Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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: 978-1-4398-0665-4 (Hardback) 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. Library of Congress Cataloging-in-Publication Data Fruit and cereal bioactives : sources, chemistry, and applications / edited by Ozlem Tokusoglu, Clifford Hall III. p. ; cm. Includes bibliographical references and index. Summary: “Presenting up-to-date data in an easy-to-use format, this comprehensive overview of the chemistry of bioactive components of fruits and cereals addresses the role of these compounds in determining taste, flavor, and color, as well as recent claims of anticarginogenic, antimutagenic, and antioxidant capabilities. It provides detailed information on both beneficial bioactives such as phenolics, flavonoids, tocols, carotenoids, phytosterols, and avenanthramides and toxicant compounds including mycotoxins; aflatoxins, ocratoxin A, patulin, citrinin, cyclopiazonic acid, fumonisin, and zearalenon. A valuable resource for current knowledge and further research, it offers critical reviews, recent research, case studies, and references”--Provided by publisher. ISBN 978-1-4398-0665-4 (hardcover : alkaline paper) 1. Fruit--Composition. 2. Grain--Composition. 3. Phytochemicals--Physiological effect. I. Tokusoglu, Ozlem, editor. II. Hall, Clifford, III, editor. [DNLM: 1. Fruit--chemistry. 2. Cereals--chemistry. 3. Dietary Supplements. 4. Phytotherapy. 5. Plant Extracts--therapeutic use. WB 430] QK865.F78 2011 664’.8--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

2010044816

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

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 I I 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 vii

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Contents

Part II I  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 I V 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 Index....................................................................................................................................................... 443

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

ix

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. xi

xii

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.

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

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

xiv 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

Part I

Introduction

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 3

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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

Stilbens

Sesamol Sesamin Sesamolin Sesamolinol

Resveratrol Piceatannol Piceid Pinosylvin Rhapontisin Tamoxiphen Derivative Phytoalexins

Tannins Hydrolyzed Condensed

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.

5

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

6

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.

Introduction to Bioactives in Fruits and Cereals

7

(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.

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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.

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 9

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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

Health Promoting Effects of Cereal and Cereal Products

11

(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

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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.

Health Promoting Effects of Cereal and Cereal Products

13

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

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

(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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Bourdon, I., W. Yokoyama, P. Davis, C. Hudson, R. Backus, D. Richter, B. Knuckles, and B. O. Schneeman. 1999. Postprandial lipid, glucose, insulin and cholecystokinin responses in men fed barley pasta enriched with beta-glucan. American Journal of Clinical Nutrition 69:55–63. Brodribb, A. J., and D. M. Humphreys. 1976. Diverticular disease: Three studies. British Medical Journal 1:424–6. Burton-Freeman, B. M., and N. L. Keim. 2008. Glycemic index, cholecystokinin, satiety and disinhibition: Is there an unappreciated paradox for overweight women? International Journal of Obesity 32 (11): 1647–54. Carr, T. P., C. L. Weller, V. L. Schlegel, S. L. Cuppett, D. M. Guderian, and K. R. Johnson. 2005. Grain sorghum lipid extract reduces cholesterol absorption and plasma non-HDL cholesterol concentration in hamsters. Journal of Nutrition 135 (9): 2236–40. Chen, F., P. Cole, Z. B. Mi, and L. Y. Xing. 1993. Corn and wheat-flour consumption and mortality from esophageal cancer in Shanxi, China. International Journal of Cancer 53 (6): 902–6. Egeberg, R., A. Olsen, S. Loft, L. Christensen, N. F. Johnsen, K. Overvad, and A. Tjonneland. 2009. Intake of whole grain products and risk of breast cancer by hormone receptor status and histology among postmenopausal women. International Journal of Cancer 124 (3): 745–50. Fki, I., Z. Sahnoun, and S. Sayadi. 2007. Hypocholesterolemic effects of phenolic extracts and purified hydroxytyrosol recovered from olive mill wastewater in rats fed a cholesterol-rich diet. Journal of Agricultural and Food Chemistry 55 (3): 624–31. Fung, T. T., F. B. Hu, M. D. Holmes, B. A. Rosner, D. J. Hunter, G. A. Colditz, and W. C. Willett. 2005. Dietary patterns and the risk of postmenopausal breast cancer. International Journal of Cancer 116 (1): 116–21. Fung, T. T., E. B. Rimm, D. Spiegelman, N. Rifai, G. H. Tofler, W. C. Willett, and F. B. Hu. 2001. Association between dietary patterns and plasma biomarkers of obesity and cardiovascular disease risk. American Journal of Clinical Nutrition 73 (1): 61–7. Goldin, B. R., H. Adlercreutz, S. L. Gorbach, J. H. Warram, J. T. Dwyer, L. Swenson, and M. N. Woods. 1982. Estrogen excretion patterns and plasma-levels in vegetarian and omnivorous women. New England Journal of Medicine 307 (25): 1542–7. Hamedani, A., T. Akhavan, R. Abou Samra, and G. H. Anderson. 2009. Reduced energy intake at breakfast is not compensated for at lunch if a high-insoluble-fiber cereal replaces a low-fiber cereal. American Journal of Clinical Nutrition 89 (5): 1343–9. Isaacson, C. 2005. The change of the staple diet of black South Africans from sorghum to maize (corn) is the cause of the epidemic of squamous carcinoma of the oesophagus. Medical Hypotheses 64 (3): 658–60. Jacobs, D. R., L. Marquart, J. Slavin, and L. H. Kushi. 1998a. Whole-grain intake and cancer: An expanded review and meta-analysis. Nutrition and Cancer—An International Journal 30 (2): 85–96. Jacobs, D. R., K. A. Meyer, L. H. Kushi, and A. R. Folsom. 1998b. Whole-grain intake may reduce the risk of ischemic heart disease death in postmenopausal women: The Iowa women’s health study. American Journal of Clinical Nutrition 68 (2): 248–57. Jacobs, D. R., M. A. Pereira, K. A. Meyer, and L. H. Kushi. 2000. Fiber from whole grains, but not refined grains, is inversely associated with all-cause mortality in older women: The Iowa Women’s Health Study. Journal of the American College of Nutrition 19 (3): 326S–30S. Jensen, M. K., P. Koh-Banerjee, F. B. Hu, M. Franz, L. Sampson, M. Gronbaek, and E. B. Rimm. 2004. Intakes of whole grains, bran, and germ and the risk of coronary heart disease in men. American Journal of Clinical Nutrition 80 (6): 1492–9. Kahlon, T. S., F. I. Chow, and D. F. Wood. 1999. Cholesterol response and foam cell formation in hamsters fed rice bran, oat bran, and cellulose plus soy protein diets with or without added vitamin E. Cereal Chemistry 76 (5): 772–6. Kasum, C. M., K. Nicodemus, L. J. Harnack, D. R. Jacobs, and A. R. Folsom. 2001. Whole grain intake and incident endometrial cancer: The Iowa Women’s Health Study. Nutrition and Cancer—An International Journal 39 (2): 180–6. Klaunig, J. E., Y. Xu, J. S. Isenberg, S. Bachowski, K. L. Kolaja, J. Z. Jiang, D. E. Stevenson, and E. F. Walborg. 1998. The role of oxidative stress in chemical carcinogenesis. Environmental Health Perspectives 106: 289–95. Koh-Banerjee, P., and E. B. Rimm. 2003. Whole grain consumption and weight gain: A review of the epidemiological evidence, potential mechanisms and opportunities for future research. Proceedings of the Nutrition Society 62 (1): 25–9.

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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.

Health Promoting Effects of Cereal and Cereal Products

17

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.

Part II

Chemistry and Mechanisms of Beneficial Bioactives in Fruits and Cereals

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 21

22

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.

24

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

OH

CH

COOH Benzoic acid derivatives p-Hydroxy benzoic acid

R1

R2

R1

R2

CH COOH

Cinnamic acid derivatives

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

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

25

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 C15H31 C17H35 C19H39 C21H43 C23H47 C25H51 C19H37

Acronym (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.)

26

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

8

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′ 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

3′

6

OH OH

OH HO

OH

O 2

Procyanidin B-1

5′

O 2

OH

6′

OH O 2

HO O

OH 8′′

4

7′′ OH OH

O

OH

Flavans 3′ 4′ Catechin OH OH

OH OH

4

OH

Procyanidin B-3

OH

HO

HO

OH

O

7′′

8′′ OH

O HO

HO

OH

OH

OH

4′

OH

O

OH

2′ 8

O

OH

O

O

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

HO

HO

O

H

HO

H HO

Epicatechin-(4βd 8;2βdO7)-catechin

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

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

28

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

HO O

Metal complexes of 3-hydroxy flavonoids HO

O O Cu+

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).

29

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.

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.

31

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

32

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

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

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).

33

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).

34

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

Alexis Baronessea Oliviaa Barley flourb

4.2 3.6 1.3 7.0

a

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.

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

36

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.

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

1 M Sodium Hydroxide

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.

38

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.

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.

40

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.

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

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

Phenolic Content (mg/kg) of Oat and Oat Products Renger and Steinhart, 2000a Phenolic Compound 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

1 M Sodium Hydroxide

4 M Sodium Hydroxide

3328 503

3282 153

26 32 29 9 57

Mattila, Pihlava, and Hellström (2005)b

Bryngelsson, Dimberg, Kamal-Eldin (2002)

Oat Bran

Oat Flakes

Precooked Oat Flakes

Raw Oatc

Raw Groatc

Raw Hull

Rolled Oat

Rolled Oat

Drum Dried Rolled Oatc

5.4 330 12 22 90 28 24 140

3.1 25 ND 16 55 20 18 110

3.6 250 ND 16 52 20 17 110

3.0 2.5 3.0

2.55 1.3 0.5

ND 1.5 6.7

1.48 2.18 0.54

ND 3.17 0.72

ND 1.4 0.5

2.5

0.75

4.6

1.24

3.46

1.8

Phytochemicals in Cereals, Pseudocereals, and Pulses

Table 3.9

84 132 47 107 96

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.

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.

45

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.)

46

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

Phytochemicals in Cereals, Pseudocereals, and Pulses

47

(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.

48

Table 3.11 Phenolic Content (mg/kg) of Rice and Rice Products Renger and Steinhart, 2000a Phenolic Compound

1 M Sodium Hydroxide

4 M Sodium Hydroxide

Long Grain White Rice

Long Grain Brown Rice

Cooked Long Grain Brown Rice

3046 310

3823 310

120 38 13 17 ND ND 9

240 76 15 20 ND 8 17

92 29 4 7 ND 3 4

ND ND ND ND ND

Sosulski, Krygier, L. Hogge (1982)c Free

Soluble Esters

Bound

2.7 1.3 1.9 ND ND 1.3

9.6 ND 2.7 ND 0.2 0.8

63.1 ND 0.4 ND ND ND

Zhou et al. (2004)d Alkaline Extraction

Enzymatic Hydrolysis

259 71

255 70

2.9 3

2.6 12

32 36 15 41 57

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. a

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

Mattila, Pihlava, and Hellström (2005)b

49

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

50

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 (<30mg/ kg) in which greater than 60% is caffeic acid (Mattila, Pihlava, and Hellström 2005). The phenolic acid composition of rye is approximately 1300–1600 mg/kg. Rye contains approximately eight phenolic acids; however, ferulic, sinapic, and p-coumaric acids are the three major phenolic acids (Table 3.13; Andreasen et al. 1999, 2000; Mattila, Pihlava, and Hellström 2005). In addition to phenolic acids, four diFA analog exist (Andreasen et al. 2000, 2001). Renger and Steinhart (2000) reported that strong alkaline hydrolysis may be required to effectively extract diFA (Table 3.14). In addition to cultivar, phenolic acid levels can be affected by the growing conditions. Andreasen et al. (2000) reported that phenolic acids were significantly lower in the 1998 harvest year compared to 1997, which was a warmer and drier year. However, these authors attributed the major differences in the phenolic acids to the rye cultivar. Weidner et al. (1999) reported differences in the phenolic acid composition in the caryopses of rye sensitive to sprouting (i.e., shallow dormancy cultivar Dańkowskie Złote) and less sensitive to sprouting (i.e., deeper dormancy cultivar Amilo). The Amilo cultivar had significantly more soluble phenolic esters (152 mg/kg vs. 90 mg/kg) than the Dańkowskie Złote cultivar. Thus, the differences in the phenolic composition of the different rye cultivars observed by Andreasen et al. (2000) may correspond to the observations made by Weidner et al. (1999). Phenolic acids are concentrated in the outer layers of the seed. Rye bran contained three times more phenolic acids than the whole rye (Andreasen et al. 2001; Mattila, Pihlava, and Hellström 2005). This difference was even greater (17 times) when the phenolic acids in bran were compared to the rye flour Table 3.13 Phytochemical Composition (µg/g) of Rye Bread Made From Flour With Different Extraction Rates Breada Phytochemical

100%

95%

90%

70%

Total phenolic contentb Total flavonoids contentc Free phenolic acids Tocopherols Tocotrienols

1720 171 55 3.42 3.38

1680 196 60 1.01 1.06

1760 177 53 1.19 0.87

1290 98 33 1.27 0.38

Source: Michalska, A., Ceglinska, A., Amarowicz, R., Piskula, M., Szawara-Nowak, D., and Zielinski, H., J. Agr. Food Chem., 55(3), 734–40, 2007. a The breads made with rye flour with extraction rates of 100, 95, 90, or 70%.

Changes in Phenolic Content (mg/kg) of Rye During Bread Making and as Affected by Alkaline Hydrolysis Adapted from Hansen et al. 2002 Stage of Bread Making Phenolic Acid Ferulic Sinapic p-coumaric Free ferulic Ferulic Dimersa 8-5-diFA 5-5-diFA 8-8-diFA 8-O-4-diFA 8-8-aryl-diFA 8-5-benzofurandiFA

Adapted from Renger and Steinhart 2000 Alkaline Hydrolysis

Rye Flour

Imitation Sourdough

Dough after Mixing

Dough after Proofing

Bread Crumb

1 M Sodium Hydroxide

4 M Sodium Hydroxide

1079 76 35 3.3

982 89 34 8.1

1022 77 33 10.4

1006 79 35 12.2

1000 82 33 16.1

7183 NR 665 NR

9193 NR 1439 NR

195 72 NR 32 NR 86

171 60 NR 22 NR 83

184 72 NR 30 NR 81

188 65 NR 30 NR 79

186 68 NR 27 NR 76

96 36 18 6 66 NR

117 123 188 124 162 NR

Phytochemicals in Cereals, Pseudocereals, and Pulses

Table 3.14

Note: NR = not reported. 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; 8-5-­ benzofuran-diFA: Ferulic acid 8-5-benzofuran-dehydrodimer; 8-8-aryl-diFA: Ferulic acid 8-8-aryl-dehydrodimer.

51

52

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 3.15 Phytochemical Composition (µg/g) of Rye Bread Made From Flour With Different Extraction Rates Breada Phytochemical Total phenolic content Total flavonoids contentc Free phenolic acids Tocopherols Tocotrienols b

100%

95%

90%

70%

1720 171 55 3.42 3.38

1680 196 60 1.01 1.06

1760 177 53 1.19 0.87

1290 98 33 1.27 0.38

Source: Michalska, A., Ceglinska, A., Amarowicz, R., Piskula, M., Szawara-Nowak, D., and Zielinski, H., J. Agr. Food Chem., 55(3), 734–40, 2007. a The breads made with rye flour with extraction rates of 100, 95, 90 or 70%. b µg ferulic acid equivalence. c µg (±)-catechin equivalence.

(Andreasen et al. 2001). Similar trends in the distribution of ferulic dimers were observed in whole rye, rye flour, and rye bran (Andreasen et al. 2001). The pericarp of rye had high levels of ferulic (654 mg/100 g) and p-coumaric (84 mg/100 g) acids (Glitsø and Knudsen 1999). Using the same rye sample, ferulic acid levels of 193, 93, and 17 mg/100 g were found in the aleurone layer, whole rye, and endosperm, respectively (Glitsø and Knudsen, 1999). Total phenolic content, as measured by µg (±)-catechin/mg extract, also supports the higher phenolic content in the pericarp (Zieliński and Kozłowska 2000). Thus, milling or fractionation could be used to enhance the phenolic contents in rye fractions. Michalska et al. (2007) reported that the flour extraction rate from rye affected the total phenolic and flavonoid contents, free phenolic acids and tocols (Table 3.15). Only the rye breads made with the 70% extraction flour had significantly lower phytochemical levels. The free phenolic levels in the breads were higher than typical rye flour. The authors attributed the free phenolic acids to the baking process (Michalska et al. 2007). Although the specific stage of the baking process was not identified, the fermentation was the likely cause for the increased free phenolic content. Hansen et al. (2002) showed that total phenolics decreased from 1575 to 1441 mg/kg using an imitation sourdough system and that other steps of the bread making process affected phenolic contents (Table 3.14). These researchers also observed significant increases in the free ferulic acid contents. Additional support that fermentation increases free phenolic acid contents was reported by Katina et al. (2007). Fermentation of the rye bran by lactic acid bacteria and yeast enhanced the free phenolic acid levels up to 90% (Katina et al. 2007). The observed increase in the free ferulic during baking (Hansen et al. 2002; Michalska et al. 2007) can be attributed to the breakage of chemical bonds between the phenolic acids and the cell wall material during heating. Gumul and Korus (2006) reported that ferulic acid contents increased in extruded rye bran compared to the original rye flour. They noted that extrudates produced at the higher extrusion moistures (20%) and temperatures (180°C) combinations had higher ferulic acid contents. However, changes (i.e., both increases and decreases) in other phenolic acids (e.g., caffeic, sinapic) appeared to be less affected by the extrusion conditions than ferulic. In fact, the changes in phenolic acid contents were cultivar dependent suggesting that bran structure might influence changes in phenolic content (Gumul and Korus 2006). However, an increase in phenolic acid content occurred after extrusion regardless of cultivar.

Phytosterols Other phytochemicals important to rye include phytosterols and alkylresorcinols. Zangenberg et al. (2004) reported the composition of phytosterols fell within a range of 761–1007 mg/kg and was cultivar dependent. Normén et al. (2002) reported the phytosterol content of common foods consumed in Sweden. Rye flour, crushed rye, and light and dark rye breads had phytosterol contents of 86, 69, 51, and 51 mg/100 g of edible portion, respectively. In all products, sitosterol (55–60%) was the major sterol followed by campesterol (20–22%) and the stanols sitostanol (10–13%) and campestanol (6–8%). Stigmasterol contents lower than 4% were also reported (Normén et al. 2002). Lampi et al. (2004) and

53

Phytochemicals in Cereals, Pseudocereals, and Pulses

Nyström et al. (2007) reported phytosterol contents of 110 and 99.5 mg/100 g of rye. In both studies, sitosterol accounted for 49–51% of the total sterols followed by campesterol (15%), sitostanol (10.6%), campestanol (7.7%), and stigmasterol (3.3%), which is in close agreement with Normén et al. (2002) and Piironen, Toivo, and Lampi (2002). Iwatsuki et al. (2003) reported that rye bran oil contained 830 mg/100 g and 2440 mg/100 g of sterol ferulates and sterols, respectively. Pearling of rye produced fractions that had phytosterol contents of 159 and 162 mg/100 g, indicating that pearling was an effective method to enhance phytosterol contents of a rye having an initial phytosterol content of 88.6 mg/100 g (Lampi et al. 2004). Nyström et al. (2007) also reported that milling was an effective means to produce phytosterol-enhanced fractions. The fractionation of the whole rye, with a 99.5 mg phytosterols/100 g, resulted in fractions with significantly higher phytosterols. The bran fraction had the highest phytosterol content at 177 mg/100 g. Regardless of the fractionation process, the individual phytosterol distribution remained consistent with that observed in the whole rye (Lampi et al. 2004; Nyström et al. 2007). Approximately 7 mg of steryl ferulates/100 g rye were also reported, along with 11–21 mg/100 g of Δ5- and Δ7-avenasterols, Δ7-stigmastenol and stigmastadienols (Lampi et al. 2004). Hakala et al. (2002) reported total steryl ferulate levels of 5.5–6.4 and 15–25 mg/100 g (wet basis) in whole rye and rye bran, respectively.

Alkylresorcinols The alkylresorcinol contents of rye have been extensively reported (Ross et al. 2001, 2003b; Chen et al. 2004b; Mattila, Pihlava, and Hellström 2005; Landberg et al. 2007). The typical alkylresorcinol contents range from approximately 500 to 1000 mg/kg and 2500 to 4200 mg/kg in whole rye and rye bran, respectively (Ross et al. 2001, 2003b; Chen et al. 2004b; Mattila, Pihlava, and Hellström 2005). However, commercial food products can range from 44 to approximately 1000 mg/kg (Ross et al. 2001, 2003b). Iwatsuki et al. (2003) reported that rye bran oil contained 3800 mg/100 g of alkylresorcinols. The most common alkylresorcinols are the C17:0, C19:0, and C21:0 homologues (Table 3.16). The distribution of the C17:0, C19:0, and C21:0 homologues is 23, 32, and 26%, respectively (Ross et al. 2001; Chen et al. 2004b; Matilla, Pihlava, and Hellström 2005; Landberg et al. 2007). These distributes remained relatively consistent in both whole rye and rye bran (Chen et al. 2004b; Table 3.16). The level of alkylresorcinols is dependent on cultivar, growing location, and extraction methodology. Ross et al. (2001) reported the alkylresorcinols compositions of 15 rye cultivars grown at two locations in Denmark. Although the authors made no conclusions about the cultivar and growing location, alkylresorcinols composition differences were observed in rye cultivars, which ranged from 621 to 1022 and 549 to 864 mg/kg in rye grown at Bjertorp and Landskrona locations, respectively. The general distribution of the individual alkylresorcinols remained consistent within and between the cultivar and growing location (Ross et al. 2001).

Table 3.16 Alkyl(enyl)resorsinol Content (mg/kg) in Whole Rye, Rye Fractions, and Bread Mattila, Pihlava, and Hellström (2005) Alkyl(enyl)resorsinol 17:0 19:0 19:1 21:0 23:0 25:0 Unknown Total a

Whole Rye 240 260 33 180 110 70 34 927

NR = Not reported or measured.

Rye Bran 1100 1300 130 850 420 260 480 4108

Rye Bread 150 150 23 100 52 29 20 524

Ross et al. (2001) Whole Rye

Pericarp/ Testa

Aleurone

Endosperm

117 168 NRa 145 61 67 NR 559

172 249 NR 230 144 163 NR 958

457 630 NR 565 239 283 NR 2174

6 10 NR 12 4 4 NR 35

54

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Differences in alkylresorcinol contents of rye reported in the literature are likely due to the extraction protocol. Typical protocols involve the extraction with organic solvents such as acetone, methanol, hot 1-propanol, and ethyl acetate (Al-Ruqaie and Lorenz 1992; Ross et al. 2001, 2003b; Chen et al. 2004b) and supercritical fluid carbon dioxide (SF-CO2) extraction (Francisco et al. 2005a, 2005b; Landberg et al. 2007). Ross et al. (2001) reported an optimized solvent extraction protocol, which involved ethyl acetate and 24-hour contact time. Acetone and methanol solvent produced similar results to ethyl acetate but depended on the material being extracted. Methanol also coextracted other materials and thus was not the preferred extraction protocol (Ross et al. 2001). Ross et al. (2003b) reported that hot 1-propanol was necessary to extract the alkylresorcinols from complex matrices such as breads. Francisco et al. (2005a, 2005b) reported that SF-CO2 extraction was better than acetone at removing alkylresorcinols. In contrast, Landberg et al. (2007) reported that ethyl acetate worked equally as well as SF-CO2 for extracting the alkylresorcinols. The alkylresorcinol contents were 1176, 2932, and 1035 mg/kg in extracts obtained by SF-CO2 extraction of the milled rye, rye bran, and aleurone layer, respectively, whereas extracts obtained from ethyl acetate for the respective fractions were 843, 2921, and 1230 mg/kg (Landberg et al. 2007). A number of reports indicated that the alkylresorcinols were heat labile. Al-Ruqaie and Lorenz (1992) reported that as high as 77% of the alkylresorcinols were lost during the extrusion of rye. No significant differences were observed between extruder conditions on alkylresorcinol retention. Winata and Lorenz (1997) also reported that rye sourdough bread processing negatively impacted alkylresorcinols. In contrast, Ross et al. (2003b) did not find reductions in alkylresorcinols in baked rye breads. In their report, they utilized hot 1-propanol as the extraction solvent. In all breads, except flour, 1.4–1.6 times higher alkylresorcinol content was determined when hot 1-propanol was used as the extraction solvent compared to ethyl acetate. The authors attributed the disruption of the bread matrix by 1-propanal as a reason for the improved alkylresorcinol extraction. The loss of alkylresorcinols during fermentation of dough is also not likely. Liukkonen et al. (2003) reported that alkylresorcinols were not affected by the fermentation of lactic acid bacteria or yeast. Katina et al. (2007) later reported the same outcome in model fermentation systems. Thus, fermentation probably has a limited impact on alkylresorcinols.

Antioxidant Activity Rye antioxidant activity has been limited to a few studies on radical scavenging activity. Kähkönen et al. (1999) reported that an 80% methanol extract of rye bran and flours had minimal antioxidant activity in a methyl linoleate system. In fact, the antioxidant activity was 10 and 100 times lower than ­vegetables and fruits, respectively. Ragaee, Abdel-Aal, and Noaman (2006) and Zieliński, Ceglińska, and Michalska (2007) similar levels of ABTS radical scavenging of 13 and 10.9 µmol/g. However, Zieliński, Ceglińska, and Michalska (2007) also reported that the ABTS radical scavenging activity ranged from 7.76 to 10.9  µmol/g for different rye cultivars. Zieliński and Kozłowska (2000) reported that 80% methanol extracts of rye were more effective antioxidants than similar extracts obtained from water. These authors also found that 80% methanol extracts of the pericarp were more active against ABTS radicals than extracts of the embryo; however, no control of phosphotidylcholine oxidation was reported for either extract. In contrast, a phosphate buffer saline extract of rye and the endosperm fraction had higher Trolox equivalent antioxidant capacity (TEAC) than 80% methanol extracts (Zieliński, Ceglińska, and Michalska 2007). However, the 80% extract of the pericarp had higher TEAC values than the phosphate buffer saline extract of the pericarp. Rye bread extracts were found to have ABTS and DPPH radical scavenging activities (Michalska et al. 2007). The breads made from rye flour with extraction rates between 90 and 100 had the best radical scavenging activity. The radical scavenging activity of the extract obtained from breads made with these extraction rates had ABTS and DPPH radical scavenge activities 40 and 25% higher, respectively, than bread extracts obtained from bread made with rye flour having an extraction rate of 70% (Michalska et al. 2007). The opposite trend was observed in superoxide radical scavenging activity assay (Michalska et al. 2007). Nyström et al. (2005) reported DPPH radical scavenging of an acetone extract (16.7 µM) of rye bran was four percentage points better than a synthetic ferulate but 17 percentage points lower than α-tocopherols. The intent of the authors was to develop extracts with high levels of steryl ferulates; thus, differences in the observed radical scavenging activities, compared to other researchers, are probably

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55

due to extract composition. The rye extract (2.58 µM) was also less effective than α-tocopherol in controlling methyl linoleate emulsion oxidation. In contrast, the 2.58 µM rye extract was more effective in bulk methyl linoleate than α-tocopherol. Andreasen et al. (2001) reported that ethyl acetate extracts of rye bran were effective in controlling LDL oxidation whereas the flour extracts were least effective. Furthermore, the ferulic dihydrodimers were more effective antioxidants than the monomers (e.g., ­ferulic acid). In general, more research is needed to characterize the antioxidant activity of rye.

Wheat Wheat is one of the most commonly used cereals and has attracted much attention due to the recent interests in whole grain foods. Wheat is a cereal that contains substantial amounts of phytochemicals located mainly in the bran and germ. Phytochemical compounds include phenolic acids (Mattila, Pihlava, and Hellström 2005), alkylresorcinols (Iwatsuki et al. 2003; Mattila, Pihlava, and Hellström 2005), tocols (Barnes 1983; Morrison and Barnes 1983), carotenoids (Adom, Sorrells, and Liu 2003), and phytosterols (Piironen, Toivo, and Lampi 2002).

Tocols The tocols in wheat have been well characterized and are highly concentrated in the germ fraction. Barnes (1983) and Morrison and Barnes (1983) provided a very good historical perspective on tocols composition in wheat. The tocols content of approximately 35–57 mg/kg are comparable to the tocols reported in recent literature. Iqbal, Bhanger, and Anwar (2006) reported average tocol levels of 89 mg/ kg for five wheat cultivars grown in Pakistan. These authors reported that tocotrienols accounted for 73% of the total tocols. Zhou, Su, and Yu (2004) reported tocopherol levels of 3.9–29.38 mg/kg in brans from various wheat cultivars and growing locations. Soft wheat cultivars had α-tocopherol level of 3.4–10.1 mg/kg, which was the only tocopherol detected (Moore et al. 2005). Tocopherol content in wheat germ oil ranges from 1947 to 4082 mg/kg in 18 wheat lines (Dolde, Vlahakis, and Hazebroek 1999). These values signify the localization of the tocopherols in the germ, which is the site of lipid storage. However, individual tocols vary depending on location. Germ oil contained 601–1396, 229–562, and 1135–2232 mg/kg of α-, β-, and γ-tocopherol, respectively (Dolde, Vlahakis, and Hazebroek 1999). Ko et al. (2003) reported similar trends in the germ, whereby α-, β-, and γ-tocopherols accounted for 182, 66, and 6 mg/kg of the 257 mg/kg total tocols. The α-tocotrienol (3 mg/kg) was the only other tocol observed in the germ. In contrast, γ-tocopherol accounted for 41 and 65% the tocols in the hull and endosperm, respectively (Ko et al. 2003). These authors also observed higher α-tocotrienol levels (9 mg/kg) in the hull than other milling fractions. Wennermark and Jägerstad (1992) reported significant reductions in tocol contents over a 60 week storage at 20°C. The whole meal had comparable tocols losses to those observed in the flour, bran, and germ. These authors also reported degradation of tocols during the baking process.

Phenolics Ferulic acid and p-coumaric acid are the predominant phenolic acids in wheat. Hard red spring wheat flour and whole wheat contain 50 and 500 mg/kg of ferulic acid, respectively (Pussayanawin and Wetzel 1987). These values typify the phenolic composition of reported recently by other researchers (Table 3.17). Wheat cultivar and fraction (i.e., bran, flour), and extraction protocols account for the wide cultivar of phenolic acids reported in the literature. Renger and Steinhart (2000) reported ferulic acid content of approximately 18,500 mg/kg in bran using an alkaline hydrolysis. Although at much lower levels, Adom and Liu (2002), Moore et al. (2005), and Liyana-Pathirana and Shahidi (2007a) supported the trend that much of the ferulic acid was bound. Adom, Sorrells, and Liu (2003) reported that 97.7–99.4% of the ferulic acid was in the bound form. Moore et al. (2006) evaluated a series of enzymes as a means to enhance phenolic acid recovery. They found that β-gluconase (Ultraflo L) promoted the release of bound phenolic acids. Phenolic acid composition differences among cultivars have been observed (Adom, Sorrells, and Liu 2003; Zhou, Su, and Yu 2004; Moore et al. 2005). Durum wheat bran contained protocatechuic acid, p-hydroxybenzoic acid, gentisic acid, caffeic acid, vanillic acid, chlorogenic acid, syringic acid,

56

Table 3.17 Phenolic Content (mg/kg) of Wheat and Wheat Products Renger and Steinhart 2000a

Phenolic Compound

Moore et al. (2005)c

Mpofu, Sapirstein, and Beta (2006)d

1M Sodium Hydroxide

4M Sodium Hydroxide

Whole Wheat Flour

White Wheat Flour

Wheat Bran

White Bread

Free Phenolic Acids

Soluble Conjugates

Bound

15,224

18,884

37 890

0 120

38 3000

0 82

1.8

38.3

519

283

338

37 7 63 13 15 280

4 2 8 3 4 26

90 22 200 32 35 1100

3 2 7 0 3 10

53 105 10 50 71

Hard White Spring

Canadian Western Red Spring 8.9 418.5 187.9 28.6

7.7 403.9 165.7 37.2

13.5 9.3

12.8 7.8

0.2

1.13

10.9

7.6 371.0 145.5 34.2

0.7 1.1

8.4 4.8

3.6 4.0

12.8 9.0

Canadian Prairie Spring White

146 205 127 182 147

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 Average value of eight soft wheat cultivars. d Includes the average from four Canadian Western Red Spring cultivars. e 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.

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Phenolic Acid Caffeic Ferulic o-coumaric 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

Mattila, Pihlava, and Hellström (2005)b

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p-coumaric acid, and ferulic acid at concentrations of 226, 124, 108, 116, 637, 84, 130, 580, and 764 mg/ kg, respectively (Onyeneho and Hettiarachchy 1992). Zhou, Su, and Yu (2004) reported similar phenolic acids in durum wheat but at lower levels. Strong interaction effects for cultivar and environment on total and individual phenolic contents were reported (Mpofu, Sapirstein, and Beta 2006). Furthermore, environmental effects were stronger than for genotype on total phenolics and ferulic acid. Similar to the phenolic acids, bound flavonoids were significantly higher than the free forms (Adom, Sorrells, and Liu 2003). The total flavonoids content, as epicatechin equivalence, was found to range from 262 to 304 µg/g in bran from different wheat cultivars grown in Pakistan (Iqbal, Bhanger, and Anwar 2006). These authors also reported that anthocyanins content varied (30.4–38.2 mg/kg) depending on cultivar.

Other Components Adom, Sorrells, and Liu (2003) reported that lutein, zeaxanthin, and β-cryptoxanthin were the three major carotenoids in wheat. Two soft wheat cultivars had the greatest lutein levels while a soft white winter and hard red spring cultivar had the highest zeaxanthin and β-cryptoxanthin levels, respectively. Lutein, zeaxanthin, and β-cryptoxanthin levels in different wheat cultivars ranged from approximately 0.26–1.43 mg/kg, 0.09–0.27 mg/kg, and 0.011–0.13 mg/kg, respectively (Adom, Sorrells, and Liu 2003). In addition to lutein, zeaxanthin, and β-cryptoxanthin, Zhou, Su, and Yu (2004) reported the presence of β-carotene in wheat bran. The β-carotene content in the bran was 0.09–0.40 mg/kg. The highest lutein and β-carotene contents were reported in the durum wheat bran (Zhou, Su, and Yu 2004). Iwatsuki et al. (2003) reported an alkylresorcinol level of 1910 mg/100g in wheat bran oil. Alkylresorcinol contents varied from 200 to 1480 mg/kg among different wheat cultivars (Ross et al. 2003b). Hengtrakul, Lorenz, and Mathias (1990) reported variations in alkylresorcinol level for 318–655 mg/kg. They reported that soft wheats contained higher alkylresorcinol levels. Winter wheat ­contained less alkylresorcinols than spring wheat cultivars grown in Sweden (Chen et al. 2004b). These authors reported that within the wheat classes the growing location had an effect on alkylresorcinol levels; however, the effect due to growing location was minimized when both winter and spring wheats were compared. In contrast, some differences in alkylresorcinol concentration of the same wheat class were observed between growing locations (Hengtrakul, Lorenz, and Mathias 1990). Ross et al. (2003b) reported that commercial wheat bran in Sweden contained 2672 mg/kg alkylresorcinols while a wheat bran cereal (All Bran) contained similar (1784 mg/kg) levels. In contrast, white wheat flour and white bread did not contain any alkylresorcinols, which is likely due to the removal of the bran fraction during the production of the white flour. This observation supports milling studies that show an increased concentration of the alkylresorcinols in the outer layers of wheat. Milling was an effective method to concentrate the alkylresorcinols Alkylresorcinol contents varied from 200 to 1480 mg/kg among different wheat cultivars (Ross et al. 2003b). The bran and short fractions contained 2500 and 1000 mg/kg alkylresorcinols, respectively, compared to the 642 mg/kg in the initial grain (Ross et al. 2003b). Landberg et al. (2007) also reported a similar trend in that the bran and aleurone layer contained higher (2932 and 1035 mg/kg, respectively) alkylresorcinol levels than the original wheat kernel (538 mg/kg). Processing of the bran by extrusion caused a 53–77% reduction in alkylresorcinols (Al-Ruqaie and Lorenz 1992). Jiang and Wang (2005) reported that wheat bran and germ contained 120 and 240 mg/100 g of phytosterols, respectively. Higher phytosterol levels were observed in the wheat bran oil (1767 mg/100 g) and wheat germ oil (2128 mg/100 g). Milling of wheat led to several fractions with high phytosterol contents. The germ fraction had the highest phytosterol content at 492 mg/100 g followed by the fine bran fraction with 207 mg/100 g (Nyström et al. 2007). Regardless of the fraction, sitosterol was the major phytosterol (Piironen, Toivo, and Lampi 2002; Jiang and Wang 2005; Nyström et al. 2007). The wheat bran contained more stanols than the wheat germ (Table 3.18; Piironen, Toivo, and Lampi 2002; Jiang and Wang 2005). Sitostanal accounted for 15.9% of the sterols in whole wheat flour and 2.9 and 15.5% in the germ and bran, respectively (Normén et al. 2002). Iwatsuki et al. (2003) reported sterol and ferulate sterol levels in wheat bran oil of 700 and 380 mg/100 g, respectively. Steryl ferulates accounted for 11.6 and 9.8% of the total sterols in wheat bran and durum wheat, respectively (Jiang and Wang 2005). Bran fractions contained approximately 30 mg/100 g of steryl ferulates while the

58

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 3.18 Phytosterol Content (mg/100 g) in Wheat Fraction Phytosterol/Phytostanol Brassicasterol Campesterol Stigmasterol Sitosterol Cycloartenol Campestanol Sitostanol 24-methylcycloartanol Δ5-Avenasterol Δ7-Avenasterol a

b

Bran

Germ

Branb

Germb

Flourb

56 366 27 453 48 174 245 301

15 470 22 1,123 69 67 83 103

25.9 1.2 253.7 16.2 33.8 39

2.4 103.4 1.9 81.4 3.4 5.4 6.8

12.3 2.1 40.5 1.2 6.9 9.2

2 3.1

13.4 9

1.2 1.2

a

a

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 Piironen, V., Toivo, J., and Lampi, A.-M., Cereal Chem., 79(1), 148–54, 2002. Data reported on a wet basis. Empty spaces indicates that no sterol was detected or that it was not determined by the reseachers.

germ contained only 2 mg/100 g (Nyström et al. 2007). The sitosteryl ferulate accounted for the highest concentration of the individual steryl ferulates (Hakala et al. 2002; Iwatsuki et al. 2003; Nyström et al. 2007). Nyström et al. (2007) reported that sitosterol glycoside accounted for 66–75% of the steryl glycosides in the whole wheat, bran, and germ. The highest concentrations (17.1 and 15.4 mg/100 g, respectively) of steryl glycosides were found in the feed flour and germ fractions.

Antioxidant Activity The antioxidant, primarily radical scavenging, activity of wheat has been correlated to total phenolic content (Zieliński and Kozłowska 2000; Adom and Liu 2002; Adom, Sorrells, and Liu 2003). The bound phenolic acids were highly correlated to radical scavenging activity in contrast to the low correlation with free phenolic acids (Adom and Liu 2002). However, these authors found that total phenolics, ferulic acid, and flavonoids all correlated highly with antioxidant activity. Mpofu, Sapirstein, and Beta (2006) also reported strong correlations between total phenolic content and antioxidant activity, and ferulic acid and antioxidant activity. These authors also observed strong interaction effects for cultivar and environment on antioxidant activity. Adom, Sorrells, and Liu (2003) reported that a white spring durum cultivar (Cham1) had both the highest phenolic content and radical scavenging activity. Ragaee, Abdel-Aal, and Noaman (2006) reported that hard wheat flours had slightly better DPPH and ABTS radical scavenging activities than soft wheat flour. Zhou, Su, and Yu (2004) observed metal chelating, and ABTS and ORAC radical scavenging activities per unit of total phenolics was greatest in Canadian hard white wheat. In this study, a low correlation existed between antioxidant activities and total phenolics. However, DPPH radical scavenging did correlate to total phenolic content (Zhou, Su, and Yu 2004). The radical scavenging activities (ABTS, ORAC, and DPPH) of wheat grown in Pakistan were related to total phenolic and anthocyanins contents while ORAC radical scavenging was also related to tocotrienol content (Iqbal, Bhanger, and Anwar 2006). Onyeneho and Hettiarachchy (1992) reported that durum wheat cultivars provide the same protection against oil oxidation. In general, the durum wheat extract was a better antioxidant than the individual phenolic acids and suggests that a synergistic effect occurs between phenolic compounds in the durum wheat extract. Zhou, Laux, and Yu (2004) reported that separation of the aleurone from the other parts of the bran was an effective method to enhance antioxidant activity. Liyana-Pathirana and Shahidi (2007a) reported ORAC values of 45–301 and 54–310 µmols Trolox equivalent/g in Canadian amber durum and red spring

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59

wheat fractions, respectively. Regardless of cultivar, the bran produced the best ORAC value followed by shorts, feed flour, whole grain, and flour. The inhibition of LDL oxidation and DPPH radical scavenging by the wheat fractions followed the same trend as in the ORAC test (Liyana-Pathirana and Shahidi 2007a, 2007b). No consistent pattern in the hydroxyl radical, superoxide anion radical, or hydrogen peroxide scavenging activities were observed; however, the bran and shorts typically had the highest activities (Liyana-Pathirana and Shahidi 2007b). Li, Pickard, and Beta (2007) reported good radical scavenging activities of alcoholic extracts of purple wheat bran. Baking of the bran caused reductions in the radical scavenging activities of extracts. Katapodis et al. (2003) reported that a ferulated oligosaccharide had good DPPH radical scavenging activity and inhibited LDL oxidation. Inhibition of LDL oxidation of wheat and wheat fractions improved after hydrolyzed at gastrointestinal pH values (Liyana-Pathirana and Shahidi 2004). Improvements in antioxidant activities were observed in hydrolyzed soft and hard wheats (Liyana-Pathirana and Shahidi 2005). Similar observations were made in enzyme-treated bran. Moore et al. (2006) reported a correlation between the levels of free phenolic acids released from the enzymatic treatment of wheat bran and antioxidant activity. The greater the release of the bound phenolic acids, the greater was the antioxidant activity. The enzyme β-gluconase (Ultraflo L) was found to promote the release of phenolics to the greatest extent (Moore et al. 2006). Calzuola, Gianfranceschi, and Marsili (2006) reported that a hydroalcoholic extract of wheat sprouts had ferricyanide reduction and superoxide scavenging activities. In contrast to the above reports, Kähkönen et al. (1999) reported that wheat had essentially no effect on linoleate oxidation while the bran fraction had low activity against linoleate oxidation. The lack of activity could be due to a number of factors such as the test system and the materials used in the testing protocol. Liyana-Pathirana et al. (2006) noted that the pearling of the wheat affected the antioxidant activity differently. However, as the pearling percentage increased, the antioxidant activity decreased. In contrast, the byproducts obtained from the 10 and 20% pearling fractions had significantly higher antioxidant activity than the whole grain and pearled wheat (Liyana-Pathirana et al. 2006). The higher antioxidant activities correlated with the higher phenolic content observed in the pearling by-products. The antioxidant capacity (ORAC test) dropped from 61 to 40% in milled bran stored at 100°C for 9 days. The reduction in DPPH radical scavenging activity between 38 and 100% was reported for the milled samples stored at elevated temperatures (Cheng et al. 2006). In contrast, no reduction in antioxidant activities of the whole grain samples was observed at an elevated temperature. This demonstrates that handling of the wheat and bran can substantially affect the antioxidant activities of wheat and wheat fractions.

