Some contain important regulatory function such
as sterols (e.g., cholesterol, phytosterol, ergosterol) produced by plants as well as
fungi such as Saccharomyces cerevisae and Gibberella fujikuroi.17 Other antioxidants
found in plant materials such as rice bran are the class of phytosterols,18 but also
found in microorganisms like algae.19 These antioxidants include tocopherols and
tocotrienols (see Figure 9.2) beneficial in lowering cholesterol as well as preventing
cardiovascular diseases.20 Tocopherols are also believed to have anticancer
effects.21,22 Many neutral lipids in mono-, di-, and triacylglycerol forms act as
functional precursors to other metabolites or act as an energy storage source. Most
lipids consumed by humans and other animals are derived from plants and animals
due to their relative abundance in these sources. Bioproduction of lipids from
microbial sources are also well known, but have only recently been established at
the commercial level for production, primarily for lipids with considerable bioactivity such as very long-chain unsaturated compounds with high degrees of omega3 fatty acids or phospholipids and sulfolipids geared toward cancer research from
algal or fungal sources.
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TABLE 9.1
Bioconversions in Order of Increasing Value and Subsequent Substrate
Purity. Pretreated Feedstocks (e.g., Mild Acid Hydrolysis) Are Converted to
Product by Enzymes from Microbial “Factories”
By-Product
Source
Primary
Substrate
Primary Enzyme
(Microbial strain)
Product (Production)
Animal waste
Corn syrup
Potato/Sweet
potato
Wood fibers
Dairy waste
Oily waste
Rice brokens
Rice straw
Switchgrass
Sugarcane
bagasse
Corn stover
Corn starch
Molasses
(sugarcane)
Corn Syrup
Complex
Glucose
Glucose
(M. flagellate)
(S. cerevisiae)
Alcohol dehydrogenase
(K. marxianus)
Xylan
(Clostridium sp. SAIV1)
Lactose
β-galactosidase
Lipids
Lipase (esterases)
Glucose
(C. acetobutylicum)
Xylose
Citrate synthase
Xylose
Lactose dehydrogenase
(L. delbrueckii)
Xylose Pyruvate Pyruvate decarboxylase
(Acetobacter sp.)
Acrylonitrile
Nitrile hydratase
D-xylulose
Xylose reductase
(C. tropicalis)
Glucose
Glucose isomerase
Sucrose
Fumarase
(B. ammoniagenes)
Glucose
(Schizochytrium sp.)
Ethanol stillage3
Glucose
Glucose
Glucose
Glucose
Glucose
Glucose
Specific
Media
Glucose
Glucose
Glucose
Glucose
Serum
Serum
Serum
Methane
Ethanol (26M tons/yr)
Ethanol (thermophilic pathway)
Ethanol
Glucose and Galactose
Fatty acids
Acetone/butanol (30/60)
Citric acid (1M tons/yr)
Lactic acid (30 M kg/yr)
Acetic acid
Acrylamide (15,000 tons/yr)
Xylitol
High-fructose corn syrup (8 M tons/yr)
L-malic acid
Oil rich in docosahexaenoic acid
($220 M/yr)
(Phaffia rhodozyma mut.) astaxanthin ($2000/kg)
AcetylCoA carboxylase L-glutamic acid (340,000 tons/yr)
(C. glutamicum)-biotin
MSG
Aspartic amino
L-phenylalanine (3000 tons/yr)
transferase (E. coli)
(Aspartame synthesis)
Aspartase (E. coli)
L-aspartic acid (4000 tons/yr)
L-aminocaprolactam
L-lysine (350,000 tons/yr) ($2/lb)
hydrolase (C. laurentii)
(P. fluorescens)
L-histidine
Penicillin amidase
6-aminopenicilloic acid (7500 tons/yr)
(P. chrysogenum)
(B. lichenformis)
Proteases ($236 M/yr)
(B. amyloliquefaciens)
Amylases ($70 M/yr)
(Rhizopus, Aspergillus)
Other enzymes ($92 M/yr)
(Anthrobacter simplex)
Prednisone
Hybridomas
Monoclonal antibodies
Human fibroblasts
Interferon
Monkey kidney cells
Polio vaccine
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Bioprocessing Technology for Production of Nutraceutical Compounds
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5
HO
H3C
H
H3C
H
2
7
8
O
Tocopherol
5
HO
CH3
CH3
2
7
8
O
Tocotrienol
FIGURE 9.2 Antioxidant sterol compounds tocopherol and tocotrienol.
9.3.1 POLAR LIPIDS
Polar lipids are often associated with vital membrane structure and function depending on the lipid class (e.g., sulfolipid, SL; phosphatidyl choline, PC; phosphatidyl
ethanolamine, PE), length and position of constituent fatty acids, and degree of
saturation.23 Sphingolipids (e.g., sphingomyelin, a phospholipids, and ceramide) are
another special class of bioactive compounds associated with cell membranes that
act as lipid mediators important to intra- and extra cellular signaling, regulation of
cell growth, differentiation, and apoptosis. Breakdown of the abundant membrane
phospholipid, sphingomyelin, by sphinomyelinases produces sphinogosine and ceramide.24 Sphininogosine serves as a precursor to sphinosine-1-phosphate, which has
cancer-promoting properties such as cell proliferation and angiogenesis, while ceramide primarily functions physiologically as a cancer preventive agent with properties
of apoptosis and growth inhibition, thus together, placing the cell into a life and
death balance.25
Cyanobacteria, like higher plants and green algae, have a glycerolipid composition that is made up of monogalactosyldiacylgylcerol (MGDG), sulfoquinovosyl
diacylglycerol (SQDG) shown in Figure 9.3, digalactosyldiacylglycerol (DGDG),
and phosphatidyl glycerol (PG).
Sulfoquinovosyl diacylglycerols are structural components of chloroplast membranes in eukaryotic algae and lamellas in prokaryotic cyanobacteria, and appear to
be the only natural lipid with sulfonic acid linkage.26 SQDG is characterized by its
unique sulfonic acid head group, a 6-deoxy-6-sulfo-glucose.27 SQDG extracted fom
cyanobacteria has been shown to inhibit HIV-1 in cultured human lymphoblastoid
T-cell lines and thus was placed as high priority for further research by the National
Cancer Institute.28 The cyanobacteria evaluated included species of lyngbya, phormidium, oscillatoria, scytonema, calothrix, and anabaeana. Sulfolipids comprised
10% by weight of the organic extractables from the cyanobacteria tested and all had
similar activity, suggesting that acyl chain length and degree of unsaturation did not
affect potency.28 Structurallyrelated acyl glycerols, lipids, and sulfonic acid derivatives did not inhibit HIV-1. SQDG was also found to reduce proliferation and
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Functional Food Ingredients and Nutraceuticals
−O SH C
3
2
HO
O
HO
OH
O
O
SQDG
O
O
O
C
R1
R2
FIGURE 9.3 Structure of SQDG where R1 and R2 represent acyl chains of different length
and degree of unsaturation.27
viability of human gastric carcinoma cells SNU-1, by induction of apoptosis to direct
necrosis at micromolar concentrations.29 These results indicate potential of whole
algae or algal extracts containing sulfolipids as chemotherapeutic or preventive
dietary supplements.
For the cyanobacterium Anabaena 7120, SQDG content in algal basis at day 20
of batch incubation was unaffected (approximately 11 mg SQDG/g algal dry weight)
by surface light intensities.30 SQDG content in the Anabaena biomass increased
throughout the exponential and decreasing exponential growth phases, indicating
SQDG may be characterized as a mixed primary and secondary product. For the
green alga Scenedesmus, the combined SQDG and PG content of algal biomass did
not differ as a function of light intensity (40 to 290 (ME m–2s–1) at day 5 of incubation,
but decreased with increased light intensity by day 25 of incubation when cells had
entered early stationary growth.31 Full characterization of the interaction between
light intensity, growth rate, and SQDG production is needed for optimization of
algal reactors.
Unlike neutral galactolipids, which are easily separated from phospholipids by
adsorption chromatography, SQDG is difficult to separate due to its polar nature.26
Quantification of SQDG in Anabaena extracts was accomplished by Archer et al.30
using total lipid extraction as per Folch et al.,32 followed by thin layer chromatography using silica gel plates and photodensitometric scanning after charring with
copper sulfate and heat. Norman et al.33 developed a method for the extraction,
separation, and analysis of SQDG from plant (spinach) biomass using the Bligh and
Dyer34 extraction method for total lipids using sodium chloride in two-phase partitioning, separation of lipid classes using silica cartridges with SQDG and phospholipids (PL) eluted using methanol, and resolution of SQDG from the PL fraction
using normal phase high-pressure liquid chromatography (HPLC) with silica column, heptane-isopropanol-0.001 M KCl 40:52:8 (v/v/v) as the mobile phase, and
detector operated at 208 nm. The sulfolipid yield was found to be 0.18 mg SQDG/g
fresh spinach.
Cell lysis of algae and cyanobacteria for extraction of lipids is more difficult
than for plant tissue, and techniques to shear cells include solvent addition with
incubation, agitation, homogenization, and sonication.35 A cell-lysis method
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developed for the green alga Scenedesmus that included quartz sand and organic
solvent addition, freeze drying, and extraction in ultrasound bath, was reported to
provide excellent extraction efficiency of pigments and fatty acids.35
Supercritical fluid (CO2) extraction (SFE) provides an alternative for the extraction and recovery of algal nutraceuticals, and avoids use of toxic organic solvents.
SFE of carotenoids and lipids from the green alga Chlorella has been found to
compare favorably to organic solvent extractions.36 Shearing of algal cells using
alternating mechanical and thermal shocking was required for efficient extraction.
Yields of carotenoids and lipids were 5% of dry weight at 35 MPa and 55°C for
whole algal cells, while yields were increased to 13.3% for disrupted cells. Similar
results were achieved using SFE for extraction of carotenoids and lipids from
Botryococcus braunii, Dunaliella salina, and Arthrospira maxima.37 SFE of sulfolipids may also prove comparable to organic solvent extraction. Recently, continuous
countercurrent supercritical carbon dioxide fractionation has been successfully
applied to fractionating low molecular fatty acids38 and antioxidants in plant oils
such as corn and rice bran oil,39 and could be applied to fungal and algal systems.
9.4 LONG-CHAIN POLYUNSATURATED FATTY ACIDS
Polyunsaturated fatty acids (PUFAs) are a class of compounds which has a variety
of important functions in biological systems. Studies have shown that the
longer-chain fatty acids, in particular EPA (eicosapentaenoic acid, C20:5n3; see
Figure 9.4), ARA (arachidonic acid, C20:4n6), and DHA (docosahexaenoic acid
C22:6n3), have important roles as biosynthetic precursors, as cellular membrane
components, and as protection against oxidative stress.40,41 The relative levels of
these compounds have been found to have profound effects on human health. Growing interest in health benefits of PUFAs has focused attention on providing suitable
sources of these compounds. Isolation of highly efficient oleaginous microorganisms
and development of related fermentation technologies may lead to fermentation as
an alternative to agricultural and animal processes.42 Some microbial species produce
high yields of certain PUFAs, which include Mortierella alpina,43–45 Mortierella
elongata,46–48 Pythium irregulare,49–52 P. ultimum,53,54 and Entomophthora exitalis.55
Pythium irregulare is regarded as one of the most promising microbial species
for possible commercial production of EPA due to its high EPA yield. This microorganism can also produce a significant amount of another important PUFA, arachidonic acid (AA).49–51 Research into fungal fermentation kinetics and modeling
have been conducted with Pythium irregulare.56 Supercritical fluid extraction and
19
17
15
13
11
9
7
5
3
1
COOH
H3C
cis-5, 8,1 1, 14, 47-eicosapentaenoic acid, 20:5 (n-3)
FIGURE 9.4 Chemical structure of EPA.
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Functional Food Ingredients and Nutraceuticals
modeling extraction behavior was implemented to develop a process for fractionation
of important components (essential fatty acids and antioxidants) from both rice bran
and fungi after fermentation.57
Beneficial health effects from consumption of certain fish oils have been attributed to the presence of the essential PUFA, EPA and docasapentaenoic acid (DHA).
These omega-3 fatty acids have been linked to a reduced risk of coronary heart
disease, arthritis, inflammation, hypertension, psoriasis, other autoimmune disorders,
and cancer.58 PUFA are currently marketed as dietary supplements at health food
stores in the form of concentrated fish oils. Supplementation into baby foods has
lately received greater interest. PUFA are also prescribed medications for humans
and pets.
Declining marine resources and an increasing demand for PUFA have prompted
the search for alternative sources of EPA and DHA. Filamentous fungi have the
potential to produce large amounts of EPA within the mycelial walls when grown
at optimal conditions.53,59 The filamentous fungus P. irregulare converts sweet whey
permeate to significant amounts of EPA (24.9 mg EPA/g dry biomass), which could
potentially add value to this food-processing by-product.50
Humans have a limited capacity to synthesize EPA, ARA, and DHA from shorterchain fatty acids. Additionally, typical American diets are extremely low in these
compounds. The importance of EPA and ARA lies in that they are the biosynthetic
precursors of the eicosanoid system that controls inflammatory and anti-inflammatory responses. There is increasing evidence that a variety of disorders, such as heart
disease and hypertension, are related to malfunctions of the eicosanoid system caused
by dietary imbalance of long-chain PUFA. DHA accumulates preferentially in the
brain where it has been found to have rules both in neuronal impulse transmission
and protecting the brain from oxidative stress. Dietary deficiencies of DHA have
been linked to bipolar disorder and schizophrenia. Connor60 noted that diets rich in
n-3 long-chain fatty acids tended to offset age-related degenerative diseases as a
result of increased n-3/n-6 ratios in the blood and fatty acid profile in the brain. As
a result, it has been found that dietary supplementation of PUFA has considerable
efficacy in the treatment of these conditions and a considerable dietary supplement
market has developed around these PUFA.61
The current commercial supplies of long-chain PUFAs primarily come from
various oil seed plants, such as flax or borage, and from marine fish oil. Plant oils
only contain the shorter-chain essential fatty acids, alpha-linolenic and linoleic acid,
and do not contain the nutritionally important long-chain PUFA. Fish oil is a good
source of EPA and DHA, which has major limitations including objectionable “fishy”
smell and taste encapsulation, and growing concern of the presence of mercury and
heavy metal accumulation in the extracted oils. While plants lack the enzymes
necessary to create long-chain PUFA greater than 18 carbon units, microbes particularly in the algal and fungal classes possess oleaginous properties and an array
of essential fatty acid desaturases required to synthesize short-chain PUFA, which
are elongated to long-chain PUFA. Currently, microalgae are being used commercially to produce EPA and DHA. Microalgal production, however, has some drawbacks. Microalgal DHA production either requires the use of expensive photobioreactors or, in the case of tropically shifted algae, the use of expensive complex media.
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Common DHA-producing organisms include the algae Gonyaulax, Gyrodinium, and
Cryptoconidium spp. and the bacteria Rhodopseudomonas and Shewanella spp.
Mortierella alpina has been commercialized for production of long-chain PUFA
primarily rich in ARA for use in infant formulas. Other ARA-producing fungi include
Mortierella enongata, Entomorphthora exitalis, Pythium ultimum, and Pythium
irregulare for use as an ingredient in infant formula to supply essential metabolic
precursors for prenatal nervous system development. P. irregulare is an attractive
candidate for PUFA production as it produces nearly equal amounts of both ARA
and EPA.57 It is relatively easy to grow, can be adapted to both solid state and
submerged culture techniques, and is capable of utilizing a wide variety of substrates.
Specifically, P. irregulare has a comprehensive complement of metabolic enzymes
and is able to be grown on substrates high in cellulose and hemicellulose. This allows
the use of inexpensive waste materials as substrates for mass production EPA and
ARA from P. irregulare.
Research has indicated that culture conditions, such as medium composition,
oxygen availability, pH, and temperature, could influence microbial lipid accumulation as well as PUFA composition.44,49,56 Due to the nature of the individual
microorganism, the optimal conditions may differ from strain to strain. Some of the
culture conditions for Pythium irregulare have been optimized.49–51,56 Glucose and
yeast extract are considered as the preferred carbon and nitrogen sources for this
microorganism.49 Nitrogen limitation is commonly believed to have significant effect
on lipid accumulation in microbial fermentation.62 However, the optimal C/N ratio
of Pythium irregulare for the target PUFA synthesis is not known, and few published
studies have addressed this issue with other species. Therefore, optimizing medium
composition and C/N ratio will be important for target PUFA production and process
control. Zhu63 and Cheng et al.51 achieved the highest yields of the EPA and ARA
(20 mg/g and 14.7 mg/g, respectively) with medium composed of 2% glucose and
0.25% yeast extract and C/N of approximately 32. Figure 9.5 shows the fungal
culture of P. irregulare and oil production from P. irregulare grown on rice bran
compared to commercial rice bran oil. Supercritical extraction of this oil compares
favorably with the highly refined oil compared to conventional hexane extracted
crude oil.64,65
Table 9.2 lists the primary fungal and algal sources considered for production
of oils containing EPA and ARA, which serve as important precursors to an important
class of metabolites including prostaglandins and leukotrienes.
9.5 LARGE-MOLECULE NUTRACEUTICALS
Large-molecular nutraceuticals are generally made up of larger polymers typically in the 103 to 106 KDa range for proteins, and as high as 1010 KDa for
polynucleotides.
9.5.1 POLYSACCHARIDE COMPOUNDS
Many polysaccharides have been identified for therapeutic activity. Poly-branched
β-D-glucans isolated from the cell walls of Saccharomyces cerevisiae76 and
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Functional Food Ingredients and Nutraceuticals
FIGURE 9.5 Photomicrograph of the mycelium of P. irregulare (1000). The mycelium contains about 20% oil rich in polyunsaturated fatty acids particularly high in EPA and ARA,
which occur in about equal amounts.
Agrobacterium sp.77 has shown potent nonspecific immune-activation of macrophage
immune cells, T-cells, NK-cells and B-cells and have shown high anti-AIDS activity
with low side effects.77 A novel sulfated polysaccharide, calcium spirulan (Ca-SP)
isolated from the cyanobacterium Spirulina platensis has been found to have both
anti viral and anti cancer properties.78,79 Chitosan is the deacetylated from of chitin
found in abundance in arthropod exoskeletal layers, but extractable chitosan is also
in various fungi such as the industrial microoganism, Rhizopus oryzae.80–82 Chitosan
is extensively marketed as a weight-loss nutritional supplement that binds to lipids,
lowers serum cholesterol, and improves low-density lipoprotein to high-density
lipoprotein (LDL/HDL) ratios. Chitosan has also shown anticarcinogenic
properties83 as well as a tissue scaffold for implants and promotes healing of
wounds.84
Purification of polysaccharides by different chromatographic processes that are
differentiated from the lab scale from milligrams or even nanograms for chemical
analysis up to the industrial scale in kilograms or tons of production for drugs and
dietary supplements. Conventional gas chromatographic technique is commonly
used for trace amounts of volatile chemical analysis, and preparative HPLC is
suitable to collect biological chemicals up to milligrams for further chemical structural determination. However, both aforementioned methods are restricted by their
loading capacities that are not suitable for industrial-scale processing of some functional carbohydrates. Recently, simulated moving bed (SMB) technique has drawn
much attention because of its highly efficient productivity and suitability for sugar
separation. The method has been found to be suitable for large-scale fractionation
of xylitol, mannitol, and other specialty sugars.85,86 In addition, SMB technology
has been used in pharmaceuticals, cosmetics, and fine chemicals. However, highly
purified polysaccharides are seldom used except for their applications in biochemical
research and in the bioprocessing industry. For instance, cellulose, agarose, and
© 2007 by Taylor & Francis Group, LLC
EPA
Fungal
Cultures
Carbon
Source
P. irregulare
P. irregulare
P. irregulare
P. irregulare
P. irregulare
M. alpina 1S-4
M. alpina
M. alpina
M. alpina M18
M. alpina 1S-4
M. alpina 1S-4
M. alpina 20-17
M. alpina
M. elongata
Saprolegnia Sp.
C. minutissima
P. cruentum
4% soy oil+
2% glucose
5% rice bran
1% glucose^
SWP
1.8% glucose~
rice bran*
6.6% glucose
10% glucose"
3% linseed oil
2% linseed oil
1% linseed
5% glucose
2% linseed oil
1% olive oil
8000 lux
ARA
Lipid in Dry
In Dry
In Dry
Biomass
In Lipids Biomass In Lipids Biomass
T °C
(% w/w)
(% w/w) (mg/g) (% w/w) (mg/g)
12
25
25
12
14
28
20
25
28
12
20
28
25
15
6
20
32
67
29
28
38
10
47
6
7
2
13
25
42
20
6.4
49
25
2
5
1
5
39
40
60
15
8
35
56
33
60
12
20
7
67
66
42
39
—
9
—
45
17
35
23
225
12
51
36
10
15
2
14
10
19
21
50
72
Reference
Cheng et al. [51]
Zhu et al. [56]
Zhu et al. [56]
Stintson et al. [49]
O’Brien et al. [50]
Higashiyama et al. [43]
Jang et al. [66]
Eroshin et al. [67]
Yu et al. [68]
Shimizu et al. [69]
Jareonkitmongkol et al. [70]
Shimizu et al. [71]
Bajpai et al. [72]
Bajpai et al. [72]
Shirasaka et al. [73]
Seto et al. [74]
Nichols and Appleby [75]
+ supplemented with 1% soy meal; ^ spiked with 0.5% glucose 2 days before harvest; ~ supplemented with 0.1% soy oil; SWPsoy whey permeate. * solid-state culture (all others are submerged culture); "supplemented with 0.8 g/L glutamate.
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TABLE 9.2
Fungal and Algal Sources of EPA and ARA Grown on Various Carbon Sources Ranging from Pure
Glucose to Complex Carbohydrates and Lipid Components
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Functional Food Ingredients and Nutraceuticals
dextran are commonly used as media of bioseparations such as electrophoresis and
chromatography.
9.5.2 PROTEINS
AND
NUCLEOTIDES
In the past decades, the advance in biotechnology has enabled production of many
desirable compounds from animals and plants. Future production of novel proteins
such as scytovirin, isolated from the cyanobacterium Scytonema varium, which
displays anticytopathic activity against laboratory strains of HIV-187 are expected to
become increasingly important as new research developments are reported. Such
controlled genetic activities involve the participation of many proteins and nucleotides. Therefore, recovery and purification of those biological macromolecules are
essential for understanding and revealing the genetic activities. Harve and Bajpai88
have reviewed the steps of isolation and purification of those biological chemicals.
In detail, common isolation steps often employ precipitation and crude adsorption
followed by purification using ion exchange, affinity, and gel chromatography.
Recently, capillary electrophoresis (CE), which is considered one of the most powerful separation technique for bioanalysts, has been commonly used regarding its
rapid, high resolution of water-soluble components. Capillary electrophoresis has
been accepted as a useful complementary tool for HPLC. However, CE and HPLC
are limited by the prerequisite of several prepurification steps that often involve
losses of useful targets. Therefore, expanded-bed adsorption has appeared to replace
the conventional packed-bed operation to simplify purification of proteins and other
biological chemicals.
Purification of antibodies, in a large degree, is similar to the purification of other
proteins. But its property in antigen specific recognition makes it also suitable for
bioaffinity chromatography. To date, monoclonal antibodies and immunoglobulins
represent by far the largest class of produced and purified proteins in numbers and
mass. Recently, aqueous two-phase extraction (ATPE) has been successfully utilized
to partition proteins into two partially immiscible phases in an aqueous environment.
Hydrophobic interactions with proteins are factors in protein folding, transport across
membranes, recognition, and enzyme catalysis. However, these hydrophobic entities
are often buried within the folded lipoprotein structure and the introduction of
hydrophobic tags (often aromatic rings) to the outer surface has been applied to
increase solvent interactions as a technique to improve bioseparation.89 Another
recent novel protein purification method particularly suitable to the class of large
glycoproteins utilizes an affinity carbohydrate-based matrix called bioskin made of
a coculture of Acetobacter xylinim, Saccharaomyces cerevisiae and Saccharomyces
pombe90 with gradient elution with potassium phosphate.91
Table 9.3 shows the relationship between bioseparation cost relative to the
general class of compound.92 Estimated global market demands for 2002 are also
represented for these classes.2,3
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TABLE 9.3
Global Demand Value and Bioseparation Cost of Various Compound
Classes
Product
Functional foods, e.g., casein
Nutraceuticals, e.g., scytovirin
Industrial enzymes, e.g., cellulases
Diagnostic enzymes, e.g., glucose oxidase
Therapeutic enzymes, e.g., urokinase
Global Demand Market
% of Total
Value (2002)
Expansion Relative Production
(US$ billion)
Rate (%)
Cost
Cost
33
47
2
8
27
10
10
6
9
14
1
2–10
5–10
50–100
50–500
10–30
30–50
30–50
50–70
60–80
9.5.3 SMALL MOLECULAR NUTRACEUTICALS
Many natural nutraceuticals are small molecules with molecular weight less than
600 Da and typical radius of 0.5 nm. These often include monomer compounds such
as sugars, fatty acids, amino acids, and organic acids. However, they are very diverse
in chemical characterization in terms of their chemical polarity, natural existence in
plants/animals, and biological activities. Many important small molecular nutraceuticals often found in plants, but also present in high concentrations within some
microbial communities include various classes of antioxidants and alkaloids that
stimulate the immune system by activation of T-cells, scavenge for free radicals,
and inhibit tremors and analgesia.93 Compounds of commercial interest include
carotenoids that function as pro-vitamin A, astaxanthin from red yeast Phaffia
rhodozyma,94 canthaxanthin, and lycopene.95
A compound known to have anticonvulsant properties is the extracellular alkaloid compound pimprinine produced by Streptomyces sp.93 and processed by filtration, chromatography, and vacuum drying. Hydroxytyrosol is a powerful antioxidant
naturally present in olive oil and is synthesized in strains of Pseudomonas aeruginosa.96 Cis -β-carotene is produced in Dunaliella salina algal culture and in fungi
Blakeslea trispora, Neurospora crassa,97,98 and Phaffiea rhodozyma when stimulated
by light.99
Due to the physicochemical and biological characteristics specific to these biomolecules, a variety of extraction and separation methods have been developed.
Solvent extraction is a traditional method to extract phytochemicals or nutraceuticals,
that is, extraction of anticancer polyphenolics from blueberry.100 Recently, supercritical fluid extraction (SFE) has been strongly recommended to extract nutraceuticals
because of the extraction efficacy of SFE and concerns of the toxicity of solvents.
But both extraction methods always bring a mixture of different classes of chemicals.
Therefore, additional steps are often required for the removal of unwanted interfering
substances. For example, solid phase extraction (SPE) and open column chromatography are now commonly used for further chemical fractionation. Successful methods of extraction of antioxidants from algae and fungi with use of supercritical
CO294,95 and absolute ethanol101 have been identified.
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Functional Food Ingredients and Nutraceuticals
Naczk and Shahidi102 reviewed the extraction and analytical methods for many
nutraceutical phytochemicals including phenolics, flavonoids, anthocynins, tannins,
and so forth. It was concluded there is no uniform or completely satisfactory procedure for extraction of all phytochemicals or a specific class of phytochemicals in
plant material. Considering the volatility of nutraceuticals, gas chromatography (GC)
and high-pressure liquid chromatography (HPLC) have been frequently used for
volatile and nonvolatile chemicals, respectively. Furthermore, different monitoring
detectors such as UV and mass spectrometer are often combined with gas and liquid
chromatography for quantitative and qualitative analysis of nutraceuticals.103,104
Enzyme-linked immunosorbent assay (ELISA) has also been frequently used in
biological and clinical research.105 As a novel emerging technique, magnetic field
combined with nanotechnology106 has been tried for drug delivery and analysis.
Nevertheless, all of the methods mentioned are limited in lab-scale research, and
only simulated moving bed (SMB) has been successfully and commercially used in
large industrial scale. In general, bioseparations are essential in the good manufacturing practice (GMP) of production of pharmaceuticals and nutraceuticals, which
generally require the products in an acceptable level of purity.
Aside from basic separation issues of antioxidants from cell biomass in production systems, extraction procedures within clinical tissues such as the liver to measure
the metabolic effect of antioxidants has hampered the analysis procedures due to
poor sample preparation prior to HPLC analysis. Burri et al.107 demonstrated that
the use of supercritical carbon dioxide without modifier proved to generate vitamin
A and carotenoid extracts from calf liver tissue that did not require further pretreatment as typically needed for organic solvent techniques.
9.6 BIOPROCESS DESIGN
Bioprocess design for nutraceutical production primarily involves the conceptual
work to implement the technology for production and bioseparations, alterations, or
scale up to the process plant design. A detailed overview of the procedures is covered
in several texts.16,108 The process design of food, pharmaceutical, and nutraceutical
production is a marriage of process analysis and cost estimation that include the life
cycle of the product, use of engineering parameters and estimates to complete a
series of mass and energy balances in a process flow simulation, and environmental
impact assessment. The economic assessment begins with an order of magnitude
estimation based on previous cost data for similar bioprocess resulting in large errors
(up to 50%108) even with experienced teams of engineers, industrial biotechnologists,
and economists. Further estimates include project planning based on major equipment needed, preliminary and detailed engineering design from process data, and
contractor estimate with estimation error usually less than 10%.108 The life cycle
analysis for products is considered to begin with preliminary research findings.
Products first undergo a feasibility analysis for screening, then a project budget
development is created, and finally, after product entry into the market, scale-up of
commercial production is assessed based on market demand.108
Bioreactor design should consider microbial growth and product formation kinetics, impact of growth-limiting substrate or products, shear sensitivity of cells,
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temperature effects, immobilized vs. suspended culture, and characterization of
products as primary, secondary, or mixed metabolite. Basic bioreactor design configurations include batch, fed-batch, and continuous-flow stirred tank reactors
(CSTR) which are covered in detail in the literature.16,109–113
9.7 PROCESS ANALYSIS
The first step of process analysis is to construct a generalized flow diagram or flow
sheet where, during initial phases of process development, the unit operations may
not be well defined. As research data containing vital biological information such
as substrate (carbon, nitrogen, oxygen, etc.), biomass and product kinetics, pH range
for the desired system (continuous, fed-batch, or batch mode), is made available,
the appropriate material and energy balances may then be applied to determine
reactor volume, product mass, temperature requirements, air flow rates, and so forth.
Furthermore, molecular size, polarity, molecular charge, and orientation (degree of
glycosylation, protein folding, etc.) of the bioproduct of interest generally determine
the type of integrated bioprocess regime needed for production and separation of
the product. Whether the biological function of the product is intracellular, extracellular, or membrane bound is another important factor to whether separation may
occur during fermentation, as done in continuous or fed-batch situations, or additional bioseparation steps are required as in the case of intracellular and membrane
bound products.
Once reactor volume, mass of products, and product properties are known,
subsequent bioseparation unit operations are placed in the flow diagram with corresponding material and energy balances conducted for each unit operation in a
similar way conducted for the bioreactor step to determine equipment sizing and
material requirements.114 Cost analysis is then integrated in the design process to
narrow down the type of reactor and separation equipment to further optimize the
process and enable some important initial order-of-magnitude cost estimates, including capital and operating costs discussed in further detail in the next section.
Figure 9.6 is an example of a process flow chart constructed in SuperPro
Designer® of a specific biorefinery concept proposed for the coproduction of fungal
oil, fiber composites, ethanol, and nanocrystalline cellulose from conventional lignocellulosic biomass. Ethanol and carbon dioxide from the simultaneous saccharification and fermentation (SSF) step may be recycled back to the oil supercritical
extraction unit as well as ethanol used to supply energy to the process.
9.8 PROCESS ECONOMICS
Economics in preliminary bioprocess design typically includes research and development cost, estimation of capital and operating costs, and profitability analysis.
Research and development cost may range in the millions of dollars depending on
the ability to bring products to market, which is often less than 10% of many products
tested. These efforts typically include screening of potential microorganisms, genetic
modification for increased productivity, testing for contaminants and toxicity, process
© 2007 by Taylor & Francis Group, LLC
S-102
P-7
Oil
S-113
S-106
S-112
S-110
P-8
Fiber composite
P-Q/GBX-101
ethanol
S-107
P-3/V-102
S-108 distillation S-109
S-105
S-103
P-6/V-101
Solid-state reactor
P-2/DX-101
SFE
S-111
P-11/CY-1
Fiber separation
P-5/MSX-101
Hydrolysis (hot water/
mild acid)
P-4/V-103
SSF or SHF
S-114
P-10
Nanocrystalline cellulose
FIGURE 9.6 Biorefinery process flow schematic for production of nutraceutical and bioenergy products from lignocellusic by-products.
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S-104
S-101
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production and bioseparation technology, process scale-up studies, clinical trials
(e.g., FDA-approved biopharma products), marketability and ethical sensitivity studies, and other research specific to handling and packaging of the product.
Capital costs are often estimated using multipliers or unit costs that include total
plant direct cost (TPDC) (i.e., installation, process piping, instrumentation and
control mechanisms, electrical, building and facility cost), equipment cost (EC), total
plant indirect cost (TPIC) (i.e., engineering and construction), and direct fixed capital
(DFC) (TPDC + TPIC + contractors fees and contingency). Building and facility
cost range from $2000/m2 for Class 100,000 process areas to as high as $8000/m2
for Class 100 areas (typical for biopharmaceutical processing subject to strict current
GMP [cGMP], GLP standards). Individual equipment cost will increase with the
key physical design scale-up parameters such as unit operation volume, membrane
surface area, or flow throughput, but generally by a factor of about 52% referred to
as the “economy of scale.”108
Operating cost include direct costs such as raw materials (10% to 80% total
operating cost), consumables (membranes, process seals, etc.), labor (20% to 50%
total operating cost), waste disposal (up to $0.5/m3), utilities (approximately
$0.1/kWh, $8/1000 kg steam, up to $50/1000 kg purified water, $0.1/1000 kcal heat
removed by refrigeration) and laboratory quality control (QC) and quality assurance
(QA) costs (10% to 20% of the labor costs). Indirect operating costs include depreciation of fixed capital investment, maintenance (up to 10% of equipment cost per
year), insurance (up to 1% of DFC), local property taxes (up to 5% of DFC), and
other overhead expenses (accounting, payroll, security, fire protection, etc. which
accounts for up to 10% DFC).108
Finally, profitability analysis includes various measures to assess profitability
and marketability of the product. These factors include return on investment (ROI)
or net profit per year per total investment value, net present value (NPV), internal
rate of return (IRR), and payback time that considers cash flows (annual revenues,
operating costs, etc.) and time value of money as a function of time of the projected
life of the processing plant.115
Simulation software like Aspen Plus® and SuperPro Designer® have extensive
capabilities to integrate both process analysis and economics for predicting cost
necessary for key decision making toward conceptualizing a processing facility,
determining scalability based on key processing properties and engineering parameters, and estimated profitability of a production plant.
9.9 CONCLUSIONS
This chapter outlined the relevance of nutraceuticals in a newly developing biobased economy that is rapidly becoming an economic player in the food and agricultural industries. Bioprocessing techniques to produce high-value specialty biochemicals from food and agricultural by-products are emphasized, and greater need
for bioenergy production to compensate for deficiencies in the present oil-based
economy is described through the biorefinery concept. Specific examples of existing
and proposed industrial bioprocesses that show substantial economic potential were
outlined in terms of lipid-based, large and small molecular nutraceutical products.
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As the world population continues to increase posing an even greater health threat
to third world countries, there is a continuing need for the integration of biology
and engineering to address the issue for optimizing functional food, nutraceutical,
and pharmaceutical production that incorporates important principles of environmentally friendly technologies and continuing education to support new advances
for a bio-based economy.
ACKNOWLEDGMENTS
The authors would like to acknowledge undergraduate students (particularly students
from the 2005 Biochemical Engineering course) and graduate assistants (Jack
Heaton, Hui Zhu, Kristy Conwel, Jared Powell, Keri Cantrell, Cheng-Yi Kuan,
Meidui Dong, and Xiaohui Yu) in the Clemson University Biosystems Engineering
Program who participated in bioprocessing projects for contributing to literature
making this chapter possible.
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Dunaliella bardawil, Plant Physiol., 116, 1239–1248, 1998.
99. Ruddat, M. and Garber, E.D., Secondary Metabolism and Differentiation in Fungi,
Bennett, J.W. and Ciegler, A., Eds., Marcel Dekker, New York, 1983, pp. 95–152.
100. Burns, K., et al., Chemopreventive potential of wild lowbush blueberry fruits in
multiple stages in carcinogenesis, J. Food Sci., 7093, S159–166, 2005.
101. Roukas, T. and Mantzouridou, F., An improved method for extraction of b-carotene
from Blakeslea trispora, Appl. Biochem. Biotechnol., 90, 37, 2001.
102. Naczk, M. and Shahidi, F., Extraction and analysis of phenolics in food, J. Chromatogr. A, 1054, 95–111, 2004.
103. Kim, H., Wu, C., Chen, F., Wang, H., and Jin, Z., Evaluation of antioxidant activity
of tea tree (melaleuca alternifolia) oil and its components, J. Agric. Food Chem., 52,
2849–2854, 2004.
104. Kim, H.J., Chen, F., Wang, X., and Rajapaks., N.C., Effect of chitosan on biological
properties of sweet basil (Ocimum basilicum L.), J. Agric. Food Chem., 53,
3696–3701, 2005.
105. Wang, X., Chen, F., Wan, P.J., and Huang, G., Development of monoclonal antibodybased enzyme linked immunosorbent assay for gossypol analysis in cottonseed meals,
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106. Son, S.J., Reichel, J., He, B., Schuchman, M., and Lee, S.B., Magnetic nanotubes
for magnetic-field-assisted bioseparation, biointeraction, and drug delivery, J. Am.
Chem. Soc., 127, 7316–7317, 2005.
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extraction and reversed-phase liquid chromatography methods for vitamin A and βcarotene heterogenous distribution of vitamin A in the liver, J. Chromatogr., 762,
201–206, 1997.
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Engineering, Oxford University Press, New York, 2003.
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Leach, C.K., and Patmore, R.A., Bioreactor Design and Product Yield, ButterworthHeinemann, Boston, 1992.
110. Grady, C.P.L., Daigger, G.T., and Lim, H.C., Biological Wastewater Treatment, 2nd
ed., Marcel Dekker, New York, 1999.
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112. Shuler, M.L. and Kargi, F., Bioprocess Engineering: Basic Concepts, Prentice-Hall,
New York, 1992.
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John Wiley and Sons, New York, 2001.
115. Peters, M.S. and Timmerhaus, K.D., Plant Design and Economics for Chemical
Engineers, 4th ed., McGraw-Hill, New York, 1991.
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10
Microbial Modeling as
Basis for Bioreactor
Design for Nutraceutical
Production
Caye M. Drapcho
Clemson University
CONTENTS
10.1
10.2
10.3
10.4
10.5
Introduction ...............................................................................................238
Xylitol........................................................................................................239
Summary of Unstructured Microbial Growth Models .............................240
Unstructured, Single Limiting Nutrient Models.......................................240
Inhibition Models ......................................................................................241
10.5.1 Substrate Inhibition ......................................................................241
10.5.2 Product Inhibition.........................................................................242
10.5.3 Inhibition by Xenobiotic Compounds .........................................243
10.6 Models for Multiple Limiting Substrates or Nutrients ............................243
10.6.1 Complementary Substrates...........................................................243
10.6.2 Substitutable Substrates ...............................................................244
10.7 Yield Parameters .......................................................................................246
10.8 Temperature Effects ..................................................................................247
10.9 Kinetic Rate Expressions ..........................................................................247
10.10 Bioreactor Design......................................................................................250
10.11 Batch Reactors ..........................................................................................250
10.12 Continuous Stirred Tank Reactors (CSTRs).............................................251
10.13 CSTR with Cell Recycle...........................................................................253
10.14 Fed-Batch Systems....................................................................................255
10.15 Bioreactor Design Strategies.....................................................................256
10.16 Modeling of Glucose/Xylose Utilization and Product
Formation by Candida ..............................................................................258
10.17 Summary ...................................................................................................261
References..............................................................................................................261
237
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Functional Food Ingredients and Nutraceuticals
10.1 INTRODUCTION
A wide variety of nutraceutical compounds, food additives, and foodstuffs may be
produced through the bioconversion of substrates contained in agricultural crops and
residues. Bioconverted products include traditional foods and beverages such as beer,
wine, vinegar, yogurt, tofu, and baker’s yeast. Bioconverted nutraceutical and food
products used in the food industry range from flavor compounds (roasted, nutty
flavor of pyrazine produced through aerobic solid-state fermentation of soybeans by
bacterium Bacillus subtilis,1 fruity flavor of citronellol, linalool, and geraniol produced by yeast Kluveryomyces lactis,2 vanillin flavoring produced by Pseudomonas
and Corynebacterium,3 and bioflavors for beer produced by Saccharomyces
cerevisiae4); essential fatty acids (omega-3 and omega-6 fatty acids eicosapentaenoic (EPA) and arachidonic (ARA) acids from aerobic submerged fungal cultures
of Pythium irregulare, Mortierella elongata, and Mortierella alpina using soybean
and rice bran substrates,5–9 and omega-3 fatty acid docosahexaenoic acid (DHA) and
EPA from algal sources such as Gonyaulax, Gyrodinium, and Cryptoconidium10,11);
organic acids (citric acid produced by aerobic fermentation of sugarcane or beet
molasses by Aspergillus niger or Candida lipolytica,12–15 lactic acid produced by
Lactobacillus helveticus from concentrated cheese whey,16 and pyruvic acid production by variety of yeasts and bacteria17); pigments (the carotenoid pigment and
antioxidant astaxanthin produced by Xanthophyllomyces dendrorhous,18 red pigments produced by fungi Paecilomyces sinclairii during aerobic fermentation of
sucrose and starch,19 and orange and red dyes produced by submerged culture of
Monascus purpureus from hard wheat substrate20); emulsifiers (biosurfactants produced by filamentous fungi Curvularia lunata,21 and high molecular weight emulsifiers produced by yeast Candida utilis22); vitamins (vitamin B12 production by
prokaryotic bacteria Propionibacterium shermanii and Pseudomonas denitrificans,23,24 or mixed cultures of methanogens,25,26 and vitamin K2-7 production by
bacterium Bacillus subtilis using fermented soybeans27); and sugar substitutes
(xylitol produced from microaerobic fermentation of D-xylose contained in hydrolysates of sugarcane bagasse, rice straw, and other lignocellulosic material and defined
media by yeasts such as Candida and Pichia28–52).
Bioprocess design involves consideration of all critical unit operations, including
feedstock pretreatment and hydrolysis, bioreactor modeling and design, and extraction and separations technologies. Bioreactor modeling and design based on microbial growth and product formation kinetics may be used to optimize production of
high-value compounds by a range of natural microorganisms such as fungi, bacteria,
yeasts, and algae and recombinant organisms. Steps to the design based on microbial
kinetics include expression of growth, decay, and product formation rates; development of mass-balance models of the bioreactor; simulation of substrate, biomass,
and product concentrations and rates of biomass and product formation for various
reactor configurations; selection of reactor configuration; and design optimization
to maximize product formation rate or product concentration based on simulation.
The focus of this chapter is on microbial modeling and bioreactor design for suspended growth, shear-tolerant, natural cells. Many bioprocessing examples could
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Microbial Modeling as Basis for Bioreactor Design
239
serve to illustrate the kinetic approach to modeling and design; we will highlight
the bioconversion of xylose to the sugar substitute xylitol.
10.2 XYLITOL
Xylitol, a 5-carbon sugar alcohol (C5H12O5), has a relative sweetness equal to
sucrose, a negative heat of solution and is nonfermentable by cariogenic bacteria.53
Once ingested by humans, 80% to 85% of xylitol consumed is metabolized by the
liver, and 20% to 80% of this portion is converted to glucose depending on metabolic
need for glucose.53 The slow conversion of xylose to glucose reduces insulin stimulation. Due to these properties, it has found many uses in the food industry,
especially in confectionaries, gums, oral hygiene products, and diabetic foods.
Xylitol is found naturally at low levels in fruits and vegetables; for example,
plums contain 1% xylitol by weight, while strawberries, raspberries, eggplant, spinach, and pumpkin contain 0.3% to 0.1% xylitiol, respectively.53 Xylitol can be
synthesized chemically through the catalytic dehydrogenation of xylose, a 5-C sugar.
However, extraction from natural foods and chemical production is costly, leading
to investigations of bioconversion processes for xylitol production as an alternative.
Bioconversion of xylose to xylitol can be accomplished by yeasts, primarily of
the genus Candida and Pichia.29–52 These yeasts can utilize pentose and hexose
sugars, with hexose sugars as the preferred substrate.34,50 The sequential use of
glucose then xylose under batch conditions has been documented extensively.32,34,37,38,41,42,46 Under aerobic conditions, both glucose and xylitol are metabolized with biomass as the main product. Under conditions described variously as
oxygen-limited or microaerobic, species of Candida can form xylitol from Dxylose29–52 and ethanol from glucose and xylose.33–35
Hemicellulosic hydrolysates from agricultural residues such as sugar cane
bagasse, rice straw and wheat straw are plentiful and contain significant quantities
of xylose and glucose in their hemicellulose and cellulose fractions. For example,
1 ton of milled sugar yields 180 to 280 kg of sugarcane bagasse, with the typical
bagasse containing 19% to 24% hemicellulose, 32% to 48% cellulose, and 23% to
32% lignin.54 This composition represents typically 71% total reducing sugars, 25%
xylitol, and 41% glucose on a dry weight basis.40 However, mild acid pretreatments
with sulfuric or hydrochloric acids result in greater hemicellulose hydrolysis than
cellulose hydrolysis.39 Varying conditions of temperature, time, and acid concentration result in greatly varied concentrations of sugars in sugarcane bagasse hydrolysate. A typical sugarcane bagasse hydrolysate may contain 17 to 105 g/L xylose and
7 to 30 g/L glucose, while a typical rice straw hydrolysate may contain 16 to 79
g/L xylose and 4 to 23 g/L glucose.39
Bioconversion of xylose can achieve xylitol yields as high as 0.7 to 0.8 g xylitol/g
xylose in hemicullulosic hydrolysates30,37,43 and 0.8 to 0.9 in defined xylose
media.35,38 The theoretical yield is 0.917 g/g. For example, batch fermentations of
sugarcane bagasse hydrolysate using Candida guilliermondii FTI20037 achieved
maximum xylitol production rates of 0.87 g/L-h with product yield of 0.67 g xylitol/g
xylose at 0.45 v.v.m. aeration rate (kLa 27 h–1).49 Reported values of maximum
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Functional Food Ingredients and Nutraceuticals
specific growth rate for Candida range widely from 0.04 to 0.52 h-1 for xylose and
glucose/xylose mixtures.38,40,50
10.3 SUMMARY OF UNSTRUCTURED MICROBIAL
GROWTH MODELS
Basic microbial growth models for suspended growth, shear-resistant cells should
consider potential impact of multiple growth-limiting substrate and inhibitory substrates, products, and xenobiotic compounds. Structured models consider individual
reactions occurring within the cell involving specific components, such as DNA or
proteins. Unstructured models view the cell as an entity and model growth and death
of the microorganism.55–63 Growth rates for unstructured models are expressed as v
(cell number specific growth rate) or µ (biomass specific growth rate), both in units
of time–1. Balanced growth occurs when v = µ and cell composition is constant with
time. Unbalanced growth occurs as cell composition changes with time, resulting
in a change in cell mass per cell; therefore v ≠ µ.56
10.4 UNSTRUCTURED, SINGLE LIMITING NUTRIENT
MODELS
The Monod model64 is a widely applied model used to describe microbial growth.
The model was developed for the growth of a single microorganism (E. coli) growing
on a media with a single limiting organic substrate (glucose) as shown below
µ=
µˆ SS
K s + SS
(10.1)
where µ = specific growth rate coefficient, h–1; µˆ = maximum specific growth rate,
h–1; SS = soluble substrate concentration, mg/L; and KS = half-saturation constant,
mg/L. The KS value is the substrate concentration at which the growth rate µ is equal
to 1/2 of µˆ .
The Monod model has been used successfully to model the growth of many
pure cultures of heterotrophic and autotrophic organisms growing on single substrates and mixed microbial cultures using mixed substrates, such as wastewater
treatment applications.59,60 Estimates of µˆ and KS parameter values may be obtained
by collecting data of specific growth rate values as a function of soluble substrate
concentration during exponential phase of batch growth, and applying a linearization
technique, such as Lineweaver-Burk or Hanes (Hofstee) equations.56,57,59
Zero and first order approximations of the Monod model (Equation 10.2 and
Equation 10.3) may be applied when the substrate concentration is high and low
relative to the KS value, respectively.
µ ≈ µˆ Zero order appropriation when SS>>KS
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Microbial Modeling as Basis for Bioreactor Design
µ≈
µˆ SS
First order appropriation when SS<< KS
Ks
241
(10.3)
Due to high substrate concentrations typically found in food/bioprocessing applications, many researchers report µˆ values and not KS .38 For Candida tropicalis utilizing
xylose under batch microaerobic conditions, µ̂ was determined to be 0.52 h–1 at
initial xylose concentrations of 50 g/L.38 However, much lower µ̂ values of 0.04 and
0.11 h–1 were reported for Candida guilliermondii41 cultures using xylose and glucose, respectively, and µ̂ values of 0.057 to 0.137 h–1 were reported for Candida
tropicalis cultures as a function of aeration rate.40 In these reports, observed specific
growth rate was apparently assumed to be the maximum specific growth rate value.
For example, Pessoa et al.40 reported that µ̂ values were determined from the growth
curve in the exponential phase. These values may represent the observed specific
growth rate for culture conditions present rather than the maximum specific growth
rate.
10.5 INHIBITION MODELS
Expansions of the Monod model to include inhibition by substrate, product, or
xenobiotic compounds have been developed. Many inhibition models for microbial
growth are empirical in nature, while others apply enzyme kinetics concepts.56 In
general, competitive inhibitors compete with substrate for binding site of the enzyme,
and may be xenobiotic compounds or products that accumulate in reactor. Uncompetitive inhibition occurs when a compound binds to the enzyme-substrate (ES)
complex. A special form of uncompetitive inhibition is substrate inhibition, where
substrate binds to an alternative site on the enzyme leading to a nonreactive ES
complex. Noncompetitive inhibitors can bind to either the free enzyme or the ES
complex. Product inhibition may be modeled as noncompetitive inhibition.56
10.5.1 SUBSTRATE INHIBITION
High substrate concentrations relative to the KS value may limit growth. Modifications of the Monod model, such as the Andrews’ Equation,65 may be used to describe
substrate inhibition:59
µ=
µˆ SS
2
K S + SS + SS K
I
(10.4)
where KI = inhibition coefficient, mg/L.
As the value of KI increases, the equation above simplifies to the Monod model. In
many cases of inhibitory substrate, the value of µ̂ may never be observed and
therefore KS cannot be calculated. In these cases, µ*, the maximum observed growth
rate, and SS*, the substrate concentration at which µ* occurs, are determined and
used to calculate µ̂ and KS :59
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Functional Food Ingredients and Nutraceuticals
µ* =
µˆ
and SS* = (KS /KI)0.5
2( K S / K I )0.5 + 1
(10.5), (10.6)
Bioprocessing studies have investigated the inhibition of cell growth rates due to
elevated substrate levels. For example, Oh and Kim38 found that increasing the initial
xylose concentration past 50 g/L for Candida batch fermentations on defined xylose
media under microaerobic conditions resulted in reduced exponential-phase specific
growth rates, from 0.52 h–1 at 50 g/L initial xylose concentration, to 0.49, 0.47, 0.35,
and 0.23 h–1 at xylose concentrations of 100, 150, 200, and 250 g/L, respectively.38
However, Equation 10.5 does not model the response well, which may indicate that
other factors, such as oxygen limitation with increasing substrate, lack of pH control,
or product inhibition, may also have influenced the specific growth rate.
For phototrophic microorganisms, Steele’s model66 is often used to describe algal
growth considering potentially inhibitory light intensity66–68

