Fruit and cereal bioactives: sources, chemistry, and applications 9781439806678, 1439806675

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Edited by

Özlem Tokus¸og˘lu  Clifford Hall III

Fruit and Cereal Bioactives Sources, Chemistry, and Applications

Fruit and Cereal Bioactives Sources, Chemistry, and Applications

Edited by

Özlem Tokus¸og˘lu  Clifford Hall III

Boca Raton London New York

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

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2011 by Taylor and Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-1-4398-0667-8 (Ebook-PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

To my mother, retired teacher Özden Tokuşoğlu & my father, retired senior colonel Armağan Tokuşoğlu, for their great emotional support and cordial encouragements.

Özlem Tokus¸ og˘lu

© 2011 by Taylor & Francis Group, LLC

Contents Preface....................................................................................................................................................... ix Editors........................................................................................................................................................ xi Contributors.............................................................................................................................................xiii

Part I  Introduction 1. Introductıon to Bioactives in Fruits and Cereals........................................................................... 3 Özlem Tokuşoğlu and Clifford Hall III 2. Health Promoting Effects of Cereal and Cereal Products............................................................ 9 Joseph M. Awika

Part II Chemistry and Mechanisms of Beneficial Bioactives in Fruits and Cereals 3. Phytochemicals in Cereals, Pseudocereals, and Pulses............................................................... 21 Clifford Hall III and Bin Zhao 4. Phenolic and Beneficial Bioactives in Drupe Fruits..................................................................... 83 Özlem Tokuşoğlu 5. Bioactive Phytochemicals in Pome Fruits................................................................................... 107 Özlem Tokuşoğlu 6. Phytochemicals in Citrus and Tropical Fruit............................................................................ 123 Mehmet Çağlar Tülbek 7. Phytochemical Bioactives in Berries............................................................................................143 Özlem Tokuşoğlu and Gary Stoner 8. Phenolic Bioactives in Grapes and Grape-Based Products.......................................................171 Violeta Ivanova and Marina Stefova 9. Nut Bioactives: Phytochemicals and Lipid-Based Components of Almonds, Hazelnuts, Peanuts, Pistachios, and Walnuts........................................................185 Biagio Fallico, Gabriele Ballistreri, Elena Arena, and Özlem Tokuşoğlu 10. Nut Bioactives: Phytochemicals and Lipid-Based Components of Brazil Nuts, Cashews, Macadamias, Pecans, and Pine Nuts.................................................213 Biagio Fallico, Gabriele Ballistreri, Elena Arena, and Özlem Tokuşoğlu 11. Bioactive Lipids in Cereals and Cereal Products...................................................................... 229 Ali A. Moazzami, Anna-Maija Lampi, and Afaf Kamal-Eldin © 2011 by Taylor & Francis Group, LLC

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Contents

Part III  Mycotoxic Bioactives of Fruits and Cereals 12. Mycotoxic Bioactives in Cereals and Cereal-Based Foods........................................................ 253 Anuradha Vegi 13. Control Assessments and Possible Inactivation Mechanisms on Mycotoxin Bioactives of Fruits and Cereals.......................................................................... 273 Faruk T. Bozoğlu and Özlem Tokuşoğlu 14. Control of Mycotoxin Bioactives in Nuts: Farm to Fork............................................................291 Mohammad Moradi Ghahderijani and Hossein Hokmabadi

Part IV Functionality, Processing, Characterization, and Applications of Fruit and Cereal Bioactives 15. Isolation Characterization of Bioactive Compounds in Fruits and Cereals............................319 Xiaoke Hu and Zhimin Xu 16. Effect of Bioactive Components on Dough Rheology, Baking, and Extrusion....................... 337 Joseph M. Awika 17. Impacts of Food and Microbial Processing on the Bioactive Phenolics of Olive Fruit Products................................................................................................ 347 Moktar Hamdi 18. Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals.........................361 Reşat Apak, Esma Tütem, Mustafa Özyürek, and Kubilay Güçlü 19. Supercritical Fluid Extraction of Bioactive Compounds from Cereals................................... 385 Jose L. Martinez and Deepak Tapriyal 20. Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives....... 409 Marina Stefova and Violeta Ivanova 21. High Pressure Processing Technology on Bioactives in Fruits and Cereals........................... 429 Özlem Tokuşoğlu and Christopher Doona

© 2011 by Taylor & Francis Group, LLC

Preface Interest in bioactive compounds of fruit and cereals has reached a new high in recent years. The scientific and commercial attention devoted to fruit and cereal bioactives has been accentuated even further by efficiency reports regarding the beneficial and toxic health effects of such compounds. The beneficial bioactives of many fruit and cereals have been declared to possess anticarcinogenic, antimutagenic effects in test animals. Recently, the strong antioxidant capacities of many edible fruits and cereals have been revealed. These many bioactive compounds are responsible for several important characteristics of fruit and cereals: taste, flavor, color alteration, and antioxidant activity. Natural toxicant bioactives as mycotoxins have also been detected in specific fruits and cereals. The specific focus for Fruit and Cereal Bioactives is on the chemistry of beneficial and nutritional bioactives (phytochemicals such as phenolics, flavonoids, tocols, carotenoids, phytosterols, avenanthramides, alkylresorcinols, some essential fatty acids) and toxicant bioactives (mycotoxins, aflatoxins, ocratoxin A, etc.) from sources such as pome, stone, and berry fruits, citrus fruits, tropical fruits and nuts, various cereals (and pseudocereals), pulses (e.g., legumes and edible beans), and so on. Overall, this book is a comprehensive and detailed reference guide to both major natural beneficial phytochemical bioactives and mycotoxic bioactives in edible fruits and cereals covering all the latest research from a wide range of experts. This book is intended for senior undergraduate and graduate students, academicians, and those in government and the fruit and cereal industry. It provides a practical reference for a wide range of experts: fruit and cereal scientists, chemists, biochemists, nutritionists, fruit and cereal processors, government officials, commercial organizations, and other people who need to be aware of the main issues concerning bioactives. Each chapter reviews dietary sources, occurrences, chemical properties, desirable and undesirable health effects, antioxidant activity, evidentiary findings, as well as toxicity of the above-mentioned bioactives and has been individually highlighted based on the fruit and cereal type. Fruit and Cereal Bioactives presents unique, up-to-date, and unified data of fruit and cereal chemistry from a biochemical standpoint.

Özlem Tokus¸ og˘lu

© 2011 by Taylor & Francis Group, LLC

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Editors Özlem Tokus¸ og˘lu, who was born in İzmir, Turkey, completed her bachelor (1992) and master (1996) degrees at EGE University from the Department of Chemistry and completed her doctorate at EGE University from the Department of Food Engineering (2001). She worked as a research assistant and Dr. Assistant at EGE University from 1993 to 2001. She was the research assistant at the Food Science and Nutrition Department at the University of Florida–Gainesville during 1999–2000. Dr. Tokuşoğlu has been an assistant professor at Celal Bayar University, Manisa, Turkey and is currently working there in the Department of Food Engineering. She is focusing on food quality control, food chemistry, food safety, and food processing technologies on traditional foods and beverages. Her specific study areas are phenolics, phytochemicals, bioactive antioxidative components, bioactive lipids, and their determinations by instrumental techniques, their effects on food and beverages quality, and the novel food processing effects on their levels. Dr. Tokuşoğlu performed academic research studies and presentations at Geneva, Switzerland in 1997; Gainesville, Florida in 1999; Anaheim–Los Angeles, California in 2002; Sarawak, Malaysia in 2002; Chicago, Illinois in 2003; Katowice-Szczyrk, Poland in 2005; Ghent, Belgium in 2005; Madrid, Spain in 2006; New Orleans, Louisiana in 2008; Athens, Greece in 2008; Anaheim–Los Angeles, California in 2009; and Skopje, the Republic of Macedonia in 2009; Chicago, Illinois in 2010; Munich, Germany in 2010. She was also a visiting professor at the School of Food Science, Washington State University, Pullman, in the state of Washington for one month during 2010. Dr. Tokuşoğlu has professional affiliations at the Institute of Food Technologists (IFT) and the American Oil Chemists’ Society (AOCS) in the United States and has a professional responsibility with the Turkey National Olive and Olive Oil Council (UZZK) as a research and consultative board member and as a Turkish Lipid Group (YABITED) founder administrative board member and consultative board member in the European Federation for Science and Technology (Euro Fed Lipid). Dr. Tokuşoğlu has 78 ­international studies containing 25 papers published in peer-reviewed international journals covered by the Science Citation Index (SIC) and 11 papers published in peer-reviewed international index covered journals, 42 presentations (as orals and posters) presented at the international congress and other organizations. She has advised two masters’ students to completion. Dr. Tokuşoğlu has several editorial assignments in international index covered journals. Clifford Hall III completed his bachelor degree in 1988 at the University of Wisconsin–River Falls; his masters (1991) and doctoral (1996) degrees at the University of Nebraska–Lincoln in the area of food science and technology. He completed a postdoctoral experience at the University of Arkansas in Fayetteville. Dr. Hall is currently an associate professor in the Department of Cereal and Food Sciences in the School of Food Systems at North Dakota State University (NDSU). He is the associate director of the Great Plains Institute of Food Safety and food science coordinator for the Food Science program at NDSU. Much of his research deals with lipid oxidation and antioxidant chemistry, stability of phytochemicals in food processing, and utilization of nontraditional ingredients in food systems. The stability of flaxseed bioactives and antioxidant activity of raisins has been his major focus recently, including the evaluation of flaxseed lignan stability in extruded bean snacks. He has published his research in 28 peer-reviewed international journals, and 12 proceedings, and has published 10 book chapters. His research has created 60 oral and poster presentations at the American Oil Chemists’ Society, Institute of Food Technologists, International Society of Nutraceutical and Functional Foods, and AACC International annual meetings. He has advised five PhD and two masters’ students to completion and currently advises two PhD and three masters’ students. He has also mentored 28 undergraduate researchers and has served on 26 graduate student committees. Professionally, Clifford has been most active in the AOCS and AACC International. © 2011 by Taylor & Francis Group, LLC

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Editors

He served as the secretary/treasurer, 2003; vice chairperson, 2004; and chairperson, 2005–2007 for the Lipid Oxidation and Quality Division of the American Oil Chemists’ Society. He served as the chair of the Best Paper Competition Committee for the Lipid Oxidation and Quality Division, 2003–2006. He has also served as the chairperson of the Education Division for AACC International, 2007–2009 and on the AACC International Foundation as a board member, 2008 to the present; and chair, 2009. He has also served as an associate editor from 1998 to 2006 and senior associate editor from 2006 to the present for the Journal of the American Oil Chemists’ Society. In addition, he is an ad hoc reviewer for Food Chemistry, Journal of Food Science, and Journal of Agricultural and Food Chemistry.

© 2011 by Taylor & Francis Group, LLC

Contributors Reşat Apak Department of Chemistry Istanbul University İstanbul, Turkey

Kubilay Güçlü Department of Chemistry Istanbul University İstanbul, Turkey

Elena Arena Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari (DOFATA) Sez. Tecnologie AgroAlimentari Università degli Studi di Catania Catania, Italy

Clifford Hall III School of Food Systems North Dakota State University Fargo, North Dakota

Joseph M. Awika Soil and Crop Science Department Texas A&M University College Station, Texas Gabriele Ballistreri Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari (DOFATA) Sez. Tecnologie AgroAlimentari Università degli Studi di Catania Catania, Italy

Moktar Hamdi National Institute of Applied Sciences and Technology University of 7th November at Carthage Laboratory of Microbial Ecology and Technology Tunis, Tunisia Hossein Hokmabadi Department of Horticulture Pistachio Research Institute of Iran Rafsanjan, Iran

Faruk T. Bozoğlu Department of Food Engineering Engineering Faculty Middle East Technical University Ankara, Turkey

Xiaoke Hu Department of Chemistry Louisiana State University Baton Rouge, Louisiana

Christopher Doona U.S. Army – Natick Soldier Research Development and Engineering Center DoD Combat Feeding Directorate Natick, Massachusetts

Violeta Ivanova Institute of Chemistry Faculty of Natural Sciences and Mathematics Ss Cyril and Methodius University Skopje, Republic of Macedonia

Biagio Fallico Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari (DOFATA) Sez. Tecnologie AgroAlimentari Università degli Studi di Catania Catania, Italy

Afaf Kamal-Eldin Department of Food Science Swedish University of Agricultural Sciences Uppsala, Sweden

Mohammad Moradi Ghahderijani Department of Plant Protection Pistachio Research Institute of Iran Rafsanjan, Iran © 2011 by Taylor & Francis Group, LLC

Anna-Maija Lampi Department of Chemistry and Applied Microbiology University of Helsinki Helsinki, Finland xv

xvi Jose L. Martinez Thar Process, Inc. Pittsburgh, Pennsylvania

Contributors Özlem Tokuşoğlu Department of Food Engineering Celal Bayar University Manisa, Turkey

Ali A. Moazzami Department of Food Science Swedish University of Agricultural Sciences Uppsala, Sweden

Mehmet Çağlar Tülbek Northern Crops Institute North Dakota State University Fargo, North Dakota

Mustafa Özyürek Department of Chemistry Istanbul University İstanbul, Turkey

Esma Tütem Department of Chemistry Istanbul University İstanbul, Turkey

Marina Stefova Institute of Chemistry Faculty of Natural Sciences and Mathematics Ss Cyril and Methodius University Skopje, Republic of Macedonia

Anuradha Vegi Department of Veterinary and Microbiological Sciences North Dakota State University Fargo, North Dakota

Gary Stoner Department of Internal Medicine The Ohio State University Columbus, Ohio

Zhimin Xu Department of Food Science Louisiana State University Agriculture Center Baton Rouge, Louisiana

Deepak Tapriyal Thar Process, Inc. Pittsburgh, Pennsylvania

Bin Zhao Kraft Foods, Inc. East Hanover, New Jersey

© 2011 by Taylor & Francis Group, LLC

Part I

Introduction

© 2011 by Taylor & Francis Group, LLC

1 Introduction to Bioactives in Fruits and Cereals Özlem Tokus¸ og˘lu and Clifford Hall III CONTENTS Phytochemicals in Fruit and Cereals........................................................................................................... 3 Phenolics in Fruit and Cereals............................................................................................................... 3 Carotenoids in Fruit and Cereals........................................................................................................... 5 Functional Lipids and Lipid Soluble Constituents................................................................................ 5 Mycotoxic Bioactives in Fruits and Cereals............................................................................................... 7 Concluding Remarks................................................................................................................................... 7 References................................................................................................................................................... 7 Fruit and cereal bioactives are classified as phytochemicals and toxicant secondary metabolites. Phytochemicals containing polyphenols, carotenoids, and functional lipids are naturally derived substances that have health-promoting, and/or nutraceutical and medicinal proper while mycotoxigenic bioactives are toxic substances that are secondary metabolites synthesized by toxigenic fungal species. A wide variety of mycotoxins are produced by various fungi, often a single fungal species can synthesize more than one type of mycotoxic bioactive under optimal conditions. Interest in the bioactive compounds of fruit and cereals has reached a new high in recent years. Especially, the scientific and commercial attention in fruit and cereal bioactives have been accentuated by efficiency reports regarding both beneficial and toxical health effects of such compounds. According to the National Institutes of Health (NIH), bioactive food phytochemicals including polyphenols, carotenoids, and functional lipids are “constituents in foods or dietary supplements, other than those needed to meet basic human nutritional needs, that are responsible for changes in health status.” Major sources of these bioactive food components are plants, especially fruits, vegetables, and cereals. But major sources of both phytochemicals and mycotoxins are fruits, nuts, and more major in cereals. In this book context, a brief description of the chemistry, sources, and applications of the abovementioned major bioactives in fruits and cereals.

Phytochemicals in Fruit and Cereals Phenolics in Fruit and Cereals As the name suggests, phytochemicals working together with chemical nutrients found in fruits, cereals, and nuts may help slow the aging process and reduce the risk of many diseases, including cancer, heart disease, stroke, high blood pressure, cataracts, osteoporosis, and urinary tract infections (Meskin et al. 2003; Omaye et al. 2000). Polyphenols occur as plant secondary metabolites. Their ubiquitous presence in plants and plant foods, favors animal consumption and accumulation in tissues. Polyphenols are widely distributed in the plant kingdom and represent an abundant antioxidant component of the human diet (Ho, Rafi and Ghai, 2007). Interest in the possible health benefits of polyphenols has increased due to the © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

corresponding antioxidant capacities (Gharras, 2009). Recent evidences show that there is a great interest to anticarcinogenic effects of polyphenolic compounds, as well as the potential to prevent cardiovascular and cerebrovascular diseases (Cheynier 2005). Polyphenols divide into several subgroups including flavonoids, hydroxybenzoic and hydroxycinnamic acids, lignans, stilbens, tannins, and coumarins that have specific physiological and biogical effects (Andersen and Markham 2006; Meskin et al. 2003; Tokuşoğlu 2001; Figure 1.1). Flavonoids are a chemically defined family of polyphenols that includes several thousand compounds. The flavonoids have a basic structure (Figure 1.2), and several subclasses of flavonoids are characterized by a substitution pattern in the B- and C-rings. The main subclasses of flavonoids include flavan-3-ols, flavonols, flavones, flavanones, isoflavones, anthocyanidins, anthocyanins, flavononols, and chalcons (Figure 1.3) that are distributed in plants and food of plant origin (Crozier, Jaganath, and Clifford 2006). Flavonoids in the circulation may protect against cardiovascular disease through their interaction with low-density lipoprotein (LDL). Biochemical and clinical studies in both humans and experimental animals have suggested that oxidized low-density lipoprotein (oLDL) has its atherogenic action through the formation of lipid hydroperoxides and the products derived therefrom. The in vivo antioxidant status of the LDL particles and the plasma are thus important determinants of the susceptibility of LDL to lipid peroxidation (Hertog et al. 1993). Many of the phytochemicals and some vitamins (vitamin E, tocopherol) have antioxidant activity in vitro, which has led to the use of the general term “antioxidants.” Phenolic compounds

Coumarins

Flavonoids Flavons Phenolic acids Isoflavons Hydroxybenzoic acids Flavonols Hydroxycinnamiz acids Flavanols Flavanones Anthocyanidins Anthocyanins Flavononols Chalcons

Lignans

Tannins

Stilbens

Sesamol Sesamin Sesamolin Sesamolinol

Hydrolyzed Condensed

Resveratrol Piceatannol Piceid Pinosylvin Rhapontisin Tamoxiphen Derivative Phytoalexins

FIGURE 1.1  Family of phenolic compounds. (From Andersen, Q. M., and Markham, K. R., Flavonoids. Chemistry, Biochemistry, and Applications, CRC Press, Taylor & Francis, Boca Raton, FL, 2006; Meskin, M. S., Bidlack W. R., Davies, A. J., Lewis, D. S., and R. K. Randolph, Phytochemicals: Mechanisms of Action. CRC Press, Boca Raton, FL, 2003; Tokuşoğlu, Ö., The Determination of the Major Phenolic Compounds (Flavanols, Flavonols, Tannins and Aroma Properties of Black Teas, PhD Thesis, Department of Food Engineering, Bornova, Izmir, Turkey: Ege University, 2001).)

3 4

2 8 7

B O

A

C

5

4

6

5 6 3

FIGURE 1.2  Chemical structure of flavonoids.

© 2011 by Taylor & Francis Group, LLC

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Introduction to Bioactives in Fruits and Cereals Flavonoids

Chalcons Flavons Apigenin Luteloin Baikalein Krysin Diosmin Genkvain Izorhoifolin Rhoifolin Tektokirisin

Isoflavons Daidzein Genistein Biokenin A Formononetin Glisitein Daidzin Genistin Glisitin 6 -O-Asetildaidzin 6 -O-Asetilgenistin 6 -O-Asetilglisitin 6 -OMalonildaidzin 6 -OMalonilgenistin 6 -OMalonilglisitin

Flavonols Quercetin Kaempferol Miricetin Quercitrin Isoquercitrin Rhamnetin Isorhamnetin kaempferid Rutin Astragalin Hiperosid

Flavan-3-ols (+)–Catechin (–)–Epicatechin (–)–Epicatechingallate (–)–Epigallocatechin (–)–Epigallocatechingallate

Flavanons Hesperetin Hesperitin Naringenin Naringin Narirutin Didimin Eriositrin Eriodiktiol Neoriositrin Neohesperitin Izosakuranetin Pinosembrin Ponsirin Prunin

Flavononols (Dihydroflavonols) Anthocyanidins Cyanidin Malvinidin Delfinidin Pelargonidin Petunidin Peonidin

Anthocyanins Grape extract

FIGURE 1.3  Flavonoid family in food plants. (Adopted from Tokuşoğlu, Ö., The Determination of the Major Phenolic Compounds (Flavanols, Flavonols, Tannins and Aroma Properties of Black Teas, PhD Thesis, Department of Food Engineering, Bornova, Izmir, Turkey: Ege University, 2001; Merken, H. M., and Beecher, G. R., J. Agric. Food Chem., 48(3), 579–95, 2000; Beecher, G. R., Antioxidant Food Supplements in Human Health, Academic Press, New York, 1999; Fennema, O. R., Food Chemistry, Marcel Dekker, New York, 681–96, 1996; Vinson, J. A., Dabbagh, Y. A., Serry, M. M., and Jang, J., J. Agric. Food Chem., 43, 2800–2802, 1995.)

Carotenoids in Fruit and Cereals Carotenoids (Figure 1.4), a group of lipid-soluble compounds responsible for yellow, orange, red, and violet colors of various fruits and cereals products, are one of the most important groups of natural pigments, owing to their wide distribution, structural diversity, and numerous biological functions (Astorg 1997; Fraser and Bramley 2004). The provitamin A activity of some carotenoid bioactives, recently, have demonstrated to be effective in preventing chronic diseases such as cardiovascular disease and skin cancer. Carotenoid bioactives are classified into four groups: carotenoid hydrocarbons, carotenoid alcohols (xanthophylls), carotenoid ketons, carotenoid acids. Hydrocarbon carotenoids are known as carotenes, and the oxygenated derivatives are termed xanthophylls (Astorg 1997; Fraser and Bramley 2004; Lee and Schwartz 2005)

Functional Lipids and Lipid Soluble Constituents There has been a great interest concerning functional lipids in cereals due to their promotion for health and preventing diseases. Fatty acids play a central role in growth and development through their roles in membrane lipids, as ligands for receptors and transcription factors that regulate gene expression, as a precursor for eicosanoids, in cellular communication, and through direct interactions with proteins. The main fatty acids in cereals are the saturated fatty acids, palmitic (16:0) and stearic (18:0), the monounsaturated fatty acid oleic acid (18:1), and the diunsaturated fatty acid inoleic acid (18:2) existing with smaller amounts of other fatty acids. These fatty acids are mainly assembled in glycerolipids; that is, triacylglycerols (TAG) and variable amounts of phospholipids (PL), glycolipids (GL), in sterol esters (SE), and waxes (or policosanols) in the different cereal grains. Lipid soluble vitamins tocopherols and amphiphilic lipids alkylresorcinols, and terpen alcohol compounds are also important bioactive constituents in cereal grains (Figure 1.5). Cereal lipids have high levels of tocotrienols that coexist with tocopherols, which are the biologically most active antioxidants © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Lycopene BCO

15

BCO-2 10′ 9′

15′ all-trans-β-carotene

α-carotene OH

HO

Lutein

HO

β-cryptoxanthin

H 3C

CH3

HO

CH3

H 3C

CH3

CH3

CH3

CH3

OH

H3C CH3

Zeaxanthine FIGURE 1.4  Major carotenoids. (Ross, C. A. and Harrison, E. H., Handbook of Vitamins, Taylor & Francis Group, Boca Raton, FL, 1–39, 2007.)

R1 HO

H

R2

H

H

H

O Tocopherols

R1

HO

H

Trematol

HO

H

Fernenol

HO R2

O

H

Tocotrienols

HO

OH

H

H

H

Isoarborinol

HO

H

H

Sorghumol R2

R1 HO

n=13-23

H

H

H

H

H

H

Alkyresorcinols

HO

HO Simiarenol Triterpen alcohols -Amyrin R1= methyl, R2 = hydrogen -Amyrin R1= hydrogen, R2 = methyl

FIGURE 1.5  Some lipid soluble constituents and cereal grains.

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Introduction to Bioactives in Fruits and Cereals

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(Peterson 2004). Alkylresorcinols have been shown to have bioactivities in vitro and in vivo experiments. They increase the γ-tocopherol level in rat liver and lung by possibly inhibiting γ-tocopherol metabolism (Ross, Kamal-Eldin, and Aman 2004). Sterols and sterol-based constituents, terpenoids play a role in traditional herbal remedies and it is reported they show antibacterial, cholesterol-lowering, antiatherogenic, and anticarcinogenic effects. Phytosterols appear not only to play an important role in the regulation of cardiovascular disease but also to exhibit anticancer properties (Jones & AbuMweis, 2009). Those beneficial bioactives of many fruits and cereals have been declared to possess anticarcinogenic and antimutagenic effects in test animals. Recently, it has also been detected in the strong antioxidant capacities of many edible fruits and cereals.

Mycotoxic Bioactives in Fruits and Cereals Mycotoxigenic bioactives are toxic substances that are produced by the secondary metabolism of various fungal species (Ho, Rafi and Ghai, 2007). Various studies have been reported about their high toxicity and the possible risk for consumer health. Fungal spoilage of cereals and mycotoxic bioactive production is most important. It has been shown that the presence of fungi on fruits is not necessarily associated with mycotoxin (aflatoxins, ochratoxin A, patulin, citrinin, T2, etc.) contamination. The mycotoxin formation depends more on endogenous and environmental factors than fungal growth does (Andersen and Thrane 2006). The studies indicated that Alternaria, and Fusarium in fruit and cereals may pose a mycotoxin risk. During spoilage of cherries and apples, Penicillum expansum is known to produce patulin. Both Alternaria and Fusarium are able to produce additional mycotoxic bioactives in moldy fruit samples: alternariols and aurofusarin. Penicillum verrucosum is known to produce Ochratoxin A in many cereals. Fusarium is able to produce zearalenone in addition to Ochratoxin A from P.verrucosum in moldy cereals. Aspergillus ochraceus, A.niger, and A.carbonarious produce Ochratoxin A in dried fruits such as raisins and currants (Iamanaka et al. 2006).

Concluding Remarks Fruit and Cereal Bioactives are comprised of the specific focus on the chemistry of beneficial and nutritional bioactives (phytochemicals such as phenolics, flavonoids, tocols, carotenoids, phytosterols, avenanthramides, alkylresorcinols, and some essential fatty acids) and toxicant biactives (mycotoxins; aflatoxins, ocratoxin A, patulin, citrinin, cyclopiazonic acid, T-2, fumonisin, deoksinivalenol, and zearalenon) from the sources of selected fleshy fruits including temperate fruits (pome, stone, and berry fruits), citrus and tropical fruits, nuts, and from various cereals (and pseudocereals), pulses (e.g., legumes and edible beans). Each chapter reviews dietary sources, occurrences, chemical properties, desirable and undesirable health effects, antioxidant activity, evidentiary findings, applications as well as toxicity of the abovementioned bioactives and have been individually highlighted based on the fruit and cereal type. Fruit and Cereal Bioactives present a unique and unified data to the fruit and cereal chemistry from a biochemical standpoint.

REFERENCES Andersen, B., and Thrane, U. 2006. Food-borne fungi in fruit and cereals and their production of mycotoxins. In Advances in Food Mycology. Vol. 571, 137–52. Berlin: Springer-Verlag. Andersen, Q. M., and Markham, K. R. 2006. Flavonoids. Chemistry, Biochemistry, and Applications. Boca Raton, FL: CRC Press, Taylor & Francis. Astorg, P. 1997. Food carotenoids and cancer prevention: An overview of current research. Trends Food Sci Tech 8:406–13. © 2011 by Taylor & Francis Group, LLC

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Beecher, G. R. 1999. Flavonoids in foods. In Antioxidant Food Supplements in Human Health, eds. L. Packer, M. Hiramatsu, and T. Yoshikawa. New York: Academic Press. Cheynier, V. 2005. Polyphenols in foods are more complex than often thought. Am. J. Clin. Nutr. 81 (Suppl): 223–9. Crozier, A., Jaganath, I. B., and Clifford, M. N. 2006. Phenols, polyphenols and tannins: An overview. In Plant Secondary Metabolites, eds. A. Crozier, M. N. Clifford, and H. Ashihara, 1–24. Oxford: Blackwell Publishing, Ltd. Fennema, O. R. 1996. Flavonoids. In Food Chemistry. 3rd ed., 681–96. New York: Marcel Dekker. Fraser, P. D., and Bramley, P. M. 2004. The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research 43: 228–65. Gharras, H. E. 2009. Polyphenols: Food sources, properties and applications—A review. Int J Food Sci and Technol. 44: 2512–8. Hertog, M. G. L., Feskens, E. J. M., Hollma, P. C. H., Katan, M. B., and Kromhout, D. 1993. Dietary antioxidant flavonoids and risk of coronary heart disease. The Zutphen Elderly Study. Lancet 342:1007–11. Ho, C. T., Rafi, M. M., and Ghai, G. 2007. Bioactive Substances: Nutraceuticals and Toxicants. In Fennema's Food Chemistry, 4th, eds. Srinivasan Damodaran, Kirk L. Parkin, Owen R. Fennema, CRC Press, Taylor & Francis, Boca Raton, FL, USA ISBN: 9780824723453, ISBN 10: 0824723457. 1160. Iamanaka, B. T., Taniwaki, M. H., Vicente, E., and Menezes, H. C. 2006. Fungi producing ochratoxin in dried fruits. In Advances in Food Mycology. Vol. 571, 181–88. Berlin: Springer-Verlag. Jones, P. J., and AbuMweis, S. S. 2009. Phytosterols as functional food ingredients: Linkages to cardiovascular disease and cancer. Curr Opin Clin Nutr Metab Care 12 (2): 147–51. Lee, J. H., and Schwartz, S. J. 2005. Analysis of carotenoids and chlorophylls in foods. In Methods of Analysis of Food Components and Additives, 179–98. New York: Taylor & Francis Group. Merken, H. M., and Beecher, G. R. 2000. Measurement of food flavonoids by high performance liquid chromatography: A review. J Agric Food Chem 48 (3): 579–95. Meskin, M. S., Bidlack W. R., Davies, A. J., Lewis, D. S., and R. K. Randolph. 2003. Phytochemicals: Mechanisms of Action. Boca Raton, FL: CRC Press. Omaye, S. T., Bidlack, W. R., Meskin, M. S., and D. K. W. Topham. 2000. Phytochemicals as Bioactive Agents. Lancaster, PA: Technomic Pub. Peterson, D. M. 2004. Barley tocols—Effects of milling, malting, and mashing. Cereal Chem 71 (1): 42–4. Ross, C. A., and Harrison, E. H. 2007. Vitamin A: Nutritional aspects of retinoids and carotenoids. In Handbook of Vitamins. 4th ed., eds. J. Zempleni, R. B. Rucker, D. B. McCormick, and J. W. Suttie, 1–39. Boca Raton, FL: Taylor & Francis Group. Ross, A. B., Kamal-Eldin, A., and Aman, P. 2004. Dietary alkylresorcinols: Absorption, bioactivities, and possible use as biomarkers of whole-grain wheat- and rye-rich foods. Nutr Rev 62 (3): 81–95. Tokuşoğlu, Ö. 2001. The Determination of the Major Phenolic Compounds (Flavanols, Flavonols, Tannins and Aroma Properties of Black Teas. PhD Thesis. Department of Food Engineering, Bornova, Izmir, Turkey: Ege University. Vinson, J. A., Dabbagh, Y. A., Serry, M. M., and Jang, J. 1995. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agric Food Chem 43: 2800–2.

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2 Health Promoting Effects of Cereal and Cereal Products Joseph M. Awika CONTENTS Introduction................................................................................................................................................. 9 Cereal Consumption and Cancer.............................................................................................................. 10 Possible Mechanisms of Cereal Grains in Chemoprevention...............................................................11 Dietary Fiber Related Mechanisms..................................................................................................11 Antioxidant Related Mechanisms....................................................................................................11 Phytoestrogen Related Mechanisms............................................................................................... 12 Mediation of Glucose Response..................................................................................................... 12 Cereal Grain Consumption and Cardiovascular Disease.......................................................................... 12 Cereal Grain Consumption in Obesity and Diabetes................................................................................ 13 Summary....................................................................................................................................................14 References..................................................................................................................................................14

Introduction Cereal grains are consumed as the primary source of energy by most humans. Consumption of whole/ unrefined cereal products is known to contribute significantly to health and chronic disease prevention. Whole cereal grains contain nutritionally significant quantities of dietary fiber, as well as various minerals and vitamins that are important for health. More recent evidence also indicates that cereals contain significant quantities of phytochemicals, like antioxidants and phytoestrogens, which may significantly contribute to reported health benefits of whole grain consumption. In most cases, these beneficial compounds are concentrated in outer layers (bran) of the grain (Table 2.1). Unfortunately, modern grain milling methods remove most of these compounds with the bran to produce refined endosperm fractions that are more appealing to consumers in most food applications. The refined grain products generally lack the health benefits that whole grains provide. At the moment, the vast majority of cereal products consumed around the world are made from refined grain. For example, in the United States, the Harris Interactive survey commissioned by the Grain Foods Foundation estimated that whole grain products constituted about 11% of total grain consumption in 2008. Additionally, only 10% of the U.S. population consumes the daily recommended whole grain intake of at least three servings per day. On the positive side, emerging strong links between unrefined grain-based diets and population health, coupled with public education, are renewing consumer interest in whole grain products. For example, various market trend data indicate that whole grain popularity is on the rise with consumers; between 2003 and 2008, the whole grain segment was among the fastest growing food product categories in the United States. The level of whole grain consumption in the United States in 2008 was 20% higher than it was in 2005. Efforts to promote whole grain consumption were until relatively recently not based on any strong epidemiological evidence of disease prevention (Slavin 1994), but mostly on recognized need for increased © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications TABLE 2.1 Antioxidant Activity and Dietary Fiber Content of Sorghum and Wheat Grain and Brana Antioxidant Activityb

Bran Dietary Fiber (% db)

Sample

Grain

Bran

Grain

Bran

Red wheat White sorghum Red sorghum Black sorghum Tannin sorghum CV %

10.6 9.8 53 104 240 3.2

36.3 30.1 230 378 890 4.3

12.6 6.3 10.3 9.8 11.1

47.6 38.3 43.9 45.3 44.5

a

b

Adapted from Awika, J. M., McDonough, C. M., and Rooney, L. W., Journal of Agricultural and Food Chemistry, 53(16), 6230–34, 2005; Awika, J. M., Rooney, L. W., Wu, X. L., Prior, R. L., and Cisneros-Zevallos, L., Journal of Agricultural and Food Chemistry, 51(23), 6657–62, 2003. µmol TE/g, measured by the ABTS method.

fiber intake that was known to improve fecal bulk and intestinal transit time, and thus believed to improve gut health. However, in the recent past, numerous epidemiological and intervention studies from around the world have demonstrated significant health benefits directly linked to whole grain consumption (Jacobs et al. 2000). Cereal grain-based products have been linked to reduced incidences of some types of cancer (Bidoli et al. 1992; Slattery et al. 1997), cardiovascular disease (CVD; Liu et al. 1999; Nettleton et al. 2009; Tighe et al. 2007), diabetes and obesity (Fung et al. 2001).

Cereal Consumption and Cancer Evidence linking grain consumption with cancer risk has been reported for some time, even though plausible mechanisms have been mostly speculative. Whole grain consumption is widely believed to help reduce cancer risk, whereas refined grain products have no beneficial effect. In fact, a few reports have linked increased consumption of some grains with an elevated risk of certain gastrointestinal cancers (Chen et al. 1993), even though such evidence could be attributed to other factors like aflatoxin (Isaacson 2005) that can be found in some grains, like corn, when grown in hot environments or handled improperly post harvest. Sorghum consumption has been particularly linked to reduced incidences of esophageal cancer in various parts of the world where this type of cancer was endemic, including parts of Africa, Iran, and China (Vanrensburg 1981). These findings were supported by epidemiological evidence linking sorghum and millet consumption with 1.4–3.2 times lower mortality from cancer of the esophagus in Sachxi Province of China (Chen et al. 1993). Interestingly, both authors reported no benefit or elevated risk of cancer of the esophagus with increased consumption of corn and wheat flour in these studies. The forms in which these grains were consumed in these regions were not reported. However, dietary patterns in these areas indicate that wheat, for example, is mostly consumed in a highly refined form in these areas. A case in point is China, where steamed bread, a major form in which wheat is consumed, is usually prized for whiteness and smooth texture, properties only possible with highly refined wheat flour. Such refined products have not been shown to contribute to chemoprevention. On the other hand the beneficial effects reported for sorghum consumption may be related to the fact that sorghum is mostly consumed with limited to no refining. Additional evidence also indicates that sorghum contains high levels of phytochemicals relative to other cereals (Awika et al. 2003). The sorghum phytochemicals may also have higher bioactivity than those found in other grains. For example, recent evidence demonstrates that some unique compounds in sorghum (e.g., 3-deoxyanthocyanins) may have stronger chemoprotective properties than their analogs from other plant sources (Yang, Browning, and Awika 2009). In the recent past, a flood of evidence (based on epidemiological and intervention studies) linking cereal grain consumption with reduced incidences of, especially, gastrointestinal cancer have emerged © 2011 by Taylor & Francis Group, LLC

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(Jacobs et al. 1998a; Kasum et al. 2001; Larsson et al. 2005; Levi et al. 2000; Schatzkin et al. 2008). In almost all cases, the positive benefits are only realized when grain is consumed in an unrefined form, or when cereal bran components are included in a diet. Thus it is safe to assume that the refined cereal endosperm products will not provide any meaningful health benefits beyond basic nutrition. For example, Larsson et al. (2005) reported a risk of 0.65 for colon cancer among those who consumed at least 4.5 servings of whole grain per day compared to those who consumed less than 1.5 servings. Levi et al. (2000) reported a significant reduction in the risk of oral, esophageal, and laryngeal cancer with increased consumption of whole grain as opposed to refined grain products. Numerous bodies of evidence that corroborate the link between whole grain consumption and gastrointestinal cancer are available in literature. Most of these investigations have, however, been conducted in developed countries. It is still not known how these data would translate to developing countries where malnutrition and presence of other confounding factors, like aflatoxin in grain, can be significant. This should be investigated since the developing countries consume a lot more cereal grain as a proportion of diet than the developed countries. Less clear is the link between whole grain consumption and some hormonally dependent cancers, such as breast cancer (La Vecchia and Chatenoud 1998). For example, a recent cohort study by Egeberg et al. (2009) failed to find a link between whole grain consumption and breast cancer risk among Danish postmenopausal women, similar to previous findings (Fung et al. 2005; Nicodemus, Jacobs, and Folsom 2001). On the other hand Kasum et al. (2001) reported that even though there was no statistical association between whole grain intake and endometrial cancer among postmenopausal women in general, a significant reduction in risk was observed when women who never used hormone replacement ­therapy were considered independently. In general, however, the link between breast and other hormonally dependent cancers and cereal grain consumption is weak. This may be due partly to the generally low levels and wide variation in phytoestrogens (usually lignans) in cereal grains. Additional evidence is needed in this regard.

Possible Mechanisms of Cereal Grains in Chemoprevention Various mechanisms have been proposed for the effects of whole grain on cancer risk based on animal and in vitro model studies. Since the strongest evidence of whole grain consumption and cancer risk are for gastrointestinal cancer, it is believed cereal components may exert their effects via direct interaction with gastrointestinal epithelial cells. The mechanisms can be summarized into four broad and generally inclusive categories: dietary fiber related mechanisms, antioxidant related mechanisms, phytoestrogen related mechanisms, and mediation of glucose response (Slavin 2000).

Dietary Fiber Related Mechanisms Dietary fiber is believed to impart its beneficial effect by two mechanisms: (1) increasing fecal bulk and reducing intestinal transit time, thus limiting interaction of potential fecal mutagens with intestinal epithelium, and (2) fermentation of soluble fiber by colon microflora to produce short chain fatty acids like butyrate, propionate, and acetate, which lower intestinal pH and promote gut health by diminishing bile acid solubility and cocarcionogenicity, and also possibly via direct suppression of tumor formation by butyrate (McIntyre, Gibson, and Young 1993). Thus, different cereal products may impact chemoprotection via different mechanisms depending on their dietary fiber composition.

Antioxidant Related Mechanisms Oxidative damage can lead to chronic cell injury, which is one of the mechanisms that may lead to cancer (Klaunig et al. 1998). Whole grains are rich in antioxidant phenolics (e.g., ferulates and flavonoids), ­vitamins (e.g., vitamin E), minerals (e.g., selenium), and other components mostly concentrated in their bran and germ. These dietary antioxidants directly suppress oxidative damage by quenching potentially damaging free radicals generated by various metabolic processes. They are also known to suppress the © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

growth of preformed cancer cells, which may contribute to elimination of cancer in early stages. Some of the antioxidants (e.g., selenium) are also cofactors of antioxidant enzymes, while others may enhance activity of protective phase II enzymes (Yang, Browning, and Awika 2009). For example, sorghum is an especially rich source of antioxidants (Table 2.1); this may partly explain the distinct chemoprotective properties against esophageal cancer reported for sorghum relative to corn or wheat.

Phytoestrogen Related Mechanisms Estrogenic effects of cereals may be produced by lignans that are found in low quantities in cereal brans. These plant lignans (e.g., secoisolariciresinol) can be metabolized by intestinal microflora into mammalian lignans like enterodiol, which are estrogenic. Other authors have suggested that dietary fiber may also interrupt enterohepatic circulation of estrogen, leading to increased fecal estrogen secretion (Goldin et al. 1982).

Mediation of Glucose Response Since a link between the cause of obesity and cancer has been suggested, it is believed that whole grains, through their effect of slowing glycemic response and thus insulin secretion, may contribute to chemoprevention (Schoen et al. 1999). See the section near the end of this chapter about cereal grain consumption in obesity and diabetes for more detail.

Cereal Grain Consumption and Cardiovascular Disease Cardiovascular disease (CVD) remains the leading cause of deaths in much of the developed world, and a major contributor to morbidity and health care costs. It has been long recognized that diets rich in unrefined grain or grain components, as well as dietary fiber can help significantly lower the risk for CVD (Trowell 1972), even though systemic evidence began emerging only in the latter part of the 1990s. A recent meta-analysis of several cohort studies estimated that an average of 2.5 servings of whole grain per day reduced the risk of CVD events by 21% compared to 0.2 servings/day of whole grain (Mellen, Walsh, and Herrington 2008). Evidence indicates that the beneficial effect of cereal grains on cardiovascular health may be related to bran components. For example, Jensen et al. (2004) reported that adding bran to a whole grain diet reduced coronary heart disease (CHD) risk by 30% compared to whole grain alone, which reduced the risk by 18% among male professionals aged 40–75 years. The authors found that the added germ had no effect on CHD risk. Similar findings have been documented in various other studies. This type of evidence initially led to the assumption that the dietary fiber in the bran part of whole grain was primarily responsible for the beneficial effect. However, other studies have found that the benefit of whole grain consumption cannot be fully explained by their dietary fiber content alone (Liu et al. 1999). Other than soluble and insoluble dietary fiber, cereal bran contains a complex mixture of antioxidant molecules, phytoseterols, policosanols, phytoestrogens, trace minerals, vitamins, and other compounds that have been associated with positive cardiovascular outcomes in controlled studies. Effects of cereal dietary fiber components on cardiovascular health are well documented. However, the exact mechanisms involved are not very clear. Some studies have reported a higher effect of insoluble cereal fiber on cardiovascular health than soluble fibers (Lairon et al. 2005), while others have reported the opposite effect. However, such inconsistencies may be due to the simple fact that it is often difficult, if not impossible, to isolate the effect of various forms of dietary fiber in cereals on cardiovascular health. In general, there is an agreement that soluble dietary fiber increases viscosity of gastric content, reducing the rate of absorption of nutrients. This may improve glycemic response and consequently reduce insulin demand and improve the blood lipid profile. The soluble fibers may also exert their effect via partial fermentation into short chain fatty acids by colon microflora; reducing colon pH and thus reducing bile acid solubility and sterol reabsorption. Some short chain fatty acids, especially butyric and propionic acid, may also directly inhibit cholesterol biosynthesis. © 2011 by Taylor & Francis Group, LLC

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Cereal bran wax components, specifically phytosterols and policosanols have been reported in various studies to reduce cholesterol absorption and biosynthesis. For example, sorghum dry distiller grain hexane extracts were shown to significantly reduce cholesterol absorption by up to 17% and non-HDL plasma cholesterol by up to 70% in animal models (Carr et al. 2005). The authors attributed the unusually potent effect of sorghum lipid extracts to the relatively high policosanol content of sorghum bran. Phenolics and other antioxidants found in cereal bran are also believed to contribute to cardiovascular health by reducing inflammation and LDL oxidation, as well as improving endothelial function, and inhibiting platelet aggregation. Some studies have also implicated phenolic compounds in cholesterol reduction (Fki, Sahnoun, and Sayadi 2007; Parker et al. 1996). Phytoestrogens found in cereal bran (mostly lignans) are hypothesized to promote favorable vascular responses to stress as well as endothelium-modulated dilation by inhibiting platelet aggregation or platelet release of vasoconstrictors (Anderson et al. 2000; Slavin, Jacobs, and Marquart 1997). It seems that the net effect of whole grain diets on cardiovascular health is a result of synergistic and complex interactions of dietary fiber with various minor components in ways that are not yet fully understood. This may also explain why isolated cellulose fiber does not produce similar cardiovascular benefits as whole grain or cereal bran (Kahlon, Chow, and Wood 1999).

Cereal Grain Consumption in Obesity and Diabetes Appetite suppression and control is the single most important mechanism to regulate calorie intake and thus affect weight gain. Satiety (longer duration between meals) and satiation (lower meal energy intake) play key roles in appetite control and energy intake. Whole grain products are believed to influence satiety and satiation due, at least partly, to their effect on glycemic response. Unrefined grain products are digested and absorbed more slowly, resulting in smaller postprandial glucose responses and insulin demand on the pancreatic β cells (Slavin, Jacobs, and Marquart 1997). By regulating insulin response, whole grain products may prevent problems associated with elevated blood insulin, including altered adipose tissue physiology and increased lipogenesis and appetite. Ludwig et al. (1999) reported that the high glycemic index (GI) foods may actually promote overeating in obese children. The authors reported that voluntary energy intake after a high GI meal was 53% higher than after a medium GI meal among obese teenage boys. On the other hand, Burton-Freeman and Keim (2008) reported that high GI meals resulted in greater satiety and suppression of hunger than low GI meals in obese women. The authors concluded that low GI diets may not be suitable for optimal appetite and satiety among overweight women. Such controversy is understandable given that satiety and GI are not by themselves precise measures of anything meaningful. Satiety is highly subjective and related to behavioral factors not fully understood. Additionally, GI in itself is highly variable depending on measuring conditions, among other factors, and its use as a predictor on the health impact of carbohydrate consumption remains very much questionable. Such variability have led some authors to propose doing away with the GI as such and evaluating meal quality based on individual and demonstrated merits like whole grain content (Sloth and Astrup 2006). All the same, glycemic response as a mechanism is useful in explaining some observations related to whole grain and dietary fiber intake. The reduced glycemic response of whole grain foods is partly attributed to the dietary fiber. Both soluble and insoluble dietary fiber found in whole grain products can provide a physical barrier to digestive enzymes, thus resulting in slow and sometimes incomplete digestion of starch. Indeed, it is known that whole grain products have higher type 1 resistant starch (physically inaccessible starch) than refined grain counterparts. The soluble part of dietary fiber may additionally increase gastric lumen viscosity that further slows digestion and macronutrient absorption. Another factor that may contribute to reduced insulin response is the reduced energy intake due to the bulking effect of dietary fiber that reduces energy density of a meal and increases satiation. However, dietary fiber alone does not explain the insulin response modulating properties of whole grain products. For example, long-term wheat bran consumption was shown to improve glucose tolerance better than pectin (Brodribb and Humphreys 1976). Other components concentrated in the bran and possibly germ, like antioxidants, vitamin E, and Mg, may also contribute to insulin sensitivity. Oxidative stress has been associated with reduced insulin-dependent glucose disposal and diabetic complications © 2011 by Taylor & Francis Group, LLC

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(Oberley 1988), whereas vitamin E and Mg may be involved in glucose metabolism (Slavin, Jacobs, and Marquart 1997). Whole grain may also affect satiation by insulin-independent mechanisms. For example, it has been shown that ingestion of whole grain products and cereal fiber may increase a secretion of the hormone, cholecystokinin, in the small intestine (Bourdon et al. 1999). This hormone is known to contribute to appetite suppression, as well as slowed gastric emptying and the inducement of satiety. Both clinical and observational studies show that an intake of whole grain is inversely associated with plasma biomarkers for metabolic syndrome and obesity, like C-peptide and leptin ­concentrations (Koh-Banerjee and Rimm 2003). Whole grain and fiber enhanced cereal products are reported to reduce overall calorie intake, and thus obesity, by suppressing appetite and via other mechanisms proposed above. For example, Hamedani et al. (2009) reported that breakfast cereal high in insoluble fiber significantly reduced short-term calorie intake in healthy individuals. Relatively recent epidemiological and some intervention studies seem to support the overall notion that whole grain consumption reduces obesity and metabolic syndrome. The Iowa Women’s Health Study found that whole grain intake was inversely correlated with body weight and fat distribution (Jacobs et al. 1998b). Pereira et al. (1998) also reported that the whole grain intake was inversely related to BMI at a 7-year follow-up of the participants of the study. Another large study of health men and women, the Multi-Ethnic Study of Atherosclerosis (MESA), reported an inverse association between whole grain intake and obesity, along with insulin resistance, inflammation, and elevated fasting glucose or newly diagnosed diabetes (Lutsey et al. 2007).

Summary Even though some controversies still remain, many studies support the link between whole grain consumption and overall health. However, most of these studies do not provide information on causality of the associations. Just like with other dietary components, it is very difficult to accurately pinpoint how and what components of a complex matrix like whole grain may impact specific health outcomes. However, given many of the rigorous studies show obvious benefits linked to whole cereal product consumption, even after correcting for various confounding variables, it is safe to conclude that whole grains should be actively promoted as a part of a healthy diet. Meanwhile more rigorous studies are needed to unravel the mechanisms by which whole grains impact health. This way, food product development efforts can be directed toward optimizing ingredient functionality to deliver health-promoting products that consumers can buy into en masse. This is especially important because no amount of preaching of health benefits will make consumers flock to a product consistently if the sensory appeal is substandard. Whole grain products, unfortunately, still largely suffer from the inferior sensory quality perception among the majority of consumers. Given that most human beings consume cereal grain-based products on a daily basis for primary nourishment, and will continue to do so into the foreseeable future, there is a tremendous opportunity to improve human health with a combination of innovative whole grain based products, public education, and cutting-edge research exposing the link between grain components and health.

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Lairon, D., N. Arnault, S. Bertrais, R. Planells, E. Clero, S. Hercberg, and M. C. Boutron-Ruault. 2005. Dietary fiber intake and risk factors for cardiovascular disease in French adults. American Journal of Clinical Nutrition 82 (6): 1185–94. Larsson, S. C., E. Giovannucci, L. Bergkvist, and A. Wolk. 2005. Whole grain consumption and risk of colorectal cancer: A population-based cohort of 60 000 women. British Journal of Cancer 92 (9): 1803–7. La Vecchia, C., and L. Chatenoud. 1998. Fibres, whole-grain foods and breast and other cancers. European Journal of Cancer Prevention 7: S25–8. Levi, F., C. Pasche, F. Lucchini, L. Chatenoud, D. R. Jacobs, and C. La Vecchia. 2000. Refined and whole grain cereals and the risk of oral, oesophageal and laryngeal cancer. European Journal of Clinical Nutrition 54 (6): 487–9. Liu, S. M., M. J. Stampfer, F. B. Hu, E. Giovannucci, E. Rimm, J. E. Manson, C. H. Hennekens, and W. C. Willett. 1999. Whole-grain consumption and risk of coronary heart disease: Results from the Nurses’ Health Study. American Journal of Clinical Nutrition 70 (3): 412–9. Ludwig, D., J. Majzoub, A. Al-Zahrani, G. Dallal, I. Blanco, and S. Roberts. 1999. High glycemic index foods, overeating and obesity Pediatrics 103: 1–6. Lutsey, P. L., D. R. Jacobs, S. Kori, E. Mayer-Davis, S. Shea, L. M. Steffen, M. Szklo, and R. Tracy. 2007. Whole grain intake and its cross-sectional association with obesity, insulin resistance, inflammation, ­diabetes and subclinical CVD: The MESA study. British Journal of Nutrition 98 (2): 397–405. McIntyre, A., P. R. Gibson, and G. P. Young. 1993. Butyrate production from dietary fiber and protection against large-bowel cancer in a rat model. Gut 34 (3): 386–91. Mellen, P. B., T. F. Walsh, and D. M. Herrington. 2008. Whole grain intake and cardiovascular disease: A ­meta-analysis. Nutrition Metabolism and Cardiovascular Diseases 18 (4): 283–90. Nettleton, J. A., J. F. Polak, R. Tracy, G. L. Burke, and D. R. Jacobs. 2009. Dietary patterns and incident ­cardiovascular disease in the multi-ethnic study of Atherosclerosis. American Journal of Clinical Nutrition 90 (3): 647–54. Nicodemus, K. K., D. R. Jacobs, and A. R. Folsom. 2001. Whole and refined grain intake and risk of incident postmenopausal breast cancer (United States). Cancer Causes & Control 12 (10): 917–25. Oberley, L. W. 1988. Free radicals and diabetes. Free Radical Biology and Medicine 5:113–24. Parker, R. A., R. L. Barnhart, K. S. Chen, M. L. Edwards, J. E. Matt, B. L. Rhinehart, K. M. Robinson, M. J. Vaal, and M. T. Yates. 1996. Antioxidant and cholesterol lowering properties of 2,6-di-t-butyl-4[(dimethylphenylsilyl)methyloxy]phenol and derivatives: A new class of anti-atherogenic compounds. Bioorganic & Medicinal Chemistry Letters 6 (13): 1559–62. Pereira, A., D. Jacobs, M. Slattery, K. Ruth, L. Van Horn, J. Hilner, and L. H. Kushi. 1998. The association of whole grain intake and fasting insulin in a biracial cohort of young adults: The CARDIA study. CVD Prevention 1: 231–42. Schatzkin, A., Y. Park, M. F. Leitzmann, A. R. Hollenbeck, and A. J. Cross. 2008. Prospective study of dietary fiber, whole grain foods, and small intestinal cancer. Gastroenterology 135 (4): 1163–7. Schoen, R. E., C. M. Tangen, L. H. Kuller, G. L. Burke, M. Cushman, R. P. Tracy, A. Dobs, and P. J. Savage. 1999. Increased blood glucose and insulin, body size, and incident colorectal cancer. Journal of the National Cancer Institute 91 (13): 1147–54. Slattery, M. L., J. D. Potter, A. Coates, K. N. Ma, T. D. Berry, D. M. Duncan, and B. J. Caan. 1997. Plant foods and colon cancer: An assessment of specific foods and their related nutrients (United States). Cancer Causes & Control 8 (4): 575–90. Slavin, J. L. 1994. Epidemiologic evidence for the impact of whole grains on health. Critical Reviews in Food Science and Nutrition 34 (5–6): 427–34. Slavin, J. 2000. Mechanisms for the impact of whole grain foods on cancer risk. Journal of the American College of Nutrition 19 (3): 300S–7S. Slavin, J., D. Jacobs, and L. Marquart. 1997. Whole-grain consumption and chronic disease: Protective mechanisms. Nutrition and Cancer—An International Journal 27 (1): 14–21. Sloth, B., and A. Astrup. 2006. Low glycemic index diets and body weight. International Journal of Obesity 30: S47–51. Tighe, P., N. Vaughan, J. Brittenden, W. G. Simpson, W. Mutch, G. Horgan, G. Duthie, and F. Thies. 2007. Effect of increased consumption of whole grain foods on markers of cardiovascular disease risk in ­middle-aged healthy volunteers. Arteriosclerosis Thrombosis and Vascular Biology 27 (6): E56–E56. © 2011 by Taylor & Francis Group, LLC

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Trowell, H. 1972. Ischemic heart disease and dietary fiber. American Journal of Clinical Nutrition 25: 926–32. Vanrensburg, S. J. 1981. Epidemiologic and dietary evidence for a specific nutritional predisposition to esophageal cancer. Journal of the National Cancer Institute 67 (2): 243–51. Yang, L. Y., J. D. Browning, and J. M. Awika. 2009. Sorghum 3-deoxyanthocyanins possess strong phase II enzyme inducer activity and cancer cell growth inhibition properties. Journal of Agricultural and Food Chemistry 57: 1797–804.

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Part II

Chemistry and Mechanisms of Beneficial Bioactives in Fruits and Cereals

© 2011 by Taylor & Francis Group, LLC

3 Phytochemicals in Cereals, Pseudocereals, and Pulses Clifford Hall III and Bin Zhao CONTENTS Introduction............................................................................................................................................... 22 Phytochemicals-Structural Characteristics............................................................................................... 22 Monophenols and Phenolic Acids........................................................................................................ 22 Tocopherols and Tocotrienols......................................................................................................... 22 Phenolic Acids................................................................................................................................ 23 Alkylresorcinols and Alkenylresorcinols............................................................................................. 25 Flavonoids............................................................................................................................................ 26 Antioxidant Activity....................................................................................................................... 27 Health Benefits................................................................................................................................ 28 Other Phytochemicals.......................................................................................................................... 29 Carotenoids..................................................................................................................................... 29 Phytosterols..................................................................................................................................... 30 Summary......................................................................................................................................... 30 Phytochemicals from Cereals and Pseudocereals..................................................................................... 30 Defining Cereals and Pseudocereals.................................................................................................... 30 Cereals..................................................................................................................................................31 Barley...............................................................................................................................................31 Phenolics......................................................................................................................................... 32 Corn................................................................................................................................................ 35 Oats..................................................................................................................................................41 Rice................................................................................................................................................. 45 Rye.................................................................................................................................................. 49 Wheat.............................................................................................................................................. 55 Pseudocereals....................................................................................................................................... 59 Amaranth and Quinoa......................................................................................................................61 Phytochemicals from Pulses: Edible beans and Legumes........................................................................ 62 Dry Peas............................................................................................................................................... 63 Tocopherol and Carotenoids........................................................................................................... 63 Phenolic Compounds...................................................................................................................... 63 Other Components.......................................................................................................................... 64 Antioxidant Activity....................................................................................................................... 64 Dry Bean.............................................................................................................................................. 65 Tocopherol...................................................................................................................................... 65 Phenolic Compounds...................................................................................................................... 65 Other Components.......................................................................................................................... 66 Antioxidant Activity....................................................................................................................... 66 Future Direction........................................................................................................................................ 67 References................................................................................................................................................. 67 © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Introduction Phytochemicals are simply bioactive plant substances that provide a health benefit. Many of these ­compounds at one time were considered antinutrients. However, an extensive study of the ­phytochemicals (e.g., phenolics, carotenoids, tocopherols) has resulted in the discovery of many health benefits. Furthermore, the usefulness of these components as food additives has been demonstrated. In this chapter, the phytochemicals from cereals (and pseudocereals) and pulses (e.g., legumes and edible beans) will be presented. Due to the diverse functionality and chemical and structural makeup of the phytochemicals, only a small number of phytochemicals will be highlighted in this chapter. The phytochemicals of interest include simple phenols, polyphenolics, phenolic acid, carotenoids, and sterols. Specific focus on the composition of phytochemicals from the various sources, effects of processing on the phytochemicals, and antioxidant activity of the phytochemicals will be highlighted. In addition, information will be presented regarding structural features of the general classes of phytochemicals. Methods for the isolation and characterization of the phytochemicals will not be presented in detail in this chapter. The author suggests that the review of the referenced literature will be of value in this regard. Important references prior to 2000 will be presented; however, the chapter material will cover research primarily from 2000 to 2007. Hall (2001, 2003) reported reviews on phytochemicals prior to 2000, and recent reviews by Awika and Rooney (2004) and Dykes and Rooney (2006) highlighted phytochemicals in several cereals, thus the reader is directed to these reviews. The authors of this chapter recognize the efforts of many researchers in the phytochemical area; however, not all of the research could be reported in this review.