Other Cereals: Millet and Sorghum Millet and sorghum are gluten free cereals that could be used in the production of low gluten foods. There have been several recent reviews on the topic of phytochemicals in millet and sorghum. Thus, the authors suggest that the reader see the reviews of Awika and Rooney (2004) and Dykes and Rooney (2006) for in-depth information on phytochemicals in these cereals. As with other cereals, carotenoids, phenolic acids, flavonoids, tannins, phytosterols, and policosanol are phytochemical components that contribute to the antioxidant activities and health benefits.

Pseudocereals The inability of humans with Celiac disease has spurred the development of gluten free products. Pseudocereals have attracted much attention as a replacement for gluten containing cereals. This replacement is likely due to the similar functionality of pseudocereals to cereal products. In addition to the functionality, the pseudocereals contain polyphenolics, phytosterols, and tocols. The three main pseudocereals include amaranth, buckwheat, and quinoa. In contrast to previous sections in this chapter, limited discussions on the broad categories of phytochemicals will be presented. Instead, focus will be placed on the phytochemical unique to the pseudocereal.

Buckwheat Common buckwheat (Fagpyrum esculentum Moench.) and Tartary buckwheat (Fagpyrum tartaricum Gaertn.) are the most commonly cultivated species of buckwheat. Phenolic acids, tocols, 3-flavanols,

60

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

and rutin are the main phytochemicals in buckwheat (Durkee 1977; Oomah and Mazza 1996; Kreft, and Kreft 1999). However, rutin has garnished the greatest attention because it can control elevated blood pressure (Matsubara et al. 1985). Buckwheat had better antioxidant activity than barley, oat, wheat, and rye (Zieliński and Kozłowska 2000). Furthermore, Mukoda, Sun, and Ishiguro (2001) reported that buckwheat hull extracts were effective in vivo antioxidants. Thus, exploitation of buckwheat as source of phytochemicals is supported by in vivo and in vitro studies.

Phenolics Zieliński and Kozłowska (2000) reported that the total phenolic contents of 80% methanol extracts of dehulled buckwheat and buckwheat hulls were 90.7 and 381.9 µg/mg lyophilized extract, respectively. Caffeic, o-coumaric, p-coumaric, ferulic, gallic, p-hydroxybenzoic, syringic, and vanillic are the major phenolic acids in buckwheat (Durkee 1977; Przybylski, Lee, and Eskin 1998; Mattila, Pihlava, and Hellström 2005). In addition to these acids, chlorogenic acid was reported in the herbal (e.g., leaves) extracts of buckwheat (Hinneburg and Neubert 2005). Przybylski, Lee, and Eskin (1998) also showed that solvent extraction with methanol produced an extract with the highest concentration (5151 mg/kg) of phenolic acids. Vanillic and caffeic accounted for 57 and 17% of the phenolic acids in this extract, respectively. In contrast, an acetone extract contained 1986 mg/kg phenolic acids with caffeic and o-coumaric each accounting for 28% of the phenolic acids while only 8% of the phenolic acids in the extract was vanillic acid (Przybylski, Lee, and Eskin 1998). Buckwheat grits contained 248 mg/kg of phenolic acids in which approximately 44 and 34% were syringic and caffeic acids, respectively, and only 2% was vanillic acid (Mattila, Pihlava, and Hellström 2005). Sun and Ho (2004) reported that acetone, ethanol, and methanol produced buckwheat extracts with 3.3, 2.3, and 2.1 g catechin equivalence/100 g. Contradiction in the phenolic data demonstrates the importance of solvent in the extraction protocol. Buckwheat contains 3870 and 13,140 mg/kg flavonoids in the seed and hulls, respectively (Oomah and Mazza 1996). In contrast, Dietrych-Szostak and Oleszek (1999) reported lower levels (188 and 740 mg/ kg, respectively) of flavonoids in the seed and hull. Watanabe, Ohshita, and Tsushida (1997) reported 360 mg/kg of flavonoids in the hull, but observed total flavonoid levels in the dried hypocotyls and cotyledons of sprouted buckwheat of approximately 2767 and 46,616 mg/kg, respectively (Watanabe 2007). Watanabe (1998) reported that buckwheat groat extracts contained (–)-epicatechin, (–)-epicatechin 3-O-p-hydroxybenzoate, (–)-epicatechin 3-O-(3,4-di-O-methyl)-gallate, and (+)-catechin 7-O-βD-glucopyranoside. Danila et al. (2007) reported rutin, catechin, epicatechin, and epicatechin gallate concentrations of 127, 330, 205, and 12.7 mg/kg in common buckwheat, respectively. Common buckwheat contained 156–202 mg/kg of (–)-epicatechin and 24–13 mg/kg of (+)-epicatechingallate whereas Tartary buckwheat lacked these polyphenolics (Morishita, Yamaguchi, and Degi 2007). In contrast, quercitrin (812–954 mg/kg) and quercetin (20–24 mg/kg) were observed in Tartary buckwheat but not in common buckwheat. However, Bajpai et al. (2005) reported that common buckwheat contained 409 mg/kg of quercetin. Common and Tartary buckwheat contain 100–770 mg/kg and 1000–2000 mg/kg of rutin, respectively (Ohsawa and Tsutsumi 1995; Oomah and Mazza 1996; Morishita, Yamaguchi, and Degi 2007). Oomah and Mazza (1996) reported rutin levels of 470 and 770 mg/kg in the seeds and hulls, respectively. Morishita, Yamaguchi, and Degi (2007) reported that Tartary buckwheat contained approximately 18,090–18,538 mg/kg of rutin whereas common buckwheat contained only 122 to 136 mg/kg. Kreft, Fabjan, and Yasumoto (2006) reported that precooked buckwheat groats had substantially lower rutin levels than the raw groats. The rutin lose was supported by the reductions in flavonoids observed by Dietrych-Szostak and Oleszek (1999). Dried buckwheat leaves, a vegetable in Asia, contained very high levels of epicatechin and rutin contents, in the range of 817–600 mg/kg and 35,840–98,194 mg/kg, respectively (Kalinova, Triska, and Vrchotova 2006). The variations were due to buckwheat cultivar and growing ­environment. The high rutin level agrees with observations made by Holasova et al. (2002). They found that the dried buckwheat leaves contained 23,443 mg/kg of rutin whereas the seed contained only 184 mg/ kg. In contrast, only 2700 mg/kg of rutin was determined in buckwheat leaf flour (Kreft, Fabjan, and Yasumoto 2006).

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Other Components Total tocopherols in buckwheat are similar to some cereal and fall in the range of 14–22 mg/kg (Kim, Kim, and Park 2002). Przybylski, Lee, and Eskin (1998) reported a tocopherol content of 539 mg/kg in a hexane extract of buckwheat. The α-tocopherol constituted 70% of the total followed by γ (20%), δ (7%), and β (3%) tocopherols. Dried buckwheat leaves contained α-tocopherol contents in the range of 299–4205 mg/kg (Kalinova, Triska, and Vrchotova 2006) while lower levels (105 mg/kg) were reported by Holasova et al. (2002). The variation was due to buckwheat cultivar and growing environment.

Antioxidant Activity The antioxidant activity of buckwheat hulls was found to be greater than the antioxidant activity in the seeds (Velioglu et al. 1998). These authors noted a correlation existed between total phenolics and antioxidant activity. Zieliński and Kozłowska (2000) reported that antioxidant activity of 80% methanol extracts of dehulled buckwheat and buckwheat hulls were 0.55 and 1.18 µmol Trolox/mg lyophilized extract, respectively. In contrast, Przybylski, Lee, and Eskin (1998) found that the extracts of buckwheat groat were significantly more effective than hull extracts. The lack of a strong antioxidant activity of the hulls was also observed in ethanol extracts of the hulls (Watanabe, Ohshita, and Tsushida 1997). Conflicting reports on the antioxidant activity in buckwheat is likely due to the extract evaluated in the testing protocol. Thus, fractionation of the hull and groat prior to the extract product is warranted. Şensoy et al. (2006) reported that roasting of the buckwheat reduced the DPPH radical scavenging activity compared to the raw flour. However, the DPPH radical scavenging activity of an extract obtained from extruded buckwheat did not differ from the scavenging activity of the raw flour. Thus, knowing the history of the buckwheat sample being evaluated is necessary when determining phytochemical composition. The solvent used in the production of the extracts also influenced the antioxidant activity. Methanol extracts provided the best protection against canola oil oxidation while ethyl acetate and acetone extracts were better DPPH radical scavengers (Przybylski, Lee, and Eskin 1998). Sun and Ho (2004) also found that acetone extracts of buckwheat produced the best DPPH radical scavenging activity while methanol extracts had the best antioxidant activity against lard oxidation and β-carotene bleaching. Gorinstein et al. (2007) also found excellent DPPH radical scavenging and inhibition of β-carotene bleaching by a methanol extract of buckwheat. Morishita, Yamaguchi, and Degi (2007) reported that Tartary buckwheat had better antioxidant activity in a DPPH radical scavenging activity assay than common buckwheat. These authors suggested that (–)-epicatechin was the major contributor to the antioxidant activity in common buckwheat. However, unknown compounds were suggested to be responsible for the majority of the antioxidant activity (Morishita, Yamaguchi, and Degi 2007). Rutin contributed little to the antioxidant activity of common buckwheat. These observations agree with the reported activity of (–)­-epicatechin by Watanabe (1998) and the lack of correlation between rutin and antioxidant activity (Oomah and Mazza 1996). Furthermore, Quettier-Deleu et al. (2000) reported that the flavanols were more active than rutin as antioxidants. Gallardo, Jimenez, Garcia-Conesa (2006) reported that a water extract had better radical scavenging activity in the ORAC test. These authors stated that the antioxidant activity was likely due to the combined effect of all phenolic compounds. In contrast, Morishita, Yamaguchi, and Degi (2007) reported that rutin was the major contributor to the antioxidant activity of Tartary buckwheat. Rutin content correlated to the inhibition of LDL peroxidation (Jiang et al. 2007). A lower correlation existed between LDL peroxidation inhibition and ­flavonoids content. Again, the Tartary buckwheat had the highest rutin and total flavonoid contents. No other components were evaluated, thus the conclusion was based only on the measure components.

Amaranth and Quinoa These pseudocereals have traditionally been consumed in specific regions of the world such as Peru and Bolivia (Nsimba, Kikuzaki, and Konishi 2008). However, the quest to develop low gluten foods has prompted a wider consumption of these commodities. In contrast to the major nutrients and functionality characteristics, limited research has been completed on the pseudocereals amaranth and quinoa as dietary sources of phytochemicals. Amaranth has attracted attention due to the potential cholesterol

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

and lipid lowering activities of this pseudocereal (Qureshi, Lehmann, and Peterson 1996; Grajeta 1999; Berger et al. 2003a; Czerwiński et al. 2004). However, the cholesterol lowering activities are still in question, but may depend on other dietary factors such as lipid intake (Grajeta 1999; Berger et al. 2003b). The DPPH and ABTS radical scavenging and β-carotene bleaching assays showed that both amaranth and quinoa had antioxidant activity (Czerwiński et al. 2004; Gorinstein et al. 2007; Nsimba, Kikuzaki, Y. Konishi 2008). Thus, the combined health benefits and antioxidant activities warrant further investigation due to the limited number of reports.

Phytochemical Components Phytosterol contents have reported to range from 561 to 834 µg/100 g in amaranth grain (Marcone, Kakuda, and Yada 2003) to 2730 mg/100 g in amaranth oil (Berger et al. 2003b). Regardless of amaranth cultivar, β-sitosterol was the major phytosterol (Marcone, Kakuda, and Yada 2003). However, León-Camacho, García-González, and Aparicio (2001) reported that clerosterol was the predominant phytosterol. Tocopherol contents of 2588 and 1788 mg/kg were observed in crude and refined amaranth oils, respectively (Berger et al. 2003b). In crude oils, δ- and γ-tocopherols accounted for 27 and 46% of the total tocopherols, respectively. The δ- and γ-tocopherols accounted for 34 and 41% of the tocopherols in refined oils, respectively (Berger et al. 2003b). Budin, Breene, and Putnam (1996) reported similar tocopherol levels and found minimal totcotrienols. In contrast, Qureshi, Lehmann, and Peterson (1996) reported that β-tocotrienol accounted for 19.5 and 498 mg/kg of the 32.7 and 1024 mg/kg total tocols in the amaranth and amaranth oil, respectively. Interestingly, γ-tocopherol accounted for only 0.3% (0.1 mg/kg) in the amaranth seed and 6.1% (63.4 mg/kg) in the oil. Furthermore, δ-tocopherols accounted for 6% (1.84 mg/kg) and 34% (355 mg/kg) of the total tocols in the seed and oil, respectively (Qureshi, Lehmann, and Peterson 1996). Lehmann, Putnam, and Qureshi (1994) reported amaranth contained α-tocopherol, β-tocotrienol, and γ-tocotrienol levels of 2.97–15.65, 5.92–11.47, and 0.95–8.69 mg/kg seed, ­respectively. Additional research is necessary to clarify the conflicting reports on tocols composition. Total polyphenolic contents of 121 µg tannic acid equivalence/g (Nsimba, Kikuzaki, Y. Konishi 2008), 148 and 415 µg gallic acid equivalence/g (Czerwiński et al. 2004; Gorinstein et al. 2007) have been reported for several cultivar of amaranth. Gorinstein et al. (2007) reported that the extraction protocol had a greater impact on total phenolics than amaranth cultivar. Anthocyanin contents of 61 and 94 mg cyanidin-3-glucoside/100 g, and flavonoid contents of 14 and 74 mg catechin equivalence/100 g amaranth have been reported (Czerwiński et al. 2004; Gorinstein et al. 2007). Similar to amaranth, quinoa had total polyphenolic, anthocyanins, and flavonoids contents of 600 µg/g gallic acid equivalence, 96 mg cyanidin-3-glucoside/100 g and 102 catechin equivalence/100 g, ­respectively (Gorinstein et al. 2007). Nsimba, Kikuzaki, Y. Konishi (2008) reported total polyphenolic contents of 94 and 148 µg tannic acid equivalence/g. The difference in total phenolics was due to cultivar. Zhu et al. (2001) reported that kaempferol and quercetin glycosides were the predominant flavonoids. Additional research is needed to clarify the health and antioxidant activities of amaranth and quinoa.

Phytochemicals from Pulses: Edible beans and Legumes Dry beans and peas are important components of the traditional diet in many Asian and African, as well as Central and South American countries. They are low in fat and are excellent sources of protein, dietary fiber, and a variety of micronutrients and phytochemicals (Naczk and Shahidi 1995; Messina 1999). The word legume is used for all leguminous plants according to the Food and Agriculture Organization (FAO) practice. For those containing only small amounts of fat, such as green peas, dry beans, and so on, the term “pulse” is used and for those containing a high proportion of fats, such as soybeans and peanuts, the term “leguminous oilseed” is used (FAO 1982; Salunkhe and Kadam 1989). Legumes also provide micronutrients, vitamins, carotenoids, and phenolic compounds (Adsule and Kadam 1989; Dueñas, Hernández, and Estrella 2006). Since phytochemicals in soybean and peanut has been reported extensively. Here phytochemicals in the low fat legume will be highlighted. Of the various food legumes

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Phytochemicals in Cereals, Pseudocereals, and Pulses

cultivated, dry beans (Phaseolus vulgaris L.), peas (Pisum sativum L.), chickpeas (Cicer arietinum L.), and lentils (Lens culinaris Mack.) are the major grown food legumes all over the world (Hartwig 1989; Smartt 1990).

Dry Peas Tocopherol and Carotenoids Krishna, Prabhakara, and Aitzetmüllerb (1997) reported that γ-tocopherol was the major tocopherol in bengal gram (Cicer arietinum L.), black gram (Vigna mungo L.), green gram (Vigna radiata L.), and horse gram (Dolichos biflorus L.). The γ-tocopherol accounted for 80.8–97.3% of the total tocopherols in these commodities. The content of tocopherols and carotenoids in the grass pea were reported to be 1.78 µg/g α-tocopherols, 53.54 µg/g γ-tocopherol, 0.21 µg/g β-carotene, and 5.68 µg/g lutein (Grela and Günter 1995). In the case of β-carotene, the highest value was reported in green split peas (160.2 mg/100 g) compared to chickpeas (29.1 mg/100 g), lentils (46.3 mg/100 g), yellow split peas (21.5 mg/100 g), and cowpeas (28.08 mg/100 g; Augustin and Klein 1989). Grela and Günter (1995) reported that increasing moisture as well as extrusion caused a significant reduction in the content of α-tocopherols, γ-tocopherol, β-carotene, and lutein in the grass pea.

Phenolic Compounds Beans, peas, and lentils contain different concentrations of phenolic acids such as protocatechuic, hydroxybenzoic, and vanillic acid (Table 3.19; Lopez-Amoros et al. 2006). Only beans contained cisferulic acid and only peas contained cis p-coumaric acid, while only lentils had a low concentration of catechin and procyanidin polymers. Free and combined phenolic acids (hydroxybenzoic acids (1.8–2.2 μg/g), free (3.2–5.7 μg/g) and combined hydroxycinnamic acids (1.4–13.5 μg/g) were mainly located in the cotyledon of lentils, while flavonoids (catechins [919–1633 μg/g]), trans-resveratrol (5.5–9.3 μg/g), and proanthocyanidins (dimer Table 3.19 Concentration (μg/100 g d.w.) of Phenolic Compounds in Legumesa Compounds Protocatechuic acid p-hydroxybenzoic acid p-hydroxybenzoic aldehyde Vanillic acid p-hydroxyphenylacetic acid trans p-coumaric acid cis p-coumaric acid trans p-coumaric acid derivative cis p-coumaric acid derivative trans ferulic acid cis ferulic acid cis ferulic acid derivative (+)-catechin Procyanidin B2 Procyanidin B3 Procyanidin C1 Procyanidin tetramer

Beans

Peas

Lentils

32.8–41.4 32.3–36.1 nd 90.9–97.9 45.8–51.6 nd nd nd nd 342–366 74.1–79.1 nd —

206–221 46.5–49.9 nd 19.4–22.2 nd 37.7–41.5 65.5–70.1 nd 31.9–33.7 9.1–10.9 nd nd —

49.9–52.3 93.6–100 13.3–15.3 73.6–79.6 nd 322–342 nd 82.8–89.6 nd 20.9–25.7 nd nd 0.1 –0.3

— — — —

— — — —

0.3–0.5 0.3–0.5 t 0.2–0.3

Source: López-Amorós, M., Hernández, T., and Estrella, I., J. Food Compos. Anal., 19, 277–83, 2006. a Values are lower and higher of the two samples analyzed in duplicate; nd: not detected; and t: trace.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

procyanidins [619–1122 μg/g], trimer procyanidins [441–498 μg/g], and dimers plus trimers of prodelphinidins [369–725 μg/g]) were concentrated in the lentil seed coat. Peas also had the similar phenolic distribution with lentils (Dueñas, Hernández, and Estrella 2002). Remiszewski et al. (2006) reported that “Ramrod” peas contained 0.86 mg GAE/g, and pea extracts contain 85 mg/g kaempferol. Mazur et al. (1998) found that the chickpea had the highest total concentration of isoflavones (1150– 3600 μg/100 g). The pea “Green split” was found to be the poorest source of all these phytochemicals. All the legumes, except the Green split pea, contained daidzein and genistein. Coumestrol was detected in most of the samples at very low concentrations (up to 10 μg/100 g; 0.4 μmol/kg). Sosulski and Dabrowski (1984) found that the total free phenolic acids (trans-ferulic, trans-p-coumaric, and syringic acids) in legumes (mung bean, fava beans, navy beans, lima beans, field peas, lentils, pigeonpeas, lupines, chickpeas, and cowpeas) ranged from 1.8 to 16.3 mg/100 g. The phenolic content of extracts will be changed significantly based on the solvent extraction technique employed to remove these compounds. For example, 50% acetone extracts from yellow pea, green pea, and chickpea had the highest total phenolic content, but acidic 70% acetone (+0.5% acetic acid) extracts from lentils had the highest total phenolic and total flavonoids contents (Xu and Chang 2007). The phenol content of the cowpeas were 0.3–1.0 mg/g, while the total phenol content of 0.4 and 1.2 mg/g were observed in white and brown pigeon pea, respectively (Oboh and Akure 2006). After soaking, the concentration of all the phenolic compounds in beans, peas, and lentils decreased. Germination of peas, beans, and lentils also changed their phenolic composition (López-Amorós, Hernández, and Estrella 2006).

Other Components The vitamin C content of the cowpeas ranged from 0.5 to 0.9 mg/100 g, while that of pigeon peas were 0.9 mg/100 g (Oboh and Akure 2006). Mazur et al. (1998) found that all the legumes analyzed contained secoisolariciresinol contents of 2.8–475.8 μg/100 g (0.08–13.5 μmol/kg). Matairesinol seems to be the rarest phytoestrogen in the leguminous plant family.

Antioxidant Activity Compared to peas and beans, lentils had the highest concentration of the total phenolic compounds and the best antioxidant capability in terms of DPPH, FRAP, and ORAC (Table 3.20; Xu, Yaun, and Chang 2007). African yam beans (23.6%), cowpea brown (29.9%), and pigeon pea brown (54.5%) had a relatively high free radical scavenging ability (Oboh and Akure 2006). The lentil seed coat had higher antioxidant activity than that of peas, which is because the lentil has larger amounts of flavonoid compounds, especially of proanthocyanidins, which contribute to the increased antioxidant activity (Dueñas, Hernández, and Estrella 2006). Amarowicz, Karamac, and Shahidi (2003) found that epicatechin and condensed tannins are mainly responsible for the antioxidant activity in the lentil seeds. The antioxidant activity of peas and beans increased after germination, but the antioxidant activity of lentils decreased after germination (López-Amorós, Hernández, and Estrella 2006). Table 3.20 Differences of Phenolics Contents and Antioxidant Activities Between Classesa Classes Yellow peas (N = 30) Green peas (N = 33) Lentils (N = 33) Common beans (N = 21)

TPC, mg GAE/g

TFC, mg CAE/g

CTC, mg CAE/g

DPPH, µmol TE/g

FRAP, mmol FE/100g

ORAC, µmol TE/g

0.94d 0.81d 6.96a 4.04c

0.13d 0.12d 3.82a 3.00b

0.39d 0.42d 6.07a 3.58b

1.95c 1.53c 19.47a 14.13b

0.71d 0.64d 11.79a 5.83c

8.35e 5.90e 80.84b 62.57c

Source: Xu, B., Yaun, S., and Chang, S., J. Food Sci., 72, S167–77, 2007. Values marked by the same letter in the same column are not significantly different (P < .05).

a

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Phytochemicals in Cereals, Pseudocereals, and Pulses

Dry Bean Tocopherol Cho et al. (2007) found four beans (black beans, kidney beans, mung beans, small red beans) contained tocopherol ranging from 86.1 to 146.8 mg/kg. The vitamin E activity was between 12 i.u./ kg for common bean and 44 i.u./kg for broad bean (Grela and Günter 1995).

Phenolic Compounds Wu et al. (2004) reported that pinto, black, red kidney, and small red beans all had significant total phenolic contents of 10.23, 8.80, 12.47, and 11.85 mg gallic acid equivalence/g beans, respectively. All these beans score very high on the oxygen radical scavenging absorbance assay suggesting the potential to be very good in vivo antioxidants. Towo, Svanberg, and Ndossi (2003) reported that the total phenolic ­content in kidney beans was 3.2–5.6 mg/g beans. The light colored beans had the greatest phenolic content whereas the brown beans had the greatest tannin content. Unlike cereals, Drumm, Gray, and Hosfield (1990) reported that free phenolic acids content was equal to bound phenolic acids in navy (cv Seafarer) and kidney (cv Montcalm) beans grown in Michigan and of pinto (cv Gala) and black turtle soup (cv Domino) were grown in Idaho (Table 3.21). Gu et al. (2004) reported similar results in that catechin and pro-pelargonidins forms were the principle type of anthocyanidins in pinto, kidney, and red beans whereas catechin form predominately in black beans. Ma and Bliss (1978) evaluated the tannin content of 29 common bean varieties with varying testa color. The growing location did not appear to have a significant impact on tannin content due in part to the large variation in data. Beans grown in the United States had an average tannin content of 2.2 and 4.6 mg catechin/g cotyledon or whole seed, respectively. Beans grown in Puerto Rico had an average tannin content of 0.9 and 7.2 mg catechin/g cotyledon or whole seed, respectively. Tannin contents of 8.9, 11.0, and 9.8 mg/g bean for black, red kidney, and white kidney beans grown in Pakistan, respectively, were observed (Rehman and Shah, 2005). Mwikya et al. (2001) reported a tannin content of 2.0 mg/g of a light kidney bean grown in Kenya. Dong, He, and Liu (2007) isolated 24 compounds including 12 triterpenoids, seven flavonoids, and five other phytochemicals from black bean seed coats. Only six flavonoids had antioxidant potential. Cho et al. (2007) found the total phenolic content of four different beans (black beans, kidney beans, mung beans, small red beans) were between 589.9 and 1370.7 mg GAE/kg. Table 3.21 Phenolic Acids (mg/100 g) Contents in Dry Beans Ferulic

Coumaric

Sinapic

Cinnamic

Free phenolic acids Navy Dark red kidney Pinto Black Turtle Soup

1.81 0.72 0.46 0.00

1.91 1.46 1.17 0.93

0.94 0.64 1.79 1.42

2.90 4.56 3.25 4.11

Phenolic acid esters Navy Dark red kidney Pinto Black Turtle Soup

3.67 6.84 7.18 6.12

2.42 2.10 2.34 2.06

1.18 2.00 3.05 3.07

4.91 2.44 3.97 3.34

Bound phenolic acids Navy Dark red kidney Pinto Black Turtle Soup

0.00 0.29 0.00 0.00

1.04 0.77 0.41 0.77

1.05 1.28 1.45 1.21

4.54 1.24 2.73 2.32

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Xu and Chang (2007) found that the black bean and red kidney bean extracts from acidic 70% acetone had the highest total phenolic content, and total flavonoids content than extracts from 50% acetone, 80% acetone, 70% methanol, and 100% ethanol extraction. Habib et al. (1976) reported that the crude extracts from the kidney bean showed that the petroleum ether extract contained sterols and/or triterpenes, while ether, chloroform, and ethyl alcohol extracts contained reducing substances. Remiszewski et al. (2006) reported that quercetin (245 mg/g) was the dominant flavonoid in the red kidney bean while luteolin (82 mg/g) was most common in the white bean. Tsuda et al. (1994b) found that delphinidin-3-O-β-Dglucoside, petunidin-O-β-D-glucoside, and malvidin-3-O-β-D-glucoside accounted for 56, 26, and 18%, respectively, of the anthocyanins in black bean.

Other Components The β-sitosterol in the winged bean can treat benign prostatic hyperplasia and lower the low density lipoprotein cholesterol levels (Becker, Staab, and Bergman 1993). Cho et al. (2007) reported that the mean carotenoid values in four beans (black beans, kidney beans, mung beans, small red beans) are 9.2 ± 10.7 mg/kg, and 47.1 mg/kg ascorbic acid. Linolenic (18:3) is from 4% in the broad bean to 22% in the kidney bean (Grela and Günter 1995).

Antioxidant Activity Freeze-dried extracts of the navy, garbanzo, and pinto bean hulls were effective at controlling the oxidation of soybean oil (Onyeneho 1990). Navy bean hull extracts were the most effective antioxidants and also had the highest phenolic acid content of 191 mg/100 g hulls. Protocatechuic acid represented 49% of the total phenolic acids while syringic and salicylic acids accounted for 12% each. The p-coumaric, p-hydroxybenzoic, caffeic, gentistic, gallic, and vanillic acids were other phenolic acids which contributed to antioxidant activity. Chou, Chao, and Chung (2003) reported that a 50% ethanol extract of red beans had very good antioxidant activity, which was supported by Wu et al. (2004) who found that red beans had very good in vivo antioxidant activity. Tsuda et al. (1994a) assessed the antioxidant of white, red, and black bean seeds (Phaseolus vulgaris L.) and found that the seed coat and germ of the white varieties had no antioxidant activity. In contrast, the red and black seed coats had good antioxidant activity. The cyanidin-3-O-β-D-glucoside (C3G) and pelargonidin-3-O-β-D-glucoside were present in extracts of the seed coats of red beans whereas delphinidin-3-O-β-D-glucoside was found in the black bean seed coat (Takeoka et al. 1997). Tsuda et al. (1994b) found that delphinidin-3-O-β-D-glucoside, petunidin-O-β-D-glucoside, and malvidin-3-O-β-Dglucoside accounted for 56, 26, and 18%, respectively, of the anthocyanins in the black bean. Ariga and Hamano (1990) reported that procyanidins B-1 and B-3 from azuki beans had radical scavenging activity. Beninger and Hosfield (2003) also reported that procyanidins could be a good source of dietary antioxidants. Lin et al. (2001) reported that all the hot water extracts of legumes had antioxidant activities. Both mung bean and adzuki bean extracts had among the highest superoxide anion scavenging activity (Table 3.22). In the case of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, the antioxidant Table 3.22 Superoxide Scavenger Activities of Different Concentrations of HWEL in the Cytochrome c Test Samples Mung bean Adzuki bean Black bean Rice bean

Concentration (mg/ml)

Scavenging Effect (%)

IC50 (mg/ml)

0.1 0.1 0.1 0.1

42.35 50.00 25.38 21.40

0.14 0.10 0.30 0.46

Source: Based on Randhir, R., Lin, Y., and Shetty, K., Process Biochem., 39, 637–46, 2004.

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Phytochemicals in Cereals, Pseudocereals, and Pulses Table 3.23 LAP Values and ORAC Values of Tested Legumes

Samples Black beans Small red beans Mung beans Cowpeas Peas Kidney beans

LAP Value (mmol α-tocopherol equivalent/kg)

ORAC Value (µmol Trolox equivalent/kg)

44.4 43.5 40.0 38.8 21.4 19.2

44.8 58.8 83.3 37.0 4.6 47.3

Source: Cho, Y.-S., Yeum, K.-J., Chen, C.-Y., Beretta, G., Tang, G., Krinsky, N., Yoon, S., Lee-Kim, Y., Blumberg, J., and Russell, R., J. Sci. Food and Agr., 87, 1096–107, 2007.

activity in mung bean sprouts was highest on day two of dark germination nine due to enhanced polymerization of simple phenols contributed to high antioxidant activity producing intermediary metabolites (Randhir, Lin, and Shetty 2004). Cho et al. (2007) also found that all the beans had the highest lipophilic antioxidant capacity (LAP) and hydrophilic ORAC values than the fruits and vegetables (Table 3.23).

Future Direction The use of DPPH and ORAC has demonstrated the radical scavenging activity of many cereals, pseudocereals, and pulses. Although this method is useful for screening purposes, additional testing should be completed in model food systems such as emulsions or in biological-based assays (e.g., human low density lipoprotein models). Furthermore, going beyond the original product tested is necessary. Research by Liyana-Pathirana and Shahidi (2004, 2005) showed that in vitro digestion of wheat extracts enhanced the antioxidant capacity of original extract. The in vitro digestion simulated the gastric digestion process; thus, represents a potential model to evaluate the antioxidant activity of cereals, pseudocereals, and pulses that have entered the gastric digestion. Testing of compounds that have been isolated in biological samples is also necessary. Kozubek and Tyman (1999) noted that the original alkylresorcinols was not the active antioxidant but instead a trihydroxy derivatives of the alkylresorcinol was the biologically active compound. Kamal-Eldin et al. (2001) supported the observation that alkylresorcinols were not very active antioxidants, although some activity was observed. These studies demonstrate that the active compound is not always the product being extracted. To better understand the true health benefits and antioxidant ­activities of cereals, pseudocereals, and pulses, research beyond simple testing of plant extracts is necessary.

References Abdul-Hamid, A., R. Raja, A. Osman, and N. Saari. 2007. Preliminary study of the chemical composition of rice milling fractions stabilized by microwave heating. J. Food Comp. Anal. 20:627–37. Adom, K., and R. Liu. 2002. Antioxidant activity of grains. J. Agric. Food Chem. 50:6182–7. Adom, K., M. Sorrells, and R. Liu. 2003. Phytochemical profiles and antioxidant activity of wheat varieties. J. Agric. Food Chem. 52:7825–34. Adsule, R., and S. Kadam. 1989. Proteins. In Handbook of World Food Legumes (Volume II), eds. D. K. Salunkhe and S. S. Kadam, 75–97. Boca Raton, FL: CRC Press. Agarwal, C., Y. Sharma, and R. Agarwal. 2000. Anticarcinogenic effect of a polyphenolic fraction isolated from grape seeds in human prostate carcinoma DU145 cells: Modulation of mitogenic signaling and cell-cycle regulators and induction of G1 arrest and apoptosis. Mol. Carcinogenesis 28:129–38. Aguilar-Garcia, C., G. Gavino, M. Baragano-Mosqueda, P. Hevia, and V. Gavino. 2007. Correlation of tocopherol, tocotrienol, γ-oryzanol and total polyphenol content in rice bran with different antioxidant capacity assays. Food Chem. 102:1228–32.

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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). 83

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Classification of common edible fruits

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 <4 m in height. They are harvested at a firm-mature stage to reduce bruising (Anonymous 2009a). Turkey is the biggest sweet cherry producer in the world. There are 7,450,000 sweet cherry trees yielding approximately 230,000 tons per year in Turkey. The other countries producing sweet cherries are the United States with 175,000 tons and Iran with 115,000 tons (Vursavuş, Kelebek, and Selli 2006).

Phenolic and Beneficial Bioactives in Drupe Fruits

85

Sweet cherries are one of the most popular spring-summer fruit species and mainly consumed as a fresh table fruit. It is known that sweet cherry have various antioxidants and its major phenolic antioxidants are anthocyanins, phenolic acids, flavonols, and flavan-3-ols (catechins). The major phenolic acids of sweet cherries are hydroxycinnamic acids (HCA; Bernalte et al. 1999; Gao and Mazza 1995; Jakobek et al. 2007a, 2007b; Usenik, Fabčič, and Štampar 2008). The main hydroxycinnamates of sweet cherries are neochlorogenic acid and p-coumarylquinic acid while sweet cherries contain a little amount of chlorogenic acid (Kim et al. 2005) and ferulic acid (Matilla, Hellström, and Törrönen 2006). Sweet cherries contain approximately 1500 mg total phenols kg–1fresh weight (FW; Gao and Mazza 1995). Usenik, Fabčič, and Štampar (2008) reported that total phenolic content ranged from 443 to 879 mg gallic acid equivalents/kg–1 FW and antioxidant activity ranged from 8.0 to 17.2 mg ascorbic acid equivalent antioxidant capacity mg/100 g FW in fruits of 13 Slovene sweet cherry cultivars: Badascony, Burlat, Early Van Compact, Fercer, Fernier, Ferprime, Lala Star, Lapins, Noire de Meched, Sylvia, Vesseaux, Vigred (red-colored), and Ferrador (bi-colored). Gonçalves et al. (2004a) reported that Portugal cultivar Saco contained the highest amounts of phenolics [2270 mg/ kg–1 FW]. It is known that phenolics contribute to the total antioxidant activity (AA) of sweet cherries. Figure 4.2 shows that the main polyphenolic compounds present in sweet cherry cultivars are derived from shikimic acid via a different metabolic route. It is stated that antioxidant activity and phenolic profiles of sweet cherries depend on the genotype (Usenik, Fabčič, and Štampar 2008; Gonçalves et al. 2004a), maturity, and are affected by climatic conditions and storage (Gonçalves et al. 2004b). The 3-glucoside and 3-rutinoside of cyanidin were found as the major anthocyanins whereas peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin 3-glucoside were also present in Portugal varieties cvs Burlat and Van by Gonçalves and coworkers (2004a). Epicatechin was found as the main monomeric flavan-3-ol with catechin present in smaller amounts in sweet cherry cultivars studied by Gonçalves et al. (2004a). It is stated that the phenolic acid contents generally decreased with storage at 1–2°C and increased with storage at 15 + /–5°C, whereas anthocyanin levels increased at both storage temperatures. The anthocyanins increased up to fivefold during storage at 15 + /–5°C in cv Van (from 47 to 230 mg/100 g of FW; Gonçalves et al. 2004b).

Tart Cherries Tart cherries (Prunus cerasus) are also called sour cherries. Tart cherries are the smallest members of the stone fruit family. Tart cherries are very juicy and pleasantly acid, making them superior for cooking compared to their sweet cherry relative. They are best known as a key ingredient in desserts; most importantly, the cherry pie and it is also used in preserved foods, salads, side dishes, and beverages. Cyanidin-3-glucosylrutinoside and pelargonidin-3-glucoside are the major anthocyanins in tart cherries. It is found that a high amount of total anthocyanin content is present (6.44 and 4.02 g/kg of dry matter; Pedisic et al. 2008), whereas there are a significantly lower quantity of flavonols, hydroxycinnamates, flavan-3-ols, and procyanidins in tart cherries (Pedisic et al. 2008). Recently, Wang et al. (1999) characterized eight polyphenolic compounds in Montmorency and Balaton tart cherries from Michigan by 1H and 13C NMR experiments. These compounds are: (1) 5,7,4′-trihydroxyflavanone, (2) 5,7,4′-trihydroxyisoflavone, (3) chlorogenic acid, (4) 5,7,3′4 ′-tetrahydroxyflavonol-3-rhamnoside, (5) ′-trihydroxyflavonol-3-rutinoside, (6) 5,7,4′trihydroxy 3′methoxyflavonol3-rutinoside, (7) 5,7,4′-trihydroxyisoflavone-7-glucoside, and (8) 6,7-dimethoxy-5,8,4′-trihydroxyflavone (Figure  4.3). The antioxidant assays revealed that 7-dimethoxy-5,8,4′-trihydroxyflavone is the most active, followed by quercetin 3-rhamnoside, genistein, chlorogenic acid, naringenin, and genistin, at 10 µM concentrations (Wang et al. 1999). Tart cherries contain powerful antioxidants called anthocyanins—which provide the distinctive red color and may hold the key to the benefits locked inside (Chandra 1992; Wang 1996, 1999). Studies suggest that these disease-fighting pigments possess antioxidant, anti-inflammatory, antiaging, and anticarcinogenic properties (Blando 2004). Tart cherries are one of the richest sources of anthocyanins. The

86

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

COO O

NH3

HO

NH2

HO

+

N

N Serotonin

5-Hydroxytryptophan

N H

H3CO

CH3

N Melatonin

H2 COO– C CH NH NH3 +

Tryptophan COO–

OH

HO OH

Shikimate –OOC +

H3N

H2 C H COOH

COOH

R3

R1 R2 Benzoic acid derivatives R OH

HO

R3 R2

R1 Cinnamic acid derivatives HO

+

O

O OH OH Anthocyanidins derivatives

HO

O

OH

R1 R2 R3

Flavonols derivatives

Figure 4.2  Main polyphenolic compounds present in sweet-cherry cultivars derived from shikimic acid via different metabolic routes. (Adapted from González-Gómez, D., Lozano, M., Fernández-León, M. F., Bernalte, M. J., Ayuso, M. C., and Rodríguez, A. B., J. Food Comp. Anal., 2009, JFCA-D-08-00550DOI: doi:10.1016/j.jfca.2009.02.008.)

unique health benefits of cherries first came to light in the 1990s, when numerous studies were published describing the antioxidant content of this fruit. Spurred by what was then anecdotal evidence that cherries alleviated the pain of arthritis and gout, researchers discovered that cherries had high antioxidant activity. Additional studies identified the active antioxidants as eight polyphenolic compounds, including anthocyanins, chlorogenic acid, gallic acid, p-coumaric acid, and quercetin (Wang et al. 1999). It was reported that during ripening, anthocyanins did not change uniformly, but in most ecotypes they were determined in higher concentrations at the last stage of maturity (3.18–19.75 g per kg of dry matter; Pedisic et al. 2010). Pedisic et al. (2010) stated that the growing region and ripening significantly influenced the accumulation of individual anthocyanins and L-value of tart cherries. Individual anthocyanins from cherry cv Balaton to its jam showed that processing caused a 90% decrease in anthocyanins. In this context, more

87

Phenolic and Beneficial Bioactives in Drupe Fruits OH

RO

O

O

HO

HO

OH O (1)

OH

(7): R=Glucose H

HO

OH O

R1 OR2

O HO (4): R1 = OH, R2 = rhamnose (5): R1 = H, R2 = rutinose (6): R1 = OCH3, R2 = rutinose

OH

O C

H

HO

HO

O

(2): R=H

(3)

OH

O COOH OH OH

HO CH3O

O

CH3O HO

O (8)

Figure 4.3  Tart cherry polyphenols. (Adapted from Wang, H., Nair, M. G., Strasburg, G. M., Booren, A. M., and Gray, J. I., J. Agric. Food Chem., 47, 840–4, 1999.)

than 73% total phenolics and more than 65% antioxidant capacity were retained after processing fruits into jams (Kim and Padilla-Zakour 2004). Currently, tart cherries are incorporated into meat products for improved nutritional qualities of meats. Meat products containing tart cherries have become available to consumers. It has been found that cooled low-fat ground beef that includes 12% tart cherries had less rancidity development (Crackel et al. 1988). It is also reported that the addition of cherry tissue to ground beef prior to frying significantly inhibited heterocyclic aromatic amine formation (Britt et al. 1998). Wang et al. (1999) stated that these protective mechanisms of the cherries may be involved in the potential antioxidant polyphenolics present in cherries.

Apricot Apricot (Prunus armeniaca L.) is a species of Prunus, classified with the plum in the subgenus Prunus. The apricot shows fleshy drupe containing a hard, stony endocarp. The endocarp contains a single seed that is toxic because of high levels of cyanogenetic glucosides (Armstrong 2008; Huxley 1992).