I 1 −
µ = µˆ L e
I opt
IL 

I opt 
(10.7)
where IL and Iopt = light and optimal light intensity, µmol/m2-s.
10.5.2 PRODUCT INHIBITION
High product concentrations may inhibit microbial growth. Product inhibition may
be modeled as
µ=
(
n
P
µˆ
µˆ S

or µ =
1−
1− P P


m
K
Pm 
KS + S

S  
 1 +

S
)
n
(10.8)
where P = product concentration, mg/L; Pm = product concentration where growth
is completely inhibited; n = coefficient determined by data. For example, yeast
growth under anaerobic conditions may be inhibited by ethanol concentrations above
5%.56,61 The growth of Candida shehatae was completely inhibited by ethanol
concentrations of 25 g/L under microaerobic fermentations33 and 37.5 g/L under
aerobic conditions.29
Another equation to describe noncompetitive inhibition by product is represented
in several different forms as shown below56,58,61
µ=
µˆ SS  K P 
µˆ
 S  1
or µ =
or µ = µˆ 


P
 K S + S 
K S + SS  K P + P 
KS  
P 

1+
 1 + S   1 + K 
Kp

S
p
(10.9)
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Microbial Modeling as Basis for Bioreactor Design
243
where Kp = product inhibition constant, mg/L.
10.5.3 INHIBITION
BY
XENOBIOTIC COMPOUNDS
A general form of uncompetitive inhibition of enzymes can be used to model
inhibition by other compounds (rearranged from Blanch and Clark56)
µ=
µˆ SS
SS 1 + I K + K S
I
(
)
(10.10)
where I = inhibitor concentration, mg/L, and KI = inhibition constant, mg/L.
Another equation used to model general inhibition is given as
 SS   K I 
µ = µˆ 
 K S + SS   I + K I 
(10.11)
Sugarcane bagasse hydrolysate contains compounds such as furfural and
5-hydroxymethylfurfural (HMF) that may inhibit yeast growth at high concentrations. Inhibition of cell growth occurred at furfural and HMF concentrations of 1.0
and 1.5 g/L, respectively, for Candida guilliermondi.30 Use of KI value of 0.01 g/L
in Equation 10.12 would result in a predicted 99% rate reduction of the specific
growth rate at furfural concentration of 1.0 g/L.
10.6 MODELS FOR MULTIPLE LIMITING SUBSTRATES
OR NUTRIENTS
Substrate or nutrients can be thought of in a broad sense as complementary or
substitutable. Complementary nutrients meet different needs for the microorganism;
for example, oxygen may serve as electron acceptor for yeast growth while glucose
serves as electron donor. Substitutable substrates meet the same need for the cell.
Glucose and xylose used by yeasts such as Candida may be viewed as substitutable
substrates.
10.6.1 COMPLEMENTARY SUBSTRATES
To model multiple complementary nutrients, an interactive form of the Monod model
may be used58–60
S2 
 S1  
µ = µˆ 
K + S 
+
K
S
2 
 s1 1   s 2
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Functional Food Ingredients and Nutraceuticals
The interactive model is based on the assumption that both substrates influence the
growth rate. This model predicts a lower specific growth rate than the noninteractive
model, particularly when substrate concentrations are small compared to their KS
values. This model is a continuous function and has been used in many applications,
including describing the growth of hybridoma cells based on substrates glucose and
glutamine with inhibition by products lactate and ammonia as follows58



Sglu
Sglut
K lact
K NH


µ = µˆ 





 K glu + Sglu   K glut + Sglut   K lact + Plact   K NH + PNH 
(10.13)
with Sglu, Sglut, Plact, and PNH = soluble concentrations of glucose, glutamine, lactate,
and ammonia, respectively, mg/L; and Kgl, Kglut, Klact, and KNH = K values for glucose,
glutamine, lactate, and ammonia, respectively.
An alternative approach to model complementary nutrients, called the noninteractive model, assumes that one nutrient limits growth59
 
S1  
S2
µ = µˆ ∗ minimum of  
, 
  K s1 + S1   K s 2 + S2
 

 
(10.14)
The predicted growth rate will be the lowest value based on each substrate concentration. This model is a discontinuous function at the transition from one nutrient
limitation to another. Both models have advantages and have been used successfully
to describe microbial growth.
Neither model was found in the bioprocessing literature investigated here to
describe Candida or Pichia growth utilizing xylose and other substrates. However,
these models could be applied for these fermentations where carbohydrate and
oxygen or nutrients may be potentially growth-rate limiting. The KS value for oxygen,
KS, O2, for most aerobic, heterotrophic bacterial cultures is quite low, and often is
less than 1 mg/L.59
10.6.2 SUBSTITUTABLE SUBSTRATES
When multiple substitutable substrates are present in the media, simultaneous use
of the substrates may occur. In this case, one compound may be preferred by the
microorganism over the other. If S1 is preferred, then its presence in the media will
inhibit the use of S2. The growth rate of the microorganism using S2 is modeled as
a function of S2 with inhibition by S1 as follows59,60
S1 

µ1 = µˆ 
 K S1 + S1 
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245

 K

S2
S1
µ 2 = µˆ 


 K S2 + S2   K S1 + S1 
(10.16)
For example, both glucose and xylose may be used by yeasts such as Candida.
Glucose is the preferred substrate, so its presence in media suppresses the use of
xylose by reversibly inactivating the enzyme xylose reductase,41 while the presence
of xylose in the media does not influence the use of glucose. The sequential use of
glucose then xylose has been documented by many researchers.34,38,40–42 Thus, the
growth rate of Candida based on glucose use may be modeled as follows

Sglu 
µ Glu = µˆ 
 K glu + Sglu 
(10.15a)
where µGlu represents the specific growth rate using glucose as organic substrate,
h–1. The growth rate of Candida based on use of xylose may be modeled as

  K glu 
SXyl
µ Xyl = f * µˆ 


K
+
S
 Xyl
Xyl   K glu + Sglu 
(10.16a)
where µXyl represents the specific growth rate using xylose as organic substrate, h–1,
and f represents factor for decreased maximum specific growth rate for nonpreferred
substrate.
These equations represent the growth of Candida as exclusively using glucose
when the glucose concentration is high. As the glucose concentration declines, the
growth rate based on glucose declines, with concomitant increase in the growth rate
based on use of xylose. Simulations of Candida specific growth rate values based
on xylose use as a function of glucose and xylose concentrations (Equation 10.16a)
using representative kinetic values are shown in Figure 10.1.
0.5
0 g/L
glucose
2 g/L
glucose
5 g/L
glucose
50 g/L
glucose
0.4
0.3
0.2
0.1
0
0
10
20
30
Xylose, g/L
40
50
FIGURE 10.1 Simulated specific growth rate (hr–1) of Candida based on xylose concentration
using representative kinetic parameter values.
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Functional Food Ingredients and Nutraceuticals
If multiple complementary and substitutable substrates are present, the terms
may be combined in multiplicative manner. The following expressions could be used
to describe growth of Candida under aerobic conditions if oxygen, glucose, and
xylose are growth-rate limiting substrates

Sglu  
SO 2

µ Glu = µˆ 


 K glu + Sglu   KO 2 + SO 2 
(10.15b)

  K glu  
SXyl
SO 2

µ Xyl = f µˆ 
 K + S 



K
+
S
K
+
S
 Xyl
Xyl  
glu
glu 
O2
O2
(10.16b)
Combining Equation 10.8 (product inhibition- ethanol), Equation 10.11 (xenobiotic
inhibition-furfural), Equation 10.12 (complementary nutrients – O2 and glucose),
and Equation 10.15 and Equation 10.16 (substitutable substrates – glucose and
xylose) results in the following growth models for Candida


Sglu
SO 2
  Pm,ETOH − PETOH
µ glu = µˆ 



PETOH
 K glu + Sglu   K O 2 + SO 2  

K furfural

  K
 furfural + S furfural 
(10.15c)

  K glu  
Sxyl
SO 2
  Pm,ETOH − PETOH 
µ xyl = f µˆ 






PETOH
 K xyl + Sxyl   K glu + Sglu   KO 2 + SO 2  

 K

K furfural

S
+
furfural
furfural 
(10.16c)
10.7 YIELD PARAMETERS
Biomass yield (YX/S) is defined as the mass of cells produced per mass of substrate
utilized. Product yield (YP/S) is defined as the mass of product formed per mass of
substrate utilized. Biomass and product yields may be determined from the stoichiometry of a balanced growth equation, or by measurement. For the balanced equation
describing anaerobic fermentation of glucose by yeast61 given below, YX/S and YEtOH/S
values are 0.078 mg biomass/mg glucose and 0.33 mg ethanol/mg glucose, respectively.
1000 C6H12O6 + 118 NH3 → 5.9 C100H174O45N20 + 1300 C2H5OH +
1540 CO2 + 430 C3H8O3 + 36 H2O
(10.17)
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Biomass and product yields are generally assumed to not vary with respect to
substrate concentration, but will vary for different electron acceptor environments.
For example, xylitol yields from xylose for Candida tropicalis were reported by
Kastner et al.35 as a function of redox potential of the culture environment as follows:
YXylitol/xylose = 0.63, 0.87, 0.15, and 0.10 g/g for redox potentials of –150, –100, –50,
and 0 mV, respectively.
10.8 TEMPERATURE EFFECTS
Microbial growth models and design should consider impact of temperature on
specific growth rate and decay constant values. Biomass yield, product yield, halfsaturation constant, and growth-associated product constant values are often considered independent of temperature. Maximum specific growth rate and decay constant
values should be corrected for temperature differences using the Arrhenius or modified Arrhenius equation shown below59
k = Ae− EA / RT Arrhenius Equation
(10.18)
where k = temperature-dependent reaction rate constant; A = Arrhenius constant,
EA = activation energy, kJ/mol; R = gas constant, kJ/mol K; T = absolute temperature,
K.
kT 1 = kT 2 θ(T1 −T2 ) Modified Arrhenius Equation
(10.19)
where kT1 and kT2 are the reaction rate constants at temperatures 1 and 2; θT =
temperature correction factor, dimensionless, and T = temperature, °C or K.
The Arrhenius expression is applicable over a wide range of temperatures. The
modified Arrhenius expression should be applied only over a narrow range of
temperatures, typically those encountered for mesophilic organisms.59
10.9 KINETIC RATE EXPRESSIONS
Once the appropriate relationship to describe the specific growth rate is determined,
the rates of utilization or formation can be developed for important reactor compounds. The rate of biomass formation is given as
rXB = µ X B
(10.20)
where rXB = rate of biomass formation, mg/L-h.
For growth rate expressions developed for cells using substitutable substrates, as in
the case of xylose and glucose use by Candida, the rates of biomass formation using
both substrates could be described as follows, following models proposed59,60 for
wastewater treatment
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rXB = µ Glu X B + µ Xyl X B
(10.20a)
Products formed from bioconversion processes were classified by Gaden69 as one
of three types: Type I, growth-associated products arising directly from metabolism
of carbohydrate; Type II, products arising indirectly from carbohydrate metabolism
that accumulate under abnormal metabolism; Type III, nongrowth-associated products formed as a result of processes other than growth/energy metabolism.
Using this classification, ethanol produced by the anaerobic fermentation of
glucose by yeasts is a Type I product and antibiotics such as streptomycin are Type
III products.69 Citric acid produced as a result of energy metabolism by aerobic
fermentation of molasses and accumulated under conditions of nutrient-limitation
of trace metals such as manganese, iron, and zinc by Aspergillus niger14 would be
characterized as a Type II product.69
This classification may be complex for some bioproducts. For example, polydroxyalkanoates (PHAs), which are internal energy/carbon storage products, are of
interest for biodegradable polymers in food packaging and films.70 PHAs can be
formed by two routes: microbes such as Alcaligenes eutrophus produce and accumulate PHAs under nutrient-limited conditions with excess carbon source, while
microbes such as Alcaligenes latus do not require nutrient limitation for PHA
production and accumulation but will accumulate greater quantities under nutrientlimited conditions.70 Therefore, PHA could be classified as a Type II or Type I
product, depending on the microbial culture employed. Many have adopted a classification that combines the Type I and Type II products described by Gaden into
one category, growth-associated (GA) products. The Type III products are nongrowth-associated (NGA) products. A middle category, called mixed products, are
those that are formed from both growth and nongrowth metabolic processes. Using
this convention, PHA would be considered a GA product.70
The rate of growth-associated product formation can be expressed as a function
of the rate of biomass formation63
rpg= kpgrXB = kpgµXB
(10.21)
where rpg = rate of growth-associated product formation, mg/L-h; and kpg = growthassociated product formation ratio, mg product/mg biomass.
For example, the kpg value for ethanol based on the anaerobic fermentation of
glucose by yeast (Equation 10.17) 61 can be determined by the stoichiometry of the
balanced equation as 4.26 g ethanol/g biomass. The value of kpg may also be
calculated as the ratio of YP/S/YX/S or measured directly.
The rate of nongrowth-associated (NGA) product formation is modeled solely
as a function of the concentration of biomass in the reactor, and not the rate of
biomass formation.63
rpn = kpnXB
where rpn = rate of nongrowth-associated product formation, mg/L-h and
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kpn = nongrowth-associated product formation constant, mg product/mg biomass-h.
Products formed from a combination of growth and nongrowth-associated means
may be modeled as 63
rP = k pg µX B + k pn X B
(10.23)
Examples of NGA products in food/agricultural bioprocessing include vitamins
such as K2-7,27 pigments produced from Monascus fermentations,20 and emulsifier
production by filamentous fungi Curvularia lunata.21 From graphs of xylose use
and xylitol formation presented by several researchers, xylitol appears to be a mixed
or NGA product.35,38,42
Other reactions that occur as a result of growth, such as substrate and oxygen
utilization, are represented also as functions of the rate of biomass formation. The
rate of organic substrate utilization is therefore
rS =
µX B
rX B
=
YX / S
YX / S
(10.24)
The rate of substrate utilization that occurs as products are formed during
nongrowth processes can be modeled as
rS =
rpn
YP /S
(10.25)
The rate of O2 utilization during aerobic growth can be expressed as a function
of a stoichiometric factor based on the balanced growth equation or measured value.
For example, in a representative equation describing organotrophic bacteria
(C5H7O2N) grown on glucose, 0.578 mg O2 are required per mg XB formed59;
therefore
ro =ratio * rX B = 0.578* µX B
(10.26)
where rO = rate of oxygen utilization, mg/L-h O2 .
If the stoichiometry is not known, then the rate of oxygen utilization can be
expressed as follows if biomass and organic substrate are measured on a chemical
oxygen demand (COD) basis59,60
ro = (1 − YX /S ) * rS =
1 − YX / S
µX B
YX / S
(10.27)
A final expression important to bioreactor modeling is the rate of microbial
decay. This rate of natural death or decay may be modeled as first order with respect
to biomass.
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rXB,d = –b XB
(10.28)
where rXB,d = rate of biomass decay, mg/L-h XB; b = decay constant, h-1.
10.10 BIOREACTOR DESIGN
The kinetic rate expressions developed above may be used in mass balance equations
with respect to substrate, biomass, and products for different bioreactor configurations. Basic bioreactor designs for suspended growth cultures are batch, continuous
(flow) stirred tank reactor (CSTR), and CSTR with biomass recycle. To develop the
mass balance equations with respect to products in the reactor, characterization of
the product as a GA, NGA, or mixed metabolite is needed. Further characterization
of the product as intracellular vs. extracellular, and “soluble” vs. “particulate” as
compared to cell separation technique is needed for CSTR with recycle designs. If
the means of cell separation (filtration, centrifugation, and settling) removes the
compound with the biomass, then the product is considered particulate. Ethanol and
xylitol are examples of soluble, extracellular products, while oils produced by
filamentous fungi Pythium and PHAs are intracellular products. Products such as
monoclonal antibodies are extracellular, “particulate” compounds, since they can be
separated by ultrafiltration.
10.11 BATCH REACTORS
The mass balance with respect to biomass for a batch reactor is given as follows,
assuming growth and decay are the only reactions
µX B − bX B =
dX B
dt
(10.29)
Although there is a temporary balancing of the growth and decay terms during
stationary phase, batch reactors never reach true steady-state conditions, where
concentrations in the reactor do not change with time. During exponential growth
in a batch reactor, decay is often assumed to be negligible. Integration of Equation
10.29 and substitution of the definition of biomass yield results in the logistic growth
equation61
K S YX / S
S
K S YX / S

 X 
(t − t o )µˆ = 
+ 1 ln  Bt  +
ln 0

 X B 0 + S0YX /S
  XB0
X B 0 + S0YX /S St
(10.30)
where t and tO = time and initial time, h; XBt and XBO = biomass concentration at
time t and tO, and SSt and SSO = soluble substrate concentration at time t and tO. This
equation describes how biomass increases while substrate decreases with time during
exponential growth. The length of incubation in a batch reactor may vary depending
on growth kinetics, accumulation of inhibitory products, and product type. Equation
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251
10.30 can also be used to determine the Monod kinetic parameters for batch growth
data, a technique that is primarily utilized in the wastewater treatment literature71,72
but is being applied to bioprocessing applications.73
10.12 CONTINUOUS STIRRED TANK REACTORS
(CSTRS)
For a simple CSTR with a single influent and effluent flow, the mass balance with
respect to biomass is
X Bi
Q
Q
dX B
− XB
+ µX B − bX B =
V
V
dt
(10.31)
where XBi = biomass concentration in influent flow, mg/L.
For cases with no biomass in the influent flow, growth based on a single limiting
nutrient and no substrate or product inhibition, the following equation results at
steady state
µ=
1
+b
τ
(10.32)
where τ = hydraulic retention time in reactor, hr.
Thus, the hydraulic retention time is the main design parameter for a CSTR,
since it controls the microbial growth rate in the reactor. A simple CSTR can be
operated at a short retention time to maintain a high specific growth rate to mimic
a batch reactor during exponential growth, with resulting sustained formation of
growth-associated products, or may be operated at a long retention time to maintain
a low specific growth rate to mimic a batch reactor during declining exponential
or stationary phases, resulting in continuous formation of nongrowth-associated
products.
Substitution of the single Monod model for µ results in
SS =
K S (1 / τ + b )
µˆ − (1 / τ + b )
(10.33)
The effluent substrate concentration is a function of the hydraulic retention time
and the kinetic parameters values, but not a function of the influent substrate
concentration.
The mass balance with respect to substrate for a simple CSTR is
SSi
Q
µX B
Q
dSS
− SS
−
=
V
V
YX / S
dt
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Functional Food Ingredients and Nutraceuticals
After substitution of Equation 10.32, the resulting steady-state concentration of
biomass is given as
XB =
YX /S (SSi − SS )
1 + τb
(10.35)
The mass balance with respect to a general product is
Pi
Q
Q
dP
−P
+ k pg µX B + k pn X B =
V
V
dt
(10.36)
The steady-state solutions for GA and NGA products are
PGA = k pg µXb τ
(10.37)
where PGA is growth-associated product concentration, mg/L; and
PNGA = k pn Xb τ
(10.38)
where PNGA is the nongrowth-associated product concentration, mg/L.
At steady state, concentrations of substrate, biomass, and product will vary as
a function of retention time. In general, the specific growth rate and effluent substrate
concentration decline and biomass concentration increases with increase in hydraulic
retention time. Thus reactor design may be based on achieving the desired effluent
concentrations. Martinez et al.36 investigated a CSTR for xylitol formation using
Candida guilliermondi cultures and sugar cane bagasse hydrolysate. In most comparisons, they found higher xylitol and biomass concentrations and lower substrate
concentrations at a longer hydraulic retention time (100 vs. 20 h).
To maximize rates of biomass or GA product formation or of substrate utilization, both a high specific growth rate and a high biomass value must be achieved.
The hydraulic retention time that maximizes these rates can be estimated from the
following63
0.5
1 ˆ   KS  
= µ 1 − 
 
τ
  K S + SSi  
(10.39)
For an NGA product, there is no single hydraulic retention time that maximizes the
rate of production. The equations above may be used for preliminary analysis and
design of a CSTR. For more expanded analysis, and for systems where multiple
substrates or inhibition occur, dynamic modeling may be used to determine the
retention time that maximizes biomass or GA formation rates, or the product concentration as a function of retention time.
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253
10.13 CSTR WITH CELL RECYCLE
A CSTR with biomass recycle includes some means to remove biomass from the
reactor effluent flow, through external or internal filter, settling device, or centrifugation, and return biomass flow to the reactor. By separating biomass from the
effluent flow, the length of time that cells remain in the reactor, the cell retention
time, θ, can be controlled independently of the hydraulic retention time. To control
θ, a portion of biomass is harvested or “wasted” from the reactor. When the harvest
flow is pumped directly from the reactor, the biomass concentration in the reactor
is equal to the concentration in the harvest flow, and θ is calculated as
θ=
VX B
V
=
QH X BH QH
(10.40)
where θ = cell retention time, h; V = reactor volume, L; XB = biomass concentration
in reactor, mg/L; XBH = biomass concentration in the harvest flow, mg/L; and QH =
harvest flow rate, L/h.
The mass balance equation with respect to biomass for a CSTR with recycle is
X Bi
Q
(Q − QH )
Q
dX B
− X B H − X Be
+ µX B − bX B =
V
V
V
dt
(10.41)
where XBe = biomass concentration in final effluent flow, mg/L. XBe ~ 0 if cell
separation step is assumed 100% efficient.
At steady state, the mass balance with respect to biomass simplifies to
µ=
1
+b
θ
(10.42)
Therefore, θ controls µ for a CSTR with recycle in the same manner as τ does for
a simple CSTR. Substitution of the single Monod model for µ results in
SS =
K S (1 / θ + b )
µˆ − (1 / θ + b )
(10.43)
The mass balance with respect to soluble substrate for a CSTR with recycle is
SSi
Q
(Q − QH ) µX B dSS
Q
− SS H − SS
−
=
V
V
V
YX / S
dt
(10.44)
Because soluble organic substrate will not be removed by the cell separation technique, the same concentration will be present in the harvest and main effluent flow.
The mass balance at steady state simplifies to Equation 10.45, which describes the
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Functional Food Ingredients and Nutraceuticals
biomass concentration as a function of both the hydraulic and cell retention times.
Influent organic substrate concentration and hydraulic retention time can be adjusted
to achieve the desired cell concentration in the reactor.
XB =
θ YX /S (SSi − SS )
×
1 + bθ
τ
(10.45)
The mass balance with respect to a general product is given below.
Pi
Q
(Q − Qw )
Q
dP
− P w − P
+ k pg µX B + k pn X B =
V
V
V
dt
(10.46)
Product concentrations at steady state will vary according to whether the product is
GA or NGA, and particulate or soluble, as shown below
(
)
P = k pg 1 θ + b X B τ if product is GA, soluble
(
)
(10.47)
P = k pg 1 θ + b X B θ if product is GA, particulate
(10.48)
P = k pn X B τ if product is NGA, soluble
(10.49)
P = k pn X B θ if product is NGA, particulate
(10.50)
In general, the specific growth rate and effluent soluble substrate concentration will
decline as cell retention time increases, and biomass and nongrowth-associated
product concentrations, especially for particulate products, will increase with
increase in cell retention time. In a CSTR with recycle, a single optimum cell
retention time that maximizes the rate of product formation for all types of products
does not exist. A model of the system based on mass balance equations may be used
to run simulations to determine the product concentration and rate of product formation at different hydraulic and cell retention times. System optimization for a
biological CSTR with recycle is a matter of determining the best combination of
cell density and production rates for the system.
A CSTR with biomass recycle inoculated with Candida guilliermondii FTI
20037 was shown to achieve xylitol volumetric productivity rates of 0.91 g/L-h at
a hydraulic retention time of 16.7 h (dilution rate of 0.060/h) and essentially infinite
cell retention time (i.e., all cell mass was filtered through 0.01-µm ultrafilter and
continuously recirculated to reactor so the only loss of cell mass was through
decay).48 This productivity was achieved with defined media containing 30 g/L
xylose and no glucose. Recycling 100% of cell mass is not typically employed in
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255
CSTR with recycle designs, since control of the specific growth rate through wasting
of cell mass is usually desired.
10.14 FED-BATCH SYSTEMS
The important difference of fed-batch compared to batch and CSTR systems is that
the specific growth rate decreases with time due to increasing volume at constant
feed rates56 as shown by
dµ d  Q 
−Q 2
= 
=