Phytochemicals-Structural Characteristics Monophenols and Phenolic Acids Tocopherols and Tocotrienols Tocopherols and tocotrienols (tocols; Figure 3.1) are a group of monophenols that have vitamin E and antioxidant activities. The antioxidant activity of the tocols has been widely documented and will not be extensively described in this chapter. However, the phenolic hydrogen at the C6 position can participate in chain breaking mechanisms, including radical scavenging (Figure 3.2). Tocopherols and tocotrienols have been well characterized as antioxidants (Yoshida, Niki, and Noguchi 2003). The research on the health benefits of tocopherols and tocotrienols is conflicting. However, some studies have supported the health benefits. The role of tocols in disease prevention has been attributed to the antioxidant activity where the tocotrienols appear to have the most benefit (Qureshi et al. 1997, 2000; McIntyre et al. 2000; Packer, Weber, R1

R1

HO

HO CH3

R2

CH3 R2

O R3

O R3

Tocopherols

Tocotrienols

R1

R2

R3

α

CH3

CH3

CH3

CH3

β

CH3

H

CH3

CH3

CH3

γ

H

CH3

CH3

H

CH3

δ

H

H

CH3

R1

R2

R3

α

CH3

CH3

CH3

β

CH3

H

γ

H

δ

H

FIGURE 3.1  The monophenols tocopherol and tocotrienols.

© 2011 by Taylor & Francis Group, LLC

Phytochemicals in Cereals, Pseudocereals, and Pulses

HO

23

R1 CH3

R2

O R3 Hydrogen abstraction

O

R1 CH3

R2

O R3 LOO trapping

O

R1 CH3

R2

O R3 OOL

FIGURE 3.2  Hydrogen donation and radical scavenging activity of monophenols.

and Rimbach 2001; Wu et al. 2005; Nakagawa et al. 2007). Halliwell, Rafter, and Jenner (2005) reported that the benefits might be related to the affects of these components in the gastrointestinal tract (GI) and the prevention of radical species formation in the GI tract. However, these authors did state that the mechanisms of action were still not clear. The anticarcinogenic activity of tocotrienols has been reported (Mizushina et al. 2006). For additional information on the health benefits of tocotrienols from rice see Hall (2003).

Phenolic Acids Similar to tocols, the phenolic hydrogen(s) of phenolic acids (Figure 3.3) contribute antioxidant activity. Phenolic acids tend to be located on the out layers (aleurone, pericarp) of cereals (Sosulski, Krygier, and Hogge 1982; Hutzler et al. 1998; Naczk and Shahidi 2006) in contrast to the higher tocol levels in the germ (Barnes 1983). Thus, the benefits of phenolic acid would be realized if the outer portions of the grain were not removed prior to the consumption. Phenolic acids can act as antioxidants through a number of different mechanisms. The chain breaking mechanisms, which include hydrogen donation and radical acceptor (i.e., radical scavenging ­activity; Scott 1985), are the most likely means by which phenolic acids act as antioxidants (Figure 3.2). Variations in the antioxidant activity of individual phenolic acids have been documented (Pratt and Birac 1979; Pratt and Hudson 1990; Cuvelier, Richard, and Berset 1992). These authors observed key structureactivity relationships that accounted for the differences in antioxidant activities. The dihydroxy forms of the phenolic acids have better antioxidant activity due the addition of a second hydroxyl group in the ortho position. This statement can be supported by the observation of Pratt and Birac (1979) that ­caffeic acid had better antioxidant than the monohydroxy phenolic acids (i.e., ferulic acid and ρ-coumaric acid). The improved antioxidant activity of caffeic was likely due to the intramolecular hydrogen bonding (Figure 3.4) that can occur in ortho substituted phenols (Baum and Perun 1962). A third hydroxyl group further enhances the antioxidant activity as trihydroxybenzoic acid (i.e., gallic acid) and is a better antioxidant than 3,4-dihydroxy-benzoic acid (i.e., protocatechuic acid; Pratt and Birac 1979). The © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications OH

OH R2

R1

R2

R1

CH

COOH Benzoic acid derivatives

CH COOH

Cinnamic acid derivatives

p-Hydroxy benzoic acid

R1 = H, R2 = H

p-Coumaric acid

R1 = H, R2 = H

Vanillic acid

R1 = H, R2 = OCH3

Ferulic acid

R1 = H, R2 = OCH3

Syringic acid

R1= OCH3, R2 = OCH3

Sinapic acid

R1= OCH3, R2 = OCH3

Dihydroxybenzoic acid

R1 = OH, R2 = H

Caffeic acid

R1 = OH, R2 = H

Gallic acid

R1 = OH, R2 = OH

OH OCH3 HO

O

O

HO

O

HO

O

OH

O

H3CO

OCH3 OH 8-8'-ferulic acid

O

CH3

OH

4-O-5'-ferulic acid

FIGURE 3.3  Common phenolic acids in cereals, pseudocereals, and legumes, including examples of diferulic compounds associated with cell walls. (Adapted from Bunzel, M., Ralph, J., Marita, J., Hatfield, R., and Steinhart, H., J. Agric. Food Chem., 48, 3166–9, 2000; Bunzel, M., Ralph, J., Marita, J., Hatfield, R., and Steinhar, H., J. Sci. Food Agric., 81, 653–60, 2001.) OH

O

O OH

OH

Hydrogen Abstraction

OH

Electron Rearrangement

H O

O OCH3

Hydrogen Abstraction

OH No H-bonding

FIGURE 3.4  Intramolecular hydrogen bonding of ortho substituted phenols. (Adapted from Baum, B., and Perun, A., Soc. Plastics Eng. Trans., 2, 250–7, 1962.)

para substitutions in the phenolic acids give mixed antioxidant results. The quinic acid substitution (i.e., chlorogenic acid) at the para position was equally effective as caffeic acid in controlling oxidation. Structurally, the only difference between the molecules was the para substitute; thus, the authors concluded that the acid proton of caffeic acid had little effect on the antioxidant activity of the cinnamic acid derivatives (Pratt and Birac 1979). In contrast, vinyl substituted phenolic acids (i.e., cinnamic acid derivatives) were more effective as antioxidants then the benzoic acid derivatives (Pratt and Hudson 1990; Cuvelier, Richard, and Berset 1992). Cuvelier, Richard, and Berset (1992) suggested that the vinyl © 2011 by Taylor & Francis Group, LLC

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Phytochemicals in Cereals, Pseudocereals, and Pulses

group could enhance the resonance stability of the phenoxyl radical whereby improving the antioxidant activity. Thus, by understanding the above relationships one can predict the antioxidant potential of a plant material containing phenolic acids.

Alkylresorcinols and Alkenylresorcinols Alkylresorcinols and alkenylresorcinols have a 1,3-dihydroxybenzene base structure and an aliphatic substitution at carbon five of the ring (Figure 3.5). The aliphatic group typically has between 17 and 25 carbons (Kozubek and Tyman 1995, 1999; Ross et al. 2001; Ross, Kamal-Eldin, and Aman 2004). When the aliphatic group is unsaturated, the compounds are generically referred to as alkenylresorcinols. However, the alkylresorcinols (i.e., saturated aliphatic group) are the most common. Furthermore, these compounds are concentrated in the bran fractions of many cereal grains and may contribute to the health benefits attributed to whole grain consumption. The interest in this group of compounds stems from the reported anticarcinogenic, antimicrobial, and antioxidant properties (Singh et al. 1995; Gasiorowski et al. 1996; Kozubek and Tyman 1999; Slavin et al. 2001). For a summary of the reported benefits, see the review by Ross, Kamal-Eldin, and Aman (2004). The bioavailability of the alkylresorcinols shows that about 60% are absorbed by the human ileostomy (Ross et al. 2003a), but only small amounts are present in the plasma (Linko et al. 2002). However, higher alkylresorcinols concentrations were present in erythrocyte membranes, which appear to be a site for alkylresorcinol storage, than plasma membranes (Linko and Adlercreutz 2005). These authors also noted that the longer chained alkylresorcinols were incorporated into the erythrocyte ­membrane at higher concentrations than short-chained alkylresorcinols. Much of the intact alkylresorcinols and metabolites 3-(3,5-dihydroxyphenyl)-1-propanoic acid and 1,3-dihydroxybenzoic acid were found in urine (Ross, Aman, and Kamal-Eldin 2004). The reader is encouraged to read the review written by Ross et al. (2004c) for more information on alkylresorcinol structural chemistry, including metabolites. The antioxidant function of alkylresorcinols and alkenylresorcinols has not been fully characterized. Compounds with the substitutions at the meta position to the hydroxyl on the benzene ring typically have poor antioxidant activity (Miller and Quackenbush 1957). Yet, several researchers have reported antioxidant effects of the alkylresorcinols in model test systems (Nienartowicz and Kozubek 1995; Winata and Lorenz 1996; Hladyszowski, Zubik, and Kozubek 1998; Litwinienko, Kasprzycka-Guttman, and Jamanek 1999). Kamal-Eldin et al. (2001) evaluated hydrogen donating and peroxy radical scavenging activity of these compounds and found very poor antioxidant activities. In fact, based on the adherence to general antioxidant definition that the compounds must be effective at low concentrations, they concluded that R

HO R

Acronym

C15H31 C17H35 C19H39 C21H43 C23H47 C25H51 C19H37

(C15:0) (C17:0) (C19:0) (C21:0) (C23:0) (C25:0) (C19:1)

OH N ame 5-n-pentadecylresorcinol 5-n-heptadecylresorcinol 5-n-nonadecylresorcinol 5-n-heneicosylresorcinol 5-n-tricosylresorcinol 5-n-pentacosylresorcinol 5-n-nonadecenylresorcinol

FIGURE 3.5  Alkyl- and alkenylresorcinols found in cereals. (Adapted from Mattila, P., Pihlava, J.-M., and Hellström, J., J. Agric. Food Chem., 53, 8290–95, 2005; Ross, A., Shepherd, M., Schüpphaus, M., Sinclair, V., Alfaro, B., Kamal-Eldin, A., and Åman, P., J. Agric. Food Chem., 51, 4111–18, 2003.)

© 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

the alkylresorcinols were not effective antioxidants (Kamal-Eldin et al. 2001) in the DPPH and sunflower triacylglycerol systems. The conversion of the alkylresorcinols to trihydroxy derivatives was proposed as a reason for the antioxidant activity (Kozubek and Tyman 1999) and not the original alkylresorcinols.

Flavonoids Flavonoids are polyphenolic compounds characterized by a C6–C3–C6 configuration (Figures 3.6 through 3.8). Flavones, flavonols, flavanones, anthocyanadins, and anthocyanins make up the largest and most diverse groups among the flavonoids. Although fruits and vegetables are the primary dietary sources 3′

2′ 8

HO

O 2

6

6′

O

c

O 2

2′

3′

4′

5′

OH H H H OH H H H

H OH OH OH H H OH OH

H OH OH OH OH OH OH OCH3

H H H OH H H H H

4′ 5′

6′ 3

6

Datiscetin Quercetin Dihydroquercetina Myricetin Morin Kaempferol Rutinb Hesperidinc

3′

2′ 8

HO

Flavonols

b

5′

OH OH

a

4′

OH

O

Flavanones 3 3′ Naringenin Naringina Taxifolin Fustinb Eriodictyol a

Dihydroquercetin has an additional H at the C-3 position due to the loss of the double bond at the C-2:C-3 position. Rutin is a glycoside in which the C-3 position contains a o-rutinose. Hesperidin contains a o-rutinose at the C-7position.

b

H,H ORh OH OH H,H

4′

H H OH OH OH

OH OH OH OH OH

Naringin is a glycoside in which the C-3 position contains a rhamnoglucose unit. Fustin lacks a C-5 OH.

OH

OH

OH

OH 3′ 2′ 8

HO

O 2

4′

HO

OH

5′ 6′

OH HO

3

6 OH

2′

6

OH OH

OH HO

O 2

OH

Procyanidin B-1

Procyanidin B-3 OH

OH OH O 2

4′ 4 5′

OH

6′

Flavans 3′ 4′ Catechin OH OH

O OH 8′′

4

7′′ OH OH

HO

OH

O

7′′

8′′ OH

O HO

HO

H

HO

H HO

Epicatechin-(4βd 8;2βdO7)-catechin

FIGURE 3.6  General flavonoids isolated from cereals, pseudocereals, and legumes.

© 2011 by Taylor & Francis Group, LLC

OH O 2

HO

O

OH OH

OH

OH

OH

HO

OH

O

OH

3′ 8

O

OH

O

O

Flavones 3′ 4′ Apigenin H OH Chrysin H OH OH OH Luteolin

HO

HO

O

OH

27

Phytochemicals in Cereals, Pseudocereals, and Pulses OH

OH OH

OH O

HO

OH

+

+

+

O

HO

O

HO

OH

OR

OR OH

OH

OGlucose

Apigeninidin 5-glucoside

Cyanidin 3-glycoside

Delphinidin 3-glycoside R

R

Cyanidin 3-glucoside Cyanidin 3-galactose

glucose galactose

OCH3

glucose rutinose

OCH3 OH

OCH3

OH +

+

O

HO

Delphinidin 3-glucose Delphinidin 3-rutinoside

OH

+

O

HO

OGlucose

O

HO

OCH3

OGlucose

OGlucose OH

OH

OH

Malvidin 3-glucoside

Petunidin 3-glucoside

Pelargonidin 3-glucoside

FIGURE 3.7  Anthocyanins isolated from pigmented corn, rice, wheat, and legumes. HO HO

O

7 5

Genistein Genistin Daidzein Daidzin

O

H2C OCOCH2COOH O OH

O

O 5

4′

7 4′ 5 OH OH OH OH OH O-glucose OH H OH OH H O-glucose

O Malonyl isoflavone derviatives. 5 6 6"-O-Malonylgenistin OH H 6"-O-Malonyldaidzin H H 6"-O-Malonylglycitin H OCH3

OH

FIGURE 3.8  Common isoflavones in edible legumes.

of flavonoids, cereals, legumes, and beans can contribute to the daily intake. Flavonoids are a group of compounds that have been well documented as hydrogen donors, radical scavengers, and metal chelators (Dziedzic and Hudson 1983; Torel, Cillard, and Cillard 1986; Husain, Cillard, and Cillard 1987; Bors et al. 1990; Das and Pereira 1990; Salah et al. 1995; Foti et al. 1999; Rice-Evans, Miller, and Paganga 1996; Cao, Sofic, and Prior 1997). Flavonoids as food antioxidants and health promoters have been reviewed extensively (Hall and Cuppett 1997; Middleton 1998; Pietta 2000; Nijveldt et al. 2001; Rice-Evans 2001; Yanishlieva and Heinonen 2001; Hall 2003; Valko et al. 2006).

Antioxidant Activity As with phenolic acids, the antioxidant activity of flavonoids is dependent on the number and location of the hydroxyl groups. Hydroxyl groups on ring B play a significant role in the hydrogen donating activity. Hydroxyl groups at the 3′, 4′, and 5′ positions on the ring B have the greatest activity followed by flavonoids with ortho hydroxyl groups on ring B (Dziedzic and Hudson 1983; Hudson and Lewis 1983; Rice-Evans, Miller, and Paganga 1996). The hydrogen donating activity greatly diminishes in flavonoids with only one B ring hydroxyl group. Similar structural features were important for radical scavenging activity (Husain, Cillard, and Cillard 1987; Bors et al. 1990; Cao, Sofic, and Prior 1997; Foti et al. 1999). Like other flavonoids, ortho hydroxyl configuration enhances radical scavenging © 2011 by Taylor & Francis Group, LLC

28

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Metal complexe of 5-hydroxy flavonoids O

HO

Metal complexes of 3-hydroxy flavonoids HO

O O Cu+

O

O

Cu+ O

HO

O

HO

O O

O

O Cu

O

Cu

FIGURE 3.9  Metal chelate complexes of flavonoids. (Adapted from Hudson, B. and Lewis, J., Food Chem., 10, 47–55, 1983.)

activity of anthocyanidins (Yoshiki, Okubo, and Igarashi 1995; Rice-Evans, Miller, and Paganga 1996; Wang, Cao, and Prior 1997). However, the presence of the hydroxyl group at the 5′ position did not improve anthocyanidin antioxidant activity (Rice-Evans, Miller, and Paganga 1996; Wang, Cao, and Prior 1997). The hydroxyl substitutions at the 5,8 and 7,8 positions on ring A improved flavonoids antioxidant activity. However, ring A dihydroxy substitutions at 5,7 positions did not influence the antioxidant activities of flavonoids. In contrast, the 7 position on the A ring did not affect the antioxidant activity of isoflavones. The addition of a hydroxyl group at the 5 position on ring A did improve the antioxidant activity (Hu et al. 1995; Wei et al. 1995). The presence of a hydroxyl group at the 3 position on ring C enhances the antioxidant activity of the flavonoids. The flavonols are generally better antioxidants than flavanones due to the presence of the hydroxyl group at the 3 position. In addition, the presence of sugar moieties on the three location of ring C diminishes the antioxidant activity of the flavanones (Das and Pereira 1990; Nieto et al. 1993). In contrast, the radical scavenging activity of the anthocyanins (glycoside form) was better than the anthocyanidins (Satué-Gracia, Heinonen, and Frankel 1997; Wang, Cao, and Prior 1997). Thus, the greater antioxidant activity of the flavones over the anthocyanidins was attributed to the carbonyl at position 4 of ring C in conjunction with the double bond at carbons 2 and 3 of ring C (Cao, Sofic, and Prior 1997; Wang, Cao, and Prior 1997). The metal chelating activity (Figure 3.9) of flavonoids can occur at two regions of the molecule. The 3′,4′-dihydroxy configuration is an important structural feature that accounts for the metal chelating properties of anthocyanins and anthocyanidins, whereas the ring C quinone at position 4 of flavones and flavonols was essential (Crawford, Sinnhuber, and Aft 1961; Pratt and Watts 1964; Letan 1966; Hudson and Lewis 1983). A loss in metal chelating activity of the flavones and flavonols was observed after the double bond at positions 2 and 3 on ring C was hydrogenation (Crawford, Sinnhuber, and Aft 1961; Letan 1966). The flavonoids have a very diverse function as a food antioxidant and these effects might contribute to the health benefits of the flavonoids.

Health Benefits The anti-inflammatory, anticarcinogenic, and antitumor activities of flavonoids have been reported (Hollman and Katan 1998; Middleton 1998; Waladkhani and Clemens 1998; Agarwal, Sharma, and Agarwal 2000). Hirano, Gotoh, and Oka (1994) reported that flavonoids had cytostatic activity against human breast carcinoma cells but did not find a structure-activity relationship. Sánchez et al. (2001) found that flavonoids lacking the C-8 methoxy substitutions had little cytotoxicity against Rhesus monkey kidney cells and rat glial tumor cells, whereas the C2’ and C5’ were an important structural ­feature. The anti-17beta-hydroxysteroid dehydrogenase activity was dependent on the C-7 hydroxyl group whereas flavonoids with C-7 methoxy or C-8 hydroxyl groups had only antiaromatase activity (Bail et al. 1998). © 2011 by Taylor & Francis Group, LLC

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Phytochemicals in Cereals, Pseudocereals, and Pulses

Bomser et al. (1999) and Zhao et al. (1999) observed antitumor activity of procyanidin B5-3′-gallate (Zhao et al. 1999). Quercetin, myricetin, and epicatechin inhibited the growth and altered the enzyme activities of MCF7 human breast cancer cells (Rodgers and Grant 1998). Flavonoids also inhibit oxidation of LDL (Meyer et al. 1997; Meyer, Heinonen, and Frankel 1998; Meyer, Jepsen, and Sórgensen 1998; Brown and Rice-Evans 1998; Heinonen, Meyer, and Frankel 1998; Hwang, Hodis, and Sevanian 2001; Porter et al. 2001) and inhibit cholesteryl ester synthesis (Borradaile, Carroll, and Kurowska 1999). Naringenin and hesperetin reduce acyl CoA:cholesterol acyltransferase activity, inhibit the activity and expression of microsomal triglyceride transfer protein, and increase LDL receptor mRNA that promote the reduction in plasma cholesterol (Wilcox et al. 2001). The inhibitions of thromboxane synthase and prostaglandin production are the reasons for the anti-inflammatory activity of flavonoids (Ishiwa et al. 2000; Skaltsa et al. 2000).

Other Phytochemicals Carotenoids and phytosterols are the final phytochemicals to be covered in this chapter. However, compounds specific to cereals or pulses will be presented under that section related to specific materials. Avenanthramides in oats, oryzanols in rice, and policosanols in sorghum are a few of examples of health promoting phytochemicals.

Carotenoids Carotenoids (Figure 3.10) are a group of compounds characterized by a conjugated polyene system. The singlet oxygen quenching characteristics of carotenoids has been well documented (Foote, Chang, and Denny 1970; Burton and Ingold 1984; Terao 1989). The presence of nine or more double bonds and oxo groups at the 4(4′) position in the β-ionone ring in the carotenoid structure greatly enhanced the singlet oxygen quenching activity (Terao 1989). The carbonyl present on the ring enhanced the stability

β-carotene

α-carotene

HO

β−cryptoxanthin

HO

Lutein

OH

OH

OH FIGURE 3.10  Carotenoids found in corn and wheat.

© 2011 by Taylor & Francis Group, LLC

Zeaxanthin

30

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

of trapped radicals; therefore, reducing the tendency of carotenoids to promote radical reactions. The polyene system can also trap radicals, thus providing additional protective activity. These activities are believed to be the cause of the health benefits of carotenoids. However, controversy also exists around the negative impact of high carotenoid levels in some populations. Dutta, Chaudhuri, and Chakraborty (2005) and Krinsky and Johnson (2005) have recently reviewed carotenoids. In the context of this chapter, the carotenoids are responsible for the yellow color in corn and durum wheat. In cereals, for example, carotenoids exist as carotenes (α- and β-carotene) and xanthophylls (β-cryptoxanthin, lutein, and zeaxanthin), where xanthophylls are typically in the highest ­concentrations. Lutein and zeaxanthin have attracted much attention due to the possible role in preventing cataracts (Knekt et al. 1992) and age-related macular degeneration, a condition that results in irreversible vision loss (Gale et al. 2003; Mozaffarieh, Sacu, and Wedrich 2003; Moeller et al. 2006; Trieschmann et al. 2007). Thus, grain consumption can contribute to the total dietary intake of carotenoids.

Phytosterols Phytosterols and phytosanols (saturated form of the sterol; Figure 3.11) are widely present in grains (Piironen, Toivo, and Lampi 2002). These compounds exist as free sterols, fatty acid, or phenolic esters, and steryl glycosides (Toivo et al. 2001; Moreau, Whitaker, and Hicks 2002). The phytosterols have limited antioxidant activity and those esterified to phenolic acids can act as chain breaking antioxidants similar to phenolic compounds. However, the ferulate esters were found to have less activity than the ferulic acid (Xu and Godber 2001). In contrast, the phytosterols were effective in controlling the oxidation of frying oils Kamal-Eldin et al. (1988) and prevention of oil polymerization (Sims, Fioriti, and Kanuk 1972; Boskou and Morton 1976; Gordon and Magos 1983; White and Armstrong 1986). The role of phystosterols in health is probably more significant than the antioxidant effects. The phytosterols have been shown to effectively reduce blood cholesterol (Fernandez et al. 2002; Gylling and Miettinen 2005), prostatic hyperplasi (Berges et al. 1995; Berges, Kassen, and Senge 2000), and colon cancer (Awad and Fink 2000). In addition, an enhanced immune function has been reported (Bouic and Lamprecht 1999). For a complete review of the benefits of phytosterols, see the recent review by Kritchevsky and Chen (2005).

Summary A varied diet of foods would be required to achieve the health benefits of the phytochemicals previously described. However, in some cases the components can be concentrated via physical methods or by solvent extractions. Thus, one must remember that in the following discussions for low levels of a component in a grain, or pulse is not necessarily a negative if the phytochemical is consumed as part of a varied diet or in a concentrated form.

Phytochemicals from Cereals and Pseudocereals Defining Cereals and Pseudocereals Cereals and pseudocereals are plant materials that have similar end uses as flours for bakery products. However, these plants are different botanically as cereals are grasses whereas pseudocereals are broadleaf plants. All of these plant materials have a cultivar of phytochemical constituents and are of interest to researchers in the health and medical fields. The cereals that have garnered attention include barley (Hordeum vulgare), corn (Zea mays), millet (Panicum milliaceum), oats (Avena sativa), rice (Oryza sativa), rye (Secale cerale), and wheat (Triticum spp). Pseudocereals of interest include amaranth (Amaranthus caudatus, A. cruentus), buckwheat (Fagopyrum esculentum), and quinoa (Chenopodium quinoa). Regardless of the plant materials, the hull or bran is usually the main source of the phytochemicals; however, the germ is also a valuable source of the lipid soluble phytochemicals. Thus, the benefit of whole grain consumption is related to the consumption of the aforementioned grain fraction. © 2011 by Taylor & Francis Group, LLC

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Phytochemicals in Cereals, Pseudocereals, and Pulses

CH3

CH3 CH3 H

CH3 H H

CH3

H

H

CH3 H H

H

HO

HO

Campestanol

Campesterol

Brassicasterol

CH2

CH2

CH3

CH3

CH3 H

H

HO

H

H

H H

HO

HO CH3

CH3

β-sitosterol

Stigmasterol

β-sitostanol CH3

CH3 CH3 H H

CH3

CH3

CH3 H

H

HO

CH3 H

H

HO

H

HO

∆5-avenasterol

∆7avenasterol Oryzanols - sterol ferulates

O

O H3C

O

O

H3C

O

HO

HO

O

O H3C

O

O

H3C

O

O

O

HO

HO

O H3C

O

O

HO

FIGURE 3.11  Phytosterolas, phytostanols, and sterol ferulates found in cereals and pseudocereals.

Cereals Barley In human foods, barley is most often used in the brewing industry. However, barley consumption as a food source has recently increased due to the reported health benefits. Barley has a number of different phytochemicals that include tocols (Peterson 1994; Goupy et al. 1999), Δ5-avenasterol (Dutta and Appelqvist © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

1996), flavonoids (Tamagawa et al. 1999), phenolic acids (Van Sumere et al. 1972; Slominski 1980; Mattila, Pihlava, and Hellström 2005), and alkylresorcinols (Mattila, Pihlava, and Hellström 2005).

Tocols The content of tocopherols varies widely among cultivars. Goupy et al. (1999) reported an average tocopherols content of 25.1 mg/kg among nine barley cultivars. The tocopherols ranged from 9.7 to 44.2 mg/kg in the Caminant and Nevada cultivars, respectively. In general, the tocopherols values were lower than the 56.7 mg/kg reported by Peterson (1994). However, in addition to tocopherols, tocotrienols were also measured in the later study. Choi et al. (2007) reported a tocols content of 19.2 mg/kg in the Ollbori barley cultivar. These studies demonstrate the influence of cultivar on tocol levels. However, the majority of the tocols are present in the germ followed by endosperm and hull, which have approximately the same tocol concentrations. Fractionation using dehulling demonstrates the simplicity of obtaining Tocol-rich fractions without solvent extraction. An increase from 36.8 to 74.8 mg/kg in the barley by-product was obtained after the dehulling process (Peterson 1994). The malting did not significantly affect the tocols. Only a slight and nonsignificant reduction in total tocols was observed in malted barley (Peterson 1994) whereas Goupy et al. (1999) found mixed results in that the tocopherol content both increased and decreased during malting. A significant increase (from 56.7 to 152.9 mg/kg) was observed in the spent grain recovered after the mashing and brewing process (Peterson 1994).

Phenolics Barley contains approximately 10 phenolic acids that occur during seed development and include sinapic, ferulic, p-, m-, and o-coumaric, syringic, vanillic, protocatechuic, salicylic and p-hydroxybenzoic acids (Slominski 1980). Recently, phenolic acids (Table 3.1) were determined in barley flour (Mattila, Pihlava, and Hellström 2005) and from three Chinese barley cultivars (Zhao et al. 2006). The extraction protocol clearly had an impact on the phenolic acid type and concentration. In particular, ferulic acid was substantially higher in extracts that utilized acid hydrolysis compared to acetone:water (4:1, v/v), presumably due to the hydrolysis of the ferulate esters from the cell walls. Furthermore, alkaline hydrolysis was used in a protocol to determine ferulic acid dehydrodimers (diFA; Table 3.2; Renger and Steinhart 2000). Other phenolics present in barley include anthocyanins, proanthocyanins, and flavonols. The anthocyanins, which include cyanidin, cyanidin 3-arabinoside, delphinidin, and delphinidin glycoside, pelargonidin, and pelargonidin glycosides, cyanidin, cyanidin 3-arabinoside, delphinidin, and delphinidin TABLE 3.1 Phenolic Acid Content (mg/kg) in Whole Barley Flour and of Several Chinese Barley Varieties Phenolic Acid

Zhao et al. (2006)b

Mattila, Pihlava, and Hellström (2005)a

Ken-3

KA4B

Gan-3

1.7 250 40 NR 3.1 1.6 11 5 7.1

7.9 12.05 1.8 2.7 NR ND NR 10.3 3.6

6.7 7.6 1.7 2.3 NR ND NR 7.8 4.5

6.3 9.4 1.4 2.6 NR ND NR 7.8 3.9

Caffeic Ferulic p-coumaric Gallic p-hydroxybenzoic acids Protocatechuic Sinapic Syringic Vanillic

Note: NR = Not reported or measured; ND = Not detected. a Phenolic acids extracted using methanol:10% acetic acid (85:15, v/v). b Phenolic acids extracted using acetone:water (80:20, v/v).

© 2011 by Taylor & Francis Group, LLC

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Phytochemicals in Cereals, Pseudocereals, and Pulses TABLE 3.2 Phenolic Content (mg/kg) of Barley Alkaline Hydrolysis Phenolic Acid

1 M Sodium Hydroxide

4 M Sodium Hydroxide

Ferulic p-coumaric

6401 151

6289 140

Ferulic Dimersas 8-5-diFA 5-5-diFA 8-8-diFA 8-O-4-diFA 8-8-aryl-diFA

44 114 25 62 121

135 197 71 163 178

Source: Adapted from Renger, A. and Steinhart, H., European Food Res. Technol., 211, 422–8, 2000. a 8-5-diFA: Ferulic acid 8-5-dehydrodimer; 5-5-diFA: Ferulic acid 5-5-dehydrodimer; 8-8-diFA: Ferulic acid 8-8-dehydrodimer; 8-O-4-diFA: Ferulic acid 8-O-4-dehydrodimer; 8-8-aryl-diFA: Ferulic acid 8-8-aryl-dehydrodimer.

glycoside, are located in the pericarp and aleurone layers of the barley kernel (Tamagawa et al. 1999). The procyanidin B-3 dimer, (+)-catechin, (–)-epicatechin and leucodelphinidin were present primarily in the aleurone (Mazza and Miniati 1993; Tamagawa et al. 1999). The location of the phenolics in the barley would indicate that milling could be used as a means to concentrate the barley phenolics. Zhao et al. (2006) reported that the (+)-catechin concentration in Ken-3 barley obtained from an extraction protocol consisting of acetone:water (4:1, v/v) was approximately 56 mg/kg followed by 46, 41, and 27 mg/kg obtained from methanol:water (4:1, v/v), ethanol:water (4:1, v/v) and water, respectively. The methanol:water (4:1, v/v) removed more (15 mg/kg) of the (–)-epicatechin from barley than acetone:water (12 mg/kg), ethanol:water (4 mg/kg), and water (3 mg/kg). A consistent pattern was observed in the extraction behavior of the solvents between cultivars thus the extraction protocol could also be a useful means to produce barley extracts with high phenolic contents. Although Zhao et al. (2006) found only minor differences in phenolic content between cultivars, phenolic contents varied widely among nine barley cultivars (Goupy et al. 1999). The variation in phenolic content is likely due to the genetic lines whereby the barley cultivars used by Zhao et al. (2006) may have been more closely linked and thus produced similar phenolic contents. Quinde-Axtell and Biak (2006) reported that hulled barley contained 533–562 mg/kg of phenolic acid whereas the hull-less cultivar contained 365–445 mg/kg. In contrast, these authors observed higher average proanthocyanidin levels in the hull-less barley genotypes than in the hulled genotype. A proanthocyanidin negative genotype contained only minor concentrations of catechin. In general, the genotype or cultivar did affect the phenolic content and discrepancies in the level of phenolics may be due to genotype or varietal differences. Regardless of the barley, the influence of cultivar on phenolic contents was minimized after the barley had been malted (Goupy et al. 1999). These authors observed significant reductions in the flavanol (62–87%) and flavonol (64–91%) contents after malting whereas phenolic acids (35–78%) were affected to a lesser extent. In contrast, the total phenolic content increased between 8 and 66% during the malting process (Maillard et al. 1996). These authors also reported increases in flavanols and flavonols of up to 300% in five different barley cultivars. Increasing concentrations of phenolic acids were also observed after the kilning process (Figure 3.12, Maillard and Berset 1995). Individual phenolic compounds also increased but to a lesser extent than the total phenolic acid concentrations. In contrast, roasting above 327ºC significantly reduced catechin levels (Duh et al. 2001).

Other Components Carotenoids, phytosterols, and alkylresorcinols are other components of barley that have antioxidant activity (Dutta and Appelqvist 1996; Kamal-Eldin et al. 2001; Ross et al. 2003b; Choi et al. 2007). © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications 800 700

mg/kg

600 500 400 300 200 100 0

Germinated kiln-50 C barley

kiln-64 C

Total phenolic acids Trans-ferulic acid

kiln-80 C

kiln-85 C

kiln-90 C

Trans-p-coumaric acid Cis-ferulic acid

FIGURE 3.12  The affects of germination and kilning on total phenolic acids, p-coumaric acid, and ferulic acids. (Data adapted from Maillard, M.-N. and Berset, C., J. Agric. Food Chem., 43, 1789–93, 1995.)

TABLE 3.3 The Alkylresorcinols (mg/kg) in Barley Alkylrescorcinol

Barley Cultivar/ Sample

17:0

Alexisa Baronessea Oliviaa Barley flourb

4.2 3.6 1.3 7.0

19:0 5.0 6.6 4.6 15.0

21:0

23:0

25:0

11.3 13.8 10.1 ND

7.1 8.2 7.6 10.0

14.7 18.9 18.5 ND

Source: Data summarized by aRoss, A., Kamal-Eldin, A., Lundin, E., Zhang, J.-X., Hallmans, G., and Aman, P. J. Nutr., 133, 2222–24, 2003; bMattila, P., Pihlava, J.-M., and Hellström, J., J. Agric. Food Chem., 53, 8290–5, 2005. Note: ND = not detected.

Carotenoids have been widely studied in fruits and vegetables whereas few studies have evaluated carotenoids in cereals. The main reason for the lack of research is that cereals generally have low carotenoids levels. Only 15 µg carotenoids/100 g barley has been reported (Choi et al. 2007). The carotenoid contents ranged from approximately 1–85 µg/100 g barley depending on cultivar (Goupy et al. 1999). Thus, carotenoids probably have little impact on antioxidant activities of barley. Lampi et al. (2004) reported that hull-less barley contained about 800 mg phytosterols/kg barley. Subsequent pearling of the barley enhanced the phytosterols to 1738 mg/kg in the barley pearl fines. Dutta and Appelqvist (1996) reported that Δ5-avenasterol accounts for 23 and 21% of the sterols in free and bound lipid, respectively. The Δ5 and Δ7-avenasterol were part of the minor sterol constituents that made up approximately 11 and 13% of the phytosterols in whole barley and barley pearling fines, respectively (Lampi et al. 2004). These authors also reported that sitosterol and campesterol were the most predominant sterols at 476 and 181 mg/kg, respectively. The Δ5 and Δ7-avenasterol have been reported as effective antioxidants in heated oils (Yan and White 1990) whereas sterols such as sitosterol have cholesterol-lowering activity. Thus, utilization of barley pearling fines could be a valuable source of food ingredients. The alkylresorcinols concentrations in barley are low compared to other cereals such as wheat and rye. Total alkylresorcinols values of approximately 30–50 mg/kg are common (Garcia et al. 1997; Zarnowski et al. 2002; Ross et al. 2003b; Mattila, Pihlava, and Hellström 2005). The cultivar and form of the barley did have an influence on alkylresorcinol levels (Table 3.3). Zarnowski et al. (2002) and Zarnowski and Suzuki (2004) observed that alkylresorcinol concentrations were affected by environmental conditions. Thus, alkylresorcinol variation mentioned above may be related to the location in which the barley was grown. © 2011 by Taylor & Francis Group, LLC

Phytochemicals in Cereals, Pseudocereals, and Pulses

35

Antioxidant Activity Although barley contains a number of phytochemical constituents, the antioxidant activity of barley extracts in methyl linoleate was relatively low (Kähkönen et al. 1999). Choi et al. (2007) reported that methanol extracts of barley had good DPPH and ABTS radical scavenging activities compared to several cereals except black rice and sorghum. Bonoli et al. (2004) observed that the solvent extraction method significantly impacted antioxidant capacities of an extract. They utilized a DPPH radical scavenging assay as a screening method in which they observed the best antioxidant activity was in an extract prepared from acetone and water (4:1, v/v). Similar trends in DPPH radical scavenging activity was reported by Zhao et al. (2006) for barley extracts obtained from 80% acetone. The radical scavenging activity of extracts obtained from 80% acetone was 30–35, 40–45, and 65 percentage points higher than scavenging activity of 80% methanol, 80% ethanol, and water, respectively. Liu and Yao (2007) also reported DPPH radical scavenging activity was best for a barley extract obtained from 70% acetone compared to the extracts from 70% ethanol or methanol extracts. The 70% acetone extract also had better peroxide inhibitory activity than the extracts obtained from the alcohol solvents (Liu and Yao 2007). In contrast, Madhujith and Shahidi (2006) identified, using response surface methodology that the highest antioxidant activity of barley extracts was obtained using 80.2% methanol as the solvent and an extraction temperature of 60.5ºC. Ragaee, Abdel-Aal, and Noaman (2006) reported that an 80% methanol extract of barley had better DPPH and ABTS radical scavenging activity than wheat and rye but less than millet and sorghum. Zhao et al. (2006) also reported that the ABTS radical scavenging activity of the 80% acetone extract of barley was significantly better than ethanol or methanol-based barley extracts; however, the differences between the degrees of radical scavenging were not as great compared to DPPH radical scavenging. In contrast, hydroxyl radical and superoxide anion radical tests and metal chelating assays showed that the acetone extract was weaker compared to the methanol and water extracts of barley (Zhao et al. 2006). Cruz et al. (2007) reported an alkaline extract of barley lignin had better DPPH radical scavenging activity than BHT, BHA, and an acid extract of barley lignin. However, the synthetic antioxidants were better at preventing β-carotene bleaching than the barley extracts. The observed radical scavenging was associated with total phenolic content and proanthocyanidins (Bonoli et al. 2004; Liu and Yao 2007). Strong correlations between (+)-catechin, (–)-epicatechin, and phenolic acids that included caffeic, ferulic, p-coumaric, syringic, and vanillic acids and the DPPH and ABTS radical scavenging were reported (Zhao et al. 2006). These authors also observed correlation between other phenolic compounds and metal chelation and other radical scavenging activities. Discrepancies in the reported antioxidant activity of the extracts could be due to a number of factors that include extraction solvent, temperature, time, and the cultivar of barley evaluated. Etoh et al. (2004) reported that 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, and isoamericanol A had better antioxidant activity than butylated hydroxytoluene (BHT) suggesting that the compounds present in ­barley and barley extract may also contributed to variations in reported antioxidant activity.

Corn Corn contains several phytochemical compounds including phytosterols (Moreau, Singh, and Hicks 2001; Moreau, Powell, and Singh 2003; Winkler et al. 2007), tocopherols and tocotrienols (Kurilich and Juvik 1999; Moreau and Hicks 2006), phenolic acids (Saulnier and Thibault 1999; Renger and Steinhart 2000; Niwa et al. 2001; Yadav, Moreau, and Hicks 2007), and carotenoids (Kurilich and Juvik 1999; Moros et al. 2002). As with other cereals, significant variations in phytochemical constituents have been reported, primarily due to cultivar differences.

Tocols Kurilich and Juvik (1999) assessed the tocopherol concentrations of 44 cultivars of sweet and dent corn lines. The total tocopherol content of 30 mg/kg was found for all cultivars with a range of 7–86 mg/kg. Regardless of the cultivar, α-, γ-, and δ-tocopherols were identified in all 44 cultivars and averaged 8, 20, and 1 mg/kg, respectively, for all cultivars (Kurilich and Juvik 1999). The distribution of the individual tocopherols was in agreement with tocopherol values reported by other researchers (Grams et al. 1970; Ko et al. 2003; Tuberoso et al. 2007). The tocopherols are found mainly in the germ (up to 90%) with the © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

pericarp and endosperm accounting for ca. 5% each (Grams et al. 1970). Moreau and Hicks (2006) reported that corn germ oil contained about 28 times more tocopherols than corn fiber oil, which ­supports previous findings. Approximately 94–96% of the corn germ oil tocopherols are α- and γ-tocopherols (Tuberoso et al. 2007), whereas γ- and α-tocotrienols account for nearly 100% of the total tocotrienols in germ oil (Moreau and Hicks, 2006). Oil tocol levels obtained from corn fiber differ from that of germ tocols. Moreau and Hicks (2006) reported that in the germ oil γ-tocopherol (79 mg/kg) accounted for ca. 69% of total tocopherols and that the remaining 31% was δ-tocopherol (35.7 mg/kg). In contrast to the germ, corn fiber oil contained δ-tocotrienol (70 mg/kg) and low levels of α-tocotrienol (15.3 mg/kg). However, γ-tocotrienol still made up the majority (378 mg/kg) of the tocotrienols (Moreau and Hicks 2006). Processing or fractionation of the corn can be a valuable tool to enhance tocols based on their distribution in corn. Corn oil obtained from the germ contains around 840 mg/kg (see the review by Hall 2001; Franke et al. 2007). Tuberoso et al. (2007) reported tocopherol levels as high as 1618 mg/kg in commercial corn oil whereas ca. 3300 mg/kg of tocols were observed in corn oil obtained from the germs (Moreau and Hicks 2006). The refining process, in particular the heating of the oil, can explain the differences. A 56% reduction in oil tocols was reported after the germ had been heated prior to oil extraction (Moreau and Hicks 2006). In contrast, no γ-tocopherol loss was observed in oils extracted from corn at 100°C (Moreau et al. 2003). The heating process did not affect δ-tocopherol (Moreau and Hicks 2006). The dry milling of corn (Pioneer Hybrid 3394) was an effective means to concentrate γ-tocopherol. The germ and bran fractions contained 68 and 4% of the γ-tocopherol, respectively (Moreau et al. 1999). In wet milling, the germ again had the highest proportion of the total γ-tocopherol. Of the 57 mg/kg, 53% was located in the germ while the fine and coarse fibers accounted for 23 and 16% of the γ-tocopherol, respectively (Moreau et al. 1999). Wang et al. (1998) reported that steeping protocols influenced the retention of tocols. The addition of ascorbic acid to the steeping water protected the tocols. Winkler et al. (2007) observed that the tocols remained high in distillers’ grains. Levels of 730–1820 mg/kg were reported depending on the extraction protocol, which is equivalent to the composition of tocols in corn oil. Furthermore, cultivar differences did have a slight impact on total γ-tocopherol (Moreau, Singh, and Hicks 2001). Wyatt, Pérez Carballido, and Méndez (1998) reported α- and γ-tocopherols of 22.6 and 58 mg/kg in raw corn found in Mexico and that cooked corn had 9.6 and 32.6 mg/kg of these same tocopherols, respectively. In contrast, only 2.9 mg/kg of γ-tocopherol was present in corn tortillas.

Phenolics Mattila, Pihlava, and Hellström (2005) reported that ferulic acid and dehydrodimers accounted for 63 and 15%, respectively, of the 601 mg/kg of phenolic acids in corn flour. Classen et al. (1990) found that (E)-ferulic and (Z)-ferulic acids accounted for 57 and 33% of the total (1143 mg/kg) phenolic acids, respectively. Sinapic acid was the only other significant phenolic acid and accounted for ca. 10% of the phenolic acids (Classen et al. 1990; Mattila, Pihlava, and Hellström 2005). Tuberoso et al. (2007) reported that commercial corn oil had vanillin, ferulic, and t-cinnamic acid at 2.8, 0.5, and 0.9 mg/kg. This data demonstrates the limited solubility of these materials in the oil and that the refining process removes phenolics. Furthermore, the phenolic compounds are typically concentrated in the outer layers of the corn, some of which are esterified to cell wall materials (Hosny and Rosazza 1997). Thus, in oil the low phenolic content is not unexpected. Although differences in the ferulic acid content of different corn cultivars has been observed, the bound ferulic make up the greatest percentage of the ferulic acid (Table 3.4; Adom and Liu 2002; de la Parra et al. 2007). The bound ferulic acid made up approximately 98% of the ferulic acid in the raw corn regardless of cultivar. The free and soluble conjugates of ferulate increased upon processing of the corn (Figure 3.13). Methods that hydrolysis the ferulic acid dehydrodimers (diFA) from the heteroxylan residues are required if accurate concentrations of diFA are to be determined (Saulnier and Thibault 1999). Yadav, Moreau, and Hicks (2007) reported the use of 1M sodium hydroxide was sufficient to hydrolyze phenolic compounds attached to arabinoxylans. Renger and Steinhart (2000) reported that diFA determination required strong alkaline conditions, where a protocol using 4 M sodium hydroxide produced high diFA levels (Table 3.5). In addition to ferulic acid, red-, blue-, and purple-colored corn genotypes/cultivars can be important sources of anthocyanins (de Pascual-Teresa, Santos-Buelga, and Rivas-Gonzalo 2002; de la Parra et al. © 2011 by Taylor & Francis Group, LLC

37

Phytochemicals in Cereals, Pseudocereals, and Pulses TABLE 3.4 The Ferulic Content (mg/kg) in Corn and Corn Products Corn Variety Product

White

Yellow

Red

Blue

High Carotenoid

Corn Masa Tortilla Chips

1,205 422 852 607

1,030 779 1,143 902

1,303 498 738 754

1,299 533 1,014 856

1,530 762 1,366 1,066

Source: Adapted from de la Parra, C., Saldivar, S., Serna, L., and Rui, H., J. Agric. Food Chem., 55, 4177–83, 2007.

Chips

Tortilla

Masa

Raw corn

0%

10%

20%

30% 40% 50% 60% 70% Percentage of ferulic acid content Free

Soluble conjugates

80%

90%

100%

Bound

FIGURE 3.13  Ferulic acid distribution in white corn and products made from white corn. (Adapted from de la Parra, C., Saldivar, S., Serna, L., and Rui, H., J. Agric. Food Chem., 55, 4177–83, 2007.)

TABLE 3.5 Phenolic Content (mg/kg) of Corn Fiber Alkaline Hydrolysis Phenolic Acid

1 M Sodium Hydroxide

ferulic p-coumaric Ferulic Dimersa 8-5-diFA 5-5-diFA 8-8-diFA 8-O-4-diFA 8-8-aryl-diFA

4 M Sodium Hydroxide

10,318 502

9440 262

117 160 45 170 186

175 239 67 255 279

Source: Adapted from Renger, A. and Steinhart, H., European Food Res. Techn., 211, 422–8, 2000. a 8-5-diFA: Ferulic acid 8-5-dehydrodimer; 5-5-diFA: Ferulic acid 5-5-dehydrodimer; 8-8-diFA: Ferulic acid 8-8-dehydrodimer; 8-O-4-diFA: Ferulic acid 8-O-4-dehydrodimer; 8-8-aryl-diFA: Ferulic acid 8-8-aryl-dehydrodimer.

© 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

TABLE 3.6 Total Anthocyanin Content (mg/kg) in Corn and Corn Productsa de la Parra et al. 2007

Del Pozo-Insfran et al. 2007b

Product

White

Yellow

Red

Blue

High Carotenoid

Mexican White

Mexican Blue

American Blue

Corn Masa Tortilla Chips

13.3 2.8 4.8 5.1

5.7 3.1 2.9 3.6

97.5 22.1 20.8 24.1

368.7 26.3 38.1 32.9

46.3 5.6 6.8 9.7

not detected

342.2 177.9 157.4 85.6

260.9 130.5 96.5 49.6

a b

Expressed as mg cyanidin-3-glucoside /kg sample. Values for the corn products are estimated values based on the percentage of retention data.

High carotenoid

Corn variety

Blue Red Yellow White 0%

10%

20% Lutein

30% 40% 50% 60% 70% Percent composition of carotenoids Zeaxanthin

β-cryptoxanthin

80%

90%

100%

β-carotene

FIGURE 3.14  Carotenoid distribution in various corn cultivars. (Adapted from de la Parra, C., Saldivar, S., Serna, L., and Rui, H., J. Agric. Food Chem., 55, 4177–83, 2007.)

2007; Del Pozo-Insfran et al. 2007). The anthocyanin content of corn is very much dependent on cultivar. Blue genotypes had significantly higher anthocyanins contents (Table 3.6; de la Parra et al. 2007; Del ­Pozo-Insfran et al. 2007). Anthocyanins such as cyanidin-3-glucoside, pelargonidin-3-glucoside, peonidin-3glucoside, and acylated forms of these have been reported in purple corn (de Pascual-Teresa, Santos-Buelga, and Rivas-Gonzalo 2002; Pedreschi and Cisneros-Zevallos, 2007). Similar to ferulates, processing affected anthocyanins (Table 3.6). Cortes et al. (2006) reported ca. 50–60% reduction in acyl-type anthocyanins but did observe increases in the agylcons cyanidin and pelargonidin. In general, the processing of corn via nixtamalization (i.e., cooking under alkaline conditions) causes reductions in the amounts of anthocyanins. However, no trend in anthocyanins stability was observed between various reports regarding the effects of subsequent processing into tortillas or chips (de la Parra et al. 2007; Del Pozo-Insfran et al. 2007).

Carotenoids Carotenoids make up a significant part of the phytochemicals of corn, unlike most cereals. The carotenoid content in corn varies widely (0.15–33 mg/kg) due to a number of factors; however, cultivar has the greatest impact (Chen and Yang 1992; Kurilich and Juvik 1999). Colored cultivars also differ significantly in total and individual carotenoids levels (Figure 3.14; de la Parra et al. 2007). Moros et al. (2002) reported that corn gluten meal contained approximately 146 mg xanthophylls/kg while corn contained 11 mg/kg thus indicting a means to concentrate xanthophylls. In general, α- and β-carotene are the major carotenes whereas β-cryptoxanthin, lutein, and zeaxanthin make up the majority of the xanthophylls. Kurilich and Juvik (1999) reported that the mean carotenoid level for all 44 cultivars was 10.4 mg/kg. © 2011 by Taylor & Francis Group, LLC

39

Phytochemicals in Cereals, Pseudocereals, and Pulses TABLE 3.7 Total Carotenoid Content (mg/kg) in Corn and Corn Products Corn Genotype / Variety Carotenoid Corn Masa Tortilla Chips

White

Yellow

Red

Blue

High Carotenoid

0.18 0.17 0.18 0.11

8.12 2.85 2.23 1.42

2.67 0.96 0.91 0.73

0.46 0.38 0.27 0.14

6.37 2.25 2.05 1.67

Source: Adapted from de la Parra, C., Saldivar, S., Serna, L., and Rui, H., J. Agric. Food Chem., 55, 4177–83, 2007.

Lutein accounted for 57% of the carotenoids while zeaxanthin and β-cryptoxanthin made up 21 and 5% of the carotenoids, respectively. Only 8% of the total carotenoids were carotenes (Kurilich and Juvik 1999). Schlatterer and Breithaupt (2005) reported β-cryptoxanthin contents of 0.21–0.39 mg/kg in canned corn and 47.9 in mg/kg in cooked. These values fall within the range of β-cryptoxanthin contents of fresh frozen sweet corn reported by Kurilich and Juvik (1999). Thus, suggesting that the heating of corn had only a slight impact on carotenoids. De la Parra et al. (2007) found lower carotenoid values in product such as chips and tortilla (Table 3.7). Much of the carotenoids loss was due to the preparation of masa, which was used to produce the tortillas and chips.

Phytosterols As with other phytochemicals, phytosterols are concentrated in the corn germ and aleurone. The ­average phytosterol content of 49 corn species was 277 mg/kg with a range from 181 to 438 mg/kg (Moreau, Singh, and Hicks 2001). Moreau et al. (1999) reported that phytosterols ferulates accounted for 98–113 mg/kg, 10.4–15.3 mg/kg, and 38–84 mg/kg of the total phytosterols in kernels, bran, and fiber, respectively, in which the coarse fiber contained the highest level (58–84 mg/kg). Jiang and Wang (2005) reported that corn fiber contained 300 mg/kg of phytosterols, which equated to 482.5 mg/kg in the corn fiber oil. Steryl ferulates accounted for 6% of the total sterols in corn fiber (Jiang and Wang 2005). Iwatsuki et al. (2003) reported sterol and ferulate sterol levels in corn bran oil of 274 and 84 mg/kg, respectively. In dry milling, 8 and 17% of the total ferulate phytosterols were obtained from the bran and germ, respectively (Moreau et al. 1999). In contrast, the germ accounted for a higher percentage of the free phytosterols (50%) and phytosterol fatty ester (43%) than the bran (9 and 11%, respectively). In wet milling, the fine and coarse fiber accounted for 25 and 67% of the ferulate phytosterols, respectively. Again, the germ accounted for 59% of the free sterols and 41% of the phytosterol fatty ester. The coarse and fine fiber wet mill fractions accounted for ca. 10% of the free sterols and 34 and 15% of the phytosterol fatty acids, respectively. The gluten fraction accounted for 18% and 10% of the free sterols and phytosterol fatty acids, respectively (Moreau et al. 1999). However, a later study by these authors showed that the aleurone layer was a significant source of phytosterols (Moreau et al. 2000; Singh, Moreau, and Cooke 2001). These authors concluded that the aleurone was the major source of the phytosterols in corn fiber oil. Moreau et al. (2000) also observed that the aleurone layers were the primary source of phytostanols. The extraction protocol is an important factor when determining phytosterol content. Moreau, Powell, and Singh (2003) found that phytosterols extraction improved with the use of hexane and methylene chloride at 100°C compared to alcohols and lower temperatures. Winkler et al. (2007) observed phytosterol contents of 8.87–17.3 mg/g (8870–17,300 mg/kg) in extracts of distillers’ grains, which corresponded to 2.91 to 1.92 mg/g in distillers grains. Soxhlet using ethanol produced the highest yields/recoveries. Ferulate phytosterols accounted for 0.35–0.53 mg/g in distillers grains with ethanol in a Soxhlet extractor giving the best extraction based on oil recovery. However, higher levels of the phytosterols in the extracts were obtained in hexane extraction protocols. A similar distribution of individual phytosterols can be observed between corn fiber and distillers’ grains (Table 3.8). The similar phytosterols were found in corn flakes (Table 3.8). Piironen, Toivo, and Lampi (2002) reported that corn flakes contained 381 mg/kg, wet basis, suggesting that processing did not negatively affect phytosterols. © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications TABLE 3.8 Phytosterol Content (mg/100 g) in Corn Products Fraction/Product Phytosterol/Phytostanol Campesterol Stigmasterol Sitosterol Cycloartenol/Δ5-avenasterold Campestanol Sitostanol 24-Me-cycloartanol/Δ7-stigmastenold a . b

c

d

Fiber

a

485 501 2,763 144 114 415 82

Distillers’ grainb

Corn Flakesc

269 89 858 81 119 294 34

5.8 1.3 22.4 not reported 1.8 6 0.9

Data adapted from Jiang, Y. and Wang, T., J. Americ. Oil Chem. Soc., 82(6), 439–44, 2005. Unknown phytosterols not presented here. Data adapted from Winkler, J., Rennick, K., Eller, F., and Vaughn, S., J. Agric. Food Chem., 55, 6482–6, 2007 for phytosterol composition from the hexane extracts. Data adapted from Piironen, V., Toivo, J., and Lampi, A.-M., Cereal Chem., 79(1), 148–54, 2002. Data reported as wet basis. Cycloartenol and 24-methylenecycloartanol reported by Jiang, Y. and Wang, T., J. Americ. Oil Chem. Soc., 82(6), 439–44, 2005; the Δ5-avenasterol and Δ7-stigmastenol by Winkler, J., Rennick, K., Eller, F., and Vaughn, S., J. Agric. Food Chem., 55, 6482–6, 2007.

Antioxidant Activity A number of reports have demonstrated the antioxidant activity of corn. Various forms of ferulate have been shown to have antioxidant activity (Ohta et al. 1994). The 5-O-feruloyl-L-arabinofuranose and O-(5-O-feruloyl-α-L-arabinofuranosyl)-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose are bound ferulates that were found to have antioxidant activity in liposomes (Ohta et al. 1994). An interesting observation was that the ferulates were more active than the free form (ferulic acid) in liposomes (Ohta et al. 1997). Another ferulate related compound, N,N′-diferuloyl-putrescine, was reported to have high DPPH and superoxide radical scavenging activities and inhibited melanin synthesis (Choi, Jeong, and Lee 2007). These authors also reported that N,N′-dicoumaroyl-putrescine had the highest hydroxyl scavenging activity among polyamine conjugates. Li et al. (2007) reported that the DPPH radical scavenging activity of whole corn meal extracts were genotype dependent. The ORAC values (18.7–24.9 g of Trolox equivalents/kg) were reported for extracts obtained from an HCL-methanol solvent extraction protocol. However, the highest ORAC values (42.9– 68.3 g of Trolox equivalents/kg) were obtained from extracts obtained from alkaline hydrolysis of corn. High amylose genotype F4-HA had the best antioxidant activity. The release of bound phenolics is likely contributed to the improved antioxidant activity of alkaline hydrolysates. Sultana, Anwar, and Przybylski (2007) reported that methanol extracts of corncobs was an effective antioxidant against DPPH radicals and linoleic oxidation. Pedreschi and Cisneros-Zevallos (2006) reported that the phenolic acids and flavanols were more potent antimutagenic compounds than the anthocyanins. The ethyl acetate fractions contained higher quercetin levels than the water extract. The higher quercetin also translated into the higher DPPH radical scavenging activity of extracts from Andean purple corn (Pedreschi and Cisneros-Zevallos 2006). Del Pozo-Insfran et al. (2007) reported that blue corn cultivars had greater antioxidant activity than a white corn cultivar. The Mexican and American blue corn cultivars had ORAC values of 29.6 and 25.6 µM trolox equivalents/g sample, respectively, whereas 17.4 µM Trolox equivalents/g sample was observed in a white cultivar. The anthocyanins and total soluble phenolic contents correlated to the antioxidant ­activity. In contrast, de la Parra et al. (2007) reported that a high carotenoid corn cultivar had the greatest activity followed by a commercial yellow corn. The white corn cultivar had equal radical scavenging activity as blue corn and better activity than a red genotype. Differences in antioxidant protocols likely lead to the differences in antioxidant activity of the raw corn. In most cases, processing of the corn resulted in lower antioxidant activity compared to the raw corn. © 2011 by Taylor & Francis Group, LLC

Phytochemicals in Cereals, Pseudocereals, and Pulses

41

The singlet oxygen quenching activity of carotenoids has been reported (Di Mascio, Murph, and Sies 1991). Age related macular degeneration is believed to be due to factors that include oxidative stress and light damage (Beatty et al. 2000; Shaban and Richter 2002). Thus, the singlet oxygen quenching of carotenoids could play a potential role in eye health. Lutein and zeaxanthin, major carotenoids in corn, have been linked to the prevention of age-related macular degenerations (Moeller et al. 2006), thus suggesting an important role as a corn phytochemical. Thus, the singlet oxygen quenching activity combined with radical scavenging makes corn a valuable source of dietary antioxidants.

Oats In earlier reviews of oat antioxidants, components such as tocols, phenolic compounds, avenanthramides, and phytic acid were discussed (Hall 2001; Peterson 2001). However, other compounds such as carotenoids, phytosterols, and β-glucans were not covered. The β-glucans should be considered a phytochemical component in oats, but in the context of the current chapter will not be discussed. For a review of β-glucans see Brennan and Cleary (2005).