Bioactives in Apricot Fruits Apricot fruits may be considered as a rich source of the bioactives, mainly polyphenols (Akın, Karabulut, and Topçu 2008; Bureau et al. 2009; Dragovic-Uzelac et al. 2005a, 2005b, 2007; Macheix, Fleuriet, and ve Billot 1990; Madrau et al. 2009; Radi et al. 1997; Rashid et al. 2007; Sass-Kiss et al. 2005; Stratil, Klejdus, and Kuban 2007; Sultana and Anwar 2008; Veberic and Stampar 2005; ) and carotenoids (Akın, Karabulut, and Topçu 2008; Dragovic-Uzelac et al. 2007; Kurz, Carle, and Schieber 2008; Radi et al. 1997; Ruiz et al. 2005; Sass-Kiss et al. 2005), which contribute significantly to their taste, color, and nutritive values (Figure 4.4). There is a noticeable interest in polyphenols and carotenoids owing to their antioxidant properties and ability to alleviate chronic diseases (Gardner et al. 2000; Rice-Evans, Miller, and Paganga 1997; Vinson 1998). The alterations of the bioactives in apricots have been studied during ripening, in relation to the geographical region (Bureau et al. 2009; Dragovic-Uzelac et al. 2007; Garcia-Viguera, Zafrilla, and TomasBarberan 1997; Radi et al. 1997), in relation to apricot genotype (cultivar) and seasonal differences (Dragovic-Uzelac et al. 2007; Radi et al. 1997; Ruiz et al. 2005; Sass-Kiss et al. 2005; Scalzo et al. 2005;

88

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Major apricot bioactives Phenolics

Carotenoids

Phenolic acids

Flavonoids

Chlorogenic acid (5-caffeoylquinic acid)

Flavonols Quercetin Kaempferol & their glucosides and rutinosides Aesculetin Scopoletin

Neochlorogenic acid Caffeic acid p-coumaric acid Ferulic acid & their esters

Flavanols (+)-Catechin (–)-Epicatechin

Carotenes Anthocyanidins Cyanidin-3-Orutinoside Cyanidin-3-Oglucoside Peonidin-3-Orutinoside

β-Carotene a-Carotene c-Carotene

Xanthophylls Zeaxanthine Lutein

Figure 4.4  Major bioactive compounds in apricots (Prunus armeniaca L.) (Compiled by Tokuşoğlu Ö.)

Veberic and Stampar 2005), after puree preparation (Dragovic-Uzelac et al. 2005a, 2005b), in relation to the drying temperature (Madrau et al. 2009), and after industrial processing and storage (Durmaz and Alpaslan 2007; Jiménez et al. 2008). The apricot varieties include different levels of polyphenols, which have been abbreviated by Macheix, Fleuriet, and ve Billot (1990). Chlorogenic acid (5-O-caffeoylquinic acid) is the dominant phenolic compound in apricots (Garcia-Viguera et al. 1994; Radi et al. 1997). The HCA (neochlorogenic acid, caffeic acid, p-coumaric acid ferulic acid, and their esters) are the most common phenolic acid bioactives (Herrmann 1973; Henning and Herrmann 1980a; Radi et al. 1997; Figure 4.4). (+)-Catechin and (–)-epicatechin are other important groups of flavanols in apricot fruits and apricot-based products (Arts, van de Putte, and Hollman 2000; Dragovic-Uzelac et al. 2005a, 2005b; Garcia-Viguera, Zafrilla, and Tomas-Barberan 1997; Herrmann, 1973; Radi et al. 1997; Rish & Herrmann, 1988; Figure 4.4). It is also reported that procyanidin B1, procyanidin B2, and procyanidin B3 were found in apricot fruits (DragovicUzelac et al. 2007). Flavonols in apricots occur mostly as glucosides and rutinosides of quercetin and of kaempferol, however, quercetin 3-rutinoside (rutin) predominates (Dragovic-Uzelac et al. 2005a, 2005b, 2007; GarciaViguera et al. 1994; Henning & Herrmann 1980). Aesculetin and scopoletin have also been determined in lower amounts in some apricot cultivars (Fernandez de Simon, Perez-Ilzabre, and Hernandez 1992; Macheix, Fleuriet, and ve Billot 1990; Resche and Herrmann 1981). In a study given by Sultana and Anwar (2008), total flavonol levels of apricots were 784.8 ± 32.6 mg kg–1 dry matter whereas individual flavonols myricetin, quercetin, and kaempferol of apricots were 406.9 ± 16.3; 322.1 ± 6.4; 5.8 ± 0.2 mg kg–1 dry matter, respectively (Sultana and Anwar 2008). The alterations in HCA levels in apricot fruits from all genotypes showed the same trend during ripening, with the highest values at the first ripening stage (immature) and the lowest values at the commercial mature stage. The decrease in HCA levels is a well-known fact during maturation (Macheix, Fleuriet, and ve Billot 1990). Only a few reports described the biochemical changes in apricot fruits at different ripening stages. Soluble and insoluble proteins were decreased during ripening apricot fruits, free amino acids varied according to the stages of maturity, while total and soluble carbohydrates increased (Sharaf, Ahmed, and El-Saadany 1989). Differences in amounts of chlorogenic acid, kaempferol-3-rutinoside, and quercetin-3-rutinoside were observed in 11 apricot fruit varieties in three stages of maturity (Garcia-Viguera et al. 1994). Apricot fruits are regarded as a rich source of carotenoids, especially b-carotene, which represents more than 50% of total carotenoid content (Radi et al. 1997; Sass-Kiss et al. 2005). Besides b-carotene, apricot fruit and its products contain smaller amounts of a-carotene, c-carotene, zeaxantin, and lutein (Fraser and Bramley 2004). The data about carotenoid changes during apricot fruits ripening are not fully reported. In apricot fruits, ripening is accompanied by enhanced biosynthesis of carotenoids (Katayama et al. 1971). The beta-carotene level was determined to be significantly different among varieties and among different regions within the same variety (Munzuroglu, Karatas, and Geckil 2003).

89

Phenolic and Beneficial Bioactives in Drupe Fruits

Research on other plant species indicated that significant changes in carotenoid amounts occur according to the stage of maturity.

Plum and Prune A plum (Prunus domesticus L.) is a stone fruit tree in the genus Prunus, subgenus Prunus. The subgenus is different from other subgenera that belong to the peach, cherry, and so on. Plums have a plump, round shape with a stem at the top. Their skin is very smooth, shiny and can be red, purple, or yellow color. The dried plum is called a prune. Plums are the most numerous and diverse group of fruit tree species (Figure 4.5) and mostly contain fruits of Prunus domestica, Prunus salicina, Prunus spinosa whereas the dried fruit prune is of some genotypes of Prunus domestica (Armstrong 2008; Blažek 2007; Huxley 1992). It is indicated that the European plums (P. domestica L.) are dried, for the table, and some canning while the Japanese plums are for the table (P. Salinica Lindl.), fresh, and canning (P. institia L.). P. cerasifera Ehrh. included in Japanese plums are commonly used as rootstock and it is consumed as fresh and canning plums (Ozcagiran 1976).

Bioactives in Plum Plums are considered a fruit class with high amounts of bioactive compounds or phytochemicals such as vitamins (A, C, and E), anthocyanins and other phenolics, and carotenoids (StacewiczSapuntzakis et al. 2001), which contribute to the antioxidant capacity. Plums have been known to contain various kinds of phenolics, containing HCA, flavonols, and anthocyanins (Figure 4.6).

var. stanley

var. papaz

var. angelina

var. formosa

var. giant

Figure  4.5  (See color insert) Various plum cultivars. (Adapted from Asli Fidancilik, İzmir, Turkey, Various plum cultivars Asli Fidancilik, İzmir, Turkey, 2009.) Major plum & prune bioactives Phenolics Flavonoids

Phenolic acids

Neochlorogenic acid (3-O-caffeoylquinic acid) Chlorogenic acid (5-O-caffeoylquinic acid) Crypto chlorogenic acid (4-O-caffeoylquinic acid) Minor phenolic acids; Gallic, protocatechuic p-hydroxybenzoic, vanillic caffeic, syringic, p-coumaric methoxycinnamic, ferulic methoxybenzoic acids.

Carotenoids & vitamins

Flavan-3ols Catechin Procyanidins Procyanidin B1 Procyanidin B2 Procyanidin B4

Flavonols

Rutin Quercetin Kaempferol & their glucosides rutinosides Galactosides Myricetin

Carotenes Anthocyanidins

β-Carotene

Cyanidin-3-Oglucosides Peonidin-3-Oglucosides Cyanidin-3-Orutinosides Peonidin-3-Orutinosides

Vitamins

Figure 4.6  Major bioactives in plum and prune fruits. (Compiled by Tokuşoğlu.)

Ascorbic acid (vitamin C) Tocopherol (vitamin E)

90

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Characteristically, plums are predominant with neochlorogenic acid (73% of total phenolics) and c­ hlorogenic acid (13%). Together, hydroxycinnamates (caffeoylquinic acids) constituted 86% of total phenolics. Minor phenolic compounds in fresh prune plums are anthocyanins (7%), flavan-3-ol catechin (5%), and flavonol rutin (2%; Macheix and Fleuriet 1998; Möller and Hermann 1983; StacewiczSapuntzakis et al. 2001).

Chlorogenic Acid and Its Derivatives in Plums and Prunes It was reported that chlorogenic acid and its isomers are major phenolic compounds in plums and prunes (Figure 4.7). Neochlorogenic acid (3-O-caffeoylquinic acid, 3-CQA) is a predominant polyphenol in the fresh fruit plum or dried fruit prune (Fang, Yu, and Prior 2002) and chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA), cryptochlorogenic acid (4-O-caffeoylquinic acid, 4-CQA) are often found as phenolic acids in plum varieties (Fang, Yu, and Prior 2002; Donovan, Meyer, and Waterhouse 1998; Herrmann, 1989; Nakatani et al. 2000; Tomàs-Barberàn et al. 2001; Figure 4.7). It is reported that 88–731 mg/kg of neochlorogenic acid (3-O-caffeoylquinic acid, 3-CQA), 15–129 mg/ kg of chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA), and 56 mg/kg of cryptochlorogenic acid (4-Ocaffeoylquinic acid, 4-CQA) are found in fresh plums in the study described by Möller and Hermann (1983). It is indicated that 807–1306 mg/kg of neochlorogenic acid (3-O-caffeoylquinic acid, 3-CQA) is in fresh prune-making plums and 144–436 mg/kg of chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA) is in pitted prunes (Donovan, Meyer, and Waterhouse 1998). It is determined that the 3-CQA ranged from 1228 to 1485 mg/kg whereas cryptochloro-genic acid (4-O-caffeoylquinic acid, 4-CQA) ranged from 288 to 351 mg/kg, and 5-CQA varied from 53 to 77 mg/kg in prunes (Prunus domestica L.) given by Nakatani et al. (2000) in a study in Osaka, Japan.

Other Phenolic Acids, Flavonols and Flavan-3-ols in Plums and Prunes It is indicated that the HCA derivatives are mostly present in the peel of a plum (Tomàs-Barberàn et al. 2001). The total HCA level is 115–375 mg/kg in the peel part of a plum whereas 16.3–194 mg/kg is in the flesh part of a plum. Dried prunes contain higher amounts of phenolic compounds (184 mg/100 g of fruit) than prune-making plums (Donovan, Meyer, and Waterhouse 1998) owing to the dehydration process concentrates of the constituents despite partial degradation. Dried plums lead to HCA degradation related to the fruit polyphenoloxidase activity; most of the HCA compounds are degraded when plums are dried at a lower temperature (Raynal, Moutounet, and Souquet 1989, Raynal and Moutounet 1989). OR5

HOOC

OH OH OR3 O R5 = H;

OH

R3 =

Neochlorogenic acid

OH O

R3 = H;

OH

R5 =

Chlorogenic acid Figure 4.7  Neochlorogenic and chlorogenic acids in plums.

OH

91

Phenolic and Beneficial Bioactives in Drupe Fruits

Small amounts of caffeic and coumaric acids (1% of phenolics) appear in dried prunes, probably as a result of cinnamate hydrolysis during processing (Stacewicz-Sapuntzakis et al. 2001). The concentrations of HCAs increase with fruit development, showing a loss when reaching maturity but show a remarkable loss when examined after removal of the harder seeds (Stöhr, Mosel, and Herrmann 1975). Neochlorogenic acid represents 71% of the total phenolics and chlorogenic acid 24%, raising the content of hydroxycinnamates to 95% of all phenolic compounds (Stacewicz-Sapuntzakis et al. 2001). It is reported that there is 1.5 mg/100 g of quercetin in the Denmark plum blue (as fresh weight; Justesen, Knuthsen, and Leth 1998) and that 564.1 mg/kg of myricetin, 0.7 mg/kg of kaempferol, and 564.8 mg/kg of total flavonols has been detected in plums at Faisalabad, Pakistan (as dry matter; Sultana and Anwar 2008). It is reported that fresh plums from Holland and Denmark include 0.9–1.5 mg/100 g of quercetin as an edible portion (Hertog, Hollman, and Katan 1992; Justesen, Knuthsen, and Leth 1998). Rutin (quercetin 3-rutinoside; Figure 4.8.) is present as 2% of all phenolics and is found in the exocarp of plums (Raynal, Moutounet, and Souquet 1989; Stacewicz-Sapuntzakis et al. 2001). Rutin is the predominant flavonol glycoside in fresh and dried plums (Fang, Yu, and Prior 2002; Raynal, Moutounet, and Souquet 1989; Stacewicz-Sapuntzakis et al. 2001). Macheix et al. (1989) reported that Prunus domestica and Prunus salicina genotypes of plums include 3-glycosides, 3-galactosides, 3-rutinosides, and 3-arabinoside-7-rhamnosides of quercetin and kaempferol (Macheix, 1990). Henning and Herrmann (1980b) determined that six examined cultivars of Prunus domestica plums contain mainly 3-rutinosides (Figure  4.9) and smaller concentrations of OH HO

O

OH

OH

HO

OH

OH

O

O

O

O H3C

O

HO HO OH Figure 4.8  Rutin (Quercetin 3-rutinoside) in plum. OH OH HO

O

OR OH R = galactoside R = glucoside R = rutinoside

O Quercetin-3-galactoside Quercetin-3-glucoside Quercetin-3-rutinoside

Figure 4.9  Quercetin glycosides in plum and prunes. (Adapted from Tomas Barberàn, F. A., Gil, M. I., Cremin, P., Waterhouse, A. L., Hess-Pierce, B., and Kader, A. A., J. Agr. Food Chem., 49, 4748–60, 2001.)

92

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

the 3-glycosides and 3-galactosides of kaempferol and quercetin and quercetin-3-rhamnoside. It is also been detected that the main flavonols are kaempferol-3,7-bisrhamnoside and kaempferol-3-arabinosyl7-rhamnoside in two examined cultivars of Prunus salicina plums in the study given by Henning and Herrmann (1980b). It has also been detected that kaempferol-3-rutinoside and quercetin- 3-rhamnoside, -xyloside, -glucoside, -galactoside, -α-l-arabino furanoside and-rutinoside, quercetin-7-rhamnoside and kaempferol-7-glucoside are in P. salicina plums in the study described by Henning and Herrmann (1980b). Flavonol glycosides were found in the range of 186–352 mg rutin/kg in a ripe plum (Tomàs-Barberàn et al. 2001). The total levels of flavonol glycosides were 20–52 mg/kg and 8–24 mg/kg in the investigated fresh plums of Prunus domestica and Prunus salicina, respectively (Henning and Herrmann 1980b). Mosel and Herrmann (1974) indicated that fresh plums contain flavan-3-ols in the range of 20–40 mg/kg. It is detected that there is 49 mg/100 g of catechin in French fresh plums (Auger et al. 2004). An average of 3.35 mg/100 g of catechin and an average of 2.84 mg/100 g of epicatechin is in the edible portion (Arts, van de Putte, and Hollman 2000). Tomàs-Barberàn et al. (2001) reported that the total flavan-3-ols level was 662–1837 mg/kg (as catechin equivalent) in the peel of plums and 138–618 mg/kg (as catechin equivalent) in fruits (Table 4.1). Figure  4.10 shows catechin compounds and proantocyanidins in plum and prunes. It also has been reported in relation to the mg per fruit, the concentrations of catechins increase progressively with fruit development, showing a loss when reaching maturity (Stöhr, Mosel, and Herrmann 1975). TomàsBarberàn et al. (2001) found that the plum cultivars Black Beaut and Angeleno were especially rich in

Table 4.1 Flavan 3-ols (mg/kg Fresh Weight) in Plum Cultivars (as Catechin)a A-Type Dimers

Cultivar

Stage

Part

PRO B1

PRO B2

PRO B4

Angeleno

mature

peel flesh peel flesh peel

80.6 ± 15.4 31.6 ± 15.0 94.9 ± 11.3 48.5 ± 10.8 291.5 ± 3.7

52.1 ± 29.3 159.0 ± 54.6 183.3 ± 21.1 165.7 ± 16.2 677.6 ± 24.0

339.9 ± 69.8 120.1 ± 42.1 343.9 ± 52.1 110.6 ± 18.2 137.3 ± 2.8

flesh peel flesh peel

136.0 ± 24.4 251.8 ± 18.0 161.9 ± 10.4 90.0 ± 16.0

136.8 ± 5.1 710.5 ± 23.4 142.8 ± 9.1 90.1 ± 21.9

88.0 ± 4.5 118.6 ± 9.2 94.4 ± 3.8 73.4 ± 10.1

flesh peel flesh peel flesh peel flesh peel flesh peel flesh

121.0 ± 37.2 81.1 ± 10.0 100.5 ± 5.4 268.3 ± 62.1 40.7 ± 11.4 115.3 ± 80.0 17.9 ± 3.1 539.9 ± 12.5 110.1 ± 17.2 473.2 ± 30.8 96.7 ± 3.9

58.0 ± 6.0 105.7 ± 12.3 59.4 ± 2.6 253.1 ± 52.4 41.8 ± 2.1 280.0 ± 58.0 43.6 ± 4.8 484.8 ± 28.2 45.7 ± 6.0 416.0 ± 35.9 41.8 ± 0.8

— 117.0 ± 51.8 77.2 ± 22.2 72.1 ± 18.6 41.4 ± 2.8 85.3 ± 15.4 39.5 ± 6.2 — — — —

75.3 ± 10.1 37.2 ± 2.8 75.5 ± 13.8 37.0 ± 4.3 77.6 ± 5.7 —

204.7 ± 33.9 20.8 ± 6.4 430.1 ± 122.8 83.5 ± 44.1 431.7 ± 34.1 —

52.4 ± 4.5 —

417.8 ± 50.0 —

ripe Black Beaut

mature

ripe Santa Rosa

mature

ripe Red Beaut

mature ripe

Wickson

mature ripe

Other

Total

256.8 ± 10.1

308.5 ± 52.2 66.8 ± 14.8 237.0 ± 46.2 60.5 ± 9.0 287.3 ± 20.8

1151.1 ± 208.0 377.3 ± 125.8 1393.6 ± 205.4 385.4 ± 53.5 1650.6 ± 19.6

103.0 ± 9.2 276.0 ± 7.0 105.6 ± 4.4 —

102.3 ± 19.8 479.9 ± 50.7 113.7 ± 23.4 408.7 ± 95.3

566.1 ± 51.0 1836.8 ± 96.2 618.3 ± 2.3 662.2 ± 133.1

—

—

33.5 ± 14.3 —

411.8 ± 55.3 —

179.0 ± 39.7 749.1 ± 124.5 237.1 ± 24.2 847.4 ± 171.1 181.9 ± 15.1 986.3 ± 202.0 221.5 ± 43.4 1533.9 ± 53.7 155.8 ± 22.5 1254.6 ± 103.2 138.5 ± 4.0

477.4 ± 66.0 564.8 ± 101.8 -

Source: Adapted from Tomas Barberàn, F. A., Gil, M. I., Cremin, P., Waterhouse, A. L., Hess-Pierce, B., and Kader, A. A., J. Agr. Food Chem., 49, 4748–60, 2001. a Standard deviations (n = 3).

93

Phenolic and Beneficial Bioactives in Drupe Fruits

Catechin Epicatechin

R1 = H,

R2 = OH

R1 = OH,

R2 = H

OH OH O

HO

OH

OH

H R1 R2

OH O

HO

H R1 R2

OH

OH OH

O

HO

OH

H R3 R4

Pro B1 R1,4 = OH, R2,3 = H Pro B2 R1,3 = OH, R2,4 = H Pro B4 R1,4 = H,

R2,3 = OH

Figure 4.10  Catechins and proantocyanidins in plum and prunes. (Adapted from Tomas Barberàn, F. A., Gil, M. I., Cremin, P., Waterhouse, A. L., Hess-Pierce, B., and Kader, A. A., J. Agr. Food Chem., 49, 4748–60, 2001.)

phenolics when grown around the Fresno area of California. Dimeric and trimeric forms of catechin were detected by HPLC-MS and quantified by Tomàs-Barberàn et al. (2001). It is reported that no clear tendency in the flavan-3-ol level of plums was observed with ripening, as Angeleno and Black Beaut increased their content, but Wickson decreased its content (TomàsBarberàn et al. 2001). Tomàs-Barberàn et al. also indicated that the results of these differences are significant from the taste point of view, as these kinds of compounds are responsible for the astringency of plums. The recent analysis given by Tomàs-Barberàn et al. has shown that the main flavan 3-ol derivatives in plums are procyanidin dimers (B1, B2, B4, and A-type dimers; Figure 4.10.), and trimers (these in smaller amounts). Tomàs-Barberàn et al. (2001) reported the occurrence of A-type dimers in some plum varieties is of some interest, as this kind of compound has been previously reported only in cranberry fruits (Porter 1994) and it may have antibacterial activity.

Anthocyanins in Plums and Prunes Cyanidin-3-glucosides and cyanidin 3-rutinosides are the major anthocyanins in plums (P. salicina) and prunes (Tomàs-Barberàn et. al. 2001; Piga, del Caro, and Corda 2003; Wu and Prior 2005). It was previously identified the cyanidin-3-glucosides, cyanidin-3-rutinosides, peonidin-3-glucosides, and peonidin-3-rutinosides in plums and sloe (Macheix, 1990; Raynal, Moutounet, and Souquet 1989; Van Buren 1970; Figure 4.11). Detected also was 3-galactosides and 3-acetylglycosides of cyanidin and peonidin apart from 3-glucosides and 3-rutinosides of cyanidin and peonidin in plums (Tomàs-Barberàn et al. 2001). According to the study given by Tomàs-Barberàn et al. (2001), the main pigmentation in plums were detected as cyanidin 3-glucoside and 3-rutinoside. However, in Red Beaut, Black Beaut, and Angeleno the presence of small amounts of cyanidin 3-acetyl glucoside was also detected, and in the case of Santa Rosa, small amounts of cyanidin 3-galactoside were found (Tomàs-Barberàn et al. 2001). Cyanidin 3-glucoside and cyanidin 3-rutinoside were identified in two Italian plum cultivars, “Sugar” and “President” in the study described by Piga, del Caro, and Corda (2003) while five anthocyanins were identified in plums sampled directly from the United States market (cyanidin 3-galactoside, cyanidin 3-glucoside, cyanidin 3-rutinoside, cyanidin 3-xyloside, and cyanidin 3-(6″’-acetoyl) glucoside (Wu and Prior 2005).

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

HO

OR OH R = glucoside R = rutinoside

Cyanidin-3-glucoside Cyanidin-3-rutinoside

Figure 4.11  Anthocyanins in plum and prunes.  (Adapted from Tomas Barberàn, F. A., Gil, M. I., Cremin, P., Waterhouse, A. L., Hess-Pierce, B., and Kader, A. A., J. Agr. Food Chem., 49, 4748–60, 2001.)

Anthocyanin [mg/100 g FW]

30 25 20 Peonidin Cyanidin

15

10 5 0

t1

t5 Jojo

t1 t5 t1 t5 Cacanska najbolja Cacanska rodna Terms cultivar

t1 t5 Valor

Figure 4.12  Average individual anthocyanin amounts for four plum cultivars at first and fifth terms of measurements (storage effects) [Average contents of total anthocyanins includes standard errors (±Se)] expect for Valor. (Adapted from Usenik, V., Kastelec, D., Veberič, R., and Štampar, F., Food Chem., 111, 830–6, 2008.)

Two anthocyanins, cyanidin rutinoside and peonidin rutinoside, were identified in plum (Prunus domestica L.) cultivars (Jojo, Valor, Čačanska rodna, Čačanska najbolja) in a Slovenia study described by Usenik et al. (2008). In that study the major anthocyanin was cyanidin rutinoside, which ranged from 0.8 to 26.6 mg/100 g FW, followed by peonidin rutinoside, ranging from 0.0 to 6.1 mg cyanidin-3­r utinoside CRE/100 g FW (Usenik et al. 2008). During the 25-day period after ripening, it was determined the increased concentration of anthocyanins (Figure 4.12) in plum varieties (Usenik et al. 2008). As expected, the anthocyanins were present only in the red cultivars, but in some cases, in over-ripe Wickson plums, a red pigmentation was also detected in the study reported by Tomàs-Barberàn et al. (2001). In the study given by Usenik, Štampar, and Veberič (2009), European plum (P. domestica L.) cultivars (Jojo, Valor, Čačanska rodna, Čačanska najbolja cultivars) were quantified for anthocyanin accumulation during a 25-day period of ripening (a 33-day period in the case of Jojo). The major anthocyanin was cyanidin 3-rutinoside, which in ripe fruits ranged from 4.1 to 23.4 mg/100 g FW (from 52.6 to 73.0%). It was followed by peonidin 3-rutinoside (from 6.5 to 37.9%), cyanidin 3-glucoside (from 1.8 to 18.4%), cyanidin 3-xyloside (from 4.7 to 7.8%), and peonidin 3-glucoside (from 0.0 to 0.4%; Usenik, Štampar, and Veberič 2009). Díaz-Mula et al. (2009) found that the levels of anthocyanins were always higher in the peel (20–40 fold) than in the pulp with significant differences among Spain cultivars (Díaz-Mula et al. 2009). The

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Phenolic and Beneficial Bioactives in Drupe Fruits

highest anthocyanin concentration was found in the peel of the Black Amber variety (4370 mg/kg) while the lowest occurred in the Black Diamond peel (1310 mg/kg) at harvest in the study reported by DíazMula et al. (2009). It was reported that the anthocyanin degradation rate increased with the increased drying temperatures (Raynal, Moutounet, and Souquet 1989). It has been suggested that anthocyanins can be transformed by a coupled oxidation mechanism involving quinones formed from chlorogenic acid under polyphenoloxidase dependence (Raynal and Moutounet 1989). According to Raynal, Moutounet, and Souquet (1989), 85% of the cyanidin 3-rutinoside degraded in plums when dried at 95°C for 1 hour.

Total Phenolics in Plum and Prunes Karakaya, El, and Taş (2001) 1435 mg/kg and 3679 mg/kg of total phenolics (as catechin equivalent) in Turkish fresh and dried black plums, respectively. Marinova, Ribarova, and Atanassova (2005) determined that the total of phenolics was 303.6 mg GAE/100g, the total of flavonoids was 136.2 mg CE/100g in Bulgarian plums (Prunus domestica), and the ratio of total flavonoids/total phenolics was 0.45 (Marinova, Ribarova, and Atanassova 2005). Chun and Kim (2004) reported the total phenolic contents of 13 plum cultivars (Beltsville Elite B70197, BY 69–339, Cacaks Best, Castleton, Early Magic, Empress, French Damson, Longjohn, Mirabellier, NY 101, NY 6, NY 9, Stanley from the New York State Agricultural Experiment Station Orchards in Geneva, New York as shown in Figure 4.13. According to the study given by Chun and Kim (2004), the total phenolics of Geneva, New York cultivars ranged from 833.6 ± 4.8 mg GAE/100 g to 138.1 ± 2.9 mg GAE/100 g as gallic acid equivalent. In that study, the average total phenolics of 13 plum cultivars calculated by CAE was 678.2 mg CAE/100 g, and it was higher than that of GAE, 368.7 mg GAE/100 g by 1.84-fold (Chun and Kim 2004; Figure 4.13). Fourteen red-fleshed plum (Prunus salicina Erhr. and hybrids) genotypes were characterized for their total phenolic contents by Cevallos-Casals et al. (2006) and it has shown that a total phenolic content range from 298 to 563 mg CGA/100 g exists for plums. Selected rich phenolic genotypes showed high antioxidant activity, stable color properties and good antimicrobial activity. Their results indicated positive correlations between phenolic compounds (r2 = 0.83) and antioxidant activity for plum fruit (Cevallos-Casals et al. 2006). The phenolic content observed in the Prunus salicina varieties in the study given by Cevallos-Casals et al. (2006) is higher than that previously reported for Prunus domestica varieties (160–300 mg/100 g; Los, Wilska, and Pawlak 2000) and for other commercial varieties reported as 14–109 mg/100 g (Gil et al. 2002) and as 125–373 mg/100 g by Kim et al. (2003). The selected rich phenolic plum genotype in the

Total phenolics (mg/100 g)

1600 GAE

1400

CAE

1200 1000 800 600 400 200

ST

1

9

10

Y

B

6

N

Y N

Y N

Plum cultivar

M

LJ

FD

EP

EM

T C

CB

BY

BE

0

Figure 4.13  The content of total phenolics by gallic acid equivalent (GAE) and chlorogenic acid equivalent (CAE) of BE stands for Beltsville Elite B70197; BY, BY69-339; CB, Cacaks Best; CT, Castleton; EM, Early Magic; EP, Empress; FD, French Damson; LJ, Longjohn; MB, Mirabellier; ST, Stanley. Content of total. (Adapted from Chun, O. K., and Kim, D. O., Food Res. Inter., 37, 337–42, 2004.)

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study given by Cevallos-Casals et al. (2006) exhibited strong antimicrobial activity against Salmonella Enteritidis and Escherichia coli O157:H7. It is reported that the selection of crops high in phenolic compounds can be related to enhanced functional properties and opens the possibility of breeding fruits with targeted functional properties for the fresh produce and processing market (Cevallos-Casals et al. 2006). The Prunus Breeding Program at Texas A&M University and the USDA Stone Fruit Breeding Program at Byron, Georgia are working at developing plums (Prunus salicina Erhr. and hybrids) with high levels of beneficial phenolic compounds for the fresh produce and processing market (Cevallos Casals et al. 2006). Cevallos Casals et al. (2006) determined that plums showed a three to fourfold higher phenolic concentration in the skin than in the flesh. According to their study, the exocarp of a plum is a concentrated source of phenolic compounds, it only represents 7–9% of the fruit weight and the total distribution of phenolic compounds in skin and flesh per fruit is 30% and 70%, respectively. Large variations in the concentration of bioactive compounds at commercial harvesting depending on the cultivar have been reported (Cevallos-Casals et al. 2006; Gil et al. 2002; Kim et al. 2003; Los, Wilska, and Pawlak 2000; Tomàs-Barberàn et al. 2001).

Antioxidant Activity of Plums and Prunes Plums have been proven to have effective antioxidant activity (CDPB 2009; Chun et al. 2003; Chun Kim, and Lee 2003; Donovan, Meyer, and Waterhouse 1998; Gil et al. 2002; Kim et al. 2003; Kim, Jeong, and Lee 2003; Murcia, Jimènez, and Martínez-Tomè 2001; Nakatani et al. 2000; Wang, Cao, and Prior 1996). Wang, Cao, and Prior (1996) demonstrated that plums had 4.4 times higher total antioxidant capacities than apples, the latter being one of the most commonly consumed fruits in a diet. They reported the total antioxidant capacity of various fruits including plums, using Trolox equivalents, where Trolox is a vitamin E analogue but not a natural compound. It is reported that selected plums BY94M1945 (Prunus salicina Erhr) rich in phenolic contents show high antioxidant activities in the study given by Cevallos Casals et al. (2006). In that study, on a wet basis, the selected plum has ∼91% of the antioxidant activity of blueberry fruit whereas on a dry basis, the plum shows ∼36% higher antioxidant activity than the blueberry (Cevallos Casals et al. 2006). Various phenolic compositions, in five different varieties of yellow and red plums, were recently analyzed by HPLC-DAD-ES IMS, but no antioxidant activity was reported (Tomàs-Barberàn et al. 2001). Plums are the most numerous and diverse group of fruit tree species (Blažek 2007). Among nutritional factors, recent observations suggest that dried plums or prunes (Prunus domestica L.) is the most effective fruit in both preventing and reversing bone loss (Hooshmand and Arjmandi 2009). Animal studies and a 3-month clinical trial conducted in our laboratories have shown that the dried plum has positive effects on bone indices (Hooshmand and Arjmandi 2009). Figure  4.14 shows the potential biological functions of prune constituents (Stacewicz-Sapuntzakis et al. 2001).

Glucose metabolism

Dietary fiber

Laxative

Sorbitol Cardiovascular health

Potassium

Antitumor

Copper Bone metabolism

Boron Phenolics

Antioxidant

Figure 4.14  Potential biological functions of prune constituents. (Adapted from Stacewicz-Sapuntzakis, M., Bowen, P. E., Hussain, E. A., Damayanti-Wood, B. I., and Farnsworth, N. R., Crit. Rev. Food Sci. Nutr., 41(4), 251–86, 2001.)

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Phenolic and Beneficial Bioactives in Drupe Fruits

Prunes, which are industrially obtained by dehydrating fresh plums at 85–90°C for 18 hours, ­contain higher levels of phenolic compounds than most other fruits. Prune phenolics have shown beneficial effects on human health (Piga, del Caro, and Cordo 2003; Stacewicz-Sapuntzakis et al. 2001).

Peach and Nectarine The peach (Prunus persica L.; Figure 4.15) is known by the species of Prunus that bears an edible juicy fruit. It is a deciduous tree growing 5–10 m (~20 feet) tall, belonging to the subfamily Prunoideae of the family Rosaceae (Armstrong 2008; Huxley 1992). Nectarines [Prunus persica (L.) Batsch, var. nectarina] [P. persica var. nucipersica] (Figure  4.16) belong to the Rosaceae family and is a variety of the peach tree (Anonymous 2009c; Salunkhe and Desai 1984). The nectarine is closely related to the peach (Brady 1993) and has a characteristic, lignified endocarp (pit or stone) that encloses the seed, a fleshy mesocarp, and a thin exocarp. However, nectarine cells have smaller intercellular spaces than peaches and are, therefore, denser (Lill, O’Donaghue, and King 1989). Nectarines and peaches are similar in appearance and color as they differ only by a single gene, the gene for skin texture. The color of nectarines varies as red, pink, yellow, or white and the skin of nectarines is smooth, fuzzless, and shiny (Anonymous 2009c),

Bioactives in Peach and Nectarine The peach is rich in ascorbic acid (vitamin C), carotenoids (provitamin A), and phenolic compounds that are good sources of antioxidants (Drogoudi and Tsipouridis 2007; Byrne 2002; Tomàs-Barberàn et al. 2001). The total phenolic content ranges from 100 to 449 mg CGA/100 g for peaches. The anthocyanin content in peaches ranged from 6 to 37 mg/100 g (Cevallos-Casals et al. 2006). It was reported that chlorogenic acid is a major phenolic compound in peaches and nectarines (Figure 4.17). It was stated that nectarines and peaches showed identical phenolic profiles, containing the same individual compounds (Tomàs-Barberàn et al. 2001).

var. florida king

var. redhaven

var. washington

var. earlyred

var. scarlet ohara

var. cardinal

Figure 4.15  (See color insert) Various peach cultivars. (var. Florida king was adapted from Aaron’s Nursery, Sumner, Georgia; var. Red Haven, var. Washington, var. Early Red, var. Cardinal were adapted from Asli Fidancilik, İzmir, Turkey; var. Scarlet Ohara was adapted from Harvey Hall, Horticultural & Food Research Institute, New Zealand.)

var. purdue

var. armking

var. lord napier

Figure  4.16  (See color insert) Various nectarine cultivars. (var. Purdue was adapted from Purdue University Horticultural Section; var. Armking was adapted from Asli Fidancilik, İzmir, Turkey; var. Lord Napier was adapted from Victoriana Nursery Gardens, London, UK.)

98

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Major peach and nectarine bioactives Phenolics

Hydroxycinnamic acids

Chlorogenic acid (5-O-caffeoylquinic acid) Neo chlorogenic acid (3-O-caffeoylquinic acid) Minor phenolic acids; caffeic acids, gallic acid

Carotenoids & vitamins Flavonoids

Flavan-3ols

Catechin Epicatechin Procyanidins Procyanidin B1 Procyanidin B3

Anthocyanidins Cyanidin-3-OQuercetin -3- glucosides galactoside Cyanidin-3-OQuercetin -3- rutinosides rutinoside Quercetin -3glucoside

Flavonols

Carotenes β-Carotene Vitamins

Ascorbic acid (vitamin C) Tocopherol (vitamin E)

Figure 4.17  Major bioactives in peach and nectarine. (Compiled by Tokuşoğlu.)

Versari et al. (2002) reported the phenolic profiles of peach juices obtained from peach cultivars (Redhaven, Suncrest and Maria Marta) grown in the Emilia-Romagna region of Italy. The catechin content of Italian cultivars varied from 20 to 34 mg/kg in the above-mentioned peach juices manufactured from Redhaven, Suncrest, and Maria Marta cultivars (Versari et al. 2002). Chlorogenic acid was found as 12–19 mg/kg in a variety of peach juices in Italy while caffeic acid was a less amount in these juices (0.5–1.5 mg/kg; Versari et al. 2002). Isoquercetin in the amount of 6.0–7.1 mg/kg was detected in peach juices from the Italian Redhaven, Suncrest, and Maria Marta varieties. According to the study by Lavelli, Pompei, and Casadei (2009), HCAs (chlorogenic and neochlorogenic) from 34 to 73 mg/kg, catechin from 0 to 24 mg/kg, quercetin 3-O-glycosides from 3.9 to 12.7 mg/kg, and cyanidin 3-O-glucoside from 0 to 9.4 mg/kg were found in Elegant Lady and Redhaven peach varieties. Cevallos-Casals et al. (2006) reported that the total phenolic content of eight selected peach [Prunus persica (Batsch) L.] genotypes ranged from 100 to 449 mg chlorogenic acid equivalent (CGA)/100 g. It was detected that the anthocyanin content of peaches was 6–37 mg/100 g (CevallosCasals et al. 2006).The main anthocyanin identified in peaches has been cyanidin 3-glucoside with contribution of cyanidin 3-rutinoside (Hsia, Luh, and Chichester 1965; Ishikura, 1975; Van Blaricom and Senn, 1967). Several hydroxycinnamates, flavan 3-ols, and flavonols, predominantly chlorogenic acid, neochlorogenic acid, catechin, epicatechin, and quercetin 3-rutinoside, have been identified in peaches (Tomàs-Barberàn et al. 2001). The obtained RAC values varied from 440 to 1784 µg Trolox/g for peaches obtained from the USDA Stone Fruit Breeding Program in Byron, Georgia (Cevallos-Casals et al. 2006). The positive correlation between phenolic content and RAC for the peach genotypes (R2 = 0.8262) in the study presented by Cevallos-Casals et al. (2006) suggests that phenolic compounds are responsible for the antioxidant activity. Effects of lye-peeling and storage on phenolics in nectarine puree and nectar were examined by Lavelli, Pompei, and Casadei (2009). Processing fruit into puree causes a decrease in phenolic and carotenoid contents (Lavelli, Pompei, and Casadei 2008) and no further degradation of these antioxidants occurred during the processing of purees into nectars (Lavelli, Pompei, and Casadei 2009). Cyanidin 3-O-glucoside, catechin, and quercetin glycosides were the least stable phenolics, whereas chlorogenic acid and neochlorogenic acid degraded at a lower rate.

Date Fruit Phoenix dactylifera, commonly known as the date palm, is in the genus Phoenix. The date palm (Phoenix Dactylifera) is a monocotyledon of the family of the Palmae, one of the genera of which are the Coryphoideae, of which one species is Phoenix Dactylifera (Amer 1994; FAO 2009)

Phenolic and Beneficial Bioactives in Drupe Fruits

99

Date palm Photo: Alanya Municipality Park, Turkey

Date cluster, Manavgat, Turkey

Figure 4.18  (See color insert) Date palm fruits (Phoenix dactylifera). (This figure adapted from the Plant Database, Ağaçlar.net galery, Turkey, 2009.)

Dates are the fruit of the date palm and tastes sweet (Figure 4.18). Dates are dark reddish brown, oval, and about 1-½ inches long. Its skin is wrinkled and coated with a sticky, waxy film (Anonymous 2009b). Dates grow in clusters below the fronds on a date palm tree. A single cluster can hold 600–1700 dates. Date trees can be 15–25 m tall and 20–40 cm in a cross-sectional radius. Date palms can grow as tall as 100 feet and stay in production for over 60 years (Amer 1994; Elnasr 2009). Often called the edible date, it has few alternate names except in regional dialects. To the French, it is dattier; in German, it is dattel; in Italian, datteri or dattero; in Spanish, datil; in Dutch, dadel; in Turkish, hurma; and the Portuguese word is tamara (Morton 1987). The date traditionally has been a staple food in Tunisia, United Arab Emirates, Egypt, Iraq, Iran, Morocco, Algeria, Pakistan, the Sudan, Libya, and Turkey. The date palm tree, Phoenix dactylifera L., is an important plantation crop for many countries extending from North Africa to the Middle East including many states of the Arabian Gulf Cooperation Countries (GCC; Erskine et al. 2004).