dt dt  V0 + Qt  (V0 + Qt )2
(10.51)
where VO = initial volume.
However, steady-state operation may be achieved by exponentially adjusting the
feed rate, Q as shown by
dV
= Q = λV
dt
(10.52)
Q = λV0 eλt
(10.53)
which is integrated to obtain
where λ is the dilution rate or 1/τ.
At steady-state operation, the expression for substrate concentration mimics that
developed for the CSTR system (Equation 10.33) as shown below excluding the
decay term
µ=
KS τ
1 ˆ SS
and SS ≅
=µ
τ
K s + SS
µˆ − (1 τ )
(10.54)
Fed-batch operation may be represented at steady-state in terms of biomass and
product formation excluding decay,61 respectively, as
X t = X0t + QYB SSi t
(10.55)
where Xt is the total biomass produced (mg) and
P = P0
© 2007 by Taylor & Francis Group, LLC
V0
t 
V
+ k p XB  0 +  t
 V 2τ 
V
(10.56)
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Functional Food Ingredients and Nutraceuticals
where P is the product concentration (mg/L).
A fed-batch bioreactor system has inherent advantages of both batch and continuous systems since the operation is semicontinuous with only substrate feed and
no effluent stream. For this reason, these systems are often employed in many
commercial applications within the food and pharmaceutical industry. The system
works well for NGA production typically accomplished by introduction of secondary
metabolite inducers, specific nutrient deprivation, or substrate inhibition at specific
times during the process. The fed-batch system is also applied to form GA and
mixed-growth-associated (MGA) products such as heterotrophic algal oils by adjusting substrate feed rate to obtain maximum conversion to oil stored in high cell
density culture. Other common examples where fed-batch systems are employed in
commercial settings include baker's yeast (GA) and penicillin (NGA) production.
Fed-batch systems have been used to produce xylitol and ethanol from mixed sugars,
glucose, and xylose, in Candida shehatae,34 Pichia stipitits, 45 and Candida boidinii50
cultures.
10.15 BIOREACTOR DESIGN STRATEGIES
Bioreactor design may be based on maximizing the rate of product formation,
biomass production, or substrate utilization, or on achieving target concentrations
of product, biomass, or substrate. As revealed by the rate expressions above, rates
of substrate, oxygen and nutrient utilization, and GA product formation will be high
when the rate of biomass formation is high, while the rates of NGA product
formation and decay will be high when the concentration of biomass is high. In
general, rates of primary product formation in a batch reactor will be highest during
mid to late exponential phase, while product concentration will be highest at end of
exponential phase. Secondary product concentration in a batch reactor will peak at
end of stationary phase. These responses are illustrated in Figure 10.2 and Figure
10.3 for representative GA and NGA products. For a GA, extracellular, soluble
product such as ethanol, a batch reactor or simple CSTR would achieve high product
formation rate. But for nongrowth-associated products or reactions that form inhibitory products, batch reactors may not be advantageous due to the low productivity
rate or elevated product concentrations that may form. Selection of a simple CSTR
allows for control of the specific growth rate through control of the hydraulic
retention time to maximize the rate of a GA product. In a simple CSTR or CSTR
with biomass recycle, the steady-state specific growth rate is inversely proportional
to the hydraulic or cell retention time, respectively, while biomass concentration is,
in general, directly proportional to the retention times. Therefore, these designs allow
for efficient production of either primary or secondary products by achieving target
specific growth rate and biomass or product concentrations through control of the
retention time. Dynamic modeling and simulation of microbial growth and product
formation are needed to optimize the design based on predicted production rates
and concentrations.
For NGA, intracellular products, such as certain lipids produced by fungi, or
NGA extracellular, particulate products, such as monoclonal antibodies produced
by hydridoma, the rates of formation and product concentrations will be maximized
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Microbial Modeling as Basis for Bioreactor Design
1: XB
60
2: GA product
257
3: NGA product
2
2
2
3
3
30
3
1
1
2
0 1
0.00
1
3
12.50
25.00
Time
1
37.50
50.00
FIGURE 10.2 Simulations of representative biomass (XB), growth-associated (GA), and
nongrowth-associated (NGA) product concentrations (g/L) vs time (h) in a batch reactor.
(Simulation conducted with STELLA® 8 software.)
1: XB form
20
2: GA product form
3: NGA product form
2
1
10
3
1
0 1
0.00
2
3
2
12.50
1 2
25.00
Time
3
1 2
37.50
3
50.00
FIGURE 10.3 Simulations of representative biomass (XB), GA, and NGA product formation
rates (g/L-h) vs. time (h) in a batch reactor. (Simulation conducted with STELLA® 8 software.)
by a CSTR with biomass recycle design with fairly long cell retention time, which
easily allows for high biomass concentration to be achieved without exposing cells
to inhibitory high substrate concentrations.
Alternating environments with respect to substrate level, electron acceptor, or
nutrients can be used to maximize product formation or selecting desired biomass
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Functional Food Ingredients and Nutraceuticals
cultures.56,59 For example, high yields of the vitamin B12 by the bacterium Propionibacterium require microaerobic conditions; however, biosynthesis of a critical
enzyme (5,6-dimethylbenzimidazole (DMBI)) requires oxygen. Therefore, biosynthesis of vitamin B12 requires two reactor environments. Martens et al.23 proposed
the use of batch reactors with anaerobic conditions for the first three days of
incubation for production of the vitamin B12 precursor cobamide, with subsequent
aeration for three days for synthesis of DMBI and ultimately, vitamin B12. These
bioprocesses would be a likely candidate for use of two-stage CSTR system with
anerobic/aerobic environment for optimizing vitamin production.
Xylitol fermentations are another example of a bioprocess where alternating
environments can be beneficial. Microorganisms such as Candida will exhibit greater
specific growth rate and greater biomass yield under aerobic conditions, while the
desired products, ethanol or xylitol, are produced only under anaerobic or microaerobic conditions. Bioreactor designs with alternating environments allow for full
utilization of glucose under aerobic conditions for biomass production, followed by
anaerobic/microaerobic conditions to allow for ethanol or xylitol production. For
example, use of a fed-batch, two-phase reactor with aerobic growth phase followed
by anaerobic conditions to produce ethanol with Candida shehatae cultures produced
yield of 0.4 g ethanol/g xylose and 75% to 100% conversion of xylose.34 Using this
design scheme, the specific growth rate for Candida shehatae under aerobic utilization of D-xylose was µ = 0.32 h–1 producing 2 × 109 cells/mL, while in the second
phase, ethanol yield was 0.23 g/g.34 Final ethanol concentrations (up to 50 g/L)
showed greater success than the genetically modified strain of Zymomonas mobilis74
which resulted in 30 g/L ethanol. With a semicontinuous process, Rodrigues et al42
used Candida guilliermondi to ferment sugar cane bagasse hydrolysate. A maximum
product yield of 0.79 g xylitol/g xylose was achieved, and a productivity of 0.66
g/L-h. The authors note that the critical first step is the rapid production of cell
mass in the culture medium, which can be achieved by aerating throughout the first
phase, then allowing oxygen levels to drop to stimulate xylitol production.42
10.16 MODELING OF GLUCOSE/XYLOSE
UTILIZATION AND PRODUCT FORMATION BY
CANDIDA
Fermentation of xylose and glucose by Candida in sugarcane bagasse hydrolysate
is an excellent example of a bioprocess with complex process kinetics that illustrates
many of the concepts discussed here. First, the sequential use of glucose then xylose
may be modeled as multiple substitutable substrates; second, the impact of xenobiotics, such as furfurals, and products such as ethanol, that have been identified as
inhibitory may be modeled; third, the ability of species such as Candida to metabolize multiple sugars under aerobic, anaerobic, and microaerobic environments, with
resulting products from each, demonstrates the advantage of using multiple phases
of batch or multiple reactors of continuous flow reactors. To illustrate the xylitol
formation by yeasts such as Candida, simulations of microaerobic batch, microaerobic CSTR, and two CSTRs in series design, with first CSTR under aerobic
© 2007 by Taylor & Francis Group, LLC
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Microbial Modeling as Basis for Bioreactor Design
259
conditions and second CSTR under microaerobic conditions, were conducted. For
simplicity, the modeling used here considered sequential use of glucose/xylose but
assumed no inhibition by substrates, xenobiotics, or products. A typical concentrated
sugarcane bagasse hydrolysate composition of 62 g/L xylose and 8 g/L glucose42
was used for these simulations. Representative kinetic and yield parameter values
used were: µ̂ = 0.5 h–1 using glucose for full aerobic conditions; f = 0.4 (assumed);
YXylitol/xylose = 0.87 g/g for microaerobic conditions.32 Xylitol formation was modeled
as a nongrowth-associated product, with kpn value of 0.3 g/g-h estimated from
reported data.32 For microaerobic conditions, YX/Glu = 0.18 g/g and YX/Xyl = 0.05 g/g
were calculated from data32 while biomass yield values for aerobic conditions (YX/Glu
= 0.4 g/g and YX/Xyl = 0.2 g/g) were assumed. Mass balance equations were solved
simultaneously using a program written with STELLA® software (High Performance
Systems, version 8.0).
As shown in Figure 10.4, the sequential use of glucose/xylose in a batch reactor
can be modeled, with xylose consumption beginning at approximately 12 h, after
glucose concentration has dropped considerably. Xylitol formation begins with use
of xylose as substrate at approximately 12 h. The overall form of the concentration
profiles match those presented for batch fermentations in mixed substrates.41,42
Results obtained by Pfeifer41 show that complete glucose utilization occurred by
~10 h of batch fermentation, and complete utilization of xylose by 30 to 35 h.
Simulations of rates of substrate utilization and product formation (Figure 10.5)
represent the sequential use of glucose/xylose and the lag in xylitol formation due
to nongrowth-associated nature of product formation.
1: Glucose
62
2: Xylose
3: Xylitol
2
2
31
1
0
0.00
3
3
1
3
1
12.50
3
1 2
25.00
Time
1
37.50
2
50.00
FIGURE 10.4 Simulations of glucose, xylose, and xylitol concentrations (g/L) vs. time (h)
in batch fermentation of sugarcane bagasse hydrolysate. (Simulation conducted with
STELLA® 8 software.)
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Functional Food Ingredients and Nutraceuticals
1: rate of Glucose util
10
2: rate of Xylose util
3: rate Xylitol form
2
5
1
3
0 1
0.00
1
2
3
1
25.00
Time
12.50
2
3
1
37.50
2
3
50.00
FIGURE 10.5 Simulation of rates of glucose utilization, xylose utilization, and xylitol formation (g/L-h) vs. time (h) in batch reactor using sugarcane bagasse hydrolysate. (Simulation
conducted with STELLA® 8 software.)
1: Glucose
62
2: Xylose
3: Xylitol
2
31
2
1
1
0
0.00
3
1
12.50
3
3
3
1 2
25.00
Time
1 2
37.50
50.00
FIGURE 10.6 Simulation of glucose, xylose, and xylitol concentrations (g/L) vs. time (h)
in CSTR at hydraulic retention time of 20 h using sugarcane bagasse hydrolysate. (Simulation
conducted with STELLA® 8 software.)
Simulation of substrate and product concentrations (Figure 10.6) for a single
microaerobic CSTR with hydraulic retention time of 20 h also represents the sequential use of glucose followed by xylose. Simulated steady-state conditions are
achieved by approximately 50 h. Simulation of substrate and product concentrations
in a two-stage, aerobic/microaerobic CSTR system with a total system hydraulic
retention time of 20 h is shown in Figure 10.7. Under aerobic conditions in the first
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Microbial Modeling as Basis for Bioreactor Design
1: Glucose1
62
2: Xylose1
2
4
3: Glucose2
261
4: Xylose2
5: Xylitol2
2
31
5
5
5
1
1
0
0.00
3
5
1
12.50
2
3 4
1
25.00
Time
3 4
1 2
37.50
3 4
50.00
FIGURE 10.7 Simulations of glucose, xylose, and xylitol concentrations (g/L) vs. time (h)
in reactors 1 and 2 of a two-stage CSTR system with hydraulic retention time of 10 h per
reactor (20 h system retention time). (Simulation conducted with STELLA® 8 software.)
reactor, no xylitol would be formed but high yields of yeast biomass would be
achieved. The simulation suggests that the two-stage CSTR system would achieve
greater xylitol concentration in the final effluent than the single-stage CSTR, through
the use of alternating environments to achieve greater product formation. Optimization of this system would be accomplished by varying hydraulic retention times
to achieve maximum concentration of xylitol.
10.17 SUMMARY
The kinetics of microbial growth, substrate utilization, and product formation can
greatly influence the effectiveness of a bioconversion process. Modeling involves
characterization of growth and product formation kinetics and development of mass
balance models of system. Dynamic modeling and simulation of the bioprocess can
be used to select, design, and optimize a bioreactor for production of nutraceutical
products.
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11
Food Pasteurization and
Sterilization with High
Pressure
Alberto Bertucco
University of Padova, Italy
Sara Spilimbergo
University of Trento, Italy
CONTENTS
11.1 Introduction ..................................................................................................270
11.2 Ultrahigh Hydrostatic Pressure Treatment (UHHP)....................................271
11.2.1 Process and Equipment Fundamentals ............................................271
11.2.2 State of the Art of UHHP ................................................................272
11.2.2.1 Microorganisms ................................................................272
11.2.2.2 Enzymes............................................................................274
11.2.2.3 Microbial Inactivation Mechanism...................................274
11.2.3 Commercial Application of UHHP .................................................278
11.3 Dense CO2 Treatment (DCO2).....................................................................280
11.3.1 Process and Equipment Fundamentals ............................................281
11.3.2 State of the Art of DCO2 .................................................................281
11.3.2.1 Microorganisms ................................................................281
11.3.2.2 Enzymes............................................................................284
11.3.2.3 Microbial Inactivation Mechanisms .................................285
11.3.3 Commercial Application of DCO2 ...................................................287
11.4 Potentials of High-Pressure Technologies and Conclusions .......................288
References..............................................................................................................289
269
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11.1 INTRODUCTION
Manufacturers of food products are currently under increasing and stringent
demands to control their production process. The entire course of manufacturing
starting from raw materials to the final packing is closely monitored and wherever
possible inspected as well. This is happening essentially because of the growing
demand for high-quality products and for energy-saving and safer production
processes. Consumers and the business class today prefer fresh or mildly processed
and user-friendly food products. Typically, such products are low in sugar, salt,
and fat, and contain as few preservatives as possible. However, these factors offer
an excellent breeding ground for microorganisms. Thus, finding the right method
for extending the shelf-life for these highly perishable products without affecting
their quality is indeed an open challenge. Microbiological stabilization of a product
is not concerned with the removal of all existing microorganisms but to reduce
the number of undesirable ones below a specific critical value during the shelflife of the product.
On the other hand, the use of food preservatives and additives is viewed with
more and more concern by consumers, as shown by the increase in restrictive
measures appearing in the food regulations of industrialized countries. The apprehension about these substances is associated with the concerns for heat treatments
that are still the most widely used procedure in food industry to microbiologically
stabilize foodstuff. Thermal pasteurization (up to 80°C) and sterilization (up to
120°C) are successful treatments in eliminating the degradation effects of enzymes
and microorganisms, but they may also decrease food quality by causing alterations
in the taste and sensory attributes of food products. As a consequence, in the future,
the food industry is expected to turn toward new and alternative technologies for
extending shelf life while retaining all original nutritional properties in the end
products. In this regard, systems based on different bactericidal agents, such as
microwave, electrical conductivity, pressure gradient, and so forth, seem to offer
new possibilities. They include high pressure (both hydrostatic and with CO2), pulsed
electric field,1 ohmic heating,2,3 pulsed x-ray,4 ultraviolet light,5 ultrasound,6 filtration,7 microwave and radio frequency processing,8 and oscillating magnetic fields.9
Although these technologies have been studied for more than one century, at the
present time none of them is ready to market.
In particular, as far as high-pressure treatments are concerned, for many years
researchers have investigated the effect of high hydrostatic pressure both on microorganism viability and on foodstuff properties.10,11 The main result has been that
such a technique can be successfully applied at room temperature, however, the high
plant costs will restrict its development for large-scale production. On the other
hand, the use of supercritical CO2 seems to be a more feasible approach, since it
allows operations at milder temperature (close to ambient) and pressure (70–300
bars).12 However, this new technique has been proposed only recently, thus no
industrial application is ready to enter the large-scale market yet.
This chapter will describe and discuss the current knowledge in the pasteurization and sterilization of foodstuff by means of UltraHigh Hydrostatic Pressure
(UHHP) method and Dense CO2 (DCO2) method. The most significant achievements
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will be summarized, including fundamental concepts, main applications in both
simple and complex solutions, and results on inactivation of different microorganisms and enzymes. At the end of the chapter the current knowledge on the different
hypotheses of bacterial inactivation mechanisms involved will be discussed and
evaluated and some conclusions about the two high-pressure techniques will be
drawn.
11.2 ULTRAHIGH HYDROSTATIC PRESSURE
TREATMENT (UHHP)
Pressure is one of nature’s fundamental forces that is known to have unique effects
on the thermodynamics of substances. Likewise, the preservation action of high
pressure on food substances has been acknowledged in the food science community
for over 100 years.13 The isostatic nature of pressure results in no shear stress within
the food and thus the food’s shape is not destroyed. However, the thermodynamic
conditions achieved under UHHP treatment are sufficiently hostile to living bacteria
that they are inactivated or irreparably damaged. In this way potential foodborne
pathogens can be destroyed and food spoilage organisms can also be greatly reduced.
Since this process is typically carried out at temperatures not far from ambient, the
food quality can be retained.
11.2.1 PROCESS
AND
EQUIPMENT FUNDAMENTALS
The use of UHHP treatment of food is carried out in batch mode. A process schematic
is reported in Figure 11.1. The UHHP equipment essentially consists of a relatively
large pressure-resistant vessel and a high-pressure generating system. A booster
pump and an intensifier pump are used to reach the required pressure. Food packaged
in waterproof sealed elastic containers ranging from bottles to bags are placed into
the vessel, which is then filled with water (or water-glycol solution) and pressurized
to typically 500–600 MPa for a time period usually between 2 and 3 minutes.
The pressure is applied by a direct or indirect compression technique but the
more widespread method is the indirect one, with a pump compressing the liquid
from the medium-pressure tank to the cell (see Figure 11.1), until the desired pressure
value is reached. The temperature control is ensured by a simple electrical resistance
if only heating is required. Otherwise a heating/cooling jacket, or a heat exchanger
inside the cell, can be applied. The form of the container is designed to minimize
the dead space inside the autoclave. It is important for reaching a high volumetric
efficiency in order to reduce the cost per unit. Under routine operation, foods are
now processed at pressures up to 600 MPa. Single plant production rates of 40
million lb/yr are already in operation. Production costs typically range from over 6
to about 4 cents/lb.14
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Functional Food Ingredients and Nutraceuticals
High-pressure cell
Pressure medium tank
Pressure booster
Pump
P
FIGURE 11.1 Schematic of typical UHHP apparatus.
11.2.2 STATE
OF THE
ART
OF
UHHP
11.2.2.1 Microorganisms
Relevant studies about the effect of high pressure on different kind of microbes and
food substrates will be briefly discussed in this section. Early experiments, dated in
the late 19th and early 20th centuries, showed that short treatments with an operating
pressure of a few thousand bars were able to reduce the microbial activity by many
orders of magnitude.13 In 1899 it was observed that pressurized milk would remain
fresh and unspoiled for a longer time than untreated milk; also microbes contained
in vegetables and fruits could be inactivated if they underwent a high-pressure
treatment for a few minutes.15,16
Many later studies also showed that short treatments with an operating pressure
of a few thousand bars were able to reduce the vegetative forms of microbial activity
by various orders of magnitude.17 For instance, Timson and Short in 1965 started a
systematic investigation to test the resistance of bacterial spores, and to try to
inactivate them completely, that is, to get sterilization. These authors studied the
behavior of spores under UHHP treatment with longer processing times at constant
pressure in a range of temperatures between –25°C and 95°C.18 With the works of
Gould and coworkers,19,20 the existence of optimal hydrostatic pressure to inactivate
spores was demonstrated. After much research on various bacterial forms, these
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authors confirmed their hypothesis about the induction of spore germination by the
action of pressure. More recently, Ludwig’s group investigated the behavior of spores
under different conditions of UHHP21 and introduced the cycle-type treatment that
proved to be more efficient than the double-level treatment.22,23
In recent years the effect of high hydrostatic pressure on the survival of different
kinds of microorganism in various food substrates under different conditions was
addressed24–33 and reviewed.26,27 Generally, the most recent results confirm the findings of early studies that food can be pasteurized under high pressure (400–600
MPa) and low to moderate temperatures (up to 60°C) but these products require
refrigeration during storage and distribution to ensure microbiological stability. The
specific operating conditions depend on the history of the bacterial cultures, that is,
the type and growth phase of the test microorganisms considered.25 Bacterial spores
were demonstrated to require more severe pressure treatments, in combination with
other preservation techniques, principally heat treatment, to achieve inactivation.31,34
Recently, high hydrostatic pressure inactivation of vegetative microorganism,
aerobic, anaerobic spores in pork Marengo (a low acidic particulate food product)
was studied by Moerman,35 who showed that Saccharomyces cerevisiae and the
Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli are more
pressure sensitive than cocci Enterococcus faecalis and Staphylococcus aureus
(Gram-positive): their inactivation at room temperature was successful only with
pressures as high as 600 MPa, whereas Gram-negative bacteria were more easily
killed at pressure of about 400 MPa. The UHHP method was also applied to solid
substrates, in particular air-dried alfalfa seeds inoculated with E. coli and L. monocytogenes. These samples were subjected to different pressure conditions (from
275–575 MPa for 2 min and 475 MPa for 2–8 min, at 40°C).36 It was shown that a
maximum reduction of 2 log can be obtained, and that treated seeds took a longer
time to germinate compared to the untreated seed. The effect of high-pressure
processing on the safety, quality, and shelf life of ready-to-eat meats (low-fat pastrami, Strasburg beef, export sausage, and Cajun beef) was investigated at 600 MPa
and 20°C for 3 min.10 After processing, samples were stored at 4°C for 98 days.
After storage their counts of aerobic and anaerobic mesophilies, lactic acid bacteria,
Listeria spp., staphylococci, Brochothrix thermosphacta, coliforms, yeasts, and
molds were undetectable or at low levels. Furthermore, sensory analyses revealed
no difference in consumer acceptability and no sensory quality degradation. An
interesting paper concerning the effect of high-pressure-induced inactivation of
Listeria innocua in buffer frozen suspension was published by Luscher et al.;37 a
cycle pressure treatment above 200 MPa resulted in inactivation of about 3 log,
probably due to the mechanical stress associated with phase transition of ice into
its different polymorphs. The effect of various pressure levels (50–600 MPa) and
holding times on color and microbiological quality of bovine muscle was also
investigated.38 The reported experiments, carried out at 10°C and pressure higher
than 300 MPa, induced modifications of meat color parameters. Pandey et al.39
studied the effect of high pressure treatment (250–400 MPa) for various holding
times (0–80 min) at 3°C and 2°C on raw milk with high count of indigenous
microflora. It was found that higher pressures, longer holding times, and lower
temperatures resulted in larger destruction of microorganisms.
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Finally, we quote an interesting paper by Linton et al.40 who found that UHHP
treatment (at 20°C and 300–600 MPa) readily inactivated psychotropic bacteria,
coliforms, and pseudomonas in different types of shellfish such as mussels, prawns,
scallops, and oyster.
Table 11.1 is a compilation of interesting UHHP applications to food that can
be found in the literature from the year 2000 on, with indication of the substrate,
the type of microorganism, the pressure conditions, and the maximum inactivation
ratio achieved.
11.2.2.2 Enzymes
Food quality deterioration is caused by a wide range of phenomena, including
physical conditions and both chemical and biochemical reactions. As far as enzymatic reactions are concerned, the effect of pressure on protein structure and functionality can vary dramatically depending on the magnitude of the pressure, the
reaction mechanism, and the overall balance of forces responsible for maintaining
the protein structure.41
To date, it is difficult to establish precisely the general effect of hydrostatic
pressure on different enzymes in various food substrates and environment. Pressure
has always been recognized as a potential denaturant of proteins, but examples of
pressure-induced stabilization have also been reported. For instance, both polyphenol
oxidases (PPO) and peroxidases (POD) which play an important role in food quality,
as they influence the visual appearance, flavor and health-promoting properties, are
known to be pressure-stable enzymes.42 This stability depends on plant source and
type of product (e.g., whole fruit, rather than puree or juice).
The global effect of pressure on enzymes is quite complex and can be explained
in terms of individual molecular interactions within proteins, including hydrophobic, electrostatic, and van der Waals interactions.41 The information available on
this topic is scarce and often contradictory; thus it is still impossible to discuss
exhaustively the effect of UHHP on most enzymes catalyzing chemical reactions
which influence quality and degree of deterioration of foodstuff. Table 11.2 provide
a summary of recent studies on the effect of pressure on enzymatic reactions
related to food, with indication of enzyme type, treatment conditions, and substrate
utilized.
11.2.2.3 Microbial Inactivation Mechanism
Microorganisms are inactivated when they are exposed to factors that substantially
alter their cellular structure or physiological functions. Structural damage includes
DNA strand breakage, cell membrane rupture, and mechanical damage to cell envelope. The reason for the effect of high pressure on microorganisms is not completely
clear. It is well known that pressures in the range of 20–180 MPa delay microbial
growth and tend to inhibit protein synthesis, while at pressures higher than 180 MPa
inactivation causes the loss of viability. Lethal UHHP treatment disrupts membrane
integrity and denatures many proteins. Another fundamental requirement for the
survival and viability of microorganisms is the regulation of the cytoplasmatic pH
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TABLE 11.1
Applications of UHHP on Microbial Cells in Simple and Complex Solutions
Reference
35
Treatment
Regime
400 MPa/30
min
Temperature Maximum
(°C)
Reduction
20–50
Bacillus subtilis
0.14 log
Bacillus
stearothermophilus
Clostridium sporogenes
Clostridium
tyrobutyricum
Clostridium
saccharolyticum
Enterococcus faecalis
Staphylococcus aureus
Escherichia coli
Pseudomonas fluorescens
Saccharomyces
cerevisiae
Alfalfa seeds
Escherichia coli
Listeria monocytogenes
0.69 log
1.63 log
1.79 log
3.35 log
6.49* log
3.51* log
575 MPa/2 min 40
475 MPa/2–8
min
300 MPa/5 min 20
400 MPa/1 min
700–800
MPa/5 min
900 MPa/1 min
1.4 log
2.0 log
10
600 MPa/180 s 20
4* log
37
200 MPa
83
>75 MPa/30
min
200 MPa/8 min
150–250 MPa
82
84
85
4* log
Subzero
3 log
temperature
37
* means total inactivation
© 2007 by Taylor & Francis Group, LLC
Solution/
Substrate
1.31 log
0.74 log
0.21 log
36
Inoculated
Microorganism
Pork marengo
Salmonella typhimurium
Escherichia col
Yersinia enterocolitica
Vibrio parahaemolyticus
Bacillus cereus
Staphylococcus aureus
Listeria monocytogenes
Anaerobic mesophiles
Lactic acid bacteria
Listeria spp.
Staphylococci
Brochothrix
thermosphacta
Coliforms yeast and
molds
Listeria innocua
0.1% Buered
peptone water
(pH 7.4)
Escherichia coli
Desoxycholate
Agar (DESO)
Escherichia coli
Not
Listeria monocytogenes
inactivated
at 250
MPa
Low-fat
pastrami
Strasburg beef
Export
sausage Cajun
beef
Buffer solution
Chilled coldsmoked
salmon
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TABLE 11.1 (CONTINUED)
Applications of UHHP on Microbial Cells in Simple and Complex Solutions
Reference
Treatment
Regime
Temperature Maximum
(°C)
Reduction
86
350–550 MPa
30–45
7* log
87
350 MPa/20
min
50
5 log
38
50–600
MPa/20–300 s
250–400
MPa/0–80 min
300–600
MPa/2 min
10
2.5
20
81
50–400
MPa/15 min
47
39
40
88
89
90
91
92
Inoculated
Microorganism
Listeria monocytogenes
Staphylococcus aureus;
Gram-negative:
Escherichia coli
Salmonella enteritidis
Alicyclobacillus
acidoterrestris
Solution/
Substrate
UHT 1% low fat
milk
Total flora
Model system
(BAM broth)
and orange,
apple, and
tomato juices
Bovine muscle
Escherichia coli
Raw milk
96%
Psychrotrophic bacteria
Coliforms
Pseudomonas
25
4 log
Total aerobic mesophilic
and psychrotrophic
bacteria
400
MPa/10 min
450
MPa/15 min
400
MPa/20 min
20
9* log
Listeria monocytogenes
40
99.97%
Room
2 log
temperature
Listeria monocytogenes
500
MPa/60 s
300–600 MPa
Room
4–5 log
temperature
Natural flora
Shellfish,
mussels,
prawns,
scallops,
oysters
Tomato puree +
natural
additives
(citric acid and
sodium
chloride)
pH 5,6 citrate
buffer
Porcine blood
plasma
ACES buffer
(N-(2-acetamido)-2aminoethanesulfonic acid)
Green beans
400–500
MPa/5–30 min
10–40
2–31
* means total inactivation
© 2007 by Taylor & Francis Group, LLC
Milk and diary
products
Red blood cells
fraction from
porcine blood
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TABLE 11.1 (CONTINUED)
Applications of UHHP on Microbial Cells in Simple and Complex Solutions
Reference
Treatment
Regime
Temperature Maximum
(°C)
Reduction
75
300, 800 MPa
93
300
MPa/15 min
94
200–600 MPa
95
50–400
MPa/1–60 min
20–80
6* log
3.67 log
2.27 log
96
1000
MPa/15 min
20
34
400
MPa/25 min
8–30
97
98
99
400
MPa/10 min
600
MPa/10 min
200 MPa/12h
545 MPa
Not
Fruit and
inactivated
vegetable
at 1000
products
MPa
0.4 log
Listeria monocytogenes
Ultrahigh
Bacillus cereus
temperature
Pseudomonas fluorescens milk
Listeria monocytogenes
Citrate buffer
and phosphate
buffer
100
600 MPa/5min
101
400 MPa/three
5-min cycle
102
400 MPa/5 min 20
103
100–500
MPa/15 min
Room
temperature
5–50
25
7
4-25-50
* means total inactivation
© 2007 by Taylor & Francis Group, LLC
4,7 log
2 log
7 log
Inoculated
Microorganism
L. plantarum
Escherichia coli
Lactic acid bacteria
Baird Parker flora
Pseudomonas sp.
Enterobacteria
Lactobacillii harmful to
beer
Bacillus subtilis
Bacillus
stearothermophilus
Streptococcus faecalis
B. cereus
Psychrophilic bacteria
Escherichia coli
Listeria monocytogenes
enteroxigenic
Staphylococcus aures
Yeast and lactic acid
bacteria
Microbial load
Solution/
Substrate
Sliced cooked
ham and
ground pork
patties
Beer
Meat batters
Tilapia fillets
Tomato-based
salsa
Beer
Chilled hake
(Merluccius
capensis)
Escherichia coli
Milk
cheese
Escherichia coli
Ringer solution
Pseudomonas fluorescens and ovine
Listeria innocua
milk
Staphylococcus aureus
Lactobacillus helveticus
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TABLE 11.1 (CONTINUED)
Applications of UHHP on Microbial Cells in Simple and Complex Solutions
Reference
104
Treatment
Regime
Temperature Maximum
(°C)
Reduction
400 MPa/10
7
min
continuous
pressure and
pulsed
pressure in two
5-min steps
5 log
Inoculated
Microorganism
H2S-producing
microorganisms
Lactic acid bacteria
Brochothrix
thermosphacta
Coliforms
Solution/
Substrate
Oysters
which plays an important role in secondary transport of several compounds; an
irreversible internal pH decrease during UHHP treatment (200–300 MPa) probably
promotes denaturation of proteins required for pH homeostasis.43 Another effect to
be taken into account is a phase transition of the cytoplasmatic membrane from the
physiological liquid-crystalline to the gel phase, which can induce leakage of sodium
and calcium ions and increase membrane permeability.44 The main structural and
functional changes in microorganisms at different pressures are summarized in
Figure 11.2.
It is worth noting that these microbial inactivation hypotheses were established
recently, thanks to the possibility of exploiting new analytical methods which can
determine structural and physiological cell modifications and loss of cytoplasmatic
content by quantitatively and qualitatively estimating viable cells. This field of
research was started because the traditional methods of analysis were not able to
assess the physiological state of damaged cells after UHHP treatment. Furthermore,
classic culture techniques were widely recognized to underestimate the number of
truly viable bacteria, especially when cells had been damaged by physical treatment.
Fluorescent staining, detection by microscopy, flow cytometry, and differential scanning calorimetry are presently the most widely used techniques.37,45–47
11.2.3 COMMERCIAL APPLICATION
OF
UHHP
In the last 15 years the food industry has been successful in marketing a number of
UHHP treated products, which meet evolving regulatory concerns for greater food
safety and a growing consumer demand for higher-quality and convenient (i.e.,
ready-to-eat) foods. Commercialization is being achieved thanks to collaboration
between food scientists, microbiologists, and high-pressure equipment providers.
Products and processes using UHHP are now available. In 1990 the Meidi−ya
Food Company of Japan, introduced the first UHHP-pasteurized products, namely
strawberry, kiwi, and apple jams. From 1993 the range of this kind of products has
become larger and larger, and a variety of jams, juices, sauces, milk-desserts, fruit
jellies, raw beef, and fish have been commercially produced. These products are
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TABLE 11.2
Applications of UHHP on Enzymes in Food Substrates
Reference
Treatment
Regime
Temperature
(°C)
105
>300 MPa/ 20 min
9
106
400, 600, 800
MPa/5, 10, 15 min
18÷22
107
108
110
111
112
74
113
114
115
116
117
Proteolytic enzymes:
Cathepsin B-like
Cathepsin B+L-like
Calpains
Beta-glucosidase
Peroxidase
Polyphenoloxidase
Pectin
methylesterase
PME
Oxidase enzymes
109
300 450 MPa/
15 min
400 MPa/10 min
300 MPa
100–400 MPa
0–450
MPa/15–30 min
207–310
MPa/0–2 min
395–445
MPa/8–11 min
0–800 MPa
118
119
50 MPa/72 h
120
Up to 700 MPa
121
600–900 MPa
122
300–400 MPa,
30–120 min
Proteolytic enzymes
Beta-lactoglobulin
7
7-40-75-100
Red raspberry and
strawberry
Fruits and vegetables
Carrots
Muscadine grape
juice
Octopus arm muscle
Oysters
Proteins
Alphalactalbumin
Beta-lactoglobulin
Microbial load and
autolytic activity
Lipase
Milk
Octopus muscles
Pacific oysters
Carrot juice
20÷65
Tomato pectin
methylesterase
polygalacturonase
Protein
Rhizomucor miehei
lipase
Lactoperoxidase
4.4 log
3.09 log
Nonflavonoid
phenolics
Pectin methyl
esterase
© 2007 by Taylor & Francis Group, LLC
Cold-smoked salmon
Polyphenoloxidase
70
÷ in the range
– separates single set points
Solution/
Substrate
Enzymes
Tomato
Cheese
Bovine milk and acid
whey
Grape juice
Orange juice
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Pressure (MPa)
300
Irreversible protein denaturation
leakage of cell content
Intracellular pH decrease
200
Membrane damage
signs of cell content leakage
Threshold of lethality
Reversible protein denaturation
compression of gas vacuoles
100
50
Atmospheric pressure
Inhibition of protein synthesis
reduction in the number of ribosomes
0.1
FIGURE 11.2 Main structural and functional changes in microorganisms as a function of
pressure (adapted from Ref. 29).
packed in plastic bags and can be stored at 4°C for two months if sealed, or for one
week after opening. At the present time, fruit, vegetables, shellfish, meat, and other
products are in commercial UHHP production.48 In Europe, at least two wellestablished UHHP pasteurized products are worth mentioning, fruit juice in France
and ham in Spain, while in Mexico companies are treating avocado puree for the
U.S. market at a processing pressure of 700 MPa.17 For the sake of completeness,
it must be mentioned that UHHP can be applied at the industrial scale to reduce the
microbial activity, as well as to modify the consistency of foodstuffs; active research
has been developed to obtain UHHP precooked foods such as meat and rice-based
products.49
11.3 DENSE CO2 TREATMENT (DCO2)
The antimicrobial effect of CO2 under pressure (or dense CO2) was discovered in
the second half of last century, but specific research began only about 20 years ago.
For instance, in the book on dense gas extraction by Quirin et al.,50 part of a chapter
is dedicated to “sterilization” by dense gases, showing considerable reduction (i.e.,
5 to 8 log) of microbial counts after appropriate CO2 pressure application at room
temperature. Soon afterward, a systematic investigation was started to exploit this
effect in order to develop a new nonthermal pasteurization technology, suitable for
applications where processing temperatures close to ambient have to be used.
As detailed in the section “state of the art” below, DCO2 was found to be lethal
to basically all forms of microorganisms to which it had been applied. In addition,
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it has been shown51 that the antimicrobial action can be exerted at pressures as low
as 7 MPa, that is, much lower than the ones required by UHHP treatment. Thus,
DCO2 qualifies as the best candidate to replace traditional thermal treatment, especially when heat-labile components need to be preserved, as often happens with
products related to food, pharmaceuticals, and cosmetics. A major limitation of the
DCO2 technology is that direct contact between CO2 and the microorganism to be
killed must be ensured. Therefore, at present, pasteurization can be achieved successfully only when liquid substrates or slurries are processed; treating solid materials by this technique remains problematic.
11.3.1 PROCESS
AND
EQUIPMENT FUNDAMENTALS
Treatment of liquid solutions and suspensions (i.e., pumpable substrates) by DCO2
is quite a simple task, which can be accomplished in many ways. The most efficient
type of equipment is a continuous contactor, where the feed to be pasteurized and
the CO2 stream can flow either cocurrently or countercurrently. A flow sheet of a
pilot unit is sketched in Figure 11.3. The plant basically consists of a CO2 surge
tank, two high-pressure pumps (one for CO2, the other for the substrate to be
pasteurized), a mixer where the two streams are suitably contacted, a thermostatically
controlled holding tube to ensure the proper retention time at the desired temperature,
a suitable depressurization system, and eventually a degasser section.
Energy requirements of this process are generally low, as liquids are easier to
pump than gases. The consumption of CO2 is quite low as well: in the case of
aqueous-based substrates the ratio between the CO2 and feed flow rates is never
more than 1:20 on a weight basis, because the solubility of CO2 in these liquids is
usually less than 0.05 (weight fraction). If desired, after depressurization, CO2 may
be recompressed and recycled back to the contactor, but this option increases substantially the energy requirement. From our experience, the retention (holding) time
to get a 6-log count reduction depends on the type of microorganism, but is usually
in the range of 5 to 30 min, and can be further reduced by a suitable design of the
holding tube.52 So far, we have processed a number of fresh fruit juices (such as
orange, apple, grape, and pear) in a unit like the one depicted in Figure 11.3, always
obtaining good results in terms of inactivation, provided that a temperature not less
than 30°C–32°C was used.
11.3.2 STATE
OF THE
ART
OF
DCO2
11.3.2.1 Microorganisms
The possibility of using CO2 under pressure to inactivate microbes was initially
addressed by Fraser in the 1950s,53 who reported the disruption of bacteria cells by
rapid release of CO2 gas from about 35 bars to ambient pressure. The first patent in
the field was obtained by Swift & Co. in 1969, who claimed that food products
could be sterilized by CO2 without degradation of their flavor at “superatmospheric”
conditions and by exposing them to relatively low radiation dosages.54
Since 1985 many papers have reported on the bacteriostatic action of CO2 and
on the growth and metabolism of different microorganisms, but it is from the paper
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TI1
VP1
A1
A2
TI2
VP 2
TI3
V-S
V1
V2
VP3
VP4
TI4
VP5
TI5
VP6
TI6
V4
TI8
P1
TI7
VP
6
PI3
VNR
TCI
P3
BT 1
PC
VL
V-3
BT2
D
PI2
PI1
PI4
V5
V3
B1
CO2
B2
V6
P2
F
C
PV
R
FIGURE 11.3 Schematic of DCO2 continuous equipment (adapted from Ref. 52); P1, P2,
P3: pumps; PV: vacuum pump; A1, A2, B1, B2, D: tanks; F: filter; V1, V2, V3, V4, V5, V6:
on-off valves; VNR: no-return valve; V-S: safety valve; VL: control valve; VP: sampling
valves; TI: temperature indicators; PI: pressure indicators; BT1, BT2: thermostatic baths; C:
chiller; R: flow meter.
by Kamihira et al. in 198755 that the inhibitory effect of CO2 under pressure toward
microbes started to be addressed systematically and quantitatively. The number of
publications on this topic has increased over the years since 1990. Many authors
have reported experimental evidence on the effect of dense CO2 on different substrates and different kinds of microbes commonly present in foodstuff, both in their
vegetative and latent forms. A review on inactivation of bacteria by the DCO2 method
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has been published recently,12 and it discusses research published in 83 articles and
patents, half of which were published from 1999 to 2003.
In the last two years several new articles have been published. They include an
article by Liu et al.56 which describes a study on the influence of compression and
decompression rate, and the concerted effects of temperature, pressure, exposure
time, water content, and initial pH on the physiology of Absidia coerula and Saccaromices cerevisiae. The paper by Erkmen57 deals with the mathematical analysis
of high-pressure CO2 regarding the inactivating effect on S. cerevisiae at different
temperatures and pressures. His study allowed the prediction of yeast inactivation
when exposed to different CO2 operating conditions. Watanabe et al.58 compared
DCO2 treatment with other methods, in particular UHHP and thermal processing,
for the inactivation of spores at different temperatures and exposure times. In the
work by Furukawa et al.,59 the effect of CO2 on the germination of spores was studied
at 65 bars, 35°C, and 120 min.
In the past two years, several patents have also been issued, including the one
claimed by Balaban,60 which deals with a continuous method to reduce microorganism and enzyme activities in liquid beer and wine products, and the one claimed by
Praxair Inc. (Burr Ridge, IL, USA), which validates a continuous DCO2 process as
a nonthermal pasteurization technology of fruit juice from lab-scale to commercialization for a feed flow rate up to 120 L/min (Better than Fresh®). In this last patent
the presence of a CIP (Cleaning In Place) equipment is evaluated as a key design
feature, to assure frequent cleaning and sanitization of the apparatus.61
Very recently, more studies have been discussed at international symposiums on
high pressure and supercritical CO2, which demonstrate the increasing interest in
this new mild pasteurization technology. Daiminger et al.62 presented a study on the
efficiency of a continuous apparatus able to ensure 8-log count reduction of different
kinds of microbes, both inoculated in orange juice and naturally present in an active
sludge. The effect of the main operating parameters (flow rate and pressure) was
discussed and an inactivation mechanism was proposed. Inactivation of Staphylococcus in liquid whole egg products by means of DCO2 in a batch stirred pilot device
was presented by Van Ginneken et al.63 and patent application on this promising
process has been filed.64 Zhang and coworkers65 investigated the synergistic effect
of DCO2 in conjunction with low levels of H2O2 to deactivate spores and found that
at least 4-log reduction can be reached at 40°C. On the inactivation mechanisms,
disruption of the exospores, morphological changes, and release of dipicolinic acid
(DPA) have been observed by means of TEM, SEM, and DPA fluorescence assay.
From these reports, it is apparent that DCO2 has been shown to be effective as
a bactericidal agent on vegetative forms of microorganisms at a near-ambient temperature and a relatively low pressure. The experiments, performed using both simple
suspensions and complex substrates, resulted in inactivation levels sufficient to assure
the pasteurization of foodstuffs. However, it should be noted that the texture of some
solid products was disrupted and the color was changed after the DCO2 treatment,66
thus further work is needed in this respect. Mainly batch devices have been used as
experimental apparatus at the laboratory scale and different operating conditions of
pressure (55–300 bars) and temperature (10°C–50°C) have been tested. The presence
of a mixing system has been shown to be beneficial in terms of inactivation efficiency.
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As far as latent forms are concerned, DCO2 alone is not suitable for practical
application in the food industry, unless higher temperatures (at least more than 60°C)
are applied.67 In this case, some hurdle approach can be beneficial.25,68
11.3.2.2 Enzymes
As mentioned above, an irreversible pressure-induced denaturation of proteins generally requires values of pressure greater than 300 MPa, much larger than the ones
used in DCO2 treatment. A different hypothesis must therefore be proposed to justify
the inactivation of enzymes caused by DCO2. Apart from pressure, a number of
factors can be taken into account when dealing with the influence of high-pressure
CO2 on enzymes: pH of the medium, temperature, processing time, surface tension
at the gas-water interface, type of microbes, and nature of the substrate.
Current research results show that DCO2 treatment can either activate or inactivate enzymes.52 The interaction between dense CO2 and enzyme molecules is
expected to cause conformational changes that will result in either loss or increase
of activity. Recent reports dealing with the influence of high-pressure CO2 on
enzymes are listed in Table 11.3 with indication of enzyme type, treatment conditions, and substrate utilized. It can be noticed that, despite the number of experimental data so far collected on enzyme activity changes after DCO2 treatment, no
correlation nor definite enzymatic inactivation hypotheses are available in the literature. For instance, the decrease of activity has been related to the loss of secondary
structures in enzyme molecules.69 Studies using gel electrophoresis showed that there
are differences in the isoelectric profiles and protein patterns between the untreated
and CO2-treated polyphenol oxidase (PPO), an enzyme causing undesirable browning on the surface of fruits and vegetables. Spectropolarimetric analysis revealed
that CO2 treatment also changes the secondary structure of this enzyme.70 An enzyme
H+-ATPase, located in the cell membrane, which is also important for the regulation
of bacterial internal pH, was shown to maintain its initial specific activity in L.
plantarum cell membrane even though viability of the cells was reduced by several
log counts after high-pressure treatment.71 In contrast, Wouters et al.72 observed
stimulation of ATPase activity in the membrane vesicles when L. plantarum was
exposed to a hydrostatic treatment at 250 MPa for 80 min. The activity assessment
of several enzymes by using the APIZYM system from B. subtilis cells before and
after DCO2 treatment was exploited to demonstrate selective enzymatic inactivation
as one of the probable cause of microbial inactivation.71 Recently, Habulin et al.73
observed that proteinase from Carica papaya latex showed improved stability at
300 bars of CO2 pressure, when compared to ambient pressure, and that addition of
water also increased the activity of this enzyme (optimum amount was between 0.5
and 0.7 g/L).
On the so-called hurdle approach, two papers have recently been published. Both
of them deal with the combined effect of UHHP and DCO2 treatments on the activity
of selective enzymes: Park and Lee74 observed that the residual activity of polyphenoloxidases, lipoxygenase, and pectinmethylesterase were less than 11.3%, 8.8%,
and 5.1%, respectively, after a combined treatment of DCO2 at 4.9 MPa and UHHP
at 600 MPa. Corwin et al. studied the UHHP processing of CO2-added solutions (at
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Food Pasteurization and Sterilization with High Pressure
TABLE 11.3
Applications of DCO
Solutions
Reference
123
69
124
2
Treatment
Regime
Treatment on Enzymes in Simple and Complex
Temperature
(°C)
31 MPa/10 min
25 MPa/30 min
Supercritical and
gaseous liquid
phase, pH 3 ÷ 6
30MPa/30 min
35, 2 MPa/15min;
62, 1 MPa/15 min
30 MPa
35–50
71
7 MPa/10 min
30
75
300–800 MPa
(0.2% CO2)
25–30
128
Micro-bubble
methods
Batch, 300 bar
125
126
127
73
285
30–60
35-45-55
25–50
35
40–55
50
Enzymes
Solution/Substrate
Pectinesterase
Pectinesterase
Acid protease, alk.
protease, papain, and
glucoamylase
Myoglobin
Lipoxygenase, peroxidase
Orange juice
Valencia orange juice
McIlvaine buffer
Aqueous solution
30% sucrose solution
Alpha-amilase
Buffer solution
acid protease
H+-ATPase, costitutive
Saline solution
enzyme in L. plantarum
Pectin methylesterase
(PME), polyphenol
oxidase (PPO)
Alpha-amilase