Tocols Worldwide, oat tocols have been well characterized. Tocol levels fall within a range of 15–50 mg/kg, with α-tocotrienol and α-tocopherol making up 86–91% (Hammond 1983; Peterson, 2001). Peterson and Qureshi (1993) reported tocol content of 19–30 mg/kg in 12 oat genotypes grown at different locations within the United States. Bryngelsson, Dimberg, and Kamal-Eldin (2002) and Bryngelsson et al. (2002) reported mean Tocol levels of 18.4 mg/kg with a range from 13.8 to 25.3 mg/kg for eight Swedish oat cultivars. Others have also reported similar findings in Hungary and the United Kingdom (Peterson 2001). In general, genotype and location affected tocols composition. In addition to genotype and location, the location of the tocols in the plant also varies. The groat (i.e., germ and endosperm) accounts for 96% of the tocols whereas the hull is a minor (4%, ca. 1 mg/ kg) source (Bryngelsson et al. 2002). The composition of individual tocols also differs between oat ­fractions. The α-tocotrienol accounts for ca. 70 and 13% of the tocols in the groat and hull, respectively. The opposite distribution occurs with α-tocopherol in which ca. 19 and 63% of the tocols in the groat and hull, respectively (Bryngelsson et al. 2002). Peterson (1995) reported that the germ and endosperm contained the majority of the tocopherols and tocotrienols, thus the groat would serve as a more balanced source of tocols. Processing can alter the composition and concentration of tocols. Peterson (1995) reported that dried groats had the highest tocol content (40 mg/kg) followed by rolled oats and flour, which had the lowest content (28 mg/kg). Bryngelsson, Dimberg, and Kamal-Eldin (2002) also reported processing of raw oats into rolled oats significantly reduced the tocols. The process for making rolled oats affected all individual tocotrienols and tocopherols; however, α-tocotrienol content decreased the most. The concentration of tocols was higher in wholemeal compared to rolled oats. Autoclaving of hulls enhanced the concentration of tocols, especially β-tocopherol, while drum drying caused reductions in tocol levels (Bryngelsson, Dimberg, and Kamal-Eldin 2002). In addition to processing, storage at room temperature dramatically reduced tocols in all samples except the undried groat (Peterson 1995).

Phenolics Sosulski, Krygier, and Hogge (1982) reported the presence of nine different phenolic acids in debranned oat flours totaling 87 mg/kg. The 66.3% of the phenolic acids were in the bound form whereas soluble esters and free forms made up 23.7 and 10% of the total phenolic acids, respectively. Regardless of the phenolic fraction, ferulic acid made up the greatest percentage of any one individual phenolic acid (Sosulski, Krygier, and Hogge 1982). The bound, soluble esters and free forms accounted for 97.8, 1.8, and 0.4% of the total ferulic acid in whole ground oat flour, respectively (Adom and Liu 2002). Differences in the distribution were likely due to the initial oat samples. Mattlia, Pihlava, and Hellström (2005) reported that oat bran contained 450 mg/kg of phenolic acids, which is substantially higher than the levels reported in debranned flour (Sosulski, Krygier, and Hogge 1982). This is not surprising since © 2011 by Taylor & Francis Group, LLC

42

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

the bran is usually high in phenolic compounds. Emmons and Peterson (1999) reported that the groat contained approximately 50% less phenolic acids than the hulls. Renger and Steinhart (2000) reported ferulic acid levels as high as 3328 mg/kg in oat bran hydrolyzed with 1 and 4 M sodium hydroxide (Table 3.9). The high concentration of alkali promoted the breakage of both ester and ether link but did not result in higher ferulic levels compared to lower alkali solution, suggesting that much of the ferulic acid is in an esterified form (Renger and Steinhart 2000). These authors also reported higher ferulic dimer concentrations in the 4 M sodium hydroxide hydrolyzed oat bran (Table 3.9). Cultivar was the main factor that affected individual phenolic concentrations; however, the location where the oat was grown also affected the phenolic ­content (Emmons and Peterson 2001). Differences in the phenolic contents can also be due to the length of the oat storage. Dimberg et al. (1996) noted that phenolic acid contents increase with increasing storage time. Furthermore, storage under high humidity also caused increases in some of the phenolic acids. For an additional review on oat phenolic acids, see the review of Peterson (2001). Caffeic acid was the most sensitive phenolic acid to heat processing (Table 3.9; Bryngelsson, Dimberg, and Kamal-Eldin 2002). Autoclaving of the oat resulted in increased ferulic, p-coumaric, and vanillin contents; however, drying of the autoclaved sample did cause some loss in these phenolic acids compared to the autoclaved samples (Bryngelsson, Dimberg, and Kamal-Eldin 2002). As with autoclaving, no caffeic acid was found in drum dried, milled rolled oats or dried wholemeal. Drum drying also reduced ferulic, p-coumaric, and vanillin contents (Bryngelsson, Dimberg, and Kamal-Eldin 2002). Dimberg et al. (2001) also found that heating caused a reduction in phenolic acids. However, only caffeic acid decreased significantly after being heated at neutral (pH 7) to alkaline (pH 12) conditions. Under acid conditions, (pH 2) minimal reduction in caffeic acid was observed (Dimberg et al. 2001).

Avenanthramides Collins (1989) was the first research to characterize fully the avenanthramides. Although 20–25 different avenanthramides were found, N-(4′-hydroxycinnamoyl)-5-hydroxyanthranilic acid (avenanthramide A), N-(4′-hydroxy-3′-methoxycinnamoyl)-5-hydroxyanthranilic acid (avenanthramide B), and N-(3′,4′dihydroxycinnamoyl)-5-hydroxyanthranilic acid (avenanthramide C) were the major avenanthramides (Figure 3.15). The health benefits of these compounds include antiatherogenic and anti-inflammatory activities (Ji et al. 2003; Chen et al. 2004a; Liu et al. 2004; Nie et al. 2006). The bioavailability of the avenanthramides is high and have in vivo antioxidant activity (Chen et al. 2004a, 2007). Thus, benefits observed in these few studies suggest additional research is needed to characterize fully the health benefits of the avenanthramides. The avenanthramides concentrations vary greatly depending on the cultivar, extraction method, and growing location (Peterson 2001). Less than 10 to 152 mg/kg have been reported (Table 3.10). As with other phytochemicals, the avenanthramides concentration varies with cultivar and ­growing conditions (Dimberg, Theander, and Lingnert 1993; Emmons and Peterson 2001; Dokuyucu, Peterson, and Akkaya 2003; Dimberg, Gissen, and Nilsson 2005). Bryngelsson et al. (2002) observed mean values of 5.3, 5.1, and 3.2 for avenanthramides C, A, and B in oat groats, respectively. They also reported that avenanthramides concentration varied widely between oat cultivars. The oat hull contained ­approximately 50% less avenanthramides than the groat (Emmons and Peterson 1999; Bryngelsson et al. 2002). However, alkaline hydrolysis promoted the release of additional avenanthramides (Peterson 2001). The various levels of avenanthramides in the hull may depend on the content of aleurone contamination, as avenanthramide content decreases away from the aleurone layer (Collins, 1989; Emmons et al., 1999). In addition to the avenanthramides, Collins, McLachlan, and Blackwell (1991) isolated avenalumic acid and the 3’-hydroxy and 3’-methoxy analogues (Figure 3.15). Dihydrodimers of avenanthramides were also present in the leaves of oats (Okazaki et al. 2004, 2007). Although not an edible part of the plant, the leaves could serve as a source of phytochemicals. Additional research is needed to characterize the activities of the avenanthramides dihydrodimers. Unlike the tocopherols and phenolic acids, avenanthramides were negatively influenced by heating and drying (Bryngelsson, Dimberg, and Kamal-Eldin 2002). Dimberg et al. (2001) found that heating caused a reduction in avenanthramides, but only at neutral (pH 7) to alkaline (pH 12) conditions. They also observed that minimal reduction in avenanthramides occurred at pH 2. The sensitivity of the individual © 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC 3282 153

84 132 47 107 96

3328 503

26 32 29 9 57

5.4 330 12 22 90 28 24 140

3.1 25 ND 16 55 20 18 110

Oat Flakes 3.6 250 ND 16 52 20 17 110

Precooked Oat Flakes

Oat Bran

4 M Sodium Hydroxide

1 M Sodium Hydroxide 2.55 1.3 0.5

0.75

2.5

Raw Groatc

3.0 2.5 3.0

Raw Oatc

4.6

ND 1.5 6.7

Raw Hull

1.24

1.48 2.18 0.54

Rolled Oat

3.46

ND 3.17 0.72

Rolled Oat

Bryngelsson, Dimberg, Kamal-Eldin (2002)

1.8

ND 1.4 0.5

Drum Dried Rolled Oatc

Note: ND = not detected, empty spaces indicated that component was not determined. a Data reported for raw oat bran fiber hydrolyzed with sodium hydroxide and solid phase extraction eluted with methanol:water (50:50 v/v). b Ferulic dimers not separated into individual components. c Estimated values from figures. d 8-5-diFA: Ferulic acid 8-5-dehydrodimer; 5-5-diFA: Ferulic acid 5-5-dehydrodimer; 8-8-diFA: Ferulic acid 8-8-dehydrodimer; 8-O-4-diFA: Ferulic acid 8-O-4-dehydrodimer; 8-8-aryldiFA: Ferulic acid 8-8-aryl-dehydrodimer.

Phenolic Acid caffeic ferulic p-coumaric p-hydroxybenzoic acids sinapic syringic vanillic Ferulic Dimersd 8-5-diFA 5-5-diFA 8-8-diFA 8-O-4-diFA 8-8-aryl-diFA

Phenolic Compound

Mattila, Pihlava, and Hellström (2005)b

Renger and Steinhart, 2000a

Phenolic Content (mg/kg) of Oat and Oat Products

TABLE 3.9

Phytochemicals in Cereals, Pseudocereals, and Pulses 43

44

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications R

HOOC

N H

O C

R1 OH

E form

R OH OH OH H H

N-(4'-hydroxycinnamoyl)-5-hydroxyanthranilic acid N-(4'-hydroxy-3'methoxycinnamoyl)-5-hydroxyanthranilic acid N-(3',4'-dihydroxycinnamoyl)-5-hydroxyanthranilic acid N-(4'-hydroxycinnamoyl)anthranilic acid N-(4'-hydroxy-3'-methoxycinnamoyl)anthranilic acid

R1 H OCH3 OH H OCH3

FIGURE 3.15  Avenanthramides found in oats. (Adapted from Collins, F., J. Agric. Food Chem., 37, 60–66, 1989.)

TABLE 3.10 Avenanthramide Content (mg/kg) of Oat and Oat Products Mattila, Pihlava, and Hellström (2005)b

Avenanthramidea A B C a

b c

Bryngelsson et al. (2002)

Peterson Oat Precooked Drum (2001) Hull/ Oat Oat Raw Raw Raw Rolled Rolled Dried Bran Flakes Flakes Oatc Groatc Hull Oat Oat Rolled Oatc Groats Hulls 4.1 4.3 4.4

8.6 9.0 9.0

8.3 8.8 9.1

3.95 2.20 3.55

3.45 2.00 3.15

2.40 1.10 1.20

1.89 2.19 3.11

6.61 6.77 11.01

2.60 2.20 2.70

54 36 52

25 17 14

Avenanthramide A: N-(4′-hydroxycinnamoyl)-5-hydroxyanthranilic acid; Avenanthramide B: N-(4′-hydroxy-3′methoxycinnamoyl)-5-hydroxyanthranilic acid; Avenanthramide C: N-(3′,4′-dihydroxycinnamoyl)-5-hydroxyanthranilic acid. Abbreviation of the avenanthramides from these authors. Estimated values from figures.

avenanthramides was different. Only avenanthramide C was completely degraded at pH 12 and only about 20% remained after heating at pH 7 (Dimberg et al. 2001). Mattila, Pihlava, and Hellström (2005) noted that oat flakes contained higher levels of avenanthramides than bran and that precooked oat flakes had only slightly lower levels of avenanthramides compared to oat flakes. Steaming and flaking had only a slight impact on the avenanthramides while drum drying caused significant reductions in these compounds (Bryngelsson, Dimberg, and Kamal-Eldin 2002). However, the avenanthramides reduction, from 7–11 mg/kg to 2–3 mg/kg, was more pronounced in the milled rolled oat (Table 3.10). In contrast, Dimberg et al. (2001) reported higher avenanthramide contents in pasta and baked products. They concluded that processing promoted the release of the bound avenanthramides. The higher concentration of avenanthramides in steeped and germinated oats was postulated as the result of a de novo synthesis (Bryngelsson, Ishihara, and Dimberg 2003).

Other Components Määttä et al. (1999) reported that oat phytosterol contents between 350 and 491 mg/kg. The β-sitosterol, Δ5-avenasterol, campesterol, Δ7-avenasterol, and stigmasterol accounted for 53, 26, 8, 8, and 4% of the total phytosterols, respectively. Dutta and Appelqvist (1996) also found similar phytosterol levels. Although differences in individual and total sterols were reported between cultivars, the percentage of individual sterols in the total sterol content was similar between cultivars (Määttä et al. 1999). Jiang and Wang (2005) reported that oat bran and hulls contained 1500 and 700 mg/kg of phytosterols, respectively. However, the oat hull oil contained higher levels (8180 mg/kg) of phytosterols than the oat bran oil (3410 mg/kg). Thus, the oat oil appears to be an effective method for delivering phytosterols into foods. © 2011 by Taylor & Francis Group, LLC

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Phytochemicals in Cereals, Pseudocereals, and Pulses

Antioxidant Activity Several reviews of the antioxidant activity oats have been published (Hall 2001; Peterson 2001). Zieliński and Kozłowska (2000) reported that 80% methanol extracts of hulls gave the highest Trolox values among different fractions tested. The epicatechin concentration was also highest in the hull fraction supporting the link between phenolics and antioxidant activity. Emmons and Peterson (2001) found that the cultivar had significant impact on the antioxidant activity of oats in a carotene-linoleic oxidation model. The antioxidant activity differences were likely due to the significant differences in phenolic composition of the cultivars. Concentration of the phenolics in pearling fractions also correlated to better antioxidant activity in a carotene-linoleic oxidation model, ORAC and DPPH radical scavenging assays, and LDL oxidation assay (Emmons, Peterson, and Paul 1999; Gray et al. 2002; Peterson, Emmons, and Hibbs 2001). Bratt et al. (2003) reported that sinapic acid had the greatest DPPH radical scavenging activity of individual oat phenols followed by caffeic, ferulic, and p-coumaric. The presence of a second OH group on ring A of the avenanthramides appeared to improve the antioxidant activity over time. However, most of the avenanthramides had antioxidant activity (Bratt et al. 2003). Peterson, Hahn, and Emmons (2000) also found that the avenanthramides had antioxidant activity in the carotene-linoleic oxidation model and DPPH radical scavenging assay. These authors also observed that avenanthramide C had the best activity in both test systems. In contrast to the observed antioxidant activity of avenanthramides, total antioxidant capacity of different oat cultivars did not correlate well with avenanthramides (Bryngelsson, Ishihara, and Dimberg 2003). The tocols content appear to have some link to antioxidant capacity of different oat cultivars but this trend was not observed with the hull. In general, the antioxidant capacity of oats is likely due to multiple components and developing concentration protocols would enhance the antioxidant capacity of oats.

Rice Rice bran has received much attention over the last two decades as an important source of phytochemicals. Thus, the discussion on rice will focus primarily on rice bran, but will also include a discussion on newer cultivars of colored rice. The phytochemicals in rice include tocols, oryzanols (Figure 3.16), phenolic acids, and anthocyanins (see reviews by Hall 2001, 2003). The interest in these phytochemicals stem from the ability of rice bran oil to modulate plasma lipid (Kahlon et al. 1996; Rong, Ausman, and Nicolosi 1997; Vissers et al. 2000; Kooyenga et al. 2001), suppress melanoma cell proliferation (He et al. 1997; Qureshi et al. 1997, 2000; Lane, Qureshi, and Salser 1999), and inhibit tumor growth (Yasukawa et al. 1998; Akihisa et al. 2000). The reader is directed to the review by Cicero and Gaddi (2001) and Smith and Kahlon (2004) for additional discussion of the health benefits of rice.

Tocols and Oryzanols A significant body of knowledge exists regarding the composition of tocols and oryzanols in rice bran oil. Thus, see the reviews by Hall (2001, 2003) for detailed information regarding these components. In O HN O

CH2CH3

O OH 4-carboethoxy-6-hydroxy-2-quinolone

FIGURE 3.16  Alkaloid antioxidant isolated from pigmented rice. (Adapted from Chung, H. S., and Shin, J. C., Food Chem., 104, 1670–7, 2007.)

© 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

general, tocols in rice bran oil ranged from 744 to 800 mg/kg, whereas oryzanol contents were much greater and ranged from 13,071 to 14,777 mg/kg (Hu et al. 1995; Hall and Proctor, 1996; Proctor and Bowen 1996; Xu and Godber 2000). The variation in composition was due primarily to extraction protocols. Significantly higher oryzanol levels were obtained from extraction protocols using isopropyl alcohol than hexane. However, comparable levels of tocols were observed in oils obtained from hexane and isopropyl alcohol extraction of rice bran. Zhao et al. (1987) reported lower tocols and oryzanol levels in oil obtained from rice bran extracted with supercritical carbon dioxide (SC-CO2) compared to hexane extraction. In contrast to rice bran oil, fewer studies have reported phytochemicals composition in the bran or rice as a whole. Aguilar-Garcia et al. (2007) reported that rice bran contained between 196 and 219 mg/ kg of tocols. The tocotrienols accounted for 72–79% of the tocols in which the variation was cultivar dependent. Choi et al. (2007) also reported that tocol levels were cultivar dependent. They observed that a black, brown, and white rice contained 93.7, 26.4, and 7.4 mg/kg total tocols, respectively. The tocotrienols again made up between 42 and 89% of the total tocols. Ko et al. (2003) reported that the germ contained the highest concentration (424.5 mg/kg) of tocols followed by the bran (237.6 mg/kg), endosperm (5.2 mg/kg), and hull (3.1 mg/kg). In contrast to previous reports, the tocopherols made up the majority of the tocols. Iqbal, Bhanger, and Anwar (2005) reported that the tocopherol and tocotrienol contents were approximately equal in rice bran. The cultivars grown in Pakistan had tocopherol and tocotrienol levels of 392–512 mg/kg and 343–478 mg/kg, respectively. The γ-oryzanol concentration was also dependent on cultivar. Aguilar-Garcia et al. (2007) reported that rice bran contained between 1550 and 2720 mg/kg of γ-oryzanol. Lilitchan et al. (2007) reported γ-oryzanol levels between 1950 and 3070 mg/kg in bran from nine rice cultivars. Iqbal, Bhanger, and Anwar (2005) found less variation in γ-oryzanol levels among five rice cultivars grown in Pakistan. These authors reported γ-oryzanol levels between 511 and 802 mg/kg. Solvent extraction protocols were shown to be an effective means to concentrate the phytochemicals. An acetone–lipid fraction from the original methanol extract of bran had 65 time more oryzanols and tocols, while 70 times more ferulic acid was found in an acetone-polar fraction of the methanol extract (Renuka Devi, Jayalekshmy, and Arumughan 2007; Renuka Devi and Arumughan 2007b). The concentrations of oryzanols and tocols in the crude methanol extract were 7832 and 146 mg/kg, respectively, whereas the acetone–lipid fraction contained 20,469 and 347 mg/kg of these same compounds, respectively (Renuka Devi, Jayalekshmy, and Arumughan 2007). The ferulic acid increases from 5786 in the crude methanol extract to 15,858 mg/kg in the acetone polar fraction (Renuka Devi, Jayalekshmy, and Arumughan 2007). The γ-oryzanol content was also concentrated in an enzyme-hydrolyzed rice bran extract. Parrado et al. (2006) reported that an enzyme extract of rice bran had 1200 mg/kg of γ-oryzanol compared to the 350 mg/kg in the original rice bran. In contrast, tocopherol contents were lower in the extract compared to the bran. Stabilization of rice bran is critical if the phytochemical components are to be utilized in food systems. Hall (2003) presented a summary of rice bran stability and the impact on the contents of the oryzanols and tocols. In summary, extrusion, microwave processing, and Gamma irradiation have been used to stabilize rice bran. Lower temperature (110°C) extrusion was deemed best as only 7 and 4% reductions in tocols and oryzanols, respectively, occurred (Kim et al. 1987a,b). These authors observed that extrusion at 140°C promoted the reduction (21 and 8%, respectively) of tocols and oryzanols. We (Hall and Proctor 1996) found that a microwave treatment did not significantly affect tocol or oryzanol levels. Oryzanol concentrations in the extracted oil decreased from 13,144 to 13,071 mg/kg after a short (1.87 sec/g rice bran) microwave treatment. In contrast, the tocols increased slightly from 734 to 769 mg/kg in the microwave treated rice bran. These results reflected the observation on microwave processing reported by Rhee and Yoon (1984) and Tao, Rao, and Liuzzo (1993). In contrast to extrusion and microwave processing, gamma irradiation was detrimental to tocols and oryzanols and thus not recommended as a stabilizing method (Shin and Godber, 1996). The refining of rice bran oil has been shown to cause significant reductions in the tocols and oryzanols (Kim et al., 1985; Yoon and Kim, 1994). Krishna et al. (2001) reported small reductions (1.1 and 5.9%, respectively) of oryzanols found in degummed and dewaxed rice bran oil. In contrast, alkali treatments removed 93.0 to 94.6% of oryzanol from the original crude rice bran oil. The soapstock contained high © 2011 by Taylor & Francis Group, LLC

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(6.3–6.9%) oryzanol levels. Thus, alkaline refining could be a method to concentrate the oryzanols if the oryzanols are recovered from the soapstock. Deodorization did not significantly affect oryzanol content; however, a reduction in tocopherol content was observed in deodorized rice bran oil (Yoon and Kim 1994; De and Bhattacharyya 1998; Krishna et al. 2001).

Phenolics As with other grains, ferulic acid is the main phenolic acid (Table 3.11). Zhou et al. (2004) reported total phenolic acid contents of 415 to 528 mg/kg in brown rice. The rice cultivar did contribute to the differences in phenolic level; however, ferulic acid was always significantly higher than other phenolic acids. The phenolic acids of rice are primarily concentrated in the bran fraction and are in the bound form (Sosulski, Krygier, and Hogge 1982; Adom and Liu 2002; Zhou et al. 2004). Hegde et al. (2005) recently reported the degradation of phenolic esters during the incubation of rice bran with Aspergillus niger. The degradation of the phenolics from the cell wall polysaccharides supports the observations that the highest percentage of the phenolic acids are of the bound type. However, treatment with α-amylase only enhanced the extraction of phenolic acids in milled rice and not in brown rice (Zhou et al. 2004). This suggested that enzymes, which hydrolyze arabinoxylans, were responsible for the observed polysaccharide-phenolic degradation reported by Hegde et al. (2005) and that the phenolic acids are most likely bound to the cell wall polysaccharide and not the starch polysaccharides. Treatment of rice bran fiber with strong alkali significantly enhanced the extraction of phenolic acids (Renger and Steinhart 2000) further supporting the cell wall-bound phenolics theory. Reduction of bound phenolic acids occurred in brown and milled rice over a six-month storage at both 4 and 37°C (Zhou et al. 2004). These authors did observe a greater phenolic acid reduction during storage at 37°C. However, the free phenolic acids that increased suggests an enzymatic release of the bound phenolic acids (Zhou et al. 2004). Rao and Muralikrishna (2007) reported that feraxans (i.e., water-soluble feruloyl arabinoxylans) from malted rice contained significantly higher concentrations of bound ferulic acid than native rice feraxans. These authors concluded that xylanase promoted the hydrolysis of arabinoxylans at either locations that lead to increased numbers of water-soluble fractions. These fractions also had higher concentrations of ferulic acid compared to the water-soluble fractions of the native rice. In recent years, there has been an increased interest in black and pigmented rice cultivars as a dietary source of phytochemicals. Cyanidin-3-O-β-glucoside and peonidin-3-O-β-glucoside were the major anthocyanins found in the black (Oryza sativa L. indica) and pigmented (Oryza sativa L. japonica) rice cultivars (Ryu, Park, and Ho 1998; Hu et al. 2003; Yawadio, Tanimori, and Morita 2007). Chung and Woo (2001) also isolated an alkaloid from pigmented rice cultivars (Figure 3.16). This alkaloid inhibited the growth of human leukemia in vitro (Chung 2002) and had antioxidant activity (Chung and Shin 2007).

Other Components On a wet basis, the phytosterols content of polished and brown rice were approximately 29 and 72 mg/ 100 g, respectively (Piironen, Toivo, and Lampi 2002). Jiang and Wang (2005) reported that rice bran contained 4500 mg/kg of phytosterols, which equated to 20,330 mg/kg in the rice bran oil (Table 3.12). The steryl ferulates accounted for 20.3% of the total sterols in rice bran (Jiang and Wang 2005). Abdul-Hamid et al. (2007) reported that carotenoids content of 0.587–2.16 mg/kg in rice bran. Choi et al. (2007) reported that carotenoid levels varied with cultivar. They noted that black rice had carotenoid levels of 0.77 mg/kg while brown and white rice had carotenoid levels of 0.14 and 0.01 mg/kg, respectively. In contrast, transgenic rice endosperm contains approximately 1.5–19 mg/kg of carotenoids (Paine et al. 2005).

Antioxidant Activity The antioxidant activity of rice has been documented extensively (Hall 2001). Recently, the radical scavenging activity of rice extracts and individual compounds has been demonstrated in DPPH, ABTS, and ORAC assays (Iqbal, Bhanger, and Anwar 2005; Nam et al. 2005; Aguilar-Garcia et al. 2007; Chung and Shin 2007). The total phenolic content (gallic acid eq.) corresponded well with antioxidant assays. © 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC

3823 310

32 36 15 41 57

ND ND ND ND ND

4 M Sodium Hydroxide

3046 310

1 M Sodium Hydroxide 120 38 13 17 ND ND 9

Long Grain White Rice 240 76 15 20 ND 8 17

Long Grain Brown Rice 92 29 4 7 ND 3 4

Cooked Long Grain Brown Rice

Mattila, Pihlava, and Hellström (2005)b

2.7 1.3 1.9 ND ND 1.3

Free 9.6 ND 2.7 ND 0.2 0.8

Soluble Esters 63.1 ND 0.4 ND ND ND

Bound

Sosulski, Krygier, L. Hogge (1982)c

255 70

2.6 12

2.9 3

Enzymatic Hydrolysis 259 71

Alkaline Extraction

Zhou et al. (2004)d

a

Note: ND = not detected or in trace amounts, empty spaces indicated that component was not determined. Data reported for raw rice bran fiber hydrolyzed with sodium hydroxide and solid phase extraction eluted with methanol:water (50:50 v/v). b Rice was the parboil type. Ferulic dimers not separated into individual components. c Reported as free, soluble esters and bound phenolic acids. d Brown rice extracted under alkaline conditions or treated with enzyme prior to extraction. e e8-5-diFA: Ferulic acid 8-5-dehydrodimer; 5-5-diFA: Ferulic acid 5-5-dehydrodimer; 8-8-diFA: Ferulic acid 8-8-dehydrodimer; 8-O-4-diFA: Ferulic acid 8-O-4-dehydrodimer; 8-8-aryldiFA: Ferulic acid 8-8-aryl-dehydrodimer.

Phenolic Acid Ferulic p-coumaric p-hydroxybenzoic acids Sinapic Syringic Vanillic Ferulic Dimerse 8-5-diFA 5-5-diFA 8-8-diFA 8-O-4-diFA 8-8-aryl-diFA

Phenolic Compound

Renger and Steinhart, 2000a

Phenolic Content (mg/kg) of Rice and Rice Products

TABLE 3.11

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Phytochemicals in Cereals, Pseudocereals, and Pulses TABLE 3.12 Phytosterol Content (mg/100 g) in Rice Fraction/Product Phytosterol/Phytostanol Brassicasterol Campesterol Stigmasterol Sitosterol Cycloartenol Campestanol Sitostanol 24-methylcycloartanol

Bran 261 180 494 333 36 84 525

a

Polished Rice

Brown Rice

4.5 3.2 20 trace 1.3 1.7 NR

14.6 10.4 37.5 4.5 1.6 1.7 NR

Note: NR = not reported. a Data adapted from Jiang, Y., and Wang, T., J. Americ. Oil Chem. Soc., 82(6), 439–44, 2005. Unknown phytosterols not presented here. b Data (wet basis) adapted from Piironen, V., Toivo, J., and Lampi, A.-M., Cereal Chem., 79(1), 148–54, 2002.

Cultivars with higher phenolic contents also had higher antioxidant activity (Iqbal, Bhanger, and Anwar 2005). Renuka-Devi and Arumughan (2007a) reported that the DPPH radical and superoxide radical scavenging activities were due to ferulic acid in the rice bran extracts. The tocopherols, tocotrienols, and oryzanols content also corresponded with the antioxidant activity of five different rice cultivars (Iqbal, Bhanger, and Anwar 2005). However, chelating activity did not correspond to the phytochemical contents. Choi et al. (2007b) reported that total phenolics and tocols corresponded well with radical scavenging assays and chelating properties. Renuka-Devi and Arumughan (2007a) reported that extracts of rice bran were more effective at controlling soybean oxidation at 60°C than individual rice components (e.g., oryzanols, ferulic acid). These authors also observed that the acetone polar fraction of a crude methanol extract had the best activity in the 60°C-heated oils. This fraction had significantly more ferulic acid, but less tocols and oryzanols, than an acetone-lipophilic fraction of the crude methanol extract, which had slightly lower antioxidant activity (Renuka-Devi and Arumughan 2007a). Sitostanyl ferulate was nearly as effective as α-tocopherol in preventing polymerization of high oleic sunflower oil during heating at 100 and 180°C (Nyström et al. 2007). Aguilar-Garcia et al. (2007) reported that the ferric reducing antioxidant power was influenced by the polyphenolics, oryzanols, and tocotrienols whereas the ORAC test results were affected by polyphenolics, oryzanols, and total tocopherols. Aguilar-Garcia et al. (2007) cautioned readers that testing protocols could influence the conclusion one makes regarding the antioxidant activity of rice bran.

Rye Rye is most commonly used in breads and flour mixes. Rye consumption is greatest in Northern Europe. In North America, rye is used in specialty breads and thus plays a lesser role in the human diet. Rye is a cereal that contains substantial amounts of phytochemicals. These compounds include phenolic acids (Andreasen et al. 1999; Mattila, Pihlava, and Hellström 2005), alkylresorcinols (Kozubek and Tyman 1995; Ross et al. 2001; Mattila, Pihlava, and Hellström 2005), tocols (Ryynänen et al. 2004), and phytosterols (Nyström et al. 2007).

Tocols The total tocols in rye are significantly lower than oilseed where most researchers have reported levels between 20 and 55 mg/kg (Barnes 1983; Piironen et al. 1986; Ryynänen et al. 2004). Regardless of the rye investigated, the total tocotrienols were higher than the tocopherols (Barnes 1983; Piironen et al. 1986; Ryynänen et al. 2004; Zieliński, Ceglińska, and Michalska 2007). The average α-tocotrienols © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

was 16.9 mg/kg followed by α-tocopherol (14.4 mg/kg) and β-tocotrienols (13 mg/kg) in 10 rye cultivars grown in Finland (Ryynänen et al. 2004). Regardless of the cultivar, the α-tocotrienols was always in the greatest composition. In contrast, Zieliński, Ceglińska, and Michalska (2007) reported similar α-tocotrienols (6.58 mg/kg) and α-tocopherol (7.68 mg/kg) levels in three Polish rye cultivars. Equal amounts of α-tocopherol and α-tocotrienols were reported among rye flours, suggesting that milling may have affected tocol contents (Piironen et al. 1986; Ryynänen et al. 2004). However, β-tocotrienols contents in rye flour were substantially higher than the β-tocopherols, following the same pattern as in the whole rye. The reduction of α-tocotrienols in rye flour can be explained by the removal of the pericarp (including testa) during rye flour production. Zieliński, Ceglińska, and Michalska (2007) observed higher α-tocotrienols in the pericarp fraction (14.5 mg/kg) compared to the endosperm (6.0 mg/kg). The average α-tocopherol contents of 2.2 and 6.29 mg/kg were found in the pericarp and endosperm, respectively. Overall, the distribution of the individual tocols remained relatively constant among data reported; however, total tocols were very much dependent on cultivars or cultivars (Piironen et al. 1986; Ryynänen et al. 2004; Zieliński, Ceglińska, and Michalska 2007).

Phenolics The contents of phenolic acids and ferulic acid dehydrodimers (diFA) in rye vary widely depending on rye cultivar or cultivar, growing location, extraction method, and grain maturity (Andreasen et al. 1999, 2000, 2001; Weidner et al. 2000). In general, free phenolic acids are in minimal concentrations ( 4)-β-D-glucans as functional food ingredients. J. Cereal Sci. 42:1–13. Brown, J., and C. Rice-Evans. 1998. Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro. Free Rad. Res. 29:247–55. Bryngelsson, S., L. Dimberg, and A. Kamal-Eldin. 2002. Effects of commercial processing on levels of antioxidants in oats (L. Avena sativa). J. Agric. Food Chem. 507:1890–6. Bryngelsson, S., A. Ishihara, and L. Dimberg. 2003. Levels of avenanthramides and activity of hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) in steeped or germinated oat samples. Cereal Chem. 803:356–60. Bryngelsson, S., B. Mannerstedt-Fogelfors, A. Kamal-Eldin, R. Andersson, and L. Dimberg. 2002. Lipids and antioxidants in groats and hulls of Swedish oats (Avena sativa L). J. Sci. Food and Agr. 82:606–14. Budin, J., W. Breene, and D. Putnam. 1996. Some compositional properties of seeds and oils of eight Amaranthus species. J. Am. Oil Chem.’ Soc. 73:475–81. Bunzel, M., J. Ralph, J. Marita, R. Hatfield, and H. Steinhart. 2000. Identification of 4-O-5′-coupled diferulic acid from insoluble cereal fiber. J. Agric. Food Chem. 48:3166–69. Bunzel, M., J. Ralph, J. Marita, R. Hatfield, and H. Steinhar. 2001. Diferulates as structural components in soluble and insoluble cereal dietary fiber. J. Sci. Food and Agr. 81:653–60. Burton, G., and K. Ingold. 1984. β-Carotene: An unusual type of lipid antioxidant. Science 224:569–73. Calzuola, I., G. Gianfranceschi, and V. Marsili. 2006. Comparative activity of antioxidants from wheat sprouts, Morinda citrifolia, fermented papaya and white tea. Inter. J. of Food Sci. Nutr. 57:168–77. Cao, G., E. Sofic, and R. Prior. 1997. Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radical Biol. Med. 22 (5): 749–60. Chen, B., and S. Yang. 1992. An improved analytical method for the determination of carotenes and xanthophylls in dried plant materials and mixed feeds. Food Chem. 44:61–6. Chen, C. Y., P. Milbury, F. Collins, and J. Blumberg. 2007. Avenanthramides are bioavailable and have antioxidant activity in humans after acute consumption of an enriched mixture from oats. J. Nutr. 137 (6): 1375–82. Chen, C. Y., P. Milbury, H. K. Kwak, F. Collins, P. Samuel, and J. Blumberg. 2004b. Avenanthramides and phenolic acids from oats are bioavailable and act synergistically with vitamin C to enhance hamster and human LDL resistance to oxidation. J. Nutr. 134 (6): 1459–66. Chen Y., A. Ross, P. Aman, and A. Kamal-Eldin. 2004a. Alkylresorcinols as markers of whole grain wheat and rye in cereal products. J. Agric. Food Chem. 52:8242–6. Cheng, Z., L. Su, J. Moore, K. Zho, M. Luther, J. Yin, and L. Yu. 2006. Effects of postharvest treatment and heat stress on availability of wheat antioxidants. J. Agric. Food Chem. 54:5623–9. Cho, Y.-S., K.-J. Yeum, C.-Y. Chen, G. Beretta, G. Tang, N. Krinsky, S. Yoon, Y. Lee-Kim, J. Blumberg, and R. Russell. 2007. Phytonutrients affecting hydrophilic and lipophilic antioxidant activities in fruits, vegetables and legumes. J. Sci. Food and Agr. 87:1096–107. Choi, S., S. Lee, E. Kim, J. Oh, K. Yoon, N. Parris, K. Hicks, and R. Moreau. 2007. Antioxidant and antimelanogenic activities of polyamine conjugates from corn bran and related hydroxycinnamic acids. J. Agric. Food Chem. 55:3920–5. Choi, Y., H.-S. Jeong, and J. Lee. 2007. Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chem. 103:130–8. Chou, S. T., W. W. Chao, and Y. C. Chung. 2003. Antioxidative activity and safety of 50% ethanolic red bean extract (Phaseolus radiatus L. var. Aurea). J. Food Sci. 68:21–5. Chung, H. S. 2002. A quinolone alkaloid, from the aleurone layer of Oryza sativa cv. Mihyangbyeo, inhibits growth of cultured human leukemia cell. Nutraceuticals Food 7:119–22. Chung, H. S., and J. C. Shin. 2007. Characterization of antioxidant alkaloids and phenolic acids from anthocyanin-pigmented rice (Oryza sativa cv. Heugjinjubyeo). Food Chem. 104:1670–7. © 2011 by Taylor & Francis Group, LLC

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Vissers, M., P. Zock, G. Meijer, and M. Katan. 2000. Effect of plant sterols from rice bran oil and triterpene alcohols from shea nut oil on serum lipoprotein concentrations in humans. Amer. J. Clin. Nutr. 72:1510–5. Waladkhani, A., and M. Clemens. 1998. Effect of dietary phytochemicals on cancer development (review). Intern. J. Mol. Med. 1:747–53. Wang, C., J. Ning, P. Krishnan, and D. Matthees. 1998. Effects of steeping conditions during wet-milling on the retentions of tocopherols and tocotrienols in corn. J. Amer. Oil Chem. Soc. 75:609–13. Wang, H., G. Cao, and R. Prior. 1997. Oxygen absorbing capacity of anthocyanins. J. Agric. Food Chem. 45:304–9. Watanabe, M. 1998. Catechins as antioxidants from buckwheat (Fagopyrum esculentum Moench) groats. J. Agric. Food Chem. 46:839–45. Watanabe, M. 2007. An anthocyanin compound in buckwheat sprouts and its contribution to antioxidant capacity. Bios. Biotech. Biochem. 71:579–82. Watanabe, M., Y. Ohshita, and T. Tsushida. 1997. Antioxidant compounds from buckwheat (Fagopyrum ­esculentum Moeench) hulls. J. Agric. Food Chem. 45:1039–44. Wei, H., R. Bowen, Q. Cai, S. Barnes, and Y. Wang. 1995. Antioxidant and antipromotional effects of soybean isoflavone genistein. Proc. Soc. Exp. Biol. Med. 208:124–30. Weidner, S., R. Amarowicz, M. Karamac, and G. Dabrowski. 1999. Phenolic acids in caryopses of two cultivars of wheat, rye and triticale that display different resistance to pre-harvest sprouting. Eur. Food Res. Tech. 210:109–13. Weidner, S., R. Amarowicz, M. Karamac, and E. Fraczek. 2000. Changes in endogenous phenolic acids ­during development of Secale cereale caryopses and after dehydration treatment of unripe rye grains. Plant Phys. Biochem. 38 (7–8): 595–602. Wennermark, B., and M. Jägerstad. 1992. Breadmaking and storage of various wheat fractions affect vitamin E. J. Food Sci. 57:1205–9. White, P., and L. Armstrong. 1986. Effect of selected oat sterols on the deterioration of heated soybean oil. J. Amer. Oil Chem. Soc. 63:525–9. Wilcox, L., N. Borradaile, L. de-Dreu, and M. Huff. 2001. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP. J. Lipid Res. 42:725–34. Winata, A., and K. Lorenz. 1996. Antioxidant potential of 5-n-pentadecylresorcinol. J. Food Proc. Pres. 20:417–29. Winata, A., and K. Lorenz. 1997. Effects of fermentation and baking of whole wheat and whole rye sourdough breads on cereal alkylresorcinols. Cereal Chem. 74:284–7. Winkler, J., K. Rennick, F. Eller, and S. Vaughn. 2007. Phytosterol and tocopherol components in extracts of corn distiller’s dried grain. J. Agric. Food Chem. 55:6482–6. Wu, D., L. Liu, M. Meydani, and S. Meydani. 2005. Vitamin E increases production of vasodilator prostanoids in human aortic endothelial cells through opposing effects on cyclooxygenase-2 and phospholipase A2. J. Nutr. 135:1847–53. Wu, X., G. R. Beecher, J. Holden, D. Haytowitz, S. Gebhardt, and R. Prior. 2004. Lipophilic and ­hydrophilic ­antioxidant capacities of common foods in the United States. J. Agric. Food Chem. 52:4026–37. Wyatt, C. J., S. Pérez Carballido, and R. O. Méndez. 1998. α- and γ-tocopherol content of selected foods in the Mexican diet: Effect of cooking losses. J. Agric. Food Chem. 46:4657–61. Xu, B., and S. Chang. 2007. A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. J. Food Sci. 72:S159–66. Xu, Z., and S. Godber. 2000. Comparison of supercritical fluid and solvent extraction methods in extracting γ-oryzanol from rice bran. J. Amer. Oil Chem. Soc. 77:547–51. Xu, Z., and J. Godber. 2001. Antioxidant activities of major components of gamma-oryzanol from rice bran using a linoleic acid model. J. Amer. Oil Chem. Soc. 78:645–9. Xu, B., S. Yaun, and S. Chang. 2007. Comparative analysis of phenolic composition, antioxidant capacity, and color of cool season legumes and other selected food legumes. J. Food Science 72:S167–77. Yadav, M., R. Moreau, and K. Hicks. 2007. Phenolic acids, lipids, and proteins associated with purified corn fiber arabinoxylans. J. Agric. Food Chem. 55:943–7. © 2011 by Taylor & Francis Group, LLC

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4 Phenolic and Beneficial Bioactives in Drupe Fruits Özlem Tokus¸ og˘lu CONTENTS Introduction............................................................................................................................................... 83 Cherries..................................................................................................................................................... 84 Sweet Cherries..................................................................................................................................... 84 Tart Cherries......................................................................................................................................... 85 Apricot...................................................................................................................................................... 87 Bioactives in Apricot Fruits................................................................................................................. 87 Plum and Prune......................................................................................................................................... 89 Bioactives in Plum............................................................................................................................... 89 Chlorogenic Acid and Its Derivatives in Plums and Prunes........................................................... 90 Other Phenolic Acids, Flavonols and Flavan-3-ols in Plums and Prunes....................................... 90 Anthocyanins in Plums and Prunes................................................................................................ 93 Total Phenolics in Plum and Prunes............................................................................................... 95 Antioxidant Activity of Plums and Prunes..................................................................................... 96 Peach and Nectarine.................................................................................................................................. 97 Bioactives in Peach and Nectarine....................................................................................................... 97 Date Fruit.................................................................................................................................................. 98 Bioactives in Date Fruits...................................................................................................................... 99 References............................................................................................................................................... 100

Introduction A phytochemical is a natural bioactive compound found in plant foods such as fruits, vegetables, and nuts that works with nutrients and dietary fiber to protect against diseases. Fruit phytochemicals are of significant interest for public health for their protective and preventive effects in several chronic diseases and the pathogenesis of a definite class of cancers (Meskin et al. 2003; Omaye et al. 2000). As the name suggests, phytochemicals work together with chemical nutrients found in fruits to help slow the aging process and reduce the risk of many diseases, including cancer, heart disease, stroke, high blood pressure, cataracts, osteoporosis, and urinary tract infections (Meskin et al. 2003; Omaye et al. 2000). Flowering plants disseminate seeds through fruit and the presence of seeds indicates that a structure is most likely a fruit, though not all seeds come from fruits (Lewis 2002). The major types of edible fruits include the following: fleshy simple fruits, fleshy aggregate fruits, fleshy multiple fruits, and dry fruits (Anonymous 2009a; Janick and Paull 2008). A classification of common edible fruits is shown in Figure 4.1. A drupe is a fruit in which an outer fleshy part (exocarp, or skin; and mesocarp, or flesh) surrounds a shell (the pit or stone) of hardened endocarp with a seed inside. Drupe fruits develop from a single carpel. A drupe has the definitive characteristic that the hard, lignified stone (or pit) is derived from the ovary wall of the flower (Armstrong 2008). © 2011 by Taylor & Francis Group, LLC

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Fleshy simple fruits 1. Drupe fruits a. [Cherry (sweet/ sour) apricot, nectarine, peach, plum, date] b. [Oily drupes: Olive, coffee, coconut] 2. Pome fruits [apple, pear, quince, medlar (loquat), rowan, hawthorn] 3. Berry fruits [grape, banana, kiwi, tomato, pomegranate] 4. Citrus fruits [lemon, orange, tangerine, mandarin, grapefruit, clementine, lime] 5. Pepo fruits [watermelon, cucumber, squash, cantelope, pumpkin, honeydew]

Fleshy aggregate fruits Common aggregate berry fruits [Strawberries, blackberries, raspberries, blueberries, elderberry, gooseberry, red/black/white current eleagnus]

Fleshy multiple fruits Common multiple fruits [figs, pineapple, red/black/white mulberries]

Tropical fruits [banana, coconut, mango, papaya, guava, jackfruit, tamarind, star fruitcarambola, passion fruit]

Dry fruits 1. Common dry fruits [raisins, plums, prunes, apricots, figs, dates] 2. Other dry fruits [mango, papaya, tomatoes, apples, pears, bananas, cranberries, peaches, pineapples]

FIGURE 4.1  Possible classification of common edible fruits. (This scheme compiled by Tokuşoğlu.)

The most common drupe fruits are sweet/sour cherry, apricot, plum, peach, nectarine, almond, and date. Oily drupes are olive, coffee, and coconut.

Cherries The word “cherry” refers to a fleshy fruit (drupe) that contains a single stony seed. Cherries are a ­member of the Rosaceae family, subfamily Prunoideae as taxonomical. They occupy the Cerasus subgenus within Prunus, being fairly distinct from their stone fruit relatives: the plums, apricots, peaches, and almonds. The subgenus is native to the temperate regions of the Northern Hemisphere, with two species in America, three in Europe, and the remainder in Asia. Cherries are typically classified as either sweet or tart. Sweet cherries Bing, Lambert, and Rainier are grown mainly in Washington State, Oregon, and Idaho. Tart cherries including Montmorency and Balaton varieties are produced principally in the Michigan area. Prunus avium L. is the sweet cherry, to which most cherry cultivars belong and Prunus cerasus L. the sour or tart cherry that is used mainly for cooking or baking.

The decrease in the proliferation of human colon cancer cells (Kang et al. 2003) has been specifically associated with cherry consumption (Serrano et al. 2005). It is stated that sweet and sour cherry phenolics have protective effects on neuronal cells (Kim et al. 2005). It is also reported that the consumption of sweet cherries alleviates arthritis and gout-related pain (Wang et al. 1999).

Sweet Cherries The sweet cherry is a vigorous tree with strong apical control with an erect-pyrimidal canopy shape; grows to about 10–15 m (12–35 feet tall). In cultivation, sweet cherries are maintained 0.03 ng/g) was found in 56 of 383 wheat samples, 11 of 103 barley samples, nine of 19 green coffee samples, and nine of 13 roasted coffee samples (Trucksess et al. 1999). Four samples of wheat and one sample of barley were contaminated with >5 ng/g OTA, indicating that cereal grains are more susceptible to OTA producing fungi (Trucksess et al. 1999). Ochrotoxin has been classified as a possible carcinogen for humans and it is a potent teratogen and hepatotoxin (Lindsey 2002; Petziner and Ziegler 2000). It has been established that OTA has an immunomodulatory effect on a human monocyte/macrophage cell line (Muller et al. 2003) and also is involved in human Balkan endemic nephropathy (Castegnaro et al. 2006). Because of their toxigenicity in animals and humans, there have been strict rules in European countries where maximum levels of 3 ng/g are set for OTA in cereal-based foods (FAO 2004). © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications (a)

O

OH O

OH

O

O

N H

CH3 Cl

(b)

OH

CH3

O

O

HO O FIGURE 12.2  Structure of (A) ochratoxin A and (B) zearalenone. (From Chem ID, Ochratoxin A, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, Zearalenone, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.)

Trichothecenes Trichothecene mycotoxins are bioactive secondary metabolites produced in infected grains by many fungi such as Myrothecium, Stachyobotrys, Fusarium, Trichoderma, and Trichothecium (Figure 12.3; Jarvis 1991; Sharma and Kim 1991). These are toxic to humans as well as animals (Prelusky et al. 1994; Wache et al. 2009). Mycotoxigenic fungi such as F. graminearum produces 8-ketotrichothecenes including deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV), and 4-acetyl nivalenol (4-ANIV), as well as the estrogenic mycotoxin zearalenone (ZEN; Mirocha et al. 1989; Seo et al. 1996). The trichothecenes are all tricyclic sesquiterpenes with a 12,13-epoxy-trichothec-9-ene ring. Trichothecenes are macrocyclic or nonmacrocyclic depending on the presence of a macrocylic ester or an ester-ether bridge between C-4 and C-15 (Chu 1998). The nonmacrocyclic trichothecenes are T-2 toxin, diacetoxyscirpenol, and DON (Jarvis 1991). Fusarium sporotrichioides is the primary species that produces mycotoxins such asT-2 toxin and diacetoxyscirpenol in cereal grains (Abramson et al. 1993). Fusarium graminearum isolates can be characterized as chemotypes based on the type of trichothecenes they produce. There are three main chemotypes (Ia, Ib, and NIV chemotypes) of F. graminearum. The chemotype Ia produces DON and 3-ADON; chemotype Ib produces DON and 15-ADON; and the NIV chemotypes produce NIV and 4-ANIV (Ichinoe et al. 1980; Moss and Thrane 2004). The NIV chemotypes are not found in North America but are reported in Africa, Asia, and Europe (Ichinoe et al. 1980, 1983). Deoxynivalenol, an important trichothecene is produced by many species of Fusarium including Fusarium graminearum, F. avenaceum, F. crookwellense, F. culmorum, F. poae, and F. sporotrichioides (Abramson et al. 1993). This trichothecene mycotoxin is phytotoxic to many cereal grains such as corn, wheat, and barley (Cosette and Miller 1995; Salas et al. 1999; Wakulinski 1989). Deoxynivalenol inhibits germination and root growth in wheat (Wakulinski 1989). Trichothecene mycotoxins such as DON, 3-ADON are also toxic to animals. Corn was contaminated with DON and ZEN in the northeastern © 2011 by Taylor & Francis Group, LLC

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Mycotoxic Bioactives in Cereals and Cereal-Based Foods (a)

H

(b)

H

H3C

H

OH

O

H

O

H3C O

OH

O

O

O CH3

OH

OH

HO CH3

HO

OH (c) CH3

O

O O

HO

CH3

CH3

CH3 CH3

O

O O

O

O

CH3

FIGURE 12.3  Structure of important trichothecene mycotoxins (A) deoxynivalenol, (B) nivalenol, and (C) T-2 toxin. (From Chem ID, Deoxynivalenol, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, Nivalenol, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, T-2 toxin, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.)

states of the United States and Canada, thus grain buyers in those regions temporarily stopped purchasing corn in 1991 (Bergstrom 1991). The neurotoxic effects of DON lead to feed refusal and reduced weight gain in swine (Prelusky 1997). A reduction in the uptake of sugars (glucose) and minerals was observed in mice fed with DON contaminated food (Hunder et al. 1991). Human immune defense cells such as macrophages are reported to be affected by DON. Immunosuppression and inhibition of cell surface activation markers of human macrophages were observed when exposed to low doses (150 µM) of DON (Wache et al. 2009). The maximum limit is set at 1 µg/g for DON in finished wheat products by U.S. Food and Drug Administration.

Zearalenone Zearalenone belongs to benzannulated macrolactones (Figure 12.2B; Brase et al. 2009; Winssinger and Barluenga 2007). This mycotoxin was first isolated from Fusarium graminearum in 1962. It coexists with other Fusarium mycotoxins (CAST 2003). Hyperestrogenism is caused by ZEN due to its similarity with 17-estradiol in the binding to cytosolic estrogen receptors (Kuiper-Goodman et al. 1987). This mycotoxin has been reported to be affecting male (decreased spermatozoa) and female reproductive systems (early puberty) in animals and human populations (Etienne and Dourmad 1994; Shier et al. 2001; Yang et al. 2007). The European Union has set regulatory limits of ZEN from 20 to 200 ng/g in unprocessed and processed cereal-based foods (FAO 2004).

Occurrence of Mycotoxic Bioactives during Cereal-Based Food Processing Fungi infect, survive, grow, and produce mycotoxins in cereal-based foods while being processed under optimal conditions. The mycotoxin levels in various cereal-based food processing stages can increase or decrease depending on the type of processing step. This section of the chapter gives brief information on a few mycotoxins and their fate during various stages of cereal-based food processing. © 2011 by Taylor & Francis Group, LLC

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Barley Malting Barley is an important cereal crop, used in the malting and brewing processes for beer production and used as livestock feed (Noots et al. 1998; Schwarz et al. 1995, 2001). The malting process is divided into three main steps including steeping, germination, and kilning (Karababa et al. 1993; Noots et al. 1998). During the steeping process, the barley is soaked in water at 12–20°C for 36–52 hours to elevate the moisture content of the barley to 42–45% (Noots et al. 1998; Schwarz 2001). During the steeping process, the grain is allowed to have brief air-rests. The germination process follows steeping and lasts for 4–5 days at 15°–20°C with controlled humidity. After germination, the green malt is subjected to higher temperatures during the kilning process for 18–24 hours. The temperatures during kilning vary over a range of 40°C–50°C to 80°C–90 oC (Hough et al. 1971; Schwarz 2001). Researchers have reported that Fusarium infection of barley kernels increased 15–90% during the steeping step of malting (Douglas and Flannigan 1988; Flannigan 1996). There was an increase in Fusarium species, colony forming units (CFU) from 300 cfu/g to 8000 cfu/g during malting (Flannigan et al. 1984). Schwarz et al. (1995) have reported that after 5 days of germination there was a significantly (p 150°C), very high pressures, and severe shear forces are used in extrusion cooking (Bullerman and Bianchini 2007; Harper 1992). In a study by Castells et al. (2006), extrusion cooking—using a single screw extruder of barley meal—reported that higher residence time (70 s) and medium temperature level (160 oC) decreased OTA.

Corn Milling A recent study by Scudamore and Patel (2009) has indicated that in dry corn milling, the endosperm of the grain contained low levels of Fusarium mycotoxins, such as deoxynivalenol, zearalenone, and fumonisins. However, embryo and outer grain layers (used mostly as animal feed) had up to five times more concentration of mycotoxins (Scudamore and Patel 2009). In a study by Castells et al. (2008), a similar trend was observed where the outer layers of corn (used in animal feed flour and corn flour) had relatively high levels of mycotoxins such as fumonisins B1, B2, and B3, and aflatoxins B1, B2, G1, and G2. Also, they observed that corn meal and flaking grits had lower levels of mycotoxins (Castells et al. 2008). Thus, in the corn milling process, the mycotoxins are not completely eliminated; however, the concentrations of these toxins differ in various fractions of corn and cornbased products. © 2011 by Taylor & Francis Group, LLC

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Worldwide Distribution of Mycotoxic Bioactives Cereals and cereal-based foods are attacked by mycotoxigenic fungi and their mycotoxins worldwide. Depending on the type of staple food used in various parts of the world, the type of mycotoxin attacking cereals and cereal-based foods also vary. In some parts of the world such as North America, cereals such as wheat and barley and their products are mostly affected by the mycotoxins, whereas in Asian regions of the world, cereals such as rice and rye are contaminated with mycotoxins (Desjardins et al. 2008; Ng et al. 2009). Deoxynivalenol producing Fusarium in cereals is common in many regions of the world. However, in some countries of Asia (such as Nepal) there has been a higher prevalence of virulent nivalenol producing Fusarium graminearum in cereals such as corn (Desjardins et al. 2008). Cereal grains, such as wheat and barley in Poland, were contaminated with OTA that was produced by both P. verrucosum and A. ochraceus, of which Penicillium species was the major source of OTA produced (93%; Czerwiecki et al. 2002). A survey of the on-farm stored cereal grains such as wheat, barley, and oats in the United Kingdom showed that 21% among 306 samples were contaminated with OTA, and barley was reported to be more susceptible to OTA contamination than wheat (Scudamore et al. 1999). A recent survey conducted in Canadian dry pasta samples (n = 274) indicated more than 0.5 ng/g of OTA present in 21, 18, and 66%, respectively, of pasta samples, collected during years 2004, 2005, and 2006. The degree of contamination with mycotoxins such as OTA is thus variable, depending on the wheat crop year (Ng et al. 2009). A study conducted in corn tortilla and masa flour samples from California, had fumonisins in all samples (n = 38). Corn-based foods are thus very susceptible to fumonisin contamination and daily consumption of highly contaminated (>1000 ng/g of fumonisins) cornbased products can be prevented to reduce the risk of disease in potentially pregnant women and their offspring (Dvorak et al. 2008).

Analytical Methods to Detect and Quantify Mycotoxins in Cereals Many methods are available that can detect and quantify mycotoxigenic bioactives in cereals. Some methods are specific for a single mycotoxin (Kabak 2009), whereas recent studies focus on methods that can analyze multiple mycotoxins in cereals and cereal-based foods (Frenich et al. 2009; Garon et al. 2006). There are different methods for analyzing mycotoxins in cereals including but not limited to microbiological, chromatographical, and polymerase chain reaction (PCR) based assays for the detection of mycotoxigenic fungi in cereals, and immunoaffinity clean-up/fluorescence detection methods for mycotoxins. However, the method of choice for many researchers depends on how quick, sensitive, reliable, specific, and cost-effective the method.

Microbiological (Culture) Methods Cereal grains if infected can be positively identified using culture methods. Validation of many modern assays to identify and quantify mycotoxigenic fungi is done primarily using traditional culture methods (Bluhm et al. 2002, 2004). To determine the mycological inhabitants on the cereal grains, direct plating of the grains can be done on the growth media. Also, surface sterilization before plating of the cereal grains as an initial step can help enumerate the internal fungi (Samson et al. 2000). Selective media can help isolate and identify specific mycotoxigenic fungi from cereal grains and cereal-based food products. Ochratoxin producing P. verrucosum can be isolated from cereal grains using dichloran yeast extract sucrose glycerol agar (DYSG; Frisvad et al. 1992; Samson et al. 2000). For isolating aflatoxin producing A. flavus and A. parasiticus species, aspergillus differential medium (ADM) is very helpful (Bothast and Fennel 1974). Similarly for Fusarium species, czapek iprodione dichloran agar (CZID) can be selectively identified in cereal foods (Abildgren et al. 1987; Samson et al. 2000). These microbiological methods are very useful in isolating and detecting © 2011 by Taylor & Francis Group, LLC

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mycotoxigenic fungi, however they are labor intensive and time-consuming. Thus, other methods have been developed that can be performed in less-time and give reliable results.

Gaseous Chromatography Methods to Detect Aspergillus, Fusarium, and Penicillium Volatiles Fungal deterioration of stored cereals can be detected at an early stage. Volatile compounds are produced by fungi in stored grains (Abramson et al. 1980). These volatiles can be detected by several chromatographic techniques such as gas chromatography–mass spectrometry (GC–MS) and other methods. For detection of the volatiles produced by the storage fungi in cereals, a sample collection is the primary step. Various methods can be applied to collect the samples. Headspace methods include direct collection of gaseous volatiles released by the fungi, onto an adsorbent such as activated carbon (Abramson et al. 1980). Sometimes the volatiles, which are released into the growth medium, should be extracted and this can be done using steam distillation and extraction or supercritical fluid extraction (Kaminski et al. 1972). Another important method known as headspace solid-phase microextraction (SPME) can be used to extract the volatiles. The volatiles from the headspace can be extracted onto a fused silica fiber coated with a polymeric organic liquid and then can be directly transferred to a gas chromatography (GC) machine and analyzed (Nilsson et al. 1996). Volatile fungal metabolites have been used as indicators of fungal growth in stored cereal grains (Borjesson et al. 1992; Tuma et al. 1989). Various volatiles found include 3-octanone, 1-octen-3-ol, and 3-methyl-1-butanol, and 3-methylfuran (Abramson et al. 1980; Borjesson et al. 1989, 1990; Tuma et al. 1989). The 3-methylfuran was produced by many fungi such as Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus during early stages of growth on wheat and oats (Borjesson et al. 1992). Some volatiles are unique to some fungal species such as thujospene, which is produced by Aspergillus, and not produced by Penicillium species. Penicillium glabrum produces 3-octanone and P. brevicompactum is reported to produce high amounts of acetone (Borjesson et al. 1992). These volatiles were reported to be correlated positively with accumulated carbon dioxide and ergosterol in cereals (Borjesson et al. 1992). Pasanen et al. (1996) showed that P. verrucosum can be differentiated into toxigenic and nontoxigenic isolates based on volatile production. High amounts of ketones are produced by ochratoxin-producing P. verrucosum species when compared to nontoxigenic isolates of P. verrucosum. Fusarium species also produce various volatiles from stored grain. Fusarium sambucinum produced sesquiterpenes such as β-farnesene, β-chamigene, β-bisabolene, α-farnesene, and trichodiene on wheat kernels (Jelen et al. 1995). These volatiles produced by fungi in stored grain were also correlated with mycotoxin production. F. sporotrichioides produced volatile terpenes that correlated with the trichothecene mycotoxins including T-2 toxin, neosolaiol, diacetoxyscirpenol, HT-2 toxin, and T-2 tetraol (Pasanen et al. 1996). Olsson et al. (2002) reported that mycotoxin contamination in grains can be detected by determining the volatiles produced by the storage fungi in barley. They found that barley samples with a normal odor had no detectable OTA, whereas the samples that had off-odor had an average OTA of 76 µg/kg and 69 µg/kg of DON. The samples with more OTA produced higher amounts of ketones such as 2-hexanone, 3-octanone (Olsson et al. 2002).