Bioactives in Date Fruits The nutritional and biochemical aspects of date fruits were reported by many researchers (Abdel Hafiz et al. 1980; Al-Farsi et al. 2005; Al-Shahib and Marshall 1993; Barreveld, 1993; Booij et al. 1992; Myhara et al. 1999; Sawaya et al. 1982; Sawaya et al. 1983; Sourial et al. 1986). The date palm fruit possesses antioxidant and antimutagenic properties in vitro (Vayalil 2002) and dates are considered high energy foods due to the sugar content. The differences in maturity stages of dates have been attributed to changes in the chemical composition (Myhara et al. 2000). Mustafa, Harper, and Johnston (1986) reported that the pectin content of dates increased on a fresh weight basis during maturation. Dates contain tannins that are made mainly of polyphenols and two groups of them (phenolic acids and condensed tannins) are thought to be important in producing the astringent sensory response (Myhara et al. 2000). The tannins are responsible for astringency of dates and according to Sawaya et al. (1982), their content decreased as maturity progressed. The main phenolic profile of the ripe date fruit in Tunisia and Spain was detected by Lorente & Ferreres, 1988; Regnault-Roger et al. 1987, respectively. Mansouri et al. (2005) reported the phenolic profiles of Algerian ripe date palm fruit (Phoenix dactylifera) by HPLC-DAD-MS data. The phenolic content was found in the range from 2.49 to 8.36 mg/100 g fresh weight in different Algerian date fruit varieties including var. Tantbouchte, var. Deglet-Nour, var. Tazizaout, and var. Ougherouss (Mansouri et al. 2005). In this same study, the Tantbouchte variety gave the highest value, followed by the variety Deglet-Nour. The Tantbouchte and Deglet-Nour varieties of dates presented the highest values for AE and EC50, respectively, and a high correlation was found between total phenolic content and antiradical activity AE (R2 = 0.975; Mansouri et al. 2005). According to Mansouri et al. (2005), since the phenolic profile of date fruit is mainly constituted of ferulic, sinapic, and p-coumaric acids, which are more active, the high antioxidant activity was expected. It was also stated that cinnamic acids and their derivatives are the most apparent compounds in all varieties of dates. The major compounds of date fruits are also ferulic, coumaric, and sinapic

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

acids and derivatives such as 5-O-caffeoylshikimic acid (Mansouri et al. 2005). The presence of 5-O­caffeoylshikimic acid (dactyliferic acid) was also observed, which is a shikimic ester. It is stated that this compound is a contributor to the browning reactions that takes place during the maturation of the fruit (Harborne et al. 1974). Coumaric acid is present as a para-form (p-coumaric form) in almost all varieties except the var. Tazerzait, var. Tafiziouine, and var. Tazizaout, where its derivatives (coumaroylquinic acids, etc.) were found in the study described by Mansouri et al. (2005). The major flavonoids were in flavones and their glycosides, however, some flavonols, flavanones, and their glycosides were also identified in dates. The concentration of flavonoids was found very low in comparison to that of cinnamic acids in date fruits (Mansouri et al. 2005). The antioxidant activity of the date palm (Phoenix dactylifera L. family Palmae), Zaghlool, consumed in Egypt ranged from 9.28 to 75.96% through a beta-carotene bleaching method (Mohamed and Al-Okbi 2005). Total phenolic content was found to be 1461 mg of gallic acid equivalent/100 g dry weight in the study given by Mohamed and Al-Okbi (2005). Sixteen cultivars of date palm (Phoenix dactylifera L.) commonly grown in Bahrain were evaluated by Allaith (2008) for their antioxidant activity, total phenolics, and ascorbic acid contents at different ripening stages using the ferric reducing antioxidant power (FRAP) assay.

References Abdel Hafiz, M. J., Shalabi, A. F. and Risk, S.Y. 1980. Chemical composition of 15 varieties of dates grown in Saudi Arabia. In Proceedings of Saudi Biological Society, 4, 180–94. Akın E. B., Karabulut, İ., and Topçu, A. 2008. Some compositional properties of main Malatya apricot (Prunus armeniaca L.) varieties. Food Chemistry 107:939–48. Allaith, A. A. A. 2008. Antioxidant activity of Bahraini date palm (Phoenix dactylifera L.) fruit of various ­cultivars. International Journal of Food Science and Technology 43:1033–40. Al-Farsi, M., Alasalvar, C., Morris, A., Baron, M., and Shahidi, F. 2005. Comparison of antioxidant activity, anthocyanins, carotenoids, and phenolics of three native fresh and sun-dried date (Phoenix dactylifera. L.) varieties grown in Oman. Journal of Agricultural and Food Chemistry, 53, 7592–9. Al-Shahib, W., and Marshall, R. J. 1993. The fruit of the date palm: Its possible use as the best food for the future? International Journal of Food and Science Nutrition 54 (4): 247–59. Amer, W. M. 1994. Taxonomic and documentary study of food plants in Ancient Egypt. Ph.D. Thesis. Cairo: Cairo University. Anonymous. 2009a. http://www.fruit.info.com. Accessed date: January 5, 2009. Anonymous. 2009b. Date-Fruit. http://www.hort.purdue.edu/ext/senior/fruits/date1.htm Anonymous. 2009c. Nectarine. http://www.hort.purdue.edu/ext/senior/fruits/necta- rine1.htm Armstrong, W. P. 2008. Identification of Major Fruit Types. Life Science Department. San Marcos, CA: Palomar College. Arts, C. W., van de Putte, B., and Hollman, P. C. H. 2000. Catechin contents of foods commonly consumed in the Netherlands. 1. Fruits, vegetables, staple foods and processed foods. Journal of Agricultural and Food Chemistry 48:1746–51. Auger, C., Al-Awwadi, N., Bornet, A., Rouanet, J. M., Gasc, F., Cros, G., and Teissedre, P. L. 2004. Catechins and procyanidins in Mediterranean diets. Food Research International 37: 233–45. Barreveld, W. H. 1993. Date Palm Products. Bulletin No. 101. Food and Agriculture Organization of the United Nations, Rome. Bernalte, M. J., Hernandez, M. T., Vidal-Aragon, M. C., and Sabio E. 1999. Physical, chemical, flavor and sensory characteristics of two sweet cherry varieties grown in ‘Valle del Jerte’ (Spain). Journal of Food Quality 22: 403–16. Blando, F., Gerardi, C., and Nicoletti, I. 2004. Sour cherry (Prunus cerasus L) anthocyanins as ingredients for functional foods. Journal of Biomedicine and Biotechnology. 2004: 253–8. Blažek, J. 2007. A survey of the genetic resources used in plum breeding. Acta Horticulturae 734: 31–45. Booij, I., Piombo, G., Risterucci, J. M., Coupe, M., Thomas, D. and Ferry, M. 1992. Study of chemical composition of dates at different stages of maturity for varietal characterization of different cultivars of dates. Fruits–Paris, 47, 667–78.

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5 Bioactive Phytochemicals in Pome Fruits Özlem Tokus¸ og˘lu Contents Introduction............................................................................................................................................. 107 Apple....................................................................................................................................................... 107 Total Polyphenols of Apples.............................................................................................................. 108 Procyanidins, Catechins, Dihdyrochalcones, and Phenolic Acids of Apples.................................... 109 Antioxidant Activity of Apples...........................................................................................................113 Specific Phenolics and their Characteristics in Apples.......................................................................113 Pear..........................................................................................................................................................114 Pear Fruit Phenolics............................................................................................................................114 Pear Juice Phenolics............................................................................................................................116 Quince......................................................................................................................................................116 Phenolic Profiles of Quince................................................................................................................117 Loquat......................................................................................................................................................117 Loquat Phenolics.................................................................................................................................117 References................................................................................................................................................119

Introduction A pome is an accessory fruit composed of one or more carpels surrounded by accessory tissue. The accessory tissue is interpreted by some specialists as an extension of the receptacle and is then referred to as “fruit cortex,” and by others as a fused hypanthium or “torus”; it is the most edible part of this fruit (Esau 1977). Pome fruits are all members of the Rosaceae, or rose family, subfamily Pomoideae (Rieger and Basra 2007). The exocarp and mesocarp of a pome fruit may have fleshy property and difficult to distinguish from one another and from the hypanthial tissue. The well-known pome fruit is the apple; other bestknown examples of pomes are the pear, loquat, quince, hawthorn, and rowan. The hawthorn and rowan is berry-like with juicy flesh (Esau 1977). Pome fruits, generally, are adapted to the cool temperate zone regions of the world and have a long storage life—up to a year for some apples. This speciality permits an extended market season by cultivation for a few varieties (Rieger and Basra 2007).

Apple Apple (Malus domestica Borkh.) is a tasty and nutritious fruit to various cultures and is widely consumed in almost all countries. The cultivated apple, Malus domestica Borkh., belongs to the Pomoideae subfamily of the Rosaceae, along with pear (Pyrus spp.), quince (Cydonia oblonga), loquat (Eriobotrya japonica), and medlar (Mespilus germanica; Rieger and Basra 2007). 107

108

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Major apple bioactives Phenolics Flavonoids

Phenolic acids Hydroxycinnamic A Chlorogenic acid (5-O-caffeoylquinic acid) Caffeic acid

Vitamins

Flavan-3ols Flavonols Dihydrochalcones (–)-Epicatechin Quercetin Phloretin (+)-Catechin Rutin Phloridzin & their Procyanidins glucosides Procyanidin B1 Procyanidin B2

Ascorbic acid (vitamin C) Anthocyanidins Cyanidin & their glucosides

Figure 5.1  Major apple bioactives.

Apples are an excellent source of several phenolic compounds and also possess high total antioxidant capacity (TAC; Biedrzycka and Amarowicz 2008; Eberhardt, Lee, and Liu 2000; Lotito and Frei 2004). These antioxidant phenolics play a role in reducing the risk of various diseases originating from oxidative stress, namely, coronary disease, immune system damage, asthma, and diabetes (Boyer and Liu 2004). The heart attack risk has been shown to decrease by 49% when apples are included in a diet at a daily amount of ≥110 g compared to those at ≤18 g (Hertog et al. 1993). Profiles and content of existed polyphenols in apples are significant due to their contribution to the sensory quality of fresh fruit and processed apple products. Morever, these phytochemicals are recognized for their health promoting antioxidant properties (Bushway, Hu, and Shupp 2002; Lea and Timberlake 1974; Sanoner et al. 1999; Van der Sluis et al. 2002). Figure 5.1 shows the major phenolics in apple and apple products. There are many health-enhancing phytonutrients including flavonoids and phenolic acids in apple skin (Boyer and Liu 2004). It is stated that apple skin constituents inhibited the human hepatocellular liver carcinoma (HepG2) cell proliferation significantly greater than that of the whole apple (Wolfe, Wu, and Liu 2003). It is reported that apple skin has unique flavonoids, such as quercetin ­glycosides, not found in the flesh part of an apple and also has three- to sixfold more flavonoids than apple flesh (Wolfe, Wu, and Liu et al. 2003; Wolfe and Liu 2003). It is stated that apples provide 20–25% of the per capita consumption of fruit polyphenols in the United States (Vinson et al. 2001). Sun et al. (2002) found that apples had the highest soluble-free phenolics when compared to 10 other commonly consumed fruits. It is stated that hydroxycinnamic acid derivatives, flavan-3-ols (flavanols; monomeric and oligomeric types of catechins), flavonols, dihydrochalcones are the major apple ­phenolics. Apples are comprised of various antioxidative phenolics: chlorogenic acid, epicatechin, procyanidin B2, phloretin, phloridzin, and quercetin as well as ascorbic acid (Awad, De Jager, and Van Westing 2000; Biedrzycka and Amarowicz 2008; Burda, Oleszek, and Lee 1990; Chinnici et al. 2004a; Guyot et al. 2002; Hunter and Hull 1993; Khanizadeh et al. 2007; Mayr et al. 1995; Tsao et al. 2005; Vanzani et al. 2005; Vrhovsek et al. 2004). Apples constitute a major part of fruit production and consumption in the world, and there is an increasing tendency to produce and consume the apple juice (Fulker 2001). The leading phenolic compounds in apple juice are chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, procyanidines (B1, B2, trimer C1), rutin, phloridzin, and also ascorbic acid (vitamin C). The type and amount of these phenolics show important variations with respect to the source varieties from which apple juice is derived (Gliszczynska-Swiglo and Tyrakowska 2003; Karaman et al. 2010; Wu et al. 2007).

Total Polyphenols of Apples It is stated that the mean level of total polyphenols in apples represent eight of the most widely cultivated varieties in western Europe between 66.2 and 211.9 mg/100 g of FW depending upon the

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Bioactive Phytochemicals in Pome Fruits Table 5.1 Total Phenolics in Apple and Apple Products Apple and Apple Products JUICES Jonathan Golden Delicious Apple juice (6 cultivar, Japan) Commercial apple juices (6 brand, U.S.) Commercial apple juice (U.K.)

Total Phenolics 12.7 mg/L 217 mg/L (as chlorogenic acid eq.) 2365 ± 525 mg/L (as chlorogenic acid eq.) 401–999 mg/L 339 ± 43 µg/mL (as gallic acid eq.)

WHOLE FRUIT OR APPLES Red Delicious (U.S.) 10 apple varieties (U.S.) Apple (cv. Fuji)

710 ± 10 mg/L; 239–440 mg/200 g FW 115–230 mg/100 g FW (as gallic acid eq.) 61 mg/100 g FW

FLESH OR PULP Red Delicious (U.S.) Golden Delicious (U.S.) Braeburn (France) Golden Delicious Pulp Gale apple pulp

430 ± 2 mg/L 624 ± 10 mg/kg FW 550 ± 24 mg/kg FW 241 ± 30.2 mg/kg FW 82 ± 3 mg/100 g FW

PEELS Red Delicious peels (U.S.) Golden Delicious skin (France) Granny Smith skin (France) Braeburn skin (France) Apple peels (2 cultivars, Poland) Apple peels (Israel) Golden Delicious peels Gale apple peel

1845 ± 4 mg/L 1764 ± 80 mg/kg FW 3149 ± 142 mg/kg FW 3212 ± 146 mg/kg FW 418–640 mg/100g FW 1.2 ± 0.12 g/100g FW 1374 ± 162 mg/kg FW 309 ± 5 mg/100g FW

Source: Adapted from Biedrzycka, E., and Amarowicz R., Food Rev. Inter., 24, 235–51, 2008.

apple variety, with flavanols (catechin and proanthocyanidins) as the major class of apple polyphenols (71–90%), followed by hydroxycinnamates (4–18%), flavonols (1–11%), dihydrochalcones (2–6%), and in red apples, anthocyanins (1–3%) (Vrhovsek et al. 2004). Table 5.1 shows the total phenolics in the studied apple and apple products. Also, it is reported that the total phenolic level of apples is above 5000 mg/kg and in some varieties, total phenolics may be 10,000+ mg/kg of fresh weight (Gorinstein et al. 2001) and ∼95% of phenolics are accumulated in the parenchyma part of the apples (Guyot et al. 2002). Lee et al. (2003) found that the average phenolic concentrations among the six apple cultivars were: quercetin glycosides, 13.2 mg/100 g fruit; vitamin C, 12.8 mg/100 g fruit; procyanidin B, 9.35 mg/100 g fruit; chlorogenic acid, 9.02 mg/100 g fruit; epicatechin, 8.65 mg/100 g fruit; and phloretin glycosides, 5.59 mg/100 g fruit (Lee et al. 2003). The polyphenol content can range from 18 to ∼152 mg/200 g of apple, with the most prevalent being hydroxycinnamic acids (10–120 mg/200 g), flavonols (4–8 mg/200 g), and monomeric flavanols (4–24 mg/200 g), including catechins and proanthocyanidins (Manach and Donovan 2004).

Procyanidins, Catechins, Dihdyrochalcones, and Phenolic Acids of Apples Apples are a typical source of proanthocyanidins from polymeric flavan-3-ols an association with flavanol catechins. It is stated that the Delicious variety of apples have the most abundant compounds being

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

OH

OH HO

OH

O

O

HO OH

OH

OH

OH Epicatechin

Catechin OH OH HO

HO

OH

OH

O O

OH

O

Glucose

Phloretin

Phloridzin

OH

OH OH

O

HO

O

HO

OH OH

OH OH HO

OH

OH

OH OH HO

O

OH

O OH

OH OH Procyanidin B1

OH Procyanidin

Figure 5.2  Catechin, dihydrochalcone and procyanidin compounds in apples.

epicatechin, procyanidin B2, and phloridzin (Chinnici et al. 2004). Figure 5.2 shows apple procyanidins, catechins, and dihydrochalcones. Polyphenol-enriched fractions, which are extracted from unripe apples (Rosaceae, Malus spp.), consisting of procyanidins (polymers of catechins) are known to have an antiallergenic effect on patients with various allergic diseases. Procyanidins are found in the whole apple fruit and their levels gradually increase from 1232 mg/kg in the seeds to 4964 mg/kg in the epidermal part of apples (Guyot et al. 1998). Sanoner et al. (1999) characterized more polymerized procyanidins in the cortex part of some apple varieties. However, Reinders et al. (2001) indicated that caffeic acid, one of the dominant apple phenolic acids, might have an impact or inhibitory effect on Escherichia coli O157:H7 in an apple juice model medium. Along with the ripening degree, the content of apple polyphenols decreases. Karaman et al. (2010) reported that the catechin, chlorogenic acid, epicatechin, caffeic acid, and phloridzin contents of certain apple varieties as Amasya, Lutz Golden, Ervin Spur, Arap Kizi, King Luscious, Sky Spur, and Granny Smith are grown in Turkey. Hydrochalcones phloridzin (phloretin 2′-βD-glucoside) and phloretin are distributed in the whole fruit and also comprise about 66% of total phenolics in the seeds of apples, while they comprise less than 3% of total phenolics in the parenchyma and epidermis (Guyot et al. 1998).

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Gökmen et al. (2001) reported the effects of different clarification treatments on the phenolic compounds (hydroxycinnamic acids, hydroxybenzoic acids, catechins, quercetin, dihydrochalcones including phloridzin, phloretin, and procyanidins) of apple juice (Tables 5.2 and 5.3). Table 5.4 shows the alterations of those phenolics of Turkish apple juices after the first process that enzymatically treated juice sequentially flocculate with 500 mg/l of gelatin and 2500 mg/l of bentonite at Table 5.2 Procyanidin, Catechin and Dihdyrochalcone Content of Apples and Apple Products Apple and Apple Products

Procyanidins, Catechins, and Dihydrochalcones

PROCYANIDINS Fresh apples with skins, average of six varieties (U.S.) Golden Delicious, Granny Smith, Braeburn flesh Peels of Golden Delicious, Granny Smith, Braeburn flesh Golden Delicious pulp (Italy) Peels of Golden Delicious pulp (Italy) CATECHINS Fresh apples with skins, average of six varieties (U.S.) Red Delicious, Granny Smith, Fuji-whole apple extract Golden Delicious, Granny Smith, Braeburn flesh Peels of Golden Delicious, Granny Smith, Braeburn flesh Golden Delicious pulp (Italy) Peels of Golden Delicious pulp (Italy) DIHYDROCHALCONES Red Delicious, Granny Smith, Fuji-whole apple extract Golden Delicious, Granny Smith, Braeburn flesh Peels of Golden Delicious, Granny Smith, Braeburn flesh Golden Delicious pulp (Italy) Peels of Golden Delicious pulp (Italy)

Procyanidin B2; 9.35 mg/100 g Procyanidin B2, 72, 134, 67 mg/kg FW Total procyanidins, 439,753, 378 mg/kg FW Procyanidin B2, 150, 241, 245 mg/kg FW Total procyanidins, 1282, 2727, 2404 mg/kg FW Total procyanidins, 89.8 ± 2.61, 147.3 ± 19.0 mg/kg FW Total procyanidins, 436 ± 56.2, 497 ± 28.9 mg/kg FW Epicatechin, 8.65 mg/100 g (Lee et al., 31) Epicatechin, 10.3, 4.8, 8.0 mg/100 g FW (as gallic acid eq.) Catechin, 4.7, 3.3, 5.3 mg/100 g FW (as gallic acid eq.) Epicatechin, 59, 96, 124 mg/kg FW Epicatechin, 124, 170, 132 mg/kg FW Flavan-3-ols, 46.1 ± 9.22, 71.1 ± 12.2 mg/kg FW Flavan-3-ols, 223 ± 26.4, 234 ± 32.2 mg/kg FW Phloridzin, 2.6, 0.3, 1.0 mg/100g FW (as gallic acid eq.) Phloridzin, 11, 6, 7 mg/kg FW Phloretin, 11, 13, 9 mg/kg FW Phloridzin, 40, 13, 34 mg/kg FW Phloretin, 42, 41, 42 mg/kg FW Total dihydrochalcones, 19.9 ± 2.61, 22.8 ± 2.47 mg/kg FW Total dihydrochalcones, 166 ± 31.7, 149 ± 37.8 mg/kg FW

Sources: Adapted from Lotito S. B., and Frei B., Free Radic. Biol. Med., 36(2), 201–11, 2004; Chinnici F., Bendini A., Gaiani, A., and Riponi, C., J. Agric. Food Chem., 52, 4684–89, 2004; Lee K. W., Kim Y. J., Kim D. O., Lee H. J., and Lee Ch.Y., J. Agric. Food Chem., 51, 6516–20, 2003; Guyot S., Le Bourvellec, C., Marnet N., and Drilleau J. F. Lebensm.-Wiss. U.-Technol., 35, 289–91, 2002.

Table 5.3 Major Phenolic Constituents of Different Originated Apple Juices (mg/L) Apple Variety

Catechin

Chlorogenic Acid

Epicatechin

Caffeic Acid

Phloridzin

King Lucious

115.30 ± 1.14 (97%)

276.36 ± 2.80 (88%)

16.63 ± 0.33 (92%)

79.09 ± 1.19 (99%)

12.43 ± 0.25 (94%)

Amasya

89.41 ± 0.91 (85%)

126.84 ± 0.13 (89%)

43.54 ± 0.65 (91%)

27.20 ± 0.41 (94%)

6.61 ± 0.13 (94%)

Ervin Spur

81.39 ± 0.85 (95%)

111.25 ± 1.14 (98%)

66.76 ± 0.70 (98%)

20.72 ± 0.31 (95%)

20.13 ± 0.41 (88%)

Sky Spur

9.09 ± 0.18 (94%)

72.99 ± 0.81 (86%)

45.28 ± 0.68 (88%)

21.80 ± 0.33 (94%)

11.80 ± 0.24 (99%)

Kızı

67.52 ± 0.69 (88%)

242.30 ± 2.50 (97%)

28.04 ± 0.42 (98%)

12.14 ± 0.24 (99%)

0.52 ± 0.01 (91%)

Lutz Golden

41.62 ± 0.62 (90%)

71.92 ± 0.74 (94%)

11.58 ± 0.17 (98%)

8.22 ± 0.16 (95%)

3.57 ± 0.07 (93%)

Granny Smith

76.77 ± 0.82 (84%)

41.10 ± 0.45 (86%)

3.38 ± 0.07 (94%)

3.04 ± 0.08 (98%)

1.15 ± 0.03 (84%)

Source: Adapted from Karaman, Ş., Tütem E., Sözgen B. K., and Apak, R., Food Chem., 120, 1201–99, 2010. a Values in parantheses represent the percentage recoveries of added antioxidant standards.

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Table 5.4 Effects of Different Clarification Treatments on the Phenolic Compounds Composition of Apple Juice Concentration of Phenolic Compounds,a mg/L Clear Juice Phenolic Compounds

Process 1

Process 2

Process 3

Process 4

Process 5

Process 6

645.5 ± 30.7

426.2 ± 18.9

246.4 ± 11.8

645.4 ± 17.7

Ferulic acid

37.9 ± 2.2 6.7 ± 0.2 33.3 ± 1.4 13.5 ± 1.7

12.5 ± 1.3 5.6 ± 0.1 29.7 ± 1.7 11.8 ± 0.9

2.7 ± 0.3 3.5 ± 0.2 15.0 ± 1.1 6.6 ± 0.4

32.8 ± 2.2 6.6 ± 0.3 30.6 ± 3.4 10.2 ± 1.6

440.3 ± 23.7 13.3 ± 3.2 6.1 ± 0.4 29.2 ± 2.8 9.9 ± 0.3

516.0 ± 21.7 21.0 ± 1.9 5.7 ± 0.5 27.3 ± 1.8 12.0 ± 0.9

364.1 ± 17.5 16.7 ± 2.0 3.9 ± 0.1 24.4 ± 1.8 8.0 ± 1.1

Hydroxybenzoic Acids Gallic acid

13.6 ± 2.2

7.0 ± 0.7

Protocatechuic acid

16.4 ± 1.1

12.4 ± 1.6

5.6 ± 0.3 5.6 ± 0.4

14.6 ± 0.9 17.2 ± 0.7

9.4 ± 1.0 14.8 ± 0.7

13.1 ± 1.1 13.2 ± 0.4

6.8 ± 0.2 10.6 ± 1.6

Hydroxycinnamic Acids Chlorogenic acid Caffeic acid p-Coumaric acid o-Coumaric acid

Flavan-3-ols Epicatechin

49.5 ± 1.1

41.1 ± 2.3

23.9 ± 1.7

48.1 ± 2.8

Catechin

20.0 ± 0.0

14.1 ± 1.9

7.2 ± 0.6

20.4 ± 1.9

46.1 ± 3.4 17.3 ± 1.2

42.4 ± 3.0 17.0 ± 1.2

33.9 ± 4.1 10.2 ± 0.4

Flavonols Quercetin

59.5 ± 3.9

51.3 ± 2.8

14.6 ± 0.7

50.4 ± 3.7

48.3 ± 2.6

34.6 ± 3.4

27.7 ± 2.4

27.8 ± 3.5 4.7 ± 0.4

27.2 ± 2.6 5.2 ± 0.2

4.5 ± 0.4 2.6 ± 0.1

28.4 ± 3.1 5.6 ± 0.2

25.8 ± 2.0 4.8 ± 0.3

21.0 ± 2.1 3.3 ± 0.1

16.1 ± 0.8 2.6 ± 0.1

Procyanidin trimer

229.4 ± 24.5 400.0 ± 30.0

214.8 ± 19.3 376.9 ± 15.8

190.3 ± 9.8 118.5 ± 14.5

Total Phenolics

1559.2 ± 36.5

1235.9 ± 12.6

647.0 ± 14.3

239.5 ± 10.8 351.0 ± 21.8 1500.4 ± 23.4

155.6 ± 8.8 418.5 ± 12.3 1239.2 ± 20.9

159.5 ± 11.1 336.0 ± 13.8 1222.1 ± 20.6

98.4 ± 7.4 254.6 ± 12.5 877.9 ± 17.3

Dihydrochalcones Phloridzin Phloretin Procyanidins Procyanidin B2

Source: Adapted from Gökmen, V., Artık, N., Acar, J., Kahraman, N., and Poyrazoğlu, E., European Food Res. Technol., 213(3), 194–9, 2001. a Phenolic concentration correction for 11.2 °Brix.

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Raw Juice

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Bioactive Phytochemicals in Pome Fruits

50°C for 2 hours prior to filtration through kieselguhr. The third process is an ultrafiltration technique that enzymatically treats juice directly by ultrafiltrating through a 10-kDa cutoff membrane applying a gauge pressure of 2 bar and the second, fourth, fifth, and sixth processes contain enzymatically treated juice from different filtration applications such as activated charcoal, adsorbent resin, or polyvinylpyrrolidone (PVP) (Table 5.4; Gökmen et al. 2001).

Antioxidant Activity of Apples Karaman et al. (2010) determined the TAC values of high performance liquid chromatography (HPLC) quantified antioxidant phenolics in apple varieties such as Amasya, Lutz Golden, Ervin Spur, Arap Kizi, King Luscious, Sky Spur, and Granny Smith and they compared with those found by cupric reducing antioxidant capacity (CUPRAC) and 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). The TAC of HPLC-quantified compounds accounted for between 40.0 and 70.6% of the observed CUPRAC capacities of apple juices with respect to apple varieties. According to a study by Karaman et al. (2010), the order of antioxidant capacity of apple juices determined by the CUPRAC and ABTS methods were: Ervin Spur > King Luscious > Sky Spur > Amasya > Arap Kızı > Granny Smith > Lutz Golden. The phenolic level and the TAC of fruits seem to be regulated by genetics (Khanizadeh et al. 2007) and also environmental, postharvest factors including fruit season, fruit maturity, light exposure, storage, and processing (Alonso-Salces et al. 2004; Amiot et al. 1992; Awad et al. 2000; Burda, Oleszek, and Lee 1990; Spanos and Wrolstad 1992; Van der Sluis et al. 2001, 2002). It was found that when compared to many other commonly consumed fruits in the United States, apples had the second highest level of antioxidant activity and also ranked second for the total concentration of phenolic compounds (Boyer and Liu 2004; Sun et al. 2002; Figure 5.3).

Specific Phenolics and their Characteristics in Apples It is reported that hydroxycinnamic acids are precursors of volatile compounds that contribute to cider aroma (Lea 1995). Several polyphenols also play an important role in browning characteristics of apple products. It has been proven that chlorogenic acid (CA), which is one of the hydroxycinnamates, is the most important substrate of polyphenol peroxidase (PPO). The CA apple is converted into its o-quinone, 200 Total antioxidant activity (µmol vitamic C equivalents/g fruit)

180 160 140 120 100 80 60 40 20

ng e ra pe fru Pi i ne t ap pl e

na

G

O

ra

r

na

Pe a

Ba

Cr an

be

rr y Ap p le Re d gr ap St e ra w be rr y Pe ac h Le m on

0

Figure 5.3  Antioxidant activity of various fruit extracts (mean ± SD, n = 3). (Adapted from Boyer, J. and Liu, R. H., Nutr. J., 3, 1–15, 2004.)

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

which further reacts with other phenolic compounds, resulting in the formation of yellow and brown pigments when subjected to oxygen and PPO (Oszmianski and Lee 1990). In apples, procyanidin phenolics are also involved in enzymatic browning and particularly their degree of polymerization is responsible for cider astringency and bitterness of apple juices and ciders (Khanizadeh et al. 2006). Moreover, the browning degree of apples was found to be dependent upon the relationship of hydroxycinnamic acids/procyanidins (Amiot et al. 1992). According to data received, 31 bitter apple cultivars present higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars (Alonso-Salces et al. 2004).

Pear The pear (Pyrus communis L.) is a typical fruit of temperate zones and is cultivated in Europe, among other regions. Owing to its nutritive properties, good taste, and low calories, it is a much preferred fruit by many consumers. Pears are placed in the Rose family (Rosaceae), subfamily Pomoideae along with apple and quince. The genus Pyrus is composed of about 22 species, found in Asia, Europe, and northern Africa. Two major species are commercially cultivated: European pear, Pyrus communis L. and Asian pear, P. Pyrifolia. The European pear is the major pear of commerce whereas the Asian pear has been increasing in popularity in the United States over the last 20 years (Rieger and Basra 2007).

Pear Fruit Phenolics Major phenolics of pears are phenolic acids (hydroxycinnamic acids) (Figure 5.4), specifically arbutin (4-hydroxy-phenyl-β-D-glucopyranoside; Figure 5.5), catechins, flavonols, and procyanidins (Oleszek, Major pear bioactives Vitamins

Phenolics

Hydroxycinnamic A Sinapic acid Syringic acid Vanilic acid p-Coumaric acid Chlorogenic acid (5-O-caffeoylquinic acid) Caffeic acid

Ascorbic acid (vitamin C)

Flavonoids

Phenolic acids Flavan-3 ols

Flavonols

(–1)-Epicatechin (+)-Catechin Procyanidins Procyanidin B1 Procyanidin B2

Glycosylated hydroquinone Quercetin Isorhamnetin Arbutin Kaempferol & their malonyl glucosides

Figure 5.4  Major pear bioactives. O O HO

OH OH

OH

OH Figure 5.5  The chemical structure of arbutin (4-hydroxy-phenyl-β-D-glucopyranoside).

Anthocyanidins Cyanidin & their glucosides

115

Bioactive Phytochemicals in Pome Fruits Table 5.5 Phenolic Acid Contents of Pearsa Moisture (%) Phenolic acid (conjugated) Gallic Protocatechuic Gentisic Caffeic Vanillic Syringic p-Coumaric Ferulic Sinapic Salicylic Phenolic acid (free) Protocatechuic p-Hydroxybenzoic

84.5 0.45 ± 0.23 2.65 ± 0.59 5.81 ± 3.81 2.81 ± 0.48 7.98 ± 1.03 33.03 ± 11.16 5.84 ± 3.28 0.10 ± 0.05 34.87 ± 32.92 1.93 ± 0.59 0.45 ± 0.24 0.48 ± 0.15

Source: Adapted from Russell, W. R., Labat, A., Scobbie, L., Duncan, G. J., and Duthie, G. G., Food Chemistry, 115, 100–4, 2009. a Conjugated phenolic acids are a summation of both the alkaliand acid-labile fractions. Values are specified on a dry weight basis in mean ± standard deviations (n = 3).

Amiot, and Aubertt 1994). Apple and pear composition appears to be constituted by hydroxycinnamic acids, ­flavan-3-ols (catechins and procyanidins), and flavonol glycosides (Suarez Valles et al. 1994). The main ­difference between both fruits is the presence of arbutin and the lack of phloretin derivatives in pears (Spanos and Wrolstad 1992). The presence of a special compound, arbutin, in the pear was first reported by Durkee and his coworkers (1968). It is found that the average level of phenolics in pears harvested at commercial maturity stage is 3.7 g/kg of fresh pulp (Ferreira et al. 2002). Table 5.5 shows the main phenolic acid profiles of pears (Russell et al. 2009). It is reported that sinapic, syringic, and vanillic are the predominant phenolic acids in pears (Russell et al. 2009). It is also found that p-coumaric, gentisic, protocatechuic, gallic, ferulic, and salicylic acids as conjugated phenolic acids and p-hydroxybenzoic and protocatechuic acids are free phenolic acids in pear fruits in the study given by Russell et al. (2009). According to Amiot et al. (1995), the main phenolic acid in pears are chlorogenic acid (2.7–14.1 mg/100 g). Procyanidins are the predominant phenolics (96%), with a mean degree of polymerization (mDP) of 13−44, whereas hydroxycinnamic acids, arbutin, and catechins are present as 2%, 0.8%, 0.7%, respectively. It is stated that the major monomer in the procyanidin structures is (−)-epicatechin (99%) and (+)-catechin and is determined as 1% (Ferreira et al. 2002). Amiot et al. (1995) reported 0.6–8.7 mg/100 g of epicatechin and ∼0.05 mg/100 g of catechin in pears. Risch and Herrmann (1988) determined that the amounts of catechin and epicatechin in pears are 0–10 and 5–60 mg/kg, respectively. Procyanidins B1 and B2 have been determined in Barlett pears (Spanos and Wrolstad 1992) and procyanidins with a mean degree of polymerization (13–44) have been determined in Portuguese pears, comprising ∼96% of the total polyphenols (Ferreira et al. 2002). Flavonol glycosides including glycosides of quercetin, isorhamnetin (3′-methylether of quercetin), kaempferol, and their malonyl glycosides make up the flavonol composition of pears (Andrade et al. 1998; Duggan 1969; Wald and Galensa 1989).

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Table 5.6 The Amounts of Phenol Compounds of Pear Juices from Different Pear Cultivars Pear Cultivars

Soluble Solids (%)

Şeker Santa Maria Passa Crassane Akça Ankara Williamson Starkrimson

8.4 11.2 10.8 13.2 9.6 11.4 13.6

Chlorogenic Acids (mg/L) 95.1 73.1 148 248 148 174 166

Epicatechin (mg/L)

Caffeic Acid (mg/L)

Coumaric Acid (mg/L)

Total Phenolics (mg/L)

17.6 11.9 37.5 21.9 21.4 34.9 81.3

3.4 4.1 9.1 2.4 10.1 11.0 11.4

— — 0.72 0.97 0.46 0.66 3.00

375 196 417 455 371 457 423

Source: Adapted from Tanrıöven, D. and Ekşi, A., Food Chem., 93, 89–93, 2005.

Oleszek, Amiot, and Aubertt (1994) isolated four hydroxycinnamic acid esters and eight flavonol glucosides in pears. Three of the glucosides were quercetin and the remaining five were isorhamnetin ­glucosides: they are quercetin 3-O-glucoside, quercetin 3-O-rutinoside, quercetin 3-O-malonylglucoside, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-rutinoside, isorhamnetin 3-O-malonylglucoside, isorhamnetin 3-O-malonylgalactoside, and isorhamnetin 3-O-galactorhamnoside. Spanos and Wrolstad (1990) stated that the phenolic content of pear depends primarily on the variety and the level of maturity. Amiot et al. (1992) reported that the phenolic content and browning tendency primarily depend on the variety and not on the year of growth or the level of maturity. Ferreira et al. (2002) reported that total amount of native phenolic compounds decreased by 64% (on a dry pulp basis) after the sun drying process. It is found that less affected compounds are arbutin and catechins after sun drying (Ferreira et al. 2002).

Pear Juice Phenolics Spanos and Wrolstad (1992) stated that the phenolics in pear juice are chlorogenic acid, epicatechin, catechin, caffeic acid, and coumaroylquinic acid and it was found that there is 6.7–16.8 mg/l of arbutin in pear juice. It is reported that the amounts of chlorogenic acid, p-coumaroylquinic acid, epicatechin, and catechin in pears are 134, 14, 26, and 3 mg/kg, respectively (Herrmann 1993). Tanrıöven and Ekşi (2005) reported the phenolic content of pear juice from seven different ­varieties. The results indicated that chlorogenic acid ranged from 73.1 to 249 mg/l, epicatechin from 11.9 to 81.3 mg/l, caffeic acid from 2.4 to 11.4 mg/l, and p-coumaric acid from 0.0 and 3.0 mg/l. It was reported that the total amount of polyphenol from pear juice samples varied between 196 and 457 mg/l and the major phenolics detected were chlorogenic acid, epicatechin, caffeic acid, and coumaric acid (Tanrıöven and Ekşi 2005; Table 5.6).

Quince The quince (Cydonia oblonga Mill.) is the only species in the genus Cydonia, which falls into the Pomoideae subfamily of the Rosaceae along with apple and pear (Rieger and Basra 2007). Quince is rarely cultivated outside of Mediterranean climates, and is sometimes confused with the common flowering quince (Chanomeles speciosa), Japanese flowering quince (Chanomeles japonica), or Chinese quince (Pseudocydonia sinensis; Rieger and Basra 2007). Quince fruit generally is used in jams, marmalade, jelly, cooking, as well as quince pudding, and rarely consumed as raw fruit. Turkey is the leading grower of quince in the world (Kaya et al. 2007). There is little production of quince fruit occuring in the United States, but 20,000 tons annually are produced in Argentina (Rieger 2010).

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Phenolic Profiles of Quince Recent studies have shown that Cydonia oblonga Miller species is an excellent low-cost natural source of phenolic acids and flavonoids, which are considered potent antioxidants (Oliveira et al. 2007; Silva et al. 2004, 2005, 2008). The quince fruit is recognized as an important dietary source of health promoting compounds, owing to its antioxidant, antimicrobial, and antiulcerative properties (Fattouch et al. 2007; Fiorentino et al., 2007, 2008; Hamauzu et al. 2005, 2006; Oliveira et al. 2007; Silva et al. 2004, 2008). Quince (Cydonia oblonga Miller) also has protective effects against oxidative hemolysis of human erythrocytes (Magalhães et al. 2009). Silva et al. (2002) and Andrade et al. (1998) reported that the total phenolic content ranges from 11.7 to 268.3 mg/kg and between 243 and 1738 mg/kg in the pulp and in the peel, respectively. In the pulp and peel of quince, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, Quercetin3-O-galactoside, Quercetin-3-O-rutinoside, Kaempferol-3-O-glycoside, ­Kaempferol-3-O-glucoside, Kaempferol-3-O-rutinoside, Quercetin glycoside acylated with p-coumaric acid, and Kaempferol glycoside acylated with p-coumaric acid were determined. It is reported that the concentration of total phenolics is 2.51 and 6.30 in the pulp and peel of quince fruit, respectively. Some specific phenolics such as lucenin-2, vicenin-2, stellarin-2, isoschaftoside, and schaftoside have been found in quince seed (Silva et al. 2004). Cyanidin 3-glucoside and cyanidin 3,5-diglucoside are the main anthocyanins detected in quince fruits with red pigmented skin. Rutin and 5-O-caffeoylquinic acids are the major phenolic compounds in the peel, while 3-O-caffeoylquinic and 5-O-caffeoylquinic acids are the main phenolics in the quince pulp (Silva et al. 2002, 2008). In the study by Alesiani et al. (2010), 59 secondary metabolites have been isolated from quince (Cydonia vulgaris) peels and characterized; among them, five metabolites, 3b-(18-hydroxylinoleoyl)-28-hydroxyurs12-ene, 3b-linoleoylurs-12-en-28-oic acid, 3b-oleoyl-24-hydroxy-24-ethylcholesta-5,28(29)-diene, tiglic acid 1-O-b-D-glucopyranoside, and 6,9-dihydroxymegastigmasta-5,7-dien-3-one 9-O-b-D-gentiobioside have been isolated.

Loquat Loquat fruit (Eriobotrya japonica Lindl.) belongs to the Pomoideae subfamily of the Rosaceae, along with apple, pear, quince, juneberry, mayhaw, and medlar. The loquat is a cold-tender evergreen, native to southeast Asia (Rieger and Basra 2007). The world loquat production has been 566,031 tons and China stands out as the main producer with Spain as the prime exporter. Turkey is one of the most important producers of loquat in the world. In 2006, Turkey ranked fourth in production with approximately 12,310 ton (Polat 2007). Brazil has some production near Sâo Paulo and commonly, it is grown as an ornamental or garden tree in the southeastern United States and California (Rieger and Basra 2007; Rieger 2010).

Loquat Phenolics It is reported that main phenolics of loquat fruits are both hydroxycinnamic and benzoic acid derivatives and cyanidine glycoside (Ding et al. 2001; Koba et al. 2007). Recently, Ferreres et al. (2009) identified the 18 compounds (eight hydroxycinnamic acid derivatives and 10 flavonoid glycosides) in loquat fruits. Total phenolic content and total flavonoid content of loquat is 199.4 ± 13.1 mg GAE/100 g FM and 14.2 ± 0.9 mg QE/100 g FM, respectively, in the study by Lin and Tang (2007). Ferreira de Faria et al. (2009) reported the 25 carotenoid profiles and vitamin A values of Brazilian loquats. It is found that all-trans-b-carotene (19–55%), all-trans-b-cryptoxanthin (18–28%), 5,6:5′,6′diepoxy-b-cryptoxanthin (9–18%) and 5,6-epoxy-b-cryptoxanthin (7–10%) are the main carotenoids.

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The total carotenoid content ranged from 196 mg/100 g (cv. Néctar de Cristal) to 3020 mg/100 g (cv. Mizumo; Ferreira de Faria et al. 2009; Table 5.7.). Polat et al. (2010) reported the total phenolic content and antioxidant capacity of loquat varieties in Hatay, Turkey. The total phenolic content ranged from 129 (“Baduna 5”) to 578 (“Hafif Çukurgöbek”) mg Gallic acid equivalent (GAE)/kg FW. Figure 5.6 shows the total phenolic of loquat varities (Baduna 5, Güzelyurt, Hafif Çukurgöbek, Ottaviani, Type 1) grown in Dörtyol, Hatay, Turkey. The “Hafif Çukurgöbek” variety is the major polyphenol source (Polat et al. 2010).