Buffer solution
acid protease
Proteinase from C. papaya
0.2% CO2 mole fraction). After treatment at 300÷800 MPa a synergistic effect on
enzyme inactivation of pectin methyl-esterase and polyphenol-oxidase in L. plantarum and E.coli was found.75
11.3.2.3 Microbial Inactivation Mechanisms
If inactivation of microorganisms in a pumpable substrate can be achieved in a
simple apparatus such as the one outlined in Figure 11.3, the reason for the ability
of DCO2 to inactivate microorganisms is not clear yet. Over the years, a number of
hypotheses have been proposed to explain the inactivation mechanisms, but to date
none appears completely satisfactory. For instance, it was demonstrated that, under
standard operating conditions, cell death is not caused by explosive decompression
of CO2 upon pressure release.76 Also, the inactivation of key enzymes as a consequence of direct interaction with CO2 has not been sufficiently proven, and remains
a possible hypothesis. On the other hand, extraction of cell wall lipids by DCO2
based on the extraction capacity of CO2 as a supercritical fluid cannot be invoked
at all as a reason for deactivation. Studies have shown that when the operating
pressure is as low as 7 MPa and the temperature is around 30°C, the solubility of
phospholipids in DCO2 is quite negligible. Thus, both intracellular acidification and
modification of the cell membrane properties remain the two main reasons for
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Functional Food Ingredients and Nutraceuticals
explaining cell deactivation. It is interesting that both reasons are associated with
the CO2 dissolution in the substrate.
To better explore this hypothesis, the data presented in Figure 11.4 may be
helpful. Let us consider the acidification aspect first. When a suitable CO2 pressure
is applied over a physiological aqueous solution where model microorganisms are
suspended, the pH of the solution is a strong function of pressure. It can be either
measured, for example, by online UV spectroscopy77 (results are shown in Figure
11.4a), or calculated according to a suitable equation of state (SAFT)78 (Figure 11.5),
so that curves such as those displayed can be obtained, depending on the system
temperature. Clearly, when a pressure between 60 and 80 bars is reached, the pH
value is lowered to about 3 by CO2 dissolution, but at higher CO2 pressures it cannot
be further reduced; this behavior is compatible and can be correlated with the trend
of CO2 solubility in water.52,78
On the other hand, a saline solution at neutral pH can be completely pasteurized
by the DCO2 treatment carried out at 80 bars of pressure, so that it might be
concluded that such a pH decrease (down to about 3) is responsible for the death
of microorganisms. However, when a different aqueous solution such as orange juice
was considered for the same experiment, no pH effect could be detected upon
application of CO2 pressure (as shown in Figure 11.4b), even though total deactivation was achieved. This is an indication that solution (i.e., external) acidification
itself is not sufficient to explain the inactivation of microorganisms. As far as
interaction with the cell membrane is concerned, it was suggested that CO2 can
dissolve into phospholipids, even at pressures of 60–80 bars.79 If the CO2 concentration in the phospholipids becomes high enough, the physical properties of the
cell membrane can be modified enough to allow the enzymes linked to the membrane
double layer to be released. On the other hand, the higher CO2 concentration in the
membrane helps penetration of CO2 into the cytoplasm, with subsequent decrease
of intracellular pH value. As a consequence of internal acidification, other phenomena such as enzyme deactivation and salt precipitation are likely to occur.
It is noteworthy that both external acidification and modification of cell membrane properties are driven by the pressure-increased chemical potential of CO2 in
the gas phase contacting the aqueous solution. If sufficient time is allowed for
equilibration after pressure is applied, thermodynamics dictates that the chemical
potential has to be the same in all the phases involved, that is the liquid bulk, the
cell membrane, and the cytoplasm, leading to the possible consequences discussed
above.
Experimental evidence of the proposed hypothesis is under current investigation.
With respect to intracellular acidification, we have recently shown that it can be
measured and correlated to the microbial inactivation level.79 As far as the property
changes of cell membrane are concerned, preliminary results suggest that when
centrifuging an E.coli suspension after DCO2 treatment, significant and unexpected
key enzyme activity can be measured in the liquid phase (supernatant), a clear
indication of the enzyme release from the cell membrane itself.52
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35°C
3.40
3.30
Water
pH
3.20
3.10
3.00
2.90
2.80
0
20
40
60
80 100 120
Pressure (bar)
140
160
180
(a)
35°C
3.60
3.50
3.40
pH
3.30
3.20
3.10
3.00
Juice
2.90
2.80
0
20
40
60
80 100 120
Pressure (bar)
140
160
180
(b)
FIGURE 11.4 Experimental extracellular pH profiles as a function of operating CO2 pressure
at 35°C in water, pH = 7 at atmospheric condition (4a) and in orange juice, pH = 3.47 at
atmospheric condition (4b).
11.3.3 COMMERCIAL APPLICATION
OF
DCO2
Despite the huge research and development efforts performed in the last 20 years,
at present no industrial applications of DCO2 to food pasteurization are known, even
though several companies are actively working in this field. It appears that commercialization could be a matter of time. In this respect it is interesting to quote the
opinion of Praxair, a company which is particularly active in developing a commercially sustainable DCO2 process,61 as reported in the proceedings of the recent Fifth
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Functional Food Ingredients and Nutraceuticals
3.35
3.30
313.15 K
pH
3.25
3.20
328.15 K
333.15 K
3.15
3.10
3.05
0
50
100
150
200
300
250
Pressure (bar)
350
400
450
500
FIGURE 11.5 Calculated pH profiles as a function of CO2 pressure at different temperatures
(taken from Ref. 78).
International Symposium on Supercritical Fluids (Orlando, FL, May 2005):80 “…
The validation of the dense phase carbon dioxide process is two-fold. First, it
demonstrated a greater than 5-log reduction of the pertinent pathogens on juice using
this process, to meet the juice HACCP guidelines set by FDA. As a result, this
technology is now considered by the FDA as an alternative to thermal pasteurization.
Second, product quality was validated by demonstrating the retention of physical,
nutritional quality and sensory profile. …” Under these circumstances, it could be
argued that the use of DCO2 as a nonthermal pasteurization treatment is going to
be reality within a short time.
11.4 POTENTIALS OF HIGH-PRESSURE
TECHNOLOGIES AND CONCLUSIONS
From the results and discussion presented in this chapter, it can be concluded that
high pressure provides efficient alternative processes for the pasteurization of foodstuffs when avoidance of thermal effects becomes an issue. It has been shown that
UHHP treatment is already a commercial technology, at least for niche products,
whereas the DCO2 process needs more scale-up development before achieving
industrialization. On the other hand, it is clear that neither UHHP nor DCO2 alone
can be exploited as sterilization techniques, and that high-pressure processing has
to be performed in combination with other physical or chemical treatments81 in order
to kill all microorganisms, including latent forms. Of course, coupling different
techniques could also be beneficial to achieve pasteurization, for instance by allowing
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the use of lower pressures and processing time, which would reduce the investments
in pressure equipment25,27 and improve the process economics. It is also interesting
that in recent works the coupled effect of DCO2 and UHHP has been given special
attention.74,75
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Food Sci., 65, 974, 2000.
101. Hurtado, J.L., Montero, P., and Borderias, A.J., Extension of shelf life of chilled hake
(Merluccius capensis) by high pressure, Food Sci. Technol. Int., 6, 243, 2000.
102. Trujillo, A.J. et al., Application of high pressure treatment for cheese production,
Food Res. Int., 33, 311, 2000.
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103. Gervilla, R., Ferragut, V., and Guamis, B., High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents, J. Dairy Sci., 83, 674, 2000.
104. Lopez-Caballero, M.E. et al., Oyster preservation by high-pressure treatment, J. Food
Protect., 63, 196, 2000.
105. Lakshmanan, R., Patterson, M.F., and Piggott, J.R., Effects of high-pressure processing on proteolytic enzymes and proteins in cold-smoked salmon during refrigerated
storage, Food Chem., 90, 541, 2005.
106. Garcia-Palazon, A. et al., The effects of high hydrostatic pressure on beta-glucosidase,
peroxidase and polyphenoloxidase in red raspberry (Rubus idaeus) and strawberry
(Fragaria x ananassa), Food Chem., 88, 7, 2004.
107. Ludikhuyze, L. et al., Effects of combined pressure and temperature on enzymes
related to quality of fruits and vegetables: from kinetic information to process engineering aspects, Crit. Rev. Food Sci. Nutr., 43, 527, 2003.
108. Ly-Nguyen, B. et al., Mild-heat and high-pressure inactivation of carrot pectin methylesterase: a kinetic study, J. Food Sci., 68, 1377, 2003.
109. Talcott, S.T. et al., Phytochemical stability and color retention of copigmented and
processed muscadine grape juice, J. Agric. Food Chem., 51, 957, 2003.
110. Hurtado, J.L. et al., Properties of proteolytic enzymes from muscle of octopus (Octopus vulgaris) and effects of high hydrostatic pressure, J. Food Sci., 67, 2555, 2002.
111. Knudsen, J.C. et al., Effect of high hydrostatic pressure on the conformation of betalactoglobulin A as assessed by proteolytic peptide profiling, Int. Diary J., 12, 791,
2002.
112. Perez-Mateos, M., Lopez-Caballero, M.E., and Montero, P., Effect of high pressure
and 4-hexylresorcinol on enzymatic activity and darkening in oysters, J. Food Sci.,
67, 2107, 2002.
113. Huppertz, T., Kelly, A.L., and Fox, P.F., Effects of high pressure on constituents and
properties of milk, Int. Diary J., 12, 561, 2002.
114. Hurtado, J.L., Montero, P., and Borderias, J., Behavior of octopus muscle (Octopus
vulgaris) under a process of pressure-time-temperature combinations, Food Sci. Technol. Int., 7, 259, 2001.
115. He, H. et al., Use of high-pressure processing for oyster shucking and shelf-life
extension, J. Food Sci., 67, 640, 2002.
116. Kim, Y.S. et al., Effects of combined treatment of high hydrostatic pressure and mild
heat on the quality of carrot juice, J. Food Sci., 66, 1355, 2001.
117. Crelier, S. et al., Tomato (Lycopersicon esculentum) pectin methylesterase and polygalacturonase behaviors regarding heat- and pressure-induced inactivation, J. Agric.
Food Chem., 49, 5566, 2001.
118. Saldo, J., Sendra, E., and Guamis, B., Hard cheese structure after a high hydrostatic
pressure treatment at 50 MPa for 72 h applied to cheese after brining, Lait, 81, 625,
2001.
119. Marie-Olive, M.N., Athes, V., and Combes, D., Combined effects of pressure and
temperature on enzyme stability, High Pressure Res., 19, 707, 2000.
120. Ludikhuyze, L.R., Claeys, W.L., and Hendrickx, M.E., Effect of temperature and/or
pressure on lactoperoxidase activity in bovine milk and acid whey, J. Dairy Res., 68,
625, 2001.
121. Castellari, M. et al., Effects of high hydrostatic pressure processing and of glucose
oxidase-catalase addition on the color stability and sensorial score of grape juice,
Food Sci. Technol. Int., 6, 17, 2000.
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122. Basak, S., Ramaswamy, H.S., and Simpson, B.K., High pressure inactivation of pectin
methyl esterase in orange juice using combination treatments, J. Food Biochem., 25,
509, 2001.
123. Balaban, M.O. et al., Inactivation of pectinesterase in orange juice by supercritical
carbon dioxide, J. Food Sci., 56, 743, 1991.
124. Ishikawa, H. et al., pH-dependent inactivation of enzymes by microbubbling of
supercritical carbon dioxide, Food Sci. Technol. Res., 6, 212, 2000.
125. Ishikawa, H. et al., Irreversible unfolding of myoglobin in an aqueous solution by
supercritical carbon dioxide, J. Agric. Food Chem., 48, 4535, 2000.
126. Tedjo, W., Eshtiaghi, M.N., and Knorr, D., Impact of supercritical carbon dioxide
and high pressure on lipoxygenase and peroxidase activity, J. Food Sci., 65, 1284,
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127. Yoshimura, T. et al., Inactivation kinetics of enzymes by using continuous treatment
with microbubbles of supercritical carbon dioxide, J. Food Sci., 66, 694, 2001.
128. Yoshimura, T. et al., Effect of CO2 flow rate on enzyme inactivation by continuous
method with microbubbles of supercritical carbon dioxide, J. Faculty Agric., Kyushu
University, 46, 345, 2002.
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12
Microencapsulation and
Delivery of Omega-3
Fatty Acids
Luz Sanguansri and Mary Ann Augustin
Food Science Australia
CONTENTS
12.1
12.2
12.3
12.4
Introduction ..................................................................................................298
Drivers for Delivery of Omega-3 Fatty Acids into Foods ..........................299
Incorporation of Omega-3 Oils into Foods .................................................300
Microencapsulation ......................................................................................301
12.4.1 Microencapsulation in the Food Industry........................................301
12.4.2 Microencapsulation of Omega-3 Oils..............................................301
12.5 Manufacture of Microencapsulated Omega-3 Ingredients ..........................302
12.5.1 Elements of Microcapsule Production.............................................302
12.5.1.1 Omega-3 Oil Core Selection ............................................302
12.5.1.2 Encapsulant Selection.......................................................304
12.5.1.3 Formulation Design ..........................................................304
12.5.1.4 Choosing the Process........................................................304
12.5.1.5 Format for Delivery ..........................................................305
12.5.2 Encapsulating Materials...................................................................305
12.5.2.1 Challenges for Selection of Materials for
Omega-3 Oil Encapsulation .............................................306
12.5.2.2 Classes of Encapsulant Materials Used for
Omega-3 Oil Encapsulation .............................................306
12.5.3 Mechanical Processes ......................................................................308
12.5.3.1 Emulsion Preparation and Stabilization ...........................309
12.5.3.2 Spray Drying.....................................................................310
12.5.3.3 Extrusion ...........................................................................311
12.5.3.4 Gelation and Particle Formation ......................................312
12.5.3.5 Secondary Coating Application........................................312
12.5.4 Chemical-Based Processes...............................................................313
12.5.4.1 Coacervation .....................................................................313
12.5.4.2 Inclusion Complexation....................................................314
12.5.4.3 Liposome Encapsulation...................................................314
297
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12.5.5 Emerging Technologies....................................................................315
12.5.5.1 Supercritical Fluid Spraying.............................................315
12.5.5.2 Nanoencapsulation............................................................315
12.6 Properties of Microencapsulated Omega-3 Ingredients ..............................315
12.7 Food as Delivery Systems for Omega-3......................................................316
12.7.1 Infant Formula..................................................................................316
12.7.2 Bakery and Cereal Products ............................................................317
12.7.3 Dairy Products..................................................................................317
12.7.4 Meat and Fish Products ...................................................................318
12.7.5 Drinks and Beverages ......................................................................318
12.7.6 Confectionery ...................................................................................318
12.8 Considerations for Successful Delivery of Omega-3 Oils into
Commercial Food Products .........................................................................318
12.8.1 Compliance with Regulatory Standards ..........................................318
12.8.2 Definition of Final Product Application to Ensure the
Correct Delivery Format ..................................................................319
12.8.3 Understanding of the Protection and Release
Requirements during Processing .....................................................319
12.8.4 Understanding of How and Where to Add the
Microencapsulated Ingredient..........................................................319
12.8.5 Understanding Possible Interactions with Other Ingredients..........319
12.8.6 Understanding the Shelf-Life, Sensory, and Physical
Properties of the Final Product........................................................319
12.8.7 Working in Partnership through Collaboration ...............................320
References..............................................................................................................320
12.1 INTRODUCTION
The functional food revolution and increasing consumer awareness of the link
between health and food have resulted in a growing demand for foods that offer
benefits for healthy living.1,2 Significant among the growing class of functional foods
are foods that are fortified with omega-3 fatty acids.
The high degree of unsaturation of omega-3 oils makes them very susceptible
to oxidation. Oxidation of omega-3 oils can occur at all stages in the supply chain,
from the handling of raw materials through to processing, storage, and handling of
the oil during food manufacturing process and subsequently in the storage and
handling of the final omega-3 enriched food products. This makes the incorporation
of omega-3 fatty acids into foods a significant challenge as their susceptibility to
oxidation and development of off-flavors affects the sensory properties of the omega3 fortified foods.3–6
To overcome some of the issues relating to stability of omega-3 oils, the use of
microencapsulation technology has been explored. Microencapsulation, which
involves the packaging of a core within a secondary material in small particles, is
proving to be a successful strategy for protecting sensitive ingredients and enabling
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their delivery into foods.7 Microencapsulation technology has proven to be a powerful technique for tailoring the properties and functionality of sensitive ingredients
for use in specific systems. It has the potential to offer novel solutions for stabilization
and improved delivery of omega-3 fatty acids in food.
This chapter reviews the issues relating to incorporation of omega-3 fatty acids
in food. It includes a discussion of microencapsulation technology and its application
for delivery of omega-3 fatty acids in the food industry, the reasons for its use, the
materials used as encapsulants, the techniques used, and examples of its use in
product applications. Trends and innovations in new materials, emerging processes
and technical challenges associated with the selection of encapsulants, and appropriate techniques for the production of microencapsulated products are discussed.
Important considerations for successful delivery of omega-3 oils into the functional
foods market are highlighted.
12.2 DRIVERS FOR DELIVERY OF OMEGA-3 FATTY
ACIDS INTO FOODS
Omega-3 fatty acids have been associated with many health benefits relating to heart
health, brain function, eye health, joint health, mood, behavior, cancer, diabetes, skin
disorders, pregnancy, and lactation.3,8–14
Interest in delivery of omega-3 fatty acids into foods has primarily been driven
by consumer awareness of the health benefits of omega-3 fatty acids and an increased
consumer demand for foods fortified with omega-3 fatty acids that have desirable
nutritional, sensory, and functional attributes.
There is also a growing recognition that modern diets are deficient in omega-3
fatty acids. The average intake of omega-3 fatty acids by consumers has been found
to be grossly inadequate due to low consumption levels of fish, one of the major
sources of omega-3 fatty acids in the diet. Major health and nutrition organizations
and scientific authorities recommend that intakes of long-chain polyunsaturated fatty
acids (PUFA) should be increased by eating two to three fatty fish meal per week,
or by supplementation. The International Society of the Study of Fatty Acids and
Lipids (ISSFAL), American Heart Association (AHA), Institute of Medicine (IOM),
and British Nutrition Foundation (BNF) all make recommendations on the daily
intake of omega-3 fatty acids. As a general guideline, the minimum recommended
daily intake for adults is approximately 500 mg of combined docosahexaenoic acid,
C22:6n-3 (DHA) and eicosapentaenoic acid, C20:5n-3 (EPA) per day for cardiovascular health, or 900 mg of combined DHA and EPA if you have coronary heart
disease.15–18
In 2000, the Food and Drug Administration (FDA) announced a qualified health
claim for dietary supplements containing EPA and DHA and the reduced risk of
coronary heart disease. The FDA recommends that consumers do not exceed more
than a total of 3 grams per day of EPA and DHA with no more than 2 grams per
day from a dietary supplement. In 2004, the FDA allowed a qualified health claim
for omega-3 fatty acids (DHA and EPA) and cardiovascular health. To qualify for
the health claim in the U.S., foods must contain a minimum of 125 mg long-chain
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omega-3 fatty acids per serving, and be part of a food that is low in fat, saturated
fat, trans fat, and cholesterol.19 In Australia and New Zealand, a nutritional claim
has been allowed for omega-3 fatty acids if the food contains a minimum 30 mg
total EPA and DHA, or 200 mg alpha-linolenic acid, C18:3n-3 (ALA) per serving,
when part of a food that is low in saturated fat and trans fatty acids.20
The accumulating evidence about the health benefits of omega-3 fatty acids, the
increasing awareness of consumers about the benefits of omega-3 fatty acids, and
advances in processing of PUFA ingredients are making omega-3 enriched foods a
reality in the market place.5,21–24 The advent of the nutritional and health claims has
opened up new opportunities for foods rich in omega-3 fatty acids. More products
enriched with omega-3 fatty acids are becoming available in the market and starting
to gain the acceptance of consumers.
12.3 INCORPORATION OF OMEGA-3 OILS INTO
FOODS
Omega-3 oils, such as fish oil, are prone to oxidative deterioration and to the
development of objectionable off-flavors and odors even when precautions are taken
such as protection from light and oxygen and low-temperature storage.25 Oxidation
also affects the nutritional quality and safety of the oil to consumers. The fishy taste
and odor associated with omega-3 oils from marine sources is another hurdle for
incorporation of omega-3 fish oils into food products.
The development of oxidative rancidity of omega-3 oils is a factor that has
limited its use in foods. The biggest challenge to the omega-3 oil manufacturers and
suppliers is overcoming the development of undesirable taste and odors in the end
products and the difficulties in using the oil as an ingredient in food and beverage
applications.26
As the oxidation of unsaturated fatty acids is an autocatalytic process, unsaturated oils used for incorporation into foods must be of the highest quality practically
achievable in order to minimize the development of off-flavors. This means that the
oils should be extracted from raw materials of good quality and the bulk oils should
be refined, stabilized, packaged, and stored under appropriate conditions, which
minimize exposure to factors that promote oxidation such as air, oxygen, and light.
Antioxidants may be added to protect the oil from oxidation. Synthetic antioxidants, such as tertiary butyl hydroquinone, butylated hyroxylanisole, and butylated
hyroxytoluene, singly or as mixtures, have been used in fish oils. The push toward
natural ingredients has resulted in natural antioxidants such as tocopherols, ascorbic
acid, carotenoids, and rosemary extracts becoming a more popular option.25,27 Mixtures of tocopherol, lecithin, and ascorbyl palmitate were found to be effective for
retarding oxidation in fish oil.28 Even with the addition of antioxidants, unprotected
oils can still oxidize very rapidly once they are exposed to a food environment.
Omega-3 oils may be added directly to foods but this approach has its limitations.
The oil is not protected from the food matrix during processing of the food product,
thereby exposing the oil to environments (e.g., air, oxygen, heat) that promote
oxidation of unprotected fats. This has undesirable consequences on the shelf
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stability of the omega-3 fortified food product. This has in part been addressed by
addition of antioxidants to the bulk oil and to the food systems.
An alternative approach is to use microencapsulated omega-3 oils.29 With the
use of microencapsulation, the oil is protected from the harsh processing environments during processing. A good microencapsulation system also protects the oil
from interactions with the food matrix and environment during storage. This allows
the omega-3 fortification of food products without affecting the taste and shelf life
of the final food.
The principles of designing microcapsules and the use of microencapsulation
and delivery technologies for incorporation of omega-3 fatty acids into foods is
discussed in more detail below.
12.4 MICROENCAPSULATION
Microencapsulation is the science of packaging components (referred to as the core
or active) within a secondary material (referred to as the encapsulant or coat) and
delivering them in small particles (i.e., microcapsules). Microencapsulation is used
as a means of isolating an ingredient from the reactions of the surrounding materials
or environment.30 It may be used for stabilizing a sensitive ingredient, masking
flavors, and for controlled delivery of active components. An associated benefit is
the improved ease of handling when microencapsulated ingredients are delivered in
a powder format.
12.4.1
MICROENCAPSULATION
IN THE
FOOD INDUSTRY
Microencapsulation technology has been used for flavor encapsulation since 1930.31
In recent years the functional food and ingredient industries have been identified as
potentially significant growth areas for the technology, especially for the encapsulation of unstable new functional ingredients. There are greater demands being made
on the integrity of the microcapsules to provide controlled delivery of the core
material at the desired time and site.7,32–36 Microencapsulation is an enabling technology that provides novel solutions for the fortification of food, while ensuring that
the taste, aroma, or texture of food is not compromised.7,34,37
12.4.2 MICROENCAPSULATION
OF
OMEGA-3 OILS
The main advantage of omega-3 oil encapsulation is the protection afforded to the
oil against oxidation during storage and processing. This is because the microencapsulated oils have a built-in barrier (i.e., the encapsulant) that isolates the oil from
the surrounding environment. Hence, the oxidation of polyunsaturated oils is inhibited, and the shelf life of these products during storage and processing may be
improved.29,38 The level of protection afforded against oxidation depends on the
encapsulant used and the integrity of the microcapsule. The oil may be protected
from direct exposure to light and oxygen during storage and from the other components in the food matrix during food processing and storage, thus reducing potentially
undesirable reactions with other food ingredients in a complex food system.
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Microencapsulation can also be used to mask the undesirable taste and odor that
may be imparted by the oil, thus enabling the production of omega-3 fortified foods
with desirable sensory properties.39
12.5 MANUFACTURE OF MICROENCAPSULATED
OMEGA-3 INGREDIENTS
The production of a microencapsulated omega-3 product involves the selection of
the omega-3 oil (i.e., the core), choosing the ingredients to be used as encapsulants,
developing the formulation comprising the required amount of omega-3 oil to encapsulant ratio, and applying an appropriate process for production.
To ensure that the microencapsulated product is used effectively for fortification
of foods, the properties of the microcapsule need to be tailored to suit the food
application. It is important to ensure that the omega-3 oil core is protected from the
harsh processing environments and that the release of the core is not triggered
prematurely. Many different encapsulant materials combined with different processing technologies have been used for the encapsulation of omega-3 oils resulting in
microcapsules with different properties.25,40–46 However, there are still limitations to
the use of microencapsulated ingredients in some applications. An integrated
approach to microcapsule production and its final product application is required
for the success of a microencapsulated ingredient (Table 12.1).
12.5.1 ELEMENTS
OF
MICROCAPSULE PRODUCTION
12.5.1.1 Omega-3 Oil Core Selection
The first step is to choose a source of omega-3 oil. It may be chosen from a range
of polyunsaturated oils that are rich in omega-3 fatty acids. The long-chain omega3 fatty acids, DHA and EPA, are found mainly in fish, such as tuna, salmon, sardines,
and mackerel, and in marine algae and seaweeds. The shorter-chain omega-3 fatty
acid, ALA, can be found in many plant sources such as linseed, canola, hemp, perilla,
and some vegetables such as spinach, green peas, and beans.3,4,47 The major commercial source of DHA and EPA is presently from fish. However, there is increasing
interest in algal oils48,49 and transgenic oil seeds50 as alternative sources of longchain omega-3 fatty acids.
It is essential to use high-quality oil with a low degree of oxidation. The specific
refining and deodorizing process applied in the processing of commercial omega-3
oil, the source of the raw material (e.g., fish oil, algal oil, plant oils, etc.), the fatty
acid composition, and addition of antioxidant mixtures to the oil will influence the
stability and the sensory properties of the microencapsulated oil product. The oxidative stability of microencapsulated omega-3 oils is dependent also on the type of
antioxidant used. Tocopherol proved to be more effective than ascorbyl palmitate
for protection of microencapsulated fish oil powder against oxidation.51
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TABLE 12.1
Elements of Microencapsulation: An Integrated Approach to the
Development of Microencapsulated Ingredients
Technological Activities
Identify core
Characterize core
Physical and chemical properties
Stability
Choose material
Use without modification
Modify to alter functionality
Characterize material
Physical and chemical properties
Stability
Production of
Microencapsules
Core
+
Encapsulant
Material
↓
Design formulation
Formulation of
Consider interactions between components Microcapsule
Stability of formulation
System
Triggers for release of core
Choose appropriate process
Conventional New/Emerging
Define unit process variables and apply
process
Assess process consistency efficiency
Determine composition
Gross composition
Proportion of encapsulated and free core
Characterize microcapsule
Physical and chemical properties
Stability
Triggers for release
© 2007 by Taylor & Francis Group, LLC
↓
Processing of
Microcapsule
Market-Related Activities
Work with a supplier of the core and
user of the ingredient
Obtain specifications
Consider cost
Establish reliable source of material
Obtain specifications
Consider cost
Consider what is available in the
market place
Range of formulations available
Limitations of existing formulations
Market need for microcapsules with
differentiated functionality
Consider need for capital investment
Processing cost
Process efficiency and reliability
Alternative processes
↓
Microencapsulated Develop product specifications and
Product
applications
Align with end users
Assess suitability
Provide technical service
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12.5.1.2 Encapsulant Selection
The encapsulant can be selected from a wide variety of natural or synthetic polymers
depending on the characteristics desired in the final microcapsule. The composition
as well as the physical and chemical properties of the encapsulant material can
influence the functional properties of the final microcapsule and the choice of
processing technologies to be used for microencapsulation. For encapsulation of
omega-3 fatty acids, it is desirable to choose materials from a natural source, which
have good oxygen barrier properties in order to afford protection of the core from
oxidation.
12.5.1.3 Formulation Design
The design of formulations for encapsulation of omega-3 fatty acids requires an
understanding of the properties of the oil and the required function of the encapsulant
materials and any other ingredients or additives chosen for the formulation. The
primary aim is to design a formulation that results in high encapsulation efficiency
and a microcapsule that gives high oxidative stability to the omega-3 oil core. A
high oil load in the microcapsule is also desirable provided this does not compromise
the quality of the microcapsule.
The development of a successful formulation for a target application requires
knowledge about the stability of the chosen oil; the encapsulating properties of the
ingredients; the likely interactions between the omega-3 oil core, the encapsulant
and other components in the formulation; and the reactivity of the microcapsule
formulation in the final product application. It has been shown that factors such as
the type of encapsulant material, addition of antioxidant, chelating agent, emulsifier,
stabilizers, and salts affect the stability of microencapsulated oil products.52.
12.5.1.4 Choosing the Process
The selection of unit processes and process conditions for omega-3 oil encapsulation requires some consideration. A number of standard unit processes suitable
for production of microencapsulated omega-3 oils are widely available in the food
industry. Standard unit processes (e.g., mixing, homogenization, and drying) have
traditionally been used for the production of microencapsulated ingredients such as
omega-3 oil powders as it usually involves the preparation of an emulsion and drying.
There are possibilities for application of emerging food-processing operations
(e.g., microfluidization, ultrasound, high-pressure processing) in the manufacture of
microencapsulated ingredients. The use of these technologies at various stages of
the microencapsulation process, in place of standard unit processes, may be used to
produce microcapsules with unique properties.
The choice of the processes used for microencapsulation will depend on the
properties required and the cost for production of microcapsules. Even with using
standard unit processes, the cost of production of microcapsules can vary significantly depending on which microencapsulation technology is being used. If nonstandard unit processes are used, there may be a need for capital investment. It is
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important to consider the essential requirements of the microencapsulated ingredient
in the final product, and whether the product can carry the additional cost.
12.5.1.5 Format for Delivery
For addition into a dry or powdered food product formulation, it is necessary to
convert the liquid microcapsules containing omega-3 oils into powder to make it
commercially feasible for dry blending. This means that a drying step is needed as
an integral part of the microencapsulation process. It is important to match the
physical properties (e.g., particle size and bulk density) of the microcapsule to that
of the powder with which it is to be dry blended in order to minimize or avoid
physical separation or segregation of the ingredients during transport and storage.
For liquid food applications, a liquid format (e.g., emulsion) is sometimes preferred,
but a powder format that can easily be rehydrated into a stable emulsion or dispersion
is also suitable.53
12.5.2 ENCAPSULATING MATERIALS
Proteins, carbohydrates, lipids, gums, polysaccharides, and cellulose materials are
commonly used for microencapsulation of omega-3 fatty acids and oils (Table 12.2).
They can be used alone or in combination to achieve certain functions either during
production or in the final product. Some of the new developments in materials for
microencapsulation of omega-3 fatty acids involve the application of clever chemistry and processing to modify the properties of the native food ingredient to achieve
new functional properties not present in their standard form.
TABLE 12.2
Materials Used as Encapsulants of Omega-3 Oil Encapsulation
Material Class
Proteins
Sugars and sugar products
Starch and starch products
Gums
Cellulose material
Other carbohydrates
Lipids
Types of Materials
Albumin, caseinate, gelatin, gluten, peptides, soy protein, whey
proteins, zein, other vegetable proteins
Fructose, galactose, glucose, maltose, sucrose, oligosaccharide, corn
syrup solids, dried glucose syrup
Maltodextrins, dextrins, starches, modified starches
Agar, alginates, carrageenan, gum acacia, gum arabic, pectins
Acetylcellulose, carboxymethyl cellulose, cellulose acetate butylate
phthalate, cellulose acetate phthalate, ethyl cellulose, methyl
cellulose, nitrocellulose
Chitosan, cyclodextrins
Acetoglycerides, beeswax, diglycerides, natural fats and oils,
fractionated fats, hardened fats, lecithin, liposomes, monoglycerides,
paraffin, tristearic acid, waxes
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A good encapsulant for omega-3 fatty acids protects the oil in the microcapsule
from degradation during processing and storage and also masks the undesirable taste
and odor associated with the microencapsulated omega-3 oil ingredient when added
into a food product. The encapsulant must be approved for food use if the formulation
is to be incorporated into food products. The encapsulant materials may be used in
combination with other components in the formulation such as oxygen scavengers,
antioxidants, chelating agents, and surfactants. Cost is also a consideration for the
selection of the encapsulant.
12.5.2.1 Challenges for Selection of Materials for Omega-3
Oil Encapsulation
It is important to consider the encapsulant that will be used in the formulation and
process for the production of the microcapsule. The ability of the encapsulant to
stabilize and protect the oil core during processing and storage of the microencapsulated omega-3 oil ingredients, and the mechanisms required for release of omega3 fatty acids at the appropriate time and site needs to be taken into account.
Other criteria that need to be considered during encapsulant selection include
the mechanical strength requirement of the microcapsule, the compatibility of the
encapsulant material with the target food product, its nutritional value, sensory
properties, and aesthetic properties.34,35
The availability of a very limited range of suitable encapsulant materials
allowed for food use still remains the biggest challenge in encapsulant material
selection, especially when more sophisticated microcapsule properties are required
by food manufacturers and consumers. The requirement for good powder flow
properties, dispersibility, and solubility are now becoming standard prerequisites
for omega-3 oil powder microcapsules, with the requirements for oxygen, moisture, heat, shear, and pH barrier properties becoming important characteristics of
the encapsulant surrounding the core. Food-grade materials commercially available
meet some of these requirements. Modification of existing food-grade materials
to achieve differentiated encapsulant functionality may be required to achieve new
properties in microcapsules and improved performance of microencapsulated
ingredients.
12.5.2.2 Classes of Encapsulant Materials Used for Omega-3
Oil Encapsulation
12.5.2.2.1 Proteins
Proteins possess many excellent functional properties. They have been used as
encapsulants for omega-3 fatty acids and oils because of their good film-forming
and emulsifying properties.54-58 The most commonly used proteins are gelatin, whey
protein, casein, soy protein, and albumin.
The good water solubility of proteins is desirable during preparation of the
encapsulant in order to achieve the required encapsulant functionality when using
an all-aqueous-based process. However, the good water solubility characteristics of
the encapsulant generally result in rapid dissolution of the encapsulant when
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the microcapsule is in contact with water in the final application. This may be
accompanied by the release of the core to the environment, which presents a disadvantage in some applications.
In order to improve the resistance of protein encapsulants to moist environments,
the solubility of protein wall materials can be modified using a number of techniques.
This may be achieved by coagulating the protein by heat or by the action of pH.59
Lee and Rosenberg60 used a double emulsification and gelation process to prepare
whey protein microcapsules (containing a milkfat core), which had limited water
solubility for controlled core release in food applications. Others have improved
stability of protein-based fish oil capsules by using transglutaminase to cross-link
soy proteins in double emulsions.61
The good oxygen barrier properties of some proteins41,62,63 makes them desirable
as a choice of encapsulant for protecting oxygen sensitive cores like omega-3 fatty
acids and oils against oxidation.
Proteins have been used in combination with carbohydrates in the formulation
of microencapsulated oil ingredients.64,65 Lin et al.44 reported that wall materials
containing gelatin, caseinate, and maltodextrin provided optimal protection against
oxidation of microencapsulated squid oil. Further, the oil stability was improved
with the addition of emulsifier (lecithin) and a stabilizer (Avicel).
12.5.2.2.2 Carbohydrates
Maltodextrins, corn syrup solids, and sugars have been used in formulations for
omega-3 oil encapsulation, but not on their own because they have poor emulsifying
and film-forming properties. Starches have also been used for encapsulation of oil
and oil-soluble cores. They have been made more suitable for encapsulating oils by
chemical modification to increase their lipophilicity and improve their emulsifying
properties. Starches, such as high amylose cornstarch, may also be used for sitespecific delivery as demonstrated by the application of high amylose starch as a
food-grade enteric encapsulant.66
Gums have traditionally been used in the flavor industry but also have applications as encapsulating materials for omega-3 oils. The gum most commonly used is
gum acacia, although it is the protein fraction in acacia gum preparations that is
responsible for its emulsification properties. Modified celluloses such as methylcellulose and hydroxypropyl methylcellulose in combination with maltodextrin have
been demonstrated to be suitable encapsulants for stabilization of fish oil in high oil
load powders with 40%–50% oil.67
Cyclodextrins, which are cyclic molecules derived enzymatically from starch,
have some unique encapsulation properties. They have the ability to form inclusion
complexes with flavors and oil-soluble nutrients and can provide stabilization, during
manufacturing and storage of some core materials that cannot be stabilized by other
methods. The use of cyclodextrin is currently limited because of its high cost.
Nevertheless, β-cyclodextrin offers advantages as a microencapsulant because it has
good controlled release properties, with binding occurring up to 200°C. The release
of the complexed material is influenced by moisture. β-cyclodextrin has been
approved by the FDA and granted GRAS status.
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12.5.2.2.3 Lipid Materials
Fats have been used mainly as a secondary coating to primary microcapsules to
improve their water barrier properties.68
Liposomes are artificially made microscopic membrane vesicles consisting of
one or more concentric layers of lipids.35, 69 These versatile materials can be used
for delivery of water- or oil-soluble core materials and are therefore suitable for
microencapsulating a range of food ingredients, including vitamins, omega-3 oils,
minerals, and flavors. Stable microcapsules, ranging from a few nanometers to
microns in diameter, may be made which makes them suitable for a range of
applications. The microcapsules produced by liposome entrapment are more versatile
and less fragile than fat-encapsulated ones. One promising area of application where
liposomes could have the advantage over other methods is the prevention of oxidation
of unsaturated fats in food emulsions such as spreads, margarines, or mayonnaise.
However this needs further development to optimize loading, protection, and delivery.
12.5.2.2.4 Protein—Carbohydrate Conjugates
An alternative to the use of physical blends of protein and carbohydrates is to form
protein-carbohydrate conjugates. Kato and Kobayashi70 demonstrated that proteindextran conjugates, developed by coupling proteins to cyanobromide-activated dextran or by linking proteins with dextran through the naturally occurring Maillard
reaction, have excellent emulsifying properties. These conjugates were superior to
the native protein, commercial emulsifiers from sucrose-fatty acid esters and polyglycerine esters, especially at high salt concentration and at acidic pH. The emulsifying properties of protein-dextran conjugates were greatly enhanced by preheating
at 100°C.
Protein-carbohydrate conjugates, formed as a result of the Maillard reaction,
have been shown to be superior to physical blends of protein and carbohydrate for
encapsulation of omega-3 oils. The conjugates have excellent film-forming, emulsifying, and oxygen scavenging properties. Omega-3 oils encapsulated within a
Maillard reaction product of film-forming protein and carbohydrate have longer shelf
life compared to those encapsulated with proteins and carbohydrate blends.71
12.5.3 MECHANICAL PROCESSES
The selection of appropriate processing technologies for microencapsulation of
omega-3 fatty acids depends on the desired properties of the final microcapsules
such as oil loading, shelf life, release properties, microcapsule format, and the final
product application.72
Microencapsulated omega-3 oil products are generally produced in powder form
for better stability and ease of handling. Liquid emulsions are also available for
more specialized liquid food applications. A generalized process used for production
of microencapsulated omega-3 oil ingredients is shown in Figure 12.1. Stable omega3 oil-in-water emulsions may be heat treated in liquid state or converted by spray
drying into free-flowing microencapsulated powders in order to improve their shelf
stability.
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Omega-3 oil + encapsulant material
Emulsification
Stable oil emulsion
(Liquid emulsion microcapsule)
Spray drying
Omega-3 oil powder
(Primary powder microcapsule)
Secondary coating in fluidized bed
(Optional process)
Omega-3 oil powder
with secondary coating
FIGURE 12.1 Generalized process for production of spray-dried microencapsulated omega3 oil powder with optional secondary coating.
12.5.3.1 Emulsion Preparation and Stabilization
Preparation of an emulsion involves the dispersion of the oil phase into very fine
droplets in which the action of an emulsifying agent at the surface of the oil droplets
provides protection or functionality to form stable oil-in-water emulsions. High shear
processes such as homogenization or microfluidization may be used for dispersing
the oil into droplets and forming the emulsion. Emulsifiers, antioxidants, and other
additives may be incorporated into both the oil phase and the aqueous phase prior
to homogenization.73 The emulsion formulation and other variables such as temperature, total solids, pH, and storage conditions influence the emulsion properties and
stability.74–76 Stable emulsion microcapsules can be dried into a powder for greater
shelf stability.
The selection of appropriate materials that can form a strong film around the oil
droplets, which will remain intact during processing and storage, is essential for
providing protection and stability to the omega-3 oil droplets. Both the physical
stability of the emulsion during processing and the storage and oxidative stability
need to be considered in the development of omega-3 oil emulsions.
A range of proteins, emulsifiers, and stabilizers have been used for the stabilization of omega-3 oil in water emulsions. The oxidative stability of omega-3 oil-inwater emulsions is dependent on the type of protein and emulsifiers used in the
formulation.77–82 This is because the oxidation of an oil-in-water emulsion droplet
is influenced by the properties of the interfacial membrane surrounding the lipid
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core as well as the properties of the bulk phase. Important determinants of oxidative
stability of emulsions include the thickness of the interfacial membrane of an emulsion droplet,83 the size of the surfactant hydrophobic tail group size,84 the type of
antioxidants, the partitioning of the added antioxidants in the emulsions,85,86 and the
level of hydroperoxides, which may be present in both lipids and surfactants.80
Advances in emulsion technology provided the possibility of designing multiple
emulsions for the controlled delivery of bioactives. An example is a method of
producing an oil-in-water-in-oil (o/w/o) emulsion where the oil or oil-soluble core
(flavors) is emulsified in a protein to form the o/w emulsion and then this emulsion
is further emulsified in a liquefied fat that is solid at room temperature. The resulting
o/w/o emulsion is then atomized into a food product as a delivery vehicle for the
core.87 Benichou et al.88 have also developed hybrids of natural polymers for entrapment and slow release of active matters in double emulsion systems.
12.5.3.2 Spray Drying
Spray drying has been generally referred to as an encapsulation technology because
it is one of the oldest encapsulation methods and has been in use since the 1930s.31
It is still the most commonly used microencapsulation method in the food industry.
The process of spray drying is efficient and cost effective, uses equipment that is
readily available, and produces particles of reasonably good quality. Food ingredients
microencapsulated by this method include fats, oils, flavors and oil-soluble materials.
Encapsulant materials are usually proteins, carbohydrates, or combinations of both.
The general process involves the dispersion of the core material into a polymer
solution, forming an emulsion or dispersion, followed by homogenization of the
liquid and atomization of the mixture into the drying chamber.89 This leads to
evaporation of the solvent (water) and the formation of matrix-type microcapsules.
The advantage of the spray drying process is that it can be operated on a continuous
basis. The microcapsules produced are soluble in water. Proper adjustment and
control of the processing conditions enables the production of a desired bulk density
and particle size distribution of the powder.
Advances in spray-dried microencapsulated ingredients have been made possible
by new dryer and atomizer designs. New dryer designs allow drying at lower outlet
temperatures (multistage dryers), longer residence time (tall-form dryer), and therefore provide milder drying conditions for minimizing deterioration of heat-sensitive
microcapsules. New atomizer designs can improve the properties of microcapsules
produced in spray dryers. New atomizer designs have been developed by Particle
Coating Technology, USA, and Niro A/S, Denmark. Both these atomizer designs
can be fitted into a spray dryer setup for the production of particles with a narrower