Polymerase Chain Reaction (PCR) Based Methods The PCR is an assay that can be used to amplify a specific deoxyribonucleic acid (DNA) fragment of a fungal species and can be used to identify the species (Niessen and Vogel 1997; White et al. 1990). Many PCR assays have been developed to detect and quantify mycotoxigenic fungi in cereal grains. Assays involving DNA can help in quick, reliable, and specific detection and quantification of fungi in cereal grains. Fusarium graminearum was detected and quantified using a PCR assay (Niessen and Vogel 1998). Fungal ribosomal DNA (rDNA) genes have been used to design DNA primers for PCR reactions. These genes are highly conserved and are species-specific (White et al. 1990). Primers © 2011 by Taylor & Francis Group, LLC

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used in PCR can be used for species-specific ­detection of mycotoxigenic fungi. F. graminearum was detected by developing a PCR assay utilizing the primers specific to the galactose oxidase gene, which is produced by very few fungal species including F. acuminatum, F. subglutinans, and F. graminearum (Barbosa-Tessmann et al. 2001; Niessen and Vogel 1997). Fusarium culmorum, F. graminearum, and F. avenaceum were detected and differentiated by using species-specific primers for the internal transcribed spacer (ITS) region of the rDNA genes (Schilling et al. 1996). Edwards et al. (2001) developed a quantitative PCR assay for the trichodiene synthase gene (Tri5) of trichothecene-producing Fusarium. The researchers found a positive correlation between Tri5 DNA and DON produced by F. culmorum and F. graminearum in winter wheat. Fungal species have also been grouped using their rDNA genes and results have been correlated with mycotoxigenicity. An A. niger species aggregate (92 isolates) was tested for OTA production. These isolates were grouped into the two species, A. niger and A. tubingensis, by using their ITS-5.8S rDNA restriction fragment length polymorphism (RFLP) patterns. Only six out of the 92 isolates studied produced OTA, and these OTA isolates were A. niger isolates (Accensi et al. 2001). Traditional PCR assays can be used to detect and quantify a single gene as well as more than one gene in a single reaction (multiplex PCR; Nicholson et al. 1998; Niessen and Vogel, 1998). Group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium was done using multiplex PCR assays in cornmeal (Bluhm et al. 2002). However, these assays are time-consuming as they involve post-PCR processes such as gel electrophoresis. Real-time PCR with SYBR Green I dye or TaqMan probes quantify PCR products in less time and do not involve gel electrophoresis (Bluhm et al. 2004; Reischer et al. 2004; Schnerr et al. 2001). Real-time PCR involving TaqMan probes has been used for specific detection and reliable quantification of fungal pathogens (Bluhm et al. 2004; Geisen et al. 2004; McDevitt et al. 2004). Figure 12.4 depicts the TaqMan probe mechanism of real-time PCR assays. These probes are oligonucleotides with a reporter dye on the 5′ end and a quencher dye on the 3′ end that attach to specific DNA sequences during the PCR cycles (Geisen et al. 2004; Heid et al. 1996; McDevitt et al. 2004). When the quencher dye is in close proximity to the reporter dye, there is no fluorescence emission. However, when the DNA polymerase enzyme comes in close contact with the probes during PCR, the 5′ nuclease activity of the enzymes cleaves the probes, separating quencher and reporter dyes on the probes and thereby increasing the fluorescence emission (Heid et al. 1996). The accumulation of PCR products is detected by monitoring the increase in fluorescence of the probe (Bluhm et al. 2004). TaqMan probes only attach to specific sequences of DNA and different quencher-reporter dye combinations can be used for different genes to be detected and quantified, thus they can be used in multiplex real-time PCR to detect more than one fungal pathogen. The amplification products formed during real-time PCR can be quantified by performing standard curve analysis. Also, correlation studies can be done to validate the real-time PCR assay data. Aspergillus flavus was detected and quantified using real-time PCR for the nor-1 gene involved in the aflatoxin biosynthetic pathway. There was a positive correlation between the copy number of the nor-1 gene determined by real-time PCR and the CFU of A. flavus in wheat (Mayer et al. 2003).

Immunological Detection Methods Enzyme-linked immunosorbent assays (ELISA) and immunoblotting techniques have been used to detect and quantify mycotoxigenic fungi (Iyer and Cousin 2003; Lu et al. 1995; Skaug 2003). An indirect ELISA was developed to detect F. graminearum and F. verticillioides in foods and the detection limits for F. graminearum and F. verticillioides were 0.1 and 1 µg/ml, respectively (Iyer and Cousin 2003). An ELISA method that was very specific for OTA producing A. ochraceus showing no cross-reactivity with Aspergillus, Penicillium, Fusarium, Mucor, and Alternaria exoantigens was developed by using rabbit antibodies that were produced against the exoantigens of A. ochraceus (Lu et al. 1995). The immunoaffinity column (IAC) can be used for sample clean-up for higher recovery of mycotoxins from cereals and cereal-based foods. A cartridge containing solid support such as agarose gel on © 2011 by Taylor & Francis Group, LLC

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Denaturation

Annealing

Primer 1 Primer 2 Taq polymerase TaqMan probe Reporter

Extension

Quencher Cleaved quencher Fluorescing reporter Cleaved nucleotides

Amplified DNA

FIGURE 12.4  Depiction of TaqMan probe mechanism in real-time PCR used to detect and quantify mycotoxigenic fungi in cereals and cereal-based foods.

which the anti-mycotoxin antibody that is immobilized is used in IAC. Visconti et al. (2005) developed a sensitive and accurate method for simultaneous detection of T-2 and HT-2 toxins in cereal grains using immunoaffinity clean-up coupled with high-performance liquid chromatography (HPLC) with fluorescence detection. The IAC in the study containing monoclonal anti T-2 antibodies helped in capturing the T-2 and HT-2 toxins of cereals such as wheat, corn, and barley. Then these toxins were eluted and quantified by reversed-phase HPLC with fluorometric detection (Visconti et al. 2005). Immunologists claim mycotoxins produced by the fungi in cereal foods can be directly detected by ELISA with less cost. However, the detection limits (0.05 ng – 1 µg) of these assays to quantify the fungi or their mycotoxins limit their use when compared to PCR assays whose detection limit is as low as 5 pg (Bluhm et al. 2004). Mycotoxin ELISA methods are also known to be cross-reactive with interfering substrates in food samples. In recent studies, immunological methods are preferred again

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as higher recovery rates and lower detection limits of the mycotoxins in cereal grains can be achieved with the IAC clean-up (Brenn-Struckhofova et al. 2009; Visconti et al. 2005).

Summary Cereal grains like barley, corn, rice, and wheat in the field, during harvest, in storage, and while processing are constantly attacked by mycotoxigenic fungi such as Fusarium, Penicillium, and Aspergillus species. There are a wide variety of the mycotoxic bioactives that are produced by fungi in cereal foods such as aflatoxins, fumonisins, ochratoxins, and trichothecenes. These mycotoxins not only affect the cereal crops by reducing yield, quality, and safety of the cereals but also affect animal and human health if they consume contaminated cereal-based foods. Worldwide many regulatory limits are present to help control the entry of mycotoxins in foods meant for human consumption. However, lack of regulatory limits on some mycotoxins such as ergot alkaloids can still be very dangerous to both animals and human if they consume very high levels of mycotoxin contaminated cereal-based foods. Methods such as headspace analysis by GC–MS can be used for earlier detection of mold growth in cereal grains. Sensitive, reliable, and specific techniques such as real-time PCR and IAC/fluorescence will be very helpful in detecting and quantifying these mycotoxigenic species and their mycotoxins in cereals and cereal-based products.

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13 Control Assessments and Possible Inactivation Mechanisms on Mycotoxin Bioactives of Fruits and Cereals Faruk T. Bozoğlu and Özlem Tokus¸ oğlu CONTENTS Introduction............................................................................................................................................. 273 Most Frequent Mycotoxin Bioactives on Fruit and Cereals: Structures and Formation........................................................................................................................ 276 Aflatoxins........................................................................................................................................... 277 Citrinin............................................................................................................................................... 279 Ochratoxin......................................................................................................................................... 279 Patulin................................................................................................................................................ 280 Ergotamine......................................................................................................................................... 280 Fusarium Mycotoxins........................................................................................................................ 280 Fumonisins.................................................................................................................................... 280 Trichothecenes.............................................................................................................................. 281 T-2 (Type A Trichothecene).......................................................................................................... 282 Zearalenone................................................................................................................................... 283 The Necessity of Inactivation Assessments............................................................................................ 283 The Inactivation Strategies on Mycotoxin Bioactives............................................................................ 285 Extrusion Process............................................................................................................................... 285 Application of Ammonia................................................................................................................... 286 Feed Additives................................................................................................................................... 286 Chlorine Dioxide................................................................................................................................ 287 Citric Acid.......................................................................................................................................... 287 Biological Detoxification................................................................................................................... 287 Sulfhydryl Compounds...................................................................................................................... 287 Miscellaneous.................................................................................................................................... 288 The Interpretations on Applicated Mycotoxin Inactivation Mechanisms on Fruits and Cereals.............................................................................................................................. 288 Summary................................................................................................................................................. 289 References............................................................................................................................................... 289

Introduction Mycotoxin bioactives occurs in raw and dried fruits, cereals, and nut products pre- and postharvest stage. Cereals are the most studied products regarding this toxin detection, but fruits and fruit-based processed products may also represent a potential source of risk with species belonging to the Aspergillus and Penicillium genera worldwide (Battilani et al. 2008; Figure 13.1). © 2011 by Taylor & Francis Group, LLC

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Aspergillus flavus

Penicillium citrinum

FIGURE 13.1  The figures on Aspergillus and Penicillum genera.

Toxic fungal metabolites known as mycotoxins contaminate maize grain and vegetables produced throughout the world and represent a major food safety problem. According to the FAO more than 25% of the world’s agricultural production is contaminated with mycotoxins. This equates to economic losses estimated at $923 million annually in the U.S. grain industry alone. Most countries have adopted regulations to limit exposure to mycotoxins, having a strong impact on the food and animal crop trade. In plant pathology, many secondary metabolites produced by bacteria and fungi have pathogenicity or virulence factors; that is, they play a role in causing or exacerbating the plant disease. The phytotoxins made by fungal pathogens of Cochliobolus (Helminthosporium) and Alternaria, for example, have well-established roles in disease development and several mycotoxins made by the Fusarium species are important in plant pathogenesis. The mycotoxin-producing mold species are extremely common, and they can grow on a wide range of substrates under a wide range of environmental conditions. Mycotoxins occur, with varying severity, in agricultural products all around the world. The estimate usually given is that one-quarter of the world’s crops are contaminated to some extent with mycotoxins. For agricultural commodities, the severity of crop contamination tends to vary from year to year based on weather and other environmental factors. Aflatoxin, for example, is usually worst during drought years; the plants are weakened and become more susceptible to insect damage and other insults. The economic consequences of mycotoxin contamination are profound. Crops with large amounts of mycotoxins often have to be destroyed. Alternatively, contaminated crops are sometimes diverted, which can lead to reduced growth rates, illness, and death. Moreover, animals consuming mycotoxin-contaminated feeds can produce meat and milk that contain toxic residues and biotransformation products. Thus, aflatoxins in cattle feed can be metabolized by cows into aflatoxin M1, which is then secreted in milk. Ochratoxin in pig feed can accumulate in porcine tissues. Court actions between grain farmers, livestock owners, and feed companies can involve considerable amounts of money. The ability to diagnose and verify mycotoxicoses is an important forensic aspect of the mycotoxin problem (Jelinek et al. 1989). The presence of mycotoxins is unavoidable and therefore testing of raw materials and products is required to keep our food and feed safe. Nevertheless, mycotoxins have also been associated with exacerbation of the energy malnutrition syndrome Kwashiorkor in human children and vitamin A malnutrition in animals and many other problems. In various animal models, in addition to being hepatotoxic, aflatoxin causes significant growth haltering and is strongly immune-suppressive at weanings. In general, mycotoxin exposure is more likely to occur in parts of the world where poor methods of food handling and storage are common, where malnutrition is a problem, and where few regulations © 2011 by Taylor & Francis Group, LLC

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exist to protect exposed populations. However, even in developed countries, specific subgroups may be ­vulnerable to mycotoxin exposure. In the United States, for example, Hispanic populations consume more corn products than the rest of the population. People who have enough to eat normally avoid foods that are heavily contaminated by molds, so it is believed that dietary exposure to acute levels of mycotoxins is rare in developed countries. Nevertheless, many mycotoxins survive processing into flours and meals. When mold-damaged materials are processed into foods and feeds, they may not be detectable without special assay equipment. It is important to have policies in place that ensure that such “hidden” mycotoxins do not pose a significant hazard to human health. Accumulation of mycotoxins is dependent upon weather conditions. Before harvest, the risk for the development of mycotoxins is greatest during major droughts. When soil moisture is below normal and temperatures are high, the number of Aspergillus spores in the air increases. These spores infect crops through areas of damage caused by insects and inclement weather. Once infected, plant stress favors the production of mycotoxins. Fungal contamination of cereals, fruit, and vegetables is usually not a problem for the Western World consumer, since contaminated products are likely to be discarded immediately. However, when the industry or farmers in both Western and Third World countries store cereals, vegetables, or fruit, moldy parts of a batch (hot spots) may not be detected and removed and possible mycotoxins may be transferred to the processed commodities. It is now well established that mycotoxicoses (the diseases caused by mycotoxins) have been responsible for major epidemics in man and animals at least during recent historic times. The first epidemic of ergotism was reported in 430 BC in Sparta. Epidemics swept through Europe in the Middle Ages; in 1673, in France, the association with bread poisoning was described by Dodart. In fact, the name of the disease is derived from the French ergot, a cockspur—which relates to the shape of kernels of contaminated grain. The most important have been ergotism that killed thousands of people in Europe in the last thousand years, alimentary toxic aleukia (ATA) that was responsible for the death of many thousands of people. The most dramatic epidemics of ATA occurred in the Soviet Union between 1941 and 1947. The enormous war casualties suffered in some areas were responsible for the autumn harvest being neglected and the grain being left under the winter snow. Near famine conditions dictated that it be used and over 10% of the population of those districts were affected by ATA. The disease was not a new entity, having been reported from Russia since the nineteenth century. It is a severe disease with a high mortality. Stachybotryotoxicosis, which killed tens of thousands of horses and cattle in the USSR in the 1930s; and aflatoxicosis, which killed 100,000 young turkeys in England in 1960 and has caused death and disease in many other animals and perhaps man as well. Each of these diseases is now known to have been caused by growth of specific molds that produced one or more potent toxins, usually in one specific kind of commodity or feed. Almost all the mycotoxins of main concern in fruits and nuts originate in the field, during crop growth, and meteorological conditions are the key factors for risk assessment and safety evaluation (Battilani et al. 2008). Although efforts to control fungal contamination of foods, mycotoxin-producing fungi are ubiquitous contaminants of nature and make their way into fruits or nuts in the orchard or growing area and at any time during harvesting, processing, storage, and marketing. Owing to their chemical and/ or physical properties, fruits and nuts are susceptible to fungal rather than microbial spoilage. In that point, their high water activity (aw), their sugar content, and their presence of organic acids that impart to the flesh of fruit a low pH (Battilani et al. 2008; Magan et al. 2004; Tokuşoğlu 2010a; Tournas and Katsoudas 2004). Figure 13.2 shows the possible mycotoxin formation causes. Harvesting and transport strategy; fruits or nuts’ genotype and genetical variety; manufacturing process of fruits or nuts; mold flora of fruits or nuts; biological influences including agrononomic, climatological, and ecological effects; storage strategy containing water activity (aw); packaging problems; environmental humidity; and environmental temperature are major possible causes of mycotoxin contamination in fruits or nuts whereas the water, the air, and fruit or nut flies are also contaminates of the mold spores (Magan et al. 2004; Tokuşoğlu 2010b; Figure 13.2). © 2011 by Taylor & Francis Group, LLC

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Mycotoxin formation in fruits or nut

Fruit or nut manufacturing stages Mold spores contamination

Water-air fruit or nut fly

Mold flora

Fruit or nut Manufacturing mycoflora process Agronomic, Fruit climatological, Fruit or nut ecological Genetical type Nut Biological effects variety effects Storage Harvesting Strategy & transport After Water activity (aw) harvesting packaging problem environment humidity environment temperature

FIGURE 13.2  The possible causes of mycotoxin formation. (Adapted from Tokuşoğlu, Ö., Special Fruit Olive: Chemistry, Quality and Technology, SİDAS Medya Ltd., Şti., İzmir, 2010.)

The defense mechanisms in fruits and nuts are fairly effective against a variety of fungi; hence, r­ elatively few genera and species are able to invade fruits or nuts. Some fungi are highly specialized pathogens, attacking only particular types of fruits or nuts whereas some have a more general adequacy to invade the fruit or nut tissue (Drusch and Ragab 2003). The occurrence of toxin-producing fungi on fruits or nuts does not assuredly imply that mycotoxins will be present owing to toxin production is influenced by various causes containing environmental conditions, type, variety, and nutritional status of fruits or nuts, the microbial load on the fruit or nuts, and fungus strain (Drusch and Ragab 2003; Sanchis and Magan 2004; Tokuşoğlu 2010a).

Most Frequent Mycotoxin Bioactives on Fruit and Cereals: Structures and Formation The ability of fungi, such as the fusaria, to convert lysergic acid containing alkaloids such as ergotainine into LSD may explain the variation in symptomatology of different outbreaks of ergotism. Such hazards to human health are nevertheless now rare, but it has been suggested that contamination of certain types of feed and vegetables is still of economic significance to livestock producers in some areas. One of these fungal infections of grain is called “ergot.” This fungal disease affects the flowering parts of some members of the grass family, mostly confined to rye. Consuming the fungus causes a nervous disorder known as St. Anthony’s fire. When eaten in large quantities the ergot alkaloids may cause ­constriction of the blood vessels, particularly in the extremities. The effects of ergot poisoning are cumulative and lead to numbness of the limbs and other, frequently serious, symptoms. The fungus bodies are hard, spur-like, purple–black structures that replace the kernel in the grain head. The ergot bodies can vary in size from the length of the kernel to as much as several times as long. They don’t crush as easily as smut bodies of other funguses. When they are cracked open, the inner broken faces can be off-white, yellow, or tan. The infected grain looks very different from ordinary, healthy © 2011 by Taylor & Francis Group, LLC

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rye grains and can be spotted easily. Ergot rarely affects other grains and will generally afflict rye only when the growing conditions are damp. Ergot is typically not a common problem and is easily spotted when it does occur. Human ­poisoning due to the consumption of rye bread made from ergot-infected grain was common in Europe in the Middle Ages. The epidemic was known as Saint Anthony’s fire, or ignis sacer. Ergot infection causes a reduction in the yield and quality of grain and hay produced, and if infected grain or hay is fed to livestock it may cause a disease called ergotism. Black and protruding sclerotia of C. purpurea are well known. However, many tropical ergots have brown or grayish sclerotia, mimicking the shape of the host seed. For this reason, the infection is often overlooked. Other grain fungi, however, are much harder to spot and also have serious consequences should they be consumed. The various species of aspergillus and fusarium molds can be a problem almost anywhere. Sometimes grain in the form of animal feed or seed grain/legumes is available (Norred et al. 1991a). Animal feeds may have a higher contaminant level than what is permissible for human consumption. Under certain circumstances, the legislations allow the sale of grain or legumes for animal feed that could not be sold for direct human food use. It may even be mixed varieties of one grain or not all one type. Seed grains, in particular, must be investigated carefully to find out what they may have been treated with. It is quite common for seed to have had fungicides applied to them, and possibly other chemicals as well. Once treated, they are no longer safe for human or animal consumption. Fusarium fungi, widely found in nature and well known as a pathogenic for plants and producers of mycotoxins, cause major damage in cereals, fruits, and vegetables. They are frequently associated with preharvest contaminated cereals (Figures 13.3 and 13.4). Wheat, barley, and maize make up almost twothirds of the world production of cereals and thus liable to contamination. Fusarium-caused diseases in cereals are worldwide and occur in all climatic conditions. The fusarium head blight or scab, a disease caused by several species of fusarium (e.g., Fusarium graminearum), chiefly in small cereals such as wheat, triticale, and barley, inhibits the formation of grains or produces wrinkled, hollow, coarse, rosy grains contaminated by trichothecenes (mainly deoxynivalenol) and zearalenone. Fusarium graminearum commonly infects barley if there is rain late in the season. It is of economic impact to the malting and brewing industries as well as feed barley. Fusarium contamination in barley can result in head blight and in extreme contaminations the barley can appear pink. The genome of this wheat and maize pathogen has been sequenced. Fusarium graminearum can also cause root rot and seedling blight. Fusarium scab, associated with deoxynivalenol production in wheat, oats, and rye, not only triggers high financial losses in the United States and Canada, but is also a great concern for animal and human health. Nevertheless, since not all F. graminearum produce deoxynivalenol, its world distribution has been mapped by phylogenic studies and molecular biology techniques. Scanty information exists in literature on the occurrence of deoxynivalenol in maize contamination and its subproducts in contaminated samples. Current research examined 24 selected F. graminearum isolates associated to the scab disease in wheat, barley, and triticale. Toxigenicity in vitro of these samples could be verified by the qualitative evaluation of trichothecenes and zearalenone production.

Aflatoxins Aflatoxins are difuranocoumarin derivatives produced by a polyketide pathway by many strains of Aspergillus flavus and Aspergillus parasiticus; in particular, Aspergillus flavus is a common contaminant in agriculture. Figure 13.5 shows the most common aflatoxigenic derivatives aflatoxin B1 (AFB1) and Aflatoxin G1 (AFG1) (Figure 13.5). From the mycological perspective, there are great qualitative and quantitative differences in the toxigenic abilities displayed by different strains within each aflatoxigenic species. For example, only about half of the Aspergillus flavus strains produce aflatoxins while those that do may produce more than 10 μg/kg. Many substrates support growth and aflatoxin production by aflatoxigenic molds. Natural contamination of cereals, figs, oilseeds, nuts, tobacco, and a long list of other commodities is a common © 2011 by Taylor & Francis Group, LLC

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FIGURE 13.3  Ergot on wheat spikes.

FIGURE 13.4  Corn ear with fusarium ear rot.

O

H

O

H

O H

O

O

O

O

O O

O

AFB1 FIGURE 13.5  Aflatoxin B1 and Aflatoxin G1.

© 2011 by Taylor & Francis Group, LLC

CH3

H

O

O

AFG1

CH3

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occurrence. Like the genetic ability to make aflatoxin, contamination is highly variable. Sometimes crops become contaminated with aflatoxin in the field before harvest, where it is usually associated with drought stress; even more problematic is the fate of crops stored under conditions that favor mold growth. In storage, usually the most important variables are the moisture content of the substrate and the relative humidity of the surroundings. Aflatoxin contamination has been linked to increased mortality in farm animals and thus significantly lowers the value of grains as an animal feed and as an export commodity. Milk products can also serve as an indirect source of aflatoxin. When cows consume aflatoxin-contaminated feeds, they metabolically biotransform aflatoxin B1 into a hydroxylated form called aflatoxin M1. In developed countries, sufficient amounts of food combined with regulations that monitor aflatoxin levels in these foods protect human populations from significant aflatoxin ingestion. However, in countries where populations are facing starvation or where regulations are either not enforced or nonexistent, routine ingestion of aflatoxin may occur. Finally, it should be mentioned that Aspergillus oryzae and Aspergillus sojae, species that are widely used in Asian food fermentations such as soy sauce, miso, and sake, are closely related to the aflatoxigenic species Aspergillus flavus and Aspergillus parasiticus. Although these food fungi have never been shown to produce aflatoxin, they contain homologues of several aflatoxin biosynthesis pathway genes.

Citrinin Wheat, oats, rye, corn, barley, and rice have all been reported to contain citrinin. With immunoassays, citrinin was detected in certain vegetarian foods colored with monascus pigments. Citrinin has also been found in naturally fermented sausages from Italy. Citrinin (CIT; Figure  13.6) is a toxic secondary metabolite, isolated from filamentous fungus Penicillium Citrinum and is also produced by other species of Penicillium Aspergillus and monascus (Betina 1989). Although citrinin is regularly associated with human foods, its significance for human health is unknown. It has since been found to be produced by a variety of other fungi that are used in the production of human foods such as cheese, sake, and red ­pigments and table olives (Tokuşoğlu et al. 2010; Tokuşoğlu and Bozoğlu, 2010). Citrinin acts as a nephrotoxin in all species; it causes mycotoxic nephropathy in livestock and has been implicated as a cause of the Balkan nephropathy and yellow rice fever in humans.

Ochratoxin Members of the ochratoxin (Figure 13.7) family have been found as metabolites of many different species of Aspergillus, including Aspergillus alliaceus, Aspergillus auricomus, Aspergillus carbonarius, Aspergillus glaucus, Aspergillus melleus, and Aspergillus niger. Although some early reports implicated several Penicillium species, it is now thought that Penicillium verrucosum, a common contaminant of barley, is the only confirmed ochratoxin producer in this genus.

OH HOOC O

CH3

O CH3

FIGURE 13.6  Citrinin.

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CH3

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications O C

OH

OH O

N H

O

O H

CH2

CH3 CI FIGURE 13.7  Ochratoxin. O

O

O

OH

FIGURE 13.8  Patulin.

Patulin Currently, Penicillium expansum, the blue mold that causes soft rot of apples, pears, cherries, and other fruits, is recognized as one of the most common offenders in patulin contamination. Patulin (Figure 13.8) is regularly found in unfermented apple juice, although it does not survive the fermentation into cider products.

Ergotamine The ingestion of these sclerotia, or ergots, has been associated with diseases since antiquity. An Asyrian tablet dated to 600 BCE, referring to a “noxious pustule in the ear of grain,” is believed to be an early reference to ergot. The human disease acquired by eating cereals infected with ergot sclerotia, usually in the form of bread made from contaminated flour, is called ergotism or St. Anthony’s fire. Two forms of ergotism are usually recognized, gangrenous and convulsive. The gangrenous form affects the blood supply to the extremities, while convulsive ergotism affects the central ­nervous system. Figure 13.9 shows the chemical formula of mycotoxin ergotamine (Figure 13.9). Modern methods of grain cleaning have almost eliminated ergotism as a human disease. Nevertheless, purported ergot poisoning occurred in the French town of Pont-St.-Esprit in 1951 and was the subject of a full-length book treatment. The principal animals at risk are cattle, sheep, pigs, and chickens. Clinical symptoms of ergotism in animals include gangrene, abortion, convulsions, suppression of lactation, hypersensitivity, and ataxia.

Fusarium Mycotoxins Fumonisins The major species of economic importance is Fusarium verticillioides, which grows as a corn endophyte in both vegetative and reproductive tissues, often without causing disease symptoms in the plant. © 2011 by Taylor & Francis Group, LLC

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O

OH

H3C

C

O

NH

N H O

O H

N

CH2

CH3 H

HN FIGURE 13.9  Ergotamine.

O

O

O

C

OH C

OH

OH

O

OH CH3

CH3 CH3

O

NH2

OH

CH3

O

C

OH C

O

O

OH

FIGURE 13.10  Fumonisin B1.

However, when weather conditions, insect damage, and the appropriate fungal and plant genotype are present, it can cause seedling blight, stalk rot, and ear rot. Most strains do not produce the toxin, so the presence of the fungus does not necessarily mean that fumonisin is also present. Although it is phytotoxic, fumonisin B1 (Figure 13.10) is not required for plant pathogenesis. It has been isolated at high levels in corn meal and corn grits.

Trichothecenes Trichothecenes are secondary metabolites produced by several genera of fungi, including fusarium and form a structurally related mycotoxin group with various degrees of cytotoxicity. They have a sesquiterpenoid structure basic ring and are classified as A (T-2 toxin; type A Trichothecene) (Figure 13.11), B, C, and D, according to the presence or absence of characteristic functional groups. The inhibition of protein synthesis, irritation of the skin, hemorrhage, diarrhea, nausea, food reflux, and vomiting are the different toxicological characteristics of trichothecenes. Deoxynivalenol (Figure 13.12), fusarenon-X, diacetoxyscirpenol, neosolaniol, and nivalenol are the most frequent trichothecenes in F. graminearum. © 2011 by Taylor & Francis Group, LLC

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T-2 (Type A Trichothecene) There is a long history of moldy grain intoxications in Japan, where disease in both human beings and farm animals has been attributed to fusarium mycotoxicoses. Fusarium graminearum (teleomorph Gibberella zeae), regularly found on barley, oats, rye, and wheat, is considered the most important plant pathogen in Japan and is believed to be the cause of red mold disease. As with all mycotoxins, depending on weather conditions, the growth of trichothecene-producing fungi and subsequent production of toxins vary considerably from year to year and from place to place. Deoxynivalenol is sometimes called vomitoxin or food refusal factor. Although less toxic than many other major trichothecenes, it is the most prevalent and is commonly found in barley, corn, rye, safflower seeds, wheat, and mixed feeds. It has been pointed out that the hypothesis for an ATA-trichothecene connection would be strengthened if T-2 toxin (Figure 13.11) were actually detected in samples of overwintered grain associated with ATA outbreaks.

O H O

OH

CH3 O

O CH3

CH3 O

O

O CH3

O

CH3 H3C FIGURE 13.11  T-2 toxin.

H H3C

H

OH

O O

O OH

CH2 OH

FIGURE 13.12  Deoxynivalenol.

© 2011 by Taylor & Francis Group, LLC

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283

Mycotoxin Control Assessments and Possible Inactivation Mechanisms HO

CH3

O

O

HO

O

FIGURE 13.13  Zearalenone.

Zearalenone Zearalenone (Figure 13.13) is a mycotoxin produced by the fungus’s secondary metabolism through the biosynthetic polycetidic pathway with estrogenic activity in mammals. Zearalenone occurs chiefly during the growing phase of several grains when the fungus attacks and preys on the seeds during periods of heavy rainfall. It proliferates in mature grains, which were not sufficiently dried, owing to humidity, during the harvest or storing period. Alternations between low (12–14ºC) and high (25–28ºC) temperatures are normally needed to start and maintain zearalenone production in grains. Probable primary biochemical lesion and early cell events in the series that direct cell toxicity or zearalenone-caused cell deregulation may be attributed to an initial lesion in the cytosolic estrogen receptor that causes hormone control damage. The zearalenones are biosynthesized through a polyketide pathway by Fusarium graminearum, Fusarium culmorum, Fusarium equiseti, and Fusarium crookwellense. All these species are regular contaminants of cereal crops worldwide.

The Necessity of Inactivation Assessments Mycotoxin formation is more affected by internal and external ambient factors than fungal growth on fruits and nuts. Figure 13.14 illustrates the interaction between intrinsic and extrinsic factors in the food chain that influences mold spoilage and mycotoxin production in stored commodities (Drusch and Ragab 2003). This schema may express the postharvest control strategies that have been developed for effective management to minimize entry of mycotoxins into the product chain. Because mycotoxin contamination is unavoidable, numerous strategies for their detoxification have been proposed. These include physical methods of separation, thermal inactivation, irradiation, solvent extraction, adsorption from solution, microbial inactivation, and fermentation. Chemical methods of detoxification are also practiced as a major strategy for effective detoxification. Control of mycotoxins is the current need, since their occurrence in foods and feeds is continuously posing threats to both health and economics all over the world. Besides the postharvest preventive measures, it is imperative that suitable detoxification methods are developed for inactivating or removing mycotoxin from the contaminated commodities, as the toxins are also produced by Aspergillus flavus and A. parasiticus even during preharvest stages of crop production. Mycotoxins are toxic mold metabolites produced by toxigenic strains of the Aspergillus species. They have an important role in the occurrence of some human diseases such as liver cancer, chronic hepatitis, and cirrhosis. When animals eat food-stuffs containing aflatoxin B1, these toxins will be metabolized and excreted as aflatoxin M1 in their milk. The A aflatoxin M1 is resistant to thermal inactivation and is not destroyed completely by ­pasteurization, autoclaving, or other food processing procedures. Understanding the mechanisms of

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Processing factors

Extrinsic factors

Agronomic practices

Climatic conditions

Implicit factors

Intrinsic factors

Fungal strains and spore load

Water activity

Pre-harvest

Interactions with insects and mites

Plant varietal differences

Microbiological ecosystem

Nature of substrate

Damage by plant disease

Nutrient composition

Time

Processing factors Drying rate Rewetting Mechanical damage Blending of grain Temperature

Time Implicit factors Interactions with insects and mites Spore load

Harvest/ drying

Extrinsic factors

Intrinsic factors Moisture content

Climatic conditions

Implicit factors

Processing factors

Time

Rapidity of drying Rewetting/hot spots Mechanical damage Atmosphere

Fungal strains and spore load Interactions with insects and mites Storage

Microbiological ecosystem

Blending of grain

Damage by plant disease

Chemical preservatives Intrinsic factors Water activity Nature of substrate Mineral nutrition Nutrient composition

Extrinsic factors

Hygienic conditions

Temperature Climatic conditions Oxygen level

FIGURE 13.14  The interaction between intrinsic and extrinsic factors in the food chain that influences mold spoilage and mycotoxin production in stored commodities. (Adapted from Magan, N., Sanchis, V., and Aldred, D., Fungal Biotechnology in Agricultural, Food and Environmental Applications, Marcell Dekker, New York, 311–23, 2004.)

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mycotoxin detoxification by physical, chemical, and microbiological methods will enable the establishment of combined treatment procedures to effectively decontaminate contaminated foods and feeds. Such treatment methods are expected to be cost effective and minimally deleterious to food constituents (Park 1993).

The Inactivation Strategies on Mycotoxin Bioactives Methods for controlling mycotoxins are largely preventive. They include good agricultural practice and sufficient drying of crops after harvest. There is considerable on-going research for methods to prevent preharvest contamination of crops. These approaches include developing host resistance through plant breeding and through enhancement of antifungal genes by genetic engineering, use of biocontrol agents, and targeting regulatory genes in mycotoxin development. As of now, none of these methods has solved the problem. Because mycotoxins are “natural” contaminants of foods, their formation is often unavoidable. Many efforts to address the mycotoxin problem simply involve the diversion of mycotoxin­contaminated commodities from the food supply through government screening and regulation programs (Basappa and Shantha 1996).

Extrusion Process Cottonseed is an economical source of protein and is commonly used in balancing livestock rations; however, its use is typically limited by protein, fat, gossypol, and mycotoxin contents. The extrusion temperature study showed that mycotoxin levels were reduced by an additional 33% when the cottonseed was extruded at 160°C as compared to 104°C . Furthermore, the multiple-pass extrusion study indicated that mycotoxin levels were reduced by an additional 55% when the cottonseed was extruded four times as compared to one time. Total estimated reductions of 55% (three stages of processing at 104°C), 50% (two stages of processing at 132°C), and 47% (one stage of processing at 160°C) were obtained from the combined equations. If the extreme conditions (four stages of processing at 160°C) of the evaluation studies are applied to the combined temperature and processing equation, the resulting mycotoxin reduction would be 76% (Buser et al. 2002). Traditional nixtamalization and an extrusion method for making the dough (masa) for corn tortillas that requires using lime and hydrogen peroxide were evaluated for the detoxification of mycotoxin. The traditional nixtamalization process reduced levels of aflatoxin B1 (AFB(1)) by 94%, aflatoxin M1 (AFM(1)) by 90%, and aflatoxin B1-8,9-dihydrodiol (AFB(1)-dihydrodiol) by 93%. The extrusion process reduced levels of AFB(1) by 46%, AFM(1) by 20%, and AFB(1)-dihydrodiol by 53%. Extrusion treatments with 0, 0.3, and 0.5% lime reduced AFB(1) levels by 46, 74, and 85%, respectively. The inactivation of AFB(1), AFM(1), and AFB(1)-dihydrodiol in the extrusion process using lime together with hydrogen peroxide showed higher elimination of AFB(1) than treatments with lime or hydrogen peroxide alone. The extrusion process with 0.3% lime and 1.5% hydrogen peroxide was the most effective process to detoxify aflatoxins in corn tortillas, but a high level of those reagents negatively affected the taste and aroma of the corn tortilla as compared with tortillas elaborated by the traditional nixtamalization process (Elias-Orozco et al. 2002). Samples of corn flour experimentally contaminated with aflatoxin B1 (AFB(1); 50 ppb) and deoxynivalenol (DON; 5 ppm) were extruded. The effects of three extrusion variables (flour moisture, extrusion temperature, and sodium metabisulphite addition) were analyzed according to a two-level factorial design. The process was effective for the reduction of DON content (higher than 95%) under all the conditions assessed, but was only partially successful (10%–25%) for the decontamination of AFB1 (Cazzaniga et al. 2001). The results show that extrusion cooking is effective for the inactivation of DON but is of limited value for AFB(1), even if metabisulphite is added. More severe extrusion conditions are needed for the detoxification of AFB(1). As contamination with DON occurs mainly in the field prior to ­harvesting and that of AFB(1) is normally produced during grain storage, maize is often contaminated with © 2011 by Taylor & Francis Group, LLC

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DON but not with AFB(1). Under these conditions, the described extrusion process can be used for the detoxification of DON. The addition of sodium metabisulphite did not significantly affect the inactivation of AFB(1). Extrusion cooking is therefore an appropriate treatment for vomitoxin-contaminated maize in countries where, because of the prevailing conditions, these are the only toxins present.

Application of Ammonia Ammoniation of corn, peanuts, cottonseed, and meals to alter the toxic and carcinogenic effects of mycotoxin contamination has been the subject of intense research effort by scientists in various government agencies and universities worldwide. Engineers have devised workable systems of treatment for whole seeds, kernels, or meals; chemists have identified and characterized products formed from the reaction of aflatoxin B1 with ammonia with and without a meal matrix; biochemists have studied the biological effects of these compounds in model systems; and nutritionists have studied animal responses to rations containing ammoniated or nonammoniated components. The results of aflatoxin/ammonia decontamination research demonstrate the efficiency and safety of ammoniation as a practical solution to aflatoxin detoxification in foods and animal feeds. Corn throughout the world is frequently contaminated by the fungus Fusarium moniliforme, which produces toxic fumonisins. Ammonia has been shown to detoxify, effectively, aflatoxins in corn and cottonseed. Since corn can be contaminated by both fumonisins and aflatoxins, the application of ammoniation of corn either cultured with or naturally contaminated by F. moniliforme showed that fumonisin B1 levels in the culture material and in naturally contaminated corn were reduced by 30 and about 45%, respectively, with the treatment. Despite the apparent reduction in fumonisin content, the toxicity of the culture material in rats was not altered by ammoniation. Reduced weight gains, elevated serum enzyme levels, and histopathological lesions typical of F. moniliforme toxicity, occurred in rats fed either the ammoniated or nonammoniated culture material. Atmospheric ammoniation of corn does not appear to be an effective method for the detoxification of F. moniliforme-contaminated corn (Norred et al. 1991). Although there was no significant change in dietary intake, body weight gain, and feed conversion ratio in chickens fed ammonia-treated aflatoxin contaminated maize, these parameters were suppressed in birds fed the aflatoxin-containing diet. These data suggest that replacement of aflatoxin-containing maize with ammoniated grains can significantly suppress aflatoxicosis, leading to an improvement in production parameters in chicken weight gain (Allameh et al. 2005). Rice, a cereal for human and animal nutrition, is susceptible to aflatoxin contamination in the field and during storage. Therefore, the goal of the research was the evaluation of the efficacy and permanence of the ammoniation process through high pressure/high temperature (HP/HT) and atmospheric pressure/ moderate temperature (AP/MT) conditions applied to rice samples artificially contaminated with aflatoxin B1. For this purpose a 2(k) design was drawn up considering the temperature, the rice moisture, and the process time as variables. Under both sets of conditions, aflatoxin B1 concentration was reduced in a range of 90–100%. In conclusion, the process efficacy and permanence were achieved through the use of high temperature and a long process time for both sets of conditions (HP/HT and AP/MT), respectively (Trujilio and Yepez 2003).

Feed Additives The possible protective effect of four feed additives against the toxic effects of T-2 toxin in growing broiler chickens was investigated in a randomized trial consisting of six dietary treatments (control with no T-2 toxin or feed additive added, 2 ppm T-2 toxin alone, 2 ppm T-2 toxin plus 2.0 g/kg Mycofix, 2 ppm T-2 toxin plus 2.0 g/kg Mycosorb, 2 ppm T-2 toxin plus 2.5 g/kg MycoAd, and 2 ppm T-2 toxin plus 3.0 g/kg Zeolex). When no feed additive was included, 2 ppm dietary T-2 toxin significantly decreased BW and increased feed:gain ratio. When 2.0 g/kg Mycofix were added to the diet, the feed additive protected against the adverse effects of T-2 toxin on BW, BW gain, and feed:gain ratio; however, no protection against the adverse effects of T-2 toxin on the final BW and then BW gain was obtained by © 2011 by Taylor & Francis Group, LLC

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supplementation of any of the other three feed additives. The results of the trial indicate that the only feed additive capable of counteracting the adverse effects on performance caused by the dietary administration of 2 ppm T-2 toxin was the additive based on the enzymatic inactivation of the 12,13-epoxide ring of the trichothecenes (Mycofix). This study also confirms previous reports showing that aluminosilicates are not effective against trichothecene mycotoxins (Diaz et al. 2005).

Chlorine Dioxide The efficacy of chlorine dioxide (ClO2) in detoxifying the trichothecene mycotoxins verrucarin A and roridin A, was evaluated. In the first experiment, verrucarin A (1, 5, or 10 mu g) and roridin A (5 or 10 mu g) were each inoculated onto square-inch sections of glass, paper, and cloth and exposed to 1000 ppm of ClO2 for either 24 or 72 hours at room temperature. In the second experiment, verrucarin A and roridin A (1 or 2 ppm in water) were treated with 200, 500, or 1000 ppm ClO2 for up to 116 hours at room temperature. Results for the first experiment showed that ClO2 treatment had no detectable effect on either toxin. For the second experiment, both toxins were completely inactivated at all tested concentrations in as little as 2 hours after treatment with 1000 ppm ClO2. For verrucarin A, an effect was seen at the 500 ppm level, but this effect was not as strong as that observed at the 1000 ppm level. Roridin A toxicity was decreased after treatment with 200 and 500 ppm ClO2, but this was not significant until the 24 hour exposure time was reached. These data show that ClO2 (in solution) can be effective for detoxification of roridin A or verrucarin A at selected concentrations and exposure times in cereals and fruits (Wilson et al. 2005).

Citric Acid Chemical inactivation of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in maize grain by means of 1 N aqueous citric acid was confirmed by the AFLATEST immunoaffinity column method, high performance liquid chromatography (HPLC), and the Ames test (Salmonella-microsomal screening system). The AFLATEST assay showed that aflatoxins in the maize grain with an initial concentration of 29  ng/g were completely degraded and 96.7% degradation occurred in maize contaminated with 93 ng/g when treated with the aqueous citric acid. Aflatoxin fluorescence strength of acidified samples was much weaker than untreated samples as observed in the HPLC chromatograms (Mendez-Albores et al. 2005).

Biological Detoxification Some toxin-producing fungi are able to degrade or transform their own products under suitable conditions. Pure cultures of bacteria and fungi that detoxify mycotoxins have been isolated from complex microbial populations by screening and enrichment culture techniques. Genes responsible for some of the detoxification activities have been cloned and expressed in heterologous hosts. The detoxification of aflatoxins, cercosporin, fumonisins, fusaric acid, ochratoxin A, oxalic acid, patulin, trichothecenes, and zearalenone in feeds and some foods by pure cultures were also reported (Karlovsky 1999; Okamoto et al. 2001).

Sulfhydryl Compounds Most food toxicants have specific groups responsible for their deleterious effects. Modifying such sites with specific acids, peptides, and proteins lessens their toxicity. Sulfhydryl (thiol) compounds such as cysteine, N-acetylcysteine, reduced glutathione, and mercaptopropionylglycine interact with disulfide bonds of plant protease inhibitors and lectins via sulfhydryl-disulfide interchange and oxidation-reduction reactions. Such interactions with inhibitors from soybeans and with lectins from lima beans facilitate heat inactivation of the potentially toxic compounds, resulting in beneficial nutritional effects. Related transformations of protease inhibitors in soy flour are also beneficial. Since thiols are potent nucleophiles, they have a strong affinity for unsaturated electrophilic centers of several dietary toxicants, © 2011 by Taylor & Francis Group, LLC

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including aflatoxins, sesquiterpene lactones such as elephantropin and parthenin, urethane, carbonyl compounds, quinones, and halogen compounds. Such interactions may be used in vitro to lower the toxic potential of the diet, and in vivo for prophylactic and therapeutic effects against oxidative damage. A number of examples are cited to illustrate the concepts and mechanisms of using sulfur amino acids to reduce the antinutritional and toxic manifestations of cereals (Friedman 1994).

Miscellaneous Aflatoxins are also sensitive to UV light and gamma radiation. Exposure of artificially contaminated milk to UV light inactivated 3.6–100% of AFM(1) in the milk depending on the exposure time; and dried figs artificially contaminated in fruits and cereals with AFB(1) reduced the toxin level by 45.7%. Toxicity of a peanut meal contaminated with AFB(1) was reduced by 75 and 100% after irradiation by gamma rays at dose of 1 and 10 kg, respectively. Solar energy is also widely used and shown to decrease the amount of aflatoxins from 30 to 80% in peanut cakes and flakes, peanut oil, and olive oils in different parts of the world . The high hydrostatic pressure application is another method to inactivate mycotoxins present in foods; however, pressure exceeding 500 MPa has detrimental effects on the food itself. Enzymatic inactivation of fungal toxins is an attractive strategy for the decontamination of agricultural commodities and for the protection of crops from phytotoxic effects of fungal metabolites. A novel approach to the prevention of aflatoxin intoxication in some animals is the dietary inclusion of aflatoxin-selective clays that tightly bind these poisons in the gastrointestinal (GI) tract, significantly decreasing their bioavailability and associated toxicities. These methods aim at preventing the deleterious effects of mycotoxins by sequestrating them to various sorbent materials in the GI tract, thereby altering their uptake and, disposition to the blood and target organs (Diaz et al. 2005).

The Interpretations on Applicated Mycotoxin Inactivation Mechanisms on Fruits and Cereals A diverse group of chemicals has been tested for the ability to degrade and inactivate mycotoxins. A number of these chemicals can react to destroy (or degrade) mycotoxins effectively but most are impractical or potentially unsafe because of the formation of toxic residues or the perturbation of nutrient content and the organoleptic properties of the product. Two chemical approaches to the detoxification of mycotoxins that have received considerable attention are ammoniation and reaction with sodium bisulfite for cereals. Many studies provide evidence that chemical treatment via ammoniation may provide an effective method to detoxify mycotoxins-contaminated corn and other commodities. The mechanism for this action appears to involve hydrolysis of the lactone ring and chemical conversion of the parent compound aflatoxin B1 to numerous products that exhibit greatly decreased toxicity. On the other hand, sodium bisulfite has been shown to react with aflatoxins (B1, G1, and M1) under various conditions of temperature, concentration, and time to form water-soluble products. The feed additive protected the animals against the adverse effects of T-2 toxin on BW, BW gain, and feed:gain ratio. The results of the trials indicate that the feed additive was capable of counteracting the adverse effects on performance caused by the enzymatic inactivation of the 12,13-epoxide ring of the trichothecenes. The aqueous extract of ajowan seeds was found to contain an aflatoxin inactivation factor (IF). Thin layer chromatography analysis of the toxins after treatment with IF showed relative reduction of aflatoxin G(1) > G(2) > B1 > B2 (Hajare et al. 1994). Studies have demonstrated that protection against AFB(1) carcinogenesis conferred by diallyl sulfide (DAS) from garlic is related to the modulation of enzymes involved in the metabolism of AFB(1). The major determinant in this chemoprotective activity appears to be the induction of rGSTA5 gene (Glutathione Transferase gene). A common feature of the GSTs is their ability to bind glutathione; another property is their ability to recognize and detoxify compounds with diverse chemical structures. © 2011 by Taylor & Francis Group, LLC

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DAS was shown to greatly reduce AFB(1) genotoxicity by enhancing mutagenic metabolite AFB1-8,9exo-epoxide (AFBO) detoxification (Lixia and Thomas 1997). The effect of aflastatin A (AsA), produced by A. parasiticus, a novel inhibitor of aflatoxin production, on melanin biosynthesis of Colletotrichum lagenarium was reported. The addition of a low concentration of AsA (0·5 µg/ml) to the culture medium almost completely inhibited the melanin production of this organism. These results indicate that aflastatin A inhibits an early step in melanin production, which suppresses the expression of PKS1. (phytochrome kinase substrate 1). It may possibly be presumed that AsA also impairs other parts in the pathway of melanin production, for example, expression of a gene encoding the reductase (PKS1) or production of malonyl-CoA (Karlovsky 1999). During research on aflatoxin biosynthesis, a hydroxyversicolorone (HVN)-accumulating mutant of A. parasiticus lost the ability to form HVN. The HVN is an orange pigment that is a precursor of aflatoxins. This bacterium, which was tentatively designated A1, also remarkably inhibited production of another precursor, norsolorinic acid (NA), in A. parasiticus strain NFRI-95, which is an NA-accumulating strain. This bacterium did not appear to inhibit growth of the fungus, as the fungus grew beyond the bacterial colony (Yan et al. 2004). Studies suggest that certain fungi, including A. parasiticus, degrade aflatoxins, possibly through fungal peroxidases. Fermentation with yeasts has also been effective in destroying patulin and rubratoxin B. Sulfhydryl (thiol) compounds such as: cysteine, N-acetylcysteine, reduced glutathione, and mercaptopropionylglycine interact with disulfide bonds of plant protease inhibitors and lectins via sulfhydryldisulfide interchange and oxidation-reduction reactions. Since thiols are potent nucleophiles, they have a strong affinity for unsaturated electrophilic centers of several dietary toxicants including aflatoxins. Such interactions may be used in vitro to lower the toxic potential of the diet.

Summary Several physical and chemical detoxification methods developed so far have been critically discussed in different reviews for their advantages and limitations based on certain adopted strategies and specific criteria. Detoxification by microbiological means is also reviewed toward knowing the status on potential microorganisms and their enzymes that can degrade mycotoxin to less toxic or innocuous end products. Understanding mechanisms of mycotoxin detoxification by physical, chemical, and microbiological methods will enable the establishment of combined treatment procedures to effectively decontaminate contaminated foods and feeds. Such treatment methods are expected to be cost effective and minimally deleterious to food constituents

REFERENCES Allameh, A., Safamehr, A., Mirhadi, S. A., Shivazad, M., Razzaghi-Abyaneh, M., and Afshar-Naderi, A. 2005. Evaluation of biochemical and production parameters of broiler chicks fed ammonia treated aflatoxin contaminated maize grains. Animal Feed Science and Technology 122 (3–4): 289–301. Basappa, S. C., and Shantha, T. 1996. Methods for detoxification of aflatoxins in foods and feeds—A critical appraisal. Journal of Food Science and Technology-Mysore 33 (2): 95–107. Battilani, P., Barbano, C., and Logrieco, A. 2008. Risk assessment and safety evaluation of mycotoxins in fruits. Chapter 1 in Mycotoxins in Fruits and Vegetables, eds. R. Barkai-Golan and N. Pastor. San Diego, CA: Academic Press. Betina, V. 1989. Mycotoxins: Chemical, Biological and Environmental Aspects. New York: Elsevier. Buser, M. D., and Abbas, H. K. 2002. Effects of extrusion temperature and dwell time on aflatoxin levels in cottonseed. Journal of Agricultural and Food Chemistry 50 (9): 2556–59. Cazzaniga, D., Basilico, J. C., Gonzalez, R. J., Torres, R. L., and de Greef, D. M. 2001. Mycotoxins inactivation by extrusion cooking of corn flour. Letters in Applied Microbiology 33 (2): 144–47. Diaz, G. J., Cortes, A., and Roldan, L. 2005. Evaluation of the efficacy of four feed additives against the adverse effects of T-2 toxin in growing broiler chickens. Journal of Applied Poultry Research 14 (2): 226–31. © 2011 by Taylor & Francis Group, LLC

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Drusch, S., and Ragab, W. 2003. Mycotoxins in fruits, fruit juices, and dried fruits. Journal of Food Protection 66:1514–27. Elias-Orozco, R., Castellanos-Nava, A., Gaytan-Martinez, M., Figueroa-Cardenas, J. D., and Loarca-Pina, G. 2002. Comparison of nixtamalization and extrusion processes for a reduction in aflatoxin content. Food Additives and Contaminants 19 (9): 878–85. Friedman, M. 1994. Mechanisms of beneficial effects of sulfur amino-acids sulfur compounds in foods. ACS Symposium Series 564:258–77. Hajare, S. S., Hajare, S. N., and Sharma, A. 2005. Aflatoxin Inactivation Using Aqueous Extract of Ajowan (Trachyspermum ammi) Seeds. Journal of Food Science 70(1): 29–34. Jelinek, C. F., Pohland, A. E., and Wood, G. E. 1989. Worldwide occurrence of mycotoxins in foods and feeds (an update). Journal Association of official Analytical Chemists 72:223–30. Karlovsky, P. 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and food ­production—a review. Natural Toxins 7 (1): 1–23. Lixia, J., and Thomas, A. 1997. Baillie metabolism of the chemoprotective agent diallyl sulfide to glutathione conjugates in rats. Chemical Research in Toxicology 10 (3): 318–27. Magan, N., Sanchis, V., and Aldred, D. 2004. Role of spoilage fungi in seed deterioration. Chapter 28 in Fungal Biotechnology in Agricultural, Food and Environmental Applications, ed. D. K. Aurora, 311–23. New York: Marcell Dekker. Mendez-Albores, A., Arambula-Villa, G., Loarea-Pina, M. G. F., Castano-Tostado, E., and Moreno-Martinez, E. 2005. Safety and efficacy evaluation of aqueous citric acid to degrade B-aflatoxins in maize. Food and Chemical Toxicology 43 (2): 233–38. Norred, W. P., Bacon, C. W., Plattner, R. D., and Vesonder, R. F. 1991a. Differential cytotoxicity and mycotoxin content among isolates of fusarium-monoliform. Mycopathologia 115 (1): 37–43. Norred, W. P., Voss, K. A., Bacon, C. W., and Riley, R. T. 1991b. Effectiveness of ammonia treatment in ­detoxification of fumonisin contaminated corn. Food and Chemical Technology 29 (12): 815–9. Okamoto, S., Sakurada, M., Kubo, Y., Tsuji, G., Fujii, I., Ebizuka, Y., Ono, M., Nagasawa, H., and Sakuda, S. 2001. Inhibitory effect of aflastatin A on melanin biosynthesis by Colletotrichum lagenarium. Microbiology 147:2623–8. Park, D. L. 1993. Perspectives on mycotoxin decontamination procedures. Food Additives and Contaminants 10 (1): 49–60. Sanchis, V., and Magan, N. 2004. Environmental conditions affecting mycotoxins. In Mycotoxins in Food: Detection and Control, eds. N. Magan and M. Olsen, 174–89. Cambridge: Woodhead Publishing. Tokuşoğlu, Ö. 2010a. Aspergillus & Penicillium mycotoxins: Analytical quality control and risk management strategies. DSA-Dr.Bakon® Magazine, ISSN: 1308–3139. Tokuşoğlu, Ö. 2010b. Special Fruit Olive: Chemistry, Quality and Technology. Seher Publishing No: 006-1B SIDAS Media Ltd. Şti., İzmir. 350. ISBN: 978-9944-5660-4-9. Tokuşoğlu, Ö., Alpas, H., and Bozoğlu, F. T. 2010. High hydrostatic pressure effects on mold flora, citrinin mycotoxin, hydroxytyrosol, oleuropein phenolics and antioxidant activity of black table olives. Innovative Food Science and Emerging Technologies 11 (2): 250–8. Tokuşoğlu, Ö., and Bozoğlu, F. T. 2010. Citrinin risk in black and green table olives: Simultaneous determination with ochratoxin-A by optimizated extraction and IAC-HPLC-FD. Italian Journal of Food Science 22 (2). In Press. Tournas, V. H., and Katsoudas, E. 2004. Mould and yeast flora in fresh berries, grapes and citrus fruits. International Journal of Food Microbiology 105:11–17. Trujilio, F. R. M., and Yepez, A. J. M. 2003. Efficacy and stability of ammonium process as aflatoxin B-1 decontamination technology in rice (Oriza sativa). Archivos Latino Americanos De Nutricion 53 (3): 287–92. Wilson, S. C., Brasel, T. L., Martin, J. M., Wu, C., Andriychuk, L., Douglas, D. R., Cobos, L., and Straus, D. C. 2005. Efficacy of chlorine dioxide as a gas and in solution in the inactivation of two trichothecene mycotoxins. International Journal of Toxicology 24 (3): 181–86. Yan, P.-S., Song, Y., Sakuno, E., Nakajima, H., Nakagawa, H., and Yabe, K. 2004. Cyclo (L-Leucyl-L-Prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Applied and Environmental Microbiology 70 (12): 7466–73.

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14 Control of Mycotoxin Bioactives in Nuts: Farm to Fork Mohammad Moradi Ghahderijani and Hossein Hokmabadi CONTENTS Introduction..............................................................................................................................................291 The Impact of Mycotoxins in Nuts......................................................................................................... 292 Control of Mycotoxin Bioactives-Case Study: Pistachio Nuts............................................................... 294 Botanical Information........................................................................................................................ 294 Pistachio Nuts and Aflatoxin.............................................................................................................. 295 Basic Conditions Influencing Aflatoxin Contamination on Pistachio Nut.............................................. 297 Weather Conditions............................................................................................................................ 297 Tree Distance..................................................................................................................................... 297 Species, Rootstock............................................................................................................................. 297 Pruning............................................................................................................................................... 298 Irrigation............................................................................................................................................ 298 Nutrition............................................................................................................................................. 299 Temperature, Kernel Moisture Content, and Environment Conditions.................................................. 299 Harvest Conditions.................................................................................................................................. 301 Processing and Storage........................................................................................................................... 303 Sorting Out Contaminated Pistachios..................................................................................................... 307 Ecology of Aspergillus Species in Pistachio Orchards........................................................................... 308 References............................................................................................................................................... 309

Introduction The infection of crops by plant pathogenic fungi impairs both quality and quantity causing huge economic losses to farmers as well as immense effects on human and animal health. This implies that fungal colonization may affect seed size, weight, seed germination rate, protein and carbohydrate contents, baking, and other quality parameters. In addition to these impairments, the most serious consequence of fungal colonization is contamination of agricultural products with mycotoxins. The term “mycotoxin” is usually reserved for the toxic chemical products formed by a few fungal species that readily colonize crops in the field or after harvest and thus pose a potential threat to human and animal health through the ingestion of food products prepared from these commodities (Scudamore 2008). Mycotoxin contamination occurs worldwide on various plant species, especially cereals, nuts, dried fruit, coffee, cocoa, spices, oil seeds, dried legumes, and fruit when there is a risk of growing molds and formation of mycotoxins (Moss 1996; Sweeney and Dobson 1998; Placinta et al. 1999; Logrieco et al. 2003, Desjardins 2006; Boermans and Leung 2007; Binder et al. 2007; Barkai-Golan and Paster 2008; Gilbert and Senyuva 2008). Mycotoxins are stable compounds as produced; therefore, the best method to manage it is prevention. Consumption or exposure to the mycotoxins or their derivatives can cause various toxic effects that are © 2011 by Taylor & Francis Group, LLC

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different according to the type of mycotoxins and target organs. Although the occurrence of mycotoxins in food and feed differ according to the variation in agro-ecological zones/regions, the exposure to mycotoxins is worldwide. Therefore, the contamination of agricultural products by producing mycotoxigenic fungal species and their mycotoxins are the most serious challenge for trade in local and international markets. This implies there is a need for monitoring and reducing mycotoxins for food and feed safety. In this regard, the application of good farming, storage, and processing practices accompanied with transportation using new technology to reduce the mycotoxin levels to the lowest levels has to be focused on, especially in the less developed countries. Several countries and international organizations have determined different guideline levels for mycotoxins in food and feed according to the potential for health risks (FAO 2003). Several fungi are able to produce mycotoxins, as secondary metabolites, particularly species of Aspergillus, Fusarium, Penicillium, Claviceps, and Alternaria. Mycotoxins comprise a group of hundreds of chemically different toxic compounds. Aflatoxins, ochratoxins, trichothecenes, zearalenone, moniliformin, enniatins, and fumonisins are the most common mycotoxins produced in agricultural products (Moss 1996; Rotter et al. 1994; Sweeney and Dobson 1998). Fusarium species are able to produce a wide range of mycotoxins; namely, trichothecenes, zearalenone, and fumonisins even though aflatoxins produced by Aspergillus spp. are the most dangerous group as well as the highest toxicity of all known mycotoxins. This explains why the maximum tolerance levels for aflatoxins were first set in the 1970s and 1980s. Several regulations have been made for different crops throughout the world. Although poisoning by mycotoxins has been common over the centuries, the mass deaths of turkeys in modern intensive livestock farming in Great Britain (turkey-X disease) at the beginning of the 1960s triggered the first systematic research into mycotoxins.