Table 5.7 Concentration (µg/100g) of Carotenoids and Vitamin A Values (µg RAE/100 g) of Loquat Cultivars Catotenolds All-trans neoxanthin All-trans-violaxanthin + 9-cis-neoxanthin All-trans-neochrome cis-5,6:5′,6′-Diepoxy-βcrytoxanthin 5,6:5′,6′-Diepoxy-βcrytoxanthin 9-cis-Violaxanthin 5,8:5′,6′ or 5,6:5′,8′ diepoxy-β-cryptoxanthin Not identified 1 cis 5,8:5,6′ or 5,6:5′,8′ diepoxy-β-cryptoxanthin All trans-lutein cis 5,6:5′,6′-Diepoxy-βcryptoxanthin 5′,6′-Epoxy-β-crytoxanthin 5,6-Epoxy-β-cryptoxanthin 13- or 13′-cis-βcryptoxanthin Phytoene 5,8-Epoxy-β-cryptoxanthin Not identified 2 cis-Phytofluene All-trans-β-cryptoxanthin All-trans-phytofluene 15-cis-β-Carotene 9- or 9′-cis-βcryptoxanthin 13-cis-β-Carotene All-trans-β-carotene 9-cis-β-Carotene Total carotenoids Vitamin A value

cv. Centenáriaa

cv. Mizautoa

cv. Mizuhoa

cv. Mizumoa

cv. Néctar de Crisrala

Not detected 9.3 ab

Not detected 12.9 a

Not detected 22.7 c

Not detected 28.2 c

0.2 4.4 b

0.3 a 3.5 a

1.5 bc Not detected

1.9 b Not detected

6.4 d Not detected

1.2 c 1.8 b

161.0 a

180.3 a

339.5 b

324.9 b

35.0 c

2.4 a 3.1 a

5.4 b 6.4 a

7.1 b 34.8 b

12.8 c 7.1 a

2.3 a 1.8 a

3.1 a Not detected

Not detected Not detected

Not detected 10.9 a

8.4 b Not detected

1.9 c 1.1 b

3.9 a 4.6 a

12.5 b 4.5 a

13.5 b 12.1 b

7.9 c 10.4 c

6.4 c 1.9 d

40.3 a 102.4 a 5.8 a

67.9 b 137.9 b 15.1 b

104.1 c 213.9 c 20.1 b

109.4 c 207.2 c 16.6 b

11.5 d 19.0 d 4.0 a

25.3 a 4.6 a Not detected 12.5 a 278.4 a 1.7 a 2.0 a Not detected

22.0 b 3.8 a Not detected 8.5 b 480.2 b 1.9 a 4.8 a Not detected

22.1 b 13.1 b Not detected 10.1 b 557.6 c 3.3 b 3.0 a Not detected

34.0 c 15.3 b Not detected 19.3 c 715.2 d 3.4 b 3.4 a Not detected

Not detected 1.6 a 1.1 Not detected 54.8 e Not detected 0.7 a 0.8

19.4 a 858.5 a 6.7 a 1548.6 a 89.0 a

42.0 b 980.9 ab 17.3 bc 2005.7 b 111.0 b

45.9 b 1090.7 b 18.0 b 2544.4 c 127.2 b

36.2 b 1441.5 c 12.3 c 3019.5 d 162.0 c

5.0 c 38.1 d 1.6 a 196.2 e 6.8 d

Source: Adapted from Ferreira de Faria, A., Hasegawa, P. N., Chagas, E. A., Pio, R., Purgatto, E., and Mercadante, A. Z., J. Food Comp. Anal., 22, 196–203, 2009. Note: Different letters (a, b, c, d, e) in the same row indicate mean difference at significant level of 5%. a Average of duplicated analysis.

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mg GAE/kg fresh weight

600 500 400 300 200 100 0

b

b

Baduna 5 Güzelyurt-1

a

b

Hafif Ottaviani Çukurgöbek

b Type 1

Figure 5.6  Total phenolic content of several loquat cultivars grown in Dörtyol, Hatay, and Turkey. (Adapted from Polat, A. A., Çalışkan, O., Serçe, S., Saraçoğlu, O., Kaya, C., and Özgen, M., Pharmacognosy Magazine, 6(21), 5–8, 2010.)

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6 Phytochemicals in Citrus and Tropical Fruit Mehmet Çağlar Tülbek Contents Introduction............................................................................................................................................. 123 Phytochemicals: Structural Characteristics............................................................................................. 124 Monophenols and Phenolic Acids...................................................................................................... 124 Citrus Fruits.................................................................................................................................. 124 Tropical Fruits.................................................................................................................................... 125 Polyphenolics.......................................................................................................................................... 125 General Distribution of Flavonoids.................................................................................................... 125 Composition of Flavonoids: Flavanones............................................................................................ 126 Composition of Flavonoids: Flavones and Flavonols........................................................................ 127 Composition of Other Phenolic Compounds..................................................................................... 128 Carotenoids............................................................................................................................................. 128 Citrus Fruit......................................................................................................................................... 128 Tropical Fruit..................................................................................................................................... 129 Bioactivity of the Phytochemicals.......................................................................................................... 130 Antioxidant Activity: Citrus Fruit...................................................................................................... 130 Antioxidant Activity: Tropical Fruit...................................................................................................131 Health Benefits....................................................................................................................................132 Processing Effects....................................................................................................................................133 Bitterness Reduction in Citrus............................................................................................................133 Enhancing Recovery of Bioactives.....................................................................................................133 Future Direction...................................................................................................................................... 134 References............................................................................................................................................... 134

Introduction The world citrus production in 2008 was approximately 122 million tonnes (Food and Agricultural Organization 2010). The 2008 production of tropical fruits was estimated at 82.7 million tonnes (Food and Agricultural Organization 2009). The production numbers represent a significant contribution of these commodities to the human diet. Thus, citrus and tropical fruits would be expected to be a significant contributor of phytochemicals in the human diet. James Lind first confirmed citrus fruits as a source of bioactive compounds in 1747. He demonstrated that limes and lemons could treat and prevent scurvy. This represents one of the first examples of the importance of citrus fruits in human health. In modern times, many citrus compounds have been identified as having possible health benefits. The bioactives in fruits fall under several broad categories that include phenolics and polyphenolics, carotenoids, vitamin C, and pectin. In this chapter, the phytochemicals from fruit will be presented. Due to the diverse functionality and chemical and structural makeup of the phytochemicals, only the phenolics, polyphenolics, and carotenoids will be presented in this chapter. Specific focus on the composition of phytochemicals from the various 123

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sources, effects of processing on the phytochemicals, and antioxidant activity of the ­phytochemicals will be highlighted. Methods for the isolation and characterization of the phytochemicals will not be presented in detail in this chapter. Furthermore, the role of pectin in glycemic and cholesterol control and the function of vitamin C in health are not covered. The authors suggest that the review of the literature will be of value in this regard. The author of this chapter recognizes the efforts of many researchers in the phytochemical area; however, not all of the research could be reported in this review given the diverse nature of fruits. The advent of new analytical tools has resulted in the identification of many unique bioactive components. Peterson et al. (2006a, 2006b), Gattuso et al. (2007) and Gonzalez-Molina et al. (2010) provide excellent reviews of bioactives in citrus, which the author recommends for additional information.

Phytochemicals: Structural Characteristics Monophenols and Phenolic Acids Citrus Fruits The monophenolic bioactives are typically the tocopherols and phenolic acids containing one hydroxyl group. Tocopherols are not commonly associated with citrus. However, the citrus seed can be a potential source of tocopherol due to the volume of seed waste from processed citrus products (Braddock 1995). Anwar et al. (2008) reported that 63–662 mg/kg of total tocopherols were found in four citrus species. The alpha tocopherol accounted for approximately 84% of the total while gamma and delta tocopherols accounted for 13 and 3%, respectively. In contrast to tocopherols, phenolic acids are more commonly found in citrus. The hydroxycinnamic acids are the primary type of phenolic acids in citrus and can exist in free or bound forms (Peleg et al. 1991). However, the ester form of the phenolic acids was present in the highest amounts compared to glycosides and bound forms (Xu et al. 2008c). The bound forms were greatly impacted by the ripening process of the citrus fruit whereas the ester forms did not change substantially during ripening (Xu et al. 2008b). Thus, the discrepancies in the literature reports on phenolic acid content might be influenced by the age of the fruit. Caffeic, chlorogenic, ferulic, sinapic and p-coumaric acids are the most common phenolic acids in citrus (Robbins 2003). Wide variations in phenolic acids have been reported in citrus. Wang, Chuang, and Ku (2007) reported that chlorogenic acid accounted for 25–40% of the phenolic acids tested. Tangerines (i.e., mandarins) contained approximately 136 µg/g (d.b.) phenolic acids of which p-­coumaric acids accounted for 37% of the phenolic acids. Sweet oranges contained approximately 75 µg/g (d.b.) phenolic acids of which caffeic, ferulic, and chlorogenic acid accounted for approximately 22, 22, and 25% of the total phenolic acids (Wang, Chuang, and Ku 2007). Lemons contained the highest phenolic acids (231µg/g (d.b.)) of which sinapic and chlorogenic acid accounted for 31 and 40% of the phenolic acids, respectively (Wang, Chuang, and Ku 2007). Xu et al. (2008a) reported phenolic acid contents of approximately 38, 69, and 56 mg/L for tangerine, orange, and lemon juices, respectively. The juice obtained was from the same cultivars (i.e., varieties) reported by Wang, Chuang, and Ku (2007). Xu et al. (2008a) reported that ferulic acid accounted for approximately 62–70% of the phenolic acids in tangerine, orange, and lemon juices. However, chlorogenic acid was not determined by Xu et al. (2008a). A follow-up study showed that chlorogenic acid was the only free phenolic acid in tangerine and grapefruit and that no bound form of chlorogenic acid was observed (Xu et al. 2008b). The variation of the phenolic acids is likely due to a number of factors. Xu et al. (2008a) reported a range of 24.8–63.7 mg/L in total phenolic acids from seven varieties of mandarins. These authors also reported a phenolic acid range of 50.4–69.2 mg/L in four varieties of sweet orange. Xu et  al. (2008b) also reported significantly higher phenolic acid levels in unripe citrus, which decreased with the ripening of the fruit. Bocco et al. (1998) reported phenolic acid contents of 144 and 183 µg/g (d.b.) in sweet orange and lemon seeds, respectively. In contrast, sour orange peels contained 2956 µg phenolic acids/g peel (d.b.). The level of total phenolic acids was nearly double in the peels of tangerine

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and grapefruits compared to the edible portion (Xu et al. 2008b). Wang, Chuang, and Hsu (2008) also observed substantially higher phenolic acid contents in the peels of tangerine, pummelo, sweet orange, and lemons compared to the edible portion. The chlorogenic acid levels in the peels and edible portions were 179–321 µg/g and 18.8–103 µg/g, respectively. In addition, p-coumaric acid contents were higher in the peels (142–346 µg/g) than the edible portion (13.1–50.3 µg/g); thus contributing to the higher total phenolic acids in the citrus peels (Wang, Chuang, and Hsu 2008). Manthey and Grohmann (2001) reported that the flavedo contained the highest level of hydrolyzable hydroxycinnamic acids followed by the albedo and membrane of oranges. Thus, small variations in phenolic acids that were reported in the literature can be attributed to the amount of contaminating peel that might occur during processing.

Tropical Fruits With a few exceptions, the phenolics of tropical fruits are less studied than those of citrus. As with citrus, tropical fruits are low in tocopherols excluding palm fruit (Sundram, Sambanthamurthi, and Tan 2003). Robles-Sanchez et al. (2009) reported between 8 and 13 µg/g of α-tocopherol in mango. Choo et al. (1996) reported that fiber press cake from palm fruit contained high (2400–3500 µg/g) levels of tocopherols and tocotrienols. These two studies illustrate the diverse nature of tropical fruits and the diverse phenolics present in tropical fruits. The palm fruit is typically used as an oil source rather than consumed as a typical fruit. However, the oil is high in tocotrienols, thus representing a potential source of bioactive compounds (Sundram, Sambanthamurthi, and Tan 2003). As observed in citrus, specific parts of the fruit will also contribute to differences in bioactive compounds. For example, the peel of a mango was reported to contain between approximately 190 and 400 µg/g (Ajila, Bhat, and Rao 2007) tocopherol while the pulp contained 0.05 µg/g (Burns, Fraser, and Bramley 2003). Phenolic acids of tropical fruit are similar to those of citrus fruits. Like citrus, the peels of tropical fruits have higher phenolic acids followed by the pulp and seeds. Quinic acid conjugates, also known as chlorogenic acid analogues are common in tropical fruit. These caffeoyl esters have been identified in many tropical fruits (Pontes et al. 2002; Ma et al. 2003). In particular, 5-caffeoyl quinic acid (5-CQA) was the major chlorogenic acid analogue and was present in levels as high as 13 g/kg (Pontes et al. 2002). Sapodilla peels and pulp contained approximately 95 and 32 mg 5-CQA/kg, respectively (Pontes et al. 2002). Ma et al. (2003) identified gallic acid and galloylchlorogenic acid analogues as other phenolic acids in Sapodilla. Mertz et al. (2009) also identified chlorogenic acid as the predominant (1060 µg/g) phenolic acid in naranjilla while dicaffeoylquininc acid accounted for 54 µg/g in this fruit. Tree tomatoes or tamarillo contained approximately 328–548 µg/g of chlorogenic acid and 210–171 µg/g of dicaffeoylquininc acid (Mertz et al. 2009). The peel, pulp, and seeds of mangos contained approximately 45, 30, and 29 µg/g of 5-CQA while guava and tamarind did not contain detectable levels of chlorogenic acids (Pontes et al. 2002). Gallic and ferulic acid were reported to be the predominant phenolic acids in durian fruit (Toledo et al. 2008). Pacheco-Palencia, Mertens-Talcott, and Talcott (2008) reported the oil from açai fruit pulp contained high levels of phenolic acids. They reported vanillic, syringic, p-hydroxybenzoic, protocatechuic, and ferulic acids in concentrations of 1616, 1073, 892, 629, and 101 mg/L oil.

Polyphenolics General Distribution of Flavonoids The flavonoids in citrus are predominantly flavanones. Minor concentrations of flavones, flavonols, and anthocyanins are present. Kawaii et al. (1999b) conducted an extensive evaluation of the flavonoids in 66 citrus species. Peterson et al. (2006a, 2006b) provided excellent reviews on the topic of flavanones in citrus. They reported that flavanones accounted for 95 and 96% of the flavonoids in sweet oranges and tangerines, respectively (Peterson et al. 2006a). Flavanones accounted for 90, 96, and 98% of the

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flavonoids in lemons, limes, and grapefruits, respectively (Peterson et  al. 2006b). The flavones and ­flavonols account for approximately 1 and 4% of the flavonoids in sweet orange, respectively. Even lower compositions in other citrus would be expected. The flavanone distribution in sweet oranges and tangerines are similar. Hesperidin and narirutin account for approximately 83–86% and 12–13% of the flavanones, respectively (Peterson et al. 2006a). In contrast to sweet orange, sour orange flavanones were approximately 40% naringin, 30% neoeriocitrin, and 23% neohesperidin. Differences in the sensory characteristics between orange varieties are likely due to the bitter nature of narigin (Soares and Hotchkiss 1998). The tangerine flavanones profile was dramatically different from that observed in tangerine-grapefruit (Tangelo) hybrids. Peterson et al. (2006a) reported hesperidin accounted for approximately 14% of the flavanones compared to 83% in tangerines. The crossing of the tangerine with the grapefruit may be one reason for the dramatic difference as hesperidin accounted for approximately 10–15% of the flavanones in grapefruit (Peterson et al. 2006b). Limes and lemons have similar flavanone profiles to those observed in sweet oranges. The distribution of flavanones in limes shows that hesperidin (90%) is again the major component while eriocitrin accounts for 8% (Peterson et al. 2006b). Hesperidin (59%) and eriocitrin (36%) are the major flavanones in lemons (Peterson et al. 2006b). Narirutin (3%) was the only other compound detected in appreciable levels. In addition to the flavones and flavonols, cyanidin glucosides are also present in citrus. Maccarone et al. (1998) identified cyanidin-3-(6″-malonyl)-beta-glucoside as a major anthocyanins in blood orange juice. Hillebrand, Schwarz, and Winterhalter (2004) noted that cyanidin-3-glucoside was the primary anthocyanins in blood oranges in addition to the malonyl derivatives. Lee (2002) reported that cyanidin3-(6”-malonylglucoside) and cyanidin-3-glucoside accounted for 44.8 and 33.6% of the anthocyanins in blood oranges, respectively. Delphinidin-3-glucoside was also observed in blood orange juice and in the plasma of subjects fed 500 mL of blood orange juice (Giordano et al. 2007). Tropical fruits also contain anthocyanins. The anthocyanins delphinidin glucosyl rutinoside, delphinidin rutinoside, cyanidin rutinoside, and pelargonidin were present in red tree tomatoes at levels of 53, 327, 121, and 1150 µg/g, respectively (Mertz et al. 2009). Cyanidin-3-rutinoside and cyanidin-3-glucoside accounted for 87 and 13% of the 2820–3030 µg/g anthocyanins in açaí fruit (DeRosso et al. 2008). Vendramini and Trugo (2004) reported total anthocyanins content in the peel of acerola fruit was 375µg/g. Cyanidin-3-rhamnoside and pelargonidin-3-rhamnoside accounted for 76–78% and 13–16%, respectively, of the anthocyanins in acerola fruit (De Rosso et al. 2008).

Composition of Flavonoids: Flavanones In general, the flavanone composition in citrus is primarily naringin, hesperidin, and neohesperiden, with only minor differences being observed between citrus products. Hesperidin contents in sweet oranges ranged between 0.28 mg/g seeds and 5.36 mg/g edible portion (Bocco et  al. 1998; Wang, Chuang, and Ku 2007). Abeysinghe et al. (2007) reported hesperidin levels between 0.63 and 246 mg/g in various edible portions of oranges. The juice of sweet oranges contained approximately 235–407 mg/L (Mouly et al. 1994) and 489 mg/L (Xu et al. 2008a) of hesperidin. Similar hesperidin values (386 mg/L) were found in tangerine juice whereas lower levels were observed in lemon (238 mg/L) and grapefruit (38 mg/L) juices (Xu et al. 2008a). Mouly et al. (1994) reported that limes and lemons contained hesperidin levels of 84–196 mg/L. Abeysinghe et  al. (2007) also reported a range (0.4– 2.93 mg/g) hesperidin contents in various edible portions of tangerines. Hesperidin levels in madefrom-concentrate orange juice averaged 441 mg/L while pasteurized orange juice contained 305 mg/L (Vanamala et al. 2006b). In contrast to hesperidin, naringin (100–800 and 113–481 mg/L, respectively) was also identified as a predominant flavanone in grapefruit (Rouseff, Martin, and Youtsey 1987; Mouly et  al. 1994). Neohesperidin (265 mg/L) and naringin (349 mg/L) were identified as the major flavanones in grapefruit juice (Xu et al. 2008a). Desiderio, De Rossi, and Sinibaldi (2005) reported naringin levels of 626 mg/L in grapefruit using capillary electrophoresis. Tangerine, sweet orange, and lemon juices did not have detectable levels of these flavanones (Abeysinghe et al. 2007; Xu et al. 2008a).

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Mouly et al. (1994) reported narirutin levels of 30–84 mg/L and 33–161 mg/L in sweet orange and grapefruit juices, respectively. Narirutin levels in juices of sweet orange, tangerines, and grapefruit were approximately 103, 104, and 94 mg/L (Xu et  al. 2008a). No narirutin was observed in lemon juice. However, lime and lemons did contain the unique flavanone eriocitrin at levels of 47–94 mg/L (Mouly et al. 1994). Vanamala et al. (2006b) reported that made-from-concentrate orange juice had higher narirutin levels than ready to drink (i.e., pasteurized) orange juice. Bocco et al. (1998) reported the flavanone contents in citrus seeds (0.04–3.28 mg/g dry basis (d.b.)) were significantly lower than levels found in peels (13.45–22.30 mg/g d.b.). They noted that naringin was substantially higher in the peels compared to seeds and supported the observations of Barthe et al. (1988) and Yusof et al. (1990). Wang, Chuang, and Hsu (2008) reported that pummelo (pomelo) peels contained 24–30 mg/g of naringin while peels of sweet orange, tangerine, and lemons contained 0.36, 0.54, and 1.51 mg/g. The hesperidin and neohesperidin contents were highest in peels of the tangerine followed by the peels of sweet orange, lemons, and pummelo (Wang, Chuang, and Hsu 2008). The fact that bitter compounds are thought to be a plant defense mechanism likely explains why naringin has high levels in the peel.

Composition of Flavonoids: Flavones and Flavonols The flavones (i.e., diosmin, luteolin, and sinsensetin) and flavonols (i.e., rutin, quercetin, and kaempferol) account for a small amount of the flavonoids in citrus (Table 6.1). The flavone and flavonol levels were significantly higher in peels than the edible fruit of tangerines and sweet oranges, except for kaempferol contents (Wang, Chuang, and Hsu 2008). The generalized trend in flavones and flavonols could not be made for lemon and pummelo, except that pummelo peels contained higher flavonols levels than the edible portion (Wang, Chuang, and Hsu 2008). Justesen, Knuthsen, and Leth (1998) reported grapefruit and lime pulp contained approximately 5 and 4 µg/g quercetin, respectively. HoffmannRibani, Huber, and Rodriguez-Amaya (2009) reported the average quercetin content in oranges was 3 µg/g whereas red and white guavas contained 10 and 12 µg/g, respectively. Quercetin concentrations between 4 and 68 µg/g were reported in durian fruit (Toledo et al. 2008). Quercetin-3-rhamnoside was detected in acerola fruit (Hanamura, Hagiwara, and Kawagishi 2005). Eight different flavonol glycosides Table 6.1 Flavones and Flavonols Content in Different Citrus Fruits Flavones (µg/g) Citrus/Author

Diosmin

Luteolin

Tangerine Fruit* Peel**

129 360

42.9 210

Sweet Orange Fruit* Peel**

84 170

Lemon Fruit* Peel** Pummelo Fruit* Peel**

Flavonols (µg/g) Sinensetin

Rutin

Quercetin

9.89 290

53 290

136 470

503 380

13.7 110

14.8 420

42 230

104 140

606 320

323 130

160 80

9.8 220

60 290

573 210

611 310

132 160

n.d. n.d.

42.8 20

90 180

61 230

12 330

Note: n.d. = not detected. *Data obtained from Wang, Y. C., Chuang, Y. C., and Ku, Y. H., Food Chem., 102(4), 1163–71, 2007. **Data obtained from Wang, Y. C., Chuang, Y. C., and Hsu, H. W., Food Chem., 106(1), 277–84, 2008.

Kaempferol

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were identified in mango (Schieber, Berardini, and Carle 2003). Mango puree concentrates contained ­quercetin-3-galactoside (22 µg/g), quercetin-3-glucoside (16 µg/g), and quercetin-3-arabinoside (5 µg/g) as predominate flavonol glycosides (Schieber et  al. 2000). Dried mango peels contained quercetin-3galactoside (651µg/g), quercetin-3-glucoside (558 µg/g), and quercetin-3-xyloside (207 µg/g) along with five other flavonol glycosides (Berardini et  al. 2005). Quercetin-3-glucoside and quercetin were also found in emblica fruits (Liu et al. 2008; Luo et al. 2009). The shell of tamarind contained 140 µg/g of luteolin (Sudjaroen et al. 2005). The polymethoxylated flavones nobiletin and tangeretin have also been identified as major flavones in tangerines. Kawaii et al. (1999) reported nobiletin and tangeretin levels of 45–128 and 15–91 µg/g in tangerines, respectively. Valencia and navel oranges contained approximately 1.8 and 3.0 µg/g nobiletin and tangeretin, respectively (Kawaii et al. 1999). These authors also reported that lemons and lime contained approximately 1–4 and 0–6 µg/g nobiletin, respectively, and 0–1 and 1.4 µg/g tangeretin, respectively. Mouly, Gaydou, and Auffray (1998) reported levels of polymethoxylated flavones in orange juices. They observed that sinsensetin and nobiletin each were present at the 2.7– 2.9 mg/L concentration. Other polymethoxylated flavones in citrus have been summarized by Gattuso et al. (2007).

Composition of Other Phenolic Compounds The flavanols and xanthones are two common classes of phenolics present in tropical fruits. The flavanol epicatechin is typically found in the pericarp or hull of many tropical fruit including mangosteen (Yu et al. 2007) and tamarind (Sudjaroen et al. 2005). Gu et al. (2003) found that procyanidin levels were up to 19 times higher than epicatechin in the pericarp of tamarind. The procyanidin levels were 4–11 times higher than epicatechin (56 µg/g) in the hull of tamarinds (Sudjaroen et al. 2005). The tamarind seeds contained significantly higher epicatechin (315–933 µg/g) content than the hulls. The procyanidin levels were 4–5 times higher than epicatechin in the tamarind seed (Gu et al. 2003; Sudjaroen et al. 2005). Several xanthone derivatives exist in mangosteen. Mangostin (α and γ derivatives) and gartanin were the primary xanthones identified (Mahabusarakam, Wiriyachitra, and Taylor 1987). Yu et  al. (2007) reported additional xanthones from the pericarp of mangosteen. The 1,3,6,7-tetrahydroxy-2,8-(3-methyl2-butenyl) xanthone had better antioxidant activity than the 1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl2-butenyl) xanthone. The only structural difference was the methoxy and hydroxyl groups at carbon 7. The xanthone glycosides mangiferin and isomangiferin were identified in mangos (Schieber et al. 2000; Schieber, Berardini, and Carle 2003). Mangiferin concentration of 4.4 µg/g was determined in a mango puree. Berardini et al. (2005) reported mangiferin and isomangiferin contents of 1690 and 134 µg/g in dried mango peels.

Carotenoids Citrus Fruit The carotenoid levels in citrus are dictated by variety or type of citrus. Pupin, Dennis, and Toledo (1999) reported total carotenoid levels in orange juice were approximately 190 and 860 mg/L. The Pera Rio variety had the highest level while the Baía variety had the lowest carotenoid levels. Mouly, Gaydou, and Corsetti (1999) reported the influence of geography on carotenoid content. They noted that Valencia juice from Spain and Belize contained 17 and 4.8 mg/L total carotenoids, respectively. Wang, Chuang, and Ku (2007) reported total carotenoids of 12.2, 5.2, and 4.3 µg/g in tangerines, sweet orange, and lemons, respectively. Gardner et al. (2000) reported carotenoid levels in orange and pink grapefruit juices were 3.0 and 8.3 mg β-carotene equivalence/L juice, respectively. The carotenoid levels in tangerine, sweet orange, lemon, and grapefruit juices were reported as 5.32, 0.39, 0.08, and 0.14 mg β-carotene equivalence/L juice, respectively (Xu et al. 2008a). Differences in carotenoid levels observed by various researchers are likely attributed to differences in variety and growing location.

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Different carotenoid levels were also reported in various edible tissues of tangerines and oranges (Abeysinghe et al. 2007). Stewart (1977) identified 10 different carotenoids in sweet oranges, which include lutein, α- and β-cryptoxanthin, α-, ξ-, and β-carotenes, antheraxanthin, and violaxanthin. Yano et  al. (2005) also observed these same carotenoids in various citrus fruits in Japan. Zeaxanthin accounted for 36.6– 40.9% of carotenoids in orange juice followed by lutein (23.5–34.2%), β-cryptoxanthin (12.0–20.7%), α-carotenes (12.6%) and β-carotenes (8.5–10.9%; Pupin, Dennis, and Toledo 1999; Wang, Chuang, and Ku 2007). Lutein (37%) and β-cryptoxanthin (33%) accounted for the majority of the carotenoids in tangerines whereas β-cryptoxanthin (61%) and β-carotenes (26.2%) were the major carotenoids in lemons (Wang, Chuang, and Ku 2007). The levels and distributions of the carotenoids in the citrus peels were different than the edible portions. Tangerine peels contained 114 µg/g carotenoids followed by peels of the sweet orange (108 µg/g), lemon (14.9 µg/g), and pummelo (2.7 µg/g) whereas the edible portions contained 12.2, 5.2, 4.3, and 2.4 µg/g, respectively (Wang, Chuang, and Ku 2007; Wang, Chuang, and Hsu 2008). Both β-cryptoxanthin (27%) and β-carotene (61%) were the two major carotenoids in tangerine peel while β-carotenes accounted for 36, 46, and 69% of the carotenoids in pummelo, sweet orange, and lemon peels, respectively (Wang, Chuang, and Hsu 2008). Vanamala et al. (2005) reported lycopene (150 µg/g) and β-carotene (45 µg/g) in Rio Red grapefruits after 4 days from harvest.

Tropical Fruit The level of carotenoids varies significantly among tropical fruits (Rodriguez-Amaya 1999). Palm fruit (Sundram, Sambanthamurthi, and Tan 2003), mango (Mercadante and Rodriguez-Amaya, 1998; Breithaupt and Bamedi 2001), and tree tomatoes (Mertz et al. 2009) have very high levels of carotenoids while trace amounts were found in fruit such as naranjilla (Mertz et al. 2009). Setiawan et al. (2001) reported that guava was a rich source of vitamin A based on retinol equivalence while mango and papaya were considered good sources. The carotenoid isomerize also vary among tropical fruit. Guava contains primarily lycopene and β-carotene levels of 47–61 µg/g and 2–25 µg/g, respectively (Padula and Rodriguez-Amaya, 1987; Wilberg and Rodriguez-Amaya, 1995; Rodriguez-Amaya 1999; Chandrika, Fernando, and Ranaweera 2009). β-Carotene levels of approximately 2–17 µg/g were observed in acerola fruit (De Rosso and Mercadante 2005). Chandrika, Fernando, and Ranaweera (2009) identified lutein (2.1 µg/g) in guava. Similar lutein levels were identified in yellow (0.98 µg/g) and red (1.25 µg/g) tree tomatoes (Mertz et al. 2009) and acerola fruit (De Rosso and Mercadante 2005). However, β-cryptoxanthin levels of 14 and 16 µg/g accounted for the majority of the carotenoids in yellow and red tree tomatoes after saponification of carotenoid extracts (Mertz et al. 2009). β-Cryptoxanthin levels in papaya varied depending on ripeness and only small differences were observed between red and orange fleshed cultivars (Wilberg and Rodriguez-Amaya 1995). In contrast, red fleshed papaya contained high (18–81 µg/g) lycopene levels, whereas orange fleshed cultivars lacked lycopene (Kimura, Rodriguezamaya, and Yokoyama 1991; Wilberg and Rodriguez-Amaya 1995). Both genotype and harvest years influenced carotenoid concentrations in acerola fruit (De Rosso and Mercadante 2005). The α- and β-carotenes account for approximately 36 and 55% of the carotenoids in palm oil (Sundram, Sambanthamurthi, and Tan 2003). β-Carotene is also one of the major carotenoids in naranjilla (Gancel et  al. 2008) and mango (Wilberg and Rodriguez-Amaya 1995; Pott et  al. 2003). However, in mango all trans-violaxanthin were determined to be in higher concentrations than β-carotene while cis-violaxanthin and β-carotene concentrations were similar (Mercadante, Rodriguez-Amaya, and Britton 1997; Mercadante and Rodriguez-Amaya 1998; Rodriguez-Amaya 1999). Literature discrepancies are likely due to a number of factors including the stage of fruit ripening and cultivar (Perkins-Veazie, Collins, and Manthey 2008). Mercadante and Rodriguez-Amaya (1998) reported increases in β-carotene and all trans-violaxanthin from approximately 2 to 7 and 5 to 18 µg/g, respectively, during ripening of the Keitt mango cultivar. Increases in β-carotene and all trans-violaxanthin to approximately 6 and 22 µg/g, respectively, were also observed during the ripening of the Tommy Atkins mango cultivar (Mercadante and ­Rodriguez-Amaya

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1998). Manthey and Perkins-Veazie (2009) reported that the Ataulfo mango cultivar had the ­significantly higher average β-carotene concentrations than other cultivars tested. Commercially processed mango juice contained comparable β-carotene to those of the fresh fruit while trans-violaxanthin was not detected thus demonstrating the influence of processing one carotenoid composition (Mercadante and Rodriguez-Amaya 1998). The concentration of β-carotene in the peel (36 µg/g) of naranjilla was 10 times those present in the edible portion (Gancel et al. 2008). Carotenoid levels can also vary based on analytical methods. Breithaupt and Bamedi (2001) reported free and esterified β-cryptoxanthin levels of 1.43 and 10.8 µg/g in papaya, respectively. Mertz et  al. (2009) reported free and esterified β-cryptoxanthin levels of 1.1 and 13.5 in yellow tree tomatoes, respectively. Slightly higher β-cryptoxanthin values were observed in red tree tomatoes. Thus, carotenoids levels may be lower than expected in some products if esters are not measured. Furthermore, variations in harvest period and cultivar may impact carotenoid levels (Tharanathan, Yashoda, and Prabha 2006).

Bioactivity of the Phytochemicals Antioxidant Activity: Citrus Fruit A number of assays have been used to demonstrate the antioxidant activity of phenolic compounds. However, mixed results have been reported for citrus fruit phenolics. Proteggente et al. (2002) reported that the antioxidant capacity of grapefruits and oranges followed only the antioxidant capacity of the anthocyanins rich fruits. Bocco et al. (1998) did not find a strong correlation between antioxidant activity and glycosylated flavanone concentrations. The flavanone accounted for only 1–20% of the antioxidant activity of the citrus seed extracts while 36–83% of the activity of the peels was related to flavanones. Minor components such as methoxylated flavones were thought to contribute to the antioxidant activity, as the flavanone glycosides and hydroxycinnamates did not have strong activity (Manthey 2004). Abeysinghe et al. (2007) reported that hesperidin accounted for 5.9–54% of the antioxidant capacity of citrus. They noted that hesperidin contributed more to the antioxidant capacity of the segment membrane tissue than ascorbic acid. Miyake et al. (2007) reported that eriocitrin had high DPPH radical scavenging activity than the lime juice. Jayaprakasha and Patil (2007) reported radical scavenging activity of several extracts of oranges obtained from various solvents. In general, the only small differences in scavenging activity were observed suggesting multiple components were responsible for the activity of the extracts. However, the solvent type did impact the reducing power of pummel and navel orange extracts. Acetone and ethanol extracts had the highest reducing potential (Jayaprakasha, Girennavar, and Patil 2008a). However, a grapefruit extracted prepared from methanol:water (80:20 v/v) had the best radical scavenging activity and reducing power among various solvent extracts of grapefruits (Jayaprakasha, Girennavar, and Patil 2008b). These authors also observed that a methanol extract of sour oranges also had high reducing power compared to other solvent extracts of sour orange. A reduction in antioxidant activity was noted after a blanching and pasteurization of blood orange juice (Lo Scalzo et al., 2004), thus indicating the sensitivity of some of the antioxidants to heat. Xu et  al. (2008a) reported that ascorbic acid and flavonoids correlated highly with ferric reducing antioxidant power (FRAP). However, only ascorbic acid correlated with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Gardner et al. (2000) also reported that ascorbic acid concentration was highly correlated to FRAP activity and accounted for 66–100% of the antioxidant capacity of orange and grapefruit juices, respectively. Ascorbic acid accounted for 27–46% of the antioxidant activity in various tissues of tangerines and oranges (Abeysinghe et al. 2007). The phenolic acids were not strongly correlated to antioxidant activity (Bocco et al. 1998; Xu et al. 2008a). In contrast, other researchers have found a correlation between antioxidant activity and phenolic content (Rapisarda et al. 1999; Gorinstein et al. 2004a,b; Xu et al. 2007). Gorinstein et al. (2004b) reported that caffeic acid and β-carotene were highly correlated to DPPH radical scavenging. Phenolic acids common to citrus did inhibit (86–97%) the oxidation of human low density lipoproteins (LDL; Meyer et al. 1998). Yu et al. (2005) also observed delayed oxidation in hamster LDL treated with flavanone ­glycosides

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whereas the DPPH radical scavenging was not as effective. Thus, indicating the s­ ystem-dependent antioxidant activity. The oxidation of LDL was delayed after the consumption of lemon flavonoids aglycone eriodictyol, homoeriodictyol and hesperetin by 10 male subjects (Miyake et al. 2009). These authors found glucuro- and sulfo-conjugates of the aglycones but no aglycones in the subject plasma. They proposed that these conjugates might be the active components. Justesen et al. (2000) observed using an in vitro human fecal fermentation model that the citrus flavonoids were metabolized into acids and aldehydes.

Antioxidant Activity: Tropical Fruit As expected, tropical fruits would have antioxidant activity based on the composition of phenolics and other antioxidant components. Leong and Shui (2002) evaluated 27 different fruits from a Singapore market. They found that sapodilla had extremely high ascorbic acid equivalent antioxidant capacity (AEAC), which was based on the 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging (ABTS) activity. Starfruit and guava ranked slightly lower than plums but were still considered to have high AEAC activity (Leong and Shui 2002). These authors observed that DPPH activities followed the same trend regarding the fruit ranking. Mahattanatawee et al. (2006) evaluated the ORAC and DPPH activities of 14 tropical fruits. Red guava and starfruit had the highest antioxidant activities while sapodilla and green papaya had the lowest antioxidant activity. Discrepancies in the observed antioxidant activities could be related to protocols used to measure antioxidant activity. Alternatively, the fruit source and stage of ripeness may have contributed to the difference in antioxidant activity. Shui, Wong, and Leong (2004) observed that the antioxidant activity and total phenolic content of sapodilla decreased upon ripening, suggesting the importance of noting the fruit age during antioxidant activity determinations. Manthey and Perkins-Veazie (2009) noted that variety had a greater impact on antioxidant components in mangos than the growing location. However, differences in vitamin C and β-carotene were observed at different harvest dates but no specific trend was observed. Pothitirat et al. (2009) observed young or immature mangosteen fruit had twice the radical scavenging activity of the mature fruit. They noted that higher total phenolic and tannins were likely responsible for the activity since these compounds were in higher concentration in the immature fruit compared to the mature fruit. Other tropical fruits such as lychee, mango, and papaya have been shown to have varying levels of antioxidant activities (Leong and Shui 2002; Mahattanatawee et  al. 2006; Lim, Lim, and Tee 2007). Lim, Lim, and Tee (2007) observed that bananas had very little radical scavenging activity but did have good chelating activity. In contrast, guava had good scavenging activity but low metal chelating activity. Barreto, Benassi, and Mercadante (2009) reported that fruits containing the highest level of phenolic compounds had the highest radical scavenging activity among 18 Brazilian fruits. The pulps with high carotenoid and ascorbic contents had lower radical scavenging activity. Gancel et al. (2008) reported that the placental tissue of naranjilla fruit on a dry weight basis had better ORAC and DPPH radical scavenging activity compared to the flesh and peel. However, on a fresh weight basis the peel had better activity followed by the placental tissue and flesh. On a fresh weight basis, a trend existed between total phenolic content and radical scavenging activity (Gancel et al. 2008). However, this trend was not observed for the plant tissue based on dry weight indicating that other compounds may contribute to radical scavenging activity. Vasco, Ruales, and Kamal-Eldin (2008) also observed similar trends in that FRAP and ABTS activities were highly correlated to phenolic acid content. However, fruits within the same family had varying antioxidant activity, which could be traced to differences in phenolic compound levels. Mertz et al. (2009) reported that Andean blackberries had the highest antioxidant activity compared to tree tomatoes and naranjilla. They also observed that acetone extracts had better ORAC values than the  exane extracts. The phenolic composition was higher in the acetone extracts and again supports the radical scavenging activity of phenols over carotenoids (Vasco, Ruales, and Kamal-Eldin 2008; Barreto, Benassi, and Mercadante 2009). Mertz et al. (2009) observed that purification of the water-washed acetone extract of Andean blackberries on a nonionic resin (XAD-7) caused a slight reduction in ORAC activity. This suggests that nonphenolic compounds had only a minor contribution to antioxidant activity. Moyer et  al. (2002) reported that total phenolic content was a better indicator of antioxidant activity than the anthocyanins.

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Luo et  al. (2009) reported that fractionation of the emblica fruit extract into purified components improved radical scavenging activity. They noted that quercetin, gallic acid, and ellagic acid were the most active compounds and were likely responsible for the scavenging activity of the emblica fruit. Yu et al. (2007) also noted that purification of a 70% methanolic extract of mangosteen by Sephadex LH-20 produced three compounds with varying antioxidant activity. The phenolic compound 1,3,6,7tetrahydroxy-2,8-(3-methyl-2-butenyl) had the best DPPH and hydroxyl radical scavenging activity while epicatechin had the best superoxide anion scavenging activity. These studies support the variability in antioxidant activity being dependent on a number of factors that include test methodology.