size distribution than conventional atomizers.
Many spray-dried formulations have involved the dispersion of liquid cores in
a carrier material prior to spray drying.90–93 For the production of microencapsulated
omega-3 oils, it is essential to design the formulation and apply the appropriate
process to obtain stable liquid microcapsules before conversion of these into powder
by drying. The formulation and processing steps that take place prior to spray drying
are important steps in the production of stable omega-3 oil powder microcapsules.
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Prior to the spray-drying step, the omega-3 oils are essentially encapsulated oil
droplets in an aqueous system (e.g., emulsion, dispersion, and coacervate systems)
and the formation of a stable liquid microcapsule is essential for the manufacture
of acceptable microencapsulated oil powders.
Omega-3 fish oils have been encapsulated using a variety of encapsulant formulations which are subsequently spray dried into powder microcapsules. These formulations include proteins (sodium caseinate and whey protein isolate),94 proteins
in combination with carbohydrates as wall materials95–97 and protein-carbohydrate
conjugates,71 which improved the stability of the omega-3 oil core against oxidation.
Spray drying can easily be substituted by any other drying method for production
of powdered omega-3 oils such as freeze drying,98-100 fluid bed drying,64 refractive
windows drying, and filtermat drying, depending on final powder properties desired
and the cost that the microencapsulated product can bear in the market.
Although drying is only one of the steps involved in the production of microcapsules, the control of the processing conditions during drying has a significant
influence on the physical properties of the final powder microcapsule.
Processing conditions such as temperature, shear, pH of the emulsion, total
solids, drying rate, and order of addition of each of the components used during
manufacture of omega-3 oil powders influence the microcapsule properties and
stability. Depending on the formulation, the drying method (e.g., spray drying, freeze
drying) used to convert emulsions and dispersions into powder can also influence
the physical structure and stability of microcapsules during storage.
12.5.3.3 Extrusion
Extrusion is used to produce high-density encapsulated products via mixing of
molten carriers with active ingredients followed by solidification. It involves projecting a dispersion or an emulsion of the core and the encapsulant through a die
or nozzle at high pressure. It has been mainly used in flavor encapsulation, but the
process has also been used for omega-3 oil encapsulation. The cost of extrusion is
believed to be twice that of spray drying;101 however, it still ranks second behind
spray drying as the most frequently used process for encapsulating flavors.102 The
use of food-grade hydrating liquid or solvent to solidify the extruded product into
pellets or powder is an important factor in considering this method for food ingredient production.
The formulation and the processing conditions during extrusion affect the properties of the microcapsule. Particle size was reduced with increased screw speed and
melt temperature during processing and increasing the hydrophilic-lipophilic balance
(HLB value) of the emulsifier in the formulation when sunflower oil was encapsulated in starch matrices via extrusion.103
There are a number of modifications to the extrusion process aimed at improving
the process and the microcapsule release properties. These include the following:
High pressure method: This method, developed by Ottens Flavors and Delta
Food Group, USA, was designed to microencapsulate key volatile compounds, flavors, and fragrances by using high pressure during the coating
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process. This process is said to keep flavors from evaporating during processing. The core is protected in matrices of carbohydrates and fats. Release
temperatures are selected such that flavors added to the food before processing are retained unaltered after cooking. The new capsule forms are
said to provide controlled release, which enables multistage release or
break-on-demand targeted delivery.
Modified extrusion process: This method, developed by Fuisz Technologies
Ltd., utilizes low-level heating to entrap a particulate dispersion within a
thin matrix. The final products are said to have enhanced flavors, or sustained-release flavor delivery, such as is required in chewing gums.
Extrusion has been used to encapsulate both particulate and liquid cores in a
protein carrier that has been heated to create a melt and then denatured by pH
adjustment and treated with an enzyme to form the microcapsule.104 Heat sensitive
and readily oxidizable cores were encapsulated by extrusion using a range of proteins
and carbohydrate materials to achieve discrete solid microcapsules with controlled
release properties.105 Cores, including lipids, have been encapsulated in a carbohydrate or protein matrix that is gelatinized or polymerized during extrusion process.
The microcapsule is claimed to release in the digestive tract.106 A recent patent
describes the use of extrusion process for protection of omega-3 oils.107
12.5.3.4 Gelation and Particle Formation
A process often used for encapsulation of oil and oil-soluble cores involves the use
of biopolymer gels as entrapment matrices. Examples include oil and oil-soluble
cores encapsulated in alginate beads by extrusion to form the particles, followed by
drying,108 and oils that are sensitive to oxygen (e.g., wheat germ oil, evening primrose
oil) encapsulated in alginate by emulsification.109
A number of methods have been developed for more efficient particle formation.
These include the following:
Centrifugal extrusion: This is one of the more established methods of microcapsule particle formation. It is generally a liquid coextrusion process utilizing
nozzles consisting of concentric orifices located on the outer circumference of a
rotating cylinder.110
Microject method: This has been developed by Ganan-Calvo111 for the production
of a core- and shell-type microcapsule particle. Ripoll et al.112 have also developed
an electrified coaxial jet method for production of micro- and nano-metric-size
microcapsule particles.
All these methods which result in the formation of wet gelled particles require
a method for segregation and collection of the microcapsules prior to drying to
produce dry free-flowing microcapsules.
12.5.3.5 Secondary Coating Application
Secondary coating application has been widely used in the flavor industry to produce
agglomerated powders or granules subsequently coated with another material,113 to
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improve shelf life and sensory properties of oil based flavors.114,115 This method has
also been used to apply a thin polymeric layer of secondary coating to spray-dried
fish oil microcapsules to increase the degree of protection and controlled release of
the primary fish oil microcapsules.116
Fluidized bed coating is the most common method used for coating applications.
It is accomplished by suspending or fluidizing particles of a core material in an
upward stream of air and applying an atomized coating material to the fluidized
particles. The coating to be applied may be a molten fat (e.g., hydrogenated fats,
stearines, fatty acids, emulsifiers, waxes) or materials from a range of film-forming
materials (such as proteins, starches, gums, or maltodextrin) dissolved in a solvent
(e.g., water). When molten fat is used, cool air is used to harden the coating. When
a soluble coating is used, hot air is used to evaporate the solvent and harden the
coating. Fat-coated core ingredients are released by an increase in temperature or
by physical fracture, whereas core ingredients coated with water-soluble materials
are released when water is added to redissolve the coating. This process is limited
to the microencapsulation of powder particles with diameters of greater than 100
microns, as particles of less than 100 microns tend to either clump to form agglomerates or get carried away in the exhaust air.117,118 This method is not suitable for
primary encapsulation of an oil or fat-based product such as omega-3 fatty acids;
however, it can be used to apply a secondary coating to the primary powder microcapsule (Figure 12.1). Fluidized bed coatings can be used to enhance, time, or tune
the release properties of the primary omega-3 oil microcapsules.
Another method used for coating application is the spinning disk system. It is
a new fast coating method for controlled release applications, developed by Particle
Coating Technologies.119 The coating materials used in fluid bed coating processes
may be applied to this system.
12.5.4 CHEMICAL-BASED PROCESSES
12.5.4.1 Coacervation
Coacervation has been defined as the separation of two liquid phases in colloidal
systems. The more concentrated phase is termed the coacervate phase and the other
phase is called the equilibrium solution. Complex coacervation occurs when two
oppositely charged colloids interact to form a complex.120
The underlying cause of coacervation is the phase separation of one or more
hydrocolloids from the initial biopolymer solution. Coacervation may be induced
by a change in temperature or by the addition of a second substance such as a
concentrated aqueous ionic salt solution or a nonsolvent. The coacervate coats the
emulsified oil droplets by phasing polymers out of solution. The coat can then be
hardened by using a chemical or enzymatic cross-linker. It is the hardening of the
shell that remains the greatest challenge, especially for food applications.121,122
Complex coacervates between proteins and polysaccharides that interact to form
an electrostatically bound complex are most commonly used in the food industry.
Protein-polysaccharide complexes have been shown to exhibit better functional
properties than that of the proteins and polysaccharide combination, in a number of
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applications.123 The complex generally has superior interfacial properties, an effect
that is attributable to the simultaneous presence of the two biopolymers, and hence
it functions well in an encapsulant capacity.
In the food industry, coacervation has typically been used for encapsulation of
flavor oils but it has potential for application to a range of other microencapsulated
food ingredients.124 Microcapsules have been produced using a plant protein (e.g.,
soy protein) and a polyelectrolyte with an opposite charge to the protein subjected
to complex coacervation and followed by hardening with cross-linking agent (e.g.,
dialdehydes and tannins). Microcapsules produced are suitable for a range of applications including food.125 A complex coacervate with gelatin-caboxymethylcellulose
cross-linked with glutaraldehyde has been used for flavor delivery.126 Chitosan coacervated microcapsules have been shown to have good diffusion release characteristics.127
This technique may be adapted for encapsulation of omega-3 fatty acids.128 Soeda
et al.129 describes a simple coacervation technique for encapsulation of flavors and
oil-soluble cores including omega-3 fatty acids, using transglutaminase as the crosslinking agent for hardening the capsule wall formed by salting out. The resulting
product has been shown to be suitable for a wide range of food applications. Oilsoluble cores (e.g., vitamins and polyunsaturated fatty acids) have been encapsulated
by cross-linking a protein, sugar, and a water-soluble salt, by heating the mixture
under specific conditions, to produce a microcapsule that is substantially insoluble
in boiling water for at least 3 minutes.130 A double emulsification and an enzymatic
gelation method using transglutaminase cross-linked protein encapsulant has been
suggested by Cho et al.61 to improve the storage stability of fish oil and achieve
controlled release.
12.5.4.2 Inclusion Complexation
Inclusion complexation is the term used when cyclodextrins are used for microencapsulating core materials. Unlike other techniques, inclusion complexation takes
place at a molecular level and utilizes cyclodextrin molecules as the encapsulating
medium.
A -cyclodextrin complex has been shown to enhance oxidation stability of onion
and garlic oils as well as mask undesirable odor and flavor.131 Inclusion complexes
using cyclodextrins have been used for the encapsulation of omega-3 fatty acids to
enhance their stability to temperature, light, oxidation, polymerization, or doublebond migration and mask undesirable taste and odor.132–134 Yoshii et al.135 demonstrated that a complex between DHA oil and cyclodextrin prepared by a twin-screw
kneader is able to produce a microencapsulated omega-3 powder with very high
resistance to oxidation in fish meal paste application even without an antioxidant in
the formulation.
12.5.4.3 Liposome Encapsulation
Liposome encapsulation means to microencapsulate or trap core materials using
liposomes. Liposomes have been used to encapsulate omega-3 fatty acids by
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dissolving the oil in phospholipid before water is added and sonicated to form the
encapsulated material. The entrapped oil is protected from oxidation and the preparation can be added directly to food products as a dispersion or it can be dried into
a free-flowing powder.136 Food approved proliposomes are now available, which can
be used for the microencapsulation of food ingredients, and are much simpler to use.
12.5.5 EMERGING TECHNOLOGIES
12.5.5.1 Supercritical Fluid Spraying
This is a relatively new technique, primarily developed for the pharmaceutical
industry, in which the core material and the microencapsulating material (typically
a polymer) are dispersed and dissolved in a supercritical fluid, such as carbon
dioxide. The supercritical fluid is ejected from a nozzle in the form of a spray. The
carbon dioxide flashes off very rapidly, leaving residual particulate material. By
careful control of the operational parameters and choice of materials, successful
microencapsulation has been achieved and the production of very uniform powder
particles is possible.137,138
12.5.5.2 Nanoencapsulation
Nanoencapsulation uses vesicular systems in which the active ingredient is confined
to a cavity surrounded by a unique polymeric membrane. The preparation of nanocapsules containing a membrane-forming molecule, a coemulsifier, and a lipophilic
component has been used to encapsulate a range of food ingredients, including
omega-3 fatty acids. The microcapsules are 40–80 nm in size and are suitable for
delivery of components into clear liquid drinks, as well as other beverages and
foods.139 Hydrophobic nanospheres encapsulated in pH- or moisture-sensitive microspheres have been shown to improve shelf life of foods and beverages and prolong
the sensation of flavor.140
12.6 PROPERTIES OF MICROENCAPSULATED
OMEGA-3 INGREDIENTS
Omega-3 powder microcapsules are small particles, typically containing 25%–60%
oil surrounded by a matrix or encapsulant. The physical properties of the microencapsulated omega-3 oil ingredients and their stability need to be defined as these
are important considerations for suppliers and users of these ingredients. The final
powder properties need to be tailored to meet the requirements in the final food
product.
Most microencapsulated omega-3 ingredients are supplied as powders that are
reconstituted and hence the ability of the powder to form a stable dispersion or
emulsion upon reconstitution is a desirable attribute. Powder particle size, encapsulation efficiency, solubility, and dispersibility can vary depending on the properties
of the emulsion or dispersion before drying, process conditions used during drying,
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and the method of drying. Particle size is an important consideration in the design
of the microcapsule for targeted delivery and application.119
Storage conditions such as temperature, humidity, oxygen availability, light, and
packaging can greatly influence the stability of the microcapsules, depending on the
core and encapsulant properties as well as the structure and morphology of the
powder microcapsule. Most of the microencapsulated omega-3 products developed
have had significant limitations in terms of storage stability and shelf life. Only very
few encapsulated products has been successfully commercialized without requiring
low temperature storage (e.g., 4°C–10°C).
Understanding lipid oxidation of microencapsulated oil ingredients is important
because oxidation results in loss of nutritional value and development of undesirable
flavors and odors during storage of the ingredient and when it is used in a wide
range of commercial food applications. The stability of microencapsulated omega3 oil powders to oxidation can be improved by appropriate selection of encapsulant
materials, processing, packaging, and storage conditions.
12.7 FOOD AS DELIVERY SYSTEMS FOR OMEGA-3
Omega-3 fatty acids and oils are key ingredients for the development of functional
food products. Higgins et al.141 have highlighted the potential of fortification with
omega-3 fatty acids in food as a vehicle for increasing omega-3 intake by consumers.
With the use of microencapsulation technology, new and innovative ways for the
protection and delivery of omega-3 fatty acids into a range of food products is made
possible.9 However, there are significant challenges involved during production and
storage of the final food product containing omega-3 fatty acids, because of the
susceptibility of the these components to oxidation and development of undesirable
flavors and odors. Developments in microencapsulation technologies and delivery
strategies have resulted in increasing numbers of successful omega-3 fortified products in the marketplace.
Omega-3 enriched products launched worldwide include pet foods, dietary supplements, dairy products, processed fish, meat and egg products, snacks, meals,
infant formula and baby foods, bakery products, and beverages. At the forefront of
omega-3 enriched foods are infant formula and baby follow-on foods, bread, dairy
products, nutrition bars, and margarines. Additionally, niche products fortified with
omega-3 oils such as soups, ice tea drinks, cakes, biscuits, and seafood products
where there has been restoration of omega-3 fatty acids are commercially available.21
12.7.1 INFANT FORMULA
Infant formula has been a major segment of focus for delivery of omega-3 fatty
acids to food because of its benefits to early childhood development.29 Fortification
of powder infant formula with omega-3 fatty acids is quite challenging, due to the
long shelf life of the product (usually 3 years from manufacture) and the limited
number of ingredients allowed in infant formula. The major challenge is to be
able to add the health benefits of omega-3 oil to infant formula without affecting
its odor, flavor, and shelf life during storage. The first successful commercial entry
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into this market was seen in Australia and New Zealand in 1998, using microencapsulated omega-3 oils that had been specially formulated using ingredients
already in use for infant formula manufacture. Omega-3 encapsulated powder was
incorporated without affecting the sensory properties, shelf life, and the ingredient
listing of the original product.142 There are now a number of commercial infant
formula products fortified with omega-3 fatty acids using a whole range of incorporation technologies.
12.7.2 BAKERY
AND
CEREAL PRODUCTS
Bread is a very popular food delivery vehicle for a number of functional ingredients
including omega-3 fatty acids. Fortification of bread with omega-3 fatty acids
requires that the omega-3 oil ingredient be protected from the liquid environment,
shear, and heat during the bread making process. As fresh bread is a short-shelf-life
product (< 1 week), long-term oxidative stability of the omega-3 oil is not required
but it is essential that the oil be not released from the microcapsule during the bread
manufacture. The incorporation of encapsulated omega-3 oils into bread has been
made possible by microencapsulation without affecting sensory properties and shelf
life.143 A number of other cereal-based products such as breakfast bars and muesli
bars fortified with microencapsulated omega-3 oils are available worldwide.
Omega-3 oils can also be delivered as a coating onto a dry food particle (soy
nuggets and cereals) where a PUFA coated particle is further coated with a sugar
solution and dried to a moisture content of the same level as the original dry food
particle.144 Encapsulated sardine oil in egg-white protein has been incorporated into
cookies without affecting the taste and quality of the final product.45
12.7.3 DAIRY PRODUCTS
There are opportunities for microencapsulated ingredients for fortification of dairy
foods.145 Dairy products have been targeted by a number of food companies as a
delivery vehicle for omega-3 fatty acids. Delivery into dairy products has been
attempted in two ways; by incorporation of encapsulated omega-3 oil into the cow
diet146 or by fortification of the dairy product during its manufacture.
Omega-3 fish oil (refined, deodorized, and hydrogenated) was incorporated with
an all-vegetable fat blend or a dairy blend mixture of butter and vegetable oils with
a view to the manufacture a longer-shelf-life, low-calorie spread (40% fat).147
The use of yogurt as a delivery vehicle for omega-3 oils is less challenging. The
sensory properties of omega-3 oil fortified yogurt were found to be superior with
the use of microencapsulated tuna oil compared to tuna oil in the free form.148 A
yogurt drink (Omega-3 laban drink) has been successfully commercialized in the
Middle East under the Live Well brand. Cheese and cheese-based products have also
been successfully fortified with omega-3 fatty acids without affecting their flavor
and shelf life.
The fortification of fresh milk with omega-3 oils is difficult because of the
delicate flavor of milk. Milk products fortified with microencapsulated nutrients
including omega-3 fatty acids have been developed, where the nutrients, which
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comprise both water-soluble and oil-soluble components, were dispersed in a hydrogenated oil containing lecithin and encapsulated in gelatin and sorbitol forming the
outer layer.149
12.7.4 MEAT
AND
FISH PRODUCTS
Microencapsulated omega-3 fatty acids have been successfully incorporated into
meat and fish products in Australia. Examples of these are the Hans low-fat range
of meat products such as Strassburg, roast chicken, and Vienna sausage, and Birds
Eye crumbed fish fillets under the “Smart Choice – Omega 3” brand with a range
of different flavors.
12.7.5 DRINKS
AND
BEVERAGES
New functional orange juice has been successfully commercialized in Australia by
Golden Circle and in the United Kingdom by a company under the Supa Jus brand.
12.7.6 CONFECTIONERY
Even with sweet and strong-flavored confectionery products the addition of omega3 fatty acids can be difficult. New orange- and mint-flavored pastille-like products
have been developed as a vehicle for omega-3 fatty acids delivery to increase intake
of omega-3 fatty acids by consumers.150 Their most promising formulation had EPA
and DHA concentration of only 0.8%–1.0% even with strong flavorings added to
mask the fishy flavor in the final product.
12.8 CONSIDERATIONS FOR SUCCESSFUL DELIVERY
OF OMEGA-3 OILS INTO COMMERCIAL FOOD
PRODUCTS
Ideally it is desirable to design omega-3 oil microcapsules that are stable in many
different applications, but in reality this is not a practical option, as the microencapsulated ingredient has to be matched to the target food application. Food ingredient
manufacturers will normally have microcapsule formulations designed for different
food category applications; for example, infant formula, bakery, dairy, and liquid
beverage applications, each of which has different requirements. There are different
challenges in the final food formulation with regards to food standard regulations,
shelf life, and release properties of the omega-3 oil microcapsules. Some of the key
issues for successful delivery of omega-3 fatty acids into commercial food products
include the following:
12.8.1 COMPLIANCE
WITH
REGULATORY STANDARDS
Prior to deciding on the microcapsule formulation, it is mandatory to check the
regulatory standards to ensure that ingredients chosen as encapsulants are approved
for food use in each country where the product will be marketed or sold. This step
will minimize the time for regulatory approval when the final product is launched
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into the market, and also avoids possible reformulation and testing as in the case
where a nonfood-grade material has been used during the initial trials.
12.8.2 DEFINITION OF FINAL PRODUCT APPLICATION
THE CORRECT DELIVERY FORMAT
TO
ENSURE
For dry blending with other food powders, it is necessary for the microencapsulated
powder to be matched in terms of water activity, bulk density, and particle size so
as to achieve a homogeneous product and avoid possible physical separation of the
different powders during transport and storage. For liquid product applications, the
rehydration and redispersion behavior of the powder is important if a powder format
is chosen. Alternatively, a liquid format for microencapsulated omega-3 ingredients
could be used.
12.8.3 UNDERSTANDING OF THE PROTECTION
REQUIREMENTS DURING PROCESSING
AND
RELEASE
Knowledge and understanding of the processing steps and unit operations involved
in the manufacture of the final food product is important to be able to design the
microcapsule properties in such a way that that the core will be protected during
the process and released at the desired time.
12.8.4 UNDERSTANDING OF HOW AND WHERE
MICROENCAPSULATED INGREDIENT
TO
ADD
THE
Familiarity with the food manufacturing plant setup and layout is also required in
order to identify the appropriate stage of addition of the microcapsule (e.g., how
and where the microcapsule can be added) without the need for any process modification.
12.8.5 UNDERSTANDING POSSIBLE INTERACTIONS
INGREDIENTS
WITH
OTHER
When the new microencapsulated omega-3 food ingredient is added to a food
product, the final food product properties and its shelf life may be affected due to
possible ingredient interactions. Sometimes these interactions may be minimized by
minor modification to the process or the formulation. However, sometimes even
small changes are not possible due to limitations in the plant layout/setup or further
modifications could add significant capital investment. Therefore formulating the
new omega-3 food ingredient has to be approached in such a way that possible
negative ingredient interactions are minimized.
12.8.6 UNDERSTANDING THE SHELF-LIFE, SENSORY, AND PHYSICAL
PROPERTIES OF THE FINAL PRODUCT
Addition of omega-3 food ingredients to short-shelf-life products with very strong
flavor is much less of a problem than addition to food products with delicate flavors
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and longer shelf life. Therefore the requirements of the final food product have to
be considered when designing the microcapsule formulation to suit the final application.
12.8.7 WORKING
IN
PARTNERSHIP
THROUGH
COLLABORATION
There is great advantage when the food ingredient supplier/company who develops
the microencapsulation technology and the food company manufacturing the final
food product can work in partnership during the development of the new food product
with added omega-3 oils.143,151 The food ingredient supplier or the company who
develops the microencapsulated omega-3 ingredient will understand the performance
and limitations of the microcapsule when added to the food product. In addition,
the food manufacturer will have a better understanding of the market and consumer
requirements of the final food product.
By choosing an appropriate encapsulant and microencapsulation process, controlled, sustained, or delayed release of omega-3 oil in the final product application
or for targeted release at specific sites in the gastrointestinal tract after consumption
may be achieved. With carefully fine-tuned controlled release properties, microencapsulation is no longer just an added-value technique, but the source of totally new
ingredients with desirable properties.124
The food industry expects increasingly complex properties from food ingredients
and such complex properties can often only be provided by microencapsulation.
With the use of appropriate microencapsulation technologies, the food industry has
the ability to develop functional omega-3 ingredients and products and improve the
nutritional content of food without affecting the taste, aroma, and texture of food,
and without reducing their bioavailability and functionality.65
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118. Balassa, L.L. and Fanger, G.O., Microencapsulation in the food industry, Crit. Rev.
Food Technol., 2, 245, 1971.
119. Labell, F., New, precise methods for encapsulation, Prepared Foods, 171, 1, 55, 2002.
120. IUPAC, Compendium of Chemical Terminology, 2nd ed., 1997, 1972, 31, 611.
121. Courts, A., Gelatin emulsions in the formation of microcapsules, Chem. Ind., 19, 943,
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122. Versic, R.J., Coacervation for flavour encapsulation, in Flavour Encapsulation, Risch,
S.J and Reineccius, G.A., Eds., ACS, Washington, 370, 1988.
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123. Schmitt, C. et al. Structure and technofunctional properties of protein-polysaccharide
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124. Gouin, S., Microencapsulation: industrial appraisal of existing technologies and
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126. Bakker, M.A.E., Galema, S.A. and Adrianus, V., Microcapsules of gelatin and carboxy
methyl cellulose, European Patent Application EP0937496-A2, 1999.
127. Pandya, Y. and Knorr, D., Diffusion characteristics and properties of chitosan coacervate capsules, Process Biochem., 26, 75, 1991.
128. Lamprecht, A., Schafer, U., and Lehr, C.M., Influences of process parameters on
preparation of microparticle used as a carrier system for -3 unsaturated fatty acid
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129. Soeda, T., Nakanishi, M., and Inoue, T., Edible microcapsules containing e.g., vitamin(s), fats and oils for addition to dried food such as soup, chewing gum and pouchpacked foods comprise edible hydrophobic core and capsule wall containing transglutaminase as cross-linking agent, U.S. Patent 6,592,916-B2, 2003.
130. Chaundy, F.K., Bower, D.K., and Killbride, T.K. Jr., Process for incorporating a
material in a crosslinked gelatin and product therefrom, U.S. Patent 5,153,177, 1992.
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133. Reichenbach, A.W., Kinetics of the oxidative stabilisation of unsaturated fatty acids
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by microencapsulation in cyclodextrins, J. Agric. Food Chem. 48, 3922, 2000.
135. Yoshii, H., et al., Oxidative stability of powdery tridocosahexanoin included in cyclodextrin and its application to fish meal paste, Biosci. Biotechnol. Biochem., 61, 1376,
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of oxidisable substances, U.S. Patent 5,015,483, 1991.
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13
Packaging Technologies
of Functional Foods
Louise Deschênes
Agriculture and Agri-Food Canada
CONTENTS
13.1 Introduction ..................................................................................................329
13.2 Packaging Practices versus the Requirements of Functional Foods...........330
13.2.1 Fruits and Vegetables .......................................................................330
13.2.1.1 Fresh Plant Products .........................................................330
13.2.1.2 Processed Plant Products..................................................331
13.2.2 Probiotics..........................................................................................331
13.2.2.1 Yogurts ..............................................................................331
13.2.2.2 Dried Cultures...................................................................332
13.2.3 Intermediate Moisture Products.......................................................332
13.2.4 Oils and Fats ....................................................................................332
13.3 Choice of Packaging Materials....................................................................333
13.4 Active Packaging..........................................................................................334
13.5 Summary ......................................................................................................335
References..............................................................................................................335
13.1 INTRODUCTION
The emerging functional foods market presents a considerable challenge to all
manufacturers to keep abreast of the use of adequate packaging materials for their
products. The interest in functional foods is intrinsically linked to the potential health
benefits of their bioactive components. Preserving these bioactive components is
therefore a crucial factor to providing the anticipated health benefits of functional
foods. However, recurring failure in maintaining the quality of functional foods has
been reported.1−3
Functional foods include a wide variety of products such as prebiotics and
probiotics, dried foods (fibers, probiotics, tea, herbs, etc.), fermented products
(yogurts, kefirs, vegetables), and fresh fruits and vegetables. Their bioactive
329
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components (minerals, vitamins, fibers, peptides, proteins, n-3 polyunsaturated fats,
antioxidants, enzymes, and symbiotics) range from very simple molecules to complex living organisms such as bacteria. The chemical nature of the bioactive components and the way functional foods are offered to consumers (in the form of dried
extracts, fresh foods, or processed foods) dramatically influence the shelf life of
these functional foods and their packaging requirements. Unfortunately, only a
limited number of documents addressing this critical issue are available. Furthermore, the data are fragmented and dispersed within the literature on studies from
other fields of research to which functional foods were incidental. A comprehensive
review of the current literature would be useful to summarize the existing knowledge
on what is required to preserve functional foods and to develop appropriate packaging guidelines.
13.2 PACKAGING PRACTICES VERSUS THE
REQUIREMENTS OF FUNCTIONAL FOODS
The effect of packaging on food composition during storage depends on the technology used and the storage conditions. Generally, functional foods packaging and
preservation practices are based on the conditions applied to products of similar
origin and format. This approach can give satisfactory results for several products
but should not be taken as a universal rule regarding all functional foods. In some
cases, the usual technologies and packaging materials could be detrimental to the
shelf life of certain functional ingredients. In other cases, minor adaptations of the
packaging technologies could likely improve the shelf life of functional foods greatly.
Some examples are given below.
13.2.1 FRUITS
AND
VEGETABLES
The effects of processing and preservation technologies on minerals and vitamins
have been extensively documented for decades.4,5 In addition to vitamins and minerals, several other important functional phytochemicals such as flavanoids, prostaglandins, and sulfur compounds are present in fresh and processed plant products.
Unfortunately, their stability under processing and storage conditions is not well
known.6
13.2.1.1 Fresh Plant Products
Minimal processing is often a way to preserve the bioactive components of functional
foods. Packaging, therefore, is a significant factor in extending the shelf life of
minimally processed food products. However, the effect of minimal processing and
of packaging technologies on the nutritional quality of functional foods is not well
established.7 Modified atmosphere packaging (MAP) is a process well known for
its capacity to enhance the shelf life of fresh fruits and vegetables.8 The improvement
in shelf life obtained using MAP is usually associated with the preservation of normal
appearance and firmness. The MAP process slows down or matches the metabolism
of packaged fresh products and microflora to avoid fermentation, thus minimizing
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microbial growth. In fresh fruits and vegetables, the level of antioxidants (vitamins,
phenolics, and carotenoids) depends on storage conditions. Gil et al. observed
constant levels of flavanoids over a seven-day period for spinach maintained at 10°C.9
However, the total antioxidant capacity of the product decreased over the same
storage period. The spinach subjected to MAP was more affected than the unpackaged spinach. These results indicate that spinach packaged in the conventional
manner — but with a shorter shelf life — could provide more functional benefits
than MAP spinach. However, the lack of data in the literature prevents the extension
of such a conclusion to the entire class of fresh fruits and vegetables.
13.2.1.2 Processed Plant Products
Plant products contain phenolic compounds, which are closely associated with the
nutritional quality of foods. Their properties are based on their antioxidant capability.10 As with other antioxidant compounds, these molecules easily oxidize in the
presence of oxygen. Phenolic compounds are present in cereals, legumes, oilseeds,
fruits, and beverages of plant origin (tea, cocoa, beer, wine, fruit juices, etc.).
Processed plant products with medium to high water activity will be the most
susceptible to browning and phenolic degradation. Conditioning and packaging these
products require controlling the oxygen level to which they are exposed.
13.2.2 PROBIOTICS
The viability of microorganisms may be affected by several factors, including packaging. The viability of both Lactobacillus and Bifidobacterium species may diminish
drastically during storage, resulting in low numbers of living cells at the time of
consumption of the food product.11 Oxygen is often reported to be a concern for the
survival of these cell cultures. Lactobacillus acidophilus is microaerophilic, and
surface growth on solid media is generally enhanced by anaerobiosis or reduced
oxygen with 5%–10% CO2. The other popular probiotics, Bifidobacteria, are usually
considered strictly anaerobic but some strains of Bifidobacterium lactis have demonstrated a relative tolerance to oxygen.11
13.2.2.1 Yogurts
Yogurts, whether they contain probiotics or not, are usually packaged in regular
high-impact polystyrene (HIPS) cups. This type of packaging is not ideal for probiotics because of their high oxygen transmission rate. Dave and Shah have demonstrated that for living cells contained in yogurts, oxygen concentration in the
product and container significantly influences the survival of probiotics.12,13 Lactobacillus and Bifidobacterium spp. are particularly sensitive to oxygen toxicity.14
Strains of these bacteria grown in defined oxygen concentrations (0%, 5%, 10%,
15%, and 21% oxygen) showed a drastic decrease in lactate production with
increased oxygen uptake.15 More recently, the Champagne research group study on
probiotics in nonfermented milks indicated that the oxygen level in these types of
products must also be controlled to ensure the survival of probiotics.16 Comparative
packaging tests showed that the use of glass hermetic containers can significantly
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increase the survival rate of probiotics in yogurts.12 However, given the cost of this
form of packaging, it is not a viable commercial alternative. The use of active
packaging, conversely, could be an option worthy of consideration. Polystyrenebased cups coated with a Nupak (gas barrier layer combined with an oxygen scavenger (Zero2™)) significantly improved the survival of Lactobacillus and Bifidobacterium spp. in yogurts.17,18
13.2.2.2 Dried Cultures
Dried probiotic cultures are also affected by storage conditions such as temperature,
moisture, light, and air.19 Alur and Grecz20 showed that problems with freeze-dried
bacteria could be attributed to surface phenomena. Other authors have also demonstrated that oxygen and moisture affect survival kinetics.21,22 Encapsulation could be
used as a primary packaging system able to contribute to the preservation of dried
probiotic cultures.23,24 As a general guideline, oxygen and water vapor should be
carefully controlled to ensure the optimal packaging conditions of dried cultures.
13.2.3 INTERMEDIATE MOISTURE PRODUCTS
Numerous food products can be classified as intermediate moisture foods (IMFs).
Typical examples of IMFs are cakes, syrups, toaster pastries, fruit rolls, and softbars.
The intermediate moisture levels in foods are generally related to a water content
of 10%–40% and a water activity of 0.60–0.9.25 Intermediate moisture nutraceutical
foods, such as semisoft food bars, belong to this category. Despite relative stability
at ambient temperatures, the rates of some deterioration processes are very high at
intermediate moisture levels.26 Browning reactions are particularly significant in
IMFs27 and lipid oxidation is also a problem, provoking rancidity and unacceptable
sensory defects.28 The rate of lipid oxidation increases as the water activity (aw)
decreases below the monolayer moisture content. IMFs would greatly benefit from
high barrier materials integrating oxygen scavengers.
13.2.4 OILS
AND
FATS
Extracted natural oils and fats contain molecules with unsaturated molecules and
natural antioxidants such as tocopherols. These molecules are easily oxidized when
exposed to oxygen and light. Consequently, light and gas barrier packaging materials
should be mandatory to preserve the functionality of these products.
Conjugated linoleic acid (CLA) is a natural fatty acid recognized for its anticancer properties.29,30 When CLA is present in its original matrix, it remains quite
well preserved within the usual shelf life of fresh products. The CLA can be found
in dairy products and meat. Lavillonnière et al. showed that the CLA level in dairy
products (e.g., yogurt, cheese, and ice cream) stored and packaged in usual conditions
does not vary significantly over a six-week period.31
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13.3 CHOICE OF PACKAGING MATERIALS
The degradation mechanisms of the bioactive components in packaged functional
foods are mainly related to ultraviolet (UV) sensitivity, water activity, microbial
load, and oxidation processes. Given these considerations, packaging technologies
should be selected based on the likelihood that they will contribute to controlling
light, humidity level, temperature, and atmosphere conditions. Table 13.1 summarizes some factors to be taken into consideration in the choice of appropriate packaging conditions for various types of functional foods. This list is not exhaustive
and specific tests are recommended to ensure the preservation of minimal levels of
bioactive components.
Common packaging technologies could be a simple solution to extending the
shelf life of functional foods. Such technologies include gas flushing, barrier, controlled atmosphere, modified atmosphere, and vacuum packaging. Further, new
advanced packaging technologies could provide additional tools to increase the shelf
life of bioactive functional food components.
TABLE 13.1
Functional Foods and Packaging Requirements
Deterioration
Factor
Oxygen
Products
Fermented milks
Oils and fats
Processed fruits
and vegetables
Moisture
Fresh fruits and
vegetables
Dried probiotics
Light
Oils and fats
Fermented milks
Protein-based
products
Microorganisms
Broccoli sprouts
Temperature
Dried herbs
© 2007 by Taylor & Francis Group, LLC
Deterioration
Effect
Packaging
Requirements
Vitamin oxidation
Decrease in survival
probiotic bacteria
Lipid oxidation
Rancidity
General nutritional
quality loss
Antioxidant
destruction
Vitamin destruction
Controlled oxygen barrier
Decrease in the active
load
Oxidation, rancidity
Oxidation of vitamins
Modification of
proteins and amino
acids
Flavor problems
Pathogen development
Moisture barrier
High barrier
High barrier
Controlled atmosphere
Light barrier
Hermeticity moisture
control
High barrier
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13.4 ACTIVE PACKAGING
Active packaging is a class of technologies in which dynamic physical and chemical
interactions between the content and the containers induce modifications of the
internal conditions resulting in shelf-life improvement. Active packaging contributes
to the preservation of functional foods by integrating sophisticated systems of oxygen
scavenging, enzyme and antioxidant release, moisture control, and antimicrobial
surface properties. Table 13.2 presents a list of various active packaging systems. A
list of commercially available active packaging systems for food applications were
published by Brody et al.32
Antioxidant release is one of the first active packaging systems to be implemented in the industry. Traditionally, hindered phenolic compounds are added to the
food contact layer and act as oxygen scavengers and chelators. The most popular
additives are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
and tertiary butyl hydroquinone (TBQH).33 The packaging material of cereals, nuts,
and oils often contains such additives to increase their storage stability.34,35 For
functional foods, complex chains of chemical interactions involving vitamins,
polyphenols, and enzymes are critical.36 The release of antioxidants naturally
involved in the biochemical processes would present a better preservation strategy
than the use of BHA and BHT. New active packaging systems are using natural
molecules as antioxidants. Deschênes et al.37 observed that the replacement of Irgafos
168 with vitamin E in the contact packaging layer increased the preservation of
vitamin C and polyphenols in sauerkraut and fermented vegetable juices by 50%.
There was a synergistic effect between the vitamin in the packaging and vitamins
C and E and polyphenols in the product, a phenomenon that was not observed when
the synthetic antioxidant was used. The advantages of using natural compounds in
packaging materials are particularly beneficial to the preservation of functional
foods.
TABLE 13.2
Active Packaging Systems
Parameter to Control
Moisture
Gas atmosphere
Mechanism of Action
Physical dehydrators
Oxygen scavengers
Ethylene absorbers
Odors absorbers
CO2 absorbers
CO2 generators
Microbial load
Bacteriocins, proteins, vegetable
extracts, metals, organic acids,
chelators, fungicides, antibiotics
© 2007 by Taylor & Francis Group, LLC
Packaging Active Constituent
Anhydrous salts, starch, clay
Ascorbates, ferrous compounds,
oxidizible polymers, enzymes
Zeolites, cristobalites, clays,
alumino-silicates, active carbon,
permanganates
Active carbon, zeolites, catalyzers
Calcium hydroxide
Ascorbic acid/sodium bicarbonate,
iron carbonate
Nisin, lactoferrin, silver, acetic
acid, imazalil, benomyl, EDTA
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With the use of active packaging, new materials can be tailored to ensure the
efficacy and safety of functional foods through the incorporation of agents to control
the internal environment.38 In addition, active packaging can enhance the stability,
bioavailability, and sensory quality of these products.39
13.5 SUMMARY
Guaranteeing significant levels of beneficial bioactive components in functional
foods is a great challenge to manufacturers. The shelf life of functional foods is
highly dependent on the storage and packaging conditions that are applied throughout the entire distribution chain. These conditions must be precisely adapted to the
specific needs of each and every functional food. Unfortunately, research on the
effect of packaging conditions on functional foods is lacking. To optimize the
preservation of these sensitive products, there is an urgent need for appropriate data.
Further, the establishment of specific labeling regulations and standards to functional
foods will, it is hoped, stimulate further research in this area. These studies should
be undertaken to supply data supporting the determination of shelf life and expiration
dates ensuring health benefit claims. Active packaging systems constitute a promising approach to improving the preservation of functional foods.
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of phenolic antioxidants, in Food Antioxidants, Hudson, B.J.F., Ed., Elsevier, New
York, 1990, pp. 85–94.
37. Deschênes, L., F. St-Germain, and R. Bélanger, Conservation de Légumes LactoFermentés, internal report, Agriculture and Agri-Food Canada, 1998.
38. Grower, J.L., K. Cooksey, and K.J. Getty, Development and characterization of an
antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes, J. Food Prot., 67, 475–479, 2004.
39. Lutter, C.K. and K.G. Dewey, Proposed nutrient composition for fortified complementary foods, J. Nutr. 133, 3011S–3020S, 2003.
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14
Biological Antioxidation
Mechanisms: Quenching
of Peroxynitrite
Aditya Kulkarni and Hideo Etoh
Shizuoka University, Japan
CONTENTS
14.1 Introduction ..................................................................................................341
14.2 Peroxynitrite Formation In Vivo ..................................................................342
14.3 Carotenoids...................................................................................................343
14.3.1 Carotenoids as Antioxidants ............................................................344
14.3.2 Reactions of Peroxynitrite with Some Important Carotenoids .......345
14.3.2.1 Reaction of Peroxynitrite with Zeaxanthin ......................345
14.3.2.2 Reaction of Peroxynitrite with Astaxanthin
(Astaxanthin Structure-Activity Relationship).................346
14.3.2.3 Reaction of Retinol and Retinyl Acetate with
Peroxynitrite......................................................................347
14.4 Conclusions ..................................................................................................349
14.5 Summary ......................................................................................................350
References..............................................................................................................350
14.1 INTRODUCTION
Peroxynitrite is a potent and versatile oxidant that can attack many types of biological
molecules and produce a rich variety of products. Because of its potential to injure
or destroy biological molecules or molecular systems, leading to the pathogenic
process of human diseases, peroxynitrite has received considerable attention worldwide. The anion of peroxynitrite (O = NOO–), which forms in vivo from nitrogen
monoxide and superoxide anion, is a powerful oxidant and known to induce breakage
of DNA strands, inactivation of enzymes, protein modification by nitration and
hydroxylation, and lipid peroxidation in LDL. In recent years, it was reported that
biological antioxidants have a tendency to readily react with peroxynitrite. However,
chemical processes and mechanism involved in these phenomena remain to be
341
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Functional Food Ingredients and Nutraceuticals
elucidated in many respects due to lack of relevant information on the nature of
peroxynitrite.
Our past research has reported some new modes of reactions exhibited by
peroxynitrite, which included quenching of peroxynitrite by lycopene in vitro, reactivity of retinol and astaxanthin with peroxynitrite, and its consecutive reaction
products and mechanisms involved. In this chapter, we have elaborated these results
further and have extended our study to include biochemical studies on peroxynitrite
and peroxynitrite quenching mechanisms by biological antioxidant systems.
14.2 PEROXYNITRITE FORMATION IN VIVO
A number of studies have indicated that peroxynitrite is formed in vivo from the
combination of nitric oxide and superoxide with a rate constant near the diffusion
controlled limit and that peroxynitrite is in equilibrium with peroxynitrous acid.
×10 M S
Oi2– + i NO 7
→ – O–O–N=O
9
–
–1 –1
O–O–N=O + H + ←
→ H–O–O–N=O
Superoxide and nitric oxide can react together to produce peroxynitrite and each
can modulate the effects of the other. For example, superoxide can diminish the
effects of nitric oxide by diverting it to form peroxynitrite. Similarly, .NO can capture