The Impact of Mycotoxins in Nuts Nuts and their commodity including pistachio, groundnut, walnut, and almond are affected by contamination with mycotoxins. In most areas, these nuts will be affected commercially. This has been considered a food safety dilemma throughout the world especially over the last two decades (Buchanan et al. 1975; Fuller et al. 1977; Morton et al. 1979; Phillips et al. 1980). Therefore, most countries have set very low levels of mycotoxins in nuts (to accept or reject) especially in EU countries and Japan (Abbas 2005). Many times EU countries have rejected shipments of nuts from other countries because of contamination to aflatoxins. A continuing embargo was placed on the importation for contaminated nuts especially for developing countries. These rejections have increased pressure to ensure that the U.S. shipments of nuts are below mandated contamination action-levels for aflatoxin (Abbas 2005). The impact for the potential of aflatoxin contamination in nuts as a food safety and international trade issue has enforced the application and development of new methods and strategies to manage aflatoxin contamination in pre- and postharvest nut products. Different factors may affect the contamination of nuts to mycotoxins in pre- and postharvest influencing the fungal infection. Among these factors, the damage from insects is a principal factor for infection of nuts in preharvest, which may lead to subsequent contamination. The insects’ damage ­provides avenues that expose the kernels to fungal infection, especially the spores of aflatoxigenic aspergilli (Phillips et al. 1980; Klonsky et al. 1990; Doster and Michailides 1995b; Doster and Michailides 1999; Schatzki and Ong 2001). Insects affecting nuts and aflatoxins are larvae of the navel orangeworm (NOW), Amyelois transitella Walker (Lepidoptera: Pyralidae), which infests kernels of almonds, walnuts, and pistachios; the peach twig borer (PTB), Anarsia lineatella Zell. (Lepidoptera: Gelechiidae), which infests meristem leaf shoots, husks, and kernels of almonds; and the codling moth (CM), Cydia pomonella (L.) (Lepidoptera: Tortricidae), which infests the husks and kernels of walnuts (Kuenen and Barnes 1981; Sibbett and Van Steenwyk 1993; Keagy et al. 1996a, 1996b). Insects facilitate the fungal infection and colonization that has been shown in the groundnut when stored in the pod and easily damaged by insects (Widstrom 1979). There is also evidence that damage to groundnuts by soil pests increase aflatoxin contamination (Widstrom 1979). To reduce the aflatoxin content, the development of new methods and insecticides are required to manage insect damage (Varela et al. 1993; Blomefield 1994; Knight et al. 1994; Sauphanor and Bouvier © 2011 by Taylor & Francis Group, LLC

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1995). The new regulations for insecticides to protect the environment mandate strict reductions on insecticide use. For example, the use of specific organophosphorous has been banned throughout world in recent years. Therefore, it is necessary to develop new insecticide or approaches to control insects on nuts. Research and development of new methods to curtail insect-feeding damage to nuts have involved a variety of approaches. Semichemicals, chemical cues insects use for communication and discerning their environment, are being exploited to disrupt insect migratory, reproductive, and host-finding behaviors (Abbas 2005). On the other hand, development of new resistance cultivar is another approach to prevent infestation of the nut kernel by insects. For the effective control of insects it is necessary to understand their relations with the environment as well as their biology in nut orchards. For example, improved orchard management could be applied to remove plant litter that act as over-wintering reservoirs for insects. This has been developed for NOW in pistachio orchards (Doster et al. 2001) In most of the nuts, spores of several species of Aspergillus, including A. flavus, can be detected in the internal and external tissues of nuts that exhibit no exterior damage and may not be contaminated with aflatoxins. This may happen in pre- and postharvest or processing. Therefore, proper postharvest handling, transportation, and storage to prevent further colonization of kernel tissues are required. For example, poor harvesting and storage conditions in groundnuts can lead to the rapid development of the fungal colonization and thus a high production of the aflatoxin (Cole et al. 1989). The infection of Brazil nuts to A. flavus may take place on the exterior of pods when still attached to the tree (Arrus et al. 2005). Later on after collecting pods, the increase in the fungal colonization can occur before the nuts reach the processing plant for final drying to safe moisture levels (Johnsson et al. 2008). The population of Aspergillus spores in nut orchards is associated with plant litter as well as animal manures that may act as substrate for Aspergillus. In the case of pistachio nuts, Aspergillus could be found frequently in fallen fruit, male flowers, and other plant litter in pistachio orchards. It should be mentioned that Aspergillus species are able to sporulate on these substrates (Doster and Michailides 1994a, 1994b; Moradi et al. 2004; Moradi and Mirabolfathy 2007b). This will be lead to an increase of air-borne spores of toxigenic Aspergilli and the probability of infected nuts while they are on the tree (Moradi et al. 2004). The contamination of nuts to Aspergillus species may occur during the development of the kernel maturation with no evidence of insect damage (Sommer et al. 1986). For example in pistachios, the hull will protect the kernel during the maturation. When this layer splits or breaks, kernels will be exposed to the air-borne spore of Aspergillus species and fungal colonization may happen as a sequence of aflatoxin productions. This type of discoloration is readily detectable and such nuts can be removed from the processing stream (Pearson 1996). The fungal infections of nuts and their ecology, especially Aspergillus flavus, have been reported in several reports (Lillard et al. 1970; Schade et al. 1975; Denizel et al. 1976; Phillips et al. 1976; Emami et al. 1977; Fuller et al. 1977; Mojtahedi et al. 1979; Phillips et al. 1979; Purcell et al. 1980; Doster and Michailides 1994a, 1994b; Doster and Michailides 1999). The studies on the fungal flora before and after harvesting as well as in supermarkets indicated that different nuts maintained a different set of fungal species as microflora, both on the surface and in internal tissues (Bayman et al. 2002a, 2002b). The infection reinforces the need for proper postharvest handling, transportation, and storage of nuts. This can lead to further kernel colonization and aflatoxin production in favorable conditions, which is a risk to human health. Ochratoxin is another important mycotoxin that produces different fungal species in various agricultural products especially nuts. The specific species in the sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus are able to produce ochratoxin. The strains of Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii have been reported to produce ochratoxin (Varga et al. 1996). The A. alliaceus has been previously identified on nuts (Doster and Michailides 1994a). Although A. ochraceus and A. melleus were also identified on some nuts, none of the strains identified produced ochratoxin (Abbas 2005). Applying fungicides or other chemicals to prevent the growth of microorganisms or to destroy aflatoxins are not feasible for ensuring that nuts remain within tolerance levels. On the other hand, the tolerance strains could occur after the first application as well as the distribution of Aspergillus species throughout world. Due to the global limitations for the usage of these substances and their side effects, and due to the high mutation of aflatoxin producing fungi, it seems that these substances could not be used. © 2011 by Taylor & Francis Group, LLC

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A more fruitful strategy, therefore, may be to find natural products within the crop that confer resistance to colonization by Aspergillus or prevent aflatoxin biosynthesis, which are natural factors that exist in nature including phytoalexins and phytoanticipins (Abbas 2005). Several factors that may affect the infection of nuts to the Aspergillus species and aflatoxin production include cracking the outer layer of nuts, environmental factors, cultural practices, insect damage, frequency and time of irrigation, plant litter, animal manures, frequency of toxigenic strains, and harvesting date (Thomson and Mehdy 1978; Mojtahedi et al. 1979, 1980; Sommer et al. 1986; Doster and Michailides 1995a, 1995b, 1999; Abbas 2005; Mirabolfathy et al. 2006; Moradi 2005; Moradi and Javanshah 2006; Moradi and Mirabolfathy 2007). In the pistachio, these factors have been shown to be critical for infection especially in early splitting cultivars where the hull (pericarp) gets split exposing the kernel to molds and insects increasing the chances of aflatoxin production and contamination (Doster and Michailides 1995a, 1995b; Moradi et al. 2004; Moradi 2005; Moradi and Javanshah 2006). Whereas the molds such as Aspergillus spp. may cause direct contamination resulting in aflatoxin production, insects may play the role of spreading fungal spores, which in turn infect exposed kernels. Deficit irrigation during April or May in pistachios will increase early split nuts (Doster et al. 2001). While deficit irrigation at later stages during nut development does not appear to affect the incidence of early splits (Doster et al., 2001; Abbas 2005; Sedaghati and Alipour 2006); hence, growers need to provide sufficient irrigation to pistachio orchards in early spring. In groundnuts, the infection can occur during the crop season as well as after harvest. In preharvest infection, drought stress increases susceptibility to fungal invasion because plants lose moisture from pods and seeds; the physiological activity is greatly reduced. Many efforts have been done to manage aflatoxin contamination in different crops using different methods (Samarajeewa et al. 1990; Chitrangada and Mishra 2000a, 2000b; Yesilcimen and Murat 2006; CFP/EFSA/FEEDAP/2009/01 2009). However, no single approach has been fully achievable. During the 1990s and recent years, atoxigenic strains of A. flavus, yeast, and bacterial strains have been applied or during progress to manage the population of toxigenic strains of A. flavus in different crops (Kimura and Hirano 1988; Brown et al 1991; Cotty and Bayman 1993; Cotty 1994; Hua et al. 1998, 1999; Horn et al. 2000; Dorner and Cole 2002; Hua 2002; Wicklow et al. 2003; Dorner 2004; Jha et al. 2005; Nesci et al. 2005; Masoud and Kaltoft 2006; Palumbo et al. 2006; United States Department of Agriculture Research Service 2007). The biological control by competitive exclusion of A. flavus is a component of the integrated management of aflatoxin contamination in nuts. This strategy involved the use of competitive and antagonist native agents that can reduce the population of toxigenic strains in soil or plant litter and subsequently the infection rates of nuts and aflatoxin could be decreased.

Control of Mycotoxin Bioactives-Case Study: Pistachio Nuts Botanical Information The commercially cultivated pistachio tree (Pistacia vera), is a species of the Anacardiacae Family. Pistacia vera is a dioecious (sexually dimorphic), and deciduas (over wintering) temperate zone tree (CRFG 2009). The mature pistachio embryo or nut kernel is enclosed by three tissue layers that serve to protect it from the external environment: A soft thin fibrous endocarp ≤0.5 mm, followed by a thicker hard calcified mesocarp ≥0.5 mm, more commonly known as the nut shell; both of which are encased in another layer of soft pithy epicarp membrane ≈1.5 mm thick, which provides both physical and biochemical protection; as it is on the outer most surface of what makes a morphologically complete nut known as the hull (Crane and Iwakiri 1982). Pistachio nuts are characterized by a split in the shell at the calyx end of the nut. This split normally occurs on the tree about a month before harvest. The hull (mesocarp) of the pistachio usually encloses the shell and remains intact through harvest, serving as protection for the kernel. On normal nuts, there is space between the hull interior and shell exterior, so the shell can split open without splitting the hull. However, about 1 to 4% of the time, the hull will adhere tightly to the shell and the hull will split open along with the shell. These nuts are called early splits. The split in the hull allows an unobstructed © 2011 by Taylor & Francis Group, LLC

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passage to the kernel for air-borne mold spores and insects or other small animals, such as mites, which might be carrying mold spores (Crane and Iwakiri 1982; Sommer et al. 1986). The individual nut grows on fruit clusters of multiple nuts, much like grapes, and is considered ripe or ready for harvest during the months of September/October in the northern hemisphere (mid-autumn), depending on local climate conditions (Crane and Iwakiri 1982). The anatomical change in nut appearance that signals ripening is the easy separation of the hull from the inner shell, which is normally accompanied by the change in the hull color of the nuts from light green to shades of red–violet in most commercially grown Iranian varieties; with a few notable exceptions where the ripe nut hull color tends toward off-white (UCD 2009).

Pistachio Nuts and Aflatoxin Contamination of the pistachio nut by Aspergillus species and their mycotoxins are the most serious challenge to pistachio production, consumption, and exportation in the world. Infection of pistachio nuts by A. flavus as well as their aflatoxins contamination during maturation at pistachio orchards has been documented (Denizel et al. 1976; Emami et al. 1977; Mojtahedi et al. 1979; Sommer et al. 1986; Doster and Michailides 1994a and b; Heperkan et al. 1994; Pearson et al. 1994; Doster and Michailides, 1995a, 1995b; Schatzki 1995a, 1995b; Schatzki and Pan 1996; Scholten and Spanjer 1996; Schatzki and Pan 1997; Mahoney and Molyneux 1998; Schatzki 1998; Doster and Michailides 1999; Schatzki and De Koe 1999; Schatzki and Ong 2000; Doster et al. 2001; Campbell et al. 2003; Kashani-Nejad et al. 2003; Yazdanpanah et al. 2005; Yazdanpanah 2006; Hokmabadi et al. 2007; Yazdanpanah et al. 2005). Factors influencing the infection of pistachio nuts include: cracking of pistachio nuts (especially early hull splitting pistachios; Sommer et al. 1986; Doster and Michailides 1994a and b), environmental factors (Denizel et al. 1976; Emami et al. 1977; Mojtahedi et al. 1979; Heperkan et al. 1994; Campbell et al. 2003; Hokmabadi et al. 2007; Moradi et al. 2010), cultural practices (Campbell et al. 2003; Fooladi and Tafti 2006; Hosseinifard and Panahi 2006; Tajabadipour 2006), frequency and time of irrigation (Doster et al. 2001; Sedaghati and Alipour 2006), plant litter (Doster and Michailides 1994; Moradi et al. 2004), animal manures (Panahi and Alipour 2003; Moradi et al. 2004), frequency of toxigenic strains (Mirabolfathy et al. 2006), distribution of aflatoxin in pistachio bulks (Pearson et al. 1994; Moradi and Javanshah 2006) and harvesting date (Crane 1978; Kader et al. 1982; Panahi et al. 2005; Esmaeilpour 2004). These factors have been shown to be critical in infection especially in early splitting cultivars where the hull (pericarp) gets split exposing the kernel to molds and insects increasing chances of aflatoxin production and contamination Whereas the molds such as Aspergillus spp. may cause direct contamination resulting in aflatoxin production, insects may play the role of spreading fungal spores, which in turn infect exposed kernels (Sommer et al. 1976, 1986; Heperkan et al. 1994; Pearson et al. 1994). Pistachio nuts are characterized by a split in the shell at the calyx end of the nut. This split normally occurs on the tree about a month before harvest. The hull (mesocarp) of the pistachio usually encloses the shell and remains intact through harvest, serving as protection for the kernel. On normal nuts, there is space between the hull interior and shell exterior, so the shell can split open without splitting the hull. However, about 1–4% of the time, the hull will adhere tightly to the shell and the hull will split open along with the shell. These nuts are called early splits. The split in the hull allows an unobstructed passage to the kernel for air-borne mold spores and insects or other small animals, such as mites, which might be carrying mold spores (Sommer et al. 1986). Insects and small animal infestation rates on early split nuts are much higher because of the easy access to the kernel. The mold, Aspergillus flavus, has been found in pistachio nuts before harvest (Thomson and Mehdy 1978). Sommer et al. (1986) showed that nearly all the Aspergillus flavus contaminated nuts also have split, insect damaged, or bird damaged hulls before harvest. Sommer et al. (1986) found that the incidence of aflatoxin contamination in early split nuts was about 50 times greater than in nonsplit nuts (one in 500 for early split nuts versus about one in 25,000 of all nuts). They also mentioned the aflatoxin contaminated in nonsplit nuts is less than 2.0  ppb, while many early split nuts contain aflatoxin concentrations greater than 20 ppb and some above 1000 ppb. The prominent physical characteristic of an early split pistachio is a distinct, dark, and smooth edged split on the suture of the hull (see Figure 14.1). When an early split occurs, the split will normally start at © 2011 by Taylor & Francis Group, LLC

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Irregular cracking pistachio

Early splitting pistachio

FIGURE 14.1  Early splitting and irregular cracking pistachio.

FIGURE 14.2  Typical early split shell stain.

the calyx end of the hull and be present on only one side of the nut. A split can occur as early as 60 days before harvest but most occur from 30 to 20 days before harvest (Doster and Michailides 1991, 1993a and b, 1995a and b). Nuts in which the hull splits 60–30 days before harvest have a much greater opportunity for Aspergillus flavus infestation and high levels of aflatoxin, since the mold has had a much longer time to grow and excrete aflatoxin. These nuts, with the split in their hull for an extended period of time, tend to be drier than normal nuts or other nuts whose hulls split closer to harvest. Doster and Michailides (1991) found that early split nuts with dry, shriveled hulls were three times more likely than other early split nuts to be infested with Aspergillus flavus. Furthermore, aflatoxin was found in 31% of the shriveled early split nuts at an average concentration of 31 ppb, and aflatoxin was seen in only 6% of the nonshriveled early split nuts at an average concentration of 0.4 ppb (Doster and Michailides 1991, 1993a and b, 1995a and b). Another kind of split that can occur on a pistachio hull shortly (less than 15 days) before harvest is called a growth split or irregular cracking pistachio. Growth splits on pistachio hulls are characterized by ragged brown edges, and the split is randomly oriented and much wider than an early split. It has been shown that these nuts do not contain aflatoxin or Aspergillus flavus at harvest time, presumably because the mold has not had time to develop (Sommer et al. 1986). Doster and Michailides (1993a) observed that early split pistachio nuts tend to have shell stains near the perimeter of the shell suture split (Figure 14.2). Furthermore, Doster and Michailides (1999) report that early split nuts with shell staining had a higher incidence of Aspergillus molds than nonstained early split nuts. They also mentioned as much as 12% of the early split nuts that were contaminated with Aspergillus flavus had only slightly visible shell stains. Figure 14.1 shows a typical early split shell stain. © 2011 by Taylor & Francis Group, LLC

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A. flavus has frequently been detected in bird and insect damaged nuts. Doster and Michailides (1995a) found that 15.4% of the insect damaged pistachio nuts had Aspergillus flavus spores while only 2.4% of the noninsect infected nuts contained spores. These nuts often have abnormally low densities and are sorted out during processing with existing quality control machines (Kader et al. 1980). On the other hand, there are some nondamaged early split nuts that are indistinguishable based on their densities from normal nuts during the processing. These pistachios cannot be sorted out using quality control machines.

Basic Conditions Influencing Aflatoxin Contamination on Pistachio Nut Weather Conditions For the establishment of the orchards, a number of factors such as the weather conditions, the plant distance, and proper species should be considered. The rainfall at harvest and the maturation period of pistachio nuts increases the relative humidity and the possibility of contamination of pistachio nuts with aflatoxin (Widstrom et al. 1990; Guo et al. 1996; Danesh et al. 1979). Growers need to get information about the potential of an orchard site, the soil composition, impact of safety concerns, available source of water suitable for irrigation, and if environmental factors are inherent (such as dust-borne contaminants and pollutants) to support the growth of the desired tree variety. These factors may affect the infection of nuts to molds, indirectly. Therefore, the growers should consult with appropriate specialists to ascertain the availability of varieties that are resistant to the various factors, especially those that have an impact on the safety and quality of nuts produced in the orchard. Studies conducted concerning drying time of pistachio nuts in processing units indicated that rainfall during sun-drying could increase drying time up to 120 hours, which favor spore germination and infection of pistachios to Aspergillus species and aflatoxin production. The application of new methods and machines for quick drying the pistachio yields are recommended (Mirdamadiha 1999). Danesh et al. (1979) showed that there will be a higher incidence of aflatoxin contamination if it rains about 1 month before harvest, when early splits frequently occur. It is evident that high humidity will enhance Aspergillus flavus growth. However, there are usually no participations in the pistachio plantation area of Iran during the harvest and processing. The weather in California’s San Joaquin Valley, where most pistachios are grown, is normally dry.

Tree Distance The number of trees in the unit of area, which affects light density, have a significant role in contamination. Several factors are affected by tree density such as early splitting, relative humidity, air freshening, and so on. Regarding the soil type, a plant distance of 3–4 meters on the rows, and 6–7 meters distance between the rows is recommended (Anonymous 2004).

Species, Rootstock Pistachio cultivars differ in the time of ripening, the abscission of ripe pistachio nuts before the harvest, the percentage of an early splitting nut, and choosing the right cultivar for a region are necessary. In the regions with rainfalls at the end of summer, early maturing cultivars such as Fandoghi and Rezaei Zoodras should be planted. In regions with chilly summers where the period of ripeness lasts until late October—due to the increased possibility of rainfalls during this period—late maturing cultivar need to be planted. Also planting mixed species of pistachios must be avoided, because the length of the growing period in early maturing species, in comparison with late-maturing species, is shorter and delay in harvest could increase the risk of contaminations. The time of early splitting formation varies in location, cultivars, and years. In Ouhadi, Kalleh-Ghuchi, and Ahmadaghaei the first early splitting pistachios belong to the cultivar of Kalleh-Ghuchi. On the other hand, the highest percentage of early splitting occurs 15 days before the harvest (Tajabadipour et al. 2006). © 2011 by Taylor & Francis Group, LLC

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Among 17 Iranian pistachio cultivars, the results showed that Ghazvini zoodras, Shahpasand, Ghazvini motevasetras, and Owhadi cultivars had no early split pistachio nuts and Italiaii, Ahmadaghaii, Akbari, Kallehghouchi, Ghazvini dirras cultivars had lower than 1% of early split pistachios. The ­greatest early split percentage belonged to Lok sirizi, Momtaz tajabadi, and Ebrahimi. Result have shown that commercial pistachio cultivars of Iran (Owhadi, Kalleh ghouchi, Ahmad aghaii and Akbari) had less than 1% of early split pistachios. Italiaii, Ghazvini zoodras, Lok sirizi Hasan zadeh, Fandoghi riz, Ahmad aghaii, Akbari, Ghazvini motevasetras, Kalleh ghouchi, Ghazvini dirras, Ebrahimi, Jandaghi cultivars had moderately low (less than 7%) irregular cracked hulls. The greatest of irregular cracked hulls belonged to the Ebrahimi cultivar (33.5%; Tajabadipour et al. 2006). Tajabadipour et al. (2006) studied the effect of rootstock and scion on the frequency of early splitting formation. They also reported that the early splitting in Ouhadi scion cultivar is significantly less than the Kalleh-Ghuchi scion, while the frequency of the cracking pistachios Ouhadi and Ahmadaghaei scions are significantly higher than the Kalleh-Ghuchi scion. In artificial inoculations with A. flavus, the susceptibility cultivars differed in kernel colonization and aflatoxin concentrations. The highest kernel colonization belonged to the Ahmadaghae and Ouhadi cultivars, while the lowest ones belong to the Akbari and Kalleh-Ghuchi cultivars. The Kal Khandan and Maghzi, and ShahPasand and Abasali cultivars had the lowest and highest content of aflatoxin kernels, respectively (Moghaddam et al. 2006). The testa may act as a barrier to kernel colonization reducing growth and aflatoxin production. There was no correlation between the amount of sugar and fat with the amount of aflatoxin Bl (Moghaddam et al. 2006). Tajabadipour et al. (2006), in a study on the effects of Pistacia vera (Ahli), Pistacia mutica (Baneh), and Pistacia atlantica (Atlantica) rootstocks on the percentage of early splitting illustrated that Pistacia mutica (Baneh) and Pistacia atlantica (Atlantica) rootstocks have the highest and Pistacia vera (Ahli) rootstocks had the lowest percentage. This was similar in the case of cracking pistachios as well as the content of aflatoxin kernels. The tree’s age also affects the early splitting formation. The results showed that the amount of early splitting is the lowest on record in the last 10 years and the highest in 30 years, while 10-year-old trees showed the highest pistachio nut cracking.

Pruning Pruning is the basic principle for growing pistachio trees. Every year the dry and infected branches, suckers, and branches growing downward or toward the center of the tree must be pruned and cut. For mechanized harvesting of the yields and reducing the fruit contamination by preventing the contact of branches with the ground surface and irrigation water as well as planting single trunk trees with a trunk height of 100–120 centimeters and in an open center form is necessary (Anonymous 2004). One of the factors affecting the ecology and biology of Aspergillus species in pistachio orchards is pistachio nuts in contact with ground surface (Moradi et al. 2004).

Irrigation Early splitting pistachios are the major source of contaminated pistachios to molds, pests, and aflatoxins. There are several factors affecting early splitting formation—especially the stress of irrigation in late spring. One of the ways to reduce the frequency of early splitting of pistachios is a regular irrigation schedule. The amount of early splitting varies from orchard to orchard and from year to year depending on soil type, nutrition, the cultivar, weather conditions, and irrigation regime. Doster et al. (2001) studied the effect of eliminating the irrigation period in June on the phenomena of early splitting formation in two regions in California. The results at harvest time showed that the percentage of early splitting in the no-irrigation in June area was significantly higher than the control. Doster and Michailides (1995a) mentioned that a deficit irrigation regime in the middle of April until the middle of June will significantly increase the percentage of early splitting among pistachio trees, while a deficit irrigation regime beginning in late July until the middle of September, will decrease the percentage of early splitting in pistachios—which is according to the period of shell hardening. On the other hand, different irrigation systems had a low effect on the early splitting phenomena, where the average of early splitting © 2011 by Taylor & Francis Group, LLC

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in orchards receiving microsprinkler, surface, and sprinkler irrigation systems were 2.8, 2.6, and 2.1, respectively. A cease in irrigation at different stages of growth during the season showed that a deficit irrigation regime in the late May significantly increases the amount of early splitting in relation with other treatments (Doster et al. 2001). It was also found that a low irrigation regime beginning at the middle of July until harvest time decreases the phenomena of early splitting. Doster and Michailides (1993a) also found that cultural practices have little effect on the quantity of early split nuts formation in the orchards. However, Michailides et al. (1993) showed that sprinkler irrigated pistachio orchards using high trajectory sprinklers (above 23°) can increase the chance of Botryosphaeria blight. This disease can cause small black lesions on the pistachio hull. The risk of Botryosphaeria blight is reduced when the sprinkler trajectory is reduced so that it does not wet the nut clusters. It is not known if there is a correlation between Botryosphaeria blight and Aspergillus flavus contamination. The experiment consisted of two treatments with irrigation intervals of 25 and 45 days. This showed that in short irrigation periods (25 days) cutting one irrigation period off would merely affect the qualitative and quantitative specifications of the yields. While in long irrigation periods (45 days), cutting one irrigation period off would regime according to the plant’s need for water not be possible; it is recommended fulfilling the irrigations steadily with regular irrigation periods during the season (Sedaghati and Alipour 2006).

Nutrition One of the most important factors affecting the cracking of the pistachio nut hull is the equilibrium of nutrition elements, which is poorly investigated in pistachio orchards and requires further studies. It may lead to reducing early splitting. Torabi and Malakooti (1998) demonstrated that the hull of cracked pistachios has lower amounts of potassium and iron and higher amounts of phosphor and zinc. They found out that the potassium–zinc ratios in cracked and intact pistachios were 32 and 49, respectively, and the iron–zinc ratio was 1.5 and 2.8, respectively, which altogether implies a significant difference between the two samples. They also stated that the observation showed considerable effects of iron in preventing pistachio hull tattering. It appears that, due to not drawing on the right methods for managing the orchard through different operations, such as irrigation, fertilization and so on, the contamination of pistachio nuts with ­aflatoxin increased. Hoseinifard and Panahi (2006) investigated the effect of poultry manure in pistachio orchards as applied in fertilizer canals, soil surface and control (no-fertilizer). Early splitting pistachios that formed in fertilizer canals and soil surface, had low amounts or no detectable aflatoxins, while the aflatoxins detected were under control. This may show that the application of poultry manure having some effects on the time of early splitting formation. Hoseinifard and Panahi (2006) determined the effects of some nutrition elements on the percentage of early splitting formation in pistachio orchards. The results of the correlation analysis showed that early splitting in pistachio nuts negatively correlated to the amount of iron in leaves as well as the ratios between iron with zinc, magnesium, and copper. This was also true for copper and its ratios with other elements. According to these results, the more concentration of iron and the less concentration of copper reduce the percentage of early splitting in different regions. They also mentioned that in sandy soils the frequency of early splitting formation is higher than clay soils where the amount of the absorbable potassium of soil is high.

Temperature, Kernel Moisture Content, and Environment Conditions Temperature, relative humidity, and kernel moisture are the main factors in colonization of pistachio kernels and aflatoxin production as well as in interactions with microflora during maturation of pistachio nuts. It has been documented that Aspergillus species can grow in lower moisture contents than most fungi (Jones et al. 1981; McGee et al. 1996; Rodriguez-del-Bosque 1996). The results showed that the density fluctuations of Aspergillus species rose from the beginning of spring reaching a peak in September (Figure 14.3). The population then gradually decreased and had little © 2011 by Taylor & Francis Group, LLC

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Number of spores per plate

8 7

Harvesting time

6 5 4 3 2 1 0

6

6

7

7

8

8

9

9 10 10 11 11 12 12 1

1

2

2

3

Month FIGURE 14.3  Monthly fluctuation of Aspergillus species in pistachio orchards of Kerman province using the settle plate method. (From Moradi. M. and Mirabolfathy, M. Modern Fungicides and Antifungal Compounds V, 15th International Reinhardsbrunn Symposium, Friedrichroda, Germany, 353–54, 2007.)

variation in relation to different periods and places (Moradi et al. 2009). This implies that Aspergillus species can successfully compete with other fungal species to colonize pistachio kernels. There was no evidence to indicate that temperature would be a limiting factor for growth and sporulation of A. flavus, and A. niger during the summer, which could be supported by the wide temperature range under which Aspergillus species grow. However, temperature could be a positive factor for the contamination of nuts by Aspergillus species in orchards (Mojtahedi et al. 1980). In the summer, other factors such as pistachio nuts falling onto the ground, cracking of pistachio green hulls, insects and bird damages, delays at the harvest time, plant debris, and animal manures from the previous year as well as horticultural practices play an important role in increasing Aspergillus species population densities in pistachio orchards (Thomson and Mehdy 1978; Doster and Michailides 1994a, 1994b; Moradi et al. 2004; Mirabolfathy et al. 2006; Moradi and Mirabolfathy 2007). Low temperature during fall and winter seasons could be one of the factors causing the decrease in spore density. Michailides et al. (1993) showed that the specific temperature requirements for some fungi explain their seasonal prevalence. For instance, Botrytis cinerea is a low temperature fungus and is more common in spring. In contrast Aspergillus niger and other Aspergillus spp. prefer high temperatures, and so are common during summer and early fall. Rodriguez-del-Bosque (1996) investigated factors affecting A. flavus infection and aflatoxins contamination on corn, and reported that aflatoxins were undetectable in all treatments in the fall growing season during 1991 and 1992, when average minimum temperatures were  98%) in less than 10 minutes. The analytical methods including gas chromatography (GC), liquid chromatography (LC), capillary electromigration techniques (CE), and immunoassay are used for isoflavone separation and quantification. Among them, chromatography and CE are definitely the most common techniques applied in this field. Because of the high resolution, efficiency, and analysis speed with a minimum reagent and sample consumption, the use of CE for analysis of isoflavones is very attractive. There are some reports of chemicals forms that are identified by CE techniques (Peng et al. 2004; Herrero et al. 2005; Vacek et al. 2008). However, the disadvantage of CE is poor reproducibility that mainly is caused by the inconsistent flow rate and injection amount. HPLC is the main method of the analysis because it requires simple preanalysis sample preparation and allows a measurement of all isoflavone chemical forms at the same time. It is widely available and has been extensively studied (Rostagno et al. 2009). The advantage of the HPLC method is highly efficient and reproducible. The reversed-phase columns with the use of MeOH or MeCN and water containing a small amount of acid as mobile phase are used to separate isoflavones (Dentith and Lockwood 2008). Another alternative to performing a high-speed separation and identification using liquid chromatography is the use of monolithic columns. The major goals of applying monolithic columns in HPLC are to achieve low column backpressure and fast mass transfer kinetic (Siuoffi 2003; Unger et al. 2008). Monolithic columns have been successfully used in some occasions for the analysis of isoflavones in soy extracts (Apers et al. 2004; Kim et al. 2006).

Carotenoids Carotenoids, a group of lipid-soluble compounds responsible for yellow and red colors of many plants and food products, are one of the most important groups of natural pigments, because of their wide distribution, structural diversity, and numerous biological functions. In addition to the provitamin A activity © 2011 by Taylor & Francis Group, LLC

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of some carotenoids, recently, they have been demonstrated to be effective in preventing chronic diseases such as CVD and skin cancer (Kohlmeier and Hastings 1995; Fraser and Bramley 2004). Carotenoids have also been successfully used for the treatment of individuals suffering from photosensitivity disease, for example, erithropoietic protoporphyria (Mathews-Roth 1986; Thurnham 1994). Carotenoids have also been reported to stimulate the immune response at different levels (Bendich and Shapiro 1986; Bendich 1989 and 1996;), enhance the gap-junction communication (Zhang et al. 1992; Acevedo and Bertram 1995), and quench the free radicals (Burton 1989).

Extraction, Purification, and Identification Methods of Carotenoids The carotenoids are polyisoprenoid compounds of a C40 primary structure that contain the conjugated double bonds and are synthesized by tail-to-tail linkage of two C20 molecules. Figure 15.2 shows the chemical structures of several common carotenoids (Oliver and Palou 2000). The carotenoids are distinguished by functional substituent groups and named alpha-carotene, beta-carotene, lutein, lycopene, and so on. The characteristic conjugated double bond system of carotenoids makes the compounds particularly unstable, especially toward light, oxygen, and heat. Therefore, the extraction of carotenoids must be carried out very carefully and quickly to avoid extensive exposure to light, oxygen, high temperatures, and prooxidant metals such as iron or copper in order to minimize autooxidation and cis-trans isomerization (Marsili and Callahan 1993; van den Berg et al. 2000). Moreover, the addition of antioxidants is one of the most common strategies to prevent the carotenoid oxidation during the extraction procedure, especially when the samples are saponified to obtain free carotenoids. Butylated hydroxytoluene (BHT), ethoxyquin, pyrogallol, ascorbic acid, and sodium ascorbate are the antioxidants used for the extraction OH HO

Lycopene

Zeaxanthin

OH O

HO

β–Carotene

Antheraxanthin

OH

O α–Carotene

O

HO

Violaxanthin

OH O β–Cryptoxanthin OH

O

Canthaxanthin

HO Lutein

OH O

OH

Capsanthin

FIGURE 15.2  Chemical structures of several all-trans carotenoids. (Adapted from Oliver, J. and Palou, A., J. Chromatogr. A, 881, 543–55, 2000.)

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or treatment procedure (Tee and Lim 1991; Sharpless et al. 1999; Oliver and Palou 2000). The analysis of carotenoids typically comprises a series of steps, such as sampling, extraction, saponification, chromatographic analysis, identification, and quantification. Originally, the separation of carotenoids was carried out by low pressure column chromatography at atmospheric pressure (Almeida and Penteado 1988). However, this method required a large sample (Su et al. 2002). With the development of HPLC technology, a number of methods, both normal- and reversed-phase columns coupled with ultraviolet-visible (UV-Vis), photodiode array detector (DAD), and even highly sensitive electrochemical array detectors have been used to separate and analysis xanthophylls and carotenes (Ferruzzi et al. 1998; Huck et al. 2000; Rozzi et al. 2002; Rodríguez-Bernaldo de Quirós and Costa 2006). In general, the extraction of carotenoids is carried out with an organic miscible solvent. The most common solvent employed in the extraction step is n-hexane (Nierenberg and Nann 1992; Olmedilla et al. 1997; Lyan et al. 2001; Gueguen et al. 2002;). Other organic solvents are also used: butanol:ethyl acetate (1:1, v/v; Lee et al. 1992), 2-propanol:dichlorometane (2:1 v/v; Barua and Olson 1998), diethyl ether (Khachik et al. 1992), and ethyl acetate (Barua et al. 1993), methanol or a mixture of methanol and other more polar solvents (Hart and Scott 1995). A SFE technique has been used as an alternative method to replace traditional liquid extraction for isolating carotenoids from food samples, since this technique has several advantages such as rapid, selective, low-cost, nonflammable, environmentally acceptable, and an easier automation method for sample preparation prior to their characterization by other analytical methods (Marsili and Callahan 1993; Vági et al. 2002). Carbon dioxide has a low supercritical temperature (31°C) making it ideal for the ­extraction of thermally labile compounds (Vági et al. 2002). Moreover, the organic solvent modifier MeOH or EtOH was added to increase CO2’s salvation power (Careri et al. 2001). Although, β-carotene was less soluble in EtOH than in hexane, when EtOH was added as a modifier, the solubility of β-carotene in CO2 increased significantly (Marsili and Callahan 1993). After the sample extraction, traditionally, the alkaline or enzymatic hydrolysis saponification procedure has been often used as a step to simplify the separation by removing substances, such as chlorophylls and lipids, which could interfere with the chromatographic detection. The degradation and loss of total carotenoid content or individual carotenoids during the saponification step have been described (Khachik et al. 1986; Granado et al. 1992). Fernandez et al. (2000) found that greater retained carotenoids by using enzymatic hydrolysis. In recent years, many chromatographic methods for the simultaneous determination of free and esterified carotenoids in fruit samples have been described to avoid the saponification step (Oliver and Palou 2000). The main problem associated with the analysis of carotenoids is the unavailability of appropriate commercialized standard compounds that could be used in quantification because of the diversity, the inherent instability, and the presence of isomers of carotenoids. Phase separation, thin-layer chromatography (TLC), LC, and preparative HPLC methods have been used to get the pure standard (Olive and Palou 2000). The AOAC official method is still based on low-pressure column chromatography (AOAC International 1995). The traditional low-pressure column chromatographic methods have been widely replaced by HPLC methods in routine analysis in most labs. The advantages of using HPLC in carotenoid analysis include automatization, shorter analysis time, smaller sample size, and so on. Reversed-phase separations (Careri et al. 2001; Lee et al. 2001; Lyan et al. 2001; Gueguen et al. 2002; Iwase 2002; Moros et al. 2002; Rozzi et al. 2002; Vági et al. 2002; Azevedo-Meleiro and Rodriguez-Amaya 2004) have been mostly used, although several normalphase methods (Hollman et al. 1993; Casal et al. 2001; Englberger et al. 2003) have also been reported in the literature. The DAD detector system applied to the HPLC system is capable of recording absorbance at the entire spectral range (from 190 to 800 nm) during analysis and makes it more suitable for the separation and quantification of different carotenoids in one analysis. However, traditional GC and GC-mass spectrometry (MS) is generally not suitable for the analysis of carotenoids, because of their inherent instability and their low volatility (Tee and Lim 1991). The LC–MS becomes an innovative and powerful analytical tool for the identification of carotenoids, which is very sensitive and provides information about the structure. Atmospheric pressure ionization interfaces (APCI) (Lacker et al. 1999; Huck et al. 2000; Fang et al. 2003) and electrospray ionization interfaces (ESI; Careri et al. 1999; Hadden et al. 1999) are widely used for carotenoid analysis. Without any doubt, the spreading use of mass spectrometry coupled to HPLC in the near future will help identify some carotenoids that have not been reported in fruit and cereal samples. © 2011 by Taylor & Francis Group, LLC

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Anthocyanins Anthocyanins are water-soluble natural red and purple colorants that have raised a growing interest due to their extensive range of colors, innocuous, and beneficial health effects. They are rich in most fruits with the red and purple color such as berries, grapes, and apples. Besides their antioxidant function, they play a vital role in the prevention of neuronal and CVD, cancer, and diabetes (Konczak and Zhang 2004). Berries contain a higher level of anthocyanins, which contri­bute to their purple color. Antioxidant capabilities and health benefits of these different berry varieties have been studied extensively. It has been reported that a positive correlation between the antioxidant capabilities and the anthocyanins content in blackberries, red raspberries, black raspberries, and strawberries; additionally, it also has been described that the berry extracts possess a high scavenging activity toward reactive oxygen species chemically generated (Heinonen et al. 1998; Wang and Lin 2000). Compared with other berries, bilberries have more than 15 different types of anthocyanins (Lätti et al. 2008). The content and composition of anthocyanins in these berries is extremely dependent on their growth environment. A high variation of anthocyanins in the bilberries harvested at different geographic areas was reported recently by Lätti et al. (2008). In red grapes, anthocyanins are also major phenolic antioxidants. Malvidin-3,5-diglucoside was identified and isolated from wild grapes and had higher antioxidant activity than α-tocopherol (Tamura and Yamagami 1994). Red wines that contain both of these important phenolics also demonstrated more effectiveness in preventing LDL lipid oxidation than tocopherol (Frankel et al. 1993). The anthocyanin fraction had greater activity in inhibiting LDL oxidation than phenolic fractions that did not contain anthocyanins (Ghiselli et al. 1998). This evidence supports that the daily intake or an appropriate amount of red wine would lower the risk of CVD. Besides anthocyanins, berries also have significant quantities of vitamins A, C, E, carotenoids, and other phenolics. These compounds and anthocyanins directly contribute to the antioxidant capability of berries. The order of antioxidant activity from high to low of different berries using a LDL oxidation model was blackberries, raspberries, blueberries, and strawberries (Heinonen et al. 1998). That study also suggested that bioavailability and bioactivity of different anthocyanins are variable. The health function of anthocyanins in preventing obesity and diabetes was also reported. Mice fed a cyanidin-3-O-glucoside diet significantly suppressed high-fat diet-induced increase in body weight gain, and white and brown adipose tissue weights after 12 weeks (Tsuda et al. 2003). Mice fed a high-fat (35%) diet plus purified anthocyanins from blueberries only had lower body weight gains and body fat than the high fat controls (Prior et al. 2008). In general, the antioxidant activity of wild berries, such as crowberry, cloudberry, whortleberry, lingonberry, rowanberry, and cranberry was higher than the cultivated berries, such as strawberry and raspberry. In a thermal stability study, degradation of the 10 anthocyanins, delphinidin, cyanidin, petunidin, peonidin, and malvidin derivatives with different conjugated sugars at heating temperatures of 80, 100, and 125°C were not significantly different from each other at the same heating temperature (Yue and Xu 2008). Degradation increased drastically, however, when the heating temperature was increased to 125°C. At that temperature, the half-lives for all anthocyanins were less than 8 minutes. The major phenolics in grapes are resveratrol, catechin, anthocyanins, and gallic acid (Carle et al. 2004). The antioxidant capability of grape extracts in inhibiting LDL oxidation has been studied intensively. Both commercial grape juices and fresh grape extracts were reported to lower human LDL oxidation (Frankel et al. 1998).

Extraction, Purification, and Identification Methods of Anthocyanins Due to the enormous potential of natural anthocyanins as healthy pigments, there is an increasing number of reports found in the literatures on the development of analytical techniques for their purification and separation (Robards and Antolovich 1997; Antolovich et al. 2000) and quantitative analysis using chromatographic and electrophoretic techniques (da Costa et al. 2000). The major food sources of anthocyanins belong to the families Vitaceae (grape) and Rosaceae (cherry, plum, raspberry, strawberry, blackberry, apple, peach, etc.). Other families containing anthocyanin pigmented food plants include the Solanaceae © 2011 by Taylor & Francis Group, LLC

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(tamarillo, aubergine), Saxifragaceae (red and black currants), Ericaceae (blueberry, cranberry), and Cruciferae (red cabbage; Hendry and Houghton 1996) (Table 15.1). The stability of anthocyanins is markedly influenced by environmental and processing factors such as temperature, pH, O2, enzymes, and condensation reactions. The anthocyanins are glycosides of different naturally occurring anthocyanidins, which are poly-hydroxy and poly-methoxy derivatives of 2-phenylbenzopyrylium (flavylium) salts. The solvent extraction is the most common of extraction methods for isolation of anthocyanins. Anthocyanins are polar molecules, thus the common solvents used in the extractions are aqueous mixtures of ethanol, methanol, or acetone (Kahkonen et al. 2001). Among these solvents, the extraction with methanol is the most efficient (Kapasakalidis et al. 2006), for example, it has been found that in R5 3′

R4

R3

8

1 + O

A

C

5 R2

4

7 6

2′

B

2 1′

6′

3

R6 4′ 5′

R7

R1

TABLE 15.1 Structural Identification of Anthocyanidins (Aglycons) Substitution Pattern Name Apigeninidin Arrabidin Aurantinidin Capensinidin Carajurin Cyanidin Delphinidin Europinidin Hirsutidin 3'-HydroxyAb 6-HydroxyCy 6-HydroxyDp 6-HydroxyPg Luteolin Malvidin 5-MethylCy Pelargonidin Peonidin Petunidin Pulchellidin Riccionidin A Rosinidin Tricetinidin

Abbreviations Ap Ab Au Cp Cj Cy Dp Eu Hs 3'OHAb 6OHCy 6OHDp 6OHPg Lt Mv 5-MCy Pg Pn Pt Pl RiA Rs Tr

R1

R2

R3

R4

R5

R6

R7

Color

H H OH OH H OH OH OH OH H OH OH OH H OH OH OH OH OH OH OH OH H

OH H OH OMe H OH OH OMe OH H OH OH OH OH OH OMe OH OH OH OMe H OH OH

H OH OH H OH H H H H OH OH OH OH H H H H H H H OH H H

OH OH OH OH OH OH OH OH OMe OH OH OH OH OH OH OH OH OH OH OH OH OMe OH

H H H OMe H OH OH OMe OMe OH OH OH H OH OMe OH H OMe OMe OH H OMe OH

OH OH OH OH OMe OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH

H OMe H OMe OMe H OH OH OMe OMe H OH H H OMe H H H OH OH H H OH

Orange N/A Orange Blue–red N/A Orange–red Blue–red Blue–red Blue–red N/A Red Blue–red N/A Orange Blue–red Orange–red Orange Orange–red Blue–red Blue–red N/A Red Red

Source: Adapted from Hendry, G. A. F. and Houghton, J. D., Natural Food Colorants, Blackie Academic & Professional An Imprint of Chapman & Hall, Bishopbriggs, Glasgow, 1996; Adapted from Castañeda-Ovando, A., Pacheco-Hernández, M. D. L., Páez-Hernández, M. E., Rodríguez, J. A., and Galán-Vidal, C. A., Food Chem. 113, 859–71, 2009. Note: N/A: not reported.

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anthocyanin extractions from grape pulp, the methanol is 20% more efficient than the EtOH and 73% more effective than only water (Metivier et al. 1980). Moreover, anthocyanins are not stable in alkaline conditions. Thus, extraction procedures have usually involved the use of acidic solvents that disrupts plant cell membranes and simultaneously dissolves the water-soluble pigments. Acidification with HCl is used to maintain a low pH and provide a favorable medium for the formation of flavylium chloride salts from simple anthocyanins. The use of acids such as HCl may alter the native form of the complex pigments by breaking associations with metals, copigments, and so on (Hendry and Houghton 1996). However, these methods are not selective for anthocyanins and are able to coextract a great number of other nonphenolic substances such as sugars, organic acids, and proteins (Coutinho et al. 2004). Therefore, a subsequent purification process is required before analysis. A wide variety of techniques have been used in the purification including solid-phase (SPE) and liquid– liquid (LLE; Donner et al. 1997; Romani et al. 1999), countercurrent chromatography (CCC; Schwarz et al. 2003), medium pressure liquid chromatography (MPLC; Nyman and Kumpulainen 2001; VivarQuintana et al. 2002; Alcalde-Eon et al. 2004), and chromatographic techniques employing insoluble polyvinylpyrrolidone (PVP; Hrazdina 1970). Anthocyanins analyses have been conducted by a mass spectrometry (MS) hyphenated techniques such as HPLC (Wang and Lin 2000; Longo et al. 2005). The MALDI-ToF-MS and Nuclear Magnetic Resonance (NMR) techniques have also been used for the anthocyanins structural elucidation, as in the case of acylated anthocyanins found in Acalypha hispida (Reiersen et al. 2003). Recently, CE has been used for anthocyanins separation, identification, and quantification. The first anthocyanin analysis by CE was reported in 1996 by Bridle et al.

Tocopherols and γ-Oryzanol Vitamin E is an important natural antioxidant in foods, especially those rich in polyunsaturated fatty acids (Kamal-Eldin and Appelqvist 1996). Vitamin E is also believed to protect our bodies against degenerative malfunctions, mainly cancer and CVD (Burton and Traber 1990). Natural vitamin E is composed of eight chemical compounds: α-, β-, γ-, and δ-tocopherols and four corresponding ­tocotrienols that have a common structure with a chromanol head and a phytyl tail (Carlucci et al. 2001; Rupérez et al. 2001). The γ-oryzanol is a mixture of compounds derived from ferulic acid with sterols or triterpene alcohols (Xu and Godber 1999). Corn is recognized as an excellent source of phytochemicals, such as tocopherols, phytosterols, and carotenoids, which generally possess the capability of preventing oxida­t ion (Truswell 2002; Martinez-Tome et al. 2004). Corn contains other carote­noids, such as α- and β-carotene, β-cryptoxanthin, and zeaxanthin, which are not found at a significant level in most other cereals. Although less than 5% of vitamin E in corn is distributed in the corn endosperm, the major vitamin E homologues here were α-tocotrienol and γ-tocotrienol (Grams et al. 1970), which are similar to rice bran. Adorn and Liu (2005) found that the total antioxidant activity of corn was highest when compared with wheat, oats, and rice. It was approximately three times higher than wheat or oats and two times higher than rice. Rice is one of the most important commodities in many Asian countries. Its edible part, the white rice kernel, is produced during rice mill processing, which removes the rice hull and rice bran from the harvested rough rice. Although rice bran makes up 10% of rice grain, it is con­sidered a waste product of rice milling and is discarded or used as animal feed. However, it was found that rice bran contains some important health-promoting compounds (Godber and Juliano 2004). Its lipid fraction consists of unsaponifiable material that seems to present positive health functions, mainly because of its high levels of α- and γ-tocopherols and γ-oryzanol (Xu and Godber 1999). Many studies have demonstrated that γ-oryzanol compounds could reduce the serum cholesterol level, the risk of tumor incidence, and inflammatory action (Rong et al. 1997; Wil­son et al. 2002; Tsuji et al. 2003). The γ-oryzanol in rice bran also exhibited significant antioxidant activity in the inhibition of cholesterol oxidation, com­pared with the four vitamin E components (Xu et al. 2001). Wheat bran also possesses various natural antioxidants that ben­efit in preventing CVD and certain can­cers (Halliwell 1996; Truswell 2002). Phenolics, tocopherols, © 2011 by Taylor & Francis Group, LLC

327

Isolation Characterization of Bioactive Compounds in Fruits and Cereals Phytyl tail

Chromanol head R1 HO 6 7 R2

5 8

4 1 O

3 2

H3C

H3C

H

CH3 CH3

CH3

CH3

H

Tocopherol

R1 HO 6 7 R2

5

4

8

1 O

CH3

CH3

3

CH3

CH3

2 CH3

CH3 Tocotrienol

Name α-tocopherol β-tocopherol γ-tocopherol δ-tocopherol

R1

R2

CH3 CH3 H H

CH3 H CH3 H

FIGURE 15.3  Structure of tocopherols and tocotrienols. (Adapted from Rupérez, F. J., Martín, D., Herrera, E., and Barbas, C., J. Chromatogr. 935, 45–69, 2001.)

and fiber in wheat bran are generally believed to be primarily responsible for its positive effects on CVD; undesirable lipid oxidation reactions in the body contribute to these disease conditions (Moller et al. 1988; Alabaster et al. 1997; Andreasen et al. 2001). As the quantity of γ-oryzanol could be up to 10 times higher than the vitamin E in rice bran, it may be a more important antioxidant of rice bran to reduce cholesterol oxidation than vitamin E, which has been traditionally con­sidered the major antioxidant in rice bran. The higher antiox­idant activities of γ-oryzanol components may be due to their structure, which is very similar to cholesterol. The analogous structure of γ-oryzanol components and cholesterol leads to similar chemical characteristics in a system. The γ-oryzanol components may have a greater ability to associate with cho­lesterol in the small droplets of an emulsion system and become more efficient in protecting cholesterol against free radical attack (Xu et al. 2001).

Extraction, Purification, and Identification Methods of Tocopherols and γ-Oryzanol Figure 15.3 illustrated the eight naturally occurring lipophilic compounds α-, β-, γ- and δ-tocopherols and tocotrienols. Food samples must be treated with some organic solvent, previous to or simultaneously with saponification or extraction process to disrupt the structures where vitamin E can be associated (membranes, lipoproteins, fat droplets, etc.) to eliminate interferences from proteins or carbohydrates. Saponification prior to extraction is classically performed by heating with KOH, frequently in EtOH or MeOH (Rupérez et al. 2001). The antioxidants such as BHT (Konings et al. 1996), ascorbic acid (AlbalaHurtado et al. 1997) and pyrogallol (Ueda et al. 1993) were used to overcome oxidation of fat-soluble vitamins caused by saponification. The soxhlet extraction with a variety of solvents, Folch extraction with chloroform–methanol (2:1), acetone, and diethyl ether are commonly used for vitamin E extraction © 2011 by Taylor & Francis Group, LLC

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(Rupérez et al. 2001). Currently, sample preparation methods are considered the key point in developing a new analysis method. Several techniques have been developed as an alternative to the liquid–liquid extraction. The use of SPE has proven to be an efficient technique for simplifying sample clean-up prior to HPLC analysis (Chase et al. 1999; Bonvehi et al. 2000). The use of supercritical fluids to perform the extraction is a relatively new and potential technology. The tocopherols were prepared by a highly selective procedure of SFE with CO2 that resulted in a comparable yield and lower amounts of interfering compounds than using other organic solvents. Many studies have been performed to develop sensitive and selective methods to determine the tocopherols in food sources. The GC-flame ionization detection (FID) is a technique that proceeded HPLC. Methods for determining vitamin E with this technique have been known since the early 1970s (Lehman and Slover 1976). HPLC separations of tocopherols provide a fast, simple, sensitive, selective, and more robust technique than GC. Most of the researchers use HPLC with UV-vis or fluorescence detection (Abidi 2000). Garlucci et al. (2001) developed the HPLC method using a Zorbax reversed-phase column with methonal–water as a mobile phase and measuring by a fluorescence detector at 303/328 (Ex/Em). Recently, the normal-phase HPLC has been reported because of the easiness of separation of β- and γ-isomeric tocopherols (Abidi et al. 1996; Kamal-Eldin et al. 2000). However, normal-phase HPLC has some disadvantages, such as the low stability of normal-phase stationary and poor reproducibility of the chromatographic parameters (Abidi 2000; Lanina et al. 2007). New reversed-phases capable of separating β- and γ-isomers such as a long-chain alkyl-bonded C30-silica (Rentel et al. 1998), nonsilica-based polyvinyl alcohol (Abidi and Mounts 1997; Rentel et al. 1998), and perfluorinated phenyl silica-based stationary phases (Richheimer et al. 1994) were introduced and more suitable for combining with sensitive electrochemical or ESI MS detection. Lanina et al. (2007) compared the applicability of both ESI and APCI in negative and positive ion modes to analyze the four tocopherol homologues and developed a simple, rapid, and sensitive LC–MS method for their simultaneous determination in food matrices using the novel perfluorphenyl silicabased stationary phases. In general, due to the sample pretreatment that requires more steps than other methods, GC is not satisfied by routine tocopherol analysis. Normal-phase HPLC is the selected tool for fat matrices when β- and γ-isomers need to be separated. The more general technique is the reversedphase HPLC that performs a better tocopherols analysis in a more rapid and simple way. Gamma-oryzanol is mainly composed of esters of trans-ferulic acid (trans-hydroxycinnamic acid) with phytosterols (sterols and triterpenic alcohols). Among these, cycloartenol, β-sitosterol, 24-methylenecycloartenol, and campesterol predominant (Xu and Godber 1999). Gamma-oryzanol also contains lower concentrations of esters of the trans-ferulic acid with Δ7-stigmasterol, stigmasterol, Δ7-campesterol, Δ7-sitostenol, campestenol, and sitostenol (Xu and Godber 1999), as well as esters of cis-ferulic (Akihisa et al. 2000) and caffeic acids (Fang et al. 2003). Gamma-oryzanol is largely lipophilic and thus extracted with rice bran oil, however, differently from tocols, γ-oryzanol is transferred to soapstock during the neutralization step of a chemical refining of rice bran oil (Narayan et al. 2006). To preserve γ-oryzanol, a physical rice bran oil refining technique was proposed by Paucar-Menacho et al. (2007). Activated and extruded rice bran obtained by the production of parboiled rice was used to extract crude rice bran oil by the expeller method. The refining consisted of acid degumming (with 85% H3PO4), centrifugation, clarification, deodorization, and winterization. Of the γ-oryzanol 97% was preserved by the proposed procedure (Paucar-Menacho et al. 2007; LermaGarcía et al. 2009) (Figure 15.4). The direct solvent extraction method that does not require specific extraction equipments has been most commonly used. Isopropanol or isopropanol:hexame (1:1 v/v) and MeOH were widely used in the extraction procedure (Emmons et al. 1999; Xu and Godber 2000). Due to the low viscosity and high diffusivity of supercritical fluids, as well as its environmental friendly merit, supercritical CO2 is much better than organic solvents. Xu and Godber (2000) compared liquid organic solvents with supercritical CO2 relative to efficiency for extracting lipids and γ-oryzanol from rice bran. Among the solvents tested, a 50:50 n-hexane/isopropanol mixture at 60°C for 45–60 minutes produced the highest γ-oryzanol yield. Without previous saponification, the yield of γ-oryzanol was approximately two times higher than that with saponification. However, using supercritical CO2 the yield of γ-oryzanol was approximately four times higher than the highest yield obtained by extraction with liquid organic solvents. The use of enzymes in rice bran processing is still today a new and © 2011 by Taylor & Francis Group, LLC

Isolation Characterization of Bioactive Compounds in Fruits and Cereals

R

Molecular Structure R O

24–methylen–cyclo– artanylferulate

Cycloartenylferulate

O

* R

O HO

Compound

*

O

HO

329

β–sitosterylferulate

*

O

Campestrylferulate

O *

FIGURE 15.4  Chemical structures of the four main components of γ-oryzanol. (Adapted from Lerma-García, M. J., Herrero-Martínez Simó-Alfonso, E. F., Mendonça, C. R. B., and Ramis-Ramos, G., Food Chem., 115, 389–404, 2009.)

relatively unexplored technology. Xylanases and cellulases have been used to help polish rice in a selective way (Das et al. 2008a, 2008b). For quantification of total γ-oryzanol in rice bran and rice bran oil, UV-spectroscopy with a normalphase HPLC method has been applied and not able to differentiate the individual steryl ferulates (Diack and Saska 1994). Separation of γ-oryzanol components has been achieved by using reversedphase HPLC (Norton 1995). The online coupling of a liquid chromatographic preseparation with capillary gas chromatography (online LC–GC) is considered an elegant and efficient approach for the analysis of sterols and/or steryl fatty acid esters in oils and fats. Miller et al. 2003 described an online LC–GC method that provided a rapid and effective isolation of γ-oryzanol from crude rice lipid extracts. Total lipids were extracted from rice and subjected to LC–GC without any prior purification. Gamma-oryzanol was preseparated by HPLC from rice lipids and transferred online to GC analysis in order to separate its major constituents. The total γ-oryzanol content could be quantified by HPLC-UV detection and distribution of γ-oryzanol constituents could be determined by online coupled GC analysis.

Summary Bioactive compounds in fruits and cereals have been shown to have antioxidant activity, antitumor, and are able to prevent the occurring of CVD. Considering the beneficial effect of these compounds, their incorporation in food products will represent an important value to the consumers. Numerous food products that contain bioactive compounds are already on the market. The implementation of better extraction, purification, and identification methodologies will have an impact on the isolation, quantification, and preservation of the compounds in food products, as well as in the development of new food products. With the rapid development of analytical science, more and more bioactive compounds in fruit and cereals will be fully studied and utilized.