Health Benefits Numerous health benefits have been linked to flavonoid consumption. An in-depth review of flavonoid bioactivity was reported by Middleton, Kandaswami, and Theoharides (2000). Gonzalez-Molina et al. (2010) provided an excellent review of the health benefits of lemon bioactives. Flavonoids and limonoids have been identified as having beneficial effects on inflammation, heart disease, and cancer. Citrus flavonoids have been linked to reducing cholesterol through the inhibition of hepatic production of cholesterol (Borradaile, Carroll, and Kurowska 1999; Wilcox et al. 2001; Kurowska and Manthey 2002; Whitman et al. 2005). Foam cell formation by macrophages is one of the early stages of atherosclerotic lesions. This process is controlled by class A scavenger receptors (SR-A) and can be measured using acetylated low density lipoproteins (acLDL, i.e., a ligand for SR-A; Whitman et  al. 2005). Nobiletin inhibited the acLDL metabolism by 50–72%, thus indicating inhibition of macrophage foam cell formation (Whitman et al. 2005). A follow-up study by the authors supported that a metabolite of nobiletin had equal or better SR-A expression than the parent compound nobiletin (Eguchi et al. 2007). All flavonoids (i.e., naringenin, hesperetin, nobiletin, and tangeretin) reduced the metabolism of β-very low density lipoproteins (VLDL), which was in agreement with previous research (Kurowska and Manthey 2002; Kurowska et al. 2004). Eriocitrin also lowered total cholesterol via VLDL and LDL reduction in rats (Miyake et al. 2006). The inhibition of cyclooxygenase-2 (COX-2) by nobiletin is another beneficial effect of this compound due to the potential reduction of inflammatory products (Murakami et al. 2000; Lin et al. 2003). Huang and Ho (2010) reported that nobiletin correlated well with anti-inflammatory activity of citrus peel extracts. Tangeretin suppressed the IL-1 beta induced COX-2 production better than nobiletin (Chen, Weng, and Lin 2007). Eriocitrin from lemons was found to inhibit 5- and 12-lipoxygenase, which also plays an important role in inflammatory processes thought to affect atherosclerosis and cancer (Nogata et al. 2007). The antiproliferation activity of flavonoids has been reported in numerous cancer cell lines (Kuo 1996; Sun et al. 2002; Xu, Go, and Lim 2003; Sergeev et al. 2007). Beneficial effects of methoxylated flavones, limonoids, and naringenin on apoptosis of breast cancer cells, neuroblastoma cells, and prostate cancer cells lines, respectively, have been reported (Poulose, Harris, and Patil 2005; Gao et al. 2006; Sergeev et al. 2006; Poulose et al. 2007). Vanamala et al. (2006a) also reported the suppression of colon carcinogenesis while Patil et al. (2009, 2010) reported the apoptosis of Panc-28 cells. Flavonoids and limonoids were believed to be responsible for the apoptosis of Panc-28 cells. Jayaprakasha et al. (2010) reported limonexic acid promoted colon cancer cell apoptosis. Citrus flavoniods have also been inhibited oral carcinogenisis in hamster cheek pouch models (Miller et al. 2008). Irwig et al. (2002) noted that β-cryptoxanthin, γ-tocopherol, and lutein + zeaxanthin had the highest diet-plasma correlations, meaning that these compounds were present in higher levels for those individuals that consumed lower levels of these compounds in the diet. They also noted that higher (more than 3/ day) tropical fruit consumption by adolescents produced higher β-cryptoxanthin than those who ate less than four servings a week. These observations follow other dietary studies regarding nutrient intake and plasma nutrient levels but demonstrate the bioavailability of carotenoid and tocopherols from tropical fruits. The health promoting properties of tropical fruits is still relatively scarce compared to other food sources such as soy. However, a number of tropical fruits have been identified as having health promoting compounds. Hogan et al. (2010) also reported the antipoliferation activity of acai extracts against C-6 rat brain glioma cells and MDA-468 human breast cancer cells. Sapodilla ethyl acetate

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extracts were cytotoxic against HCT-116 and SW-480 human colon cancer cell lines (Ma et al. 2003). These authors reported the cytotoxicity of the s­apodilla ethyl acetate extracts was due to methylO-galloylchlorogenate and 4-O-galloylchlorogenic acid. The xanthone α-mangostin extracted from mangosteen inhibited the 7,12-dimethylbenz[α]anththracene (DMBA)-induced preneoplastic lesions in mouse mammary culture assays (Jung et al. 2006). They proposed that the peroxynitrite scavenging activity was responsible for the inhibition of DMBA-induced preneoplastic lesions in vitro. Several mangosteen xanthones inhibited cell growth of the human colon cancer cell line DLD-1 (Matsumoto et al. 2005) and human breast adenocarcinoma cells (Lee et al. 2010). Ito et al. (2003) observed that 7-O-methylgarcinone had the strongest inhibitory activity against Epstein-Barr virus early antigen (EBV-EA) followed by α- and β-mangostin. Collectively, there is a growing body of knowledge supporting the bioactivity of mangosteen (Pedraza-Chaverri et al. 2008).

Processing Effects Bitterness Reduction in Citrus The reduction in citrus bitterness due to the presence of naringin has been studied extensively. Adsorption (Ribeiro, Silveira, and Ferreira-Dias 2002) and enzymatic hydrolysis (Puri et al. 1996; Puri and Banerjee 2000; Chien, Sheu, and Shyu 2001; Prakash, Singhal, and Kulkarni 2002; Pedro et al. 2007; Ribeiro and Ribeiro 2008) are the two methods for removing bitter compounds. However, enzymatic hydrolysis using naringinase has advantages over adsorption such as better flavor and sweetness retention (Ribeiro and Ribeiro 2008). Ribeiro and Ribeiro (2008) reported 65% reduction in naringin in grapefruit juice after 120 minutes of exposure to naringinase immobilized on κ-carrageenan. Naringenin results from the hydrolysis of the rhamnose unit of naringin causing a reduction in bitterness. Ribeiro and Ribeiro (2008) observed the formation of 46.3 µg/mL naringenin during the hydrolysis of naringin in grapefruit juice. Naringenin is nearly tasteless (Puri et al. 1996) and thus might be more suitable for flavonoid fortification than naringin. Girennavar et al. (2008) reported that electron beam irradiation above 10 kGy increased naringin in grapefruit juice indicating a potential increase in bitterness. Deacidification through the removal of citric acid has been reported in orange juice (Couture and Rouseff 1992) and tropical fruit juices (Vera et al. 2003a, 2003b, 2007). Vera et al. (2003a) reported that titrable acidity was significantly reduced and 60 and 85% of the organic and inorganic anions were eliminated using electrodialysis. Comparison to the traditional calcium precipitation and ion exchange deacidifications showed that electrodialysis was comparable to both methods (Vera et al. 2003b). However, passion fruit juice scored better on sensory tests compared to the ion exchange deacidified juice. The improvement of a sensory score on deacidified juices warrants additional investigations into how bioactive compounds are affected by the electrodialysis process.

Enhancing Recovery of Bioactives The recovery of bioactive compounds from citrus waste can add value to the citrus industry because the bioactives can be sold for health purposes and the waste disposal cost can be reduced (Schieber, Stintzing, and Carle 2001). Coll et al. (1998) reported that 13 kg of flavonoids could be extracted per ton of waste lemons. The production of a citrus molasses occurs during the peel processing (Manthey and Grohmann 2001). The molasses has been found to contain concentrated levels of bioactive compounds (Hasegawa et al. 1996; Manthey and Grohmann 1996, 2001). With the exception of lemons, ferulic acid was the major hydroxycinnamic acid in citrus molasses. Tangerine molasses contained approximately 1206 µg/mL ferulic acid (Manthey and Grohmann 2001). The ferulic acid levels in citrus peels are approximately 75–150 µg/g; thus the production of citrus molasses can substantially increase phenolic acid content. The polymethoxylated flavones were also concentrated in the molasses. Nobiletin content of 711 µg/mL was observed in Dancy tangerine molasses (Manthey and Grohmann 2001) while levels of 1.5–3.5 µg/mL tangerine juice have been reported

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(Mouly, Gaydou, and Auffray 1998; Kawaii et al. 1999). The nobiletin content (88–95 µg/mL) in orange peel molasses (Manthey and Grohmann 2001) was also higher than the content (1.9–4.6 µg/mL) in orange juice (Pupin, Dennis, and Toledo 1998; Kawaii et  al. 1999). These examples demonstrate the potential for obtaining concentrated bioactives from waste products such as peels. Ma et  al. (2008a, 2008b, 2008c, 2009) proposed the use of ultrasound to enhance the recovery of citrus peel bioactives. They observed that the total phenolic acid content of extracts increased from 1065 µg/g in the maceration extract (8 hours) to 1935 µg/g in the ultrasound (10 minutes) promoted extraction. The flavanone glycosides in the maceration and ultrasound promoted extracts also increased from 753 to 1373 µg/g, respectively (Ma et al. 2008a). The 40°C extraction temperature during ultrasound exposure enhanced flavanone glycoside extraction whereas phenolic acid extraction was favored at 30°C (Ma et al. 2008a). The optimal extraction conditions for the cinnamic and benzoic acids were 40 minutes at 30°C and 10 minutes at 40°C, respectively (Ma et al. 2009). Ultrasound power of 30 W also was found to maximize extraction of phenolic acids. Khan et  al. (2010) also reported that 40°C at 150 W sonication power using ethanol:water (4:1 v/v) was the most effective ultrasound assisted extraction conditions. Hayat et al. (2009) reported microwave assisted extraction using a microwave power of 152 W, 66% methanol at a 16–1 (peels) for 49 seconds produced extracts with similar total phenolic acid concentrations to the ultrasound (200 W) promoted extraction using 80% methanol at a 20:1 v/v and 60 minutes at room temperature. Rodrigues and Pinto (2007) and Rodrigues, Pinto, and Fernandes (2008) utilized the ultrasound as a means to concentrate phenolic compounds from coconut shells. The best phenolic extraction conditions were 50% ethanol in water at a 50:1 solvent to solid ratio and 15 minutes at 30°C. The ultrasound intensity was 4.87 kW/m 2. Under these conditions an extract with 22.44 mg phenolics/g was produced from coconut shells (Rodrigues, Pinto, and Fernandes 2008).

Future Direction There has been substantial progress in understanding phytochemicals in citrus and tropical fruits. There is general agreement about the chemical constituents in fruits. However, discrepancies in concentration and antioxidant activities confound our understanding of the true components responsible for bioactivity. Furthermore, correlation between antioxidant tests such as DPPH radical scavenging and in vivo bioactivity is difficult to ascertain. The LDL antioxidant testing of citrus bioactives might be better suited for comparing in vivo and in vitro antioxidant testing. Attempts have been made to characterize the bioactivity of the citrus carotenoids, phenolic acids, and flavonoids. Future directions in this area should attempt to evaluate mixtures of compounds, including combinations of different classes of bioactives in citrus or tropical, in animals and cell lines. The role of essential oils combined with phenolics should be considered because recent data suggests that limonoids can contribute to apoptosis of pancreatic cell lines. Sugiura et al. (2002) reported that serum β-cryptoxanthin correlated to serum consumption. Although some research on the bioavailability of citrus and tropical fruits has been conducted, additional research that determines baseline serum bioactives should be completed. Thus, the scientific community might be able to make better recommendations regarding the consumption of citrus. Furthermore, the investigation regarding the metabolic forms of the bioactives should be determined. Multiple reports have indicated that the intact bioactive is not always found in plasma or the blood and therefore is not likely to be the active components. Identification of the metabolic products and testing those compounds using in vitro cell lines should be completed.

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Ribeiro, I. A., and Ribeiro, M. H. L. 2008. Naringin and naringenin determination and control in grapefruit juice by a validated HPLC method. Food Control 19 (4): 432–8. Ribeiro, M. H. L., Silveira, D., and Ferreira-Dias, S. 2002. Selective adsorption of limonin and naringin from orange juice to natural and synthetic adsorbents. European Food Res Tech 215 (6): 462–71. Robbins, R. J. 2003. Phenolic acids in foods: An overview of analytical methodology. J Agric Food Chem 51 (10): 2866–87. Robles-Sanchez, R. M., Islas-Osuna, M. A., Astiazaran-Garcia, H. Vazquez-Ortiz, F. A., Martin-Belloso, O., Gorinstein, S., and Gonzalez-Aguilar, A. 2009. Quality Index, Consumer Acceptability, Bioactive Compounds, and Antioxidant Activity of Fresh-Cut “Ataulfo” Mangoes (Mangifera indica L.) as Affected by Low-Temperature Storage. J. Food Sci. 74:S126–34. Rodrigues, S., and Pinto, G. 2007. Ultrasound extraction of phenolic compounds from coconut (Cocos nucifera) shell powder. J Food Eng 80 (3): 869–72. Rodrigues, S., Pinto, G., and Fernandes, F. 2008. Optimization of ultrasound extraction of phenolic compounds from coconut (Cocos nucifera) shell powder by response surface methodology. Ultrasonics Sonochemistry 15 (1): 95–100. Rodriguez-Amaya, D. 1999. Latin American food sources of carotenoids. Archivos Latinoamericanos De Nutricion 49 (3): 74S–84S. Rouseff, R. L., Martin, S. F., and Youtsey, C. O. 1987. Quantitative survey of narirutin, naringin, hesperidin, and neohesperidin in citrus. J Agric Food Chem 35 (6): 1027–30. Schieber, A., Berardini, N., and Carle, R. 2003. Identification of flavonol and xanthone glycosides from mango (Mangifera indica L, cv, “Tommy Atkins”) peels by high-performance liquid chromatography­electrospray ionization mass spectrometry. J Agric Food Chem 51 (17): 5006–11. Schieber, A., Stintzing, F. C., and Carle, R. 2001. By-products of plant food processing as a source of functional compounds—Recent developments. Trends Food Sci Tech 12 (11): 401–13. Sergeev, I. N., Ho, C. T., Li, S. M., Colby, J., and Dushenkov, S. 2007. Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells. Molecular Nutr Food Res 51 (12): 1478–84. Sergeev, I. N., Li, S. M., Colby, J., Ho, C. T., and Dushenkov, S. 2006. Polymethoxylated flavones induce Ca2 + -mediated apoptosis in breast cancer cells. Life Sciences 80 (3): 245–53. Setiawan, B., Sulaeman, A., Giraud, D., and Driskell, J. 2001. Carotenoid content of selected Indonesian fruits. J Food Comp Anal 14 (2): 169–76. Shui, G., Wong, S., and Leong, L. 2004. Characterization of antioxidants and change of antioxidant levels ­during storage of Manilkara zapota L. J Agric Food Chem 52 (26): 7834–41. Soares, N., and Hotchkiss, J. 1998. Bitterness reduction in grapefruit juice through active packaging. Packaging Techn Sci 11: 9–18. Stewart, I. 1977. Provitamin-A and carotenoid content of citrus juices. J Agric Food Chem 25 (5): 1132–7. Sudjaroen, Y., Haubner, R., Wurtele, G., Hull, W., Erben, G., Spiegelhalder, B., Changbumrung, S., Bartsch, H., and Owen, R. 2005. Isolation and structure elucidation of phenolic antioxidants from Tamarind (Tamarindus indica L,) seeds and pericarp. Food Chem Tox 43 (11): 1673–82. Sugiura, M., Kato, M., Matsumoto, H., Nagao, A., and Yano, M. 2002. Serum concentration of beta­cryptoxanthin in Japan reflects the frequency of Satsuma mandarin (Citrus unshiu Marc) consumption. J Health Sci 48 (4): 350–3. Sun, J., Chu, Y. F., Wu, X. Z., and Liu, R. H. 2002. Antioxidant and anti proliferative activities of common fruits. J Agric Food Chem 50 (25): 7449–54. Sundram, K., Sambanthamurthi, R., and Tan, Y. 2003. Palm fruit chemistry and nutrition. Asia Pacific J Clinical Nutr 12 (3): 355–62. Tharanathan, R., Yashoda, H., and Prabha, T. 2006. Mango (Mangifera indica L.) “The king of fruits”—An overview. Food Reviews Intern 22 (2): 95–123. Toledo, F., Arancibia-Avila, P., Park, Y.-S., Jung, S.-T., Kang, S.-G., Heo, B., Drzewiecki, J., et  al. 2008. Screening of the antioxidant and nutritional properties, phenolic contents and proteins of five durian cultivars. International J Food Sci Nutr 59 (5): 415–27. Vanamala, J., Cobb, G., Turner, N. D., Lupton, J. R., Yoo, K. S., Pike, L. M., and Patil, B. S. 2005. Bioactive compounds of grapefruit (Citrus paradisi Cv Rio red) respond differently to postharvest irradiation, ­storage, and freeze drying. J Agric Food Chem 53 (10): 3980–5.

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Vanamala, J., Leonardi, T., Patil, B. S., Taddeo, S. S., Murphy, M. E., Pike, L. M., Chapkin, R., Lupton, J., and Turner, N. 2006a. Suppression of colon carcinogenesis by bioactive compounds in grapefruit. Carcinogenesis 27 (6): 1257–65. Vanamala, J., Reddivari, L., Yoo, K. S., Pike, L. M., and Patil, B. S. 2006b. Variation in the content of bioactive flavonoids in different brands of orange and grapefruit juices. J Food Comp Anal 19 (2–3): 157–66. Vasco, C., Ruales, J., and Kamal-Eldin, A. 2008. Total phenolic compounds and antioxidant capacities of major fruits from Ecuador. Food Chem 111 (4): 816–23. Vendramini, A., and Trugo, L. 2004. Phenolic compounds in acerola fruit (Malpighia punicifolia, L.). J Brazilian Chem Soc 15 (5): 664–8. Vera, E., Ruales, J., Dornier, M., Sandeaux, J., Sandeaux, R., and Pourcelly, G. 2003a. Deacidification of ­clarified passion fruit juice using different configurations of electrodialysis. J Chem Techn Biotech 78 (8): 918–25. Vera, E., Ruales, J., Dornier, M., Sandeaux, J., Persin, F., Pourcelly, G., Vaillant F., and Reynes, M. 2003b. Comparison of different methods for deacidification of clarified passion fruit juice. J Food Eng 59 (4): 361–7. Vera, E., Sandeaux, J., Persin, F., Pourcelly, G., Dornier, M., and Ruales, J. 2007. Deacidification of clarified tropical fruit juices by electrodialysis, Part I, Influence of operating conditions on the process performances. J Food Eng 78 (4): 1427–38. Wang, Y. C., Chuang, Y. C., and Hsu, H. W. 2008. The flavonoid, carotenoid and pectin content in peels of citrus cultivated in Taiwan. Food Chem 106 (1): 277–84. Wang, Y. C., Chuang, Y. C., and Ku, Y. H. 2007. Quantitation of bioactive compounds in citrus fruits cultivated in Taiwan. Food Chem 102 (4): 1163–71. Whitman, S. C., Kurowska, E. M., Manthey, J. A., and Daugherty, A. 2005. Nobiletin, a citrus flavonoid ­isolated from tangerines, selectively inhibits class A scavenger receptor-mediated metabolism of acetylated LDL by mouse macrophages. Atherosclerosis 178 (1): 25–32. Wilberg, V., and Rodriguez-Amaya, D. 1995. HPLC quantitation of major carotenoids of fresh and processed guava, mango and papaya. Food Sci Techn-Lebensmittel-Wissenschaft Technologie 28 (5): 474–80. Wilcox, L. J., Borradaile, N., de Dreu, L., and Huff, M. 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. Xu, B., Yaun, S., and Chang, S. 2007. Comparative analysis of phenolic composition, antioxidant capacity, and color of cool season legumes and other selected food legumes. J. Food Sci. 72:S167–77. Xu, G. H., Chen, J. C., Liu, D. H., Zhang, Y. H., Jiang, P., and Ye, X. Q. 2008a. Minerals, phenolic compounds, and antioxidant capacity of citrus peel extract by hot water. J Food Sci 73 (1): C11–18. Xu, G. H., Liu, D. H., Chen, J. C., Ye, X. Q., Ma, Y. Q., and Shi, J. 2008b. Juice components and antioxidant capacity of citrus varieties cultivated in China. Food Chem 106 (2): 545–51. Xu, G. H., Ye, X. Q., Liu, D. H., Ma, Y. Q., and Chen, J. C. 2008c. Composition and distribution of phenolic acids in Ponkan (Citrus poonensis Hort ex Tanaka) and Huyou (Citrus paradisi Macf Changshanhuyou) during maturity. J Food Comp Anal 21 (5): 382–9. Xu, J. G., Go, M. L., and Lim, L. Y. 2003. Modulation of digoxin transport across Caco-2 cell monolayers by citrus fruit juices: Lime, lemon, grapefruit, and pummelo. Pharmaceutical Res 20 (2): 169–76. Yano, M., Kato, M., Ikoma, Y., Kawasaki, A., Fukazawa, Y., Sugiura, M., Matsumoto, H., Oohara, Y., Nagao, A., and Ogawa, K. 2005. Quantitation of carotenoids in raw and processed fruits in Japan. Food Sci Tech Res 11 (1): 13–8. Yu, J., Wang, L. M., Walzem, R. L., Miller, E. G., Pike, L. M., and Patil, B. S. 2005. Antioxidant activity of citrus limonoids, flavonoids, and coumarins. J Agric Food Chem 53 (6): 2009–14. Yu, L., Zhao, M., Yang, B., Zhao, Q., and Jiang, Y. 2007. Phenolics from hull of Garcinia mangostana fruit and their antioxidant activities. Food Chem 104 (1): 176–81. Yusof, S., Ghazali, H., and King, G. 1990. Naringin content in local citrus-fruits. Food Chem. 37:113–21.

7 Phytochemical Bioactives in Berries Özlem Tokus¸ og˘lu and Gary Stoner Contents Introduction to Berry Fruits.....................................................................................................................143 Phytochemical Bioactives in Berry Fruits.............................................................................................. 144 Blackberry.......................................................................................................................................... 144 Blueberry............................................................................................................................................145 Raspberry............................................................................................................................................147 Red Raspberry................................................................................................................................147 Black Raspberry.............................................................................................................................152 Strawberry...........................................................................................................................................153 Bayberry............................................................................................................................................. 154 Chokeberry......................................................................................................................................... 156 Currant............................................................................................................................................... 157 Cranberry........................................................................................................................................... 158 Elderberry.......................................................................................................................................... 160 Gooseberry..........................................................................................................................................161 References................................................................................................................................................162

Introduction to Berry Fruits Berry fruits, commonly called aggregate fruits, have clusters of one-seeded drupelets, each cluster of drupelets developing from a single flower. The drupelets are typically eaten as a cluster and not individually (Rieger 2006b). The origin of berries is very complicated and there are numerous cultivated varieties that have been developed through the centuries. Figure  7.1 shows major bioactives in berry fruits. Additionally, specific bioactives can be found in specific berries. Genotype-variety is the major factor in determining fruit nutritional quality, but it is also affected by crop conditions (environmental and cultivation techniques), ripening season, preharvest and postharvest conditions, shelf-life, and processing (Wang, 2007; Connor et al. 2002; Prior et al. 1998; Proteggente et al. 2002; Wang, Cao, and Prior 1996). A multitude of phenolic compounds have been detected in berries (Hertog et al. 1992b; Justesen et  al. 1998; Schuster and Herrmann 1985; Wildanger and Herrmann 1973), their content being highly variable in different berries. Recent studies have shown that extracts of berries, in particular strawberries and berries of the genus Vaccinium, have antioxidative (Costantino et al. 1992; Kähkönen et al. 2001; Kalt et al. 1999; Prior et al. 1998; Wang et al. 1996) and anticarcinogenic (Stoner et al. 2010a; Bomser et al. 1996) effects in vitro, which are partly thought to be due to phenolic compounds.

143

144

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Major bioactives in berry fruits Other phytochemicals

Phenolics

Phenolic acids

Flavonoids

Ellagic acid Flavan-3ols Anthocyanidins Caffeic acid Flavonols (+)-Catechin & anthocyanins p-coumaric acid Quercetin (–)-Epicatechin Ferulic acid Cyanidin Rutin Gallic acid Procyanidins Peonidin Myricetin p-hydroxybenzoic acid Pelargonidin Procyanidin B1 Kaempferol Cinnamic acid Procyanidin B2 Kaempferidin & their & their esters & their dimers & their glucosides Salicylic acid rutinosides rutinosides glucosides galactosides xylosides acylated derivatives

Carotenoids Lutein Zeaxanthine β-and α−Carotene β-Cryptoxanthin Vitamins Ascorbic acid (vitamin C) Organic acids Citric acid Malic acid Fiber

Figure 7.1  Major bioactives in berry fruits.

Phytochemical Bioactives in Berry Fruits Blackberry Blackberries (Rubus fruticosus sp.) are a species of fruit belonging to the subgenus Eubatus in the genus Rubus and are very complex in terms of genetic background, growth characteristics, and number of species (Figure 7.2). It has been shown that blackberries contain higher amounts of anthocyanins and other antioxidants than other fruits (Halvorsen et al. 2006; Cho et al. 2005; Moyer et al. 2002; Pantelidis et al. 2007). The blackberry fruits can be used in the industry for ice cream, juice, jam, marmalade, cake, and so on (Türemiş et al. 2003). Blackberry extracts have been shown to have various bioactivities including protection against endothelial dysfunction and vascular failure in vitro (Serraino et al. 2003), attenuating the injury caused by LPS-induced endotoxic shock in rats (Sautebin et al. 2004), and exhibiting cytotoxic effects on human oral, prostate (Seeram et al. 2006), and lung (Feng et al. 2004) cancer cells. In a previous report, it was shown that an anthocyanin-containing extract (ACE) from Hull cultivar grown in Kentucky inhibited HT-29 colon cancer cell growth and reduced lipid A-induced interleukin-12 release from murine dendritic cells (Dai et al. 2007). Seven wild and 10 cultivated blackberries (Arapaho, Bartin, Black Satin, Bursa I, Bursa II, Cherokee, Chester, Jumbo, Navaho, and Ness) were analyzed for total anthocyanins, total phenolics, and antioxidant activity as ferric reducing antioxidant power (FRAP). The respective ranges of total anthocyanin and total phenolic contents of blackberries were 0.95–1.97 and 1.73–3.79 mg/g, respectively (Koca and Karadeniz 2009). In the study of Koca and Karadeniz (2009), FRAP values of blackberries varied from 35.05 to 70.41 mmol/g. Kafkas et al. (2006) reported the ascorbic acid (vitamin C) levels of blackberry cultivars: C. Thornless, Bursa II, and Loch Ness as 2.5 ± 0.3 mg/g extract, 4.6 ± 0.4 mg/g extract, and 14.9 ± 2.7 mg/g extract, respectively (Kafkas et al. 2006). Cyanidin-3-glucoside (cyn-3-glu) is the major anthocyanin in blackberries (Cho et al. 2004; FanChiang & Wrolstad 2005; Koca and Karadeniz 2009; Wada and Ou, 2002). Cyn-3-glu ranged from 77 to 90% of total anthocyanidins and the other minor anthocyanins that were identified included cyanidin3,5-diglucoside (cyn-3,5-di glu), peonidin-3-glucoside (peo-3-glu), pelargonidin-3-glucoside (plg-3-glu), and cyanidin-3-rutinoside (cyn-3-rut; Koca and Karadeniz 2009). Dai et al. (2009) reported the anthocyanin contents in puree and powder from selected U.S. blackberry cultivars (Table 7.1). It is clear that blackberries are good sources of anthocyanins.

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Phytochemical Bioactives in Berries

Figure 7.2  Blackberry (Yalova).

Table 7.1 Total Anthocyanin and Total Phenolics in Selected Blackberry Cultivars Raw Material Puree

Powder

Cultivar

Total Anthocyaninb (mg/g)

Total Phenolicsa

Hull 2005∂ Hull 2006 Chester 2006 Black Satin 2006 Hull 2005 Chester 2006 Black Satin 2006

5.34 ± 0.19 7.54 ± 0.14 7.95 ± 0.15 7.16 ± 0.22 4.51 ± 0.50 7.88 ± 0.23 7.67 ± 0.51

24.07 ± 2.70 25.78 ± 0.56 25.31 ± 1.80 22.91 ± 1.24 12.00 ± 0.77 14.61 ± 1.48 14.81 ± 1.59

Source: Adapted from Dai, J., Gupte, A., Gates, L., and Mumper, R. J., Food Chem. Toxicol., 47, 837–47, 2009. a The extraction was repeated at least two times. All assays were carried out in triplicate. b Total anthocyanins are expressed as cyanidin 3-glucoside equivalent. c Total phenolics are expressed as gallic acid equivalent.

Blueberry The blueberry fruit belongs to the family Ericaceae and the genus Vaccinium (V). The family includes both “highbush” (V. corymborsum and V. ashei) (Figure 7.3) and “lowbush” (native American: V. augustifolium) blueberries. All blueberries originated from wild berries. Highbush blueberries represent 57% of the total North American blueberry production (USHBC 2009). Blueberries have great health benefits due to their high antioxidant properties that are attributed to bioactive compounds such as the anthocyanins, phenolic acids, flavonols, flavan-3-ols, and ascorbic acid (Faria et al. 2005; Cho et al. 2005; Gosch, 2003; Kähkäonen et al. 2003; Kalt, 2006; Prior et al. 1998; Taruscio et al. 2004). The Vaccinium family of blueberries includes more than 450 plants. The plant family grows wild around the world and there are many names given to different blueberries. Different varieties of blueberries are found in the United States: (a) V. corymbosum (Northern highbush), which grows in the

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Figure 7.3  (See color insert) Blueberry (highbush blueberry [V. corymbosum]) in the United States.

Figure 7.4  (See color insert) Blueberry (V. myrtillus) in Eastern Black Sea Region, Turkey.

forests of North America and, along with V. Ashei, are used for cultivation of the modern highbush and cultivated blueberry industry; (b) V. ashei (called Southern rabbiteye), which thrive in the southern United States; (c) V. Angustifolium (lowbush or wild blueberries), which are very cold hardy and reach a height of only 1 or 2 feet. These berries grow in the wild from the Arctic to Minnesota and in the mountains of New York, New Hampshire, and Maine; and (d) V. Myrtilloides, a sour-tasting velvet-leaf blueberry found in the wilds of New England and the western United States (USHBC 2009). Another variety of blueberries (V. myrtillus) commonly called by several different names are “Yaban Mersini,” “Çalı Çiçeği,” “Mavi Çilek,” “Mavi Yemiş” that in Turkey grows naturally in the Eastern Black Sea region (Giresun, Ordu region) (Figure 7.4) and in Western Turkey (Uludağ region). Traditional names for this berry are “likapa” in Rize, “kaskanaka” in Rize-Pazar, “çera” or “çela” in Ardeşen, “ligarba,” “lifos,” or “trabzon” in Trabzon, “morsivit” or “mahabak” in Artvin, or “bear berry” (ayı üzümü), tea berry (çay üzümü), or “bucolie berry” (çoban üzümü) in the Black Sea Region (Çelik 2005; ISHS 2009).

Blueberry Bioactives The blueberry contains a group of phytochemicals that have been implicated as mediators of cardiovascular protection. Recently, blueberries have been shown to prevent bone loss in an ovariectomized rat model of postmenopausal osteoporosis (Devareddya et al. 2008).

Phytochemical Bioactives in Berries

147

The phytochemicals present in blueberries include high quantities of flavonoid anthocyanins, p­ redominately in their glycosylated and acylated forms; flavonols such as quercetin, kaempferol, and myricetin; flavan-3-ols such as ( + ) catechin, (–) epicatechin, and their oligomeric forms; and proanthocyanidins and phenolic acids such as gallic, p-hydroxybenzoic, chlorogenic, p-coumaric, caffeic, ferulic, ellagic, benzoic, and cinnamic acids. The content and profiles of phenolic bioactives in blueberries vary widely, based on heredity, the geographic region where the berries are grown, and the environmental conditions during the growing season and during the process of fruit maturation and ripening (Giovanelli et al. 2009; Taruscio et al. 2004; Kalt et al. 2003, 2001a, 2001b; Sellappan et al. 2002; Prior et al. 2001, 1998; Hakkinen and Torronen 2000; Hakkinen et al. 1999a; Kader et al. 1996; Kalt and McDonald 1996; Gao and Mazza 1994; Stohr and Hermann 1975). The published data on blueberry composition usually refers to the cultivated highbush varieties such as Vaccinium corymbosum; there are relatively few reports concerning the composition of lowbush varieties, although they generally contain higher amounts of total phenolics and anthocyanins than the highbush varieties (Sinelli et al. 2008; Lee et al. 2004a, 2004b; Moyer et al. 2002; Kalt et al. 2001a; Prior et al. 1998). Blueberry breeding includes investigating the germplasm of wild species to identify phenolic-rich species from which to breed cultivars with enhanced levels of bioactive components such as the anthocyanins (Scalzo et al. 2005).

Anthocyanins and Total Phenolics in Blueberry According to data given by Ehlenfeldt and Prior (2001), total phenolic and total anthocyanin contents were evaluated in fruit tissues of 87 highbush blueberry (Vaccinium corymbosum L.) and speciesintrogressed, highbush blueberry cultivars. Average values for phenolic acids and anthocyanins in the berries were 1.79 mg/g (gallic acid equivalents) and 0.95 mg/g (cyanidin-3-glucoside equivalents), respectively. Kalt et al. (1999) reported that total phenolic and total anthocyanin values were 27.7 and 4.35 µmol g–1 for the lowbush blueberry and 22.7 and 2.67 µmol g–1 for the highbush blueberry, respectively. Zheng and Wang (2003) found that the total anthocyanin and phenolic contents in blueberries were 1.20 and 4.12 mg g–1, respectively. Based on Table 7.2, 1435.2–8227.2 mg kg–1 of total anthocyanins are in various blueberry fruits including A-98, Bluecrop, Ozarkblue, US-497, US-720 (Cho et al. 2004). It is stated that individual anthocyanin levels of US-497 is higher than the other genotypes. Blueberries are among the fruits that are best recognized for their anthocyanin and flavonoid content and for their potential health benefits. Marinova and Ribarova (2007) reported the presence of carotenoids in blueberries (Vaccinium myrtillus L.) obtained from Bulgarian markets. However, the highest levels of zeaxanthin (29 mg/100 g), beta-cryptoxanthin (30.1 mg/100 g), and beta-carotene (101.4 mg/100 g) carotenoids were found in blackberries in this study.

Raspberry The raspberry is the edible aggregate fruit of a multitude of plant species in the genus Rubus and the subgenus Idaeobatus. The name originally referred to the European species Rubus idaeus (with red fruit), which is still used as its standard English name (IF 2009; Figures 7.5 through 7.7). Raspberries are members of the Rosaceae family, are grown as perennial crops, and are made of many drupelets and a hollow center where the fruit detaches from the receptacle. Raspberries are soft and juicy with a distinct aroma and are a good source of natural antioxidants including anthocyanin, phenolic acids, and other flavonoids (Mullen et al. 2002a). Additionally, raspberries have high levels of vitamins and minerals (Heinonen et al. 1998; Wang and Lin, 2000)

Red Raspberry The European subspecies of red raspberry (Rubus idaeus L.) is designated R. idaeus subsp. vulgatus Arrhen, whereas the North American subspecies is termed R. idaeus subsp. strigosus Michx., or more

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 7.2 Antocyanin Content of Blueberry Genotypes (as mg kg–1 Fresh Weight) ∗ Genotype Compound Delphinidin 3-galactoside Delphinidin 3-glucoside Cyanidin 3-galactoside Delphinidin 3-arabinoside Cyanidin 3-glucoside Petunidin 3-galactoside Cyanidin 3-arabinoside Petunidin 3-glucoside Peonidin 3-galactoside Petunidin 3-arabinoside Malvidin 3-galactoside Malvidin 3-glucoside Peonidin 3-arabinoside Malvidin 3-arabinoside Delphinidin 3-acetylglucoside Petunidin 3-acetylglucoside Malvidin 3-acetylglucoside Unknown acylated anthocy.b Total anthocyaninsc

Bluecrop

Ozarkblue

US-497

US-720

730.5 ± 56.1d 14.3 ± 1.2 155.1 ± 8.8 362.8 ± 40.8 7.1 ± 0.8 544.5 ± 48.5 64.2 ± 5.0 14.3 ± 1.0 58.7± 3.6 243.5 ± 23.5 715.6 ± 49.2 43.8 ± 2.4 68.2 ± 9.6 352.1 ± 25.6 68.7 ± 8.6

188.5 ± 22.1 126.4 ± 15.2 35.6 ± 3.4 163.1 ± 19.7 20.2 ± 2.0 113.3 ± 14.5 26.1 ± 2.6 111.4 ± 14.2 13.8 ± 0.9 81.9 ± 12.1 159.8 ± 25.0 153.3 ± 22.0 6.4 ± 0.5 144.0 ± 22.0 27.9 ± 4.1

389.8 ± 41.2 8.1 ± 0.8 112.7 ± 12.3 185.8 ± 17.1 4.0 ± 0.4 228.6 ± 23.8 46.7 ± 4.7 7.5 ± 0.9 18.4 ± 2.1 113.7 ± 10.2 195.7 ± 20.9 18.7 ± 2.3 2.4 ± 0.8 105.2 ± 9.5 5.3 ± 1.2

1519.0 ± 92.6 50.4 ± 4.2 762.5 ± 57.0 659.8 ± 49.4 38.0 ± 1.6 1133.2 ± 109.6 349.4 ± 29.2 57.4 ± 4.2 335.8 ± 22.4 365.9 ± 52.6 1792.6 ± 83.8 195.9 ± 20.9 34.8 ± 2.5 718.6 ± 66.7 44.2 ± 3.8

973.1 ± 88.2 28.2 ± 3.8 319.8 ± 33.3 540.0 ± 76.6 13.0 ± 1.4 766.4 ± 85.7 137.5 ± 18.8 33.6 ± 4.0 62.4 ± 6.8 330.1 ± 40.5 681.3 ± 80.3 67.0 ± 6.8 ND 357.0 ± 41.8 6.4 ± 0.3

190.9 ± 20.2

15.8 ± 3.3

ND

114.9 ± 9.9

ND

17.4 ± 1.5

36.1 ± 4.6

ND

10.0 ± 0.5

ND

39.5 ± 4.7 3691.2b

11.6 ± 2.2 1435.2c

1.3 ± 0.1 1443.9c

44.9 ± 4.2 8227.3a

3.6 ± 0.3 4319.4b

A-98

a

Note: ND, not detected * All data was adapted from Cho, M. J., Howard, L. R., Prior, R. L., and Clark, J. R., J. Sci. Food Agric., 84, 1771–82, 2004. a Breeding selection not available for sale or present in commerce at the time of writing. b Data expressed as delphinidin 3-monoglucoside equivalents. c Values with similar letters are not significantly different (LSD, p > 0.05). d Standard deviation (n = 3).

Figure 7.5  (See color insert) Black raspberry in the United States. (From Oregon Berry Packing Company, Hillsboro, Oregon, USA, 2010. With permission.)

Phytochemical Bioactives in Berries

149

Figure 7.6  (See color insert) Red raspberry in Turkey, Blacksea Region. (Agaclar.net, Turkey, Jelsoft enterprises Ltd.)

Figure 7.7  (See color insert) Stockholm originated yellow raspberry (allgold) in Turkey. (Adapted from Agaclar.net, Turkey, Jelsoft enterprises Ltd.)

simply R. idaeus (European) and R. strigosus (North American), whereas black raspberry (R. ­occidentalis L.) is fairly straight-forward, being a good species on its own. Its range overlaps that of R. strigosus, but extends further to the south (Rieger 2006a). Red raspberries are the most important commercial raspberries. They are produced in 37 countries worldwide on about 184,000 acres (FAOSTAT 2007). The five countries with the highest production (percentage of world raspberry production) include Russia (24%), Serbia and Montenegro (23%), the United States (13%), Poland (11%), and Germany (7%; FAOSTAT 2007). In Turkey, the raspberry known as R. idaeus is called “ahududu” or “framboise.” For the fresh market, red raspberries are best harvested when bright red. They can be stored at 0°C for only a few days due to the breakdown of berry components such as vitamin C. About 42% of the total crop of red raspberries are quick frozen for the frozen market, 35% are converted to juice, and 25% are freshly consumed (Akdag 2008). The red raspberry has high free radical scavenging capacity owing to its numerous bioactive compounds with potential health benefits. In this context it is an economically important berry fruit (De Ancos et al. 2000a).

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Anthocyanins in Red Raspberries Red raspberries (R. idaeus L.) have a high free radical scavenging capacity and are rich in both vitamin C and total phenolics (De Ancos, et al. 2000a). They contain a distinct spectrum of 11 different anthocyanins, the most abundant being cyanidin-3-sophoroside, cyanidin-3-(2G-glucosylrutinoside), and cyanidin-3-glucoside (Mullen et al. 2002a). De Ancos et al. (1999) reported the content of anthocyanins in four Spanish raspberry cultivars; Autumn Bliss, Heritage, Rubi, and Ceva and the highest levels (expressed as cyanidin-3-glucoside) were found in the late cultivars, Rubi (96.08 mg/100 g fresh weight or f.w.) and Ceva (122.88 mg/100 g f.w.). These investigators also measured vitamin C in these cultivars and found the highest amount (31.14 mg/100 g f.w.) in Spanish Rubi. In the same study, the organic acids were found to be the fruit constituents responsible for color quality. Citric acid was the main nonvolatile organic acid (90%) in all raspberry cultivars and the Rubi cultivar had the highest total level of nonvolatile organic acids (2003 mg/100 g f.w.). Hunter color CIE values showed that Rubi was the reddest raspberry cultivar.

Factors Influencing Levels of Anthocyanins in Red Raspberries The levels of anthocyanins in red raspberries are influenced by the genotype, environmental conditions in which the berries are grown, ripeness at the time of harvest and storage conditions after harvest. The anthocyanin content depends on the genotype such that late cultivars appear to have a higher content of anthocyanins than the early ones (De Ancos et al. 1999). Anttonen and Karjalaine (2005) reported that the level of anthocyanins and other phenolics varied widely and significantly between raspberry cultivars grown in northern European conditions (i.e., in Finland). The total anthocyanin content varied from close to 0 (yellow cultivars) to 51 mg/100 g f.w. (cv Gatineau) whereas the content of total phenolics varied from 192 (cv Gatineau) to 359 mg/100 g f.w. (cv Ville). Full mature raspberries (100%) have higher total anthocyanin contents when compared with less ripe (50% maturity) berries, and this higher degree of ripeness is associated with their increased antioxidant activity (Wang et al. 2009). Wang et al. (2009) also reported that raspberries harvested at greener stages (5% and 20% ripeness) also consistently showed higher antioxidant activities and total phenolics than those harvested at 50%. Kalt et al. reported that temperature above 0°C have been shown to increase the anthocyanin contents of red raspberries on storage (Kalt et al. 1999). Freezing increased the total anthocyanin content in some raspberry cultivars, but decreased it in others (De Ancos et al. 2000b).

Ellagitannins in Raspberries Daniel et al. (1989) found that ellagitannins occur in high concentrations in both red and black raspberries. These berries contain about three times more ellagic acid than walnuts and pecans. Red raspberries contain two ellagitannins (sanguiin H-6 and lambertianin C) in significant quantities with sanguiin H-6 being the most abundant (Mullen et al. 2002a). Figures 7.8 and 7.9 illustrate the structures of Lambertianin C and Sanguiin H-6, respectively. The ellagitannins contribute significantly to the antioxidant activity and vasodilation properties of raspberries. It is stated that Sanguiin H-6 was a major contributor to the antioxidant capacity of raspberries together with vitamin C and the anthocyanin compounds (Mullen et al. 2002a). The ellagitannins in berries are hydrolyzed to ellagic acid (Figure 7.10), a bioactive compound that has been reported to have antiviral activity (Corthout, et al. 1991) and provide protection against cancers of the colon, lung, esophagus, and skin (Stoner et al. 2007). As with other polyphenolic antioxidants, ellagic acid has a chemoprotective effect in cellular models by reducing oxidative stress, and has antioxidant and antiproliferative properties in a number of in vitro and small-animal models (Ross et al. 2007; Seeram et al. 2005; Vattem and Shetty, 2005). The anti-initiation properties of ellagic acid have the ability to directly inhibit the DNA binding of certain carcinogens, including nitrosamines and polycyclic aromatic hydrocarbons (PAHs) (Stoner et al. 2010b; Mandal and Stoner 1990; Teel et al. 1986).