superoxide and divert its effects, even to the extent of acting as an antioxidant. A
number of systems are now known in which the presence of nitric oxide can alter,
or even protect against, the effects of superoxide and superoxide-derived reactive
oxygen species.
A very high concentration of nitric oxide is found in cigarette smoke. In fresh
smoke, .NO concentration can be as high as 500 ppm, and as smoke ages, the nitric
oxide is oxidized to nitrogen dioxide. Cigarette smoke can also initiate lipid peroxidation, producing lipid peroxyl radicals. In smoke systems, the lipid peroxyl radicals
can react with either nitric oxide or nitrogen dioxide, thus inhibiting lipid peroxidation. These reactions produce either peroxynitrite esters or peroxynitrate esters.
Other studies have also revealed that nitric oxide can scavenge superoxide in
vivo. In the DNA of macrophages, nitric oxide and superoxide induce oxidative
damage in addition to deamination. Beckman et al.1,2 showed independent existence
of peroxynitrite in vivo using immunohistochemical techniques. They revealed the
presence of nitrotyrosine residues in atherosclerotic human coronary arteries. This
was major evidence for peroxynitrite formation in arterial plaques, since tyrosine is
nitrated by peroxynitrite but not by nitric oxide itself. Figure 14.1 represents potential
sources and reactions of nitric oxide (Figure 1a) and superoxide (Figure 1b).
The peroxynitrite anion is relatively unreactive, and its conjugate acid known
as peroxynitrous acid (HO-O-N=O), which is an inorganic peroxyacid, is more
reactive. Peroxyacids, while having a peroxide O-O bond, do not usually dissociate
to form radicals. The reasons for this include the fact that the O–O bond in these
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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite
1
Arg
NOS
1
NADPH oxidase
O2
Cigarette
smoke
•NO
2
•NO
+e−
Nitrosation
(e.g., RS–NO)
8 O2
NO−
−OONO
11 H+
9 O2•−
−
14 Hb
OONO
O2•−
QH2
3 H2O
NO2−
O2
6 H+
4 −e−
5
+NO
7
343
10 H+
HOONO
12
13
S
NO2−
NO3−
+SO
2O2•−
2
O2
Xenobiotics
3 2H+
H2O2
4 Fe
HO•
O2
5
Oxidation (e.g., catechols)
&
Reduction (e.g., ferretin)
NO
6
−OONO
Met Hb
(a)
(b)
FIGURE 14.1 Representations of sources and reactions of nitric oxide and superoxide.
(Reprinted with permission from The American Psychological Society, (701, 1995.)
compounds is relatively strong for peroxides, and peroxyacids are polar with a
tendency for undergoing even-electron, nonradical reactions. One of the striking
features of peroxynitrite chemistry is that a scavenger has not yet been found that
can capture 100% of the reactive intermediate purported to be formed: HO. or
HOONO*. Because HOONO* would be less reactive than the hydroxyl radical, a
mechanism involving HOONO* as an intermediate can also explain the inability of
hydroxyl-radical scavengers to completely block peroxynitrite reactions.
14.3 CAROTENOIDS
The carotenoids are yellow, orange, or red pigments that are widely distributed in
the plant and animal kingdom. They are the major pigments of certain yellow, orange,
or red flowers, vegetables, berries, mushrooms, insects, bacteria, feathers, and egg
yolk. In plants, they are found in leaves together with chlorophyll, and in animals
they are dissolved in fats or combined with protein in the aqueous phase; ordinarily,
they are fat soluble.
Color and antioxidant activities of carotenoids are a consequence of their unique
structure, an extended system of conjugated double bonds. Carotenoids are tetraterpenes formed by tail-to-tail linkage of two C-20 units, and in many carotenoids the
end-groups are modified into five- or six-membered rings giving monocyclic or dicyclic
compounds. There are two main groups of carotenoids, the hydrocarbons (carotenes)
and the xanthophylls (a range of which are shown in Figure 14.2). The xanthophylls
have oxygenated functional groups most often as either hydroxy-, methoxy-, epoxy-,
carboxy-, or carbonyl groups. Most of the substituents are usually on the ring positions
1−6 and 1’−6’, although the 5,8 epoxides are the main exceptions.
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Functional Food Ingredients and Nutraceuticals
β-carotene (carrot, mango)
Lycopene (tomato)
α-carotene (orange)
OH
HO
Lutein (orange, egg yolk)
OH
HO
Zeaxanthin (mango, egg yolk)
O
OH
HO
O
Astaxanthin (salmon, lobster)
FIGURE 14.2 Structures of selected carotenoids.
14.3.1 CAROTENOIDS
AS
ANTIOXIDANTS
Carotenoids have the capacity to quench or inactivate excited states of molecules.
It was originally thought that carotenes and xanthophylls served as light filters for
chlorophyll or functioned as protectors of enzymes in cells. Some of the well-established functions of carotenoids are their pro-vitamin A and antioxidant activity, and
their abilities to quench singlet oxygen and inhibit the growth of certain tumors.
Research on the antioxidant activity of carotenoids was sparked by the description of their singlet-oxygen-(1O2)-quenching properties and their ability to trap
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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite
345
peroxyl radicals. Singlet oxygen quenching by carotenoids occurs via physical or
chemical quenching. The efficacy of physical quenching greatly exceeds that of
chemical quenching and involves the transfer of excitation energy from 1O2 to the
carotenoid, resulting in ground-state oxygen and excited triplet-state carotenoid. The
energy is dissipated through rotational and vibrational interactions between the
excited carotenoid and the surrounding solvent to yield the ground-state carotenoid
and the thermal energy. In the process of physical quenching, the carotenoid remains
intact and can undergo further cycles of singlet oxygen quenching, thus acting like
a catalyst. The quenching activity of carotenoids mainly depends on the number of
conjugated double bonds and is influenced to a lesser extent by carotenoid end
groups (cyclic or acyclic) or the nature of substituents in carotenoid containing cyclic
end groups.
14.3.2 REACTIONS OF PEROXYNITRITE
CAROTENOIDS
WITH
SOME IMPORTANT
14.3.2.1 Reaction of Peroxynitrite with Zeaxanthin
Two carotenoids have been identified in the macular area of the human retina: the
xanthophylls zeaxanthin and lutein. Humans accumulate both β-carotene and xanthophylls, oxygenated carotenoids, in plasma and tissues, but zeaxanthin and lutein
appear to be specifically concentrated in the macula. The biological function of
xanthophylls has not yet been clearly established; for example, compared to βcarotene, xanthophylls have little pro-vitamin A activity.3 The oxygenated carotenoids zeaxanthin and lutein may offer protection against or repair of oxidative
damage associated with the degenerative diseases of aging. Macular pigments like
zeaxanthin, could protect the highly unsaturated lipids of retina cell membranes
from oxidation by peroxynitrite. β-carotene, cantaxanthin, and zeaxanthin have been
reported to inhibit macrophage-mediated low-density-lipoprotein oxidation,4 in
which peroxynitrite presumably plays a role. Scheidegger et al.5 studied the kinetics
of the reaction of peroxynitrite with zeaxanthin in liposomes constructed from the
fully saturated lipid L-α-dimyristoyl-phosphatidylcholine (C14: O) and from the
unsaturated egg lecithin and its absorbance spectrum in liposomes strongly resembles
in shape and amplitude that of zeaxanthin dissolved in methanol. They found that
zeaxanthin reacts with peroxynitrous acid slowly in a second order reaction. The
formation of hydrogen bonds between peroxynitrous acid and the distal hydroxyl
groups of zeaxanthin may limit the rate of reaction.
The research revealed that the carotenoid, zeaxanthin, could be easily
incorporated into liposomal vesicles prepared from egg lecithin or L-α-dimyristoylphosphatidylcholine, dimyristoyl (C14: O). In electron micrographs, liposomes containing zeaxanthin have had a rougher appearance than liposomes prepared in the
absence of zeaxanthin. Gabrielska and Gruszecki6 have shown by proton NMR that
incorporation of zeaxanthin in the lipid bilayer is associated with rigidity of the
membrane which is not observed in liposomes prepared with β-carotene; they suggest that zeaxanthin molecules adopt a regular orientation in the membrane, with
the polar ends contacting the aqueous milieu and the hydrophobic chain in the
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Functional Food Ingredients and Nutraceuticals
membranes the hydrophobic core, and that β-carotene, by contrast, which has no
polar moiety, becomes randomly organized in the membrane.
In the reaction between zeaxanthin and peroxynitrite, the change in the membrane spectrum resembled spectral changes found in the trans- to cis-isomerization
of β-carotene.7 However, after longer reaction times, that is, 5 s, the cis-like absorbance is lost, and at very long reaction times (over days), spectral changes in
zeaxanthin resulting from reaction with peroxynitrite are similar to those found with
photo-oxidation reactions, although the peroxynitrite itself is isomerized to nitrate
within seconds of initiating the reaction. Kinetic fits of the spectral data indicate an
A to B to C type reaction, consistent with initial production of one or more cisisomers of zeaxanthin followed by further reaction, possibly including cleavage of
the carbon chain. The subsequent reactions cannot be attributed to further reaction
with peroxynitrite, but most likely result from the initial formation of a reactive
intermediate.
The overall reaction of zeaxanthin with peroxynitrous acid is second order. The
order of the reaction with respect to zeaxanthin could not be determined directly
from a linear plot of the apparent first order rate constant as a function of zeaxanthin
concentration since that dependence was negative. The negative dependence could
be attributable to hydrogen bonding interactions between peroxynitrous acid and the
distal hydroxyl groups of zeaxanthin. The pH profile indicates that the reaction with
zeaxanthin involves peroxynitrous acid and not the conjugate anion.
Solubilization of zeaxanthin in liposomes allowed the observation of the reaction
between the water insoluble carotenoid and water-soluble peroxynitrite, which is
one of the first demonstrations of a reaction between a carotenoid and peroxynitrite.
Liposomes represent a good model system for biological membranes, and are very
suitable for in vitro studies of membrane-associated biological antioxidants. It can
be estimated that zeaxanthin plays a major role in protection of macular tissue from
oxidative damage. Other studies have also shown that zeaxanthin is more active than
other carotenoids in preventing lipid oxidation, and that peroxynitrite plays a pivotal
role in mediating oxidative damage in vivo is becoming increasingly clear. It can be
guessed that the direct reaction of peroxynitrite with zeaxanthin and other carotenoids is less important in vivo than reactions of peroxynitrite with other targets in
the lipid bilayer. However, carotenoids may play a role in repair of other target
molecules damaged through reaction with peroxynitrite or other reactive oxygencontaining species.
14.3.2.2 Reaction of Peroxynitrite with Astaxanthin
(Astaxanthin Structure-Activity Relationship)
Many carotenoids such as zeaxanthin, canthaxanthin, and astaxanthin are known to
inhibit lipid peroxidation significantly. Of these, astaxanthin, which is found as a
common pigment in algae, fish, and birds, is reported to be more effective than βcarotene in preventing lipid peroxidation in solution and various biomembrane
systems, such as egg yolk phosphatidylcholine liposomes and rat liver microsomes.
However, the reason why the antiperoxidation effect of astaxanthin is more than that
of β-carotene is not well understood. Many attempts were made in order to solve
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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite
347
MDA (nmol/ml)
0.6
0.4
0.2
0
0
1
2
Carotenoids (mol%)
3
FIGURE 14.3 Effects of β-carotene (●) and astaxanthin () on the formation of lipid peroxides in terms of MDA in liposomes.
this question. In a recent study done by Goto et al.8 the effects of astaxanthin and
β-carotene on lipid peroxidation of liposomes induced by ADP/ Fe2+ under various
conditions were examined.
For this, a certain amount of carotenoid was added to an etherial solution of
EyPC (egg yolk phosphatidylcholine) and BhCL ( cardiolipin from bovine heart) in
a molar ratio of 9:1 (EyPC/BhCL liposomes), and then the liposomes were prepared
by reverse-phase evaporation method at pH 7.4. Lipid peroxidation was initiated
with 1mM ADP and 0.1 mM Fe2+. Progress of lipid peroxidation was determined
as the amount TBARS (thiobarbituric acid-reactive substances) measured in terms
of MDA concentration 45 min after addition of ADP/ Fe2+. At this time, peroxidation
without carotenoids had completely stopped. As shown in Figure 14.3, both
β-carotene and astaxanthin inhibited liposomal lipid peroxidation. The production
of lipid peroxides was inhibited progressively by these carotenoids with increases
in their amounts in the liposomal membrane up to 1 mol%, and their effects almost
leveled off at 1 mol%. At 3 mol%, astaxanthin was almost two times more effective
than β-carotene, being consistent with its relative radical trapping effects in organic
solvents and antiperoxidative effects in membrane systems, such as EyPC liposomes
and rat liver microsomes.
14.3.2.3 Reaction of Retinol and Retinyl Acetate with
Peroxynitrite
Retinol is a fat-soluble vitamin that plays an important role in the visual cycle and
is essential for the normal growth of bones, reproduction, embryonic development,
and in differentiation of epithelial tissues. Retinol is a circulating form of vitamin
A and is generated from either β-carotene or retinyl esters. Retinol is stored as retinyl
esters in the liver. Retinols are sensitive to oxidation.9−13 The extended polyene
structure of retinols is reactive toward peroxyl radical addition reactions, and is
thought to trap lipid-peroxyl radicals, since they inhibit lipid peroxydation. However,
the peroxynitrite reaction mechanism of retinol is not yet understood. Some studies
have been done in this direction to understand this mechanism. In this regard, retinyl
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Functional Food Ingredients and Nutraceuticals
OCH3
OCOCH3
OH
14-hydroxy-13-methoxyretinyl acetate
O
OCOCH3
O
5, 6, 11, 14-diepoxyretinyl acetate
OCOCH3
O
5, 6-epoxyretinyl acetate
O
OCOCH3
11, 14-epoxyretinyl acetate
FIGURE 14.4 Structures of retinyl acetate and reaction products.
acetate, an acetate derivative of retinol, has been subjected to reactions with an
alkylperoxyl radical.13
Retinyl acetate was reacted with an alkylperoxyl radical in two solvent systems,
methanol and benzene. The alkylperoxyl radical was generated by the thermal
decomposition of a free radical initiator, 2, 2’-azobis (2, 4-dimethylvaleronitrile)
(AMVN), at 37°C. The main product of the reaction was 14-hydroxy-13-methoxyretinyl acetate in addition to some minor products. The reaction with benzene gave
some products with low yield. They were 5,6-epoxyretinyl acetate, 11,14-epoxyretinyl acetate, and 5,6,11,14-diepoxyretinyl acetate (Figure 14.4). These results also
show that the lipid peroxidation initiated by AMVN is inhibited by retinyl acetate.
The inhibition of the peroxidation by conjugated polyene compounds has been
attributed to their lipid peroxyl radical-trapping ability. These compounds seem to
exert antioxidant activities by a mechanism in which the chain-propagating peroxyl
radical is trapped by addition to the conjugated polyene system rather than the
mechanism of hydrogen donation. On the other hand, conventional chain-breaking
antioxidants such as tocopherols trap peroxyl radicals by donating hydrogen atoms.
Samokyszyn et al.14 have demonstrated that peroxyl radicals preferentially reacted
with 13-cis-retinoic acid via addition and not by hydrogen-atom abstraction reactions. The lack of a distinct induction period during the inhibition of methyl linoleateperoxidation by retinyl acetate, in contrast to α-tocopherols, can be explained by
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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite
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the idea that the reaction rate of retinyl acetate with peroxyl radicals is similar to
the rate of chain propagation of methyl linoleate peroxidation. The reaction in
benzene gave only epoxy compounds, demonstrating that the peroxyl can attack
every double bond of the polyene structure.
The reaction of conjugated polyenes with an alkylperoxyl radical can be accomplished by several processes. The alkylperoxyl radical (AOO.) adds to the double
bond of polyenes in Reaction 14.1.
(1)
AOO. +
C
C
AOO
C.
C
Reaction 14.1
Addition can be followed by the unimolecular decomposition of the β-alkylperoxylalkyl radical, affording epoxide and an alkoxyl radical (AO. , Reaction 14.2), or
by the reaction with oxygen, giving polyperoxides (Reaction 14.3).
(2)
O
AOO
C
C.
AO. +
C
C
C
C
Reaction 14.2
(3)
AOO
C
C. + O2
AOO
OO.
Reaction 14.3
The results indicate that the alkylperoxyl radical reacts with retinyl acetate to
form epoxides by Reaction 14.1 and Reaction 14.2. Low yields of the epoxy compounds in benzene indicate these compounds are unstable and decompose into some
other products. The oxygenated products obtained in this study suggest that retinyl
acetate is capable of scavenging lipidperoxyl radicals by its conjugated polyene
system. Keeping in mind the reaction of peroxyl radicals with retinyl acetate, further
studies on reaction of retinol with peroxynitrite are under way in our laboratory.
14.4 CONCLUSIONS
The versatility of the reaction mode is suggestive of the involvement of several
different active species in the reaction of peroxynitrite. There are many yet unidentified products, the identification of which may provide additional new reaction
modes of reaction of peroxynitrite with various biological antioxidation systems.
These reactions would probably be involved in vivo and contribute to the degradation
of biological systems, eventually leading to the pathogenic process of diseases. Better
understanding about the behavior of peroxynitrite toward a wide variety of biological
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Functional Food Ingredients and Nutraceuticals
antioxidation systems should enable us to predict the role of peroxynitrite in vivo
and should provide valuable information on its physiological significance.
14.5 SUMMARY
Peroxynitrite, the reaction product of superoxide and nitric oxide, is a powerful
oxidant produced by macrophages and neutrophils. Peroxynitrite and its decomposition products are known to induce DNA strand scission, protein modification by
nitration and hydroxylation, and lipid peroxidation in LDL. Recently, it was reported
that biological antioxidants readily react with peroxynitrite. In this review we present
results of biochemical studies on peroxynitrite and peroxynitrite quenching mechanism by biological antioxidant systems. We hope to clarify peroxynitrite quenching
mechanisms by considering interactions with antioxidants, intracellular localization,
and biological concentrations.
REFERENCES
1. Beckman, J.S., Ye, Y.Z., Anderson, P., Chen, J., Accavitti, M.A., Tarpey, M.M., and
White, C.R., Extensive nitration of protein tyrosines in human atherosclerosis detected
by immunohistochemistry, Biol. Chem. Hoppe-Syler, 375, 81, 1994.
2. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J.C., Smith, C.D., and Beckman,
J.S., Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase,
Arch. Biochem. Biophys., 298, 431, 1992.
3. Weiser, H. and Kormann, A.W., Provitamin A activities and physiological functions
of carotenoids in animals: relevance to human health, in Carotenoids in Human
Health, New York Academy of Sciences, New York, 1993, p. 213.
4. Carpenter, K.L.H., van der Veen, C., Hird, R., Dennis, I.F., Ding, T., and Mitchinson,
M.J., The carotenoids β-carotene, canthaxanthin and zeaxanthin inhibit macrophage
mediated LDL oxidation, FEBS Lett., 401, 262, 1997.
5. Scheidegger, R., Pande, A.K., Bounds, P.L., and Koppenol, W.H., The reaction of
peroxynitrite with Zeaxanthin, Nitric Oxide, Biol. Chem., 2, 8, 1998.
6. Gabrielska, J. and Gruszecki, W.I., Zeaxanthin (dihydroxy-β-carotene) but not βcarotene rigidifies lipid membranes: a 1H-NMR study of carotenoid-egg phosphatidylcholine liposomes, Biochem. Biophys. Acta Bio-Membr., 1285, 167, 1996.
7. Frank, H.A., Physical and chemical properties of carotenoids, in Carotenoids in
Human Health, New York Academy of Sciences, New York, 1993, p. 1.
8. Goto, S., Kogure K., Abe, K., Kimata, Y., Kitahama, K., Yamashita, E., and Terada,
H., Efficient radical trapping at the surface and inside the phospholipid membrane is
responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin,
Biochim. Biophys. Acta, 1512, 251, 2001.
9. Jungalwala, F.B., and Cama, H.R., Preparation and properties of 5,6-monoepoxyvitamin A acetate, 5,6-monoepoxyvitamin A alcohol, 5,6-monoepoxyvitamin A aldehyde and their corresponding 5,8-monoepoxy (furanoid) compounds, Biochem. J.,
95, 17, 1965.
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Biological Antioxidation Mechanisms: Quenching of Peroxynitrite
351
10. Takashima, Y., Nakajima, T., Tanaka, S., Washitake, M., Anmo, T., and Matsumaru,
H., Stability of retinal analogs, IX, stability of vitamin A acetate in aqueous ethanolic
solutions and quantitative analysis of its decomposition products by high performance
liquid chromatography, Chem. Pharm. Bull., 27, 1553, 1979.
11. Ikeda, A., Iwahashi, H., Negoro, Y., and Kido, R., Detection of radical species in
mixtures of some retinoids with hematin by using the ESR spin-trapping technique,
J. Biochem., 101, 743, 1987.
12. Oyler, A.R., Motto, M.G., Naldi, R.E., Facchine, K.L., Hamburg, P.F., Burinsky, D.J.
Dunphy, R., and Cotter, M.L., Characterization of autoxidation products of retinoic
acid, Tetrahedron, 45, 7679, 1989.
13. Yamauchi, R., Miyake, N., Kato, K., and Ueno, Y., Peroxyl-radical reaction of retinyl
acetate in solution, Biosci. Biotech. Biochem., 56, 1529, 1992.
14. Samokyszyn, V.M. and Marnett, L.J., Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid, Free Radic. Bio. Med., 8, 491, 1990.
© 2007 by Taylor & Francis Group, LLC
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15
Stability of Lycopene
During Food Processing
John Shi1 , Yukio Kakuda2, Yueming Jiang3,
Gauri S. Mittal4
1
Agriculture and Agri-Food Canada
University of Guelph
3 Chinese Academy of Sciences
4 University of Guelph
2
CONTENTS
15.1 Introduction ..................................................................................................353
15.2 Physical and Chemical Properties of Lycopene ..........................................354
15.3 Lycopene Degradation during Processing ...................................................356
15.3.1 Effect of Temperature on Lycopene Degradation ...........................356
15.3.2 Effect of Light Irradiation on Lycopene Degradation.....................360
15.3.3 Effect of Oxygen on Lycopene Degradation...................................361
15.4 Lycopene Isomerization in Food Processing...............................................364
15.5 Summary ......................................................................................................366
References..............................................................................................................368
15.1 INTRODUCTION
Lycopene is a natural pigment that imparts red color to tomato, guava, rosehip,
watermelon, and pink grapefruit.1 Tomatoes (especially deep-red fresh tomato fruits)
and tomato products are considered to be the most important sources of lycopene
in the human diet.2,3 Recent interest in the consumption of lycopene-rich foods as
a means of reducing the risk of cancer has prompted researchers to investigate the
level of lycopene in foods frequently consumed by people. The ability of lycopene
to function as an antioxidant may contribute to a reduction in disease risk.4 Increasing
clinical evidence supports the role of lycopene as an important micronutrient, since
it appears to provide protection against prostate cancer, lung cancer, and a broad
range of epithelial cancers.5–7 With increasing interest and awareness of the health
benefits of lycopene, lycopene concentrates and lycopene-rich food products have
353
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been used in functional foods, and its stability during food processing and storage
has drawn more and more attention. In order to ensure lycopene stability, there is a
need to understand the influence of processing parameters and conditions on the
oxidation and isomerization of lycopene.
Lycopene belongs to the carotenoid family and exists in nature in the all-trans
form. Heat, light, oxygen, and different food matrices are factors that have a marked
effect on its isomerization and autoxidation. Lycopene may isomerize to mono- or
poly-cis forms and reisomerization can take place during storage. The oxidation of
lycopene splits the molecule, which causes a loss in color and the production of offflavors. It is generally accepted that the all-trans isomer has the highest bioactive
stability, and the cis-isomers have the lowest bioactive stability. Bioactive potency
is dependent on the extent of isomerization and oxidation. A true assessment of the
nutritional quality of tomato products depends not only on the total lycopene content
but also on the distribution of the lycopene isomers. The reports concerning the
effects of heat, oxygen, and light on the stability of lycopene are generally in
agreement in much of the literature; however, in some areas controversy still exists
such as the exact conditions required for the isomerization reaction.
15.2 PHYSICAL AND CHEMICAL PROPERTIES OF
LYCOPENE
Lycopene is the major carotenoid pigment found in tomatoes The chemical structure
of lycopene is shown in Figure 15.1. Lycopene is a polyene hydrocarbon characterized by a symmetrical and acyclic structure containing 13 double bonds of which
11 are conjugated double bonds arranged in a linear array and having a molecular
formula of C40H56.
Color and antioxidant activities of lycopene are a consequence of its unique
structure, an extended system of conjugated double bonds. In nature, lycopene exists
in the all-trans form, the most thermodynamically stable form. However, under
certain conditions, seven of these bonds can isomerize from the trans form to the
less stable mono- or poly-cis form. Lycopene is soluble in oils and apolar organic
solvents. In aqueous systems they tend to aggregate and precipitate as crystals; such
reactions are thought to hinder the bioavailability of lycopene in humans. In ripe
tomato fruits, lycopene takes the form of elongated, needle-like crystals, which are
responsible for the typical bright red color of ripe tomato fruits. Lycopene is more
soluble in chloroform, benzene, and other organic solvents than in water. Lycopene
is also very sensitive to light, heat, oxygen, acids, and some metallic ions such as
Cu 2+, Fe 3+ which catalyzes its oxidation.
FIGURE 15.1 Molecular structure of lycopene.
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Stability of Lycopene During Food Processing
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TABLE 15.1
Comparison of Antioxidant Activities of Carotenoids:
Singlet Oxygen Quenching
Lycopene
(a) Singlet oxygen quenching, Kq × 109 (m–1s–1)
(b) Radical scavenging (Trolox equivalents)
(c) Reaction of carotenoid radical anions with O2; k × 108 (m–1s–1)
31
2.9
2
Other carotenoids’ singlet oxygen quenching, 109 × Kq (m–1s–1)
γ-carotene
α-carotene
β-carotene
Lutein
Astaxanthin
Bixin
Canthaxanthin
Zeaxanthin
25
19
14
8
24
14
21
10
Source: Data from Di Mascio et al.8,11; Conn et al.9,12; Miller et al.13
Lycopene exhibits many unique and distinct biological properties. One of its
main activities is to function as an antioxidant. Through its conjugated double-bound
system, lycopene is able to quench efficiently the energy of deleterious forms of
oxygen (singlet oxygen) and to scavenge a large spectrum of free radicals. Lycopene
is among the most efficient singlet oxygen quenchers of the natural carotenoids.8,9
The quenching activity of the different carotenoids depends essentially on the number of conjugated double bonds. It is modulated by the end groups or the nature of
the substituents in the carotenoids that contain cyclic end groups.10 There are considerable differences in the quenching rate constants (Kq) for various carotenoid
species (Table 15.1). The antioxidant activities of lycopene and other carotenoids
are highlighted by their singlet oxygen quenching properties and their ability to trap
peroxyl radicals.14
Lycopene is known to exist in a variety of geometric forms, including all-trans,
mono-cis, and poly-cis forms. The all-trans isomer of lycopene is the predominant
geometrical isomer in fresh tomatoes, and is the most thermodynamically stable
form. However, lycopene can undergo trans-to-cis isomerization during tomato
processing and storage. In various tomato-based foods, the all-trans isomer is composed of 35% to 96% of total lycopene.15 In general, cis isomers are more polar
than their all-trans counterparts, and are less prone to crystallization due to their
kinked forms. The cis isomers are also more soluble in oil and hydrocarbon solvents
than the all-trans isomers. The bioactive potency of the cis isomers is not the same
as the all-trans isomers, because of the differences in their structural shapes.
Most stability studies of lycopene in food systems focused on its degradation.
Lycopene is a conjugated polyene and is susceptible to at least two reactions during
tomato processing, that is, isomerization and oxidation. Lycopene may be partially
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Functional Food Ingredients and Nutraceuticals
destroyed in food products by heat, light, oxygen, or the presence of metallic ions
(Cu2+, Fe3+, etc.). The isomerization of lycopene occurs when pure lycopene is treated
and when the lycopene in food products is subjected to processing conditions. On
the other hand, the conversion of the cis isomer to the trans isomer, which is the
more favorable reaction, occurs during the storage of the product. The cis isomers
are the less stable form, while the trans isomers are in the more stable ground state.
15.3 LYCOPENE DEGRADATION DURING
PROCESSING
More than 80% of the tomatoes produced are consumed in the form of processed
products such as tomato juice, paste, puree, ketchup, sauce, and salsa. The lycopene
content in concentrated tomato products is generally lower than expected, because
of losses during tomato processing.16 The main causes of lycopene degradation in
food processing are isomerization and oxidation. It is widely assumed that lycopene
in general undergoes isomerization during thermal processing. These changes in
lycopene content and in the distribution of trans-cis isomers result in a change in
biological activity. Physical and chemical factors known to degrade lycopene include
elevated temperature, exposure to light, oxygen, extremes in pH, and active surfaces.17
15.3.1 EFFECT
OF
TEMPERATURE
ON
LYCOPENE DEGRADATION
Lycopene content, mg/100 g
The native lycopene is located within the tomato cell matrix which gives it some
protection from degradation during heat processing. The effect of heating on total
lycopene and cis-isomer content in tomato puree are shown in Figure 15.2. Lycopene
retention in tomato juice is shown in Table 15.2. The length of cooking time has
less effect on the degradation of lycopene if the heating temperature is less than
8
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
Time (hr)
90°C
100°C
120°C
150°C
FIGURE 15.2 Effect of heat treatment on total lycopene degradation.18 (a) HPLC chromatogram of lycopene in oil solution after being treated at 25°C for 90 min. (b) HPLC chromatogram of lycopene in oil solution after being treated at 100°C for 90 min. (c) HPLC
chromatogram of lycopene in oil solution after being treated at 180°C for 90 min.
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Stability of Lycopene During Food Processing
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TABLE 15.2
Lycopene Loss Rate in Tomato Juice during Heating
Lycopene Loss (%)
Heating
Temperature (°C)
Heating time 1 min
Heating time 3 min
Heating time 7 min
90
100
110
115
118
121
124
127
130
0.6
0.9
2.2
2.7
3.7
4.6
5.5
6.5
7.4
0.9
1.4
3.2
4.5
6.0
7.3
8.5
9.9
11.5
1.1
1.7
4.4
7.0
9.1
10.6
12.5
14.6
17.1
Source: Data from Miki and Akatsu.19
100°C. However, temperatures greater than 120°C will result in more lycopene loss.
It has also been reported that serious losses of lycopene can occur when the holding
times at high temperature are long (Table 15.2).
The changes in trans- and cis-isomer concentrate during heat treatment are
shown in Figure 15.3. Increasing the temperature from 100°C to 180°C increased
the degradation of both the trans and cis isomers of lycopene. It was observed that
the cis isomers increased with thermal treatment at 100°C, but dropped significantly
with treatment at 180°C. An increase in temperature from 100°C to 180°C caused
a 76% decrease in total lycopene content (Figure 15.3). A 90-min treatment at 180°C
resulted in lycopene degradation and a greater loss in total lycopene, compared to
cis-isomer formation. Total lycopene concentration decreased at all treatment times,
but the cis isomers appeared only during the first hour of heating. After 1 h of
heating, the rate of cis-isomer accumulation decreased. It was observed that increasing the temperature from 100°C to 180°C or increasing thermal treatment time would
increase the degradation of trans isomer and cis isomer of lycopene (Figure 15.3).
It was suggested that degradation of lycopene was the main mechanism of lycopene
loss when heated above 100°C, and that lycopene in general underwent isomerization
with thermal processing. An increase in temperature from 90°C to 150°C caused a
35% decrease in total lycopene content. The high temperature and large amount of
air incorporated in the tomato juice during the breaking and straining operations can
quickly destroy substantial amounts of lycopene. The results suggest that length of
heating is a critical factor controlling the degradation of lycopene. It appears that
deaeration and “high temperature-short duration” heat treatment of tomato juice can
have beneficial effects on tomato juice quality.
Research by Agarwal et al.21 showed that subjecting tomato juice to cooking
temperatures in the presence of corn oil resulted in the formation of cis isomeric
forms. According to Ax et al.,22 total lycopene content in oil-in-water emulsions
decreased during thermal treatment with and without exposure to oxygen. Higher
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Functional Food Ingredients and Nutraceuticals
40
cis-isomers
mAU
30
all-trans
20
10
5-cis
0
0
5
10
15
20
Min
(a). HPLC chromatogram of lycopene in oil solution after being treated at 25°C for 90 min
40
mAU
30
20
cis-isomers
all-trans
10
5-cis
0
0
5
10
15
20
Min
(b). HPLC chromatogram of lycopene in oil solution after being treated at 100°C for 90 min
FIGURE 15.3 Lycopene oxidation and isomerization during thermal treatment.20
temperatures were directly correlated with increasing lycopene losses, and thermal
treatment led to a significant decrease in the concentrations of all-trans and 13-cis
isomer, while the concentration of the 9-cis isomer increased.
Zanoni et al.23 performed a study on the loss of lycopene during air drying.
Tomato halves were dried in a cabinet air dryer and the drying was carried out at
80°C and 110°C, with an airflow rate of 1.5 m/s. Lycopene losses of 12% occurred
at 110°C. According to Sharma and LeMaguer,24 heating tomato pulp at 100°C for
120 min decreased lycopene content from 185.5 to 141.5 mg/100g total solid, and
the freeze drying and oven drying (25°C to 75°C) of tomato pulp solids did not
cause any loss in lycopene content. Anguelova and Warthesen25 found that 70% of
all-trans lycopene were retained in tomato products at 6°C, but storage at 45°C
affected lycopene stability to a greater extent as only about 40% of all-trans lycopene
were retained under the same conditions of six weeks of light irradiation.
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Stability of Lycopene During Food Processing
359
all-trans
40
mAU
30
20
cis-isomers
10
5-cis
0
0
5
10
15
20
Min
( c). HPLC chromatogram of lycopene in oil solution after being treated at 180°C for 90 min
FIGURE 15.3 (Continued).
Lycopene in the pure form, without plant matrix protection, will undergo
degradation more quickly than in tomato tissue. Lee et al.26 studied the stability of
lycopene by heating lycopene (lycopene dissolved in hexane) under 50°C, 100°C,
and 150°C, respectively. For the 50°C treatment, there was no significant change in
the all-trans lycopene content within the first 12 h; however, the content began to
decline thereafter. The levels of the mono-cis forms of lycopene were found to
decrease with increasing heating time, which indicates that the degradation rate of
mono-cis lycopene may be greater than the formation rate. Unlike the mono-cis
forms of lycopene, the percentage change of two di-cis isomers showed increasing
trends, which were probably due to the conversion of mono-cis lycopene. There was
a significant decrease in total lycopene after heating at 150°C for 9 h. The result
revealed that isomerization was the main reaction during heating in the first 9 h,
after which the degradation reaction dominated. Similar results were observed for
the concentration change of all-trans lycopene and its cis isomers during heating at
100°C, the level of all-trans lycopene decreased by 78% after 120 min heating. The
mono-cis forms of lycopene showed a decreasing trend. The levels of two di-cis
isomers were found to rise in the first 60 min and then decreased, implying that dicis lycopene might be converted to the mono-cis lycopene or undergo degradation
after prolonged heating. The result indicated that the isomerization reaction was
favored at the beginning and then the degradation dominated after prolonged heating
at 150°C. When comparing the results, it appears that with increasing temperature
and heating time, degradation dominated over isomerization.
Shi et al.20 dissolved extracted lycopene into canola oil, and heated the sample
at 25°C, 100°C, and 180°C, respectively. It was observed that increasing the temperature from 100°C to 180°C or increasing thermal treatment time increased the
degradation of trans isomer and cis isomer of lycopene (Figure 15.4). Compared
with the treatment at 25°C, the cis isomers increased with thermal treatment at
1005°C, but dropped significantly with treatment at 180°C.
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Functional Food Ingredients and Nutraceuticals
Lycopene
CHO
O
2-methy-2-hepten –6-one
O
Pseudo-ionone
Geranial
O
6-methyl-3.5-heptadien-2-one
CHO
Neral
FIGURE 15.4 Molecular structural changes of lycopene during heating. (Reprinted with
permission from J. Agric. Food Chem., 38, 1238–1242, 1990. Copyright 1990 American
Chemical Society.)
27
It is also possible to form peroxyl radical capable of acting as a prooxidant and
propagate autoxidation. The proposed degradation pathway of lycopene is shown in
Figure 15.3. It is widely assumed that lycopene in general undergoes isomerization
with thermal processing. This isomerization results in the conversion of the all-trans
isomer into cis isomers. The cis isomers are present in processed food samples and
their concentration increased with increasing temperature and time of thermal treatment. A large loss of lycopene during processing would indicate a longer and more
drastic thermal procedure. The changes in lycopene content and the formation of
cis isomers may result in a reduction in bioactive potency.28,29,30
15.3.2 EFFECT OF LIGHT IRRADIATION ON LYCOPENE DEGRADATION
Increasing illumination increases the loss of lycopene. Lee et al.26 studied lycopene
stability by dissolving standard lycopene in hexane and illuminating (illumination
intensity ranged 2000 to 3000 lux) the samples for 6 days at 25°C. The content of
all-trans lycopene was found to decrease with increasing illumination time. After
144 h of exposure to light the losses amounted to 94% (Figure 15.5). All the monocis isomers of lycopene showed an inconsistent change. For instance, the level of
5-cis-lycopene was found to increase in the beginning and then decrease after the
illumination time reached 2 h. Likewise, a similar trend was observed for 9-cis-, 13cis-, and 15-cis lycopene. These results appear to indicate that isomerization and
degradation of lycopene and its cis isomers, during illumination, were proceeding
simultaneously. The increased level of mono-cis lycopene was probably due to the
initial conversion of all-trans lycopene. With continued illumination, a decrease in
the mono-cis lycopene could occur due to a further conversion to another cis form
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Stability of Lycopene During Food Processing
361
0.50
0.00
In (CA/CAD)
−0.50
y = −0.0176x + 0.1435
R2 = 0.9609
−1.00
−1.50
−2.00
−2.50
−3.00
0
20
40
60
80
Time (h)
100
120
140
160
FIGURE 15.5 First-order plot for the degradation of total amount of lycopene during illumination at 25°C for 144 h.26
through intermediate all-trans lycopene or undergoing degradation. The percentage
changes of all-trans lycopene and its cis isomers showed a different trend when
compared to the changes in the total amount of lycopene. The all-trans lycopene
may have been isomerized to form mono-cis or di-cis lycopene. These results may
account for the percentage increase of all the mono-cis and di-cis forms of lycopene
during illumination.
The effects of light irradiation on the content of total lycopene, trans isomers
and cis isomers in pure lycopene samples under different light irradiation intensities
of 2010 (outdoor), 900, 650, and 140 (indoor) µmol m–2 s–1 for 24 h are shown in
Figure 15.6. The results indicate that isomerization and degradation of lycopene and
its cis isomers proceed simultaneously during illumination. The loss in total lycopene, trans-I somers and cis isomers increased significantly as the intensity of the
light irradiation increased. The small amount of cis isomer formed during light
irradiation indicates that either less cis isomers were formed, or that the oxidation
reactions were predominating. It is possible that any cis isomer formed was quickly
degraded into oxidative by-products, indicating that the rate of cis-isomer oxidation
was much greater than formation of cis isomers by light irradiation. This would
suggest that cis-isomer oxidation is the main reaction pathway. The content of total
lycopene, trans isomers, and cis isomers in lycopene concentrates decreased under
all light irradiation conditions, which indicates that light irradiation induces lycopene
oxidation.
15.3.3 EFFECT
OF
OXYGEN
ON
LYCOPENE DEGRADATION
Henry et al.31 studied the effect of oxygen on the degradation of lycopene in an
aqueous model system. The standard lycopene was exposed to continuous flow of
water saturated with oxygen at 30°C, and the result showed that 90% of the lycopene
was lost after 2 h. Ax et al.22 dissolved lycopene in the oil phase of an oil-in-water
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17.5
15
mAU
12.5
10
7.5
cis-isomers
5
all-trans
2.5
5-cis
0
0
5
10
15
20
Min
(a). HPLC chromatogram of lycopene in oil solution after irradiation by light at 2010 μmol
m−2 s−1 intensity (outdoor) for 24 hrs
17.5
15
mAU
12.5
10
7.5
cis-isomers
5
all-trans
5-cis
2.5
0
0
5
10
15
20
Min
(b). HPLC chromatogram of lycopene in oil solution after irradiation by light at 900 μmol
m−2 s−1 intensity (outdoor) for 24 hrs
FIGURE 15.6 The changes of all-trans-and cis-isomers of lycopene of oxidation and isomerization during sun-light irradiation.20 (a) HPLC chromatogram of lycopene in oil solution
after irradiation by light at 2010 µmol m–2 s–1 intensity (outdoors) for 24 h. (b) HPLC
chromatogram of lycopene in oil solution after irradiation by light at 900 µmol m–2 s–1 intensity
(outdoors) for 24 h.
emulsion, and the emulsions were poured inside a glass flask with a sintered-glass
frit allowing continuous flushing with either synthetic air or nitrogen gas, thus
providing oxygen saturation or oxygen-free conditions. At 25°C, about 25% of the
lycopene was degraded within 30 h in the oxygen-removed emulsions, whereas 80%
was lost in the oxygen-saturated emulsion. In the presence of oxygen, lycopene
destabilization was about three times higher than in the absence of oxygen. A
nitrogen or argon headspace can be employed to keep the exposure to atmospheric
oxygen to a minimum.17 However, according to Ribeiro et al.,32 removing oxygen
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Stability of Lycopene During Food Processing
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17.5
15
mAU
12.5
10
all-trans
7.5
cis-isomers
5
5-cis
2.5
0
0
5
10
15
20
Min
(c ). HPLC chromatogram of lycopene in oil solution after irradiation by light at 650 μmol
m−2 s−1 intensity (indoor) for 24 hrs
17.5
all-trans
15
mAU
12.5
10
7.5
cis-isomers
5
5-cis
2.5
0
0
5
10
15
20
Min
(d). HPLC chromatogram of lycopene in oil solution after irradiation by light at 140 μmol
m−2 s−1 intensity (indoor) for 24 hrs
FIGURE 15.6 (c) HPLC chromatogram of lycopene in oil solution after irradiation by light
at 650 µmol m–2 s–1 intensity (indoors) for 24 h. (d) HPLC chromatogram of lycopene in oil
solution after irradiation by light at 140 µmol m–2 s–1 intensity (indoors) for 24 h.
in water by flushing the system with nitrogen achieved no improvement of stability.
Instead, lycopene degradation was accelerated. On the other hand, complete exclusion of oxygen using the enzyme glucose oxidase was shown to produce far better
stability.
Anguelova et al.33 subjected tomato powder to three treatments: light exposure
at room temperature, 6°C, and 45°C in the dark. Differences among the storage
treatments are not obvious from the data, but the amount of cis isomers as a
percentage of the total lycopene increased to the 14% to 18% range, regardless of
the storage conditions (Table 15.3). The treatments at 6°C did not have less 5,5′ dicis lycopene than the other two treatments; this was explained as the degradation of
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TABLE 15.3
Contents of Presumptive 5, 5’ di- cis Lycopene in Tomato
Powders (Percentage of Total Lycopene in Sample after Given
Storage Period under Fluorescent Light 38,500 lux) 33
Tomato Powder T1
Weeks
0
1
2
3
4
5
6
Light Exposure
4.3
5.6
5.8
7
8.6
8.1
14.2
6°C
45°C
4.3
4.8
5.4
5.5
9.4
11
18.4
4.3
6.5
5.3
5.4
9.1
12.8
14.6
Tomato Powder T2
Light Exposure
6.2
6.7
5.2
5.5
7.7
11.8
14.1
6°C
45°C
6.2
6.1
7.6
6.4
6.8
11.7
18.2
6.2
5.9
6.3
7
7.8
12.1
14.1
cis isomer due to autoxidation, since cis isomers were more susceptible to autoxidation than the all-trans isomer.34
15.4 LYCOPENE ISOMERIZATION IN FOOD
PROCESSING
Nguyen and Schwartz17 assessed the effect of several different heat treatments on
lycopene’s isomeric distribution in a variety of tomato products (Table 15.4). Schierle
et al.18 showed that heating tomato-based food in oil caused increased lycopene
isomerization than heating in water as shown in Table 15.5. Lycopene isomerization
TABLE 15.4
Lycopene Isomers in Various Thermally Processed
Tomato Products
Sample
Total Lycopene
(mg/100g, dry basis)
Cis Isomers
(%)
Peeled tomato
Tomato juice (hot-break)
Tomato juice (retorted)
Tomato (whole, retorted)
Tomato paste (concentrated)
Tomato paste (retorted)
Tomato soup (retorted)
Tomato sauce (retorted)
149.89
161.23
180.10
183.49
174.79
189.26
136.76
73.33
5.37
5.98
3.56
3.67
5.07
4.07
4.34
5.13
Source: Data from Nguyen and Schwartz.35
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Stability of Lycopene During Food Processing
365
TABLE 15.5
Effect of Heating Treatment on Lycopene Trans to Cis
Isomerizationin Aqueous and Oily Dispersions of Tomato
Paste (70°C)
Heating Time
(min)
All-trans
(%)
5-cis
(%)
9-cis
(%)
15-cis
(%)
Other cis
(%)
In Water
0
15
30
60
120
180
92.6
92.3
88.1
87.1
86.2
83.4
4.5
4.4
5.1
5.2
5.5
6.1
0.9
0.9
2.1
2.2
2.7
3.6
1.6
1.6
2.3
2.7
2.6
3.2
0.5
0.5
2.5
3.0
3.1
3.8
In Olive Oil
0
30
90
120
180
87.4
85.2
83.5
80.3
76.7
4.8
5.8
6.2
7.0
8.1
4.3
5.5
5.9
6.9
8.8
3.0
2.9
3.3
3.2
3.1
0.5
0.5
1.2
2.6
3.3
* Reprinted from Food Chem., 59(3), Schierle, J., Bretzel, W., Buhler, I., Faccin,
N., Hess, D., Steiner, K., and Schuep, W., “Content and isomeric ratio of
lycopene in food and human blood plasma,” 459–465, 1996, with permission
from Elsevier. Source: Data from Schierle et al.15
and the amount of cis isomers increased as a function of processing time during heating
of tomatoes. Heat treatment clearly increased the percentage of the cis isomers. It is
obvious from these results that food processing can enhance cis isomerization in
tomato-based foods. This indicates that not only the duration and temperature of heat
treatment, but also the food matrix components such as oil or fat, influence lycopene
isomerization. It was observed that lycopene loss and the rate of thermal isomerization
was less while heating tomato pulp than when heating pure lycopene in an organic
solution . The tomato tissue matrix may offer protection for lycopene.
It is generally accepted that the all-trans form of lycopene has the highest
stability and the cis isomers have the lowest stability. Bioactive potency depends on
the extent of isomerization and oxidation as well as the stability.4,28,29 Thus isomerization would lead to degradation of lycopene. Although the typical processes such
as cooking, freezing, or canning do not usually cause significant changes in total
lycopene content, it is widely assumed that lycopene undergoes isomerization upon
thermal processing. Heat, light, acids, and other factors have been reported to cause
isomerization of lycopene.15,20,35 The changes in lycopene content and the distribution
of trans-cis isomerization will result in a reduction in biological potency when
lycopene concentrates are subjected to processing.28,29,30
The key question now is how to maintain high bioactive properties during food
processing and storage. The isomerization and oxidation of lycopene greatly depends
on the treatments used since each treatment produces a different form of energy
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All-trans-isomers
Cis-isomers
Oxidized products
FIGURE 15.7 Pathway of lycopene degradation.
(such as heat and light, respectively). It was found that the light irradiation caused
more losses in total lycopene than the heating treatment. In all treatments, the rate
of trans-isomer loss was greater than the cis-isomer formation, which suggests that
degradation through oxidation of lycopene was the predominant mechanism, rather
than the isomerization of trans isomer to cis isomer. The losses could be coupled
to the conversion of trans- to cis-isomers, followed by the direct degradation into
smaller oxidized by-products which are not isomers of lycopene.36 Because lycopene
is a highly unsaturated molecule, comprising many conjugated double bonds, it is
very susceptible to oxidation. The pathway of lycopene degradation is described in
Figure 15.7.
Bioactive potency depends on the content of total lycopene and the extent of
isomerization.4,28,29 Thus isomerization can lead to a change in the bioactivity of
lycopene. Characterization and quantification of lycopene isomers would provide a
better understanding of the potential bioactive properties and health benefits of the
lycopene-based products. Controlling isomerization and oxidation of lycopene during the preparation of a lycopene-based food would be of benefit in improving
product quality.
A true assessment of the relationship between nutritional quality and health
benefits of dietary lycopene depends not only on the total lycopene content, but also
on the distribution of lycopene isomers. Characterization and quantification of lycopene isomers would provide better insight into the potential nutritional quality and
healthy benefit of the processed tomato products, and more accurately predict the
lycopene bioactivity than just a total lycopene content with no knowledge of its
isomeric composition. The control of lycopene oxidation and isomerization during
production and storage can be of benefit in improving the retention of biological
activity and health-promoting effect. Lycopene isomers in various thermally processed tomato products are listed in Table 15.6.
15.5 SUMMARY
Consumers, researchers and the leaders in the food industry have become dramatically more interested and aware of the health benefits of lycopene from tomatoes.
Lycopene in the human diet has significant protective and beneficial physiological