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16 Effect of Bioactive Components on Dough Rheology, Baking, and Extrusion Joseph M. Awika CONTENTS Introduction............................................................................................................................................. 337 Effect of Bioactive Components on Dough Properties........................................................................... 338 Dietary Fiber...................................................................................................................................... 338 Mechanisms by which Fiber Affect Dough Rheology and Product Texture..................................... 338 Insoluble Fiber Components......................................................................................................... 338 Soluble Fiber Components............................................................................................................ 339 Overcoming Negative Effects of Whole Grain Fiber.................................................................... 339 Resistant Starch............................................................................................................................. 340 Antioxidants....................................................................................................................................... 340 Redox Status and Dough Rheology.............................................................................................. 340 Whole Grain Antioxidants and Dough Rheology..........................................................................341 Effect of Baking on Whole Grain Antioxidants............................................................................ 342 Cereal Bioactive Compounds in Extrusion............................................................................................. 342 Effect of Extrusion on Cereal Antioxidants....................................................................................... 343 References............................................................................................................................................... 344

Introduction In the general sense, a material is considered bioactive (biologically active) if it has interaction with or effect on any cell tissue in the human body. By this broad definition, starch, which constitutes about 70% of cereal dry matter, would be the most bioactive component of cereals. However, the common usage of the term “bioactive” in the food and nutrition field typically refers to compounds with beneficial effects related to promoting health and preventing or mitigating effects of a disease. In the medical field, bioactivity or a close relative, pharmacological activity, is used to define the beneficial or adverse effects of a compound in treating or preventing a disease. In this chapter, we will use the term bioactivity and its variants to refer to the beneficial effect of food constituents in promoting health and reducing disease risk. Cereal grains contain a diverse mixture of compounds that are considered bioactive. The most obvious and abundant group of compounds associated with bioactivity in cereals is the dietary fiber, which typically constitutes between 8 and 20% of whole cereal grain depending on species. This group includes cell wall polysaccharides found mostly in the bran (outer part of grain), and numerous other nondigestible components like resistant starch. Until recently, beneficial physiological effects of whole grain consumption were largely attributed to the dietary fiber. However, newer evidence shows that the benefits of whole grain consumption cannot be attributed to dietary fiber alone, and the presence of various phenolic compounds, waxes and lipids, phytoestrogens, vitamins, minerals, phytate, among others are crucial to the observed biological effects. Thus in describing the effect of cereal bioactives in processing, it is most appropriate to consider whole grain as a unit, as well as isolated components with demonstrated biological effects. © 2011 by Taylor & Francis Group, LLC

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The increased recognition of the role food plays in disease prevention has necessitated a search for strategies to improve the health profile of food. Food scientists and processors are constantly searching for ways to increase the levels of beneficial dietary components in foods people consume every day. Cereal grain is the single commodity group that is most widely and consistently consumed by humans around the world, and thus present excellent opportunity to promote healthy eating. Additionally, whole grain-based products have been associated with various health benefits (discussed in a separate chapter). Unfortunately, most consumers prefer refined grain products that are of low nutritional quality. The challenge is thus to transform whole grain-based products into foods that meet consumer sensory expectations. An understanding of how the health-promoting whole grain components behave during processing is key to optimizing their use in food products.

Effect of Bioactive Components on Dough Properties As mentioned above, bioactives in cereal are a complex mixture of compounds. These compounds work together in ways that are not fully understood to affect dough properties and final product quality. Among the most prominent components of cereal bran that have been studied in isolation for their impact on processing are dietary fiber components and antioxidants. We will look at each of these components briefly as they impact dough properties and product quality. Despite the well-known health benefits of consuming whole or unrefined grain products and a generally well aware consumer base, whole grain consumption remains low. Most consumers prefer refined cereal products primarily due to inferior sensory profiles of the whole grain products; especially a dull appearance, firm or coarse/gritty texture, and harsh flavor. Food manufacturers are constantly struggling to improve sensory profile of these products. Another problem food processors have to contend with is that the whole grain constituents (e.g., dietary fiber) that are known to produce health benefits typically also have a negative impact on product handling during processing. For example, whole wheat or wheat bran fortified flour produces dough that is difficult to handle and process. Common problems include altered water absorption, which can impact dough stickiness and stiffness, as well as reduced ability of key ingredients, proteins and starch to form the desired network and consistency.

Dietary Fiber The bulk of the undesirable characteristics of the whole wheat dough system can be attributed to soluble and insoluble fiber components (mostly nonstarch polysaccharides) of the bran. Proper gluten network formation via disulfide cross-linking during mixing is critical for the viscoelastic properties of the dough that allows it to trap gases during proofing and produce the desirable texture of bread during baking. Any ingredient that dilutes available gluten or alters its ability to cross-link will invariably affect dough rheology and product quality. Bread and related systems are highly dependent on proper gluten network formation for their quality, and are generally more adversely affected by bran components than other products like cakes and cookies that do not require a strong gluten network. An obvious effect of bran dietary fiber components on dough is the dilution of gluten, which reduces effective gluten concentration and thus its ability to form a proper viscoelastic network during mixing. However, research shows that the detrimental effect of bran fiber components on wheat dough rheology and subsequent product quality (e.g., loaf volume) is generally higher than what would be expected of the dilution effect on gluten alone (Lai, Hoseney, and Davis 1989). This indicates that the bran components are involved in both physical and chemical interactions during processing.

Mechanisms by which Fiber Affect Dough Rheology and Product Texture Insoluble Fiber Components Effect of whole wheat dietary fiber is twofold. Insoluble fiber particles (mostly lignocelluloses) are fairly rigid and do not hydrate easily thus can weaken dough by cutting gluten strands or interfering with their © 2011 by Taylor & Francis Group, LLC

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formation. As expected, the more coarse the fiber particles, the more they affect gluten network formation and products quality. Fine grinding of wheat bran has long been known to significantly improve its functionality as an ingredient in baking (Lai, Hoseney, and Davis 1989); this may be partly due to an increased surface area for more rapid hydration and thus improved pliability, as well as reduced ability to physically interfere with gluten strands. The knowledge of improved functionality of finer particle size bran was recently employed by major commercial bakers in the United States to produce the relatively successful “whole wheat” white bread, which combined ultrafine grinding of wheat bran with improved nonpigmented wheat varieties.

Soluble Fiber Components The other major effect of dietary fiber on dough rheology is related to water absorption properties of the soluble fiber components. One of the most obvious initial effects of bran addition to cereal dough systems is the increased demand for water to form workable dough. Cereal brans, depending on the source, contain significant quantities of soluble fiber that is capable of binding large amounts of water relative to its weight. For example, in commercial refined wheat flour the water soluble fiber components typically constitute less than 1% by weight, but are believed to account for 20–30% of dough water absorption. In whole grain flour or flour fortified with wheat bran where the soluble fiber proportion level is much higher, the effect on dough water absorption is usually more pronounced. On the surface, the dough water absorption problem should be easy to fix by just adding more water. However, it is not this simple for two reasons: First, bran particles are difficult to finely grind and thus tend to produce relatively large particles. The large bran pieces will absorb water relatively slowly; that is, diffusion of water to the center of each particle will take a long time. Secondly, the soluble fiber components, like highly branched arabinoxylans, or mixed linkage β-glucans (commonly designated “β-glucans”) are usually part of a cell wall structure and are thus embedded in an insoluble cell wall matrix; this further slows their rate of water absorption. The consequence of the above scenarios is that if the theoretical correct amount of water (based on known absorption potential of bran constituents) is added to flour at the beginning of mixing, the dough will be very sticky and difficult to work and process since there is effectively too much free water in the system that is not yet taken up by bran soluble fiber. On the other hand, the addition of less than the correct amount of water will lead to a dough that may be workable at the beginning, but will stiffen and feel dry with time (e.g., during proofing) due to continued slow absorption of water by the bran soluble fiber. The soluble fiber effectively competes with gluten for moisture and thus leads to the loss of viscoelastic properties of gluten. An experiment by Lai, Hoseney, and Davis (1989) neatly demonstrated this concept using bread dough as a model. These authors reported that when the slow water absorption property of wheat bran was partially overcome by fine grinding and presoaking the bran for several hours in water, the resulting loaf volume was significantly improved to a level equivalent to what would be expected of inert fiber (i.e., by dilution effect alone). They further demonstrated that the effect of wheat bran on water absorption was the biggest contributor to reduced loaf volume of bran fortified flour. Similar effects are seen in most dough systems that involve substituting cereal endosperm with whole grain or bran components. However, since different cereal grains have different soluble fiber composition, effects can vary widely. In general, the net effect of bran fiber components on gluten network will be to impede extensibility of dough and its functionality during the baking process, which results in adverse effects like reduced loaf volume, increased rate of firming of the bread, reduced flexibility of tortillas, reduced spread in cookies, and so on. The bran fiber components may also impede starch swelling and gelatinization by limiting available water, and also interfere with starch reassociation after baking, thus further impacting texture.

Overcoming Negative Effects of Whole Grain Fiber To overcome the above mentioned problems typically require the use of additional ingredients to improve product quality. Gluten isolate can be used to partially overcome the diluting effect of whole grain/bran components and also compensate for some of the lost functionality of flour gluten due to the presence of © 2011 by Taylor & Francis Group, LLC

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fiber. Commercial hydrocolloids (water soluble polysaccharides) of known functionality (e.g., carboxymethylcellulose) are also commonly used to help overcome the problem of poor texture (e.g., crumbly tortillas) and dry mouth feel of bran/fiber-enhanced grain products. These commercial soluble polysaccharides have advantage over whole grain soluble polysaccharides in that they are of known composition and predictable functionality. But even more importantly, they are designed to solubilize rapidly and thus can provide highly controlled functionality. The main effects include improving product moistness, yield, softness, pliability, and so on, all of which are related to their ability to retain moisture. Surfactants like sodium stearoyl lactylate (SSL) will also generally improve dough handling and product texture by partially bonding with and “blocking” the water soluble NSP hydroxyl groups, creating hydrophobic regions that limit the NSPs ability to cross-link via hydrogen bonds or to trap and retain water. This mode of action of the surfactants also reduces the ability of gelatinized starch to retrograde and form junction zones after baking, which contributes to the longer shelf life of whole wheat bread and related products.

Resistant Starch Another strategy that is becoming increasingly popular, is the use of resistant starch as an ingredient to boost dietary fiber in baked products. Resistant starch obtained via physical (usually by annealing or controlled temperature treatment) or chemical means (usually by cross-linking) have been shown in limited studies to produce similar physiological effect as soluble dietary fiber. In terms of processing, resistant starch offers a huge advantage over other forms of dietary fiber in that it is bland, very white, and of fine particle size, thus physically near identical to refined wheat flour. Additionally, resistant starch can be custom-made to behave like inert fiber (i.e., very limited water absorption capacity), and thus will generally not produce any adverse effects in dough handling or baked product quality beyond that expected from gluten-diluting effect. The diluting effect can be readily overcome by using stronger gluten flour or adding gluten to the system as some studies indicate. In the recent years, technology has allowed for the development of resistant starch that is virtually 100% dietary fiber (Woo, Maningat, and Seib 2009); this provides significant room for flexibility in terms of level of incorporation to achieve desired dietary fiber level in the product. Some authors have reported that products made with resistant starch can be of better quality (measured by textural profile and consumer acceptance) than products made with refined flour alone (Sharma, Yadav, and Ritika 2008). They are thus becoming attractive as potential ingredients to “stealthily” boost dietary fiber intake and health profile of refined cereal grain products.

Antioxidants Redox Status and Dough Rheology The viscoelastic properties of wheat gluten make wheat a unique and hard to replace commodity utilized in many cereal products. The viscoelastic properties of gluten are primarily due to the interaction of glutenin and gliadin fractions of the wheat protein. Development of the dough during mixing requires the formation of disulfide bonds between thiol groups of cysteine residues of gluten. Disulfide bonds act to stabilize the gluten network by enhancing protein folding and thus lowering its entropy. These bonds can also enhance protein–protein hydrophobic interactions by enhancing local concentration of protein residues and thus lowering the effective local concentration of water molecules; this in turn lowers the ability of water molecules to attack amide–amide hydrogen bonds and break up secondary protein structure. The extent of disulfide cross-linking during dough development is enhanced in oxidizing environment, where the thiols are readily oxidized to disulfides as illustrated in Equation 16.1.

2 R-SH + Br2 → R-S-S-R + 2 HBr.

(16.1)

For this reason, oxidizing agents such as potassium bromate, calcium peroxide, and ascorbic acid are often used to improve dough strength and loaf volume. In fact ascorbic acid (or more precisely its oxidized form, dehydroascorbic acid) has long been recognized for its ability to strengthen dough and enhance © 2011 by Taylor & Francis Group, LLC

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loaf volume ( Melville and Shattock 1938; Meredith 1965). On the other hand, the disulfide bonds are generally unstable in reducing the environment. Consequently reducing agents, such as sodium bisulfite or cysteine can significantly reduce dough mixing tolerance and strength. The effect of reducing agents on dough rheology is easy to demonstrate: the excess addition of cysteine to wheat dough formulation will lead to a completely inelastic gooey mass after a relatively short period of mixing. This is because the excess cysteine will readily reduce all disulfide linkages formed during mixing to form cystine residues and thus prevent the gluten from forming the viscoelastic network. The reducing effect of cysteine is sometimes applied in tortilla manufacturing to slightly weaken the gluten in the relatively strong bread flour commonly used in these formulations. Adding just the right amount of reducing agent will ensure that only a limited number of the disulfide linkages are reduced and so the dough will retain most of its elasticity. A slightly weakened gluten is important in tortilla processing to prevent the spring-back effect and produce a large diameter product. Spring-back is where the dough tends to shrink back after being pressed into a disk, usually due to too much elasticity. An added benefit of slightly weakening the gluten is that the product is less chewy.

Whole Grain Antioxidants and Dough Rheology Among the most valuable bioactive compounds in whole grain cereals are the phenolic antioxidants. The compounds, mostly concentrated in bran, are believed to contribute significantly to health benefits reported for whole grain products, including promotion of cardiovascular health and chemoprotective properties (discussed in a separate chapter). In wheat, like most commonly consumed cereal grains, ferulic acid and its derivatives, is the most abundant phenolic compound, and probably the most widely studied for its effect on dough properties. As stated earlier, the presence of wheat bran generally has a negative effect on dough rheology and handling, which is partly due to the soluble and insoluble fiber. However, another component of wheat bran that negatively affects dough handling is the phenolic group of compounds, primarily ferulates. Through their action as antioxidants, phenolic compounds have long been recognized for their reducing reaction on gluten disulfide cross-linkages, which induces dough breakdown during mixing, thus reducing dough stability (Dahle and Murthy 1970; Weak et al. 1977). This in turn results in reduced loaf volume and overall product quality. Other structurally different phenolic compounds, like flavonoids, whether inherent in cereal grains or added to flour (e.g., catechin; Wang et al. 2006), have been shown to negatively impact dough quality and loaf volume in a similar manner as the ferulates. This confirms that the antioxidant mechanism that is beneficial from a health perspective is detrimental to dough properties and product quality. With the growing recognition of the importance of antioxidants in diet and the need to enhance antioxidant profile of cereal-based foods, considerable research has gone into devising mechanisms to reduce the negative impact of antioxidants on dough rheology and product quality. One mechanism that has been considered promising is the induction of new gluten cross-linkages that are independent of the disulfide–sulfhydryl (disulfide–thiol) interchange reaction, and thus not sensitive to the redox state of the system. The use of transglutaminase, an enzyme that catalyzes acyl-transfer reactions, producing covalent cross-linking among proteins via the formation of inter- and intramolecular glutamyl and lysine isopeptide bonds, has been considered in this regard. Even though this enzyme is widely used in the meat industry as a protein binder (e.g., for making imitation crab meat), its use in the baking industry has not been investigated very much. Various reports have shown that the use of transglutaminase can strengthen dough and improve its mixing properties in ways that are somewhat similar to the effect of oxidizing agents (Bauer et al. 2003). In fact transglutaminase has been suggested as a modifier of protein functionality in gluten-free bread (Moore et al. 2006). However, a recent study did not find any significant improvement in dough rheology (mixing tolerance and elasticity) by transglutaminase when ferulic acid was also added as the antioxidant at 250 ppm, even though the transglutaminase did seem to reduce dough stickiness (Koh and Ng 2009). The resulting loaf quality (volume and rate of firming) were also not improved by transglutaminase. The authors indicated that their transglutaminase use level might have been too low to produce the desired effect. In fact other studies have indicated that a higher level transglutaminase than used by these authors © 2011 by Taylor & Francis Group, LLC

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may be necessary to enhance dough quality (Bauer et al 2003). However, among the problems that will need to be overcome in optimizing transglutaminase functionality is the fact that it tends to stiffen the dough (reduces dough extensibility), which often can result in reduced loaf volume and rapid firming of bread. In dough systems that are less dependent on a proper gluten network formation for product quality (e.g., in cookie dough), antioxidants generally have minimal effect on dough quality and product textural properties (Nanditha, Jena, and Prabhasankar 2009). Such products would thus seem to be an easier target for antioxidant fortification.

Effect of Baking on Whole Grain Antioxidants Even though much is known about how antioxidants affect dough rheology, what happens to the grain antioxidants during baking is not clear. Most studies do indicate that the level of antioxidants is reduced significantly by the baking process, the net reduction of which is dependent on the severity of the heat treatment as well as the processes preceding the heat treatment (Awika et al. 2003b). However, the baking process, or any heat treatment for that matter, transforms the proteins, starch, and other components of the food matrix in a way that completely alters extractability of most antioxidant molecules by common laboratory methods. The altered extractability may significantly confound measured antioxidant activity in a product. For example some studies indicate that levels of phenolic acids, especially ferulic acid and diferulate esters, and antioxidant activity of some whole grain products, including wheat products (Moore et al. 2009), increases after baking, probably due to the ability of heat to disrupt cellular matrix and cell wall polysaccharide integrity leading to the release of ferulate esters and other bound phenolic compounds. Such reports of increased antioxidant compounds after processing are also available for some vegetable products. On the other hand, other studies report a reduction or no change in phenols or antioxidant activity due to processing (Alvarez-Jubete et al. 2010). The separating effect of processing on the antioxidant molecules and effect of altered extractability remains a challenge. For example, even though it is known that some phenolic compounds are heat labile and will degrade over prolonged heating in model systems, such data has limited application in the true food system, especially complex systems like cereal-based products. This is because in such systems molecules will behave very differently due to their ability to interact with other molecules in ways that can alter their structure or significantly influence their stability. In general, available evidence suggests that altered extractability may account for a large part of the reported change in antioxidant properties of cereal products after thermal processing. For example, in comparing different baked products made with sorghum brans of different phenolic composition, Awika et al. (2003b) reported that white sorghum with very low levels of extractable phenols and antioxidant activity did show an increase in antioxidant activity after baking, whereas sorghum brans high in easily extractable phenols (flavonoids) and antioxidants showed reduced activity after processing. They attributed the increase in antioxidant activity of white sorghum bran-enriched products to increased extractability of phenolic acids, the primary antioxidants in white sorghum. This supports other findings for whole wheat (Moore et al. 2009), as well as other low antioxidant whole grain products. Thus it seems measured phenol content and antioxidant activity in baked cereal products is dependent on a balance between the enhanced release of bound phenolics and their breakdown by heat, as well as how the altered food matrix affects their extractability. Another problem is that the measured in vitro extractability of the phenolics or their antioxidant activity does not correlate with any known physiological properties. Hence in vitro changes in cereal phenolics during processing may not predict their health benefits. There is plenty of room for research in this arena.

Cereal Bioactive Compounds in Extrusion Extrusion in the grain industry is mostly used to produce ready to eat snacks and related products. Since snacks are traditionally among the least healthy food products (traditional extruded products © 2011 by Taylor & Francis Group, LLC

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are predominantly made from cereal endosperm or starches), significant research effort has gone into transforming them into healthy products. The use of whole grain, as well as the incorporation of fiber, antioxidants, and other health promoting components are among the popular strategies that have been employed to enhance the health profile of extruded products. Socioeconomic factors have also driven the research to increase the incorporation of fiber and antioxidants in extruded products. For example, the recent spike in fuel oil prices necessitated a drive to develop alternative renewable energy sources, the most popular of which has been ethanol derived from cereal grain fermentation. This process creates significant quantities of spent brewers grain (also called distillers grain), which had limited economic value but are rich in proteins, dietary fiber, and other health promoting compounds associated with whole grain. In general extrusion is largely dependent on extensive starch gelatinization and melting under high heat and pressure, and then rapidly retrograding to form expanded structure when steam is released as pressure is dropped. Thus any factors that dilute the starch or interfere with starch melting and reassociation will likely lead to a reduced product expansion (increased bulk density) and thus harder texture, which is undesirable in snacks. This has been observed in various studies that utilize whole grain or spent brewers’ grain. Generally, the higher the level of substitution of the starchy component (e.g., endosperm) with a predominantly nonstarch component (e.g., bran) the lower the extrudate expansion. However, the reduced expansion of a fiber enhanced product can be compensated somewhat by optimizing extrusion conditions. For example, increasing screw speed from 100 to 300 rpm was reported to reduce bulk density of extruded snack fortified with 10–30% brewers spent grain by an average of 2.5 times (Ainsworth et al. 2007). Also, as observed in baking, fine grinding of bran before incorporation can significantly reduce its negative effect on product expansion. This might be due to reduced ability of fine bran pieces to cut through starch polymers and destroy their network, similar to an effect of bran on the gluten network during baking. Another component of whole grain, bran and spent brewers grain that has a major effect on extrusion, is the lipid. Lipids can form complexes with starch during extrusion (De Pilli et al. 2008), which would affect the ability of starch to reassociate and form junction zones immediately after expansion. Thus, all other factors constant, high lipid content tends to cause a reduced expansion and harder texture in product. The effect of fat is usually much more evident in single screw extrusion than twin screw extrusion. This is because single screw extrusion is more dependent on friction to generate the heat that will cause starch to melt and plasticize. Lipids will tend to produce lubricity that reduces friction and the ability of starch to melt.

Effect of Extrusion on Cereal Antioxidants Data is mixed on how extrusion affects phenolic content and antioxidant activity. Some authors show a decrease (Dlamini, Taylor, and Rooney 2007) while others show an increase or no change (Stojceska et al. 2009) for both phenols and antioxidant activity. Again, the problem here is likely similar to what is mentioned above for baked products; differences in the food matrix in question and types, levels, and extractability of the antioxidants in the raw material will affect what is measured before and after extrusion. This may be illustrated by some experiments that have demonstrated those conditions that promote extrudate expansion (e.g., increased screw speed or reduced moisture) generally result in reduced antioxidant activity in the product (Ozer et al. 2006). This hints at reduced extractability as a major contributor to the change in the antioxidant profile of extruded snacks. Another experiment that demonstrates how types of phenols in raw material may affect the measured effect of extrusion was reported by Awika et al. (2003b). The authors reported that under similar extrusion conditions, whole grain white sorghum extrudate had 18% higher antioxidant activity than its raw material, whereas black (high in 3-deoxyanthocyanins) and tannin sorghums showed decrease in antioxidant activity of 56 and 36% respectively (Table 16.1). Some reports also indicate that extrusion may partly depolymerize some high molecular weight polyphenols like proanthocyanidins into lower MW forms (Awika et al. 2003a). These authors reported that monomers to tetramers of sorghum proanthocyanidins increased whereas the higher MW oligomers and polymers decreased during © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications TABLE 16.1 Effect of Whole Grain Extrusion on Phenol Content and Antioxidant Activity of Different Sorghum Types Sorghum Type

Sample Type

Phenol Content (mg/g, db)

Antioxidant Activity (µmol TE/g, db) ORAC

TEAC

White

Grain Extrudate

0.9 1.2

22 26

Black

Grain Extrudate

6.3 4.7

219 94

57 37

Tannin

Grain Extrudate

13.1 6.1

454 286

108 90

CV%

6.0

6.8

5.6 6.9

3.5

Source: Adapted in part from Awika, J. M., Rooney, L. W., Wu, , X. L., Prior, R. L., and Cisneros-Zevallos, L., J. Agric. Food Chem., 51(23), 6657–62, 2003. Note: ORAC = both lipophilic and hydrophilic oxygen radical absorbance capacity; TEAC = Trolox equivalent antioxidant capacity measured by the ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) method.

the single screw extrusion process. How such change may affect bioactivity of the products remains unknown.

REFERENCES Ainsworth, P., S. Ibanoglu, A. Plunkett, E. Ibanoglu, and V. Stojceska. 2007. Effect of brewers spent grain addition and screw speed on the selected physical and nutritional properties of an extruded snack. Journal of Food Engineering 81 (4): 702–9. Alvarez-Jubete, L., H. Wijngaard, E. K. Arendt, and E. Gallagher. 2010. Polyphenol composition and in vitro antioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and baking. Food Chemistry 119 (2): 770–8. Awika, J. M., L. Dykes, L. W. Gu, L. W. Rooney, and R. L. Prior. 2003a. Processing of sorghum (Sorghum bicolor) and sorghum products alters procyanidin oligomer and polymer distribution and content. Journal of Agricultural and Food Chemistry 51 (18): 5516–21. Awika, J. M., L. W. Rooney, X. L. Wu, R. L. Prior, and L. Cisneros-Zevallos. 2003b. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. Journal of Agricultural and Food Chemistry 51 (23): 6657–62. Bauer, N., P. Koehler, H. Wieser, and P. Schieberle. 2003. Studies on effects of microbial transglutaminase on gluten proteins of wheat. II. Rheological properties. Cereal Chemistry 80 (6): 787–90. Dahle, L. K., and P. R. Murthy. 1970. Some effects of antioxidants in dough systems. Cereal Chemistry 47 (3): 296–303. De Pilli, T., K. Jouppila, J. Ikonen, J. Kansikas, A. Derossi, and C. Severini. 2008. Study on formation of starchlipid complexes during extrusion-cooking of almond flour. Journal of Food Engineering 87 (4): 495–504. Dlamini, N. R., J. R. N. Taylor, and L. W. Rooney. 2007. The effect of sorghum type and processing on the antioxidant properties of African sorghum-based foods. Food Chemistry 105 (4): 1412–9. Koh, B. K., and P. K. W. Ng. 2009. Effects of ferulic acid and transglutaminase on hard wheat flour dough and bread. Cereal Chemistry 86 (1): 18–22. Lai, C. S., R. C. Hoseney, and A. B. Davis. 1989. Effect of wheat bran in breadmaking. Cereal Chemistry 66:217–9. Melville, J., and H. T. Shattock. 1938. The action of ascorbic acid as a bread improver. Cereal Chemistry 15:201–5. © 2011 by Taylor & Francis Group, LLC

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Meredith, P. 1965. Oxidation of ascorbic acid and its improver effect in bread doughs. Journal of the Science of Food and Agriculture 16 (8): 474–80. Moore, J., M. Luther, Z. H. Cheng, and L. L. Yu. 2009. Effects of baking conditions, dough fermentation, and bran particle size on antioxidant properties of whole-wheat pizza crusts. Journal of Agricultural and Food Chemistry 57 (3): 832–9. Moore, M. M., M. Heinbockel, P. Dockery, H. M. Ulmer, and E. K. Arendt. 2006. Network formation in glutenfree bread with application of transglutaminase. Cereal Chemistry 83 (1): 28–36. Nanditha, B. R., B. S. Jena, and P. Prabhasankar. 2009. Influence of natural antioxidants and their carry-through property in biscuit processing. Journal of the Science of Food and Agriculture 89 (2): 288–98. Ozer, E. A., E. N. Herken, S. Guzel, P. Ainsworth, and S. Ibanoglu. 2006. Effect of extrusion process on the antioxidant activity and total phenolics in a nutritious snack food. International Journal of Food Science and Technology 41 (3): 289–93. Sharma, A., B. S. Yadav, and B. Y. Ritika. 2008. Resistant starch: Physiological roles and food applications. Food Reviews International 24 (2): 193–234. Stojceska, V., P. Ainsworth, A. Plunkett, and S. Ibanoglu. 2009. The effect of extrusion cooking using different water feed rates on the quality of ready-to-eat snacks made from food by-products. Food Chemistry 114 (1): 226–32. Wang, R., W. B. Zhou, H. H. Yu, and W. F. Chow. 2006. Effects of green tea extract on the quality of bread made from unfrozen and frozen dough processes. Journal of the Science of Food and Agriculture 86 (6): 857–64. Weak, E. D., R. C. Hoseney, P. A. Seib, and M. Biag. 1977. Mixograph studies. 1. Effect of certain compounds on mixing properties. Cereal Chemistry 54 (4): 794–802. Woo, K. S., C. C. Maningat, and P. A. Seib. 2009. Increasing dietary fiber in foods: The case for phosphorylated cross-linked resistant starch, a highly concentrated form of dietary fiber. Cereal Foods World 54 (5): 217–23.

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17 Impacts of Food and Microbial Processing on the Bioactive Phenolics of Olive Fruit Products Moktar Hamdi CONTENTS Introduction............................................................................................................................................. 347 Olive Fruit Composition and Bioactive Phenolics Content.................................................................... 348 Effect of the Postharvest of Olive Fruit on the Phenolics....................................................................... 350 Effect of the Table Olive Processing on the Bioactive Phenolics............................................................351 Impact of Olive Oil Processing on the Bioactive Phenolics....................................................................353 Conclusions..............................................................................................................................................355 References................................................................................................................................................355

Introduction The olive tree, Olea europea L., is the only species of the Oleacea with edible fruit. Cultivation began in the Mediterranean countries more than 6000 years ago, developed in Andalucia by Arabs and was then introduced to America. In the last decades, cultivations were promoted in Asia, Australia, and South Africa. Among the 1500 olive cultivars catalogued in the world, only approximately 100 are classified as a main cultivar producing varieties and classified according to the use of their fruits: oil extraction, table olive processing, and dual use cultivars. The oleicol olive world heritage counts more than 800 million olive trees that occupy about 8711 ­thousand acres. Of that land, 99% is located in the Mediterranean basin (Luchetti 1993). Olive oil represents the main product of the olive tree, since 91% of harvested olives are destined to be pressed into oil (Luchetti 1999). The Mediterranean area alone provides 98% of the total surface area for olive tree culture and 97% of the total olive production. The largest olive oil producers are Spain, Italy, Greece, Turkey, and Tunisia. A number of olive cultivars are being cultivated in the Mediterranean countries for processing as table olives (IOOC 2000). Some Spanish and Italian cultivars such as Gordal Sevillana, Manzanilla de Sevilla, and Ascolana have been exported to the other countries (including Argentina, Australia, United States, and Israel) to produce table olives. The world production of table olives is estimated to surpass 1.5 million tons per year, with the Mediterranean countries being the main producers. There has been an increased demand for fermented green and black table olives in recent years in all regions of the world because of their nutritional and functional foods proprieties. The International Olive Oil Council statistical data for 1989/1990 and 2000/2001 shows that the production of table olives increased in the majority of countries during the last decade (IOOC 2000). Spain and Turkey are the main producers of green olives and naturally black olives, respectively. The improvement in nutritional value of various plant food commodities, by increasing their content of biologically active polyphenolic and phytochemicals, has become a challenge for scientists and

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technologists. Saija and Uccelle (2000) suggested that an understanding of olive growing and ­processing technologies, and the measurement of extra virgin olive oil and table olives macrobio-, microbio-, and techno-components behavior, after the raw material has been subjected to appropriate harvesting, milling, malaxation, extraction, and debittering treatments, is important in order to communicate the hedonic-sensory quality and functional quality of olive agrifood to consumers. The bioactive phenolic of olive fruit and their concentrations in the olive fruit products depend on the olive cultivars, harvesting, processing, and storage.

Olive Fruit Composition and Bioactive Phenolics Content The cultivar, the region of production, the degree of drupe maturation, and the postharvest conditions determine the nutritional quality, the functional and technological potential of the olive fruit (Figure 17.1). The olive tree gives an oval fruit, which is fleshy green drupe, and consists of a pulp and a stone representing 70–90% of the olive weight and the pit is the other 10–30% (Fernández Diéz et al. 1985; Rejano 1977). The pulp consists mainly of oil (10–25%) and water (60–75%). The oil fraction includes mainly triglycerides, diglycerides, monoglycerides, free fatty acids, sterol esters, terpenes alcohol, and phospholipids. The olive fruit texture is attributed to the presence of fiber fraction (1–4%; Gullen et al. 1992) and the pectic substances (0.3–0.6%; Minguez-Mosquera et al. 2002). Sugars and polyols represent 20% of the fresh pulp weight. The green color of olives is attributed to chlorophylls (1.8–13.5 mg/100 g fresh pulp) and carotenoid pigments (0.6–2.4 mg/100 g fresh pulp; MinguezMasquera and Garrido-Fernandez 1989; Roca and Minguez-Masquera 2001). The variations of concentrations of most nutrients are influenced by the type of cultivar, the growing conditions, and the degree of ripeness. During ripening processes, the ratio between chlorophylls and carotenoids change because the chlorophylls decrease. In the development of the olive fruit, three phases are usually distinguished (Soler-Rivas et al. 2000): a growth phase, during which accumulation of oleuropein occurs; a green maturation phase, coinciding with a reduction in the levels of chlorophyll and oleuropein; and a black maturation phase, characterized by the appearance of anthocyanins and during which the oleuropein levels continue to fall. Saija and Uccelle (2000) summarized the phenolic compound structures that range from quite simple compounds to highly polymerized substances such as the tannins. Their content in olive fruit can vary between 1 and 2% and are represented mainly by the oleuropein. Indeed, the bitter taste of olives is largely ascribed to the content of oleuropein (García et al., 2001; GutiérrezRosales et al., 2003). During maturation, oleuropein is partially converted into demethyloleuropein, which becomes the major phenol in black olives (Romero et al. 2002). The most important changes

FIGURE 17.1  Olive fruit.

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TABLE 17.1 Simple Phenolic Compounds in Olive Flesh Taken at Three Degrees of Ripeness: Green (GO), Varicolored (VO), and Black (BO) in mg/100 g Dwt Phenolic Compounds Gallic acid Protocatechuic acid Hydroxytyrosol Tyrosol p-hydroxy-benzoic acid p-hydroxy-phenyl-acetic acid Vanillic acid Caffeic acid Syringic acid Vanillin p-coumaric acid Ferulic acid m-coumaric acid Benzoic acid o-coumaric acid Oleuropein Total

Fresh GO

Fresh VO

Fresh BO

6.1 ± 0.7 7.2 ± 0.3 256.8 ± 12 7.2 ± 0.4 2.7 ± 0.05 35.9 ± 1.3 3.4 ± 0.1 ND 1.7 ± 0.02 ND 4 ± 0.2 5.4±0.4 ND 55.3 ± 3.5 ND 266 ± 11.2 651.8 ± 30.17

ND 28.1 ± 1.7 165 ± 8.2 5 ± 0.03 ND 41.3 ± 1.9 1.1 ± 0.01 0.5 ± 0 ND 2.7 ± 0.02 1.9 ± 0.01 4.7 ± 0.3 0.7 ± 0.01 19.4 ± 0.2 1.7 ± 0.02 111.8 ± 4.2 384.1 ± 16.6

ND 19.4 ± 0.5 135.2 ± 3.2 2.9 ± 0.02 ND 31.7 ± 1.7 ND ND ND 1.6 ± 0.01 2 ± 0.03 3.8 ± 0.05 2 ± 0.02 54.8 ± 1.5 0.5 ± 0.01 56.9 ± 2.8 311 ± 9.84

Source: From Ben Othman, N., Roblain, D., Thonart, P., and Hamdi, M., J. Food Sci., 73(4), 235–40, 2008.

in the phenolic fraction are due to the depletion and partial conversion of oleuropein during the olive fruit ­development and the concentration increase of tyrosol and hydroxytysol (Ferreira et al. 2002; Piga et al. 2001; Ryan et al. 1999; Servili et al. 2006 ). The major phenolic compounds present in table olives are tyrosol, hydroxytyrosol, and oleanolic acid and the concentration of these compounds is dependent upon the degree of maturation and the method of treatment of olive drupe till they become edible (Blekas et al. 2002; Owen et al. 2003; Romero et al. 2002, 2004). The concentration of simple phenolic compounds change in olive flesh when taken at three degrees of ripeness: green, varicolored, and black (Table 17.1). Green olives due to their higher phenolic content have a higher antioxidant activity; however, oleuropein has a bitter taste and represents 40.8% of simple phenolic compounds (Ben Othman et al. 2008). Table olive products are a principal functional food and the most important components of the Mediterranean diet. The benefit of olive oil has been investigated for many years more than table olives. Olives are an essential source of linoleic acid and monounsaturated fatty acids having a high biological and nutritive value. In addition to monounsaturated fatty acids, polyphenols, chlorophylls, and carotenoids contribute to the nutritional benefits and biological functions of olive products. Olive products contribute to the daily intake of nutritional antioxidants, since they contain an array of polyphenolic phytochemicals, including various hydroxytyrosol derivatives (e.g., oleuropein) and flavone glycosides (Romero et al. 2002; Saija and Uccelle 2002). The consumption of table olives in combination with the consumption of olive oil, provide a large amount of natural antioxidants as compared to the 23 and 28 mg of flavones and flavanones intake per day for the Netherlands and Denmark, respectively, and of 115  mg per day for the United States, as reviewed by Ross and Kasum (2002). Table olives have a similar phenolic profile with polyphenols in different quantities; varying according to type and about 5–10 table olives might cover the daily intake of polyphenols. Recently, Bouskou et al. (2006) mentioned that the consumption of table olives is considered to have a high intake of antioxidants, mainly polyphenols, and so will provide a health benefit for the prevention of many diseases. In fact, the table olives are a good source of antioxidants, mainly polyphenols that protect the body ­tissues against oxidative stress (Bouskou et al. 2006). Moreover, polyphenol intake is beneficial for © 2011 by Taylor & Francis Group, LLC

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human health because their antioxidant activity has been associated with a lower risk of coronary heart disease, some types of cancer, inflammation, and inhibition of platelet-activating factor activity (Boskou and Visioli 2003). The consumption of standardized aqueous olive pulp extract is considered safe at levels up to 20 mg/kg/day. Among the polyphenols found in the extract, the major constituent of biological significance is hydroxytyrosol (50–70%; Soni et al. 2006).

Effect of the Postharvest of Olive Fruit on the Phenolics The harvested mature-green or black olives need to be stored and transported by methods that minimize physical damage, chemical contamination, and microbiological deterioration. A great deal of variation on storability between cultivars has been observed in the same growing area. For example, fresh Chondrolia green olives are very sensitive to chilling injury, and would lose their capacity to develop skin color and ripen after 2–4 weeks of cold storage showing excessive internal browning, resulting in pitting and external discoloration (Nanos et al. 2002). Mature-green olives are chill sensitive when kept long enough at temperatures below 5 C, while the fruit of some cultivars can be damaged at temperatures as high as 10 C (Maxie 1963). In the altered zone of the olive fruit, the surrounding components of pigments were affected. The main chilling injury symptoms of olives include internal browning of the flesh around the pit, pitting appearing as a dull skin color and, progressively, external browning (Kader 1986). The enzymatic and oxidative degradation of the phenols in the olive fruit increases with the harvest period probably as a consequence of the senescence process of the fruit tissue (Oueslati et al. 2009). The storage of the olive fruits induces the increase in the total phenolic compounds of aqueous phase from 492 to 1517 mg of gallic acid/l, and a decrease in the total simple phenols of olive oil from 85.7 to 16.4 mg of pyrogallol/l (Kachouri and Hamdi 2006). The increase of the aqueous phase’s phenols can be explained by the polymerization of simple phenolic compounds by auto-oxidation on contact with the oxygen. In fact, phenolic compounds of olives became blacker during storage because of the autooxidation and subsequent polymerization giving dark colored phenolic compounds (Hamdi 1993). It was reported that the greatest oxidation was observed with oleuropein whose concentration was reduced, in approximately one minute, to a level below 40% of its initial content. The addition of ascorbic acid prevented the oxidation of the hydroxytyrosol and a change in the total concentration of phenols over time. As a result, the solution did not darken and the oxygen consumption was minimal (Segovia-Bravo et al. 2009). Microbial invasion of fruit tissue by bacteria and fungi can occur when storage conditions are favorable. The postharvest handling can produce breaks in the tissue allowing microorganisms to enter and affect the fruit quality and odor. The different resistance of each olive variety to the microorganisms attack could determine important differences in the formation of guaiacol, 4-ethylphenol, and 4-ethylguaiacol during olive fruit storage, and their consequent concentration in virgin olive oils (Vichi et al. 2009). Specific species of microorganisms, Aspergillus, Geotrichum, and Penicillium— which are able to grow in the olive fruit and may cause spoilage and poisoning—are able to degrade phenolic compounds of olives (Garcia Garcia et al. 2000; Hamdi 1993). Whereas, the phenolic compounds have the capacity to inhibit or delay the growth rate of several bacteria and microfungi (Saija and Ucelle 2002). The quality of the table olives and olive oil depend on the skin color and flesh firmness of the raw product at the time of processing. Several studies for the effects of the controlled atmosphere during low temperature storage on olive fruit have been done to improve olive storage and allow for an extension of the processing period using the Spanish method for fresh green olives (Kader 1986). Dourtouglou et al. (2006) showed that postharvest storage of olives under a CO2 atmosphere for a period of 12 days resulted in color and flavor development and reduced bitterness. The gradual loss of bitterness observed during storage under CO2 may be due to oleuropein decomposition. The antioxidant characteristics were lower in olives stored under air than under the CO2 atmosphere, which is an indication that the functional properties of olives may be enhanced after CO2 storage. © 2011 by Taylor & Francis Group, LLC

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Effect of the Table Olive Processing on the Bioactive Phenolics The production of the fermented foods is based on the microbial activity, which induces beneficial changes and produces flavor ingredients that give the final product its distinctive taste. Olives are consumed only after fermentation because it makes the olive more digestible and reduces the bitterness and toxicity of phenols. Fermented olives preserve vast quantities of functional compounds in a wide diversity of flavors, aromas, and phenolics that enrich the human diet. Table olive preparation is mainly conducted by three processing methods: the Greek, the Spanish, or the Californian style (Hamdi 2008). The olive fruits are harvested at different degrees of ripeness: yellowish-green, turning color, and black for Spanish style, Californian style, and Greek style, respectively. The hydrolysis of oleuropein and the polymerization of anthocyanin pigments are the main changes observed in the Greek-style black olives (Romero et al. 2004; Vlahov and Solinas 1993). The harvesting period of green olives is short followed by storage in brine before final processing. Olives destined for Californian and Greek style processing are harvested mature when skin color is not important. Among the lactic acid bacteria of the natural flora, Lb. plantarum is the predominant microorganism for successful green olive fermentation (Adams and Moss 1995; Chammem et al. 2005; Garrido Fernández et al. 1997). The Lb. plantarum population generally coexists with a yeast population until the end of the fermentation process and during storage (Ruiz-Barba et al. 1994; Vaughn 1982). The Greek style method of treatment is mild and includes washing, natural fermentation in brine, airoxidation for color improvement, sizing, and packing. The hydrolysis of oleuropein and the polymerization of anthocyanin pigments are the main changes observed in the Greek-style black olives (Romero et al. 2004; Vlahov and Solinas 1993). The Spanish and Californian style processes include pretreatment with lye, which hydrolyses the bitter glycoside oleuropein and increases the permeability of the olive skin, resulting in the efflux of flesh nutrients into the surrounding liquid (Garrido Fernández et al. 1997). Oleuropein aglycones diminished considerably, while tyrosol and hydroxytyrosol increased markedly (Marsilio et al. 2001). Further investigation for the development of table olive processing that will enable fast olive debittering with minimal environmental impact is required (Dourtoglou et al. 2006). Postharvest storage of olives under a CO2 atmosphere for a period of 12 days resulted in color and flavor development and reduced bitterness to provide a natural debittering without the use of chemicals (e.g., alkaline solutions, brine). The selected strain of Lb. pentosus (1MO) allowed the reduction of the debittering phase period to 8 days (Servili et al. 2006). The naturally black dry salted olives are obtained by the fermentation of olives under high osmotic pressure (40 g/100 g) for 40–60 days (Panagou 2006). Several solutions have been proposed such as the use of starter (such as Lb. plantarum, Lb. pentosus, Enterococcus casseliflavous, and bacteriocin producing strains), addition of sugars, extra salt supplement, and acidification of the brine in order to improve fermentation kinetics and to control the quality of the table olive product (De Castro et al. 2002; Montano et al. 2006; Ruiz-Barba et al. 1994; Sánchez et al. 2001). Inoculation is strongly advisable to control all stages of the fermentation and reduce the risks of microbial alterations. The total number of the lactobacilli in the inoculated fermentors was similar to that in the spontaneous process (Chammem et al. 2005; Ruiz-Barba et al. 1994; Ruiz-Barba and JímenezDíaz 1995). Inoculation with the Lb. plantarum starter culture leads to a faster pH decrease in green table olive processing with respect to the spontaneous one and this may help to reduce the risk of spoilage during the first days of fermentation (Garrido Fernández et al. 1997; Leal-Sánchez et al. 2003). Green olives were fermented also with starter cultures of Enterococcus casseliflavus and Lb. pentosus. This is the first report dealing with the presence of E. casseliflavus in table olive brines and their utilization as the starter culture (De Castro et al. 2002). The temperature-controlled fermentation of Leccino cv. olives resulted in obtaining ready-to-eat, high-quality table olives in a reduced-time process. However, fermented olives showed a decrease of oleuropein and an increase of the hydroxytyrosol concentration (Ben Othman et al. 2009; Servili et al. 2006). All table olive processing results in a decrease of the total amount of phenols (Table 17.2). The phenolic fraction of table olives is very complex and can vary both in the quality and quantity of phenolic

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© 2011 by Taylor & Francis Group, LLC

466.81

88.2

ND

2.99 ± 0.2

ND

ND

ND

2.08 ± 0.02

1.69 ± 0.03

ND

6.5 ± 1.1

ND

14.4 ± 0.02

24.99 ± 0.9

35.55 ± 1.3

ND

ND

Meski M2

355.25

ND

1.29 ± 0.07

ND

ND

ND

1.86 ± 0.02

3.27 ± 0.4

ND

7.98 ± 1.3

ND

20.21 ± 5.2

11.25 ± 1.1

274.8 ± 9.7

34.6 ± 1.6

ND

Meski M3

387.29

ND

ND

ND

ND

ND

ND

2.48 ± 0.8

ND

6.05 ± 1.2

25.04 ± 5.2

30.71 ± 4.6

10.35 ± 1.9

283.2 ± 9.8

29.45 ± 3.8

ND

Meski M4

Source: From Ben Othman, N., Roblain, D., Thonart, P., and Hamdi, M., J. Food Sci., 73(4), 235–40, 2008. Note: ND: not detected. * Results are expressed as mean ± standard deviation of three determinations.

Total

ND

Oleuropein

213 ± 25.2

Benzoic acid

ND

1.35 ± 0.02

m-Coumaric acid

o-Coumaric acid

4.1 ± 0.35

ND

p-Coumaric acid

Ferulic acid

ND

Caffeic acid

Vanillin

4.41 ± 0.2

1.35 ± 0.01

Vanillic acid

ND

5.9 ± 0.1

p-Hydroxy-benzoic acid

p-Hydroxy-phenyl-acetic acid

16.9 ± 2.4

Tyrosol

ND

219.8 ± 3.2

Protocatechuic acid

Hydroxytyrosol

ND

Meski M1

Gallic acid

Phenolic Compound

169.38

ND

ND

ND

1.04 ± 0.05

2.12 ± 0.25

11.02 ± 3.8

ND

2.32 ± 0.01

2.33 ± 0.6

ND

10.61 ± 1.9

56.60 ± 2.7

83.34 ± 5.8

ND

ND

Chemlali CH

48.65

ND

ND

ND

ND

1.43 ± 0.06

3.91 ± 0.04

ND

ND

3.27 ± 0.7

11.5 ± 0.9

ND

28.52 ± 4.7

ND

ND

ND

Besbessi B

Simple Phenolic Compounds in Olive Flesh of the Seven Types of Tunisian Table Olives Expressed in mg per 100 g of Dry Weight

TABLE 17.2

132.13

ND

ND

ND

ND

ND

1.17 ± 0.09

ND

1.21 ± 0.6

5.56 ± 1.7

ND

ND

24.62 ± 6.9

85.78 ± 9.7

13.79 ± 6.6

ND

Tounsi T

352 Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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compounds (Uccella 2001), is dependent upon the processing method (Romero et al. 2004), upon the cultivar (Romani et al. 1999), upon irrigation regimes, and upon the degree of drupe maturation (Patumi et al. 2002). The concentration of phenolic compounds decreased slightly during the fermentation process, particularly hydroxytyrosol, which was found in high concentration in wastewaters (De Castro and Brenes 2002). Whereas, the sterilization step did not change tyrosol and hydroxytyrosol contents of the processed olives (Marsilio et al. 2001). An analysis carried out on 48 samples showed that olives with changed color in brine had the highest concentration in polyphenols (approximately 1200 mg/kg), whereas oxidized olives had the lowest (approximately 200 mg/kg; Romero et al. 2004). Owen et al. (2003) investigated the antioxidant capacity of two Italian brined olive drupe varieties (one black and one green) and showed that black olives, which contain higher concentrations of phenolic compounds, present higher antioxidant activity compared to the green olive extract. The evolution of phenolic compounds has been studied during fermentation of the chétoui cultivar olives that are taken at three degrees of ripeness: green, varicolored, and black (Ben Othman et al. 2009). Both spontaneous and controlled fermentations led to an important loss of total phenolic compounds with a reduction rate of 32–58% and the antioxidant activity decreased 50–72%. During olive fermentation phenolic loss is essentially due to the diffusion of these compounds in brine, the main phenolic compounds identified and quantified in brine was hydroxytyrosol. After fermentations, hydroxytyrosol and caffeic acid concentrations increased, while protocatechuic acid, ferulic acid, and oleuropein concentrations decreased. The hydroxytyrosol concentration in black olives increased from 165 to 312 and 380 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. The oleuropein concentration in green olives decreased from 266 to 30.7 and 16.1 mg/100 g dry weight, respectively, after spontaneous and controlled fermentation. To preserve antioxidant quality of table olives it is necessary to use an innovative process to minimize the phenolic compound loss. The survived probiotic bacterial species should contribute to the preservation of the antioxidative activity of bioactive phenolic compounds. Survival studies in table olives of Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium bifidum, and Bifidobacterium longum, demonstrated that Bifidobacteria and one strain of L. rhamnosus showed a good survival rate at room temperature (Lavermicocca et al. 2005).

Impact of Olive Oil Processing on the Bioactive Phenolics Olive oil is an essential part of human diets, for its nutritional worth and its biological effects on human health. The basic aspect that distinguishes olive oil from other vegetable oils is its high proportion of monounsaturated fatty acid (i.e., oleic acid) and the modest presence of polyunsaturated fatty acids (Delplanque et al. 1999). Moreover, olive oil contains natural antioxidants such as tocopherols, carotenoids, sterols, and phenolic compounds that represent 27% of the unsaponifiable fraction (Boskou 1996). Olive oil is mainly obtained by a three processing extraction influencing its bioactive phenolics concentration (Garcia et al. 1996; Gimeno et al. 2002; Salvador et al. 2003). The main phenols identified in olive oil are gallic, caffeic, vanillic, p-coumaric, syringic, ferulic, homovanillic, p-hydroxybenzoic and protocateuric acids, tyrosol, and hydroxytyrosol (Montedoro et al. 1992). Other phenolic compounds have been identified in olive oil including oleuropein and ligstroside aglycons and the dialdehydic forms of decarboxymethyl oleuropein and ligstroside aglycons (Mateos et al. 2001; Montedoro et al. 1993). Recently 2-(3,4-dihydroxyphenyl) ethyl acetate (hydroxytyrosyl acetate; Brenes et al. 1999; Espartero et al. 1999) and two lignans, pinoresinol and 1-acetoxypinoresinol (Brenes et al. 2000; Owen et al. 2000) have been identified as components of the phenolic fraction in olive oils. The flavones luteolin and apigenin were detected many years ago (Vazquez-Roncero et al. 1976). The phenolic compounds of olive oil have multiple biological effects, including the oxidative stability in extra virgin olive oil during storage. It has been claimed that hydroxytyrosol is the most active antioxidant compound in virgin olive oil (Chimi et al. 1988; Tsimidou et al. 1992). In addition, the phenolic compounds prevent the oxidation of the triglycerides of olive oil during preservation. © 2011 by Taylor & Francis Group, LLC

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Concerning total phenols, Arbequina cultivars produced similar amounts in Tunisia as well as in Spain under a high-density planting system. Whereas, Arbosana cultivated in Tunisia produced lower amounts of total phenols as compared to its original growing area (Allalout et al. 2009). In the arid Tataouine zone, the water shortage tends to generate a stress situation in the olive tree that induces phenol production ranging from a minimum of 290 to a maximum of 907 mg/kg. As with oxidative stability, the total polar phenol contents of Chemlali Tataouine, Fakhari Douirat, and Zarrazi Douirat virgin olive oil were among the highest of all the Tunisian varieties, comparable to that reported for the second main Tunisian Chétoui variety and for some virgin olive oil derived from Spanish varieties (Oueslati et al. 2009). The Chétoui cultivar fruit is medium to large with a fat yield of about 20–30% of fresh weight and the oil is characterized by a good content of total phenols, o-diphenols, tocopherols, and good resistance to oxidation (Ben Temime et al. 2006). The nutritional, biological, and organoleptic value is intimately linked to the quality of the oil and especially to the oil’s content in certain phenols. Unfortunately, phenolic compounds are autoxidized during the harvest, and more important, during the storage of the fruits before triturating. It has been reported that the concentration of phenolic compounds, particularly orthodiphenols, decrease in oil during malaxation (Servili et al. 1999), mainly due to oxidation reactions. The effects of the crushing systems, stone mill, and hammer crusher all have to be taken into account for the chemical changes of virgin olive oil when considering antioxidant compounds (Veillet et al. 2009). The application of Lactobacillus plantarum to the olive fruits increased the simple phenols in extracted virgin olive oil (Table 17.3). This increase could be explained by the depolymerization of phenolic compounds existing in the olive fruits by Lactobacillus plantarum. In fact, Ayed and Hamdi (2003) showed that Lactobacillus plantarum has the capacity to reduce the redox potential and to realize the inverse reaction of auto-oxidation of phenolic compounds to tannins present in olive mill wastewaters (OMW) by reductive depolymerization. Lin and Chang (2000) found that some intestinal lactic acid bacteria (Lactobacillus acidophilus), inhibiting linoleic acid oxidation, revealed significant antioxidative activity. Kullisaar et al. (2000) also found that two Lactobacillus fermentum have a high antioxidative activity. As a regard to the quality parameters of the oils, the acidity is inferior when the olive fruits were inoculated with Lactobacillus plantarum. Oils from inoculated olive fruits tend to have a lower K 232 TABLE 17.3 Effect of the Application of Lactobacillus Plantarum to the Olive Oil Process on the Phenolic Composition (mg/kg) of Virgin Olive Oils Compound Hydroxy-tyrosol Tyrosol Vanillic acid Vanillin p-coumaric acid Hydroxy-tyrosol -AC m-coumaric acid Hydroxy-tyrosol -EDA Tyrosol -AC Pinoresinol 1-Acetoxypinoresinol Luteolin Orthodiphenolsa Non-orthodiphenolsb

Season 2001/2002 Control

Inoculated

Season 2002/2003 Control

3.4 12.0 0.4 0.5 0.2 — 0.3 31.0 — 1.3 3.2 3.5 37.9 17.9

2.1 19.7 0.7 0.8 0.4 — 0.5 56.0 — 5.8 4.5 10.1 68.2 32.4

3.6 7.4 0.2 0.1 0.1 1.8 0.0 12.3 0.8 1.9 3.4 7.9 25.6 13.9

Inoculated 3.0 10.9 0.4 0.5 0.2 0.1 0.3 34.9 1.6 3.8 3.4 4.5 42.5 21.1

Source: From Kachouri, F. and Hamdi, M., J. Food Eng., 77(3), 746–52, 2006. Sum of Hy, Hy-AC, Hy-EDA and luteolin. b Sum of Ty, vanillic acid, vanillin, p-coumaric, m-coumaric, Ty-AC, 1-Acetoxypinoresinol and pinoresinol. a

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and K270 value than oils from uninoculated olive fruits. This difference may be due to the capacity of Lactobacillus plantarum to inhibit the linoleic acid oxidation. It has been shown that Lactobacillus acidophilus and Bifidobacterium longum have an inhibitory effect on linoleic acid peroxidation (Lin and Yen 1999). The average sensory attributes—fruity, bitterness, rancissement, and piquant—showed that the application of Lactobacillus plantarum has significant statistical differences between oils extracted from inoculated and uninoculated olive fruits (Kachouri and Hamdi 2006). Phenolic compounds found in virgin olive oils responsible for stability come from olive fruit during the extraction process, but a large part of these compounds is lost in the OMW. In the Mediterranean area, the oil manufacturing process produces millions of tons per year of OMW, which cause considerable pollution to the environment (Hamdi 1993; Niaounakis and Halvadakis 2004). Incubation of olive oil samples with fermented OMW by L. plantarum caused the decrease of polyphenols in OMW and their increase in oil with multiple biological effects (Kachouri and Hamdi 2004). The lower total phenolic content in fermented OMW of 845 mg/l in comparison to OMW control with 1247 mg/l resulted from the depolymerization of phenolic compounds of high molecular weight by L. plantarum. Fermentation with L. plantarum induced reductive depolymerization of phenolic compounds of OMW (Ayed and Hamdi 2003), which are more soluble in olive oil. The total simple polyphenol content of olive oil mixed with OMW and fermented by L. plantarum was higher (703 mg/l) than an olive oil control mixed with OMW (112 mg/l). Simple polyphenol content was increased in olive oil when L. plantarum was added to OMW, especially for oleuropein, p-hydroxyphenylacetic, vanillic and ferulic acids, and tyrosol. The effect with the addition of individual phenolic compounds and OMW extract to refined olive and husk oils showed that 3,4-dihydroxyphenyl acetic acid, hydroxytyrosol, and the OMW extract possess useful antioxidant properties and may be used as alternatives in the search for a natural replacement of synthetic antioxidant food additives (Fki et al. 2005). In fact, natural antioxidants extracted from OMW are highly effective for oxidative stabilization of lard and can be considered as a novel food additive for human health benefits (De Leonardis et al. 2007).

Conclusions The bioactive phenolic concentrations are higher in the table olive than the olive oil (Tables 17.2 and 17.3). The improvement of the bioactive phenolic content of the table olive and the olive oil requires a more innovative approach and technology to control olive harvesting, processing, storage, and by-products reuse. The depolymerization of phenolic compounds and their conversion by Lb. plantarum (Ayed and Hamdi 2003; Kachouri and Hamdi 2004, 2006; Lin and Yen 1999) should be an interesting way to improve the functional proprieties of bioactive phenolic olives and its derivative products. In fact, it has been found that the higher the molar mass of tannin molecules is, the stronger the antinutritional effects and the lower the biological activities are (Chung et al. 1998). The management commitment, proper personal and process hygiene should be improved in order to avoid undesirable contamination of the olives products with the establishment of a good manufacturing practice (GMP).