Factors Influencing Ellagitannin Levels in Raspberries Anttonen and Karjalaine (2005) reported that the ellagitannin levels in raspberries varied with the cultivar. Ellagic acid content varied from 38 (cv Gatineau and cv Nova) to 118 mg/100 g f.w. (cv Ville) in

151

Phytochemical Bioactives in Berries OH HO

HO

HO O C C O O O O

HO

OH

HO

HO O C C O O O O

HO HO HO

O O

O C

OH

O

O

C O

OH

OH OH

O

O

O

O C

O C

O

OH

OH

O

OH

C OH

C O

OH HO

C O

O C

OH HO

OH OH

OH

OH HO

OH

O HO

O

OH HO

C

HO HO

O

O

HO

HO HO

O C C O O O O

HO

OH

OH OH

OH

OH

Figure 7.8  Lambertianin C in raspberries. (Adapted from Beattie, J., Crozier, A., and Duthie, G. G., Curr. Nutr. Food Sci., 1:71–86, 2005.)

OH HO O C

HO

OH

C O O O O

HO

HO O C

HO

C O O

O O

O

O

HO OH

OH

O

C

C

O

O

HO

HO

OH

O

O

O

O

C

C

O

OH

C

OH OH

O

OH HO

C

O

OH HO

OH HO

OH

OH

HO HO

OH HO

OH

Figure 7.9  Sanguiin H-6 in raspberries. (Adapted from Beattie, J., Crozier, A., and Duthie, G. G., Curr. Nutr. Food Sci., 1:71–86, 2005.) OH O

O

OH

O

HO

O

OH Figure 7.10  Ellagic acid in raspberries. (Adapted from Beattie, J., Crozier, A., and Duthie, G. G., Curr. Nutr. Food Sci., 1:71–86, 2005.)

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Finland raspberry cultivars. In addition, processing red raspberries by freezing and storing them frozen decreased the total level of ellagic acid and its derivatives (De Ancos et al. 2000a; Zafrilla et al. 2001). The effect of storing the berries frozen on ellagic acid content appears to vary with different cultivars (De Ancos et al. 2000a).

Flavonols in Red Raspberry Mullen et al. (2002a) identified the three quercetin conjugates (quercetin-3-rutinoside, quercetin-3­glucoside, and quercetin-3-glucuronide) and also kaempferol glucuronide in red raspberry. The levels of kaempferol are lower than that of quercetin (Mullen et al. 2002b).

Factors Influencing Flavonol Levels in Raspberries Anttonen and Karjalaine (2005) reported that flavonol levels varied significantly between Finland ­raspberry cultivars. The quercetin level ranged from 0.32 (yellow cultivar) to 1.55 mg/100 g f.w. (cv Balder). Flavonol compounds are quite stable when berries are heated during jam processing (Zafrilla et al. 2001). Flavonols can decrease significantly when berries are stored at 20°C over a period of 6 months. However, Häkkinen et al. (2000) reported no loss in quercetin levels when raspberries were stored frozen for 9 months. Anttonen and Karjalaine (2005) also found that the environment in which raspberries are grown has a considerable effect on the quercetin concentration. In this context, breeding material should be evaluated for its content of bioactives in different regions to identify those areas that provide the highest levels of bioactives.

Black Raspberry Black raspberries have been investigated extensively for their ability to prevent cancer in rodents and, potentially, in humans (see reviews by Stoner 2009; Stoner et al. 2007, 2008a,b). Freeze-dried black raspberry powder has been shown to prevent chemically induced cancer in the rodent oral cavity, esophagus, and colon when provided in a synthetic diet at concentrations of 5 and 10%. In addition, the oral consumption of black raspberry powder (20 g/3 × /day) in a slurry of water for an average of 3 weeks led to a reduction in the growth rate and an increase in the apoptotic rate of colon cancer cells in cancer patients. In patients with a hereditable disease termed familial adenomatous polyposis (FAP), the oral consumption of black raspberry powder (20g/3 × /day) coupled with the intrarectal administration of black raspberry suppositories (1.4 g/day) led to a 36% median regression rate of rectal polyps. Mechanistic studies have shown that the berries positively modulate the expression levels of genes associated with proliferation, apoptosis, inflammation, angiogenesis, cell cycling and adhesion, differentiation, and multiple metabolic processes (Stoner et al. 2008; Wang et al. 2008, 2009; Huang 2006). Black raspberries exhibit high antioxidant activity due, in part, to their high levels of anthocyanins, ellagitannins, and other phenols (Stoner et al. 2009; Tian et al. 2006; Kresty et al. 2001). The four major anthocyanins in black raspberries are: cyanidin-3-glucoside, cyanidin-3-rutinoside, cyanidin-3xylosylrutinoside, and cyanidin-3-­sambubioside (Tulio et al. 2008; Tian et al. 2005). Biofractionation studies provide evidence that the anthocyanins in the alcohol/water soluble fraction of black raspberries are responsible for an appreciable amount of their chemopreventive activity in vitro (Hecht et al. 2006) and in vivo in rat esophagus (Wang et al. 2009). However, the alcohol/water insoluble fraction is also chemopreventive in rat esophagus and studies are underway to identify the active components in this fraction. Pharmacokinetic studies in rodents and in humans indicate that the anthocyanins and ellagic acid in black raspberries are poorly absorbed into the blood (Stoner et al. 2005). Black raspberries also contain numerous other components with chemopreventive potential including vitamins A, C, E, and folic acid; calcium, selenium, and zinc; ellagic, ferulic, p-coumaric, and chlorogenic acids; and quercetin and phytosterols such as ß-sitosterol and stigmasterol (Kresty et al. 2001 and Stoner et al. 2009).

Phytochemical Bioactives in Berries

153

Except for red and black raspberries, the yellow variety tastes quite spectacular. It has a normal raspberry taste but much sweeter than normal red varieties. The yellow variety is available in December, and its breeding may be carried out. Figure 7.7 shows Stockholm originated yellow raspberries (allgold) in Turkey.

Strawberry The cultivated strawberry, Fragaria X ananassa Duch., is a member of the family Rosaceae, subfamily Rosoideae, along with blackberries and raspberries (Rieger 2009). Strawberries are produced in 73 countries worldwide on about 529,000 acres (FAOSTAT 2007). The top 10 producing countries (as a percentage of world strawberry production) include the United States (27%), Spain (9%), Japan (7%), Korea (7%), Mexico (5%), Italy (5%), Russia (5%), Turkey (5%), Poland (4%), and Germany (3%; FAOSTAT 2007). Strawberries (Fragaria x ananassa Duch.; Figure 7.11) are very popular among the available berry types and are widely consumed as fresh fruit and as preserves, jams, yogurts, and ice creams. In California, the major cultivars of strawberries are Camarosa, Diamante, Aromas, and Selva; and in Florida they are Camarosa, Selva, Oso Grande, Sweet Charlie, and Rosa Linda. Strawberry fruits have antioxidant, anticancer, anti-inflammatory and antineurodegenerative biological properties (Hannum 2004). Specific antioxidants present in strawberries include quercetin, kaempferol, chlorogenic acid, p-coumaric acid, ellagic acid, and vitamin C (Shin et al. 2007; Olsson et al. 2004; Maas et al. 1991a). Studies in our laboratory (Stoner 2009) indicate that freeze-dried strawberries are nearly as effective as freeze-dried black raspberries in preventing chemically induced cancer in the rodent esophagus (Carlton, et al. 2001).

Ellagic Acid in Strawberry Häkkinen et al. (1999a) reported that ellagic acid is the main phenolic compound in strawberry fruit (51% of the phenolic compounds analyzed; Figures 7.12 and 7.13). It is present in strawberries, and in other berry types, in the form of ellagitannins, which release the ellagic acid upon hydrolysis. The ellagic acid content of different cultivars of strawberries can vary widely, ranging from 19.9 to 522 µg/g f.w. (Gil et al. 1997; Häkkinen & Törrönen 2000). Häkkinen et al. (2000) found that total ellagic acid content ranged from 40 to 52 mg/100 g f.w. in six strawberry cultivars from Finland. Da Silva Pinto et al. (2008) reported that the total ellagic content among fully ripened strawberry fruits of the cultivars Dover, Camp Dover, Camarosa, Sweet Charlie, Toyonoka, Oso Grande, and Piedade, grown in Sao Paulo, Brazil varied significantly from 17 to 47 mg/100 g f.w. Finally, the total ellagic acid content in nine cultivars: Pocahontas, Dorit (216), Douglas, Seascape, Selva, Camarosa, Oso Grande, Red Chief,

Figure 7.11  Strawberry in the United States. (FDA, Food and Drug Administration, USA.)

154

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

C

HO

C O

HO

O

O

O

O OH

OH

O

O

C

O OH

O

OH OH

HO HO

OH

OH

OH

Figure 7.12  Ellagitannin in strawberry.

O HO

O OH

HO O

OH O

Figure 7.13  Ellagic acid in Strawberry.

and Honeoye grown in Turkey ranged from 58 to 66 mg/100 g f.w. (Tokusoglu and Yemis 2010). Each cultivar of strawberry has a distinct chemical composition, and is bred for improved quality including high phenolic content and antioxidant capacity. The major anthocyanins in strawberries are pelargonidin-3-glucoside and cyanidin-3-glucoside (which are responsible for the red color of the berries) with lesser quantities of pelargonidin-3-rutinoside and pelargonidin 3-glucoside-succinate (Kahkonen et al. 2001, Wang et al. 2002). The total anthocyanin concentrations in ripe strawberries are significantly higher than in nonripe (green) berries, but the concentrations of total phenolics are higher in green berries than in ripe berries (Wang and Lin 2000). The total antioxidant activity of strawberries increases with the degree of ripeness, presumably due to the increased levels of anthocyanins.

Bayberry Bayberry (Myrica rubra Sieb. et Zucc.) belongs to the family Myricaceae, and is widespread in tropical, subtropical, and temperate regions of the world (Chen et al. 2004; Kuang et al. 1979; Figure 7.14.) Most cultivars of bayberry fruits ripen in the hot and rainy seasons of June or July and can be kept fresh with an attractive dark red color for 3 days at 20–22°C and for 9–12 days at 0–2°C (Xi and Zheng 1993). Besides being consumed fresh, bayberry fruits are often processed into juice, juice concentrate or wine, and consumed as jam and canned fruit; the pomace of the berry has also been evaluated as a value-added food product (Chen et al. 2004; Fang et al. 2007). Three bayberry cultivars, Xiangshan, Biqi, and Dongkui, were evaluated for content of phenolic acids using a hyphenated technique of HPLC coupled with photodiode array and electrospray ionization mass spectrometry (HPLCDAD-ESIMS) by Fang et al. (2007; Table  7.3). The berries were found to contain gallic acid, protocatechuic acid, quercetin 3-glycoside, and seven flavonol glycosides including

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Phytochemical Bioactives in Berries

Figure 7.14  Bayberry.

Table 7.3 Phenolics in Bayberry Fruits Bayberry Cultivars Phenolic Compounds Phenolic acids Gallic acid Protocatechuic acid Total phenolic acids Flavonol glycosides Myricetin hexoside Myricetin dehexoside Myricetin dehexoside-gallate Total Quercetin 3-glucoside Quercetin hexoside-gallate Quercetin deoxyhexoside Total Kaempferol hexoside Total flavonol glycosides Total phenolicsa Total phenolicsb

Xiangshan

Biqi

2.6 2.3 4.9

7.0 3.1 10.1

9.6 55.3 6.2 71.1 15.3 4.3 15.5 35.1 4.6 110.8 115.7 4460.9

ND 33.4 1.9 35.3 44.2 4.1 69.5 117.8 3.7 156.8 166.9 3602.6

Dongkui

2.7 ND 2.7

ND 51.1 2.1 53.2 5.3 ND 42.7 48.0 3.1 104.3 107.0 3821.8

Source: Fang, Z., Zhang, M., and Wang, L., Food Chem., 100, 845–52, 2007. Note: All flavonol glycosides are calculated as quercetin 3-glucoside. ND = Not detected. a Sum of the individual phenolic compounds in ethyl acetate extracts. b Extracted in acid acetonitrile, estimated by the Folin-Ciocalteau method and expressed as mg GAE/ Kg FW.

two myricetin hexoside and two myricetin deoxyhexoside derivatives, quercetin hexoside and quercetin deoxyhexoside derivatives, and a kaempferol hexoside derivative. Gallic acid (2.6–7.0 mg/kg f.w.) was the major phenolic acid in all analyzed cultivars. Myricetin glycosides (71.1 mg/kg f.w.) were the major flavonol glycosides in the Xiangshan cultivar and quercetin glycosides (117.8 mg/kg f.w.) were the major ones in the Biqi cultivar.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Figure 7.15  Black chokeberry. (USDA, Washington, DC, 2007.)

Fang et al. (2007) reported that the content of total flavonol glycosides in the three bayberry cultivars was 104.3–156.8 mg/kg f.w., and that of the total phenolics ranged from 3602.6 mg GAE/ Kg f.w. to 4460.9 mg GAE/ Kg f.w. (Table 7.3). Zhou et al. (2009) examined five Chinese cultivars of bayberry (i.e., Myrica rubra, Biqi, Wandao, Dongkui, Dingao, and Zaodamei) for content of anthocyanins and phenolic acids. The main anthocyanin was cyanidin-3-O-glucoside (3073.3–6219.2 mg/kg dry weight [DW]) and the main flavonol was quercetin-3-O-glucoside (296.2–907.9 mg/kg dry weight). Quercetin and myricetin were also found in bayberry pomaces, and both quercetin and myricetin deoxyhexosides were tentatively identified in pomace. The dominant phenolic acids were gallic acid (102.9–241.7 mg/kg dry weight) and protocatechuic acid (29.5–57.2 mg/kg dry weight).

Chokeberry Chokeberries, also called aronia berries (Aronia melanocarpa), are native to eastern North America (Figure 7.15). They belong to the Rosaceae family (Jeppsson 1999). Chokeberries (kuş kirazı) are eaten by birds, which then disperse the seeds in their droppings. The raw fruits of chokeberries are inedible, owing to their astringent taste, but they are widely used in the food industry (e.g., in juice, soft drinks, wine, jam, syrup, food coloring, natural health products, and as tea and tinctures; Anon 2009; Bermúdez-Soto and Tomás-Barberán 2004). The chokeberries are often mistakenly called chokecherries, which is the common name for Prunus virginiana (Miller and Miller 1999). Although chokeberries originate from North America, they are extensively cultivated in Denmark, Eastern Europe, Russia, and Turkey. Chokeberries are rich in polyphenols, mostly anthocyanins and procyanidins (Bermúdez-Soto and Tomás-Barberán 2004; Wu et al. 2004). Extracts of chokeberry exhibit antiproliferative and anticarcinogenic effects on human colon cancer cells (Bermúdez-Soto et al. 2007; Malik et al. 2003; Zhao et al. 2004). Bermúdez-Soto et al. (2007) reported that repetitive exposure (2 hours a day for a 4-day period) of human colon cancer cells to chokeberry (Aronia melanocarpa) juice containing a mixture of polyphenols resulted in an antiproliferative effect as well as increased apoptosis (Bermúdez-Soto et al. 2007). Zapolska-Downar et al. 2006 found that coincubation with an extract from chokeberry in a concentration of 10, 50, and 100 mg/ml decreased oxLDL-induced both early (anexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose dependent manner. In a concentration of 100 mg/ml extract from the chokeberry reduced early and late stages of apoptosis induced by oxLDL. They

Phytochemical Bioactives in Berries

157

stated that the inhibition of apoptosis by the extract might preserve the function of the human endothelium and contribute to the prevention of atherosclerosis (Zapolska-Downar et al. 2006). The contents of flavonones in chokeberries have been investigated. The flavonone, eriodictyol 7-O-βglucuronide, together with five quercetin derivatives; 3-O-(6″-O-β-arabinosyl-β-glucoside), 3-O-(6″-αrhamnosyl-β-galactoside), 3-O-(6″-α-rhamnosyl-β-glucoside), 3-O-β-galactoside, and 3-O-β-glucoside have been detected in the fruit and flower umbels of chokeberries (Slimestad et al. 2005). Black chokeberries were found to contain > 71 mg flavonols per 100 g fresh weight. The dark color of chokeberries is due to their content of four anthocyanins: cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-O-arabinoside, and cyanidin-3-O-xyloside (Oszmiánski and Sapis 1988; Strigl et al. 1995). An 80% ethanol extract of chokeberry (Aronia Melanocarpa) contained 403.4 mg/g anthocyanins including 39.1% monomers (21.7% cyanidin 3-0 galactoside, 1.2% cyanidin 3-0 galucoside, 15% cyanidin 3-0-arabinoside, 1.2% cyanidin 3-0-xyloside) and 60.9% polymers (Oszmiánski and Sapis 1988). Recently, black chokeberries have been evaluated for potential use as a food colorant (Bridle and Timberlake 1997).

Currant Red, pink, and white currants belong to three European species (Ribes rubrum, R. Petraeum and R. ­sativum). Black currants (cassissier, gadellier noir, and groseillier noir are French; Schwarze Johannisbeere is German; kuş üzümü is Turkish) are related to European (Ribes nigrum) and Asian (Ribes ussuriense) species of ribes berry (Meades 2009; Figure 7.16). Blackcurrant (Ribes nigrum) berries are healthy for humans owing to their high levels of antioxidants; they are widely cultivated for use in beverages (Costantino et al. 1993). Black currants are rich in phenolics, notably anthocyanins and hydroxycinnamic acids (Häkkinen and Auriola 1998; Koeppen and Herrmann 1977). The black coloration or very dark red coloration of the berries has been attributed to their very high level of anthocyanins. The water-soluble anthocyanins in black currants are found mainly in the fruit skin, and the total level of anthocyanins is at least 2000 mg/kg (as f.w.; Demina 1974; Koeppen and Herrmann 1977). Currant seeds contain the anthocyanins, delphinidin, and cyanidin rutinosides and glucosides (Lu and Foo 2003). They also contain flavonols and derivatives (myricetin, quercetin rutinosides and glucosides, kaempferol glucosides, dihydroquercetin, aureusidin (Mikkonen et al. 2001) Figure 7.17.) It is reported that different maceration treatments influence enhancement of anthocyanins and other phenolics in black currant juice (Landbo et al. 2004).

Figure 7.16  (See color insert) Red currant (left) and black currant (right).

158

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

R HO

OH

OH

+ O

OH O OH

OH O

HO

O

O

OH

OH

HO

O CH

OH OH

Oglc OH

OH OG

OG OH O

HO

O

HO OH

OH

OH

O

O

Figure 7.17  Anthocyanins and flavonols in blackcurrant fruits and seeds. (Lu, Y. and Foo, L. Y., Food Chem., 80:71–6, 2003. With permission from Elsevier.)

Figure 7.18  (See color insert) Cranberries (Kızılcık-Yalova Şenköy).

Cranberry Cranberries (Vaccinium macrocarpon) belong to the family Ericaceae, and are small, dark red fruits from a shrub that is native to eastern North America (Winston et al. 2002) (Figure 7.18). Cranberries (kızılcık), widely consumed as juice and sauce, are a source of antioxidant flavonoids and phytochemicals. They have possible benefits to the cardiovascular and immune systems, as well as anticancer potential (Chen et al. 2001; Chen and Zuo 2007; Seifried et al. 2007; Zhang and Zuo, 2004;). Cranberries have been used in folk medicine since the 19th century (Thomos 1900). The American cranberry is a prominent food crop in Canada, Massachusetts, Wisconsin, Michigan, New Jersey, Oregon, and Washington, and the crop size is approximately 500 million pounds annually (NASS 2001). Cranberries are processed into three major product categories; fresh (5%); sauce products, concentrate, and various value-added applications (35%); and juice drinks (60%). Recent research indicates that cranberry juice inhibits chemically induced bladder cancer in rats (Prasain et al. 2008) and may inhibit the development of human breast cancer (Zuo et al. 2003). In vitro and ex vivo studies show that cranberry products prevent adhesion of bacteria to the cell walls of the urinary tract, therefore preventing urinary tract infections (Di Martino et al. 2006; Liu et al. 2006). Specifically, cranberry proanthocyanidins have been shown to inhibit the adherence of E. coli with P-type fimbriae to the wall of the urinary tract (Howell et al. 1998). Cranberry phenolics also improve the human lipoprotein profile (Ruel et al. 2006) and protect LDLs from oxidative injury (Wilson et al. 1998).

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Phytochemical Bioactives in Berries

Peptic ulcers—open sores in the lining of the stomach, esophagus, or duodenum (the first part of the intestine—are common. Helicobacter pylori (H. pylori), a type of bacteria, is responsible for most ulcers. This organism weakens the protective coating of the stomach and first part of the intestine and allows damaging digestive juices to eat away at the sensitive lining below. Foods containing flavonoids like cranberries (including cranberry juice) may inhibit the growth of H. pylori. The studies concluded that regular consumption of cranberry and cranberry juice can suppress Helicobacter pylori infection in endemically afflicted populations and may reduce the risk of stomach and duodenum ulcers (Burger et al. 2002; CI 2009; Lin et al. 2005; Zhang et al. 2005). Cranberry consumption may also have anti-inflammatory effects similar to aspirin (Duthie et al. 2005). Table 7.4 shows the profile and content of phenolics in cranberries. Singh et al. (2009) compared a cranberry crude extract and its fractions with known antioxidants (ascorbic acid, propyl gallate, and quercetin) for their relative ability to scavenge DPPH radicals. It is shown that cranberry extracts are strong antioxidants. The results of this study are shown in Table 7.5.

Table 7.4 The Phenolic Profiles of Cranberries (as µg/g dry wt)a µg/g DWT

Cranberry Phenolics Proanthocyanidins Epicatechin (monomers) Dimers (epicatechin-(4β→8, 2β→O→7)-epicatechin Trimers (epicatechin-(4β→8)-epicatechin-(4β→8, 2β→O→7)-epicatechin Flavonols Myricetin-3-β-galactoside Myricetin-3-α-arabinofuranoside Quercetin-3-β-galactoside Quercetin-3-glucoside Quercetin-3-rhamnoside

18.4 42.8 20.0

12.0 6.0 100 1.0 42.4 5.0

Q-3-O-(6″-p-benzoyl)-β-galactoside

Source: Adapted from Singh, A. P., Wilson, T., Kalk, A. J., Cheong, J., and Vorsa, N., Food Chem., 116, 963–8, 2009. a Based on comparisons with authentic standards and LC-MS mass fragmentation pattern.

Table 7.5 Percentage Scavenging Capacitiesa of Cranberry Crude Extracts and Various Low Molecular Weight Antioxidants Toward DPPH Radicala Concentration Treatment Ascorbic acid n-Propyl gallate Quercetin Crude extract

1 µM

2.5 µM

5 µM

10 µM

6.0 ± 0.2 14.1 ± 0.3 12.1 ± 0.2 14.5 ± 2.9

11.4 ± 1.2 30.3 ± 0.4 29.5 ± 0.4 29.9 ± 0.5

19.2 ± 2.9 54.4 ± 0.7 58.4 ± 1.2 53.5 ± 4.9

41.6 ± 3.8 90.5 ± 2.2 94.1 ± 0.2 90.4 ± 2.4

Source: Adapted from Singh, A. P., Wilson, T., Kalk, A. J., Cheong, J., and Vorsa, N., Food Chem., 116, 963–8, 2009. a Percent scavenging capacity = [1-((Abs of cranberry crude extract/fractions-Abs of blank)/ Abs control)] × 100; The final concentration of DPPH radical was 43 µM.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Elderberry Elderberry or elder (Sambucus sp.) is a genus of between 5 and 30 species of shrubs or small trees in the Moschatel family, native in temperate-to-subtropical regions of both the Northern and the Southern Hemispheres (USDA 2005; Figure 7.19). The European elder (Sambucus nigra) is found in the warmer parts of Europe and North America; it is also known as black elderberry or common elder. The American or “sweet” elder (Sambucus canadensis) is found in eastern North America. The Florida elder (Sambucus simpsonii) grows in the southeastern United States and is known as blue-black elderberry. Black-dark blue elderberries are most frequently used in recipes, retail extracts, syrups, for juice, juice concentrate, wine, and in the manufacture of jellies (Jensen et al. 2001; USDA 2005). Elderberry (S. nigru) anthocyanins are all based on cyanidin-3-sambubioside (β-D-xylose-(1-2)glucose) (Figure 7.20), cyanidin (3-glucoside, 3-sambubioside-5-glucoside and 3,5-diglucoside; Bronnum-Hansen and Hansen 1983). Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-­glucoside

Figure 7.19  Dark blue elderberry (Sambucus canadensis) (USDA, Germplasm Resource Information Network, United States Department of Agriculture, 2005–10–13, 2005.) OH OH Cl– O+

HO

O OH

O

OH

O O

HO HO OH

OH OH

Figure 7.20  Special anthocyanidin in elderberry; Cyanidin-3-sambubioside. (Polyphenols Laboratories As, Sandnes, Norway, 2010. With permission.)

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and other acylated pigments have been identified in other elderberry varieties (Johansen et al. 1991; Lamaison et al. 1979; Nakatani et al. 1995). Elderberry has been employed in herbal medicine for treating many illnesses ranging from asthma and colds to constipation and arthritis (Kilham 2001). In Turkey e­ lderberries, or mürver, are used as a natural medicine and are prepared with the elder extract and honey (Başer 2007). Cyanidin-3-sambubioside and cyanidin-3-glucoside together account for ∼85% of the anthocyanin content of the elderberry with the sambubioside being the most predominant (Goiffon et al. 1999). The extraction yields of anthocyanins ranged from 1.4 to 2.4 mg/g wet weight elderberry mash equivalent to a span of concentrations of anthocyanins in the juices of 2123–3273 mg/L (Landbo et al. 2007). Other acylated pigments have also been identified in other elderberry varieties (Johansen et al. 1991; Lamaison et al. 1979; Nakatani et al. 1995). Netzel et al. (2005) and Landbo et al. (2007) reported the yields of total phenols in elderberry and found that they ranged from ∼3.0 to 6.0 mg GAE/g wet weight, equivalent to phenolic levels in the juice of 5895–8215 mg GAE/L. Recently, the antioxidant effects of elderberry anthocyanins (and coexisting phenolics) were studied in humans (Netzel et al. 2005). The research indicated that both total phenolics and the antioxidant capacity of plasma significantly increased after 1 hour of ingesting 400 ml of elderberry juice.

Gooseberry Gooseberry (Ribes uva-crispa; syn. Ribes grossularia), native to Europe, northwestern Africa, and southwestern Asia, is a species of Ribes and its bushes produce an edible fruit (Figure 7.21). Gooseberry contains high levels of phenolic acids (Table 7.6; Russell et al. 2009). The p-coumaric acid, caffeic acid, protocatechuic acid, and p-benzoic acid are the major phenolic acids in gooseberry fruits locally produced in Scotland (Russell et al. 2009). Pantelidis et al. (2007) reported levels of solids, anthocyanins, and phenolics in gooseberry (Ribes grossularia) cultivars obtained from a commercial farm in Northern Greece. The soluble solid contents were 8.5 ± 1.4% and 8.5 ± 2.1%, respectively, for gooseberry varieties Whinham’s Industry (red) and White Smith (yellow). Anthocyanin levels varied from 2.4 to 43.3 mg/100g

Figure 7.21  Gooseberry (R. grossularia), (Bektaşi üzümü). (Adapted from Meyveler.gen.tr, Turkey.)

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 7.6 Phenolic Acids in Gooseberries Phenolic Acids

Gooseberry∗

Free Phenolic Acids Gallic Protocatechuic Gentisic Caffeic

1.27 ± 0.50 27.22 ± 2.13 2.01  ± 0.40 13.23 ± 1.45

Conjugated Phenolic Acids Gallic Protocatechuic p-Hydroxybenzoic Gentisic Caffeic Vanillic p-Coumaric Ferulic

43.59 ± 15.63 389.80 ± 100.52 120.75 ± 19.75 41.28 ± 6.30 354.51 ± 60.82 48.21 ± 6.07 504.81 ± 62.83 67.27 ± 5.37

Source: Russell, W. R., Labat, A., Scobbie, L., Duncan, G. J., and Duthie, G. G., Food Chem., 115, 100–4, 2009. With permission. Moisture (%) of gooseberry: 86.4. Conjugated phenolic acids are a summation of both the alkaliand acid-labile fractions. Values are specified on a dry weight basis in mg kg_1 and are given as mean ± standard deviations (n = 3).

f.w. (as cyanidin-3-glucoside equivalents) while total phenolics varied from 1257 to 1321 mg/100 g DW (as gallic acid equivalents). The ascorbic acid (vitamin C) content in gooseberries ranged from ­20.3 to 25.4 mg/100g f.w. (Pantelidis et al. 2007).

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NASS. 2001. National Agricultural Statistics Service. Agricultural Statistics Board. Cranberries. Annual Reports, Fr Nt 4, Washington, DC: USDA. Netzel, M., Strass, G., Herbst, M., Dietrich, H., Bitsch, R., Bitsch, I., and Frank, T. 2005. The excretion and biological antioxidant activity of elderberry antioxidants in healthy humans. Food Research International 38 (8–9): 905–10. Olsson, M. E., Ekvall, J., Gustavsson, K. E., et al. 2004. Antioxidants, low molecular weight carbohydrates, and total antioxidant capacity in strawberries: Effects of cultivar, ripening, and storage. Journal of Agricultural and Food Chemistry 52:2490–8. Oszmiánski, J., and Sapis, J. C. 1988. Anthocyanins in fruits of Aronia melanocarpa (Chokeberry). Journal of Food Science 53 (4): 1241–2. Pantelidis, G. E., Vasilakakis, M., Manganaris, G. A., and Diamantidis, G. 2007. Antioxidant capacity, phenol, anthocyanin and ascorbic acid contents in raspberries, blackberries, red currants, gooseberries and cornelian cherries. Food Chemistry 102:777–83. Prasain, J. K., Jones, K., Moore, R., Barnes, S., Leahy, M., Roderick, R., Juliana, M. M., and Grubbs, C. J. 2008. Effect of cranberry juice concentrate on chemically-induced urinary bladder cancers. Oncol Rep. 19 (6): 1565–70. Prior, R. L., Cao, G., Martin, A., Sofic, E., McEwen J., O’Brien, C., et al. 1998. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity and variety of Vaccinium species. Journal of Agricultural and Food Chemistry 46:2686–93. Prior, R. L., Cao, G., Martin, A., Sofic, E., McEwen, J., O’Brien, C., Lischner, N., et al. 1998. Antioxidant capacity as influenced by total phenolic and anthocyanin content, maturity, and variety of vaccinium species. Journal of Agricultural and Food Chemistry 46:2686–93. Prior, R. L., Lazarus, S. A., Cao, G., Muccitelli, H., and Hammerstone, J. F. 2001. Identification of procyanidins and anthocyanins in blueberries (Vaccinium spp.) using high-performance liquid chromatography/mass spectrometry. Journal of Agricultural and Food Chemistry 49:1270–6. Proteggente, A. R., Pannala, A. S., Pagana, G., Van Buren, L., Wagner, E., Wiswman, S., et al. 2002. The ­antioxidant activity of regular consumed fruit and vegetables reflects their phenolic and vitamin C composition. Free Radical Research 36:217–33. Rieger, M. 2006. Introduction to Fruit Crops. Binghamton, NY: The Haworth Press. Rieger, M. 2006a. Blackberries and Raspberries (Rubus spp.) http://www.uga.edu/fruit/ rubus.html. Retrieved March 2010. Ross, H. A., McDougall, G. J., and Stewart, D. 2007. Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts. Phytochemistry 68:218–28. Ruel, G., Pomerleau, S., Couture, P., Lemieux, S., Lamarche, B., and Couillard, C. 2006. Favourable impact of low-calorie cranberry juice consumption on plasma HDL cholesterol concentrations in men. British Journal of Nutrition 9:357–64. Russell, W. R., Labat, A., Scobbie, L., Duncan, G. J., and Duthie, G. G. 2009. Phenolic acid content of fruits commonly consumed and locally produced in Scotland. Food Chemistry 115:100–04. Sautebin, L., Rossi, A., Serraino, I., Dugo, P., Di Paola, R., Mondello, L., Genovese, T., Britti, D., Peli, A., Dugo, G., Caputi, A. P., and Cuzzocrea, S. 2004. Effect of anthocyanins contained in a blackberry extract on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat. Planta Med. 70: 745–52. Scalzo, J., Politi, A., Pellegrini, N., Mezzetti, B., and Battino, M. 2005. Plant genotype affects total antioxidant capacity and phenolic contents in fruit. Nutrition 21:207–13. Schuster, B., and Herrmann, K. 1985. Hydroxybenzoic and hydroxycinnamic acid derivatives in soft fruits. Phytochem. 24:2761–4. Seeram, N. P., Adams, L. S., Henning, S. M., Niu, Y., Zhang, Y., Nair, M., and Heber, D. 2005. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. Journal of Nutritional Biochemistry 16 (6): 360–7. Seeram, N. P., Adams, L. S., Zhang, Y., Lee, R., Sand, D., Scheuller, H. S., and Heber, D., 2006. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. Journal of Agricultural and Food Chemistry 54, 9329–39. Seifried, H. E., Anderson, D. E., Fisher, E. I., and Milner, J. A. 2007. A review of the interaction among dietary antioxidants and reactive oxygen species. Journal of Nutritional Biochemistry 18 (9): 567–79.

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Serraino, I., Dugo, L., Dugo, P., Mondello, L., Mazzon, E., Dugo, G., Caputi, A. P., and Cuzzocrea, S. 2003. Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure. Life Science 73(9):1097–114. Sellappan, S, Akoh, C. C., and Krewer, G. 2002. Phenolic compounds and antioxidant capacity of Georgiagrown blueberries and blackberries. Journal of Agricultural and Food Chemistry 50:2432–8. Shin, Y., Liu, R. H., Nock, J. F., Holliday, D., and Watkins, C. B. 2007. Temperature and relative humidity effects on quality, total ascorbic acid, phenolics and flavonoid concentrations, and antioxidant activity of strawberry. Postharvest Biology and Technology 45:349–57. Simirgiotis, M. J., Theoduloz, C., Caligari, P. D. S., and Schmeda-Hirschmann, G. 2009. Comparison of ­phenolic composition and antioxidant properties of two native Chilean and one domestic strawberry genotypes. Food Chemistry 113:377–85. Sinelli, N., Spinardi, A., Di Egidioa, V., Mignanib, I., and Casiraghia, E. 2008. Evaluation of quality and nutraceutical content of blueberries (Vaccinium corymbosum L.) by near and mid-infrared spectroscopy. Postharvest Biology and Technology 50:31–6. Singh, A. P., Wilson, T., Kalk, A. J., Cheong, J., and Vorsa, N. 2009. Isolation of specific cranberry flavonoids for biological activity assessment. Food Chemistry 116:963–68. Slimestad, R., Torskangerpoll, K., Nateland, H. S., Johannessen, T., and Giske, N. H. 2005. Flavonoids from black chokeberries, Aronia melanocarpa. Journal of Food Composition and Analysis 18:61–68. Stoner, G. D., Sardo, C., Apseloff, G., Mullet, D., Wargo, W., Pound, V., Singh, A., Sanders, J., Aziz, R., Casto, B., and Sun, X. L. 2005. Pharmacokinetics of anthocyanins and ellagic acid in healthy volunteers fed freeze-dried black raspberries daily for 7 days. Journal of Clinical Pharmacology 45:1153–64. Stoner, G. D., Zikri, N., Wang, L.-S., Chen, T., Hecht, S. S., Huang, C., Sardo, C., and Lechner, J. F. 2007. Cancer prevention with freeze-dried berries and berry components. Seminars in Cancer Biology 17:403–10. Stoner, G. D., Dombkowski, A. A., Reen, R. K., Cukovic, D., Salagrama, S., Wang, L.-S., and Lechner, J. F. 2008a. Carcinogen-altered genes in rat esophagus positively modulated to normal levels of expression by both phenethyl isothiocyanate and black raspberries. Cancer Research 68:6460–4. Stoner, G. D., Wang, L.-S., and Casto, B. C. 2008b. Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries. Carcinogenesis 29:1665–74. Stoner, G. D. 2009. Foodstuffs for cancer prevention: The preclinical and clinical development of berries. Cancer Prevention Research 2:187–92. Stoner, G. D., Wang, L. S., Sardo, C., Zikri, N., Hecht, S. S., and Mallery, S. R. 2010a. Cancer prevention with berries: Role of anthocyanins. In: Bioactive Compounds and Cancer. Eds. Milner, J. A., and Romagnolo, D. F. 2010, Humana Press; Totawa, New Jersey, 882p. Stoner, G. D., Wang, L. S., Seguin, C., Rocha, C., Stoner, K., Chiu, S., and Kinghorn, A. D. 2010b. Multiple berry types prevent N-nitrosomethylbenzylamine-induced esophageal cancer in rats. Pharm. Res. 27(6):1138–45. Stöhr, H., and Hermann, K. 1975. Phenolics in fruits. VI. Phenolics of currants, gooseberries and blueberries and the changes in phenolic acids and catechins during the development of blackcurrants. Zeitschrift fuer Lebensmittel-Untersuchung und-Forschung 159:31–7. Strigl, A. W., Leitner, E., and Pfannhauser, W. 1995. Qualitative and quantitative analysis of the anthocyanins in black chokeberries (Aronia melanocarpa Michx. Ell.) by TLC, HPLC and UV/VIS-spectrometry. Zeitschrift fuer Lebensmittel-Untersuchung und-Forschung 91:177–80. Taruscio, T. G., Barney, D. L., and Exon, J. 2004. Content and profile of flavanoid and phenolic acid compounds in conjunction with the antioxidant capacity for a variety of northwest Vaccinium berries. Journal of Agricultural and Food Chemistry 52:3169–76. Teel, W., Babcock, M. S., Dixit, R., and Stoner, G. D. 1986. Ellagic acid toxicity and interaction with benzo[a] pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells. Cell Biology and Toxicology 2:53–62. Thomos, J. D. 1900. Cranberry harvest: A history of cranberry growing in Massachusetts. New Bedford: Spinner Publication. Tian, Q., Aziz, R. M., Stoner, G. D., and Schwartz, S. J. 2005. Anthocyanin determination in black raspberry (Rubus occidentalis) and biological specimens using liquid chromatography-electrospray ionization tandem mass spectrometry. Journal of Food Science 70:C43–C47.

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8 Phenolic Bioactives in Grapes and Grape-Based Products Violeta Ivanova and Marina Stefova Contents Introduction..............................................................................................................................................171 Flavonoids................................................................................................................................................172 Anthocyanins......................................................................................................................................172 Flavan-3-ols........................................................................................................................................174 Flavonols and Dihydroflavonols.........................................................................................................177 Nonflavonoids..........................................................................................................................................177 Hydroxybenzoic Acids........................................................................................................................177 Hydroxycinnamic Acids and Derivatives............................................................................................178 Stilbenes..............................................................................................................................................178 Evolution of Polyphenols During Winemaking and Aging.....................................................................179 References................................................................................................................................................181

Introduction Polyphenols are a large and complex group of compounds responsible for the characteristics, quality, and color of grape and wine, especially for red ones. The polyphenolic composition has been extensively studied and a large family of structures have been identified in grapes and wine (Ribereau-Gayon 1965; Somers 1971; Nagel and Wulf 1979; Slinkard and Singleton 1984; Bourzeix et al. 1986; Cheynier and Rigaud 1986; Da Silva et al. 1991b; Prieur et al. 1994; Souquet et al. 1996) These compounds can affect the appearance, taste, mouth-feel, fragrance, and antimicrobial properties of wine. In addition, polyphenols were confirmed to be the key compounds responsible for the antioxidant potential of wine (Burns et al. 2000). Wines and grapes contain a number of polyphenolic constituents classified as flavonoids and nonflavonoids that contribute to wine sensory characteristics, especially to color, flavor, and astringency and therefore, to the differences between red and white wines. The family of wine flavonoids includes anthocyanins, flavan-3-ols, flavonols, and dihydroflavonols, whereas the nonflavonoids include phenolic acids (hydroxybenzoic and hydroxycinnamic acids (HCA) and their derivatives) and stilbenes. Red wines contain all the above phenolics, while white wines contain mainly phenolic acids and flavanols. In particular, flavan-3-ols (monomeric flavan-3-ols and proanthocyanidins) confer the astringency and structure to the wine (Sarni-Manchado et al. 1999) and anthocyanins, as red pigments, are responsible for the color of the wines (Wulf and Nagel 1978). The grape phenolic composition and content is affected by several factors such as grape variety, ripening stage, climate, soil, place of growing, and vine cultivation. In addition, wine-making practices (maceration time, temperature, intensity of pressing, yeast, SO2-doses) affect the extraction of phenolic compounds from grapes and also enological treatments and aging when subsequent reactions in wine occur to influence the wine phenolic composition. 171

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Flavonoids Flavonoids exist as free or polymerized with other flavonoids, sugars, and nonflavonoids, or in combination with nonflavonoids, such as HCA derivatives and occur esterified to sugars, organic acids, or various alcohols. The main structure of flavonoids that consists of two benzene rings (A and B) linked by an oxygen-containing pyrane ring condensed with the A ring, is presented in Figure 8.1. Flavonoids may be divided into the following groups.

Anthocyanins Anthocyanins are red compounds, responsible for the color of red grapes and wines. They are mainly located in the vacuoles of grape skins, with the exception of the teinturier varieties that contain anthocyanins in the pulp. Anthocyanins identified in grape skins and wines from Vitis vinifera L. are based on five main anthocyanidins—delphinidin, cyanidin, petunidin, peonidin, and malvidin. The most abundant anthocyanins are the 3-O-glucosides and among them, malvidin-3-glucoside is the major compound. Acylated monoglucoside anthocyanins: 3-O-acetylglucosides, 3-O-p-coumroyl­glucosides, and 3-Ocaffeoylglucosides have also been identified in grapes and wines. Acylation occurs in the C-6 position of the glucose molecule with acetic, p-coumaric, and caffeic acid (Ribereau-Gayon 1965; Somers 1971; Wulf and Nagel 1978; Baldi et al. 1995; Núñez et al. 2004;, Alcalde-Eon et al. 2006). The amount of acylated anthocyanins is largely influenced by the grape variety. The non-V. Vinifera grapes, such as V. labrusca, V. rotundifolia, and V. upestris and hybrid grape varieties contain anthocyanin 3,5-diglucosides, as well as 3-(p-coumaroylglucoside)-5-glucosides (Giusti et al. 1999; Favretto and Flamini 2000). The structure of antocyanins is presented in Figure 8.2. Enological practices affect the extraction of phenolic compounds from grapes and their subsequent reactions in wine. For red wine production, the grapes are crushed after the separation of the leaves and stalks and then maceration is applied on the pomace. Maceration means contact between the grape juice and pomace (hard parts of the grape skins and seeds) and its duration varies depending on the grape that is processed and type of wine that is produced. During the maceration period when the grape juice and pomace are in contact, anthocyanins and the other phenolic compounds are transferred from the solid parts of the grape to the must. After a few days of fermentation, anthocyanins reach a maximum level followed by a decrease of their concentration as a result of precipitation with tartaric salts in a form of colloidal material, adsorption on yeast cell walls, or elimination during filtration and fining. Anthocyanin pigments extracted from the grapes give the color of the young wines, but during the winemaking and aging of wine, anthocyanins may be modified to create stable, C4-substituted pigments through reactions involving pyruvic acid (Bakker et al. 1997; Fulcrand et al. 1998) and vinyl phenol derivatives (Fulcrand et al. 1996). A number of oligomeric pigments resulting from a condensation reaction involving acetaldehyde and from direct reactions of anthocyanins with flavanols have also been described (Somers 1971; Fulcrand et al. 1996; Berg and Akiyoshi 1975; Bakker and Timberlake 1986). Recently, the existence of anthocyanins acylated with lactic acid, formed in wine, has been reported (Alcalde-Eon et al. 2006). Despite all these changes, the wine anthocyanin profile in some studies has also been used as criteria for characterizing and classifying grape varieties in order to establish differences between them (González-San José et al. 1990; Arozarena et al. 2000). The color of anthocyanins is directly linked to the pH of the medium, as several studies have shown the presence of colored and uncolored forms of anthocyanins depending on the pH (Somers 1971; Brouillard and Dubois 1977; Brouillard et al. 1978). Flavylium cations are mainly present in highly acidic media, which lose their color as pH increases by formation of colorless carbinol bases as a result of addition of water and loss of proton. Carbinol bases are in equilibrium with the open ring chalcone yellow form. The equilibrium between Carbinol and chalcone forms is very slowly attained at room temperature in a slightly acidic media, while increased temperature displaces the equilibrium toward the chalcone forms. In neutral and alkaline medium, as a result of deprotonation (acid-base reaction), the flavylium is transformed into a purple quinoidal anhydrobase (Figure 8.3).