effects, because lycopene has many unique and distinct biological properties. Among
these is its strong antioxidant activity which plays an important role in improving
human health and providing protection against a broad range of epithelial cancers.
The antioxidant activities of lycopene and other carotenoids are highlighted by
their singlet oxygen quenching properties and their ability to trap peroxyl radicals.
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Stability of Lycopene During Food Processing
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TABLE 15.6
Lycopene Isomers in Various Commercial Tomato Products
Sample
Total Lycopene
(mg/100g
All-trans 5-cis 9-cis 13-cis Other cis
wet basis)
(%)
(%) (%) (%)
(%)
Tomato paste
(“Tomatenmark,” Panocchia, Italy)
Tomato paste
(“'Maracoli,” Kraft, Germany)
Tomato Ketchup
(“Hot Ketchup,” Del Monte, Italy)
Tomato Ketchup
(“Hot Ketchup,” Heinz, USA)
Instant Meal
(“Eier-Ravioli,” Hero, Switzerland)
Sauce
(“Hamburger Relish,” Heinz, The
Netherlands)
Sauce
(“Sauce Bolognaise,” Barilla, Italy)
Canned tomatoes
(“Chris,” Roger Sud, Italy)
52
96
4
<1
<1
<1
3.7
91
5
1
2
<1
9.5
88
7
2
3
1
3.0
77
11
5
7
1
0.6
76
8
5
6
5
3.0
93
5
<1
3
<1
9.2
67
14
6
5
8
7.1
84
5
3
5
3
* Reprinted from Food Chem., 59(3), Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner,
K., and Schuep, W., “Content and isomeric ratio of lycopene in food and human blood plasma,”
459–465, 1996, with permission from Elsevier. Source: Data from Schierle et al.15
Lycopene is known to exist in a variety of geometric forms, such as the all-trans,
mono-cis, and poly-cis isomers. The all-trans isomer of lycopene is the most predominant geometrical isomer in fresh tomatoes. Lycopene can undergo trans-to-cis
isomerization during tomato processing and storage. Lycopene can undergo at least
two changes during tomato processing, that is, isomerization and oxidation. Lycopene may be partially destroyed in processed tomato products by heat, light, oxygen,
and by the presence of metallic ions. The opposite reaction, the conversion of the
cis isomer to the trans- form can occur during the storage of the product. Cis isomers
are less stable than the all-trans isomer. Lycopene is a very efficient antioxidant that
quenches highly reactive singlet oxygen (O2.) and traps peroxyl radicals (ROO.). In
processed tomato products, oxidation is a complex process that depends upon many
factors, such as processing conditions, moisture, temperature, and the presence of
pro- or antioxidants.Certain steps can be taken to minimize lycopene degradation
during food processing and storage. Products containing lycopene need to be protected from excessive heat, extreme pH conditions, exposure to oxygen, and light.
Processing technology should be optimized to prevent lycopene oxidation. The
reduction of lycopene content and trans-cis isomerization result in a reduction in
biological activity. It is generally accepted that the all-trans form of lycopene has
the highest stability and the cis isomers have the lowest stability. Bioactive potency
depends on the extent of isomerization and oxidation. A true assessment of the
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Functional Food Ingredients and Nutraceuticals
nutritional quality and healthy benefit of processed tomato-based food depends not
only on the total lycopene content, but also on the distribution of lycopene isomers.
Lycopene can undergo degradation via isomerization and oxidation under different
processing conditions, which impacts on its bioactivity and reduces its health-promoting functionalities. How to maintain the high bioactive properties of lycopene
during food processing and storage is a key question. Controlling isomerization and
oxidation of lycopene during tomato processing or other lycopene-based food products can improve product quality. The mechanism of lycopene destruction depends
on many parameters during food processing and storage. The main cause of damage
to lycopene during food processing and storage is oxidation. Low storage temperature, low oxygen content, low light, low water activity, and low moisture content
will help to limit the oxidation of lycopene.
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Douglass, L.W., Gebhardt, Susan H.D., and Schakel, S., Carotenoid content of U.S.
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E and C, -carotene, and other carotenoids, Ann. N. Y. Acad. Sci., 669, 7, 1992.
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6. Olson, J., Carotenoid, vitamin A and cancer, J. Nutr., 116, 1127, 1986.
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Y., Lycopene is a more potent inhibitor of human cancer cell proliferation than either
β-carotene or -carotene, Nutr. Cancer, 24, 257, 1995.
8. Di Mascio, P., Kaiser, S., and Sies, H., Lycopene as the most efficient biological
carotenoid singlet oxygen quencher, Arch. Biochem. Biophys., 274, 532, 1989.
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interaction. J. Photochem. Photobiol. B. Biol., 11, 41, 1991.
10. Stahl, W. and Sies, H., Uptake of lycopene and its geometrical isomers is greater
from heat-processed than from unprocessed tomato juice in humans, J. Nutr., 122,
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11. Di Mascio, P., Murphy, M.C., and Sies, H., Antioxidant defense systems, the role of
carotenoids, tocopherols and thiols, Am. J. Clin. Nutr. Suppl., 53, 194, 1991.
12. Conn, P.F., Lambert, C., Land, E.J., Schalch, W., and Truscott, T.G., Free Radicals,
Res. Commun., 16, 401, 1992.
13. Miller, N.J., Sampson, J., Candeias, L.P., Bramley, P.M., and Rice-Evans, C.A.,
Antioxidant activities of carotenes and xanthophylls, FEBS Lett., 384, 240, 1996.
14. Burton, G.W. and Ingold, K.U., -carotene: an unusual type of lipid antioxidant,
Science, 224, 569, 1984.
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15. Schierle, J., Bretzel, W., Buhler, I., Faccin, N., Hess, D., Steiner, K., and Schuep, W.,
Content and isomeric ratio of lycopene in food and human blood plasma, Food Chem.,
59, 459, 1996.
16. Tavares, C.A. and Rodriguez-Amaya, D.B., Carotenoid composition of Brazilian
tomatoes and tomato products, Lebensm.-Wiss. U.–Technol., 27, 219, 1994.
17. Nguyen, M.L. and Schwartz, S.J., Lycopene: chemical and biological properties, Food
Technol., 53,38, 1999.
18. Shi, J., LeMaguer, M., Bryan, M., Kakuda,Y., Kinetics of lycopene degradation in
tomato puree by heat and light irradiation, J. Food Proc. Eng., 25, 485, 2003.
19. Miki, N. and Akatsu, K., Effect of heating sterilization on color of tomato juice, J.
Japan. Food Ind., 17, 175, 1970.
20. Shi, J., Wu, Y., Bryan, M., and LeMaguer, M., Oxidation and isomerization of
lycopene under thermal treatment and light irradiation in food processing, Nutraceut.
Food, 7, 179, 2002.
21. Agarwal, A., Shen, H., Agarwal, S., and Rao, A.V., Lycopene content of tomato
products: its stability, bioavailability and in vivo antioxidant properties, J. Med. Food,
4, 9, 2001.
22. Ax, K., Mayer-miebach, E., Link, B., Schuchmann, H., and Schubert, H., Stability
of lycopene in oil-in-water emulsions, Eng. Life Sci., 4, 199, 2003.
23. Zanoni, B., Peri C., Nani, R., and Lavelli, V., Oxidative heat damage of tomato halves
as affected by drying, Food Res. Int., 31, 395, 1999.
24. Sharma, S.K. and LeMaguer, M., Kinetics of lycopene degradation in tomato pulp
solids under different processing and storage conditions, Food Res. Int., 29, 309, 1996.
25. Anguelova, T. and Warthesen, J., Lycopene stability in tomato powders, J. Food Sci.,
65, 67, 2000.
26. Lee, M.T. and Chen, B.H., Stability of lycopene during heating and illumination in
a model system, Food Chem., 78, 425, 2002.
27. Kanasawud, P. and Crouzet, J.C., Mechanism of formation of volatile compounds by
thermal degradation of carotenoids in aqueous medium, 2, Lycopene degradation, J.
Agric. Food Chem., 38, 1238, 1990.
28. Emenhiser, C., Sander, L.C., and Schwartz, S.J., Capability of a polymeric C30
stationary phase to resolve cis-trans carotenoids in reversed phase liquid chromatograph, J. Chromatogr. A., 707, 205, 1995.
29. Wilberg, V.C. and Rodriguez-Amaya, B.D., HPLC quantitation of major carotenoids
of fresh and processed guava, mango and papaya, Lebensmittel-Wissenschaft und Technol., 28, 474, 1995.
30. Stahl, W. and Sies, H., Perspectives in biochemistry and biophysics, Arch. Biochem.
Biophys., 336, 1, 1996.
31. Henry, L.K., Catignani, G.L., and Schwartz, S.J., Oxidative degradation kinetics of
lycopene, lutein, 9-cis and all-trans beta-carotene. J. Am. Oil Chem. Soc., 75, 823,
1998.
32. Ribeiro, H.S., Ax, K., and Schubert, H., Stability of lycopene emulsions in food
systems, J. Food Sci., 68, 2730, 2003.
33. Anguelova, T. and Warthesen, J., Lycopene stability in tomato powders, J. Food Sci.,
65, 67, 2000.
34. Abushita, A.A., Daood, H.G., and Biacs, P.A., Change in carotenoids and antioxidant
vitamins in tomato as a function of varietal and technological factors, J. Agric. Food
Chem., 48, 2075, 2000.
35. Nguyen, M.L. and Schwartz, S.J., Lycopene stability during food processing, Exp.
Biol. Med., 218, 101, 1998.
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36. Boskovic, M.A., Fate of lycopene in dehydrated tomato products: carotenoid isomerization in food system, J. Food Sci., 44, 84, 1979.
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16
Fruits with High
Antioxidant Activity as
Functional Foods
Shiow Y. Wang
U.S. Department of Agriculture
CONTENTS
16.1 Introduction ..................................................................................................372
16.2 Antioxidants in Fruits ..................................................................................373
16.3 Effect of Preharvest Factors.........................................................................374
16.3.1 Genotypes and Maturity...................................................................374
16.3.2 Geographical Variation and Year-to-Year Differences.....................375
16.3.3 Climate, Temperature, and Light .....................................................376
16.3.4 Cultural Practices .............................................................................376
16.4 Effect of Postharvest Handling....................................................................378
16.4.1 Storage Duration and Temperature..................................................378
16.4.2 Controlled Atmospheres...................................................................380
16.4.3 Heat Treatment and Irradiation........................................................383
16.4.4 Treatment with Natural Compounds or Chemicals.........................385
16.5 Effect of Processing .....................................................................................386
16.5.1 Heat ..................................................................................................387
16.5.2 Freezing ............................................................................................389
16.5.3 Sugar, Ascorbic Acid, and Other Chemicals ...................................389
16.5.4 Drying and Dehydration ..................................................................391
16.5.5 Extraction Techniques ......................................................................392
16.5.6 High Pressure ...................................................................................393
16.5.7 Microwave and pH...........................................................................394
16.5.8 Oxygen and Light ............................................................................395
16.5.9 Enzymes ...........................................................................................395
16.6 Genetic Engineering Techniques .................................................................396
16.7 Conclusions ..................................................................................................397
References..............................................................................................................398
371
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16.1 INTRODUCTION
Foods that we eat supply our bodies with nutrients essential for good health. Foods
that are classified as functional foods contain components that impart health benefits
beyond basic nutrition. An example of a functional food is fruits, which are good
sources of antioxidants. In addition to the usual nutrients such as vitamins and
minerals, fruits are also rich in anthocyanins, flavonoids, and phenolic acids. Today’s
consumers are more educated and increasingly demand functional foods. Though
the mechanisms through which they work are not always clear, when eaten on a
regular basis as part of a varied diet, functional foods may lower the risk of developing certain diseases. It is estimated that more than 5000 phytochemicals have been
identified. However, a large percentage remains unknown and needs to be identified
before their health benefits are fully understood.1 Some of these beneficial chemicals
appear to work alone. Others may work in conjunction with vitamins and other
nutrients to stimulate protective enzymes or to block various hormonal actions and
metabolic pathways that are associated with the development of cancer and heart
disease.
Active oxygen species are generated as by-products of normal metabolism.
Exposure to free radicals from external sources such as cigarette smoke, pollutants,
chemicals, and environmental toxins may enhance the production of active oxygen
species. Increased levels of these active oxygen species or free radicals can create
oxidative stress, which leads to impairment of metabolism and DNA damage. The
different antioxidant components found in fruits provide protection against harmful
free radicals and have been associated with lower incidence and mortality rates of
various diseases, in addition to a number of other health benefits.2,3
Fruits are considered excellent functional foods because they contain high levels
of natural antioxidants such as carotenoids, vitamins, phenols, flavonoids, dietary
glutathionine, and endogenous metabolites. These antioxidants are capable of performing a number of functions including free radical scavengers, peroxide decomposers, singlet and triplet oxygen quenchers, enzyme inhibitors, and synergists.4
Antioxidants can also delay or prevent the oxidation of lipids or other molecules by
inhibiting the initiation or propagation of oxidizing chain reactions. Growing evidence suggests that eating more fruits and vegetables can aid in preventing stomach,
lung, mouth, esophagus, colon and rectal cancers. The incidence of other chronic
diseases, such as coronary heart disease, atherosclerosis, and stroke may also be
reduced through increased fruit and vegetable consumption. Fruits and vegetables
may also play a preventive role in birth defects, cataract formation, hypertension,
asthma, diverticulosis, obesity, and diabetes.5−9 Excellent reviews on the health
benefits of fruits and vegetables are contained in recent articles by Birt et al.,10
Heber,11 Le Marchand,12 Wise,13 and IARC handbooks of cancer prevention.14
Research is in full force to find the best genotypes, optimum cultural practices, and
most desirable postharvest handling and processing techniques to enhance and preserve
the natural antioxidants in fruits and vegetables for their use as functional foods. This
chapter summarizes the antioxidants in various fruits and describes the factors affecting
these antioxidant content and activities. The possible use of genetic engineering and
biotechnology techniques to enhance phytonutrients in fruits is also discussed.
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16.2 ANTIOXIDANTS IN FRUITS
Antioxidants are compounds that can delay or inhibit the oxidation of lipids, nucleic
acids, or other molecules by inhibiting the initiation or propagation of oxidizing
chain reactions.15 In general, there are two basic categories of antioxidants, natural
and synthetic. Recently, interest has been increasing considerably in finding naturally
occurring antioxidants for use in foods or medicinal materials to replace synthetic
antioxidants, which are being restricted due to their potential carcinogenicity.16 Fruits
are good sources of natural antioxidants and contain thousands of phytonutrients.7,9,17
The phytonutrients in fruits responsible for antioxidant activity can largely be attributed to phenolic compounds such as anthocyanins, phenolics, and to other flavonoid
compounds. These compounds may act independently or in combination as anticancer or cardio-protective agents by a variety of mechanisms. The antioxidant activities
of phenolic compounds are mainly due to their redox properties which can play an
important role in absorbing and neutralizing free radicals, quenching singlet and
triplet oxygen, or decomposing peroxide.4
Fruits showed a remarkably high scavenging activity of chemically generated
active oxygen species. The range of hydrophilic antioxidant capacities (H-ORACFL)
was large among the different fruits, such as prunes, raisins, blackberries, black
currants, blueberries, raspberries, strawberries, grapes, pomegranates, and some
varieties of apples.18−22 H-ORACFL for all of the melons was relatively low. Among
the small fruits, black raspberries and blackberries have higher antioxidant activities
than red raspberries, while strawberries generally have lower values of total antioxidants.21 Häkkinen et al.23 also found that extracts of crowberry, cloudberry, whortleberry, cowberry, aronia, rowanberry, and cranberry had high antioxidant activities,
while red currant, black currant, and strawberry had relatively low activities. These
results correlate with the findings that black raspberries and blackberries contain
high amounts of cyanidin glycosides, a strong antioxidant, while strawberries contain
pelargonidin 3-glucoside and ascorbic acid, which are weak antioxidants.24−26 Among
the anthocyanins, the relative antioxidant strength in preventing oxidation of human
low-density lipoprotein is as follows: delphinidin > cyanidin > malvidin > pelargonidin.27 Among the dried fruits, such as figs, raisins, and dates, the H-ORACFL
value was about 30 µmol TE/g, whereas those of prunes were nearly three times
higher.19,20 Major antioxidant compounds in prunes are caffeoylquinic acid isomers,
which have been shown to have a high antioxidant capacity.28,29
There is also a large range for lipophilic antioxidant capacities (L-ORACFL) for
different fruits. Compared to H-ORACFL in fruits, L-ORACFL values were generally
low.19,20 L-ORACFL values for the dried fruits were usually ranged from 0.30 to 1.8
µmol of TE/g. L-ORACFL values for fresh fruits could be as low as 0.07 µmol of
TE/g fresh weight for tangerines and as high as 5.52 µmol of TE/g fresh weight in
avocados. Avocado contains linoleic acid and vitamin E, but which lipophilic constituents contribute to the high lipophilic antioxidant capacity is largely unknown.19,20
Among all berry fruits, cranberries, raspberries, and blackberries have the highest
L-ORACFL values with >1µmol of TE/g fresh weight.19,20
The peroxyl radical is the most common free radical in human biology, but
radicals of superoxide radicals (O2• –), hydrogen peroxide (H2O2), hydroxyl radicals
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(•OH), and singlet oxygen (1O2) are all present in biological systems. However,
relatively little information is available on the scavenging of these radicals. Fruits
such as blackberries, blueberries, cranberries, raspberries, and strawberries not only
possess peroxyl radical (ROO•) absorbance capacity, but also have antioxidant activities against superoxide radicals (O2• –), hydrogen peroxide (H2O2), hydroxyl radicals
(•OH), and singlet oxygen (1O2).30 Different species or different cultivars showed
varying degrees of scavenging capacity on different active oxygen species (O2•-,
H2O2, •OH, and 1O2). Among all the species and cultivars, blackberries had the
highest inhibition of active oxygen species (O2• –, H2O2, •OH, and 1O2) production.30
Antioxidant enzyme activities are usually positively correlated to antioxidant
activities. The production of antioxidant enzymes in fruits is a complex process that
is not yet totally understood. The antioxidant enzyme defense system consists of
hundreds of different substances and mechanisms. The main antioxidant enzymes
in fruits are catalase, glutathione-peroxidase (GSH-POD), superoxide dismutase
(SOD), guaiacol peroxidase (G-POD), ascorbate peroxidase (AsA-POD), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR).31 Antioxidant enzymes have the capacity to lower the free
radical burden and neutralize excess free radicals created by stress conditions.
Antioxidant enzymes serve as catalysts that can act at one or more of the three stages
of free radical formation: initiation, propagation, and termination. Therefore, it is
possible that antioxidant enzymes can prevent cellular and tissue damage in the
human body.
16.3 EFFECT OF PREHARVEST FACTORS
16.3.1 GENOTYPES
AND
MATURITY
Genetic factors play an important role in determining antioxidant capacity in crops.
For many crops, a large variety of genotypes exist and thus there is potential for
genetic variability relating to antioxidant activity. The influence of variety on the
content of phenolic compounds and antioxidant activity in the berries of the genus
Vaccinium, Ribes, Rubus, and Aronia have been studied by several researchers.15,18,21,32−47 In blackberry, ‘Hull Thornless’ cultivar yields higher antioxidant values (ORAC) compared to ‘Chester Thornless,’ and ‘Triple Crown’ cultivars. For
raspberry fruit, ‘Jewel’ (a black raspberry) has higher values compared to red
raspberry cultivars (‘Autumn Bliss,’ ‘Canby,’ ‘Sentry,’ and ‘Summit’). ORAC, anthocyanin, and total phenolic contents are also different among the different cultivars
of strawberry fruits. ‘Earliglow’ has the highest ORAC value. There is a linear
relationship between both ORAC and total phenolics and between ORAC and anthocyanins of ripe berry fruits.18,21 Various species and cultivars of blueberry and bilberry
also have different antioxidant capacities.18
The effect of genotype variation on antioxidant activities has also received
much attention. in grapes,48−50 prunes, and plums,51−55 sea buckthorn,56,57 apples,58–61
sweet cherries,62 citrus,63 guavas,64 and nectarines and peaches.54 The cultivated
varieties of apricot possess significantly higher vitamin C, vitamin E, β-carotene,
and selenium contents than the wild type.65 The antioxidant potential is significantly
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higher in the grapefruit hybrids than in white grapefruit.66 Different cultivars of
clingstone peaches also have different antioxidant activities.67
Antioxidant capacity also varies considerably with different stages of maturity.
Many phytonutrients are synthesized in parallel with the overall development and
maturation of fruits. Fully ripened fruits offer not only the ultimate in taste and
flavor but also maximum color intensity and high antioxidant activities. Blackberry,
raspberry, and strawberry fruits harvested during their ripe stage consistently yield
higher antioxidant values than those harvested during the pink stage.21 Unripe strawberries contain lower concentrations of chlorogenic acid, p-coumaric acid, quercetin,
and kaempferol compared with ripe strawberries.68 Immature blueberries harvested
at an early stage (immediately after turning blue) have lower ORAC values and total
anthocyanin content when compared to more mature blueberries harvested 49 days
later.18 Kalt et al.69 found anthocyanin content was substantially higher in highbush
blueberries of more advanced stages of ripeness than in berries of less ripeness. In
grapes, the content of all polyphenolics generally increase as fruit ripen, the highest
concentrations being found in the skins.48 Free ellagic acid, ellagic acid glycosides,
and total ellagic acid increase during maturation in the skin of grapes.48 Antioxidant
activities also increase with ripening in Rubus L. hybrids.43 Ripening also results
in an increase of total antioxidant activity and vitamin C content in both peel and
flesh of ‘yuzu’ citrus.70 However, while the amounts of all total phenolics, hesperidin,
and naringin increased with ripening in peel, they decreased slightly in flesh.70 Both
ascorbic acid and total carotenoids increase with maturation and ripening in ‘Clingstone’ peaches and ‘Solo’ papayas.71,72
16.3.2 GEOGRAPHICAL VARIATION
DIFFERENCES
AND
YEAR-TO-YEAR
The effect of geographical variation and year-to-year difference on the antioxidant
capacity of various fruits is well described and is likely due to environmental
conditions. Anthocyanin production can be stimulated by cold growing conditions.73
Abiotic conditions (temperature, moisture, irradiation, soil fertility) can vary markedly from year to year and affect the content of phenolic components.74 Kalt and
McDonald75 found that seasonal variation in anthocyanin content among lowbush
blueberry cultivars over seven seasons was quite marked in fruit harvested from the
same site. Anthocyanin content varied by up to 2.4-fold for ‘Blomidon,’ 1.8-fold for
‘Cumberland,’ and 2.0-fold for ‘Fundy.’75 Connor et al.76 found that antioxidant
content varied significantly in blueberry fruit harvested in Minnesota, Michigan, and
Oregon among the cultivars across location and years. ‘Bluegold’ and ‘Northland’
had the highest overall antioxidant activity in Minnesota and Oregon. The antioxidant
activities of blueberries were substantially lower in Michigan than in Minnesota and
Oregon. Prior et al.18 found no substantial difference in antioxidant activities in
blueberry ‘Jersey’ harvested at sites in Oregon, Michigan, and New Jersey in a single
year, but Connor et al.76 showed differences between these locations and also showed
variability between years. This may reflect differences in climate and cultural practices among locations, including differences in ultraviolet radiation, temperature, or
water stress, or mineral nutrient availability.76
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Different origins and harvesting dates affect vitamin C, tocopherols, tocotrienols,
and phytosterols content in sea buckthorn.56,57 Apricot fruit from different geographical regions had different vitamin C content. Cultivars of apricot grown in highaltitude regions had significantly higher vitamin C content than the same cultivars
grown in other regions.65 Goncalves et al.77 found that flavonol and flavan-3-ol
contents in cherry fruit remained quite constant, but the level of phenolic acids were
higher in 2001 and anthocyanin levels were higher in 2002. Ascorbic acid content
in strawberries is also highly affected by climate conditions and growing area.78
Olsson et al.68 showed some variation in antioxidant activity, ascorbic acid content,
and ellagic acid content between 1999 and 2000 within each strawberry cultivar.
The total folate content in 13 strawberry cultivars harvested in 1999 and 2001 were
higher than in the harvest from 2000. These data suggest a significant influence of
climatic conditions on these compounds and antioxidant activity. The effect of
geographical variation and year-to-year difference on ORAC values will be described
more in the following sections.
16.3.3 CLIMATE, TEMPERATURE,
AND
LIGHT
Growing temperature and light intensity have been shown to influence the nutrient
content of fruits, especially carotenoids, thiamine, ascorbic acid, and riboflavin.
Increased light intensity resulted in higher ascorbic acid content in strawberry fruit
than of the same varieties produced under lower light intensity conditions.71 Strawberries grown under high temperature conditions showed significantly higher content
of flavonoids and antioxidant capacities. Plants grown in cool day and night temperatures generally had the lowest antioxidant capacity in fruit.79 Month-to-month
variability in vitamin C content dependent on growing conditions has been documented in processed Florida citrus products such as orange and grapefruit juices.80
One explanation for this difference could be related to different flavonoid concentrations.79 The composition of flavonols in red raspberry juice was also influenced
by cultivar, processing, and environmental factors.81
16.3.4 CULTURAL PRACTICES
Soil types, composts, mulching, and fertilization influence the water and nutrient
supply to the plant and can affect the nutritional composition and antioxidant activity
of the harvested fruit. Increasing the nitrogen and/or phosphorus supply to citrus
trees results in somewhat lower acidity and ascorbic acid content in citrus fruits,
while increasing potassium fertilization increases their acidity and ascorbic acid
content.82 Strawberry plants grown with different soil nutrients also show differences
in ascorbic acid content. Plants grown in low-organic-matter and low-cationexchange-capacity sandy soil amended with calcium, magnesium, and nitrogen
produce more ascorbic acid in their fruit than plants without supplemental fertilizer.83
In green-flesh honeydew muskmelons, the total ascorbic acid and folic acid are
higher when grown on clay loam vs. sandy loam soil.84 Sandy loam soil produces
less β-carotene as compared with sandy loam soils in orange-fleshed muskmelon
fruit.85
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Cultural practices, such as conventional, organic cultivations or use of compost
as a soil supplement can all affect antioxidant capacity in fruits. Compost as a soil
supplement increases organic matter in the soil, which also enhances antioxidant
content in strawberries. Compost significantly enhances content of ascorbic acid
(AsA), glutathione (GSH), and the ratios of AsA/dehydroascorbic acid (DHAsA)
and the GSH/oxidized glutathione (GSSG) in fruit of strawberry.86 The oxygen
absorbance capacity for peroxyl radical, as well as the superoxide radical, hydrogen
peroxide, the hydroxyl radical, and singlet oxygen in strawberries increases significantly with increasing compost use.86 Plants grown with compost yield fruits with
high levels of phenolics, flavonol, and anthocyanin content.86 It is possible that
compost causes changes in soil chemical and physical characteristics, increases
beneficial microorganisms, and increases nutrient availability and uptake, thus favoring plant and fruit growth. Woese et al.87 reported that the optimal conditions for
plant growth generally result in the highest levels of antioxidants.
Using different mulches for growing strawberries also affects strawberry fruit
quality. Different mulches probably lead to differences in canopy temperature, soil
temperature, and moisture content, and the quantity and quality of light transmitted,
reflected, or absorbed. These differences in turn affect plant growth, development,
fruit quality, and carbohydrate metabolism in strawberry plants. Fruit from a hill
plasticulture (HC) production system have higher flavonoid content and antioxidant
capacities compared to fruit from plants grown in a matted row (MR) system.88 In
general, phenolic acid and flavonol content, as well as cyanidin- and pelargonidinbased anthocyanins and total flavonoids are greatest in the HC system. Fruits from
plants grown in the MR system generally have the lowest content of phenolic acids,
flavonols, and anthocyanins. Fruit grown under HC conditions have the highest
peroxyl radical absorbance capacity.88
Although food products from organic farming are believed to be healthier than
the corresponding conventional foods, clear experimental evidence supporting this
assumption is still lacking and assessment of the nutritional potential of these products
requires further research.87 Carbonaro et al.89 found a parallel increase in polyphenol
content and polyphenoloxidase activity of organic peaches and pears compared with
the corresponding conventional samples. Ascorbic and citric acids were higher in
organic than conventional peaches and α-tocopherol also increased in organic pears.
Lombardi-Boccia et al.90 grew plums in conventional (tilled soil) and organic cultivations (soil covered with trifolium, and soil covered with natural meadow) and found
that differences in macronutrients were marginal, whereas antioxidant vitamins and
phenolic compounds concentration markedly differed among cultivations. Ascorbic
acid, α-, γ-tocopherols, and β-carotene were higher in organic plums grown on soil
covered with natural meadow. The highest phenolic acid content was detected in plums
grown on soil covered with trifolium. Total polyphenols and quercetin were higher in
conventional plums, but myrecitin and kaempferol were higher in organic plums.
Asami et al.91 showed that higher levels of total phenolics were consistently found in
organically and sustainably grown cultivations of marionberry and strawberry as compared to those produced by conventional agricultural practices. These data provide
evidence that an improvement in the antioxidant defense system of the plant occurred
as a consequence of the organic cultivation practice. Therefore, the type of soil
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management turned out to be of primary importance in influencing the concentration
in these health-promoting compounds.
Carbon dioxide concentrations may also have an effect on antioxidant capacity.
Higher CO2 (300 and 600 µmol mol–1 above ambient) concentrations in the field
result in increases in anthocyanins, phenolics, AsA, GSH, and ratios of AsA to
DHAsA and GSH to GSSG in strawberry fruit.92 These increases are associated with
increased antioxidant capacity and increased removal of free radicals. Growing
strawberry plants under CO2 enrichment conditions significantly enhances fruit
flavonoid content as well.92 Fruit of strawberry plants grown in CO2 enrichment
conditions also have higher oxygen radical absorbance activity against ROO•, O2• –,
H2O2, •OH, and 1O2 radicals.92 This indicates that strawberry fruit grown with CO2
enrichment have high scavenging activity for chemically generated active oxygen
species.
Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MJ) are a class of
oxylipins derived from the lipoxygenase-dependent oxidation of fatty acid. Both
compounds have been found to occur naturally in a wide range of higher plants.
JA/MJ play key roles in plant growth and affect a great diversity of physiological
and biochemical processes.93 Preharvest spray of MJ significantly enhances anthocyanin, total phenolic, flavonoid content, and antioxidant capacity in raspberries.94
MJ results in stimulation of anthocyanin biosynthesis in strawberry ripening.95 MJ
vapor also promotes β-carotene synthesis and chlorophyll degradation in Golden
Delicious apple peel.96
16.4 EFFECT OF POSTHARVEST HANDLING
16.4.1 STORAGE DURATION
AND
TEMPERATURE
Concentrations of nutrients in fruits tend to decrease during postharvest handling
and storage. Losses of vitamins and other nutrients in fruits vary by nutrient, type
of fruit, physical damage, temperature, and storage environment. Fresh fruits are
perishable because they are composed of living tissues. These tissues must be kept
alive and healthy throughout marketing. Respiration is one of the essential processes
of living cells. High respiration rates reflect fast consumption of energy and rapid
depletion of reserves such as carbohydrates, organic acids, amino acids, proteins,
fatty acids, and lipids. Refrigeration is the best method of slowing down respiration
and retarding the deterioration. Strawberries respire about 10 times as fast at 20°C
as at 0°C.71 High temperature also increases moisture loss and promotes growth of
decay-causing microorganisms. Therefore, rapid removal of field heat and prompt
cooling of the harvested fruits by applying precooling procedures immediately
following harvest are essential for quality maintenance. Postharvest handling can
also affect phytonutrient levels in fruits. Most fruits reach their maximum vitamin
and nutrient content when fully mature. However, many of them are usually harvested at a slightly immature stage with firmer texture in order to facilitate handling
and transportation and to minimize mechanical damage of the commodities. Therefore, those fruits could continue to mature and increase in antioxidant and nutrient
values in storage.
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In cranberries, postharvest storage temperatures between 0°C and 15°C increase
antioxidant capacity, anthocyanins, and total phenolic content.97 Strawberries stored
at 4°C retain vitamin C well but less chlorgenic acid and quercetin.68,98,99 Kalt et al.99
found that anthocyanin concentration and surface color increases in strawberries during
storage. Temperature, and to a lesser extent light, affects the rate of strawberry color
development. At 20°C anthocyanins accumulate rapidly and at 30°C the pigments form
more slowly than at 20°C and there is a significant increase in the surface color rating
of white-harvest berries after storage in the light.99 Gil et al.26 reported that the content
of ellagic acid in strawberries increase slightly during storage for five days in 5°C.
Strawberries and raspberries stored at temperatures greater than 0°C also result in an
increase in antioxidant capacity, anthocyanin, and total phenolic content, and the
magnitude of this increase is related to storage temperature. There are no ascorbate
losses in strawberries or highbush blueberries during eight days of storage at the various
temperatures.100 Increases in anthocyanin content during storage have also been
reported for lowbush blueberries (V. angustifolium Aiton),75 rabbiteye blueberries (V.
asheii Reade),101 pomegranates,102 highbush blueberries,100 and other cultivar of strawberries.103−−105 Connor et al.106 demonstrate that increases in antioxidant activity, total
phenolic, and anthocyanins content in blueberry during cold storage are cultivar dependent. Tomas-Barberan et al.105 found an increase in anthocyanin concentration in
nectarine, cherry, grape, and strawberry fruits and an increase in ellagic acid in strawberries during air storage whereas flavonoids and hydroxycinnamic acid derivatives
remained constant. Goncalves et al.77 studied the changing of cherry phenolic profiles
during storage at 15 ± 5°C and 1°C to 2°C and found phenolic contents generally
decreased with storage at 1°C to 2°C and increased with storage at 15 ± 5°C. Anthocyanin levels increased at both temperatures. In ‘Van’ cherry, anthocyanin levels
increased up 5-fold during storage at 15 ± 5°C (from 47 to 230 mg/100g of fresh
weight). Kalt et al.69 found ORAC was positively correlated with total phenolic content,
but not with anthocyanin in highbush blueberry cultivars Bergitta, Bluegold, and
Nelson during ripening and storage. Kiwifruit slices stored at 5°C and 10°C exhibited
a gradual decrease in ascorbic acid and an increase in dehydroascorbic acid content.
The total vitamin C was 8%, 13%, or 21% lower than initial values in slices kept for
six days at 0°C, 5°C, or 10°C, respectively.107
Piretti et al.108 found that the most important phenolics in ‘Granny Smith’ apple
skin, epicatechin, quercetin glycosides, and procyanidins decreased from day 100
to the end of storage at day 205, both in air and controlled atmosphere (CA) storage
(1.0% O2 + 2.0% CO2). Further decreases were found during one week of shelf life
at 20°C. However, Curry109 found the level of antioxidants in ‘Delicious’ apple
increased 2 to 10 times during the first two months of storage at −1°C and in ‘Granny
Smith’ apples, whereas antioxidant content significantly decreased during the following four months of storage. Leja et al.110 found that increase of soluble peroxidase
(POD) activity was much stronger in apple kept in air than in CA, while after
subsequent seven-day storage at high temperature, a further increase in enzyme
activity was observed in all treatments. Ju et al.111 found ‘Delicious’ and ‘Ralls’
apple, held for four or five months in cold storage, showed no changes in the
concentrations of simple phenols, flavonoid, and anthocyanin. However, during seven
days at 20°C following storage, simple phenols and flavonoids decreased rapidly.
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Coseteng and Lee112 have shown the total phenol concentration in apples is relatively
stable during storage. Burda et al.113 reported that the concentration of epicatechin,
procyanindin B2, and phloridzin in ‘Golden Delicious,’ ‘Empire,’ and ‘RI Greening’
in both the flesh and the skin remained relatively constant during six months of cold
storage. Lin et al.114 found that cyaniding 3-galactoside in ‘Starkrimson’ apple stored
at 2°C and 73% relative humidity for about seven months still remained constant.
Reay115 also reported that both anthocyanin and quercetin glycoside concentrations
did not change significantly in ‘Gala’ apple during storage at 1°C for about five
months. Perez-Ilzarbe et al.116 found that in ‘Granny Smith’ apple stored at 4°C for
10 days, the concentration of phenolic compounds increased in the skin but not in
the flesh. During rewarming the fruit at 22°C for 21 days and during cold storage,
their concentration remained stable in the skin while it decreased in the flesh. Golding
et al.117 found that individual phenolics in the peel of ‘Granny Smith,’ ‘Lady Williams,’ and ‘Crofton’ apples remained relatively stable after storage in air at 0°C up
to nine months. However, Zhang et al.118 reported that flavan-3-ols monomers and
dimers and cyanidin 3-glucoside decline in litchi fruit following storage at ambient
temperature (20°C to 25°C) for seven days and at 4°C for 35 days.
Postharvest treatment of strawberries with a high electric field has been reported
to increase their shelf life.119 High electric field exposure for short periods was
reported to suppress the respiration rate in some fruits and vegetables.120 Antioxidant
capacity of ‘Irwin’ mango fruit stored at low temperature after high electric field
pretreatment remained unchanged for 20 days of storage at 5°C, but decreased
thereafter. Total phenol and carotenes increased during storage, and antioxidant
capacity of fruits was significantly correlated to ascorbic acids in fruit.121
16.4.2 CONTROLLED ATMOSPHERES
Controlled atmospheres (CA) means removal or addition of gases resulting in an
atmosphere different from that of air. The potential benefits from this method are
retardation of senescence, suppression of biochemical and physiological changes,
reduction of physiological disorders (e.g., chilling injury) in some fruits, and retardation of microbial growth, maintenance of quality, and extension of storage life of
fresh produce. This storage technique is considered a supplement to refrigeration.
The storage life of certain fruits can be greatly extended by this storage technique.
The potential benefit of using controlled atmosphere is dependent upon commodity,
variety, physiological age, atmosphere composition, and temperature and duration
of storage.
Increasing the carbon dioxide concentration around fruits inhibits the postharvest
increase in anthocyanin, by affecting its biosynthesis, degradation, or both.26,102,103
Carbon dioxide-enriched atmospheres (10% to 20%) are especially effective in
retarding decay and softening of strawberries. However, exposure to high concentrations of carbon dioxide can adversely affect the color change in strawberry fruit.26
In strawberries, high carbon dioxide storage does not affect anthocyanin content in
external tissues, but causes a reduction in red color intensity and a decrease in
anthocyanin content of internal tissue of strawberry fruit. It is possible that high
carbon dioxide causes an increase in pH which in turn affects the stability of
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anthocyanins. As carbon dioxide levels increase, the concentration of pelargonidin
glycosides in the internal tissue decreases.26 In red raspberries, the ascorbic acid
increases slightly while red pigment shows no significant changes after CA (10%
O2 + 15% CO2, 10% O2 + 31% CO2) storage for seven days at 2°C.122 Agar et al.123
stored berry fruits in high CO2 (10% to 30% CO2), with or without a parallel
reduction in O2, and found a decrease in vitamin C content, particularly in strawberries. This reduction in vitamin C was moderate in black currants and blackberries
and almost absent in raspberries and red currants when compared with strawberries.
Ascorbic acid was more diminished at high CO2 than dehydroascorbic acid. This
suggests a stimulating effect of high CO2 concentrations on the oxidation of ascorbic
acid and/or an inhibition of mono- or dehydroascorbic acid reduction to ascorbic
acid. Agar and Streif 124 also reported a decrease in ascorbic acid content in red
raspberries in CA storage. ‘Stevens’ cranberries had a higher phenolics content and
total antioxidant activity than ‘Pilgrim’ fruits. The storage atmosphere did not affect
the content of total phenolics or flavonoids. The total antioxidant activity of the
fruits increased overall by about 45% in fruits stored in air. This increase was
prevented by storage in 30% CO2 plus 21% O2.125
Holcroft and Kader103,104 and Holcroft et al.102 reported that concentrations of
ellagic acid, catechin, quercetin, and kaempferol derivatives in strawberries increased
during air storage, but remained relatively stable in high CO2 storage. Vitamin C
content of fresh-cut kiwifruit slices decreased by 14%, 22%, or 34% when kept in
5, 10, or 20 kPa CO2, respectively, and decreased by 7%, 12%, or 18% when kept
in 0.5, 2, or 4 kPa O2, respectively, for 12 days at 0°C.107
Veltman et al.126 found that “Conference” pears stored in CA (2 kPa O2 + 10
kPa CO2) resulted in 60% loss in ascorbic acid, but the concentration of total
phenolics was not affected by storage conditions.127 Larrigaudiere et al.128 also
showed that total ascorbate and glutathione in “Conference” pears decreased rapidly
during storage, especially when the fruit was stored in CA (2% O2 and 5% CO2).
Fruit exposed to CA showed a sharp burst in ascorbate peroxidase and glutathione
reductase activity immediately after storage. A significant increase in superoxide
dismutase activity and a late decrease in catalase were also found in the CA-stored
fruits. Increased lipoxygenase activity and higher amounts of hydrogen peroxide
were found in storage especially when the fruit were exposed to CA.128 Enhanced
losses of vitamin C in response to CA may be due to their stimulating effects on
oxidation of ascorbic acid and inhibition of dehydroascorbic acid reduction to ascorbic acid.107 In ‘Selva’ strawberries, Holcroft and Kader103,104 reported the activities
of phenylalanine ammonia lyase (PAL) and UDP glucose: flavonoid glucosyltransferase (GT) decreased in both external and internal tissues of strawberries stored in
air + 20% KPa CO2 and the effects were more obvious in the internal tissues.
Bangerth 129 found that losses of ascorbic acid in apples and red currants were
reduced by storage in reduced O2 atmospheres, but these losses were accelerated by
storage in elevated CO2 atmospheres. Mareczek et al.130 reported that the level of
total phenolic in the peel of ‘Golden Reinders’ and ‘Gala Must’ apples were similar
in fruit stored for about seven months at 0°C in air and in CA (3% O2 + 5% CO2),
but an additional eight days at 16°C reduced phenolics concentration only in the
peel of ‘Gala Must’ fruit previously stored in air. Leja et al.110 found that the increase
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of soluble peroxidase (POD) activity was much stronger in apple kept in air than in
CA (2% O2 and 2% CO2) at 1°C, but after a subsequent seven days’ storage at high
temperature, a further increase in enzyme activity was observed in all treatments.
‘Jonagold’ and ‘Sampion’ apple showed a slight decrease in anthocyanins stored in
air, while the CA (2% CO2 and 2% O2) treatment did not cause any significant
changes, and antioxidant capacity of fruit peel increased significantly after cold
storage and in CA.110 Additional storage at 16°C caused a slight further increase in
total phenols, antioxidant activity and radical scavenging activity of ‘Jonagold’ fruit
stored previously in a regular cold chamber. This was probably due to synthesis of
phenols in the peel.131,132 However, Mazza and Miniati133 observed that anthocyanins
in apples were relatively stable when stored at 2°C, but decreased in low O2 and
high CO2. Awad and de Jager134 reported that during storage of ‘Jonagold’ apple for
three, six, and eight months and ‘Elstar’ apple for two, four, and six months plus
one or two weeks shelf life, the concentrations of cyanidin 3-galactoside and quercetin glycosides were relatively constant, while the concentrations of catechins,
phloridzin, and chlorogenic acid showed only minor decreases. Moreover, there were
no significant differences in the concentration of flavonoids and chlorogenic acid
between fruits stored at 1°C under ultralow oxygen storage (1.2% O2 + 5% CO2)
compared to air conditions. It was concluded that flavonoids present in apples are
stable, following harvest.
Zheng et al.135 studied the effects of superatmospheric O2 treatments (40%, 60%,
80%, or 100% O2 at 5°C) on ‘Duke’ highbush blueberries and showed that the
antioxidant levels were markedly increased by 60% to 100% O2 treatments as
compared with 40% O2 treatment or air control during 35 days of storage. Elevated
O2 between 60% and 100% also promoted increases of total phenolics and total
anthocyanins, in particular, malvidin-based anthocyanins, as well as the individual
phenolic compounds. Stewart et al.136 also reported that soft fruit stored under
elevated O2 levels exhibited good antioxidant capacity over the first four days of
storage, but then declined with prolonged storage, possibly due to O2-promoted
oxidation of the main antioxidants including anthocyanins and other phenolic compounds.136 This was confirmed by Pérez and Sanz137 who found that, in comparison
with fruits stored in air, strawberries held in 80% O2 + 20% CO2 had significantly
higher levels of total anthocyanins during the first four days of storage, but significantly lower levels of total anthocyanins at the end of storage. It seems that the
effect of high O2 on total phenolics, total anthocyanins, and ORAC value may vary
depending on the commodity, O2 concentration, and storage time and temperature.
Data obtained in these studies suggest that high-oxygen treatments may improve the
antioxidant capacity of fruits.
Taira et al.138 treated ‘Hiratanenashi’ persimmom fruit after harvest with 15%
ethanol vapors at 20°C or with 80% CO2 at 20°C and showed that the soluble tannin
decreased during treatment and fruits were not astringent after seven days of ethanol
treatment or three days of the CO2 treatment. Zavrtanik et al.139 treated ‘Hachiya’
and ‘Fuyu’ persimmons with CO2 or N2 at 99.9% for 24 h at 20°C , then stored the
fruits at 1°C. The total phenol concentration decreased very quickly and fruits were
edible after one day of the CO2 treatment; N2 treatment reduced polyphenol content
less quickly and fruit were edible after 5 to 7 days while control fruit lost polyphenols
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slowly and were edible after 10 days of storage. Wright and Kader140 showed CA
(2% O2, air + 12% CO2 or 2% O2 + 12% CO2) had no significant effect on changes
in total ascorbate content for persimmons and strawberries after seven to eight days
at 5°C. Al-Redhaiman 141 also found that total tannin content in ‘Barhi’ date fruit
did not change significantly after 26 weeks at 0°C under 20% CO2 storage.
Ozone, the triatomic form of oxygen (O3), is an unstable compound that decomposes either spontaneously, producing hydroxyl radicals and other free radical species, or upon contact with oxidizable surfaces. Ozone has been used in different
applications in the food industry, and has been recommended to be used as a GRAS
(generally recognized as safe) disinfectant or sanitizer for foods in the United
States.142 Ozone, applied as a gas or as ozonated water, has been tested for postharvest
treatment of fruits and vegetables. Pérez et al.143 reported that at the end of cold
storage, the vitamin C content of ozonated strawberries was three times that of
control fruits. A decrease in the anthocyanin content of treated and nontreated
samples was observed after three days at 2°C, with a significantly lower value in
ozonated fruits (639. 08 ± 11.01 nmol/g fresh weight) than in nontreated fruits
(811.34 ± 6.81 nmol/g fresh weight). When strawberries were placed at 20°C, a
slight increase in anthocyanin accumulation was detected in both treated and nontreated fruits. No significant differences were found after four days at 20°C.143
Blackberry fruit stored over 12 days at 2°C in a 0.3 ppm ozone atmosphere showed
a sharp decrease in anthocyanin levels.144 Artés-Hernández et al.145 showed an
increase of up to twofold in total stilbenoid content in ‘Napoleon’ grapes after shelf
life for CA (5 kPa of O2 + 15 kPa of CO2 + 80 kPa of N2) and O3 (8 ppm) treatments