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Montedoro, G., Servili, M., Baldioli, M., and Miniati, E. 1992. Simple and hydrolysable phenolic compounds in virgin olive oil. 1. Their extraction, separation, and quantitative and semiquantitative evaluation by HPLC. J. Agric. Food Chem. 40:1571–6. Montedoro, G., Servili, M., Baldioli, R., Selvaggini, E., Miniati, E., and Macchioni, A. 1993. Simple and hydrolyzable compounds in virgin olive oil. Spectroscopic characterization of the secoiridoids derivatives. J. Agric. Food Chem. 41:2228–34. Nanos, G. D., Kiritsakis, A. K., and Sfakiotakis, E. M. 2002. Preprocessing storage conditions for green ‘Conservolea’ and ‘Chondrolia’ table olive Postharvest. Biol. Technol. 25 (1): 109–15. Niaounakis, M., and Halvadakis, C. P. 2004. Olive Mill Waste Management. Literature Review and Patent Survey. Athens, Greece: Typothito–George Dardanos. Oueslati, I., Anniva, C., Daoud, D., Tsimidou, M. Z., and Zarrouk, M. 2009. Virgin olive oil (VOO) production in Tunisia: The commercial potential of the major olive varieties from the arid Tataouine zone. Food Chem. 112:733–41. Owen, R. W., Haubner, R., Mier, W., Giacosa, A., Hull W. E., and Spiegelhalder, B. 2003. Isolation, structure, elucidation and antioxidant potential of the major phenolic and flavonoid compounds in brined olive drupes. Food Chem. Toxico. 41:703–17. Owen, R. W., Mier, W., Giacosa, A., Hull, W. E., Speigelhalder, B., and Bartsch, H. 2000. Identification of lignans as major components in the phenolic fraction of olive oil. Clin. Chem 46:976–88. Panagou, E. Z. 2006. Greek dry-salted olives: Monitoring the dry-salting process and subsequent physicochemical and microbiological profile during storage under different packing conditions at 4 and 20°C. LWT—Food Sci. Technol. 39 (4): 323–30. Patumi, M., d’Andria, R., Marsilio, V., Fontanazza, G., Morelli, G., and Lanza, B. 2002. Olive and olive oil quality after intensive monoclone olive growing (Olea europaea L., cv. Kalamata) in different irrigation regimes. Food Chem. 77:27–34. Piga, A., Gambella, F., Vacca, V., and Agabbio, M. 2001. Response of three Sardinian olive cultivars to Greekstyle processing. Ital. J. Food Sci. 13:29–40. Rejano, L. 1977. El aderezo de lasaceitunas in El cultivo delolivio, eds. D. Barranco, R. Fernandez-Escolar, and L. Rallo, 565–86. Madrid: Junta de Andaluciaand Ediciones Mundi-Prensa. Roca, M., and Minguez-Masquera, M. I. 2001. Changes in chloroplast pigments of olive varieties during fruit ripening. J. Agri. Food Chem. 49:832–9. Romani, A., Mulinacci, N., Pinelli, P., Vincieri, F. F., and Cimato, A. 1999. Polyphenolic content in five Tuscany cultivar of Olea europaea L. J. Agric. Food Chem. 47:964–7. Romero, C., Brenes, M., Yousfi, K., Garcia, P., Garcia, A., and Garrido, A. 2004. Effect of cultivar and processing method on the contents of polyphenols in table olives. J. Agric. Food Chem. 52 (3): 479–84. Romero, C., Garcia, P., Brenes, M., Garcia, A., and Garrido, A. 2002. Phenolic compounds in natural black Spanish olive varieties. Euro. J. Lipid Sci. and Technol. 215:489–96. Ross, J. A., and Kasum, C. M. 2002. Dietary flavonoids: Bioavailability, metabolic effects, and safety. Annual Reviews Nutrition 22:19–34. Ruiz-Barba, J., Cathcart, L., Warner, D. P., and Jímenez-Díaz, R. 1994. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture of Spanish-style green olive fermentations. Appl. Environ. Microbiol. 60:2059–64. Ruiz-Barba, J. L., and Jímenez-Díaz, R. 1995. Availability of essential B-group vitamins to Lactobacillus plantarum in green olive fermentations brines. Appl. Environ. Microbiol. 61:1294–7. Ryan, D., Robards, K., and Lavee, S. 1999. Changes in phenolic content of olive during maturation. International J. Food Sci. Technol. 34:265–74. Saija, A., and Uccella, N. 2000. Olive biophenols: Functional effects on human wellbeing. Trends in Food Sci. Technol. 11:357–363. Salvador, M. D., Aranda, F., Gómez–Alonso, S., and Fregapane, G. 2003. Influence of extraction system, production year and area on Cornicabra virgin olive oil: A study of five crop seasons. Food Chem. 80:359–66. Sánchez, A. H., Rejano, L., Montaño, A., and de Castro, A. 2001. Utilization at high pH of starter cultures of lactobacilli for Spanish-style green olive fermentation. Int. J. Food Microbiol. 67:115–22. Segovia-Bravo, K. A., Jarén-Galan, M., Garcia-Garcia, P., and Garido-Fernandez, A. 2009. Browning reactions in olives: Mechanism and polyphenols involved. Food Chem. 114:1380–5.

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Servili, M., Baldioli, M., Maiotti, F., and Montedoro, G. F. 1999. Phenolic composition of olive fruit and virgin olive oil: Distribution in the constitutive parts of fruit and evolution during oil mechanical extraction process. Acta Horticulturae 474:609–13. Servili, M., Settanni, L., Veneziani, G., Esposto, S., Massitti, O., Taticchi, A., Urbani, S., Montedoro, G. F., and Corsetti, A. 2006. The use of Lactobacillus pentosus 1MO to shorten the debittering process time of black table olives (Cv. Itrana and Leccino): A pilot-scale application. J. Agric. Food Chem. 54 (11): 3869–75. Soler-Rivas, J. C., Espín, H., and Wichers, H. J. 2000. Oleuropein and related compounds. J. Sci. Food Agric. 80:1013–23. Soni, M. G., Burdock, G. A, Christian, M. S, Bitler, C. M., and Crea, R. 2006. Safety assessment of ­aqueous olive pulp extract as an antioxidant or antimicrobial agent in foods. Food Chem. Toxicol. 44 (7): 903–15. Tsimidou, M., Papadopoulos, G., and Boskou, D. 1992. Phenolic compounds and stability of virgin olive oil— Part I. Food Chemistry 45:141–4. Uccella, N. 2001. Olive biophenols: Novel ethnic and technological approach. Trends Food Science Technol. 11:328–39. Vaughn, R. H. 1982. The fermentation of olives. In Industrial Microbiology. 4th ed., ed. G. Reed, 206–36. Westport, CT: AVI. Vazquez-Roncero, A. Janer, C., and Janer, M. L. 1976. Phenolic components in olive fruits. III. Polyphenols in olive oil. Grasas Aceites 27:185–91. Veillet, S., Tomao, V., Bornard, I., Ruiz, K., and Chemat, F. 2009. Chemical changes in virgin olive oils as a function of crushing systems: Stone mill and hammer crusher. C.R. Chimie 12:895–904. Vichi, S., Romero, A., Gallardo-Chacon, J., Tous, J., Lopez-Tamames, E., and Buxaderas, S. 2009. Volatile phenols in virgin olive oils: Influence of olive variety on their formation during fruits storage. Food Chemistry 116:651–6. Vlahov, G., and Solinas, M. 1993. Anthocyanins polymerisation in black table olives. Agric. Med. 123:7–11.

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18 Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals Reşat Apak, Esma Tütem, Mustafa Özyürek, and Kubilay Güçlü CONTENTS Introduction..............................................................................................................................................361 Definitions of Oxidative Stress, Antioxidant, and Prooxidant Terms..................................................... 362 Primary (Chain Breaking) Antioxidants............................................................................................ 363 Secondary Antioxidants..................................................................................................................... 363 Total Antioxidant Capacity (TAC) Assays Applied to Phenolics in Fruits and Cereals......................... 364 HAT-Based Assays............................................................................................................................. 364 ET-Based Assays................................................................................................................................ 365 Original CUPRAC (Cupric Ion Reducing Antioxidant Capacity) Method........................................ 368 Some Modifications of the CUPRAC Method.............................................................................. 369 Other Antioxidant Activity Tests........................................................................................................ 372 Antioxidant Capacities of Regularly Consumed Fruits.......................................................................... 372 Antioxidant Capacities of Regularly Consumed Cereals........................................................................ 376 The Basic Difficulties Encountered in the TAC Assays of Cereals................................................... 376 The Contribution of Antioxidants Bound to insoluble Fractions and of High Molecular-Weight Polyphenols to the Measured TAC....................................................376 Possible Losses of Antioxidants During Various Treatments of Cereals such as Heat- and Physical-Processing and Alkaline Hydrolysis................................................. 376 Summary of TAC Measurements in Individual Cereal Samples....................................................... 377 References............................................................................................................................................... 380

Introduction Plant polyphenols are aromatic hydroxylated compounds, commonly found in vegetables, fruits, and many food sources that form a significant portion of our diet, and are among the most potent and therapeutically useful bioactive substances. Phenolic derivatives represent the largest group known as “secondary plant products” synthesized by higher plants probably as a result of antioxidative strategies adapted in evolution by respirative organisms starting from the precursors of cyanobacteria. Many of the phenolic compounds are essential to plant life, for example, by providing a defense against microbial attacks and by making food unpalatable to herbivorous predators (Bennick 2002). Over eight thousand naturally occurring phenolic compounds are known (Balasundram et al. 2006). These substances contain at least one aromatic ring with one or more attached –OH groups in addition to other substituents (Bennick 2002), and can be divided into 15 major structural classes (Harborne and Simmonds 1964). The major classes of plant phenolics with “the type of carbon skeleton, class name (example)” format include: C6, simple phenols (resorcinol); C6 –C1, phenolic acids (p-hydroxy­benzoic acid); C6 –C2, acetophenones and phenylacetic acids; C6 –C3, hydroxycinnamic acids (caffeic acid); C6 –C4, hydroxyanthraquinones (physcion); C6 –C2–C6, stilbenes (resveratrol); C6 –C3–C6, flavonoids © 2011 by Taylor & Francis Group, LLC

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TABLE 18.1 Antioxidant Composition of Different Types of Cereals Cereals

Antioxidant Compounds

Wheat

Vanillic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, p-coumaric acid, caffeic acid, sinapic acid, tocols (β-tocopherol, and α-tocopherol), lysophosphatidylcholine Choline, betain p-coumaric acid, syringic acid, vanillic acid, protocatechuic acid, caffeic acid, sinapic acid, α-tocopherol Vitamin E, γ-oryzanol (Gamma oryzanol is a mixture of substances derived from rice bran oil, including sterols and ferulic acid), tocols (γ-tocotrienols, γ-tocopherol, and α-tocopherol), phosphatidylcholine, sterols (β-sitosterol) similar cysteine and methionine Cyanidin-3-glucoside, peonidin-3-glucoside Phytic acid, avenanthramides (alkoloids containing phenolic groups), tocols (α-tocotrienols, and α-tocopherol), phenolic acids (vanillic acid, and p-hydroxybenzoic acid), phosphatidylcholine, similar cysteine, methionine, phytic acid Benzoic and cinnamic acid derivatives (ferulic acid), proanthocyanidins, quinines, flavonols, chalcones, flavones, flavanones, amino phenolic compounds, similar cysteine and methionine Isoferulic acid, coumaric acid, syringic acid, p-hydroxybenzoic acid, caffeic acid, sinapic acid, dimer 8-O-4-di ferulic acid, phosphatidylinositol, tocols (β-tocopherol, and α-tocopherol), similar cysteine and methionine Tannins, anthocyanins (apigeninidin, luteolinidin), apigenin, luteolin, vanillic acid, p-hydroxybenzoic acid, naringenin, carotenoids (lutein, zeaxanthin, β-carotene), α-tocopherol, lysophospholipid Flavones (C-glycosylvitexin, vitexin, and glycosylorientin), tocols (α-tocotrienols, and α-tocopherol), lysophosphatidylcholine, and phosphatidylcholine

Toasted wheat Corn Rice

Black rice Oat

Barley

Rye

Sorghum

Millet

Major Component Ferulic acid

Ferulic acid trans-Ferulic acid

Ferulic acid and caffeic acid

Ferulic acid and p-coumaric acid Ferulic acid

p-coumaric acid and ferulic acid Ferulic acid , p-coumaric acid, cinnamic acid and gentisic acid

Source: White, P. J. and Xing, Y., Natural Antioxidants: Chemistry Health Effects, and Applications, 25–63, Champaign, IL: AOCS Press, 1997.

(quercetin); (C6 –C3)2, lignans (matairesinol); (C6 –C3–C6)2, biflavonoids (agathisflavone); (C6 –C3)n, lignins; (C6 –C3–C6)n, condensed tannins (procyanidin) (Harborne and Simmonds 1964). Fruits and vegetables are usually mentioned as primary sources of phenolic compounds in food but different cereals may be a good source of phenolic compounds as well. The cereals of primary economic and nutritional importance in developed countries include wheat, rye, barley, oat and rice, whereas corn, millet, and sorghum (that are more consumed in developing countries) are consumed much less (Stratil et al. 2007). Whole grain cereals contain a much wider range of compounds with potential antioxidant effects than do refined cereals (Table 18.1). These include vitamin E (mainly in the germ), folates, minerals (iron, zinc), trace elements (selenium, copper, and manganese), carotenoids, phytic acid, lignin and other compounds such as betaine, choline, sulfur amino acids, alkylresorcinols, and lignans found mainly in the bran fraction. Some, such as vitamin E, are considered to be direct free radical scavengers, while others act as cofactors of antioxidant enzymes (selenium, manganese, and zinc), or indirect antioxidants (folates, choline, and betaine). Whole-grain cereals are a major source of polyphenols, especially phenolic acids such as ferulic, vanillic, caffeic, syringic, sinapic, and p-coumaric acids (Fardet et al. 2008).

Definitions of Oxidative Stress, Antioxidant, and Prooxidant Terms Halliwell’s perception of oxidative stress is somewhat vague, and defines it as “the biomolecular damage that can be caused by direct attack of reactive species” (Halliwell and Whiteman 2004). Oxidative stress © 2011 by Taylor & Francis Group, LLC

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is caused by an imbalance between the production of reactive oxygen species (ROS; including hydroxyl and superoxide anion radicals, hydrogen peroxide, and singlet oxygen) and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage to all components of the cell, including biological macromolecules like proteins, lipids, and DNA (Halliwell 2007). Oxidative stress, as defined by Sies (1985, 1986), is a serious imbalance between oxidation and antioxidants, “a disturbance in the prooxidant–antioxidant balance in favor of the former, leading to potential damage.” An antioxidant may be defined as “any substance that when present at low concentrations, compared with those of the oxidizable substrate, significantly delays or inhibits oxidation of that substrate” (Gutteridge 1994). Thus antioxidants are health-beneficial compounds that may prevent chronic diseases resulting from oxidative stress. For convenience, antioxidants have been traditionally divided into two classes; primary or chain-breaking antioxidants and secondary or preventative antioxidants (Madhavi et al. 1996). On the other hand, prooxidants are chemicals that induce oxidative stress, either through creating ROS or inhibiting antioxidant systems (Puglia and Powell 1984).

Primary (Chain Breaking) Antioxidants Chain-breaking mechanisms are represented by:

L• + AH → LH + A•

(18.1)



LO• + AH → LOH + A•

(18.2)



LOO• + AH → LOOH + A•

(18.3)

Thus radical initiation (by reacting with a lipid radical: L•) or propagation (by reacting with alkoxyl: LO• or peroxyl: LOO• radicals) steps are inhibited by the antioxidant: AH.

Secondary Antioxidants Secondary (preventive) antioxidants retard the rate of oxidation. For example, metal chelators (e.g., ironsequesterants) may inhibit Fenton-type reactions (represented by Equation 18.4) that produce hydroxyl radicals (Ames et al. 1993): Fe2+  + H2O2 → Fe3+  + •OH + OH– (18.4) • •– • One important function of antioxidants toward free radicals such as OH, O 2 , and ROO is to suppress free radical-mediated oxidation by inhibiting the formation of free radicals and/or by scavenging radicals. The formation of free radicals may be inhibited by reducing hydroperoxides and hydrogen peroxide and by sequestering metal ions (Niki 2002) through complexation/chelation reactions. Radical scavenging action is dependent on both the reactivity and concentration of the antioxidant. In a multiphase medium (such as an emulsion), the localization of the antioxidant at the interphases may be important. The evaluation of antioxidant activity is complicated by the prooxidative effect of antioxidants in the presence of unsequestered metal ions such as iron and copper. The lower oxidation states of these metals [i.e., Fe(II) and Cu(I)] should not be present at significant levels in tests measuring antioxidant status so as not to initiate Fenton-type reactions exemplified in Equation 18.4. The prooxidative effect of phenolic antioxidants (ArOH), generally induced by transition metal ions like Cu(II) in the presence of dissolved oxygen, gives rise to oxidative damage to lipids, and can be demonstrated by the following reactions (Huang et al. 2005):



Cu(II) + ArOH → Cu(I) + ArO• + H + 

(18.5)



ArO• + LH → ArOH + L•

(18.6)



L• + O2 → LOO•

(18.7)



LOO• + LH → LOOH + L•

(18.8)



Cu(I) + LOOH → Cu(II) + LO• + OH–

(18.9)

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A reducing agent may even be a prooxidant if it reduces oxygen to free radicals or converts transition metal ions to lower oxidation states that may give rise to Fenton-type reactions (Halliwell and Whiteman 2004). Currently, prooxidant activity assay methods are by no means adequate, as they are primarily based on the measurement of reducibility of transition metal ion-complexes that give rise to reactive species. The prooxidant activity of flavonoids is generally accepted to be concentration-dependent, and both the antioxidant and the copper-initiated prooxidant activities of a flavonoid depend on the number and position of –OH substituents in its backbone structure (Cao et al. 1997). Flavones and flavanones, which have no –OH substituents, showed neither antioxidant nor Cu-initiated prooxidant activities in the automated ORAC assays set for the purpose (Cao et al. 1997). It was also observed that Cu(II)-induced prooxidant activity of Ar–OH proceeds via intra- and intermolecular electron transfer reactions accompanying ROS formation, and copper complexation followed by oxidation of resveratrol analogues (e.g., 3,4-dihyroxystilbene) ending up with quinone (Ar = O) products (Zheng et al. 2006).

Total Antioxidant Capacity (TAC) Assays Applied to Phenolics in Fruits and Cereals The chemical diversity of phenolic antioxidants makes it difficult to separate and quantify individual antioxidants (i.e., parent compounds, glycosides, and many isomers) from the plant-based food matrix. Moreover, the total antioxidant power as an “integrated parameter of antioxidants present in a complex sample” (Ghiselli et al. 2000) is often more meaningful to evaluate health beneficial effects because of the cooperative action of antioxidants. Therefore it is desirable to establish and standardize methods that can measure the total antioxidant capacity (TAC) level directly from plant-based food extracts containing phenolics. By means of standardized tests for TAC, the antioxidant values of foods, pharmaceuticals, and other commercial products can be meaningfully compared, and variations within or between products can be controlled. By considering the changes in TAC values of human serum measured by standardized methods, one can detect diseases and monitor the course of medical treatments. For the sake of simplicity, only spectrophotometric or fluorometric assays using molecular probes (i.e., UV-Vis absorbing or fluorescent probes) will be discussed in this work. Due to complexity and limitations of directly following reaction kinetics of the inhibited autoxidation of lipids, molecular spectrometric assays that may or may not apply a suitable radical, but without a chain-propagation step as in lipid autoxidation will be discussed. Antioxidant capacity assays may be broadly classified as ET (electron transfer)-based assays and HAT (hydrogen atom transfer)-based assays (Huang et al. 2005; Prior et al. 2005), though in some cases, these two mechanisms may not be differentiated with distinct boundaries. In fact, most nonenzymatic antioxidant activity (e.g., scavenging of free radicals, inhibition of lipid peroxidation, etc.) is mediated by redox reactions (Pulido et al. 2000). In addition to these two basic classes considering mechanism, ROS scavenging assays will also be taken into account.

HAT-Based Assays The HAT-based assays measure the capability of an antioxidant to quench free radicals (generally peroxyl radicals) by H-atom donation (Table 18.2). The HAT mechanisms of antioxidant action in which the hydrogen atom of a phenol (Ar–OH) is transferred to an ROO• radical can be summarized by the reaction:

ROO• + AH/ArOH → ROOH + A•/ArO•,

(18.10)

where the aryloxy radical (ArO•) formed from the reaction of antioxidant phenol with peroxyl radical is stabilized by resonance. The AH and ArOH species denote the protected biomolecules and phenolic antioxidants, respectively. Effective phenolic antioxidants need to react faster than biomolecules with free radicals to protect the latter from oxidation. Since in HAT-based antioxidant assays, both the © 2011 by Taylor & Francis Group, LLC

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Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals TABLE 18.2 HAT-Based Antioxidant Capacity Methods and Basic Principles Method

Principle

ORAC (oxygen radical absorbance capacity) assay

TRAP (total radical trapping antioxidant parameter) PCL (photochemiluminescence) assay β-carotene/linoleate system Crocin based assay

Calculating the net protection area under the time recorded fluorescence decay curve of red-phycoerythrin or β-phycoerythrin Measuring the consumed oxygen Measurement of chemiluminiscence of luminol radical Measurement of bleaching of β-carotene Measurement of bleaching of crocin

fluorescent probe and antioxidants react with ROO•, the antioxidant activity can be determined from competition kinetics by measuring the fluorescence decay curve of the probe in the absence and presence of antioxidants, and integrating the area under these curves (Huang et al. 2005; Prior et al. 2005). HAT-based assays include oxygen radical absorbance capacity (ORAC) assay (Cao et al. 1995), total peroxyl radical-trapping antioxidant parameter (TRAP) assay using R-phycoerythrin as the fluorescent probe developed by Wayner et al. (1985) and further developed by Ghiselli et al. (1995, 2000), Crocin bleaching assay using AAPH as the radical generator (Bors et al. 1984), and β-carotene bleaching assay (Burda and Oleszek 2001), although the latter bleaches not only by peroxyl radical attack but by multiple pathways (Prior et al. 2005). In general, HAT reactions may be considered to be relatively independent from solvent- and pHeffects, and are completed in a short time (at the order of sec-min) as opposed to ET-based assays. On the other hand, the ET mechanism of antioxidant action is based on the reaction:

ROO• + AH/ArOH → ROO – + AH•+ /ArOH•+

(18.11)



AH•+ / ArOH•+ + H2O ↔ A• / ArO• + H3O +

(18.12)



ROO  + H3O  ↔ ROOH + H2O

(18.13)

–

+

where the reactions are relatively slower than those of HAT-based assays, and are solvent- and pHdependent. The aryloxy radical (ArO•) is subsequently oxidized to the corresponding quinone (Ar = O). The more stabilized the aryloxy radical is, the easier the oxidation will be from ArOH to Ar = O due to the reduced redox potential. Oxygen radical absorbance capacity (ORAC) assay (Cao et al. 1995) applies a competitive reaction scheme in which antioxidant and substrate kinetically compete for thermally generated peroxyl radicals through the decomposition of azo compounds such as ABAP (2,2′-azobis(2-aminopropane) dihydro­ chloride) (Huang et al. 2005; Prior et al. 2005). The net area under curve (AUC), found by subtracting the AUC of blank from that of antioxidant-containing sample (the fluorescence decay of which is retarded), is an indication of the total antioxidant concentration of the sample in the ORAC method. The fluorescent probes used in the ORAC assay were initially β-phycoerythrin (Cao et al. 1993; Ghiselli et al. 1995; Glazer 1990), and later fluorescein (Ou et al. 2001), though TAC results obtained with the latter probe are much higher than those reported with the former. The ORAC measures the inhibition of peroxyl radical induced oxidations by antioxidants and thus reflects classical radical chain-breaking antioxidant activity by H-atom transfer (Ou et al. 2001; Prior et al. 2005). The reaction was reported to go to completion so that both inhibition time and inhibition degree are considered in the quantification of antioxidants (Cao et al. 1995).

ET-Based Assays In most ET-based assays, the antioxidant action is simulated with a suitable redox-potential probe; that is, the antioxidants react with a fluorescent or colored probe (oxidizing agent) instead of peroxyl radicals. Spectrophotometric ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced (Table 18.3). The degree of color change (either an increase or © 2011 by Taylor & Francis Group, LLC

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TABLE 18.3 ET-Based Antioxidant Capacity Methods and Basic Principles Method

Basic Principle

DPPH (2,2- diphenyl-1-picrylhydrazyl) assay TEAC (Trolox equivalent antioxidant capacity)/ABTS [2,2′-azinobis-(3ethylbenzothiazoline-6-sulphonic acid)] assay FRAP (Ferric reducing ability of plasma) assay Folin method CUPRAC (Cupric ion reducing antioxidant capacity) method

Evaluation of scavenging activity of antioxidants by measurement of change in absorbance at 515–517 nm Measurement of inhibition of the absorbance of ABTS•+ radical cation by antioxidants at 415 nm Measurement of blue color of reduced [Fe2+-TPTZ tripyridyltriazine] at 593 nm at low pH Measurement of reduction of Mo(VI) to Mo(V) Measurement of orange-yellow color of reduced [Cu+-Neocuproine] at 450 nm at pH 7

decrease of absorbance at a given wavelength) is correlated to the concentration of antioxidants in the sample. ABTS/TEAC (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid/trolox-equivalent antioxidant capacity) (Miller et al. 1993; Re et al. 1999) and DPPH (2,2-diphenyl-1-picrylhydrazyl) (Bondet et al. 1997; Brand-Williams et al. 1995; Sánchez-Moreno et al. 1998) are decolorization assays, whereas in Folin total phenols assay (Folin and Ciocalteu 1927; Singleton et al. 1999), FRAP (ferric reducing antioxidant power) (Benzie and Strain 1996; Benzie and Szeto 1999), and CUPRAC (cupric ion reducing antioxidant capacity) (Apak et al. 2004, 2005) there is an increase in absorbance at a prespecified wavelength as the antioxidant reacts with the chromogenic reagent (i.e., in the latter two methods, the lower valencies of iron and copper, namely Fe(II) and Cu(I), form charge-transfer complexes with the ligands, respectively). The basic chromophores used in Folin, ABTS/TEAC, FRAP, ferricyanide, ferric-phenanthroline, DPPH, and CUPRAC assays are shown in Figure 18.1. There is no visible chromophore in the Ce4 + -reducing antioxidant capacity assay developed recently by Özyurt et al. (2007), as the remaining Ce(IV) in dilute sulfuric acid solution after polyphenol oxidation under carefully controlled conditions was measured at 320 nm (i.e., in the UV region of the electromagnetic spectrum). These assays generally set a fixed time for the concerned redox reaction, and measure thermodynamic conversion (oxidation) during that period. ET-based assays, namely ABTS/TEAC, DPPH, Folin–Ciocalteu (FCR), FRAP, ferricyanide, and CUPRAC (though ABTS/TEAC, DPPH are considered as mixed HAT–ET-based assays by some researchers) use different chromogenic redox reagents with different standard potentials. Although the reducing capacity of a sample is not directly related to its radical scavenging capability, it is a very important parameter of antioxidants. The reaction equations of various ET-based assays can be summarized as follows:

Folin: Mo(VI) (yellow) + e – (from AH) → Mo(V) (blue)

(18.14)

(λmax = 765 nm) where the oxidizing reagent is a molybdophosphotungstic heteropolyacid comprised of 3 H2O – P2O5 – 13 WO3 – 5 MoO3 – 10 H2O, in which the hypothesized active center is Mo(VI).

FRAP: Fe(TPTZ)23+  + ArOH → Fe(TPTZ)22+  + ArO• + H+ 

(18.15)

(λmax = 595 nm) where TPTZ: 2,4,6-tripyridyl-s-triazine ligand.

Ferricyanide/Prussian Blue: Fe(CN)63– + ArOH → Fe(CN)64– + ArO• + H+

(18.16)



Fe(CN)64– + Fe 3+  + K+ → KFe[Fe(CN)6] (λmax = 700 nm)

(18.17)



ABTS/TEAC: ABTS + K2S2O8 → ABTS•+ (λmax = 734 nm)

(18.18)



ABTS•+ + ArOH → ABTS + ArO• + H+

(18.19)

where ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and TEAC is Trolox-equivalent antioxidant capacity (also the name of the assay). Although other wavelengths such as 415 and 645 nm © 2011 by Taylor & Francis Group, LLC

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N N

N N

N

N

N

Fe (II)

N N

Fe(II)/3

N

Tris(1,10-phenanthroline) iron(II)

N

N N

N

FRAP: [Fe(II)(TPTZ)2]2+

(Ferrous tripyridyltriazine cation)

O2N N

NO2

N

N

O2N

N

H3C

DPPH radical

CH3

Cu(I)/2

CUPRAC: Bis(neocuproine)copper(I) chelate cation + HO3S

3H2O-P2O5-13WO3-5MoO3-10H2O

S

S N

SO3H

N

N

N

Et Et ABTS•+ radical cation

Folin reagent

FIGURE 18.1  Basic chromophores used in TAC (total antioxidant capacity) assays.

have been used in the ABTS assay (Prior et al. 2005), the 734 nm peak wavelength has been predominantly preferred due to less interference from plant pigments.

DPPH: DPPH• + ArOH → DPPH + ArO• + H+ 

(18.20)

(λmax = 515 nm), where DPPH•. is the 2,2-diphenyl-1-picrylhydrazyl stable radical.

CUPRAC: 2 n Cu(Nc)22+  + Ar(OH)n → 2 n Cu(Nc)2+  + Ar( = O)n + 2 n H + 

(18.21)

(λmax = 450 nm), where the polyphenol with suitably situated Ar–OH groups is oxidized to the corresponding quinone, and the reduction product [i.e., bis(neocuproine)copper(I) chelate] shows absorption maximum at 450 nm. It should be noted that not all phenolic –OH are reduced to the corresponding quinones, and the efficiency of this reduction depends on the number and position of the phenolic –OH groups as well as on the overall conjugation level of the polyphenolic molecule. The ABTS–TEAC assay was first reported by Miller et al. (1993), which is based on the scavenging ability of antioxidants to the long-life radical anion ABTS•+. In this assay, ABTS is oxidized by peroxyl radicals or other oxidants to its radical cation, ABTS•+, and the TAC is measured as the ability of test compounds to decrease the color reacting directly with the ABTS•+ radical. Originally, this assay used metmyoglobin and H2O2 to generate ferrylmyoglobin, which then reacted with ABTS to form ABTS•+ (Miller et al. 1993). ABTS•+ can be generated by either chemical reaction (e.g., potassium persulfate; Re et al. 1999) or enzyme reactions (e.g., horseradish peroxidase; Arnao et al. 1996). Generally, the chemical generation requires a long time (e.g., up to 16 hours for potassium persulfate generation), whereas enzymatic generation is faster and the reaction conditions are milder. In this assay, 415 and 734 © 2011 by Taylor & Francis Group, LLC

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nm were adopted by most investigators to spectrophotometrically monitor the reaction between the antioxidants and ABTS•+. In terms of quantification methods, most recently revised methods measure the absorbance decrease of ABTS•+ in the presence of a testing sample or Trolox at a fixed time point (1–6 min), and then antioxidant capacity was calculated as Trolox equivalents. The FRAP assay was first developed by Benzie and Strain (1996). At a low pH, reduction of ferric tripridyltriazine (Fe(III)-TPTZ) complex to ferrous form (which has an intense blue color) is monitored by measuring the change in absorbance (increase in absorbance) at 593 nm. The original Folin–Ciocalteu (F–C) method was first developed in 1927 and originated from chemical reagents used for tyrosine analysis (Folin and Ciocalteu 1927) in which the oxidation of phenols by a molybdotungstate reagent yields a colored product with λmax at 745–750 nm and designed to determine the total content of phenolics (total phenols) (Singleton et al. 1999). The DPPH method was first reported by Brand-Williams et al. (1995). The DPPH• radical bearing a deep purple color is one of the few stable organic nitrogen radicals. This is a free radical scavenging assay involving decoloration based on the measurement of the reducing ability of antioxidants toward DPPH•. This assay spectrophotometrically measures the loss of DPPH color at 515 nm after a reaction with antioxidant compounds.

Original CUPRAC (Cupric Ion Reducing Antioxidant Capacity) Method The CUPRAC assay was developed in our laboratories and expanded with some modifications. The chromogenic redox reagent used for the CUPRAC assay was bis(neocuproine)copper(II) chelate. This reagent was useful at pH 7, and the absorbance of the Cu(I)-chelate formed as a result of redox reaction with reducing polyphenols was measured at 450 nm. The color was due to the Cu(I)-Nc chelate formed (see Figure 18.2). The reaction conditions such as the reagent concentration, pH, and oxidation time at room and elevated temperatures were optimized (Apak et al. 2004, 2005). The chromogenic oxidizing reagent of the developed CUPRAC method; that is, bis(neocuproine) copper(II) chloride (Cu(II)-Nc), reacts with antioxidants (AOX) acting as reductants in the following manner. In this reaction, the reactive Ar–OH groups of polyphenolic antioxidants (AOX) are oxidized to the corresponding quinones (Ar = O) and Cu(II)-Nc is reduced to the highly colored Cu(Nc)2+ chelate showing maximum absorption at 450 nm. Although the concentration of Cu2+ ions is in stoichiometric excess of that of neocuproine in the CUPRAC reagent for driving the redox equilibrium reaction represented by Figure 18.2 to the right, the actual oxidant is the Cu(Nc)22 + species and not the sole Cu2 + , because the standard redox potential of the Cu(II/I)-neocuproine is 0.6 V, much higher than that of the Cu2 + / Cu+ couple (0.17 V; Tütem et al. 1991). As a result, polyphenols are oxidized much more rapidly and efficiently with Cu(II)-Nc than with Cu2 + , and the amount of colored product (i.e., Cu(I)-Nc chelate) 2+

(a)

N H3C H3C

N Cu

CH3 CH3

(b)

AOX

+

Oxidized AOX Product

N H 3C H3C

CH3

Cu N

Light blue CUPRAC reagent

N CH3

+H

+

N

Yellow–orange product, λmax = 450 nm

FIGURE 18.2  The CUPRAC reaction and chromophore: Bis(neocuproine)copper(I) chelate cation. (Protons liberated in the reaction are neutralized by the NH4Ac buffer).

© 2011 by Taylor & Francis Group, LLC

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369

emerging at the end of the redox reaction is equivalent to that of reacted Cu(II)-Nc. The liberated protons are buffered in an ammonium acetate medium. The CUPRAC reagent is capable of oxidizing suitably situated phenolic –OH groups to the corresponding quinones as long as the corresponding conditional quinone–phenol potential is less than or close to that of cupric/cuprous-neocuproine in neutral medium. In the normal CUPRAC method (CUPRACN), the oxidation reactions were essentially complete within 30 minutes. Flavonoid glycosides required acid hydrolysis to their corresponding aglycons for fully exhibiting their antioxidant potency. Slow reacting antioxidants needed elevated temperature incubation so as to complete their oxidation with the CUPRAC reagent (Apak et al. 2004, 2005). Special precautions to exclude oxygen from the freshly prepared and analyzed solutions of pure antioxidants were not necessary since oxidation reactions with the CUPRAC reagent were much more rapid than with dissolved O2 (i.e., the latter would not appreciably occur during the period of CUPRAC protocol since there is a spin restriction for the ground state triplet of dioxygen molecule to participate in fast reactions). However, plant extracts should be purged with N2 to drive off O2, and should be kept in a refrigerator if not analyzed on the day of extraction, since complex catalyzed reactions with unpredictable kinetics may take place in real systems. Additionally, the oxidation of ascorbic acid with dissolved oxygen may take place more rapidly than with polyphenolics, especially in the presence of transition metal salts. As a distinct advantage over other ET-based TAC assays (e.g., Folin, FRAP, ABTS, DPPH), CUPRAC is superior in regard to its realistic pH close to the physiological pH, favorable redox potential, accessibility, and stability of reagents, flexibility, simplicity, low-cost, and applicability to lipophilic antioxidants as well as hydrophilic ones. CUPRAC gives additive responses to antioxidants in regard to their contribution to TAC, and perfectly linear calibration curves (of absorbance vs. concentration) over a relatively wide concentration range of antioxidants. An example of the calculation of TAC for apricots with respect to the CUPRAC method is given below (Güçlü et al. 2006):

TAC (in µmol TR/g) = (Af  /ЄTR) (Vf  /Vs) r (Vi  /m) × 103,

where ЄTR = 1.67 × 104 Lmol–1cm–1 (CUPRACN method); Vi = initial extract volume; m = grams of solid apricot sample; r = extract dilution ratio; Vs = sample volume for analysis; Vf = final volume; Af = sample absorbance.

Some Modifications of the CUPRAC Method It should be remembered that the CUPRAC assay does not merely measure the TAC of an antioxidant sample, but gives rise to many other modified assays of radical scavenging or activity measurement that may be useful for antioxidant research (Demirci Çekiç et al. 2009; Özyürek et al. 2007, 2008a, 2008b, 2009). In this regard, CUPRAC should be perceived as a train of antioxidant measurement methods in varying media, one evolving from the other. This resembles the highly popular Russian stacking doll, “Matrushka” (Figure 18.3).

Simultaneous Measurement of Lipophilic and Hydrophilic Antioxidants Lipophilic and hydrophilic antioxidants can be assayed simultaneously by solubilizing lipophilic compounds such as β-carotene, vitamin E, and oil-soluble synthetic antioxidants and hydrophilic compounds such as vitamin C and phenolic antioxidants as “host–guest” complexes with 2% methyl-β-cyclodextrin (M-β-CD; w/v) in 90% aqueous acetone (Özyürek et al. 2008a). This method eliminates the wide variability in apparent antioxidant capabilities arising from different levels of accumulation of oil- and watersoluble antioxidants at emulsion interfaces, and assigns an objective TEAC value to each antioxidant that depends only on its chemical character (i.e., electron donating ability).

Determination of Ascorbic Acid by the Modified CUPRAC Method with Extractive Separation of Flavonoids-La(III) Complexes The modified CUPRAC method (Özyürek et al. 2007) for ascorbic acid: AA (vitamin C) determination is based on the oxidation of AA to dehydroascorbic acid with the CUPRAC reagent of TAC assay; that © 2011 by Taylor & Francis Group, LLC

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FIGURE  18.3  CUPRAC assay resembles the famous Russian stacking doll, Matrushka, as the mother CUPRAC method of TAC measurement has given rise to many other modified CUPRAC methods for activity/radical scavenging determination.

is, Cu(II)-neocuproine (Nc), in ammonium acetate-containing medium at pH 7, where the absorbance of the formed bis(Nc)-copper(I) chelate is measured at 450 nm. The flavonoids (essentially flavones and flavonols) normally interfering with the CUPRAC procedure were separated with a preliminary extraction as their La(III) chelates into ethylacetate (EtAc). The Cu(I)-Nc chelate responsible for color development was formed immediately with AA oxidation.

Hydroxyl Radical Scavenging Assay of Phenolics and Flavonoids with a Modified CUPRAC Method

A salicylate probe was used for detecting • OH generated by the reaction of iron(II)-EDTA complex with H2O2. The produced hydroxyl radicals attack both the salicylate probe (see the formulas of dihydroxybenzoic acids: DHBAs produced from salicylate under hydroxyl radical attack, Figure 18.4) and the hydroxyl radical scavengers that are incubated in a solution for 10 minutes. Added radical scavengers compete with salicylate for the •OH produced, and diminish chromophore formation from Cu(II)-neocuproine. At the end of the incubation period, the reaction was stopped by adding catalase, and the reaction products were quantified with both CUPRAC and HPLC (see Figure 18.5 for the HPLC quantification of DHBAs). With the aid of this reaction, a kinetic approach was adopted to assess the hydroxyl radical scavenging properties of polyphenolics, flavonoids, and other compounds (e.g., ascorbic acid, glucose, and mannitol). A second-order rate constant for the reaction of the scavenger with • OH could be deduced from the inhibition of color formation due to the salicylate probe (Özyürek et al. 2008b).

Measurement of Xanthine Oxidase Inhibition Activity of Phenolics and Flavonoids with a Modified CUPRAC Method Since some polyphenolics have a strong absorption from the UV to the visible region, XO inhibitory activity of polyphenolics was alternatively determined without interference by directly measuring the formation of uric acid and hydrogen peroxide using the modified CUPRAC spectrophotometric method at 450 nm (Özyürek et al. 2009). The CUPRAC absorbance of the incubation solution due to the reduction of Cu(II)-neocuproine reagent by the products of the X–XO system decreased in the presence of polyphenolics, the difference being proportional to the XO inhibition ability of the tested compound. © 2011 by Taylor & Francis Group, LLC

371

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals CO2H OH OH 2, 3-Dihydroxybenzoic acid CO2H

CO2H

OH

OH

Salicylic acid

OH 2, 4-Dihydroxybenzoic acid CO2H OH HO 2, 5-Dihydroxybenzoic acid

FIGURE 18.4  Major hydroxylation products formed from a salicylic acid probe upon the attack of • OH radicals.

123.95

d

Response (mAU)

103.95

83.95

63.95

43.95

23.95

a

c b

3.95 0

2

4

6

8

10

12

14 16 Time (min)

18

20

22

24

26

28

FIGURE 18.5  The HPLC chromatogram for salicylate and its hydroxylation products in the absence of hydroxyl ­radical scavengers. The retention times were (a) 2,5-DHBA 9.38 min; (b) 2,4-DHBA 9.78 min; (c) 2,3-DHBA 11.20 min; and (d) salicylate 17.65 min.

Modified Cupric Reducing Antioxidant Capacity (CUPRAC) Assay for Measuring the Antioxidant Capacities of Thiol-Containing Proteins in Admixture with Polyphenols In most assays measuring a TAC, proteins are not taken into account (e.g., in assays carried out in the hydrophilic fraction of human serum) and remain in the precipitate (obtained by using perchloric acid, trichloroacetic acid, ammonium sulfate, etc.). Modified CUPRAC assay for proteins has © 2011 by Taylor & Francis Group, LLC

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verified that the contribution of proteins, especially thiol-containing proteins, to the observed TAC is by no means negligible (Demirci Çekiç et al. 2009). Various protein fractions (egg white, whey proteins, gelatin) and peptides (like glutathione: GSH) may either respond to TAC assays directly via their free –SH groups, or indirectly after protein denaturation through their exposed (originally buried) thiol groups. An 8 M urea buffer was used to expose these thiols to TAC assays. Urea—in combination with SDS—maximized the reactivity of thiols and disulfides that may be buried within the protein matrix. Urea partly denaturated proteins and significantly lowered the reduction potential of disulfide/thiol couples in peptides facilitating thiol oxidizability. Among the tested TAC assays, only CUPRAC and ABTS/H 2O2/HRP possessed the property of optical absorbance additivity (i.e., obeying Beer’s law). This study has reported, for the first time, the measurement of the TAC of thiolcontaining proteins in admixture with phenolic antioxidants after taking up the protein fractions with a suitable buffer that neither causes the precipitation of proteins nor interferes with the selected antioxidant assay (specifically CUPRAC assay), and is expected to be useful in estimating the TAC values and hence food qualities of dairy products and other protein-containing food varieties in further studies. The comparison of methods for assessing antioxidant capacity summarizing the experiences of our analytical chemistry laboratory at Istanbul University is presented in Table 18.4.

Other Antioxidant Activity Tests As for molecular probes used in the colorimetric/fluorometric detection of ROS, nitro blue tetrazolium (NBT) has been used for superoxide anion (O2•–), scopoletin for hydrogen peroxide (H2O2), deoxyribose/thiobarbituric acid (TBA) or modified CUPRAC reagent for hydroxyl radicals (• OH), and tetratert-butylphtalocyanine for singlet oxygen (1O2) (Huang et al. 2005). Ewing and Janero developed a superoxide dismutase (SOD) microassay based on the spectrophotometric assessment of O2•– –mediated NBT reduction by an aerobic mixture of NADH and phenazine methosulfate, which produces superoxide chemically at a nonacidic pH (Ewing and Janero 1995). Hydrogen peroxide has been assayed by its ability to oxidize scopoletin, a naturally occurring fluorescent compound, in the presence of horseradish peroxidase as catalyst, to a nonfluorescent product, and the decrease in fluorescence is an indication of H2O2 at nanomolar levels (De la Harpe and Nathan 1985). Hydroxyl radicals generated from a Fenton-reaction (Equation 18.4) were most frequently detected by means of their oxidative attack on a deoxyribose probe producing malondialdehyde (MDA) as the end product; MDA was colorimetrically detected by the formation of colored products with TBA, forming the basis of the TBARS (thiobarbituric acid–reactive substances) method (Gutteridge 1981; Halliwell and Gutteridge 1981). Bektaşoğlu et al. (2006) used p-aminobenzoate, 2,4- and 3,5-dimethoxybenzoate probes for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II) + EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37°C-incubated solutions for 2 hours. The CUPRAC absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of •OH scavengers, the difference being proportional to the scavenging ability of the tested compound (Bektaşoğlu et al. 2006).

Antioxidant Capacities of Regularly Consumed Fruits Phenolic substances can be extracted from fruits using a sequence of solvents with divergent polarity. In general, useful solvents with a decreasing order of polarity are: water, 80% methanol or 70% ethanol, 80% acetone, and ethyl acetate. Among antioxidant phenolics, certain classes of compounds such as phenolic acids, hydroxycinnamic acids, flavonoids, and carotenoids require a decreasing order of solvent polarity for extraction, respectively, although suitable solvent combinations may be tailored for specific purposes. Moreover, the dielectric constant of the solvent, intra-/intermolecular hydrogen bonding associations and standard redox potential of phenolics and derived aryloxy radicals in a given solvent may be important for electron transfer kinetics in antioxidant assays (Huang et al. 2005; Prior et al. 2005). © 2011 by Taylor & Francis Group, LLC

Difficult and expensive

Simple

Simple

Rather difficult and expensive

Rather difficult

Simple (but exact composition and redox potential of Folin reagent unknown)

FRAP

CUPRAC

ABTS/TEAC

DPPH

Folin

Simplicity

ORAC

Antioxidant Assay

Interference Quality of β-PE probe variant from lot to lot, recent fluorescein probe can bind to some antioxidants, dependent on temperature and oxygen concentration Nonresponsive to thiols, oxidation of hydroxycinnamic acids incomplete Only strong reductants and strong copper chelators (both of which are not antioxidants) interfere Too dependent on the radical generation method, pH- and solvent-dependent Too much influenced by light, oxygen concentration, pH and solvent Nonspecific oxidant for all phenols and other substances (sugars, amino acids, etc.)

Comparison of Methods for Assessing Antioxidant Capacity

TABLE 18.4

© 2011 by Taylor & Francis Group, LLC ET

ET (or mixed HAT–ET)

ET (or mixed HAT–ET)

ET

ET

HAT

Mechanism

Fixed time

Fixed time

Fixed time

Fixed time

Fixed time

Fixed time

End-Point

Increase in absorbance

Decrease in absorbance

Decrease in absorbance

Increase in absorbance

Increase in absorbance

Decrease in florescence integrated value lag time

Property Measured

–

+

+

+

–

+

Lipophilic and Hydrophilic AOC

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals 373

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Although it is difficult to define a universally acceptable solvent, 80% MeOH and 70% EtOH are generally the most preferred solvents for phenolics extraction from plants. In antioxidant tests carried out by TEAC, ORAC, and FRAP methods on regularly consumed fruits and vegetables available on the U.K. market, fruits and vegetables rich in anthocyanins (e.g., strawberry, raspberry, and red plum) showed the highest TAC values, followed by those rich in flavones (e.g., orange and grapefruit) and flavonols (e.g., onion, leek, spinach, and green cabbage), while the hydroxycinnamic acids—rich ones (e.g., apple, tomato, pear, and peach) exhibited the lower values. The antioxidant capacities (in TEAC units, on fresh weight basis) followed the hierarchic order: strawberry >> raspberry = red plum >> red cabbage >>> grapefruit = orange > spinach > broccoli > green grape ≈ onion > green cabbage > pea > apple > cauliflower ≈ pear > tomato ≈ peach = leek > banana ≈ lettuce (Proteggente et al. 2002). For plant foods consumed in the Italian market, spinach was the highest TAC exhibiting vegetable, followed by peppers and asparagus, while in fruits, berries (i.e., blackberry, redcurrant, and raspberry) showed the highest capacities; coffee and citrus juices among beverages, and soybean oil and extra virgin olive oil among the vegetable oils, were the richest in antioxidants (Pellegrini et al. 2003). For fruits consumed in the American diet, Vinson et al. (2001) made a distinction between free phenols (sample extracted with 50% MeOH, incubated at 90°C, cooled and centrifuged) and total phenols (sample extracted with 1.2 M HCl in 50% MeOH), and found that, on a fresh weight basis, cranberry had the highest total phenols, and was distantly followed by red grape; fruits had significantly better quantity and quality of phenolic antioxidants than vegetables. Only a few fruits (avocado, cranberry, honeydew melon, and orange) had a large portion of their phenolic contents in free form; the other fruits had a high percentage (31–94%) of the phenols conjugated (Vinson et al. 2001). In the American food market, the phenolic antioxidant capacity—analyzed with the DPPH method—of various plant food were as follows: fruits, 600–1700 μmol Trolox equivalent (TE)/100 g, with a high 2200 TE for plums; berries averaged 3700 TE and vegetables averaged 450 TE with a high 1400 TE for red cabbage; whole grain breakfast cereals analyzed 2200–3500 TE (Miller et al. 2000). A meal containing a 100 g serving of breakfast cereals, fruits, and vegetables provided an average antioxidant content of 2731, 1200, and 447 TE, respectively (Miller et al. 2000). Güçlü et al. (2006) determined the TAC values of five varieties of apricots harvested in Malatya (Turkey), namely Hacihaliloglu, Cologlu, Kabaasi, Soganci, and Zerdali using three different assays: CUPRAC, ABTS/persulfate, and Folin (the TAC values in the units of μmol TE g–1 reported in this order for the assays): fresh apricot (3.62 ± 0.65; 3.47 ± 0.60; 10.1 ± 1.27), sun-dried apricot (14.2 ± 3.1; 14.1 ± 2.9; 36.2 ± 5.8), and desulfited apricot that was originally sulfite-dried (13.6 ± 2.7; 13.8 ± 2.4; 40.3 ± 3.5). In this study, the CUPRAC test was performed for the assay of both TAC and sulfite content of apricots; sulfite, normally contributing to the color measured in the CUPRAC method, could be removed prior to assay on a strongly basic anion exchanger at pH 3 in the form of HSO3 –, without affecting the analytical precision of phenolic TAC determination. The CUPRAC results correlated well with those of ABTS and Folin (r = 0.93). The tests also showed that the sun-dried Malatya apricot completely preserved its antioxidant values unlike some other dried fruits, and gave very close TAC values to the desulfited samples that were originally sulfite-dried (Güçlü et al. 2006; see Figure 18.6 for visualizing the tested apricots). Taking 100 g fresh weight of the fruit, 150 mL glass beverage, and 500 mL glass beer as the standard serving amounts, Paganga et al. (1999) reported that the hierarchic equalities of TE—total antioxidant activities of some beverages and fruits were: 1 glass of red wine = 12 glasses of white wine = 2 cups of tea = 4 apples = 5 portions of onion = 5.5 portions of eggplant = 3.5 glasses of blackcurrant juice = 3.5 glasses of beer = 7 glasses of orange juice = 20 glasses of apple juice (long-life). Naturally these values are the results of in vitro TAC tests, and are not associated with the in vivo levels of antioxidants when these food sources are ingested as a diet. Velioğlu et al. (1998) state that when all plant materials are included in statistical analysis, there is a positive and highly significant relationship between total phenolics content (Folin) and antioxidant activity (β-carotene bleaching); however, for plants with phenolics content largely consisting of anthocyanins, there may not be a significant correlation between these two assay results. Among edible plant materials, Kähkönen et al. (1999) found remarkably high antioxidant activity and high phenolic content (gallic acid equivalents > 20 mg g–1) for berries, though these two parameters did not have the same meaning for all samples. A list of © 2011 by Taylor & Francis Group, LLC

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FIGURE 18.6  (See color insert) The tested apricots: (left side) sun-dried apricot and (right side) sulfite-dried apricot.

TABLE 18.5 Major Phenolic Components, Total Phenolic Content, and Total Antioxidant Capacity of Some Selected Fruits

Fruit Strawberry Raspberry Red plum Orange

Banana Apple

Major Phenolic Componentsa Pelargonidin-3-glucoside Cinnamoyl glucose Cyanidin-3-sophoroside Cyanidin-3-glucoside Cyanidin-3-glucoside 3´-Caffeoylquinic acid Hesperidin Narirutin Neohesperidin Quercetin-3-glucoside/ conjugates 5´-Caffeoylquinic acid Rutin Quercetin-3-glucoside/ conjugates

Total Phenols 330 ± 4a 133 ± 20b 228 ± 6a 320 ± 12a 226 ± 20b 126 ± 6a 41 ± 23b 38 ± 4a 325 ± 61b 48 ± 1a 186 ± 26b

TEAC (µmol TE/100 g Fw)

ORAC (µmol TE/100 g Fw)

FRAP (µmol Fe2+/100 g Fw)

2591 ± 68a 1134c 1846 ± 10a 1679c 1825 ± 28a 511c 849 ± 25a 874c

2437 ± 95a 3577e 1849 ± 232a 4925e 2564 ± 185a 6239e 1904 ± 259a 1814e

3352 ± 38a 2800c 2325 ± 53a 4303c 2057 ± 25a 1279c 1181 ± 6a 2050c

181 ± 39a 64c 343 ± 13a 159c 640 ± 270d

331 ± 59a 879e 560 ± 18a 2936e

164 ± 32a 228c 394 ± 8a 384c

As mg gallic acid equivalent (GAE)/100 g Fw from Proteggente, A. R., Pannala, A. S., Paganga, G., et al., Free Radic. Res., 36, 217–33, 2002. b As mg catechin equivalent (CE)/100 g Fw from Vinson, J. A., Su, X., Zubik, L., and Bose, P., J. Agric. Food Chem., 49, 5315–21, 2001. c From Pellegrini, N., Serafini, M., Colombi, B., et al., J. Nutr., 133, 2812–19, 2003. d From Paganga, G., Miller, N., and Rice-Evans, C. A., Free Radic. Res., 30, 153–62, 1999. e From Wu. X., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Gebhardt, S. E., and Prior, R. L., J. Agric. Food Chem., 52, 4026–37, 2004. a

selected fruits, of which the total antioxidant contents were assayed using different methods, are tabulated in Table 18.5. Park et al. (2006) used five different antioxidant assays: FRAP, CUPRAC, TEAC, DPPH, and Folin methods to find the peak of the kiwifruits antioxidant activity during the first 10 days of ethylene treatment (100 ppm at 20°C). It was found by all applied methods that kiwifruit samples had the highest content of polyphenols and flavonoids and the highest antioxidant activity on the sixth day of the ethylene treatment. The correlation coefficients between polyphenols, flavonoids, and antioxidant activities of kiwifruit methanol extracts with TEAC and CUPRAC, were 0.81 and 0.63, and 0.23 and 0.17, respectively, and showed that the free polyphenols correlation coefficients were higher than that of the flavonoids. Park et al. (2008) also studied ethylene-treated kiwifruit (Actinidia deliciosa) cultivar “Hayward” which was compared with the air-treated kiwi. The correlation coefficients between total polyphenols and the © 2011 by Taylor & Francis Group, LLC

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antioxidant capacities measured by ABTS/TEAC (Trolox equivalent antioxidant capacity), DPPH, and CUPRAC assays for ethylene-treated kiwifruits were 0.74, 0.93, and 0.98, in comparison with air-treated samples of 0.72, 0.88, and 0.97, respectively. CUPRAC produced the most consistent measurements for ethylene-treated kiwifruit. Arancibia-Avila et al. (2008) investigated the antioxidant properties of durian fruit (Durio zibethinus Murr., cv Mon Thong) at different stages of ripening using fluorometry, UV spectroscopy, and HPLC/ DAD analyses. They found that total polyphenols, flavonoids, anthocyanins, and flavanols in ripe durian were significantly higher (p   oat flakes > wholemeal wheat > natural rice > smooth wheat flour > semismooth wheat flour > wholemeal wheat flour > to millet hulled. Unlike the case in fruits, conjugated phenolic compounds predominated in all the types of cereals analyzed, and they formed 68%–85% of the total phenolic compounds. The antioxidant activity of cereals determined by TEAC in decreasing order for free phenolic compounds was: peeled barley (25.2 mmol/kg DM) > oat flakes > natural rice > wholemeal wheat > semismooth wheat flour > smooth wheat flour > millet > coarse wheat flour (6.76), and for total phenolic compounds: buckwheat (88.3) > wholemeal wheat > oat flakes > natural rice > smooth wheat flour > millet > semismooth wheat flour >  > coarse wheat flour (48.3). Essentially the higher content of phenolic compounds and the antioxidant activity of wholemeal flour, in addition to the higher content of vitamins, mineral substances, phospholipids, and fibers, can be an additional favorable factor for human health and a significant reason for its preferable consumption instead of white flours (Stratil et al. 2007). The antioxidant capacity for cereals determined by FRAP method was (in decreasing order): buckwheat > wholemeal wheat flour > oat flakes > natural rice > smooth wheat flour > millet > semismooth wheat flower ≈ coarse wheat flour (Stratil et al. 2007). The values of antioxidant activities determined with the DPPH method were lowest, despite this method giving the same values as the TEAC method. The DPPH method gave several times lower values for extracts than TEAC. This significant difference in values could be explained by a relatively higher stability of the DPPH © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications TABLE 18.6 Antioxidant Activity and Total Phenolics Content of Methanolic Extracts of Grain Products Measured by β-Carotene Bleaching and Folin Methods, respectively Sample Buckwheat hulls Wheat germ Buckwheat seed Fibrotein MK22E3 Fibrotein MK11-DDG Fibrotein MK43 Fibrotein MK37

Antioxidant Activity (%)

Total Phenolics (mg/100 g)

94.9 64.9 63.7 95.1 82.3 63.4 56.0

3900 349 726 — 1241 169 213

Source: Veliog˘lu, Y. S., Mazza, G., Gao, L., and Oomah, B. D., J. Agric. Food Chem., 46, 4113–17, 1998.

radical, which may result in significantly lower reactivity. This radical would evidently react only with the more reactive phenolic substances. Therefore, it was not expected to detect the less reactive phenolic substances, which still could have antioxidant activity in the human organism (Stratil et al. 2007). The antioxidant activities and total phenolics of 28 plant products, including sunflower seeds, flaxseeds, buckwheat seeds and hulls, four wheat products, and several fruits, vegetables, and medicinal plants were determined by Velioğlu and coworkers (Table 18.6; 1998). Antioxidant activities of buckwheat seed and hulls were 63.7 and 94.9%, respectively. The significantly high activity of the hulls reflected the higher phenolic content of the hull, 3900 mg of phenolics/100 g in hull versus 726 mg/100 g in seed. Wheat germ had moderate antioxidant activity, consistent with its moderate content of phenolic compounds. Some of the fibrotein samples included in this study showed very strong activity (fibrotein MK22E3), and others showed medium-to-high activities. The antioxidant activities of these products probably result from the combined action of phenolics and proteins in the samples (Velioğlu et al. 1998). A statistically significant relationship was observed between total phenolics and antioxidant activity of cereal products (R2) 0.905; p  barley > oat > wheat ≅ rye. The antioxidant activity was observed in extracts prepared from separated parts of buckwheat and barley. In respect to hulls, the antioxidant hierarchy was as follows: buckwheat > oat > barley. The correlation coefficient between total phenolic compounds and total antioxidative activity of the extracts was −0.35 for water extracts and 0.96, 0.99, 0.80, and 0.99 for 80% methanolic extracts originated from whole grains, hulls, pericarb with testa fractions, and endosperm with embryo fractions, respectively (Zieliński and Kozłowska 2000). Adom and Liu used a modified total oxyradical scavenging capacity (TOSC) assay (Eberhardt et al. 2000; Winston et al. 1998) for determining the TAC of cereal extracts. In this assay, peroxy radicals formed from 2,2′-azobis-amidinopropane (ABAP) oxidize α-keto-β-methiolbutyric acid (KMBA) to form ethylene gas, which was measured by gas chromatographic headspace analysis. The degree of inhibition of ethylene gas formation by sample extracts was used as the basis for calculating the TAC. The dose required to cause 50% inhibition (EC50) for each sample was used to calculate the total antioxidant activity, which was expressed as micromoles of vitamin C equivalent per gram of grain (Adom and Liu 2002). Ferulic acid in sample extracts was quantified using a RP-HPLC procedure, and the total phenolic content of each extract was determined using a Folin method described by Singleton et al. (1999). Corn had the highest total phenolic content (15.55 ± 0.60 μmol of gallic acid equiv [GAE]/g of grain) of the grains tested, followed by wheat (7.99 ± 0.39 μmol GAE/g of grain), oats (6.53 ± 0.19 μmol GAE/g of © 2011 by Taylor & Francis Group, LLC

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grain), and rice (5.56 ± 0.17 μmol GAE/g of grain). The major portion of phenolics in grains existed in the bound form (85% in corn, 75% in oats and wheat, and 62% in rice), although free phenolics were frequently reported in the literature. Ferulic acid was the major phenolic compound in grains tested, with free, soluble-conjugated, and bound ferulic acids present in the ratio 0.1:1:100, respectively. Corn had the highest total antioxidant activity (181.42 ± 0.86 μmol of vitamin C equiv/g of grain), followed by wheat (76.70 ± 1.38 μmol of vitamin C equiv/g of grain), oats (74.67 ± 1.49 μmol of vitamin C equiv/g of grain), and rice (55.77 ± 1.62 μmol of vitamin C equiv/g of grain). Bound phytochemicals were the major contributors to the total antioxidant activity: 90% in wheat, 87% in corn, 71% in rice, and 58% in oats. Bound phytochemicals could survive stomach and intestinal digestion to reach the colon. This may partly explain the mechanism of grain consumption in the prevention of colon cancer, other digestive cancers, breast cancer, and prostate cancer, which is supported by epidemiological studies (Adom and Liu 2002). Another study on the importance of insoluble-bound phenolics to antioxidant properties of cereals describes the antioxidant capacity of free, soluble esters, and insoluble-bound phenolics isolated from soft and hard whole wheats, brans, and flours (Liyana-Pathirana and Shahidi 2006). The content of total phenolics was determined with the Folin reagent according to a modified version of the procedure described by Singleton and Rossi (1965), and expressed as micrograms of ferulic acid equivalents (FAE) per gram of defatted material. The antioxidant activity of phenolic fractions was evaluated using ABTS/ TEAC, DPPH radical scavenging, reducing power, and ORAC. The bound phenolic content in the bran fraction was 11.3 ± 0.13 and 12.2 ± 0.15 mg FAE/g defatted material for hard and soft wheats, respectively. The corresponding values for flour were 0.33 ± 0.01 and 0.46 ± 0.02 mg FAE/g defatted sample. The bound phenolic content of hard and soft whole wheats was 2.1 (±0.004 or ±0.005) mg FAE/g defatted material. The free phenolic content ranged from 0.14 ± 0.004 to 0.98 ± 0.05 mg FAE/g defatted milling fractions of hard and soft wheats examined. The contribution of bound phenolics to the total phenolic content was significantly higher than that of free and esterified fractions. In wheat, phenolic compounds were concentrated mainly in the bran tissues. In the numerous in vitro antioxidant assays carried out, the bound phenolic fraction demonstrated a significantly higher antioxidant capacity than free and esterified phenolics. Thus, the inclusion of bound phenolics in studies related to quantification and antioxidant activity evaluation of grains and cereals is essential (Liyana-Pathirana and Shahidi 2006). With the aim to expand the Italian TAC database, the TAC values of 18 cereal products were determined using three different assays considering the contribution of bound antioxidant compounds by Pellegrini and coworkers (Table  18.7; 2006). Grains (barley, white and whole meal rice, and spelta kernels), flours (whole meal buckwheat, corn, whole meal oat, whole meal rye, white and whole meal wheat, durum wheat), cereal products (white and whole meal pastas), and breakfast cereals (barley [puffed], cornflakes, oat [whole meal, puffed with honey], rice [white, puffed], wheat bran [extruded]) were studied. In another study Pérez-Jiménez and Saura-Calixto (2005) aimed to conduct an assessment of the antioxidant capacities of cereals using both chemical extraction and in vitro digestive enzymatic extraction of antioxidants. The samples selected were raw rice, boiled rice, wheat flour, French bread, wheat bran, and oat bran. Wheat flour and rice are the chief sources of cereal foods in the diet, while wheat and oat bran are increasingly used in ready-to-eat breakfast cereals and as ingredients in dietary fiberenriched foods. Boiled rice and French bread (whose main ingredient is wheat flour, plus water, salt, and some additives) are two of the most common ways of consuming cereals (Pérez-Jiménez and SauraCalixto 2005). Two complementary methods were used to determine the antioxidant capacities in these samples: FRAP, which measures the sample’s ferric reducing power, and DPPH, which measures free radical scavenging capacity, and total phenolics were determined according to the Folin–Ciocalteau procedure. The most efficient antioxidant extraction was achieved by using successively acidic methanol/ water (50:50, v/v, pH 2) and acetone/water (70:30, v/v). The antioxidant capacity in these extracts ranged from 1.1 to 4.4 μmol Trolox equivalent (TE)/g dw. A significant amount of hydrolyzable phenolics with a high antioxidant capacity (from 5 to 108 μmol TE/g dw) was found in the residues of this aqueousorganic extraction. The antioxidant capacities of these nonextractable polyphenols are usually ignored in the literature, although they may have an antioxidant role in the gastrointestinal tract, especially after colonic fermentation, and may be fermentated to active metabolites. On the other hand, the analysis of in vitro digestive enzymatic extracts suggests that the antioxidant activity of cereals in the human gut may © 2011 by Taylor & Francis Group, LLC

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications TABLE 18.7 TAC Values of Some Selected Cereal Extract with Respect to the Three Different Antioxidant Capacity Assays Cereal

FRAP (mmol Fe2+/kg)

TRAP (mmol TR/kg)

TEAC (mmol TR/kg)

Grains Barley Rice (white) Rice (brown) Spelta

18.97 7.91 16.83 14.16

4.16 3.74 4.64 4.64

4.59 2.20 3.85 4.01

Flours Buckwheat (whole meal) Corn (white) Durum wheat (white) Oat (whole meal) Rye (whole meal) Wheat (white) Wheat(whole meal)

55.32 11.52 13.09 12.18 23.29 10.45 20.23

16.29 2.69 2.09 2.54 8.51 1.10 4.14

26.22 3.01 2.70 2.79 11.64 1.93 4.58

Source: Pellegrini, N., Serafini, M., Salvatore, S., Del Rio, D., Bianchi, M., and Brighenti, F., Mol. Nutr. Food Res., 50, 1030–8, 2006.

be higher than what might be expected from literature data based on measurements of aqueous-organic extracts (Pérez-Jiménez and Saura-Calixto 2005), emphasizing the difference between in vitro and in vivo TAC measurement (though no such assay is currently available for the latter). Serpen et al. (2008) developed a new procedure with a methodology to place the solid cereal sample and the radical reagent solution in direct contact, skipping all the extraction steps. Using this approach, the soluble moiety of the cereal product exerts its antioxidant capacity by quenching the ABTS radical cation present in the solvent matrix according to the usual liquid–liquid type reaction. At the same time, the insoluble parts of the cereal sample exert their antioxidant capacity as a result of the surface reaction occurring at the solid–liquid interface, where the solid phase is represented by the antioxidant group bound to the insoluble polysaccharide fraction (Serpen et al. 2007b). Four dehulled covered grains (emmer, oat, millet, and barley), four naked grains (rice, rye, wheat, and corn), a pseudocereal (buckwheat), a cereal ingredient (wheat germ), and two different milling fractions of durum wheat bran were studied. The direct measurement procedure was based on a previous study (Serpen et al. 2007b), and an extraction/hydrolysis procedure was the second procedure applied to measure the TAC of cereal samples. For all the cereal samples, data obtained by the direct measurement procedure were comparable or slightly higher than those obtained by the sequential extraction procedure. In some cases, the differences became particularly relevant: for emmer and wheat followed by barley and rice, the TAC values were between 40 and 100% higher when the direct procedure was applied. On the other hand, for corn, millet, and oat, no significant differences were found (Serpen et al. 2008).

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19 Supercritical Fluid Extraction of Bioactive Compounds from Cereals Jose L. Martinez and Deepak Tapriyal CONTENTS Introduction............................................................................................................................................. 385 Fundamentals of Supercritical Fluid Technology................................................................................... 386 Supercritical Fluid Extraction: Process Description.......................................................................... 387 Supercritical Fluid Extraction of Compounds from a Solid Matrix.................................................. 388 Processing Parameters in the Supercritical Extraction of Solids.................................................. 390 Supercritical Fluid Extraction of Compounds from a Liquid Feed................................................... 392 Supercritical Fluid Processing of Cereals............................................................................................... 393 Amaranth........................................................................................................................................... 393 Barley................................................................................................................................................. 394 Corn................................................................................................................................................... 394 Oats.................................................................................................................................................... 397 Rice.................................................................................................................................................... 398 Rye..................................................................................................................................................... 400 Wheat................................................................................................................................................. 401 Supercritical Fluid Extraction: Industrial Process Implementation for Cereal Lipids............................ 403 Conclusions............................................................................................................................................. 404 References............................................................................................................................................... 405

Introduction In the last decade new trends in the food industry have emerged. These trends include an increased preference for natural products over synthetic ones, and stricter regulations related to nutritional and toxicity levels of active ingredients. Additionally, consumers are taking more proactive roles in maintaining their health, which has driven a new generation of products on to the market addressing disease prevention. As a consequence, over the past few years the functional foods and nutraceutical market has become one of the fastest-growing markets. These trends have made supercritical fluid technology a primary alternative to traditional solvent extraction for the extraction and fractionation of active ingredients. This chapter is intended to give an overview of the fundamentals of the supercritical fluid technology and its applications to extract and fractionate cereal oil components. A chemical and quality comparison of the products obtained by conventional and supercritical processing methods is discussed. Additionally, process economics for industrial process implementation of the cereal lipids is also included.

© 2011 by Taylor & Francis Group, LLC

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Fundamentals of Supercritical Fluid Technology

Pressure

The different physical states of a pure substance can be illustrated in a pressure–temperature diagram, as shown in Figure 19.1. The lines separate the different physical states (solid, liquid, and vapor) and represent the equilibrium between two phases. The vapor–liquid equilibrium line represents the vapor pressure. It starts in the triple point, where the solid, liquid, and vapor phase coexist, and ends at the critical point. The critical point of a pure substance is defined as the temperature and pressure at which the liquid and gas phases become indistinguishable. When both pressure and temperatures are above the critical values, the substance is considered to be in a supercritical phase. In the supercritical phase no phase transition will occur regardless of any increase or decrease in pressure or temperature. For instance, it will not be possible to liquefy a vapor that is above its critical temperature by increasing the pressure nor to gasify a liquid that is above its critical pressure by increasing the temperature. The critical points are specific parameters for each substance. Table 19.1 lists the critical parameters of some fluids considered as supercritical fluids. Carbon dioxide (CO2) and propane have low critical temperatures, while water and methanol have high critical temperatures. There are significant differences in solvent power and selectivity between these fluids. Generally propane is the solvent used for commodity products, because the critical pressure is low. However CO2 is the preferable solvent because it is nontoxic, nonflammable, inexpensive, environmentally friendly, inert to most materials, widely available, and has convenient critical parameters. Water is commonly used in environmental applications, such as remediation of dilute aqueous waste streams or a sewage sludge treatment. This process is known as supercritical water oxidation. Supercritical methanol has gained popularity in recent years as a reactant to produce biodiesel by transesterification reactions of oils. The physicochemical properties of supercritical fluids are intermediate of the gaseous and liquid state. It exhibits gas-like transport properties of diffusivity and viscosity, directly related to mass transfer and hydrodynamic properties, and liquid-like density, directly related to solvent power. Additionally, the surface tension is negligible allowing easy penetration into solid matrices.

Supercritical phase

Solid phase

Pc

Critical point

Liquid phase

Gas phase

Tc

Temperature

FIGURE 19.1  P–T diagram of a pure substance.

TABLE 19.1 Critical Parameters of Fluids of Interest in Supercritical Processes Fluids Carbon dioxide Propane Methanol Water

Critical Temperature (°C)

Critical Pressure (MPa)

Critical Density (kg/L)

30.98 96.74 239.45 373.95

7.38 4.25 8.1 22.06

0.47 0.22 0.28 0.32

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A unique characteristic of supercritical fluids is the ability to tune their density by manipulating the pressure and temperature. The density is directly related to the solvent power. As a general rule for supercritical fluids, the solvent power increases with density at a given temperature. However, the effect of temperature is more complicated. When temperature is increased at a constant pressure, two opposite effects occur. The density of the solvent decreases and therefore the solvent power decreases. On the other hand, the vapor pressure of the solute increases with temperature. The result depends on which of these is the dominant factor. Normally at high pressures, density changes with temperature are more moderate. At low pressures, a loss in solvent power induced by a lower density prevails. Solvent power is not only a function of solvent density but it also depends on the chemical properties of the solute such as polarity and molecular interaction.

Supercritical Fluid Extraction: Process Description A supercritical fluid extraction process consists of two steps: extraction of the component(s) soluble in a supercritical fluid and separation of the extracted compound(s) from the solvent. As mentioned earlier, one of the main advantages of supercritical fluids is the ability to modify their selectivity by varying the pressure and temperature (i.e., modifying their fluid density). Because of this, supercritical fluids are often used to selectively extract or separate specific compounds from a mixture. One method of doing this is by a fractional extraction process. With this method, the extraction is carried out in two stages. During the first stage, a relatively low fluid density is selected that allows extraction of the compound(s) that are soluble at the low pressure. Then, the residue is further extracted at the high fluid density to recover heavier compounds. An example of this method is in the dealcoholization of cider (Medina and Martinez 1997). Another example of fractional extraction involves selective modification of the polarity of the solvent. The removal of nonpolar fractions takes place in the first stage by using a supercritical solvent. The removal of a more polar fraction from the residue in the second stage is carried out by using a cosolvent. An example of this method is in the extraction of active ingredients from grape seed (Martinez et al. 2003). Another procedure to selectively separate specific compounds from a mixture is by sequential depressurization (Stahl et al. 1988). In this case, both fractions (light and heavy) are simultaneously extracted by using high density fluid. After the extraction, the supercritical solvent and the extract pass through multiple depressurization steps allowing fractional separation. In the first depressurization stage, the heavier fraction is collected, while the volatile or light fraction is collected in the last stage. Two depressurization steps are generally used, although in specific cases three separation steps have been used. This type of process has been successfully applied in a wide variety of products. In some cases both fractions are desirable: oleoresin and essential oils, color, and pungent fractions; while in others only one of the fractions has commercial interest: oils and free fatty acid/water fraction. In regards to the separation of soluble compounds from the supercritical fluid, this can be carried out by modifying the thermodynamic properties of the supercritical solvent or by the use of an external agent (Figure 19.2). In the first case, the solvent power is modified by manipulating the operating pressure and/ or temperature. The more common method decreases the operating pressure by an isoenthalpic expansion, which provides a reduction of the fluid density and therefore a reduction of the solvent power. If the separation takes place by manipulating the temperature, two situations may occur depending on the solubility of the dissolved compounds. If the solubility increases with temperature at a constant pressure, a decrease in the temperature will decrease the solubility and separate the compound(s) dissolved in the supercritical solvent. If the solubility decreases with an increase in temperature at a constant pressure, the increase in temperature will separate the compound(s) from the supercritical fluid solvent. If the separation is carried out by an auxiliary agent, such as an adsorbent, there is not a significant pressure change, so the differential pressure across the pump is much lower. This type of process implies lower operating costs; however, the recovery of the extract from the adsorbent is often very difficult and can result in a high level of losses of the extract. To prevent this, the adsorption step may be replaced by an absorption step. The extract dissolved in the supercritical solvent is absorbed by a wash fluid in a countercurrent flow using a packed column or spray tower under pressure. The separation of solute(s) by adsorption and absorption has been applied in the decaffeination of coffee (Zosel 1974, 1981). A novel © 2011 by Taylor & Francis Group, LLC

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Reducing pressure Modifying temperature

By modifying thermodynamic properties

By membranes

Extraction vessel

Absorption vessel

Adsorption vessel

By external agents

FIGURE 19.2  Basic schemes for separation of soluble compounds from supercritical fluid.

approach is by using membrane technology as the auxiliary agent. The two major advantages of this approach are (1) the recovery of the extract without depressurization and (2) the separation of compounds with similar solubility in the supercritical fluid based on molecular weight difference. However, the following main issues need to be addressed: proper membrane module design for high pressures with robust sealing for the feed and CO2 streams, and a good understanding of physical/chemical interactions between membrane, supercritical CO2, and the compounds to be separated. Supercritical extraction can be applied to a solid, liquid, or viscous matrix. Most of the development and industrial implementation in supercritical fluid extraction has been performed on solid feed materials. More emphasis will be presented in this chapter on the extraction of compounds from a solid matrix, as most applications of supercritical fluids on cereals are done with a solid matrix.

Supercritical Fluid Extraction of Compounds from a Solid Matrix A general flow diagram of an industrial supercritical extraction process from solids is shown in Figure 19.3. The solvent is subcooled prior to the pumping to assure a liquid phase and to avoid cavitation issues. It is then pressurized and heated above its critical point to the desired extraction pressure and temperature prior to entering the extraction vessel. The extraction vessel, which is filled with the feed material, is electrically or water heated to maintain the extraction temperature. The supercritical solvent flows through the fixed bed and the soluble compounds are extracted from the feed material. The supercritical fluid with the dissolved extract leaves the extraction vessel from the top, and passes through a pressure reduction valve. As the solvent power decreases with pressure reduction, the compound(s) precipitate. To assure total precipitation, the supercritical solvent is heated above the saturation temperature to reach the gas phase. Under those conditions the solvent power is negligible. The extracted material is collected in a separator while the solvent in a gas phase leaves the separator vessel from the top and is © 2011 by Taylor & Francis Group, LLC

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Condenser Back pressure regulator

Vaporizer

Separator Receiver

Extraction vessel

Cosolvent pump

Preheater

Supercritical fluid pump

Precooler

FIGURE 19.3  Flow diagram of a supercritical extraction from solids.

recirculated through the CO2 recycling system and back to the extraction vessel. Once the raw material is fully extracted, the following steps are required in the extraction vessels: • • • • • •

Depressurization Opening of the extraction vessel Unloading the spent material Loading with fresh material Closing the extraction vessel Pressurizing to operating conditions

Currently, supercritical fluid extraction of a solid feed material can only be carried out in a batch process. Generally the solid feed material is handled by using preloaded baskets. From an industrial or commercial point of view, the use of only one extraction vessel, even with a quick opening closure allowing for a rapid opening and closing, is not recommended. Therefore multiple extraction vessels operating in parallel or in sequence are preferred. Figure 19.4 shows a general scheme of a cascade extraction with three extraction vessels. As the CO2 passes sequentially through the vessels, fresh supercritical solvent will first extract the raw material in the first vessel, and then pass through the second. Once the first vessel is fully extracted, it will be taken off-line and the third vessel will be brought online. In this way, fresh solvent will then pass through the second vessel and then into the third while the first is being depressurized, emptied, and recharged with fresh material. This cascade flow allows for higher solvent loading (amount of material extract/amount of solvent). The objective is to maximize the solvent loading (i.e., maintain the supercritical solvent saturated or close to its saturation point). Since the individual extractors are operated batch wise, it is critical to minimize the charge and discharge cycle times. Therefore a cap automation mechanism with a quick opening closure, coupled with fast depressurization, and an efficient unloading/loading sequence are the critical process and design aspects of a supercritical extraction plant. The equipment design and selection for a supercritical fluid processing plant requires consideration of some parameters and specifications that are unique to this © 2011 by Taylor & Francis Group, LLC

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Supercritical fluid with or without cosolvent FIGURE  19.4  Schematic diagram of a cascade operation of multiple extraction vessels for supercritical extraction of solids.

type of plant. Many components of the plant are not available or not easily found for the specific applications, operating, or design conditions of the plant or process requirements. Unique conditions may require special material of construction. Martinez and Vance (2008) have reviewed in detail the design of the main components of the supercritical processing plants.

Processing Parameters in the Supercritical Extraction of Solids Parameters affecting the supercritical fluid extraction of solids are listed in Table 19.2. The influence of the process parameters can be summarized as follows: • Solubility of compounds increases by increasing the extraction pressure at constant temperature. • At pressure close to the critical pressure, the solubility of the compounds increases by decreasing the temperature. However at high pressures, the solubility of compounds increases by increasing the temperature. This crossover effect is due to the competing influences of the reduction in solvent density and the increase of the vapor pressure. The latter has a marked influenced at higher pressures. Additionally, high temperatures decrease the viscosity of the solvent and liquids that favors the mass transfer rate. The pressure at which the crossover effect occurs depends on the type of compounds to extract. The crossover range for most of the compounds takes place between 20 and 35 MPa. • Separation parameters. In general the separation conditions are carried out at 5–6 MPa. However the operating conditions will depend on the solubility of the compounds at different pressures and temperatures as well as whether a fractionation of extract is carried out by sequential depressurization steps. For essential oils or volatile fractions, the separation takes place at 3–5 MPa and low temperatures to maximize the recovery of the top-note components. For oils, the separation can take place at 15–20 MPa due to their low solubility in supercritical CO2 under those conditions. This will have a direct impact on the operating costs. © 2011 by Taylor & Francis Group, LLC

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TABLE 19.2 Processing Parameters in the Extraction of Solids Related to feedstock • Particle morphology and size • Moisture • Chemical reactions to release the compounds to be extracted • Cell disruption • Pelletization Related to process parameters • Extraction conditions • Pressure • Temperature • Batch time • Solvent flow rate • Solvent/feed ratio • Linear velocity • Separation conditions • Pressure • Temperature Related to operation • Extraction • Constant conditions • Fractional extraction • Sequential pressure increase • Sequential solvent polarity increase • Separation • Modifying thermodynamic properties of the solvent • Single separation stage • Sequential depressurization • By external agent • Adsorption • Absorption

• Solvent/Feed ratio. This will depend on many factors, such as concentration of the solute in the feed material, solubility in the supercritical solvent, type of feed material, and distribution of the compound in the feed material. Low solvent/feed ratios imply lower operating costs and higher production capacity. Generally the industrial processes target solvent/feed ratios lower than 30. However, a higher solvent/feed ratio will be justified for high value-added products. In specific cases, solvent/feed ratios higher than 100:1 have been reached for commercial applications. • Solvent flow rate. High solvent flow rates imply high operating and capital costs. However, high flow rates could increase production capacity. The solvent flow rate or the residence time of the solvent in the extraction vessel must be optimized. A high residence time implies a long batch time. Conversely, a short residence time may result in a shorter contact time between the solvent and solute resulting in a loading of the solvent much lower than the saturation concentration at the selected operating conditions. Linear velocities ranging from 1 to 5 mm/s are commonly used in the supercritical fluid extraction process. • Particle size/shape. The size and morphology of the solid material have a direct effect on the mass transfer rate. In general an enlargement of the surface area will increase the extraction rate. Therefore the smaller particle size or geometry such as flakes will favor higher mass transfer, decreasing the batch time as well as the diffusion controlled process. If the soluble substances are located in rigid structures inside of the solid matrix, the size reduction will break this structure so it will be easily accessible for the solvent. However, very small particles can result in a channeling effect, which will decrease the extraction rate. The particle size will © 2011 by Taylor & Francis Group, LLC

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need to be evaluated case by case based on the type of material to be processed. In the case of processing spices and seeds, the particle size generally used is between 30 and 60 mesh. • Moisture. As with particle size, moisture content will need to be evaluated case by case. Generally high moisture content is not desirable, as it will act as a mass transfer barrier. On the other hand, the moisture will expand the cell structure facilitating the mass transfer of the solvent and the solute through the solid matrix (e.g., in seeds and beans). The influence of moisture between 3 and 10% has generally negligible impact on mass transfer of edible oil from seeds.

Supercritical Fluid Extraction of Compounds from a Liquid Feed When the feed material is in a liquid state, the extraction is typically carried out in a countercurrent column. The dense material (liquid) is introduced from the middle or the top of the column while the material with lower density (solvent) is introduced from the bottom of the column. This continuous process leads to lower operating costs compared to the extraction from a solid matrix. A general process flow diagram is shown in Figure 19.5. The separation steps and regeneration of the solvent are similar to the extraction from solids.

Solvent recirculation

Separator Reflux

Extract Countercurrent column

Feed Supercritical fluid

Raffinate FIGURE 19.5  Flow diagram of a supercritical extraction from liquid.

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Similar to the conventional column processes, the contact between phases is enhanced by random or structured packing material. Additionally, a reflux of the extract will improve the selectivity in the extraction process. The extract and solvent leave the column from the top, while the heavier material or raffinate is collected from the bottom. The countercurrent column is heated electrically or with a water jacket, and the extraction process can take place at a constant temperature or with a controlled temperature gradient. The latter process will provide an internal reflux that will increase the selectivity. Process design is based on phase equilibrium data, which determine the number of theoretical stages necessary to perform a specific separation. The height of the column, which is related to mass transfer or height equivalent to a theoretical plate (HETPS), and diameter of the column determine the capacity. The latter parameter is related to hydrodynamic behavior of the mixture in contact with the packing. In cases where the viscosity of the liquid is very high, the extraction process requires intensive and uniform contact between the feed and the solvent. This contact can be carried out by mechanical mixing or by nebulizing the viscous material through a nozzle (Martinez and Vance 2008).

Supercritical Fluid Processing of Cereals There is a wide variety of bioactive compounds in cereals. They are mainly concentrated in the bran and germ of cereal grains, while the endosperm, which is the main component of the refined products, is virtually devoid of these compounds (Kamal-Eldin 2007). The main focus will be on polyunsaturated fatty acids (PUFAs), tocols, sterols, sterol esters, and carotenoids. These compounds are of particular interest as they are soluble in CO2, the preferable supercritical solvent. In order to determine if a compound is extractable with a supercritical solvent or if the supercritical solvent is sufficiently selective to fractionate or separate a mixture of compounds, the following thermodynamic data are required: the solubility of the specific compound in the supercritical fluid as a function of pressure, temperature, and solute concentration; partition coefficients; and selectivity or separation factors. The solubility is strongly dependent on the operating pressure and temperature (i.e., solvent density); as well as the physical and chemical properties of the solute, such as molecular weight, vapor pressure, molecular structure or polarity. Another important factor to be considered is the molecular interaction in the supercritical phase as well as within the phase being extracted. A very extensive database of phase equilibria and solubility data for binary systems has been generated over the last two decades. Guclu-Ustundag and Temelli (2000, 2004) have reviewed and correlated the solubility of major and minor lipid compounds.

Amaranth Amaranth grain contains significant amounts of squalene. It is considered to be a significant alternative to marine animal sources of squalene. A study of amaranth grain of 104 genotypes from 30 species revealed oil content ranging from 1.9 to 8.7% (average 5%). The average content of the major fatty acids in amaranth oil were 21.3% of palmitic acid, 28.2% of oleic acid, and 46.5% of linoleic acid. The concentration of squalene in amaranth oil ranged from traces to 7.3% (average 4.2%) and the average concentration of squalene in seeds was 2.13 mg/g seed (He et al. 2003). Although amaranth grain is an excellent source of high quality protein, the application of supercritical fluid technology in the processing of amaranth has been focused in the extraction of amaranth oil and the concentration of squalene. He et al. (2003) studied the pressure (15–30 MPa), temperature (40–70°C), CO2 flow rate (1–5 L/ min) as well as the particle size effect on the extraction of oil and squalene from amaranth grain. The highest extraction yield (91%) was obtained using the smallest particle size ( 87% were achieved in all cases. The supercritical defatted meal had a significantly higher flavor score than the untreated CDG. The difference in the flavor score could be related to fat content. Untreated CDG had a moderate fermented flavor, while in all the supercritical treated samples the intensity of the fermented flavor was reduced. The highest flavor score was obtained when CO2 + water was used at 68 MPa and 80°C. Similar results were reported by Wu et al. (1994) in reducing the flavor intensity of the fermentation off-flavor from corn gluten meal, a product of the wetmilling process, by using supercritical CO2. This meal has a high protein content (>60%); however, the undesirable flavor prevents its use in food. Processing the meal at 68 MPa and 80°C with a particle size of 637 µm obtained the lowest fermented flavor intensity. Rónyai et al. (1998) studied the effect of supercritical extraction (CO2, CO2 + ethanol) on the chemical composition of extracted corn germ oils and meals from wet-milled corn germ, as well as the functional properties (foaming activity and stability, emulsifying properties, water and oil absorption, and protein solubility) of corn germ protein isolates obtained from defatted meals with alkaline extraction. The use of ethanol as a cosolvent improved the functionality of the protein and is related to the phospholipid content. Studies on the extraction of corn oil using supercritical CO2 are summarized in Table 19.3. Based on the data reported on the extraction of corn oil, extraction efficiency, chemical composition, and quality of the supercritical extracted oil is summarized below. Extraction yield. List et al. (1984) compared the crude oil obtained from wet-milled and dry-milled corn germ with hexane prepress and expeller press, respectively. Supercritical extracted oils had lower neutral oil losses and lighter color than the conventionally processed oils. Extraction yield of supercritical extracted oil from wet-milled corn germ and dry milled corn germ was 43.3 and 22.9%, respectively. Winkler et al. (2007) observed comparable extraction yields of oil from CDG using supercritical CO2 (12.5%) and hexane (12.67%). However, the higher extraction yield was achieved using ethanol (32.73%) due to its polar nature, which results in the extraction of lipid and non-lipid components. Oil extracted with ethanol became very viscous and nonhomogenous after solvent removal. The oil displayed a dark brown color, while the supercritical and hexane extracted oils had a bright yellow color. Moreau et al. (1996) carried out the extraction from commercial corn fiber using hexane and supercritical carbon dioxide. Slightly lower yields were obtained with CO2 (2.91%) compared to hexane (3.33%). Fatty acid composition. There is no significant difference in the fatty acid composition of CO2 extracted oil and commercial oil. Free fatty acid. Free fatty acid content in supercritical extracted oil from wet-milled (1.15%) and dry-milled (0.5%) corn meal is lower compared to the hexane prepress (1.2%) and expeller (0.7%), respectively (List et al. 1984). Phospholipids. Supercritical extracted oil has a negligible amount of phospholipids, while hexane or expeller oils needs to be refined. © 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC

— 420 — — 10–48

2.5 60 1000 1000 1000

8 — 13 8 —

Moisture (%)

— 90 2000 2000 —

Vessel Volume (ml)

a

Percentage as compare to hexane extratable oil.

Particle Size (mm)

Sample Size (g)

Com

Supercritical Extraction of Corn Oil

TABLE 19.3

55 63, 68, 81 55, 82 55 30

Pressure (MPa) 80 85, 100 50 50 42

Temperature (°C) 2 L/min 45.75 g/min 33 g/min 18 L/min —

CO2 Flow Rate

Process Parameters Studied

60 30 — — —

Run Time (min) 12.5 99%a 21, 43 24.00 95%a

(%, w/w)

Extraction Yield

1.71 — 0.8–1.8 — —

Tocols (mg/g oil)

15.8 — — — —

Sterol (mg/g oil)

Bioactive Compounds

Reference Winker et al., 2007 Wu et al., 1990 List et al., 1984 Christianson et al., 1984 Ronyai et al., 1998

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Phytosterols. Similar content of phytosterols were obtained in the oil extracted with supercritical (15.8 mg/g of oil) and hexane (16.2 mg/g of oil) from CDG. Ethanol extracts had slightly lower concentration (9.9–11.4 mg/g; Winkler et al. 2007). Ferulate phytosterol ester (FPE). Similar concentrations of FPE were obtained in the supercritical (3.75 mg/g of oil) and hexane (3.99 mg/g of oil) extracted oil from CDG, while lower concentrations were obtained with ethanol (1.62–1.98 mg/g of oil) (Winkler et al. 2007). In the case of corn fiber, hexane extracted oils had higher PFE content (6.77%) as compared to supercritical extracted oil (5.38 %; Moreau et al. 1996). Taylor and King (2000) applied a fractional extraction method to corn fiber for the enrichment of FPE. The first step used CO2 to extract most of the trigylcerides and fatty acid phytosterol esters. The next steps used CO2 + ethanol progressively increasing the amount of ethanol (1–10%). In the first step most of the triglycerides (85% of the fraction collected) and fatty acid phytosterol esters (15% of the fraction collected) were extracted. The last fraction mainly consisted of FPE, free fatty acid, free sterols, and minor amounts of diglycerides. The concentration of FPE in the last fraction was 53.36% (101 mg of FPE). Tocols. Only 18% of the tocols content in the whole grain is typically recovered after wet milling, while 73% is typically recovered after dry milling. The tocol content from wet-milled corn germ was slightly lower with CO2 (0.89 mg/g of oil) as compared to hexane (1.0 mg/g of oil) while the tocol content from dry-milled corn germ was higher with CO2 (1.84 mg/g of oil) as compared to expeller (1.69 mg/g of oil; List et al. 1984). Similar quantities of total tocols were present in oil extracted from CDG by CO2 (1.71 mg/g) and hexane (1.8 mg/g; Winkler et al. 2007).

Oats The primary nutritional interest in food oats and health effects has been related to total dietary fiber and β-glucan content in oat products (Kaukovirta-Norja and Lehtine 2008). However, there are other nutritional compounds such as oat oils comprising essential fatty acids, sterols, tocols, and phospholipids that could enhance the nutritional interest of oats. Oat contains the largest amount of lipid of the cereal grains, normally between 7 and 10% (Przybylski 2006). However the lipid content and composition of oat grain depend on multiple factors such as environmental effects, storage, processing, as well as the extraction method used (Zhou et al. 1999). About 80% of the nonstarch lipids are free lipids (i.e., extractable with nonpolar solvents), while the remaining 20% are bound lipids (i.e., extractable with polar solvents). Bound lipids are mainly phospholipids and galactolipids. The majority of the lipids are dispersed in the endosperm (53%), with small quantities in the bran and germ. The fatty acid composition, primarily consists of palmitic (15–26%), oleic (27–48%), and linoleic acid (31–47%; Przybylski 2006). Total tocol concentration ranges from about 15 to 48 mg/kg with α-tocotrienol as the predominant tocol. Both α-tocopherol and α-tocotrienol combined account for 86–91% of the total tocols (Peterson 2001). Most of the tocotrienols are located in the oat endosperm, whereas almost all the tocopherols were located in the oat germ. The application of supercritical fluid technology in the processing of oats has been primarily focused in the following areas: extraction of oat oil, manufacturing of oat lecithin, and characterization of defatted oat bran products. The first reference on supercritical extraction of oat oils was in 1990 (Fors and Eriksson 1990). They compared the oil obtained from two dehulled and milled oat varieties (Magne and Chihuauhuam) by supercritical CO2 and hexane. The fatty acid compositions of the supercritical CO2 and hexane were similar, while the phosphorous content of the hexane-extracted oil was significantly higher, ranging from 380 to 1120 ppm. Alkio et al. (1991) carried out the extraction of neutral lipids with supercritical CO2 from oat crude oil previously obtained by alcohol extraction (2-propanol). The objective was to produce lecithin from oat oil. The crude oat oil contained about 20% of polar lipids. They compared the extraction efficiency between crude oat oil only and crude oil adding a carrier (wheat flour and dextrose). The results indicated that the addition of carriers will not increase the extraction yield, possibly due to channeling. Under © 2011 by Taylor & Francis Group, LLC

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the pressure and temperature ranges studied, 25–60 MPa and 40–55°C, respectively, the lecithin yield obtained ranged from 20.3 to 23.5%. Regarding quality attributes, the oat lecithin had a very pleasant aroma and was crystalline or semiplastic. Chemically, the oat lecithin had significantly higher acetone insolubles and lower phospholipid content compared with crude soy lecithin, 80% versus 60% and 31% versus 50%, respectively. Although the phospholipid content is lower, the antioxidative power was superior. On the other hand, the tocopherols were extracted along with the neutral lipids, which prevent the possible synergistic antioxidative function of tocopherols, phenolic acid compounds, and phospholipids in the lecithin. Kaukovirta-Norja et al. (2008) developed a fractionation process where the lipid removal is carried out by supercritical extraction and the dry fractionation takes place using the defatted raw material. The main fractions obtained were: an oat bran concentrate with a β-glucan content of 40%; a starchy endosperm flour with a protein content of 20–25% and low content in β-glucan (0.4–1.5%); a low fat protein fraction with a protein content up to 75%; an endosperm cell wall concentrate with a β-glucan content higher than 50%; and oat oil and lipid fractions with a total extraction yield of 5.6%. Aro et al. (2007) carried out the extraction and characterization of oat lipids by supercritical fluid technologies. The process consisted of three steps. The first step consisted of the extraction of the nonpolar lipids by using supercritical CO2. In this step they used two types of materials: groated oat and oat flakes at constant operating conditions, 45 MPa and 70°C. The extraction efficiency on oat flakes was 1.7 times higher than using groated oats. Approximately 74% of the original lipid content was extracted in this step. In the second step they used CO2 with ethanol as a cosolvent (90:10 mass ratio) at 40 MPa and 70°C to extract the polar lipids. An additional 13% of the original lipid content was extracted in this step. The solution collected was concentrated by evaporation prior to the last step. The last step consisted of a supercritical antisolvent process to precipitate the polar lipids by removing the ethanol using supercritical carbon dioxide at 23 MPa and 70°C. The estimated percentages of phospholipids and glycolipids in the precipitated fraction were 40% and 60%, respectively. Where 70% of the glycolipid fraction corresponds to digalactosyl digylceride, and 30% of the phospholipid fraction corresponds to phosphatidylcholine. Based on the fatty acid profile of the oat flakes, defatted oat flakes, and the antisolvent precipitated, linoleic acid was the major fatty acid in the glycolipids fraction. Stevenson et al. (2007) studied the structures and functional properties, such as crystallinity, thermal, pasting properties, and the water-holding capacity of oat bran concentrate, Nutrim-OB; a jet-cooked oat bran product, with or without lipids removed by supercritical CO2 extraction. In a later work (Stevenson et al. 2008) carried out studies comparing oat bran concentrate that was defatted by supercritical CO2, its subsequent pin-milling and separation into five particle size fractions by air classification.

Rice Supercritical fluid technology has mainly focused on the extraction of oil and bioactive compounds from rice bran. Rice bran is a by-product obtained during the milling process used to produce white rice. It is widely used in the food industry due to its nutritional value. It contains about 12–16% proteins, 7–11% crude fiber, 34–52% carbohydrates, 7–10% ash, and 15–20% lipids (Juliano and Hicks 1996). The saponifiable fraction in rice bran oil consists of about 81–84% triglycerides, 2–3% diglycerides, 1–2% monoglycerides, 2–10% free fatty acids, and 3–4% wax (Xu and Godber 1999), while the unsaponifiable fraction (1.8–3%) comprises γ−oryzanol, tocols, sterols, ferulic acid, and squalene (McCaskill and Zhang 1999; Saunders 1985). The tocols and oryzanol contents are about 800 mg/kg of oil (Qureshi et al. 2000) and 14,000–15,000 mg/kg of oil (Cheruvanky 2003), respectively. In order to commercialize the rice bran oil as edible oil, the inactivation of lipase is required. These active enzymes are released in the milling process and promote the hydrolysis of the oil, producing free fatty acids and glycerol. However, if the enzymes are deactivated, the rice bran is stable and the oil is not degraded. The free fatty acid content of rice bran oil obtained from stabilized bran is below 2%. By using proper refining steps, the free fatty acid content will meet edible oil requirements. Supercritical fluid extraction has been primarily focused in the extraction of rice bran oil, the concentration of tocols and oryzanol fractions, as well as in the deacidification of rice bran oil. The work reported in the first two topics is summarized in Table 19.4. Based on the data reported © 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC

— — — — — — —

20 20 150 300 5 10, 35 35

— 3.10 — 8.48 6.00 10.00 —

Moisture (%)

50 250 1000 1000 — 250 —

Vessel Volume (ml)

35, 52, 69 15, 35 30 24 48–63 23, 35 30

a

40, 60, 80 40 35 40 70–100 40, 50 40

Temperature (°C) 1.08 g/min 6–10 L/min 20.5 g/min 3.5 kg/h — 10–14 g/min —

CO2 Flow Rate

Process Parameters Studied

Pressure (MPa)

Percentage as compare to hexane extratable oil.

Particle Size (mm)

Sample Size (g)

Ricebran

Supercritical Extraction of Rice Bran Oil

TABLE 19.4

60, 120, 240 20, 80 60 240 90 — —

Run Time (min) 24.65 22 17.98 96a 19–20.4 16–19 16, 17

(%, w/w)

Extraction Yield

1050–1228 284 — 420 — — —

Tocols (mg/g oil)

18 11 — 18.08 — 10, 13 15

Oryzanol (mg/kg oil)

— — 7.25 27.53 — — —

Sterol (mg/ kg oil)

Bioactive Compounds

Perretti et al. 2003 Zhao et al. 1987 Ramsay et al. 1991 Shen et al. 1997 Kuk et al. 1998 Wang et al. 2008 Chen et al. 2008

Reference

Supercritical Fluid Extraction of Bioactive Compounds from Cereals 399

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extraction efficiency, chemical composition, and quality of the supercritical extracted oil are summarized below. Extraction yield. Rice bran oil can be effectively extracted using supercritical CO2, however, it is conventionally extracted with hexane. The extraction yield by hexane extraction is about 20%, while the supercritical extracted oil is about 17–18% (Ramsay et al. 1991). Selecting high operating pressures and temperatures as process parameters results in shorter extraction time and less CO2 required. For instance, by operating at 69 MPa and 80°C, 95% of the oil was extracted in 1 hour. In order to get a similar extraction yield at 34.5 MPa and 40°C, a 4 hour batch was required (Perretti et al. 2001). Additional solvents, such as pure isopropanol, have been studied showing comparable extraction yields to hexane (Proctor and Bowen 1996). Fatty acid composition. There is not a significant difference in the fatty acid composition between hexane and supercritical extracted oil. Free fatty acid (FFA). FFA content in the hexane extracted oil is higher as compared to supercritical extracted oil. Due to the solubility difference between FFA and triglycerides in supercritical CO2, it is possible to reduce significantly the FFA content in the rice bran oil. Zhao et al. (1987) carried out fractional extraction by applying a gradient pressure (14–34 MPa) at a constant temperature (40°C). The FFA was primarily extracted at low pressures. The FFA content at 34 MPa was only 4.4% as compared to oil extracted by one CO2 step, which was 8.8% or hexane extracted oil which was 11.9%. Shen et al. (1997) reduced the FFA content by sequential depressurization. The lighter components, such as FFA and water, were collected in the second separator. By using this method, they were able to reduce the FFA content to 50%. Another alternative to remove the FFA of crude rice bran oil is by using a countercurrent column. Operating at low pressure and high temperature achieves the removal of FFA while minimizing triglycerides and phystosterol losses (Dunford and King 2000). Oryzanols. These compounds are derivatives of phytosterols and ferulic acid. The four major γ-oryzanols are cycloartenyl ferulate, 24-methylenecycloar-tanyl ferulate, campesteryl ferulate, and β-sitosteryl ferulate (Xu and Godber 1999). Concentrations of 18 mg/g of oil were reported by Perretti et al. (2003) and Shen et al. (1997). Concentrations of oryzanol three times higher than that found in high oryzanol rice bran oil commercially available was obtained by a phystosterol enrichment process using a countercurrent column (Dunford and King 2000). Tocols. The higher concentration of tocols is obtained at high pressures and temperatures. Tocopherol concentration increased from 1176 mg/kg of oil at 34.5 MPa and 40°C to 1228 mg/ kg of oil at 69 MPa and 80°C (Perretti et al. 2001). At even lower operating pressures (24 MPa) about 90% (wt extracted by CO2/wt extracted by hexane) of α-tocopherol was extracted (Shen et al. 1997).

Rye Rye has been mainly consumed in the form of bread or flour. Rye mainly consists of starch (73%), protein (7%), lipids (1%), and ash (1%; Verwimp 2004) Interest in rye has grown because of the presence of alkylresorcinols. Alkylresorcinols are phenolic lipids that occur in rye at concentrations of 360–3200 µg/g of dry matter (Ross et al. 2003). Francisco et al. (2005a) reported the extraction of the lipid fraction and alkylresorcinols from rye bran using supercritical CO2. The extraction was done on rye flakes using supercritical CO2 at 35 MPa and 55°C. The extract obtained was only 35% as compared to the acetone extraction but the alkylresorcinol content was negligible. Alkylresorcinols are very polar and high molecular weight compounds. Therefore they have very low solubility in pure CO2. In order to extract alkylresorcinols, the use of cosolvent is required. Extract yield increased by 75–80% by using 10% methanol or ethanol, respectively, at 30 MPa and 55°C as compared to acetone. However the alkylresorcinols content was lower by 23% when using 10% ethanol and 31% when using 10% methanol as compared to acetone. In a later work, Francisco et al. (2005b) carried out an extraction at 35 MPa and 55°C with two fractionation steps at 10 and 5 MPa. © 2011 by Taylor & Francis Group, LLC

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They used a binary mixture of CO2 and 5% ethanol as a solvent and the extraction yield was 80% as compared to acetone. Further, alkylresorcinols homologues were separated in two cyclones by using sequential depressurization. The first depressurization step took place at 10 MPa and 40°C while the second depressurization step took place at 5 MPa. About 47% of alkylresorcinols (C23 or higher) were collected in the first separator and about 27% were collected in the second separator.

Wheat Supercritical fluid technology has mainly focused on the extraction of oil and bioactive compounds from wheat germ. Wheat germ is a by-product of the wheat-milling industry. Wheat germ is separated to minimize the possibility of rancidity and increase the storage shelf-life of the flour and its palatability. Wheat germ constitutes about 2–3% of the wheat grain and contains about 8–14% oil (average 10%; Megahad and El Kinawy 2002). The bioactive compounds include tocols, phenolics (ferulic acid and anillic acid in free form and glycoflavones), and carotenoids (lutein, zeaxanthin, and β-carotene; Gelmez et al. 2009). Wheat germ oil has a high content of unsaturated fatty acids (about 80%). In the total fatty acid content, 15–17% corresponds to oleic acid, 57–58% to linoleic acid, and 6–7% to linolenic acid. Additionally wheat germ oil has a high content in unsaponifiable matter (3.5–6%; Bockisch 1998). The unsaponifiable matter consists mainly of phytosterols and tocols. The most common sterols in cereals are β-sitosterol and campesterol. Wheat germ oil is reported as the vegetable oil richest in tocol, up to 2500 mg/kg of oil, which is about 60% α-tocopherol (Kamal-Eldin 2007). Generally the extracted wheat germ oil needs to be refined, due to the high FFA content. This could vary from 5% up to 25% depending on germ separation conditions, germ storage, and oil extraction (Wang and Johnson 2001). Megahad and El Kinawy (2002) reported that the FFA content increased from 3.86 to 6.1% during the storage of wheat germ for three weeks. On the other hand, they did not observe significant oil oxidation during germ storage prior to extraction. Studies on the extraction of wheat germ oil using supercritical CO2 are summarized in Table 19.5. Based on the data reported, extraction efficiency, chemical composition, and quality of the supercritical extracted oil are summarized below. Extraction yield. Higher extraction yields were obtained by hexane than supercritical CO2 extraction. This difference is attributed to the lower selectivity of hexane than CO2, leading to a higher concentration of bioactive compounds in the supercritical CO2 extract. The extraction yield of supercritical extracted oil increases with an operating pressure at a constant temperature. No significant difference on extraction yield was observed based on the particle size. Fatty acid composition. There are no significant differences in fatty acid composition of hexane, liquid, and supercritical CO2 extracted oil. Free fatty acid. Hexane extracted oil has slightly higher (21–27%) FFA content than that extracted with supercritical CO2 (Eisenmenger et al. 2006; Zacchi et al. 2005). However, in both cases a refining step to decrease the fatty acid content is required. Phospholipids. As expected, phospholipid content in supercritical CO2 extracted oil was below the detection limit because solubility of phospholipids in supercritical CO2 is negligible. Therefore the degumming step is not required. The highest content of phosphorous was found in pressed wheat germ oil followed by oil extracted with hexane (Zacchi, et al. 2005). Eisenmenger and Dunford (2008) reported that the highest content of phospholipids in commercial wheat germ oil was phophatidyl inositol + phosphatidic acid with 61% of the total phospholipids (19.8 mg of phospholipids/g of oil). Tocols. Supercritical extracted oil has a higher tocols content than hexane extracted oil. The selectivity of the supercritical extraction process allows tailoring of the extracts, so that wheat germ oil with a high content of tocopherol or high yields could be obtained. By manipulating the extraction pressure, temperature, and solvent/feed ratio it is possible to obtain extracts with different tocol concentrations. The highest selectivity of the extraction process for tocols takes © 2011 by Taylor & Francis Group, LLC

© 2011 by Taylor & Francis Group, LLC

0.505

5

—

Small flakes Small flakes 0.18–0.25

35

450

 (400 MPa) > (500 MPa). Zhang et al. (2005a) reported a higher extractability of flavonoids from propolis by HPE. Similar results were reported in the extraction of anthocyanins from grape by-products (Corrales et al. 2008), and flavones and salidroside from Rhodiola sachalinensis using HPE (Zhang et al. 2007). Prasad et al. (2009b) indicated that effects of HPE on the extraction yield, total phenolic content, and the antioxidant activity of longan fruit (Dimpcarpus longan Lour.) pericarp. The different solvent effects, solvent concentration (25–100%, v/v), solid-to-liquid ratio (1:25−1:100, w/v) were individually determined using these optimum extraction conditions. With utilizing the various pressures of HPP (200−500 MPa), durations (2.5−30 min), and temperatures (30−70°C), the extraction yield, total phenolics, and scavenging activities of superoxide anion radical and 1,1-dipheny l-2-picrylhydrazyl (DPPH) radical by HPE were determined and compared with those from a conventional extraction. The HPE provided a higher extraction yield and required a shorter extraction time compared to CE. In addition, the total phenolics and the antioxidant activities of HPE were higher than those produced by CE. Table 21.4 shows the effect of thermal (TP) and high pressure treatments on anti-radical power, total phenols, total carotenoid content in tomato purées. © 2011 by Taylor & Francis Group, LLC

439

High Pressure Processing Technology on Bioactives in Fruits and Cereals R1 3‘ B

⊕ O

OH 7

A

C

OH

4‘ 5‘

R2 OH

3

OH OH

O

5

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CH2OR

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FIGURE 21.5  Anthocyanins in grape by-products. (Adapted from Corrales, M., Toepfl, S., Butz, P., Knorr, D., and Tauscher, B., Innov. Food Sci. Emerg. Technol., 9, 85–91, 2008.)

Tokuşoğlu et al. (2010) reported that the total phenolics of table olives increased (2.1–2.5)-fold after HPP (as mg gallic acid equivalent/100 g). Phenolic hydroxytyrosol in olives increased on average (0.8–2.0)-fold, whereas oleuropein decreased on average (1–1.2)-fold after HPP (as mg/kg dwt). Antioxidant activity values varied from 17.238−29.344 mmol Fe2+ /100 g for control samples, and 18.579–32.998 mmol Fe2+ /100 g for HPP-treated samples. In the HPP application of olives, total mold was reduced 90% at 25°C, and it was reduced 100% at 4°C based on the use of the RoseBengal Chloramphenicol Agar (RBCA). Total aerobic-mesofilic bacteria load was reduced 35–76% at 35 ± 2°C based on the use of plate count agar (PCA). Citrinin load was reduced 64–100% at 35 ± 2°C. Citrinin contamination (CITcont) at concentrations of 2.5 ppb and less in table olives degraded by 56%, whereas concentrations of 1 ppb CITcont in table olives degraded 100% (Tokuşoğlu et al. 2010). Corrales et al. (2008) examined the extraction capacity of anthocyanins from grape by-products (Figure 21.5) assisted by HPP and other techniques. The HPP at 600 MPa showed feasibility and selectivity for extraction purposes. After 1 hour of extraction, the total phenolic levels of grape by-product samples subjected to this novel HHP technology was 50% higher than in the control samples (Corrales et al. 2008). From a nutritional prospective, HPP is an excellent food processing technology that has the potential to retain the bioactive constituents with health properties in fruits, cereals, and other foods. HPP-treated foods retain more of their fresh-like features and can be marketed at a premium over their thermally processed counterparts.

ACKNOWLEDGMENT Mention of trade names and commercial products in this publication is solely for the purpose of providing specific information and does not constitute nor imply recommendation or endorsement by the U.S. Army Natick Soldier RD&E Center or any other federal government entity. © 2011 by Taylor & Francis Group, LLC

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REFERENCES Ahmed, J., and Ramaswamy, S. 2006. High pressure processing of fruits and vegetables. Stewart Postharvest Review 1:1–10. Barbosa-Cánovas, G. V., Pothakamury, U. R., Palou, E., and Swanson, B. 1998. Emerging technologies in food preservation. In Nonthermal Preservation of Foods, 1–9. New York: Marcel Dekker. Barbosa-Cánovas, G. V., Tapia María, S., and Pilar Cano, M. 2005. Novel Food Processing Technologies. New York: Marcel Dekker. Cheftel, J. C. 1995. Review: High pressure, microbial inactivation and food preservation. Food Science and Technology International 1:75–90. Cheftel, J. C., and Culioli, J. 1997. Effects of high pressure on meat: A review. Meat Science 46:211–36. Chung, Y. K., Malone, A. S., and Yousef, A. E. 2008. Sensitization of microorganisms to high-pressure processing by phenolic compounds. Chapter 7 in High Pressure Processing of Foods, eds. C. J. Doona and F. E. Feeherry. Blackwell Publishing Ltd, Oxford: U.K. Corrales, M., Toepfl, S., Butz, P., Knorr, D., and Tauscher, B. 2008. Extraction of anthocyanins from grape ­by-products assisted by ultrasonic, high hydrostatic pressure or pulsed electric fields: A comparison. Innovative Food Science and Emerging Technologies 9:85–91. Deliza, R., Rosenthal, A., Abadio, F. B. D., Silva, C. H. O., and Castillo, C. 2005. Application of high pressure technology in fruit juice processing: Benefits perceived by consumers. Journal of Food Engineering 67:241–6. Doona, C. J., Feeherry, F. E., and Baik, M.-Y. 2006. Water dynamics and retrodegradation of ultrahigh pressurized wheat starch. Journal of Agricultural and Food Chemistry 54:6719–24. Doona, C. J., Feeherry, F. E., and Ross, E. W. 2005. A quasi-chemical model for the growth and death of microorganisms in food by non-thermal and high-pressure processing. International Journal of Food Microbiology 100:21–32. Doona, C. J., Feehery, F. E., Ross, E. W., Corradini, M., and Peleg, M. 2008. The quasi-chemical and Weibull distribution models of nonlinear inactivation kinetics. Chapter 6 in High Pressure Processing of Foods, eds. C. J. Doona and F. E. Feeherry. Blackwell Publishing Ltd, Oxford: U.K. Doona, C. J., Ross, E. W., and Feeherry, F. E. 2008. Comparing the quasi-chemical and other models for the high pressure processing inactivation of Listeria monocytogenes. Acta Horticulturae 802:351–7. Dornenburg, H., and Knoor, D. 1993. Cellular permeabilization of cultured plant tissues by high electric field pulses or ultra high pressure for the recovery of secondary metabolites. Food Biotechnology 7:35–48. Feeherry, F. E., Doona, C. J., and Ross, E. W. 2005. The quasi-chemical kinetics model for the inactivation of microbial pathogens using high pressure processing. Acta Horticulture 674:245–51. Gassiot, M., and Masoliver, P. 2010. Commercial high pressure processing of ham and other sliced meat products. In Case Studies in Novel Food Processing Technologies: Innovations in processing, packaging, and predictive modelling. eds. C. J. Doona, K. Kustin and F. E. Feeherry. Woodhead Food Series No. 197, Woodhead Publishing Ltd, Abington Hall, Abington, Cambridge: UK. Guerrero-Beltráni, J. A., Barbosa-Cánovas, G. V., and Swanson, B. G. 2005. High hydrostatic pressure processing of fruit and vegetable products. Food Reviews International 21:411–25. Hayashi, R. 1997. High-pressure bioscience and biotechnology in Japan. In High Pressure Research in the Biosciences and Biotechnology, ed. K. Heremans, 1–4. Leuven: Leuven University Press. Hogan, E., Kelly, A. L., and Sun, D-W. 2005. High pressure processing of foods: an overview. Emerging Technologies for Food Processing, ed. Sun Da Wen, 3–31. Academic Press. Hoover, D. G., Metrick, A. M., Papineau, A. M., Farlas, D. F., and Knorr, D. 1989. Biological effects of high hydrostatic pressure on food microorganisms. Food Technology 43:99–107. Mertens, B., and Knorr, D. 1992. Developments of non-thermal processes for food preservation. Food Technology 46:126–33. Ohio State University. 2009. Extension Factsheet—High Pressure Processing. Available at http://ohioline.osu.edu Patras, A., Brunton, N., Da Pieve, S., Butler, F., and Downey, G. 2009a. Effect of thermal and high pressure processing on antioxidant activity and instrumental colour of tomato and carrot purées. Innovative Food Science and Emerging Technologies 10:16–22. Patras, A., Brunton, N. P., Pieve, S. D., and Butler, F. 2009b. Impact of high pressure processing on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of strawberry and blackberry purées. Innovative Food Science and Emerging Technologies 10:308–13. © 2011 by Taylor & Francis Group, LLC

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Patterson, M. F. 2005. A review: Microbiology of pressure-treated foods. Journal of Applied Microbiology 98:1400–9. Patterson, M. F., Linton, M., and Doona, C. J. 2008. Introduction to high pressure processing of foods. Chapter 1 in High Pressure Processing of Foods, eds. C. J. Doona and F. E. Feeherry. Blackwell Publishing Ltd, Oxford: U.K. Prasad, K. N., Yang, B., Zhao, M., Ruenroengklin, N., and Jiang, Y. 2009a. Application of ultrasonication or high-pressure extraction of flavonoids from litchi fruit pericarp. Journal of Food Process Engineering 32:828–43. Prasad, K. N., Yang, E., Yi, C., Zhao, M., and Jiang, Y. 2009b. Effects of high pressure extraction on the extraction yield, total phenolic content and antioxidant activity of longan fruit pericarp. Innovative Food Science and Emerging Technologies 10:155–9. Qiu, W., Jiang, H., Wang, H., and Gao, Y. 2006. Effect of high hydrostatic pressure on lycopene stability. Food Chemistry 97:516–23. Richard, J. S. 1992. High Pressure Phase Behavior of Multi Component Fluid Mixtures. Amsterdam, The Netherlands: Elsevier. Ross, E. W., Taub, I. A., Doona, C. J., Feeherry, F. E., and Kustin, K. 2005. The mathematical properties of the quasi-chemical model for microorganism growth/death kinetics in foods. International Journal of Food Microbiology 99:157–71. Taub, I. A, Feeherry, F. E, Ross, E. W., Kustin, K., and Doona, C. J. 2003. A quasi-chemical kinetics model for growth and death of Staphylococcus aureus in intermediate moisture bread. Journal of Food Science 68:2530–7. Tokuşoğlu, Ö., Alpas, H., and Bozoğlu, F. T. 2010. High Hydrostatic Pressure Effects on Mold Flora, Citrinin Mycotoxin, Hydroxytyrosol, Oleuropein Phenolics and Antioxidant Activity of Black Table Olives. Innovative Food Science and Emerging Technologies. 11(2), 250–8. Yen, G. C., and Lin, H. T. 1996. Comparison of high pressure treatment and thermal pasteurisation on the quality and shelf life of guava puree. International Journal of Food Science and Technology 31:205−13. Zhang, S., Junjie, Z., and Changzhen, W. 2004. Novel high pressure extraction technology. International Journal of Pharmaceutics 278:471–4. Zhang, S., Xi, J., and Wang, C. 2005a. High hydrostatic pressure extraction of flavonoids from propolis. Journal of Chemical Technology and Biotechnology 80:50–4. Zhang, S., Xi, J., and Wang, C. 2005b. Effect of high hydrostatic pressure on extraction of flavonoids in ­propolis. Food Science and Technology International 11:213–6.

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FOOD SCIENCE AND TECHNOLOGY

Fruit and Cereal Bioactives Sources, Chemistry, and Applications

Interest in bioactive compounds of fruit and cereals has reached a new high in recent years, accentuated by efficiency reports regarding both beneficial and toxical health effects of such compounds. Presenting up-to-date data in an easyto-use format, Fruit and Cereal Bioactives: Sources, Chemistry, and Applications focuses on the chemistry of beneficial and nutritional bioactives and toxicant bioactives from sources of pome, stone, berry, citrus, and subtropical fruits and nuts as well as from various cereals and pulses. It provides a comprehensive and detailed reference guide to major natural beneficial phytochemical bioactives and mycotoxic bioactives in edible fruits and cereals from a biochemical standpoint. FEATURES: • Examines and classifies the bioactive compounds of fruit and cereal types • Includes phytochemical and lipid-based bioactives in fruits and cereals, pseudocereals, and pulses • Covers the mycotoxin bioactives in fleshy and dried fruits, nuts and cereals, and cereal-based snacks • Reviews relevant legal regulations on bioactives of fruits and cereals • Evaluates the function of these bioactive compounds against certain effects in humans Each chapter reviews dietary sources, occurrence, chemical properties, desirable and undesirable health effects, antioxidant activity, evidentiary findings, and toxicity of bioactives based on the fruit and cereal type. The beneficial and nutritional bioactives covered include phytochemicals such as phenolics, flavonoids, tocols, carotenoids, phytosterols, avenanthramides, alkylresorcinols, and some essential fatty acids, while the toxicant bioactives include mycotoxins. A valuable resource for current knowledge and further research, the book offers critical reviews, recent research, case studies, and references.

K10332

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