up to 38 days at 0°C. Piceid concentration remained unaltered or slightly changed,
whereas large increases were observed after shelf life for resveratrol, even up to
three- and four-fold for O3-treated grapes and two-fold for CA-treated ones. Therefore, improved techniques for the keeping quality of ‘Napoleon’ table grapes during
long-term storage seem to maintain or enhance their antioxidant compound content.
16.4.3 HEAT TREATMENT
AND IRRADIATION
One of the postharvest techniques that has been demonstrated to effectively maintain
quality of fresh fruits is prestorage heat treatment. In addition to controlling diseases
and insects, adequate heat treatment can also retard senescence or degradative
processes in fresh produce. Application of thermal treatments reduced the softening
rate of apples, avocado, peach, papaya, pear, and banana.146−149 Civello et al.150 also
found that heat treatments delay ripening and postharvest decay of strawberry fruit.
Strawberries were treated for 1 to 5 h at temperatures ranging from 39°C to 50°C .
After treatments, fruits were placed at 0°C overnight and then held at 20°C for three
days. Most of the heat treatments improved strawberry shelf life, with best results
obtained for fruit heated at 42°C and 48°C for 3 h. However, both treatments reduced
anthocyanin accumulation and PAL activity relative to the controls. Civello et al.
also showed that heat treatment (48°C air, 3 h) reduced anthocyanin accumulation
in strawberries. According to these results, the lower fungal development and the
slower ripening rate shown by heat-treated fruits suggest that this physical method
could be useful to extend the postharvest shelf life of strawberries. In contrast,
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Yoshikawa et al.151 treated ‘Chandler’ strawberries with humid air at 43°C and 46°C
for 80 min and found severe damage in the fruits. These contradictory results could
be due to cultivar-dependent responses of strawberries to heat treatments. This
phenomenon has been described for other fruits as well.148,152
Red pigmentation of apples can be improved after harvest using artificial light.
A range of wavelengths from UV-B (280 to 320 nm) to red light (680 to 780 nm)
is effective.153,154 Red light alone is only slightly effective in stimulating anthocyanin
production, UV-B has considerable effect, and both together have synergistic
effects.153 Marais et al.155 showed that postharvest irradiation with high-pressure
sodium light-enhanced anthocyanin synthesis in apples, but not in pears. Postharvest
irradiation with UV rays at 2.35 W m−2 alone markedly promoted red color development and increased the anthocyanin content in the skin of ‘Satohnishiki’ sweet
cherries within 48 h.156 In grapes, accumulation of anthocyanin occurred in the skins
when berry sections were exposed to UV irradiation.157 It was proposed that UV
irradiation induced and activated decay-resistance mechanisms, for example, by an
increase in antifungal compounds in the fruit peel. An additional positive effect of
UV treatment is the enhancement of levels of anthocyanins in strawberries and red
apples.154,158 Accumulation of anthocyanins in apple skins occurred from de novo
synthesis of PAL and chalcone isomerase following UV-B treatment, and it was
suggested that there could be a similar effect of short exposure to high-energy UVC irradiation. By lowering the temperature during irradiation from 23°C to 11°C ,
the effectiveness of UV irradiation was suppressed. The decrease in temperature
may cause a suppression of the enzymatic activities related to phenylpropanoid
synthesis which was stimulated by UV radiation.156 Irradition (2 to 3 kGy) combined
with refrigeration has been shown to extend the shelf life of strawberries.159 During
storage, ascorbic acid significantly increased while dehydroascorbic acid decreased
in irradiated strawberries.159
Stilbenes are nonflavonoid phenolics that are mainly present in grape, and they
have been reported to have a number of health-beneficial properties.160,161 The induction of stilbenes in response to stress, such as preharvest and postharvest UV-C
irradiation has been described.162−−165 Cantos et al.160 demonstrated that postharvest
UV-C irradiation (510 W, 40 cm, 60 s) enhances stilbene content in red grape
varieties ‘Flame,’ ‘Red Globe,’ ‘Crimson,’ and ‘Napoleon,’ as well as the white
varieties ‘Superior,’ ‘Dominga,’ and ‘Moscatel Italica.’ The most inducible stilbenes
in grapes were trans-resveratrol, trans-piceatannol, and viniferins. The net resveratrol induction ranged from 3.4-fold (‘Flame’) to 2315-fold (‘Red Globe’). The
highest viniferins content was observed in the variety ‘Flame’ (0.73 mg/100 g fresh
weight), although the variety ‘Red Globe’ presented the highest viniferins induction
(175-fold). The highest content and induction of piceatannol (0.17 mg/100 g fresh
weight and 173-fold, respectively) was observed in the variety ‘Flame.’ Cantos et
al.162 found that the content of most phenolics in ‘Napoleon’ grapes remained quite
constant during postharvest refrigerated storage (10 days at 0°C) while the resveratrol
derivatives increased twofold. Postharvest treatment of grapes with UV-C and UVB light induced a large increase in resveratrol derivatives (three- and twofold,
respectively).163−−165 The biological activity of resveratrol as an anticarcinogenic and
an antioxidant has been reported.166,167 These results showed that UV irradiation of
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table grapes can be beneficial in terms of increasing the content of potentially healthpromoting resveratrol.163−−165
Patil et al.168 demonstrated that the response of ‘Rio Red’ grapefruit to irradiation
depended on harvest time. The early season grapefruit exposed to low doses of
irradiation (70 and 200 Gy) followed by storage (35 days) at 10°C had significantly
higher naringin, narirutin, and total flavanone concentrations compared to the initial
flavanone concentrations. This increase may be attributed to an increase in PAL
activity during low-temperature storage and low dose irradiation exposure.169,170 The
early-season grapefruit also had higher levels of β-carotene after 35 days of storage
than at their initial (0 day) level, irrespective of the irradiation. On the contrary, in
late-season fruit, irradiation had no effect on β-carotene in grapefruit before or after
45 days of storage conditions. Higher doses of irradiation (400 and 700 Gy) and 35
days of storage had detrimental effects on quality of early-season grapefruit; however, no significant effect was observed on the quality of late-season fruit.168 Oufedjikh et al.171 also reported that the irradiated fruit (300 Gy) had lower concentration
of flavanone glucoside and polymethoxylated flavones. The decrease in flavanone
content was ascribed to their role in counteracting the oxidative stress induced by
the gamma irradiation. Variation in the flavanone content at different doses of
irradiation treatment may be due to equilibrium between gamma irradiation-induced
stress and de novo synthesis of flavonoids by increased PAL activity.171 The UV-C
(9.2 kJ m−2) and heat treatments (45°C) retained fruit quality and antioxidant activity
better than in control fruit of boysenberries.172 The effect of UV rays on promoting
the accumulation of anthocyanin has also been reported in apples, grapes, sweet
cherry, and several kinds of fruits.24,25,105,153−−158,162
16.4.4 TREATMENT
WITH
NATURAL COMPOUNDS
OR
CHEMICALS
A naturally occurring compound, methyl jasmonate (MJ), was found to reduce
chilling injury, retard decay, and improve storage quality of several fruits.173,174 The
MJ treatment maintained higher levels of ORAC in blueberries than the untreated
control fruit, especially during the latter part of storage.175 The high antioxidant
activities were associated with better overall quality of MJ-treated raspberries, which
included high levels of sugars, organic acids, and low incidence of decay.176 Chanjirakul et al.177 studied the effect of various natural volatiles such as methyl jasmonate
(MJ), allyl isothiocyanate (AITC), essential oil of Melaleuca alternifoli (tea tree oil
or TTO), and ethanol (EtOH) on antioxidant capacities and antioxidant enzymes in
berry fruits and found that strawberries and blackberries treated with MJ had the
highest antioxidant capacity expressed as oxygen radical absorbance capacity
(ORAC) values after seven days of storage (40.77 and 67.70 µmol TE/g fresh weight,
respectively).177 Moreover, MJ treatment enhanced antioxidant capacity in strawberries and blackberries as measured by the radical DPPH• and ABTS•+ scavenging
activity in both 7 and 14 days after storage. The ED50 of free radical DPPH•
scavenging capacity ranged from 31.79 to 36.94 mg after 7 days of storage and from
32.57 to 42.72 mg after 14 days of storage in strawberries, but only from 4.29 to
6.65 mg and from 5.10 to 7.73 mg in blackberries after 7 and 14 days of storage,
respectively. The MJ-treated fruits showed the highest percent inhibition for DPPH
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radicals among all the treatments in both strawberries and blackberries. The MJ
treatment also increased scavenging capacities on (ROO•) superoxide radicals (O2•–),
hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2) in
strawberries and blackberries, except for O2•– scavenging capacity in blackberries
stored for 14 days. Treatment with TTO or EtOH enhanced most of these free radical
scavenging capacities, except for H2O2 in strawberries, and for O2•– and 1O2 in
blackberries. It is possible that the elevated capacity in scavenging various free
radicals by these natural volatile compounds increased the resistance of tissues to
decay.
In raspberries, treatment with MJ enhanced the activity of several antioxidant
enzymes, including SOD, G-POD, AsA-POD, GSH-POD, GR, MDAR, and DHAR.
Moreover, raspberries treated with MJ showed the highest amount of AsA, DHAsA,
GSH, and GSSG compared to other treatments. Treatments with TTO or EtOH also
enhanced the antioxidant system. Although AITC treatment promoted the O2•– scavenging capacity in blackberries after 14 days of storage, it had little effect on
scavenging capacities of other radicals or the antioxidant enzyme activities. These
results indicate that MJ may increase the resistance of tissues to decay through
enhancing their antioxidant system and their free radical scavenging capability, while
AITC may retard the decay directly by its antimicrobial properties. Therefore, it is
possible to enhance the antioxidant system, reduce decay, and extend storage life of
berry fruits by treatment with natural volatiles.177
Calcium dips may be used to reduce physiological disorders and maintain firmness in apples, cherries, and other fruits. Bangerth178 observed an increase in vitamin
C content of apples treated with calcium chloride. When dipped in 1% CaCl2 after
cutting, and kept in an ethylene-free atmosphere, kiwifruit slices had a slightly higher
ascorbic acid content than those treated with 1% CaCl2 only.107
Diphenylamine (DPA) has been used in commerical postharvest treatment to
suppress the storage disorder superficial scald during long-term storage of apples.
Superfical scald is a serious physiological disorder that affects some susceptible
apple cultivars, such as ‘Red Delicious’ and ‘Granny Smith.’ Golding et al.117 treated
apple (‘Granny Smith,’ ‘Lady Williams,’ and ‘Crofton’) with DPA at harvest then
stored in air at 0°C for nine months and found that the concentrations of benzoic
acid derivatives, cinnamic acid derivatives, flavan-3-ols, flavonols, and procyanidins
of stored fruit were higher in the treated peel than in the control peel. However,
Duvenage and DeSward 179 showed that DPA inhibited both the synthesis and oxidation of flavonols during storage.
16.5 EFFECT OF PROCESSING
Many changes in physical and chemical properties occur in fruits during processing.
Several types of oxidative reactions may take place in which electrons are removed
from atoms/molecules leading to the formation of an oxidized form. These reactions
cause browning reactions, loss or changes to flavor or odor, changes in texture, and
loss of nutritional value from destruction of vitamins and essential fatty acids.180
The health-promoting capacity of processed foods in the diet is still not completely
known. The concentration of nutrients may be changed, influencing the overall
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antioxidant potential of fruits by the processing methods.181,182 In general, fruit
processing may be responsible for a decrease, increase, or no change in content and
functionality of phytochemicals. Additional information on the influence of processing on antioxidants can be found in Klein and Kurilich,183 Nicoli et al.,184 and
Pokorný and Schmidt.185
16.5.1 HEAT
It is generally recognized that processing can negatively affect the original antioxidant properties of raw material. Generally, thermal processing results in a decrease
in ascorbate, anthocyanins, and tocopherols. But recent research has shown that food
processing also has some positive effects on the quality and health properties of the
processed food. The most notable positive effect of processing on the overall quality
or health capacity of fruit is increased bioavilability of β-carotene, resulting in an
increased antioxidant status. Carotenoid terpenes are plant pigments that are present
in fruits such as oranges, pink grapefruit, mango, autumn olive, and papaya. Different
carotenoids have different degrees of sensitivity to heat treatment. For example,
epoxy carotenoids were sensitive to heat treatment, while lutein, neurosporene, αand β-carotene, lycopene, phytofluene, and phytoene survived heat treatment.186
Thermal processing has been shown to convert all-trans forms of β-carotene to cis
forms.187 These compounds enhance immune response and protect skin cells against
the sun's ultraviolet radiation.
Loss of vitamin C after heat treatment has been widely reported.188 Freshsqueezed orange juices are higher in ascorbic acid and anthocyanins than processed
juices.189 Fruits are high in vitamin C content and processing tends to cause loss
from as little as 10% to almost 100% during cooking or canning.190 Vitamin A loss
in canning totals about 10%, whereas the individual nutrients in 13 fruits and
vegetables were 35% to 96% less for fat-soluble nutrients (96% tocopherols, 42%
total carotenoids, 35% vitamin) as compared with frozen fruits.191 The canned fruits
also showed about twice as much vitamin C, niacin, and riboflavin loss and over
three times as much thiamin loss compared with frozen fruit.
Blueberry puree was relatively high in its antioxidant capacity, with values close
to those in intact fresh fruit and frozen fruit samples. The antioxidant capacity
(ORAC) of blueberry pie filling prepared from canned fruit had only about one-third
of the ORAC of fresh fruit, suggesting that substantial damage and/or losses of
antioxidants occurred during canning processing.192 Lo Scalzo et al.193 showed that
thermal treatments reduced ascorbic acid in blood orange juice. However, an increase
in anthocyanins, hydroxycinnamic acids, and antioxidant capacity in thermally
treated juice was found.193 Kalt et al.192 showed that extraction of lowbush blueberry
at 60°C resulted in higher anthocyanins content and antioxidant capacity, compared
to extracts obtained at 25°C. Subsequent room temperature storage resulted in losses
in anthocyanin and antioxidant capacity only in those extracts obtained at 60°C. On
the other hand, Jackman et al.194 indicated that heat is one of the most destructive
factors of anthocyanins in berry juices. Increasing storage temperatures from 20°C
to 100°C decreased half-life time of strawberry preserves. In dry powder of
elderberry extracts, the stability of anthocyanins increased six to nine times when
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temperature was reduced from 20°C to 4°C.195 Anthocyanin degradation in aqueous
solution increased from 30% to 60% after 60 days when storage temperatures were
increased from 10°C to 23°C.196 Therefore, high-temperature, short-time processing
is recommended for maximum anthocyanin retention of foods containing anthocyanins.197 Asami et al.198 demonstrated that both storage and thermal processing
conditions had a profound impact on the levels of polyphenolics in clingstone
peaches and showed a dramatic decrease in procyanidins in thermally processed
peaches. Cold storage at 4°C for 14 days or freezing and storing at −12°C for three
months produced no loss in total phenolics and procyanidins. Peaches stored at 30°C
for 24 h resulted in a 1.7-fold increase in total phenolics. Peaches processed at
temperatures of 100°C for 40 min, 104°C for 10 min or 110 °C for 2.4 min showed
that processing above 100°C decreased levels of both total phenolics (up to 21%)
and procyanidins (up to 100%). Processing at 100°C for 40 min produced no
significant loss in total phenolics, but a 30% to 43% loss in phenolic levels was
found during the first three months in storage after canning. This result indicated
that freezing has relatively little impact on total phenolics and procyanidins, whereas
peeling and canning using high temperatures typically employed in commercial
sterilization have detrimental impacts on both total phenolics and procyanidins
levels.198 The processing temperatures at 120°C also showed reduction of the free
radical scavenging capacity in red grape pomace peels (28.3%) and white grape
pomace peels (22.9%) sample extracts as compared to those at 20°C.199 Skorikova
and Lyashenko200 obtained a negative correlation between the heating period and
the polyphenol content of apple and pear juices, with the leucoanthocyanins being
the least stable polyphenol fraction. Similarly, Sato et al.201 found a correlation
between wine color and the ability of the wine constituents to scavenge superoxide
radicals. Specifically in red grape pomace peels, losses in the total polyphenols,
color, and antioxidant capacity were reported when they were dried at 100°C and
140°C, with the anthocyanins being more affected than other compounds.202 Bioactive compounds (such as polyphenols, flavonoids, etc.) that are associated with the
antioxidant capacity of the samples are usually oxidized at high temperatures.203
Therefore, a lower reduction in the free radical scavenging capacity of white grape
pomace peel extracts with processing temperature as compared to that from red
grape pomace peels, suggests that the bioactive constituents of white grape pomace
peel extracts are more heat resistant than those from red grape pomace peels.
Food processing, especially if heat-treatment based, can result in undesired
reactions, mostly of a chemical nature. For instance, oxidation can result in antioxidant and vitamin depletion. On the other hand, heating can also induce the formation
of compounds with new antioxidant properties, as occurs during the development
of the Maillard reaction in which a reducing sugar reacts with amino groups.
Antioxidant depletion in thermally treated fruits may also be attributed to consumption of ascorbic acid, polyphenols, sugar and amino acids as reactants in the Maillard’s reaction.184,204 Maillard reaction products have an effect on the nutritive value
(loss of the essential amino acid lysine), as well as the development of new compounds, which improve flavor and color in many food products during baking,
cooking, roasting, or frying.205 Although they have been extensively studied, it is
not still clear whether the Maillard reaction produces mutagenic or antimutagenic
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compounds or substances, which favor or block microbial growth or enzymatic
activity.206,207
Jeong et al.208 showed that the antioxidant activities of citrus peel extracts
increased as heating temperature increased. Heat treatment of citrus peels at 150°C
for 60 min increased the total phenolic content, radical scavenging activity, and
reducing power. Several low-molecular-weight phenolic compounds such as 2, 3diacetyl-1-phenylnaphthalene, ferulic acid, p-hydroxybenzaldoxime, 5-hydroxyvaleric acid, 2,3-diacetyl-1-phenylnaphthalene, and vanillic acid were newly formed
in the citrus peels heated at 150°C for 30 min. These results indicated that the
antioxidant activity of citrus peel extracts was significantly affected by heating
temperature and duration of treatment on citrus peels, and that the heating process
can be used as a tool for increasing the antioxidant activity of citrus peels.208 The
formation of new phenolic compounds with antioxidant properties in the citrus peels
heated at 150°C for 30 min may be attributed to the development of Maillard reaction
products.
16.5.2 FREEZING
Overall, fruits retain more nutrients if they are frozen rapidly, stored in airtight
packaging, and thawed rapidly.209 Freezer temperature should be −18°C (or 0°F) or
lower. If a constant temperature of −22°C is maintained, many foods will only lose
about 25% vitamin C in one year.210 Mullen et al.211 studied the effects of storage
and freezing on raspberries and showed that the antioxidant capacity was not significantly different in fresh and frozen berries. It was also demonstrated that the
freezing process had little effect on the ellagic acid released by acid hydrolysis, total
phenol, vitamin C content, and antioxidant capacity.212 However, during long-term
frozen storage (12 months), the level of ellagic acid declined 14% to 21% and
vitamin levels fell 33% to 55%, although the antioxidant capacity of the berries was
unchanged.212 Ancos et al.212 also analyzed the anthocyanins of raw, just frozen, and
long-term frozen storage at −20oC of four raspberry cultivars (cvs. ‘Heritage,’
Autumn Bliss, Zeva, and Rubi) and found that the stability of anthocyanins after
freezing and frozen storage was dependent on the seasonal period of harvest. Heritage and Autumn Bliss (early cultivars) were less affected by processing and longterm frozen storage (one year), and the total pigment extracted increased 17% and
5%, respectively. ‘Rubi’ and ‘Zeva’ (late cultivars) exhibited a decrease in total
anthocyanin content of 4% for “Rubi” and 17.5% for “Zeva.” Cyanidin 3-glucoside
degraded the most during processing and storage. Bushway et al.213 evaluated five
raspberry cultivars harvested at the red-ripe stage and during long-term frozen
storage at −20°C. They rated color by instrument and by a sensory panel and found
that the effect of freezing on instrumental hue and sensory color was not significant
for any of the five varieties investigated.
16.5.3 SUGAR, ASCORBIC ACID,
AND
OTHER CHEMICALS
High sugar concentrations (> 20%) are protective of anthocyanins.214 Pasteurized
ascorbic-acid-enriched chokeberry extracts with 65% sucrose had three times longer
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anthocyanin half-life compared with sucrose-free extracts. Moderate sucrose levels
(13%) also showed slightly improved anthocyanin stability.215 Glucose, sucrose, and
maltose are more effective for this protection than fructose, arabinose, lactose, and
sorbose.197 Strawberries frozen with added sugar retain more vitamin C than unsweetened strawberries.209 Heat processing did not result in a loss of total anthocyanins,
total phenolics, and antioxidant activity when the values in syrup and cherries were
combined. There was little loss of total anthocyanins with canning of cherries, but
about 50% of anthocyanins and polyphenolics were redistributed to the syrup. About
half of anthocyanins and polyphenolics were leached from fruits into the brine
solution.216 This may be due to increased extraction efficiency in the softened fruits.
High temperature may increase membrane permeability in the macerated peel tissue
facilitating phenolic extraction.217 Moreover, with the breakdown of the cellular
constituents, bound phenolic compounds may be released.218
The phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries (bayberry, black currant, blackberry, blueberry, cowberry, cranberry, raspberry, red currant, and strawberry) remained stable
during jam processing.219 This indicated that flavonoids and phenolic acid retained
their conjugate forms in the samples during jam processing. Zafrilla et al.220 found
the flavonol content of raspberry jams decreased slightly with processing and more
markedly during storage of the jams. The ellagic acid derivatives remained quite
stable with processing and during six months of jam storage. The content of free
ellagic acid increased 2-fold with processing, and it continued increasing up to 3.5fold after one month of storage of the jam. The increase observed in ellagic acid
could be explained by a release from ellagitannins with the thermal treatment. The
industrial processing of pomegranates juices showed an increase of their antioxidant
capacity and phenolics content,22 whereas strawberry processing to produce jams
decreased the total ellagic acid content by 20%221 and the flavonols by 15% to
20%.222 Cooking also affects flavonoid content. Cooking strawberries with sugar to
make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking
of bilberries with water and sugar, 40% of quercetin was lost.222 Strålsjö et al.223
found almost no losses of folate when strawberries were cooked to jam or stewed
to strawberry desserts from frozen berries. This finding is contradictory to data
reported in four European food tables, which indicate that only 3% to 30% of folate
was retained in various strawberry products such as jam and stewed desserts.224
Fruits commonly lose nutrients when they are used to make juices.191 Important
variables affecting nutrient content are temperature, pH, exposure to air or light,
type of storage container, and presence of antioxidants or preservatives.225 It was
reported that vitamin C is most stable in pineapple juice and less stable in apple
juices blended with other fruits, and carbonated fruit beverages. Vitamin A is quite
stable when added to juices and other beverages, but least stable in pineapple juice
and pineapple juice drinks. Carotenoids are very stable in juices and the B-vitamins
are stable in frozen beverages.191,225 Common food additives (such as sodium nitrate)
cause folate destruction.226 Sulfur dioxide and sulfite have been widely used in fruit
processing. Sulfur dioxide enhances the extraction of anthocyanins from fruit tissues.227 At low concentrations (30 ppm), it inhibits the enzymatic degradation of
anthocyanins; at moderate concentrations (500 to 2000 ppm), it forms a reversible,
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colorless SO2-anthocyanin complex; at high concentrations (0.8% to 1.5%), it forms
an irreversible complex, as in the bleaching of red cherries during brining.228 Use
of sulfite in food processing to inhibit browning reactions can lead to extensive
losses of thiamine.226 The presence of vitamin C in food can improve folate retention
during heating whereas the presence of metals can increase loss.226 Antioxidant
activity of whole apple juices which were fortified with ascorbic acid declined after
10 days of storage at room temperature or at 4°C and unfortified whole apple juice
remained relative stable.229
16.5.4 DRYING
AND
DEHYDRATION
Dry anthocyanin powders are stable for several years when stored in hermetically
sealed containers.197,230 Zajac et al.195 reported that anthocyanin content of dry black
currant powder decreased 14% after 15 months of storage at 20°C, but when starch
syrup or maltodextrins were included as carriers during the drying of extracts, the
anthocyanin losses were reduced to 2% and 3%, respectively.
Larrauri et al.202 found that polyphenolic content, color, and antioxidant activity
of red grape pomace peels were not significantly affected when dried at 60°C.
However, when drying temperature was either100°C or 140°C, a significant reduction in both total extractable polyphenols (18.6% and 32.6%) and condensed tannins
(11.1% and 16.6%) were observed, as well as a decrease of 28% and 50%, respectively, in the antioxidant activity of the samples. Hue angle and total color difference
in the samples dried at 140°C were significantly higher than in the freeze-dried
reference material. This may be due to the thermal degradation of the phenolic
compounds at high temperatures.202 Dried blueberries were varied in their ORAC
levels, and appeared to be lower in fruit that had been dried more thoroughly. The
ORAC value of juice concentrate was only about 65% that of the fresh.192 Vacuum
and osmotic concentration of red raspberry juice decreased total quercetin-forms
compared to unconcentrated juice.81 Freeze drying, vacuum microwave drying, air
drying, and a combination of air drying and vacuum microwave drying all reduced
total phenolics and anthocyanin contents in addition to reduced antioxidant activities,
as compared with fresh frozen Saskatoon berries.231 The air-dried and freeze-dried
apple peels had the highest total phenolic, flavonoid, and anthocyanin content as
compared to the oven dried at 40°C, 60°C, or 80°C.232 Piga et al.233,234 dehydrated
fresh plums to prunes and found that drying destroyed anthocyanins and flavonols.
High temperatures and high oxygen concentrations involved in the air-drying process
lead to the rapid degradation of the anthocyanins.133 During drying, the balance
between the various types of anthocyanins shifts toward either the quinoid base or
the colorless chalcones, which are destroyed by different oxidation mechanisms to
produce different types of compounds.235 A marked decrease in ascorbic acid content
was found after the dehydration process. The fruits dried at 85°C showed significantly higher losses, ranging from a minimum of 61% to a maximum of 80%. Total
polyphenol values decreased in the samples dried at 60°C, but were two and a half
times higher in ‘President’ plums dried at 85°C. No significant changes were found
in the chlorogenic and neochlorogenic acid when processing was conducted at the
higher temperature (85°C), whereas a decrease was found in the prunes obtained at
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60°C. It has been reported that during the dehydration process, the polyphenoloxidase (PPO) activity remains high for long periods when the drying temperature is
around 55°C, whereas only moderate activity is observed at temperatures higher
than 75°C.236 Raynal et al.236 also found a decrease of 25% and 30% in chlorogenic
and neochlorogenic acids, respectively, for plums dried at 85°C. The antioxidant
capacity tended to increase significantly with the higher drying temperature in the
‘Sugar’ variety, while in the ‘President’ plum sample, dried at 60°C, antioxidant
capacity was significantly lower than in the fresh fruits. Processed plums, dried at
85°C, exhibited doubled antioxidant activity in both cultivars (‘Sugar’ and ‘President’).233,234 This may be due to high temperature stabilization procedures that may
lead to the formation of new compounds with higher antioxidant activity. This is
essentially the case in the Maillard reaction, in which Maillard reaction products
(MRPs) are created with markedly higher antioxidant power, often by a chainbreaking type mechanism.237−239
Osmotic dehydration has been used as a prestep to further processing.240 These
processes use a sequence of technological steps to achieve controlled changes of the
original properties of the raw material. Torreggiani and Bertolo241 found that the
pigment retention of strawberry halves was significantly higher in osmotic dehydration than that observed in the fruits frozen without a concentration pretreatment. An
osmotic step could also improve the stability of vitamin C and color during air drying
and frozen storage of osmodehydrofrozen apricot cubes by the modification of sugar
composition.241,242 Therefore, the quality characteristics of processed fruit could be
improved through the application of an osmotic step.
16.5.5 EXTRACTION TECHNIQUES
Processing apples into juice had resulted in large flavonoid losses. Conventional
apple juice production (straight pressing of apple pulp or pressing after pulp enzyming) resulted in a juice poor in flavonoids with only 3% to 10% of the antioxidant
activity of the original fruit.243 However, by applying an alcoholic extraction step,
either on the pulp or on the pomace, the levels of flavonoids and chlorogenic acid
in enriched juice are between 1.4 (chlorogenic acid) and 9 (quercetin glycosides)
times higher than in conventional apple juice. In enriched juice the antioxidant
activity was five times higher than in conventional apple juice, with 52% of the
antioxidant activity of the originating fruits.244 Häkkinen et al.35 showed that traditional preservation of crushed lingonberries in their own juice caused a considerable
(40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in
unprocessed berries were retained in juices made by common domestic methods
(steam-extracted black currant juice, unpasteurized lingonberry juice). Cold pressing
was superior to steam extraction in extracting flavonols from black currants. During
nine months of storage at −20°C, quercetin content decreased markedly (40%) in
bilberries and lingonberries, but not in black currants or red raspberries. Myricetin
and kaempferol were more susceptible than quercetin to losses during storage. When
juices were made by common domestic processing methods, considerable losses of
flavonols were observed. Cold pressing was superior to steam extraction in extracting
the flavonols. Also, crushing the berries resulted in a considerable loss of quercetin.
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Rommel and Wrolstad 81 reported that the red raspberry juice produced by highspeed centrifugation contained the most total quercetin forms and much more than
juices made by other techniques. Juice following high-speed centrifugation, combined with addition of pectinases, contained less total quercetin forms (ca.190 ppm)
than the juice prepared without pectinases (280 ppm). Diffusion-extracted juice
contained even less total quercetin forms. This was probably due to deglycosylation
of flavonol glycosides to less-stable aglycones in the presence of pectinases. In the
crowberry juices, such an effect was not observed. Rommel and Wrolstad41 also
found that juices of red raspberry made by diffusion extraction and a standard process
had the highest concentration of ellagic acid and its forms. High-speed centrifugation
reduced total ellagic forms by half compared to diffusion extraction and depectinization and concentration decreased total forms even further. Spanos et al.217 reported
that a high temperature during initial diffusion extraction produced up to a fivefold
increase in the recovery of phloretin glucosides as compared to that obtained in a
conventionally pressed juice without temperature elevation.
Gil-Izquierdo et al.245 evaluated phenolic compounds, vitamin C, and antioxidant
capacity in orange juices by different extraction processing techniques on commercial (squeezing, mild pasteurization, standard pasteurization, concentration, and
freezing) and domestic squeezing (a hand processing technique) and found that
commercial squeezing extracted 22% more phenolics than hand squeezing. The
freezing process caused a dramatic decrease in phenolics, whereas the concentration
process caused a mild precipitation of these compounds into the juice cloud. In pulp,
pasteurization led to degradation of several phenolic compounds, that is, caffeic acid
derivatives, vicenin 2 (apigenin 6,8-di-C-glucoside), and narirutin (5,7,4′-trihydroxyflavanone-7-rutinoside) with losses of 34.5%, 30.7%, and 28%, respectively.
Orange juice produced by commercial squeezing contained 25% more vitamin C
than by domestic squeezing. Mild and standard pasteurization slightly increased the
total vitamin C content as the contribution from the orange solids parts, whereas
concentration and freezing did not show significant changes. The content of Lascorbic acid provided 77% to 96% of the total antioxidant capacity of orange juice.
Mild pasteurization, standard pasteurization, concentration, and freezing did not
affect the total antioxidant capacity of juice, but they caused a 47% reduction in pulp.
16.5.6 HIGH PRESSURE
Food processing under high pressure and low to moderate temperature (below 70°C)
conditions has been introduced as an alternative to high temperature preservation.246,247 High pressure has been used to inactivate pectinesterase and pectin methylesterase for bypassing the use of extreme heat during processing which results in
a loss of fresh fruit flavor in the juice.248,249 There are a few studies that have been
published on the effect of high hydrostatic pressure on postprocessing total antioxidant activity of orange juice or other food systems. Polydera et al.250−252 studied the
total antioxidant activity of high pressure processed (500 MPa, 35°C, 5 min or 600
MPa, 40°C, 4 min) compared to thermally pasteurized (80°C, 30 s or 80°C, 60 s)
fresh orange juice as a function of storage at different isothermal conditions (0°C
to 30°C) and found higher antioxidant activities for high-pressure pressurized
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compared to thermally pasteurized orange juice during their storage. This may be
due to high-pressure treatment leading to better ascorbic acid retention compared to
conventional pasteurization. Nienaber and Shellhammer253 found that stabilization
of fresh orange juice at pressures of 800 MPa and 25°C for 1 min and use of thermally
pasteurized pulp yielded the lowest level of residual pectinmethylesterase activity
(3.9%) and good cloud stability at 4°C and 37°C over a period of more than two
months. Ascorbic acid loss was less than 20% after storage for three months at 4°C
or two months at 15°C. Color values were stable during storage at 4°C, 15°C, and
26°C. DeAncos et al.254,255 reported stability or even improvement of radical scavenging activity of persimmon fruit purees after high hydrostatic pressure treatment.
This may be due to the stability of carotenoids which are related to the increase of
vitamin A. Sancho et al.256 surveyed the retention of ascorbate on ultra-high-pressurized strawberry coulis over 30 days, and found that vitamins B1 and B6 showed
no significant losses but vitamin C degraded slightly after ultra-high-hydrostatic
pressure treatment. Therefore, high-pressure processing of fruit products offers the
chance of producing food of high quality, greater safety, and increased shelf life.
16.5.7 MICROWAVE
AND PH
When foods are microwave cooked at low power, nutrient retention is equal to or
better than that of the same food prepared by conventional methods or held hot in
food service operations.257−259 Vitamin retention improves if small amounts of water
are used.259 Low-power warming techniques show equal or better retention of thiamine, riboflavin, vitamin B6, folate, and ascorbic acid when compared with foods
that are conventionally reheated.257 To date, no information on the effects of microwave cooking on flavonoid content in fruits has been reported.
The red color of anthocyanins is present only at low pH in aqueous solution.
By raising the pH above 4, yellow, colorless, and blue compounds are produced.
Anthocyanins exhibit the highest stability as the red flavylium cation around pH 1
to 2, whereas the other forms, especially the chalcone, are unstable and eventually
lead to the degradation of the anthocyanins. In juices and wines, where the pH
typically is between 2.5 and 4, anthocyanins are present as their most stable red
flavylium cation, due to copigmentation with various other compounds.260 Nielsen
et al.261 found that the stability of anthocyanins (cyanidin 3-ο-β-glucoside, cyanidin
3-ο-β-rutinoside, delphinidin 3-ο-β-glucoside, and delphinidin 3-ο-β-rutinoside) in
black currant juices are pH dependent. More than 90% of each anthocyanin remained
intact up to pH 3.3. At pH 3.8 a minimum instability was detected, suggesting the
presence of an unstable intermediate, and at pH > 4.5 the stability rapidly decreased.
In acidic foods, vitamin C and thiamine are more stable, while flavonoids are also
less stable if the pH is alkaline.226
Cabrita et al.196 also studied the effect of pH on anthocyanin stability. Anthocyanin 3- glucoside solutions in the range of pH 1 to 12 were stored in dark over 60
days at 10°C and 23ºC. At pH 1 to 3, more than 70% of initial anthocyanin
concentrations remained after 60 days at 10ºC, while considerable losses (> 90%)
occurred at pH 5 to 6 after 8 days. Similar stability patterns occurred at the higher
temperature of 23ºC, although the rate of anthocyanin degradation was higher. Only
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40% of the initial anthocyanins were detectable after 60 days. Kalt et al.192 showed
that anthocyanin content and antioxidant capacity in lowbush blueberry fruit were
greatest in pH 1 extracts, compared to extracts at pH 4 and 7.
16.5.8 OXYGEN
AND
LIGHT
Atmospheric oxygen is responsible for most losses of vitamin C during storage.194,225
Oxygenation is also detrimental to both anthocyanins and antioxidant capacity in
fruit products.192 It is known that anthocyanins stored under vacuum or nitrogen are
more stable than when exposed to oxygen.194 Kalt et al.192 found that in vials completely filled with blueberry juice that had little oxygen-containing head space, there
was no loss in anthocyanins after 6 h, while in the half-full vessels, 76% of monomeric anthocyanins were lost during the same period. Total phenolic content and
antioxidant capacity did not decrease in sealed vials, but decreased 30% and 46%,
respectively, in those with air, over the same period. In strawberry juice at 45°C,
degradation of pelargonidin 3-glucoside is very weak from pH 2.0 to 4.5 in the
absence of O2, whereas degradation increased considerably at pH 4.0 in the presence
of O2.228 It is generally advisable that oxygen should be eliminated during processing
and storage of juice or when anthocyanins are used as a food colorant. In cranberry
juice, elimination of oxygen tripled the half-life of color.262 Addition of ascorbic
acid can protect anthocyanins from oxidation and degradation of juice color.263
Flavonoids are highly sensitive to degradation in the presence of light, visible
and UV.183,197Light has been reported to be a major cause of anthocyanin degradation
in elderberry extract.264 The short wavelength had been proven to have a more
deteriorating effect. Packing material with proper light barriers in visible and the
ultraviolet range of spectrum showed protective effects. Therefore, fruit juices and
fruit-based beverages should be kept in the dark during storage. Glycosidation,
acylation, and copigmentation have been reported to improve light stability for
anthocyanins.265,266 Light has also been shown to reduce carotene and to convert all
trans form of β-carotene to cis forms of β-carotene, which are then oxidized. The
rate of cis-isomer oxidation was much greater than the rate of formation under heat
and light treatment, resulting in loss of carotenoid activity.187 Light also destroys
vitamin C. Therefore, thawing fruits in the dark can protect vitamin C.
16.5.9 ENZYMES
In beverages, as in other food products, enzymes may occur naturally or may be
added. Enzymes perform many functions in beverages. They can help increase yields,
provide nutrients for the fermentation process, facilitate processing, and affect the
color, flavor, and clarity of the finished product. Adding pectinase in making grape
wine can accelerate the increase of pigments from the grape skin. In juice processing,
pectin methyl esterase, polygalacturonase and pectinase are mainly used to increase
yield and clarify the juice.267 Nariginases (a combination of enzymes containing αrhamnosidase and β- naturally) have been used to reduce naringenin by total hydrolysis or decrease naring 7-β-D-glucoside, a compound with low bitterness in citrus
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juice, particularly grapefruit juice.268 Limonin, another bitter citrus compound, may
also be present in citrus. This too can be enzymatically broken down.269
Enzymes can affect the anthocyanin content in fruits and juices. Polyphenoloxidase is the major enzyme causing anthocyanin destruction during blueberry juice
processing.270,271 Skrede et al.271 used frozen blueberries to process into juice and
concentrate, and to monitor the changes of anthocyanin and polyphenolics (cinnamates, procyanidins, flavonol glycosides). While juice yield was 83%, only 32% of the
anthocyanins were recovered in juice. Flavonol, procyanidin, and chlorogenic acid
recoveries in juice were 35%, 43%, and 53%, respectively. The proportion of
polyphenolics remaining in the press-cake residue ranged from 1% (chlorogenic
acid) to 18% (anthocyanins). Pronounced losses of anthocyanins and polyphenolics
during milling and depectinization are believed to be due to polyphenol oxidase.
Losses during concentration ranged from 1.5% (anthocyanins) to 20% (procyanidins). Malvidin glycosides were the most stable and delphinidin glycosides the
least.271 Peroxidase activity has been shown to be responsible for undesirable browning reactions, which appear in processed canned syrup strawberries after three
months. This undesirable browning reaction has been associated with turnover and
degradation of anthocyanin pigments.272,273 Glycoside enzymes can also be very
destructive of anthocyanins in berry fruits.214,274
16.6 GENETIC ENGINEERING TECHNIQUES
Genetic engineering techniques are considered to be among the most powerful and
economically promising techniques for use in many areas. Using traditional genetic
modification methods, such as cross-fertilization, scientists can produce desired
traits. But in doing so, they mix thousands of genes, requiring many attempts over
many years to remove the unwanted traits that occur. Genetic engineering techniques
are more precise, predictable, and sometimes faster. By controlling the insertion of
one or two genes into a plant, scientists can give it a specific new characteristic
without transferring undesirable traits. Examples of genetically engineered foods
that have been established so far include: rice, corn, and soybeans engineered to
contain altered levels of nutrients; corn, soybeans, tomato, and canola plants that
withstand herbicide application; corn, tomatoes, and potatoes that have their own
“built-in” pesticides; corn, tomatoes, potatoes, soybeans, grapes, cantaloupe, canola,
and other plants that have been manipulated to resist plant viruses; tomatoes, peas,
peppers, and fruits engineered to improve processing and extend shelf life; and
various enzymes used to make wine, fruit juice, sugar, beer, and oil.275
While little information is available on modifying phytonutrients in fruits, successful genomic-based approaches to metabolically engineer the vitamins in other
crops has been reported. A good example is the engineering of high levels of βcarotene in the endosperm of “golden rice.”276−278 In the “golden rice,” daffodil
phytoene synthase and lycopene β-cyclase were placed under the control of the
endosperm-specific glutelin promoter, while phytoene desaturase was placed under
the control of the CaMV 35S promoter (35S::tp::crtI). As a result, phytoene synthase
and phytoene desaturase were sufficient not only for the synthesis of lycopene, but
also for that of β-carotene and zeaxanthin. This led to “golden rice” which contained
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up to 200 µg β-carotene per 100 g (i.e., a tenth of the RDA in a daily intake of 300
g of rice).276−278 Although this is far from the RDA, it will have a high dietary impact
on populations of Asia, Africa, and Latin America where avitaminosis A is widespread and rice consumption is high.
Tomatoes contain lycopene and a variable amount of β-carotene. “Good” tomato
cultivars contain >10 mg per 100 g fresh weight lycopene, which is a dietary
antioxidant reported to lower the risk of certain cancers.279 Another aim of genetic
engineering has been to increase the content of lycopene in tomatoes. Mehta et al.280
demonstrated that the lycopene levels in engineered tomatoes increased 2 to 3.5
times compared to the nonengineered tomatoes.
Canola is another example. Canola seeds and oil contain negligible amounts of
β-carotene. In this case, the simple overexpression of a bacterial phytoene synthase,
crtB, fused to a plastidic transit peptide and under the control of a seed-specific
napin promoter, was sufficient to boost β-carotene levels 300-fold, to a level similar
to that found in oil palm.281 Therefore, it seems to be possible to enhance the
flavonoid content in fruits by genetic modification.
These research findings show great promise for increasing the flavonoids and
vitamins of agricultural crops and are a good example of the potential that exists
for manipulating phytonutrients in fruits. Thus, the genetics of the fruits can sometimes be altered to create plants that offer higher levels of phytonutrients, so-called
“designer functional foods” for enhancing human health.282,283 Furthermore, the
advent of the human genome project has led to the concept of “nutrigenomics,”
where a person’s nutritional intake would be custom-tailored to their DNA profile.
Therefore, the foods of the future are emerging. Rather than taking out certain
ingredients, we can begin to engineer new nutrients that have been clinically proven
to help protect against diseases and can be individualized according to a person’s
needs. In the future, fruits and other functional foods will be seen as a powerful new
tool that consumers can use to safeguard against diseases.283−285
16.7 CONCLUSIONS
Of the 10.3 million new cases of cancer diagnosed around the world each year, as
many as two-thirds may be caused by dietary and lifestyle factors. Diets rich in
fruits and vegetables may decrease the risk of many types of cancer and other
degenerative diseases. The reason fruits and vegetables have such health benefits is
because they contain abundant phytonutrients. Many of these phytonutrients have
antioxidant capacity. Recent advances in structural and functional genomics, as well
as technical advances in plant breeding, bioengineering, and biotechnology make it
possible now more than ever to create designer foods for consumers. Carrots have
been bred to contain elevated levels of β-carotene, and tomatoes have been selected
for increased concentrations of lycopene. New hybrids of various fruits with high
phytonutrient content and high antioxidant capacity can be achieved for providing
the protective effects they may confer in cellular detoxification processes. Therefore,
enhanced antioxidant capacity of fruit crops can be achieved through plant breeding
and genetic engineering and biotechnology techniques.
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Selection and breeding of fruit crops from a diverse genetic base with known
concentrations of phytonutrients is a key step in developing an improved food supply.
In addition, preharvest factors may influence the concentration and stability of
phytonutrients with nutritional value. For example, climate, temperature, and light
all affect antioxidant activity in various fruit crops. The stage of crop maturity also
significantly influences the antioxidant capacity of various fruits. One of the most
nutritionally significant applications of modern technologies may well be to retard
the softening process so that fruits can be harvested and marketed at a more mature
stage, when more of the phytocompounds have already been biosynthesized. Postharvest evaluation of the phytochemical composition of fruit crops is an important
component in assessing the impact of handling procedures on the nutritional content
of a fruit. New research is being initiated to develop a better understanding of how
transport and storage influence their chemical composition. Research is needed to
develop the best processing techniques to reduce the degradation of nutritive factors
such as flavonoids, carotenoids, catechins, and bioactive peptides. Despite increasing
media attention in the past few years, the science of phytonutrients is still an
embryonic discipline. A number of priority research areas on phytonutrients need
to be emphasized and evaluated. Future research should include: (a) improved
selection criteria among different fruit crops including variety selection, molecular
genetic breeding, and maturity determination at time of harvest; (b) investigation of
the influence of production practices on the formation of selected phytonutrient
compounds; and (c) evaluation of postharvest conditions of transport, storage, and
processing techniques on the stability of phytonutrient compounds in fruits. Knowledge gained from these research studies will be helpful in improving human health
by optimizing the nutritional content and quality of fruits in the diet.
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