173

Phenolic Bioactives in Grapes and Grape-Based Products 3'

(a)

2' 8

1'

O1

7 A

C

5

4

6

2

4' B

5'

6'

3

(b)

O *

O *

O Flavanone

Flavan O

O Flavone

O

O

* *

OH

O Dihydroflavonol

O Flavonol

O * *

OH

Flavan-3-ol

O * * OH Flavan-4-ol Figure 8.1  (a) Flavonoid structure, (b) flavonoid groups.

O * *

OH

OH Flavan-3,4-diol

OH

174

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications R1 OH O+

HO

R2 HO O

OH O

OH

OH

OR3

Anthocyanins Delphinidin-3-glucoside Cyanidin-3-glucoside Petunidin-3-glucoside Peonidin-3-glucoside Malvidin-3-glucoside

R3:

CO CH3

CO CH CH

R1

R2

Mr

OH OH OCH3 OCH3 OCH3

OH H OH H OCH3

465 449 479 463 493

OH

OH

CO CH CH OH

-acetyl

-p-coumaroyl

-caffeoyl

Figure 8.2  Structures of anthocyanins. (Adapted from Giusti, M. M., Rodriguez-Saona, L. E., Griffin, D., and Wrolstad, R. E., J. Agric. Food Chem., 47, 4657–64, 1999; Adapted from Favretto, D., and Flamini, R., Am. J. Enol. Vitic., 51, 55–64, 2000.)

Anthocyanins can act as electrophiles in their flavylium form through the C-2 and C-4 positions (C-ring) accepting an electron pair in a chemical reaction. In addition, they can act as nucleophiles in hemiketal form through their C-6 and C-8 positions (A-ring) by donating electrons to the reaction partner, forming a chemical bond.

Flavan-3-ols Flavan-3-ols are a large family of polyphenolic compounds mainly responsible for the astringency, bitterness, and structure of the wine (Arnold et al. 1980). These compounds can be found as monomers, but also as oligomers and polymers. The major flavan-3-ol monomers in grapes are (+)-catechin and (–)-epicatechin, and, to a lesser extent, the gallic ester of (–)epicatechin and (–)-epicatechin-3-O-gallate (Su and Singleton 1969). In Vitis vinifera grapes, gallocatechin has been detected and also catechin-3-Ogallate and gallocatechin-3-O-gallate have been identified (Piretti et al. 1976; Lee and Jaworski 1987). The structure of flavan-3-ol monomers is presented in Figure 8.4. Flavanol oligomers and polymers are better known as condensed tannins or proanthocyanidins. The term tannin also refers to their capacity to interact or react with proteins and precipitate them out. When heated under acidic and oxidative conditions, these molecules release red anthocyanidin pigments by an acid-catalyzed cleavage (Bate-Smith 1954), hence the term proanthocyanidins is used. Grape and wine proanthocyanidins belong to two groups, procyanidins and prodelphinidins that release cyanidin and delphinidin, respectively. Procyanidins consist of catechin- and epicatechin-based polymers, and prodelphinidins contain gallocatechin and epigallocatechin units in addition to catechin and epicatechin (Czochanska et al. 1979; Porter et al. 1986). Proanthocyanidins composed of flavan-3 -ol constitutive units containing a single interflavan linkage and linked by C4-C8 and/or C4-C6 bonds are named B-type (Da Silva et al. 1991a) whereas, A-type proanthocyanidins contain double interflavan linkages, with the C2-O-C7 or C2-O-C5 bond in addition to the C4-C6 or C4-C8. Figure 8.5 shows the structure of procyanidin dimers identified in grapes and wines.

175

Phenolic Bioactives in Grapes and Grape-Based Products R1 OH O

HO

OH R2

Quinoidal anhydrobase (A)

OG1c OH Deprotoanation (–H+) R1 OH O+

HO

R2

Flavylium cation (AH+)

OG1c

Hydration reaction (+H2O/–H+)

OH

R1 OH O

HO

OH R2

R1 OH

OG1c OH

OHO

HO

Tautomeric reaction

R2

Chalcone (C)

OG1c OH Figure 8.3  Anthocyanin transformations depending on pH of the medium. (Adapted from Somers, T. C., Phytochemistry, 10, 2175–86, 1971; Adapted from Brouillard, R., and Dubois, J. E., J. Am. Chem. Soc., 99, 1359–64, 1977; Adapted from Brouillard, R., Delaporte, B., and Dubois, J. E., J. Am. Chem. Soc., 100, 6202–5, 1978.)

R OH OH

OH

O

OH

OH R2

OH

OH

R1

O –gallate

Flavan-3-ols (+)-Catechin (–)-Epicatechin (+)-Gallocatechin (–)-Epigallocatechin (–)-Epicatechin-3-gallate Figure 8.4  Structures of flavan-3-ol monomers.

R

R1

R2

Mr

H H OH OH H

OH H OH H H

H OH H OH OGallate

290 290 306 306 442

176

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

HO

2

OH R2

4 OH

OH O

HO

4 HO

8

OH

OH R2

OH

6

HO

R1

R1

R1

OH

O

O

R2

OH R2

HO

R1

OH

OH

C4-C8 bond

C4-C6 bond B-type procyanidins OH O

HO

OH R2 R1

OH

O

O OH HO

R1

R2

HO A-type procyanidin Figure 8.5  Structures of flavan-3-ol dimmers.

Determination of proanthocyanidins is generally based on acid-catalyzed depolymerization in the presence of strong nucleophilic reagent (Thompson et al. 1972; Foo et al. 1983) followed with formation of adducts, which are analyzed by reversed phase high-performance liquid chromatography (Shen et al. 1986; Rigaud et al. 1991; Koupai-Abyazani et al. 1992). In acidic media, proanthocyanidins are depolymerized, releasing terminal subunits, as flavan-3-ols and extensional subunits, as electrophilic flavan-3-ol intermediates (Figure 8.6). These electrophilic intermediates can be trapped by a nucleophilic reagent, followed by formation of adducts (Kennedy and Jones 2001). The most commonly used electrophilic reagents are phloroglucinol (Kennedy and Jones 2001) and benzylhydrosulfide (Remy et al. 2000). After the performed acid-catalyzed depolymerization and HPLC analysis, the proanthocyanidin composition, the mean degree of polymerization (mDP), and  the concentration of released units can be determined. According to literature data, the mDP in grape skins is around 30 and in seeds and stems around 10 (Souquet et al. 2000).

177

Phenolic Bioactives in Grapes and Grape-Based Products OH OH HO

O

R1

Extension (“upper”)

OR2

OH

OH R1 = H or OH

OH

HO

O

R1

R2 = OH or gallate

OR2

OH

OH

OH

Terminal (“bottom”) subunit

HO

O

R1 OR2

OH Figure 8.6  Basic structure of condensed tannins.

Flavonols and Dihydroflavonols Flavonols, which are located in epidermal and hypodermal grape skin vacuoles (together with the anthocyanins present in red grapes), absorb the UV radiation and play a protective role. In this regard, the flavonol biosynthesis is increased in the grapes highly exposed to daylight (Spayd et al. 2002). Flavonols present in red grape Vitis vinifera L. varieties, are mainly 3-glucosides of myricetin, quercetin, kaempferol, and isorhamnetin (Cheynier and Rigaud 1986; Figure 8.7). Recently, the methoxylated flavonols, laricitrin and syringetin, and their 3-glucoside forms have been identified (Wang et al. 2003; Amico et al. 2004; Castillo-Muñoz et al. 2007). The presence of dihydroflavonols astilbin (dihydroquercetin-3-O-rhamnoside) and engeletin (dihydrokaempferol-3-O-rhamnoside) has been confirmed in grape and wine (Trousdale and Singleton 1983). Another compound from this group, dihydromyricetin-3-O-rhamnoside, has been reported in wine by Vitrac et al. (2001).

Nonflavonoids The main nonflavonoid phenols in grape and wine that contain only one aromatic ring are derivatives of HCA and hydroxybenzoic acid. Stilbenes and stilbene glycosides are another class of nonflavonoids.

Hydroxybenzoic Acids The commonly present hydrozybenzoic acids are gallic acid, gentisic acid, p-hydroxybenzoic acid, protocatechuic acid, salicylic acid, syringic acid, and vanillic acid, which are mainly found in conjugated forms as esters and glycosides in grapes (Figure 8.8). The free forms of these compounds mainly

178

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications R1 OH HO

O

R2 OR3

OH Flavonols Myricetin Myricetin-3-glucoside Myricetin-3-glucuronide Quercetin Quercetin-3-glucoside Quercetin-3-glucuronide Kaempferol Laricitrin-3-glucoside Syringetin-3-glucoside Isorhamnetin Isorhamnetin-3-glucoside

O R1

R2

R3

Mr

OH OH OH OH OH OH H OCH3 OCH3 OCH3 OCH3

OH OH OH H H H H OH OCH3 H H

H glucoside glucuronide H glucoside glucuronide H glucoside glucoside H glucoside

318 480 494 302 464 478 286 494 508 316 498

Figure 8.7  Structures of flavonols.

prevail in the wine because of hydrolysis or heat breakdown reactions of the complex molecules, such as ­anthocyanins, that take place. Gallic acid is the dominant hydroxybenzoic acid in wine. It originates from the grapes and also can be formed by hydrolysis of hydrolyzable and condensed tannins (i.e., the gallic acid esters of flavan-3-ols).

Hydroxycinnamic Acids and Derivatives The main HCA present in the wine are caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid (Figure  8.9a). They can be present in cis- and trans- forms, but the trans forms are more stable and therefore more prevalent (Häkkinen et al. 1998). In wine, HCA are predominant in a form of esters of l-(+)-tartaric acid and among the derivatives of HCA, caffeoyltartaric (caftaric) acid, ­p -coumaroyltartaric (p-coutaric) acid, and feruloyltartaric (fertaric) acid are main compounds ­present in grape and wine (Figure 8.9b). Caftaric and coutaric acids are the most abundant in the wine, which are highly oxidizible components that cause the browning of white must (Cheynier et al. 1989, 1990). Another compound from the group of HCA, named 2-S-glutathionylcaffeoyltartaric acid (also, known as GRP or grape reaction product) has been identified. This compound is formed as a result of the nucleophilic addition of glutathione onto the caffeoyltartaric acid quinone that arises from enzymatic oxidation of coutaric and caftaric acid, catalyzed by polyphenoloxidases in musts (Singleton et al. 1985).

Stilbenes Stilbenes, located in grape skin and pips, are a subclass of phenolic compounds in grape and wine that can be biosynthesized by grapevines as a defense response to stress, such as microbial infection and UV

179

Phenolic Bioactives in Grapes and Grape-Based Products COOH

R1

R2 OH

Hydroxybenzoic Acids

R1

R2

Mr

Gallic acid p-Hydroxybenzoic acid Protocatechuic acid Syringic acid Vanillic acid

OH H OH OCH3 H

OH H H OCH3 OCH3

170 138 154 198 168

COOH R1

R2

Gentistic acid Salicylic acid

R1

R2

Mr

OH OH

OH H

154 138

Figure 8.8  Structures of hydroxybenzoic acids.

irradiation. During the winemaking process, they are transferred from the grapes into the wine in very low quantities (Sun et al. 2006; Amira-Guebailia et al. 2006; Viñas et al. 2008). One of the most relevant and extensively studied stilbenes is trans-resveratrol (3,5,4′-trihydroxystilbene). In nature, resveratrol exists in two isomeric forms (cis- and trans-configurations) in the free, as well as in ß-gluconjugated forms. The 3-O-ß-D-glucosides of cis- and trans-resveratrol are called piceids (Figure  8.10). The levels of resveratrol depend on the grape variety; in general, red wines contain a higher concentration of resveratrol than white wines (Romero-Pérez et al. 1996).

Evolution of Polyphenols During Winemaking and Aging For red wine production, studies are primary focused on the influence of maceration on the extraction of grape pigments and tannins. Anthocyanins are the first components to be extracted from the grape skins together with the skin tannins at the beginning of fermentation. According to the literature ­(Gil-Muñoz et al. 1997, 1999; Bautista-Ortín et al. 2004; Ivanova et al. 2009), anthocyanins reach the maximal values during the first days of vinification followed by decreasing till the end of the fermentation. Longer maceration time is very often accompanied by oxidative polymerization of monomeric anthocyanins and their complexation with other phenolics, whereupon oligomeric and polymeric pigments are formed that can precipitate and decrease the anthocyanin contents and the red color, enhancing the brown color of wine (Somers 1971). Tannin extraction from seeds, which is more dependent on ethanol content because of their lower solubility in water, starts toward the midpoint of alcoholic fermentation and continues until pressing during the postfermentation phase (Canals et al. 2005). In

180

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

O

OH

R1

R2 OH Hydroxycinnamic Acids Coumaric acid Caffeic acid Ferulic acid Sinapic acid (b)

R1

R2

Mr

H OH OCH3 OCH3

H H H OCH3

164 180 194 224

OH HOOC O

COOH O

R OH Derivatives of Hydroxycinnamic Acids Coutaric acid Caftaric acid Fertaric acid

R1

Mr

H OH OCH3

296 312 326

Figure 8.9  (a) Structures of hydroxycinnamic acids, (b) structures of derivatives of hydroxycinnamic acids. R

OH

HO OH

OH R 1-R: OH

2-R: OH

3-R: OGlc

4-R: OGlc

Figure 8.10  Structures of stilbenes: (1) trans-resveratrol, (2) cis- resveratrol, (3) trans-piceid, and (4) cis-piceid.

Phenolic Bioactives in Grapes and Grape-Based Products

181

fact, flavan-3-ols in seeds are protected with a lipidic layer, which is disrupted when appropriate content of alcohol is formed, allowing their extraction from the seeds. Therefore, extraction of seed tannins occurs at later phases of vinification, and increasing of their concentrations during a longer maceration time is expected. Sulphur dioxide is naturally present in wine, produced at concentrations up to 64 mg/L by the yeast metabolism (Larue et al. 1985), but most of the yeasts cannot produce more than 10 mg/L SO2, so that contents of SO2 higher than 30 mg/L usually are added during the vinification. The use of SO2 in winemaking is due to its ability of an effective antioxidant, preventing the activity of oxidases. Also, it has significant activity as antimicrobial agent, as well as a potential for bleaching the ­pigments and elimination of unpleasant odors (as a result of oxidation). SO2 can selectively act against the wild yeasts that come from the grape skin or equipment in the winery and can stop their activity, since yeasts are very sensitive to SO2 (also, to other stress factors). Sulphur dioxide can be added in the form of a salt, potassium metabisulphite (K 2S2O5), which can be ionized in acid media, releasing gaseous SO2. Higher doses of SO2 in the must can lead to faster and more efficient precipitation, especially SO2 in free form contributes to better extraction of phenolic components present in grape skins and seeds. The potential of yeast to affect the extraction of grape phenolic compounds has already attracted the scientist’s attention. The first study of the influence of yeast selection on the phenolic profile of Burgundy wine (Cuinier 1988) presented small effects on color intensity and total phenolics. Other studies focused on the effect of two yeast strains used for vinification of Pinot noir, as well as the Merlot wines, showed that no differences in total phenolics, anthocyanins, and color intensity were observed in the wines fermented with both yeasts (Gil-Muñoz et al. 1999; Girard et al. 2001). Recent studies showed that anthocyanins can be adsorbed at the yeast cell walls (Morata et al. 2003; Mazauric and Salmon 2005). During maturation—aging and storage of wines, colored and noncolored phenolics have an important role on the color and taste of wine and they undergo a number of reactions during aging that result in changes in the sensory characteristics. Those changes are mostly due to the conversion of grape anthocyanins to derived pigments (Somers 1971) through reactions of anthocyanins with pyruvic acid (carboxy-pyrano-anthocyanins) and acetaldehyde (pyrano-anthocyanins) leading to pyranoanthocyanin formation (Timberlake and Bridle 1976; Glories 1984; Bakker and Timberlake 1986; Fulcrand et al. 1996; Remy et al. 2000). Another possible mechanism of the evolution of phenolics during wine aging that takes place spontaneously is the formation of direct condensation adducts between anthocyanins and flavonols (Timberlake and Bridle 1976), as well as, formation of ethyl linked pigments formed by acetaldehyde-mediated condensation between anthocyanins and flavan-3-ols. All of these formed adducts contribute to the color stability of wine and decrease in astringency.

References Alcalde-Eon, C., Escribano-Bailón, M. T., Santos-Buelga, C., and Rivas-Gonzalo, J. C. 2006. Changes in the detailed pigment composition of red wine during maturity and ageing. A comprehensive study. Anal. Chim. Acta 563:238–54. Amico, V., Napoli, E. M., Renda, A., Ruberto, G., Spatafora, C., and Tringali, C. 2004. Constituents of grape pomace form the Sicilian cultivar “Nerello Mascalese.” Food Chem. 88:599–607. Amira-Guebailia, H., Chira, K., Richard, T., Mabrouk, T., Furiga, A., Vitrac, X., Monti, J. P., Delaunay, J. C., and Mérillon, J. M. 2006. Hopeaphenol: The first resveratrol tetramer in wines from North Africa. J. Agric. Food Chem. 54:9559–64. Arnold, R. A., Noble, A. C., and Singleton, V. L. 1980. Bitterness and astringency of phenolic fractions in wine. J. Agric. Food Chem. 28:675–8. Arozarena, I., Casp, A., Marín, R., and Navarro, M. 2000. Multivariate differentiation of Spanish red wines according to region and variety. J. Sci. Food Agric.70:1909–17. Bakker, J., Bridle, P., Honda, T., Kuwano, H., Saito, N., Terahara, N., and Timberlake, C. F. 1997. Identification of an anthocyanin occurring in some red wines. Phytochemistry 44:1375–82. Bakker, J., and Timberlake, C. F. 1986. The mechanism of color changes in aging port wine. Am. J. Enol. Vitic. 37:288–92.

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Baldi, A., Romani, A., Mulinacci, N., Vincieri, F. F., and Casetta, B. 1995. HPLC/MS application to anthocyanins of Vitis-vinifera L. J. Agric. Food Chem. 43:2104–9. Bate-Smith, E. C. 1954. Leuco-anthocyanins. 1. Detection and identification of anthocyanidins formed from leuco-anthocyanins in plant tissues. Biochem. J. 58:122–5. Bautista-Ortín, A. B., Fernádez-Fernádez, J. I., López-Roca, J. M., and Gómez-Plaza, E. 2004. Wine-making of high coloured wines: Extended pomace contact and run-off of juice prior to fermentation. Food Sci. Technol. Int. 10:287–95. Berg, H. W., and Akiyoshi, M. A. 1975. On the nature of reactions responsible for color behavior in red wine: A hypothesis. Am. J. Enol. Vitic. 26:134–43. Bourzeix, M., Weyland, D., and Heredia, N. 1986. Etude des catechines et des procyanidols de la grape de raisin, du vin et d’autres derives de la vigne. Bull OIV 59:1171–254. Brouillard, R., Delaporte, B., and Dubois, J. E. 1978. Chemistry of anthocyanin pigments. 3. Relaxation amplitudes in pH-jump experiments. J. Am. Chem. Soc. 100:6202–5. Brouillard, R., and Dubois, J. E. 1977. Mechanism of structural transformations of anthocyanins in acidic media. J. Am. Chem. Soc. 99:1359–64. Burns, J., Gardner, P. T., O’Neil, J., Crawford, S., Morecroft, I., Mc Phail, D. B., Lister, C., et al. 2000. Relationship among antioxidant activity, vasodilation capacity, and phenolic content of red wines. J. Agric. Food Chem. 48:220–30. Canals, R., Llaudy, M. C., Valls, J., and Canals, J. M. 2005. Influence of ethanol concentration on the extraction of color and phenolic compounds from the skin and seeds of Tempranillo grapes at different stages of ripening. J. Agric. Food Chem. 53:4019–25. Castillo-Muñoz, N., Gómez-Alonso, S., García-Romero, E., and Gutiérrez I. H. 2007. Flavonol profiles of Vitis vinifera red grapes and their single-cultivar wines. J. Agric. Food Chem. 55:992–1002. Cheynier, V., Basire, N., and Rigaud, J. 1989. Mechanism of trans-caffeoyltartaric acid and catechin oxidation in model solutions containing grape polyphenoloxidase. J. Agric. Food Chem. 37:1069–71. Cheynier, V., and Rigaud, J. 1986. HPLC separation and characterization of flavonols in the skins of Vitis vinifera var. Cinsault. Am. J. Enol. Vitic. 37:248–52. Cheynier, V., Rigaud, J., Souquet, J. M., Duprat, F., and Moutounet, M. 1990. Must browning in relation to the behavior of phenolic compounds during oxidation. Am. J. Enol. Vitic. 41:346–9. Cuinier, C. 1988. Influence des levures sur les composés phénoliques du vin. Bull. OIV 689–690:596–601. Czochanska, Z. Y., Newman, R. H., Porter, L. J., Thomas, W. A., and Jones, W. T. 1979. Direct proof of a homogeneous polyflavan-3-ol structure for polymeric proanthocyanidins. J. Chem. Soc. Chem. Commun. 8:375–7. Da Silva, J. M. R., Bourzeix, M., Cheynier, V., and Moutounet, M. 1991a. Procyanidin composition of Chardonnay, Mauzac and Grenache blanc grapes. Vitis 30:245–52. Da Silva, J. M. R., Rigaud, J., Cheynier, V., Cheminat, A., and Moutounet, M. 1991b. Procyanidin dimers and trimers from grape seeds. Phytochemistry 30:1259–64. Favretto, D., and Flamini, R. 2000. Application of electrospray ionization mass spectrometry to the study of grape anthocyanins. Am. J. Enol. Vitic. 51:55–64. Foo, L. Y., McGraw, G. W., and Hemingway, R. W. 1983. Condensed tannins: Preferential substitution at the interflavanoid bond by sulphite ion. J. Chem. Soc. Chem. Commun. 12:672–3. Fulcrand, H., Benabdeljalil, C., Rigaud, J., Cheynier, V., and Mountounet, M. 1998. A new class of wine ­pigments generated by reaction between pyruvic acid and grape anthocyanins. Phytochemistry 47:1401–7. Fulcrand, H., Doco, T., Es-Safi, N. E., Cheynier, V., and Moutounet, M. 1996. Study of the acetaldehyde induced polymerisation of flavan-3-ols by liquid chromatography-ion spray mass spectrometry. J. Chromatogr. A 752:85–91. Girard, B., Yuksel, D., Cliff, M. A., Delaquis, P., and Reynolds, A. G. 2001. Vinification effects on the sensory, colour, and GC profiles of Pinot noir wines from British Colombia. Food Res. Int. 34:483–99. Giusti, M. M., Rodriguez-Saona, L. E., Griffin, D., and Wrolstad, R. E. 1999. Electrospray and tandem mass spectroscopy as tools for anthocyanin characterization. J. Agric. Food Chem. 47:4657–64. Gil-Muñoz, R., Gómez-Plaza, E., Martínez, A., and López-Roca, J. M. 1997. Evolution of the CIELAB and other spectrophotometric parameters during wine fermentation. Influence of some pre and postfermentative factors. Food Res. Int. 30:699–705.

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Gil-Muñoz, R., Gómez-Plaza, E., Martínez, A., and López-Roca, J. M. 1999. Evolution of phenolic compounds during wine fermentation and post-fermentation: Influence of grape temperature. J. Food Compos. Anal. 12:259–72. Glories, Y. 1984. La couleur des vins rouges. Mesure, origine et interprétation. Partie I. Connaiss. Vigne Vin 18:195–217. González-San José, M. L., Barrón, L. J. R., and Díez, C. 1990. Evolution of anthocyanins during maturation of Tempranillo grape variety (Vitis vinifera) using polynomial regression models. J. Sci. Food Agric. 51:337–43. Häkkinen, H., Kärenlampi, S. O., Heinonen, I. M., Mykkänen, H. M., and Törroönen, A. R. 1998. HPLC method for screening of flavonoids and phenolic acids in berries. J. Sci. Food Agric. 77:543–51. Ivanova, V., Stefova, M., and Vojnoski, B. 2009. Assay of the phenolic profile of Merlot wines from Macedonia: Effect of maceration time, storage, SO2 and temperature of storage. Maced. J. Chem. Chem. Eng. 28:141–9. Kennedy, J. A., and Jones, G. P. 2001. Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J. Agric. Food Chem. 49:1740–6. Koupai-Abyazani, M. R., McCallum, J., and Bohm, B. A. 1992. Identification of the constituent flavonoid units in sainfoin proanthocyanidins by reversed-phase high-performance liquid chromatography. J. Chromatogr. 594:117–23. Larue, F., Park, M. K., and Caruana, C. 1985. Quelques observations sur les conditions de la formation d’anhydride sulfureux en vinification. Connaiss. Vigne Vin 19:241–8. Lee, C. Y., and Jaworski, A. W. 1987. Phenolic compounds in white grapes grown in New York. Am. J. Enol. Vitic. 38:277–81. Mazauric, J. P., and Salmon, J. M. 2005. Interactions between yeast lees and wine polyphenols during simulation of wine aging: I. Analysis of remnant polyphenolic compounds in the resulting wines. J. Agric. Food Chem. 53:5647–53. Morata, A., Gómez-Corovés, M. C., Suberviola, J., and Bartolomé, B. 2003. Adsorption of anthocyanins by yeast cell walls during the fermentation of red wines. J. Agric. Food Chem. 51:4084–8. Nagel, C. W., and Wulf, L. W. 1979. Changes in the anthocyanins, flavonoids and hydroxycinnamic acid esters during fermentation and aging of Merlot and Cabernet Sauvignon. Am. J. Enol. Vitic. 30:111–6. Núñez, V., Monagas, M., Gómez-Cordovés, C., and Bartolom, B. 2004. Vitis vinifera L. cv. Graciano grapes characterized by its anthocyanin profile. Postharvest Biol. Technol. 31:69–79. Piretti, M. V., Ghedini, M., and Serrazanetti, G. 1976. Isolation and identification of the polyphenolic and ­terpenoid constituents of Vitis vinifera. v. Trebbiano variety. Annali di Chimica 66:429–37. Porter, L. J., Hirtstich, L. N., and Chang, B. G. 1986. The conversion of procyanidins and prodelphinidins to cyanidins and delphinidins. Phytochemistry 25:223–30. Prieur, C., Rigaud, J., and Cheynier, V. 1994. Oligomeric and polymeric procyanidins from grape seeds. Phytochemistry 36:781–4. Remy, S., Fulcrand, H., Labarbe, B., Cheynier, V., and Moutounet, M. 2000. First confirmation in red wine of products resulting from direct anthocyanin-tannin reactions. J. Sci. Food Agric. 80:745–51. Ribereau-Gayon, P. 1965. La couleur des vins. Aliment Vie 53:232–48. Rigaud, J., Perezilzarbe, J., Da Silva, J. M. R., and Cheynier, V. 1991. Micro method for the identification of proanthocyanidin using thiolysis monitored by high-performance liquid-chromatography. J. Chromatogr. 540:401–5. Romero-Pérez, A. I., Lamuela-Raventós, R. M., Buxaderas, S., and de la Torre-Boronat, M. C. 1996. Resveratrol and piceid as varietal markers of white wines. J. Agric. Food Chem. 44:1975–8. Sarni-Manchado, P., Cheynier, V., and Moutounet, M. 1999. Intereaction of grape seed tannins with salivary proteins. J. Agric. Food Chem. 47:42–7. Shen, Z., Haslam, E., Falshaw, C. P., and Begley, M. J. 1986. Procyanidins and polyphenols of Larix gmelini bark. Phytochemistry 25:2629–35. Singleton, V. L., Salgues, M., Zaya, J., and Trousdale, E. 1985. Caftaric acid disappearance and conversion to products of enzymic oxidation in grape must and wine. Am. J. Enol. Vitic. 36:50–6. Somers, T. C. 1971. The polymeric nature of wine pigments. Phytochemistry 10:2175–86. Slinkard, K. W., and Singleton, V. L. 1984. Phenol content of grape skins and the loss of ability to make anthocyanins by mutation. Vitis 23:175–8.

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9 Nut Bioactives: Phytochemicals and LipidBased Components of Almonds, Hazelnuts, Peanuts, Pistachios, and Walnuts Biagio Fallico, Gabriele Ballistreri, Elena Arena, and Özlem Tokus¸ og˘lu Contents Introduction to Nuts and Nut Bioactives..................................................................................................185 Almond....................................................................................................................................................188 Phenolics.............................................................................................................................................188 Neutral Lipids.....................................................................................................................................189 Polar Lipids.........................................................................................................................................191 Phytosterols and Tocols......................................................................................................................191 Hazelnut.................................................................................................................................................. 192 Phenolics............................................................................................................................................ 192 Neutral Lipids.................................................................................................................................... 193 Polar Lipids........................................................................................................................................ 194 Phytosterols and Tocols..................................................................................................................... 195 Peanut...................................................................................................................................................... 195 Phenolics............................................................................................................................................ 196 Neutral Lipids.................................................................................................................................... 197 Polar Lipids........................................................................................................................................ 197 Phytosterols and Tocols..................................................................................................................... 197 Pistachio.................................................................................................................................................. 198 Phenolics............................................................................................................................................ 198 Neutral Lipids.................................................................................................................................... 199 Polar Lipids........................................................................................................................................ 201 Phytosterols and Tocols..................................................................................................................... 201 Chlorophylls and Xanthophylls......................................................................................................... 201 Walnut..................................................................................................................................................... 202 Phenolics............................................................................................................................................ 202 Neutral Lipids.................................................................................................................................... 202 Polar Lipids........................................................................................................................................ 203 Phytosterols and Tocols..................................................................................................................... 203 References............................................................................................................................................... 205

Introduction to Nuts and Nut Bioactives Tree nuts were long perceived as an unhealthy food due to their high fat content and caloric value. The energy content of nuts ranges from 23.7 to 29.3 kJ/g and the fat content is ~ 0.51–0.73 g/g. Nuts that are higher in energy are higher in fat. However, clinical trials and epidemiological studies have established that nuts contribute to improved health and well-being in humans (Willet 2001) (Figure 9.1). 185

186

Almond (Prinus dulcis)

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Hazelnut (Corylus avellana L.)

Peanut (Arachis hypogaea L.)

Pistachio (Pistacia vera L.)

Walnut (Juglans regia L.)

Figure 9.1  (See color insert) Almond, hazelnut, peanut, pistachio, and walnut.

Table 9.1 Fatty Acid Composition of Selected Nuts and Selected Oils as Percentage of Total Fat by Weighta Fatty Acid Total Fat

10:0

12:0

14:0

16:0

18:0

18:1

18:2

18:3

% of total fat by wt Nuts Almonds Hazelnuts Peanuts Pistachiosb Walnuts

52.2 62.6 49.2 50.0 56.6

0.0 0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0 0.0

0.6 0.2 0.1 0.0 0.0

6.6 5.0 10.5 9.9 3.7

1.9 2.0 2.2 2.1 2.5

63.7 77.7 48.1 69.6 21.0

20.1 9.3 31.6 15.4 59.2

0.7 0.2 0.0 0.5 5.8

Mean

54.1

—

—

0.2

7.1

2.1

56.0

31.7

1.4

Oils Olive oil Canola oil Safflower oil

100 100 100

0.0 0.0 0.0

0.0 0.0 0.0

0.0 0.0 0.0

11.0 4.0 4.8

2.2 1.8 1.3

72.5 56.1 75.3

7.9 20.3 14.2

0.6 9.3 0.0

Data reported by: Kris-Etherton, P., Yu-Poth, S., Sabate, J., Ratcliffe, H., Zhao, G., and Etherton, T., Amer. J. Clin. Nutr., 70, 504s–511s, 1999. b Arena, E., Campisi, S., Fallico, B., and Maccarone, E., Food Chem., 104, 403–8, 2007. a

Several large prospective studies have found an inverse relationship between nut consumption and cardiovascular disease (CVD) risk (Fraser et al. 1992; Fraser 1999; Hu et al. 1998; Ellswort et al. 2001; Albert et al. 2002). Regular consumption of nuts is also associated with favorable plasma lipid profiles, enhancement of immune function, reduced risk of cancer, stroke, type-2 diabetes, inflammation, and several other chronic diseases (Grunwald 1975; Phillips et al. 1999; Moreau et al. 2002). These beneficial effects of healthy diets containing specific tree nuts are attributed to their high levels of monounsaturated or polyunsaturated fats, known for their favorable effects on blood lipids and low levels of saturated fats. Compared with high-oleic-acid vegetable oils (olive, canola, and safflower), nuts have less saturated fatty acids (SFA) than olive oil and slightly more SFA than canola and safflower oils on average. The oleic-acid content of nuts is similar to that of canola oil, but less than that of olive oil and safflower oil. Canola oil and nuts contain similar amounts of linoleic acid, and these amounts are appreciably greater than those present in olive oil and safflower oil (Kris-Etherton et al. 1999). The fatty acid profiles of selected nuts and vegetable oils are shown in Table 9.1. Besides having a healthy lipid composition, nuts contain dietary fiber, plant proteins, folic acid, various micronutrients including manganese, copper, magnesium, phosphorus, zinc, and bioactive constituents (“phytochemicals”) that may confer additional beneficial effects (Kris-Etherton et al. 1999). They are also rich sources of several antioxidant substances such as antioxidant vitamins, phenolic compounds, and phytosterols (Table 9.2). Phenolic compounds in nuts are mainly located in the skin or testa (Milbury et al. 2006; Monagas et al. 2007), which is usually removed by blanching or roasting for the use of

Bioactive Substances of Selected Nuts per 100 g of Dry Weight* Nut Item (specie) Almonds (Prunus dulcis) Hazelnuts (Corylus avellana L.) Peanuts (Arachis hypogea L.) Pistachios (Pistacia vera L.) Walnuts (Juglans regia L.)

Anthocyanins

Flavonoids

Stilbenes

Total Phenols

Phytoestrogens

Tocopherols

Chlorophylls

Xanthophylls

(2.46)a

(28.7) 0.018–93.5b (37.9) 0.027–113.7f (94.9) 0.094–189.8j (26.7) 0.033–143.3p (333.4) 0.031–744.8v

nd

(90.0) 12.4–239c (210.9) 8.0–425g (357.9) 8.0–645.9l (335.0) 15.8–867r (814.5) 21.0–1625w

(40.0) 0.112–120d (0.093) 0.080–0.107h (110.0) 0.173–220m (36.14) 0.062–108s (35.93) 0.139–107.5x

(23.6) 17–27.44e (30.6) 15.36–45.5i (9.7) 4.8–16n (24.6) 8.4–53.0t (24.3) 14.9–31.72y

nd

nd

nd

nd

nd

nd

(14.3)u

(3.8)u

nd

nd

(6.7)a Nd (26.2) 24.3–28.1o Nd

nd (0.096) 0.002–0.2k (0.24) 0.009–0.71q nd

187

Note: Data are expressed as means (in parentheses) and range. nd, not detected. * Data reported by Ballistreri, G., Arena, E., and Fallico, B., Acta Horticulturae, in press, 2010a. Data reported by: a  Harnly, J. M., Doherty, R. F., Beecher, G. R., Holden, J. M., Haytowitz, D. B., Bhagwat, S., and Gebhardt, S., Journal of Agricultural and Food Chemistry, 54, 9966–77, 2006. b,c  Milbury, P. E., Chen, C. Y., Dolnikowski, G. G., and Blumberg, J. B., Journal of Agricultural and Food Chemistry, 54, 5027–33, 2006. b,d,f,h,p,s,v,x  Thompson, L. U., Boucher, B. A., Liu, Z., Cotterchio, M., and Kreiger, N., Nutrition and Cancer, 54, 184–201, 2006. b,d,f,h,j,m,p,s,v,x  Kuhnle, G. G. C., Dell’Aquila, C., Aspinall, S. M., Runswick, S. A., Mulligan, A. A., and Bingham, S. A., Journal of Agricultural and Food Chemistry, 56, 7311–5, 2008. b,c,f,g,j,l,p,r,v,w  Yang, J., Liu, R. H., and Halim, L., LWT—Food Science and Technology, 42, 1–8, 2009. c,e,g,i,l,n,r,t,w,y  Kornsteiner, M., Wagner, K., and Elmadfa, I., Food Chemistry, 98, 381–7, 2006. c,e,g,i,r,t,w,y  Miraliakbari, H., and Shahidi, F., Food Chemistry, 111, 421–7, 2008. c,r,v  Arcan, I., and Yemenicioglu, A., Journal of Food Composition and Analysis, 22, 184–8, 2009. d,e,i,m,n,s,t,x,y  Kocygit, A., Koylu, A. A., and Keles, H., Nutrition, Metabolism & Cardiovascular Diseases, 16, 202–9, 2006. e,g,i,l,n,r,t,w,y  Arranz, S., Cert, R., Perez-Jimenez, J., Cert, A., and Saura-Calixto, F., Food Chemistry, 110, 985–90, 2008. k,q  Tokuşog ˘lu, O., Unal, M. K., and Yemis, F., Journal of Agricultural and Food Chemistry, 53, 5003–9, 2005. k,q  Baur, J. A., and Sinclair, D. A., Nature Reviews Drug Discovery, 5, 493–506, 2006. k  Hurst, W. J., Glinski, J. A., Miller, K. B., Apgar, J., Davey, M. H., and Stuart, D. A., Journal of Agricultural and Food Chemistry, 56, 8374–8, 2008. o,u  Bellomo, M., and Fallico, B., Journal of Food Composition and Analysis, 20, 352–9, 2008. o,p,q,r,t  Ballistreri, G., Arena, E., and Fallico, B., Molecules, 14, 4358–69, 2009. p  Seeram, N. P., Zhang, Y., Henning, S. M., Lee, R., Niu, Y., Lin, G., and Heber, D., Journal of Agricultural and Food Chemistry, 54, 7036–40, 2006. p,q,r,t  Gentile, C., Tesoriere, L., Butera, D., Fazzari, M., Monastero, M., Allegra, M., and Livrea, M. A., Journal of Agricultural and Food Chemistry, 55, 643–8, 2007. q  Grippi, F., Crosta, L., Aiello, G., Tolomeo, M., Oliveri, F., Gebbia, N., and Curione, A., Food Chemistry, 107, 483–8, 2008.

Nut Bioactives

Table 9.2

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the kernel in the bakery and confectionary industry. Nut skins and other by-products derived from the processing of nuts have traditionally been used for livestock feed and as a raw material for energy production. But several studies have confirmed that they are an inexpensive, valuable source of natural antioxidants for nutraceutical and pharmaceutical applications (Wijeratne et al. 2006; Shahidi et al. 2007; Yu et al. 2007). The amount of dietary fiber is ~9 g/100 g of nuts, of which ~25% is soluble fiber. Soluble fibers reduce total- and LDL-cholesterol concentrations and improve glycemic control (Anderson et al. 1994). There is some evidence suggesting that even arginine, the second most abundant amino acid found in nut proteins, has a hypocholesterolemic effect (Kurowski and Carroll 1992). Folic acid is also found in nuts. An adequate consumption of folic acid is important for ­preventing carotid-artery stenosis (Selhub et al. 1995) and 100 g of nuts provides ~16% of the daily intake of folic acid that is 400 μg/d. On average, one serving of nuts (~30 g) contains ~18% of the daily intake for copper (Kris-Etherton et al. 1999) and therefore nuts can be a significant source of this essential mineral (Allen et al. 1977). Copper plays a key role in hematopoiesis and its lack has been associated with adverse changes in lipids, glucose tolerance and blood pressure (Klevay 1993). Almost all nuts are good sources of magnesium, providing ~8–20% of the daily intake (400 mg) for this essential mineral in a serving. Low magnesium status can contribute to dysrhythmias, myocardial infarction, and hypertension. Phenolic compounds are considered nonnutrient, biologically active compounds (Shahidi and Naczk 1995). The functionality of these compounds is expressed through their action as an inhibitor or an activator for a large variety of enzyme systems, and as metal chelators and scavenger of free oxygen radicals (Sanchez-Moreno et al. 1999; Russo et al. 2000; Garbisa et al. 2001). Oxygen free radicals are involved in many pathological conditions such as cancer and chronic inflammation (Briviba and Sies 1994). Epidemiologic studies (Hertog et al. 1993, 1995) have also shown that phenolic intake is ­significantly and inversely associated with coronary heart disease (CHD) mortality. Even vitamin E reduces CHD risk, but only in high doses (>100 IU/d). Nuts are a rich source of tocopherols, although the quantities obtained from typical nut consumption are far less than the amounts shown to have beneficial effects on CHD. Nonetheless, nut consumption is still an effective means of increasing vitamin E intake. Phytosterols (~110 mg/100 g of nuts) have been shown to reduce blood cholesterol, as well as to decrease the risk of certain types of cancer and enhance immune function (Ling and Jones 1995; Awad and Fink 2000; Bouic 2001; Moreau et al. 2002; Ostlund 2004). Other nutrients present in notable quantities in most nuts include thiamine, niacin, riboflavin, selenium, potassium, and iron.

Almond Almond (Prunus dulcis) is one of the most popular nut crops. The United States is the first largest producer of almonds in the world followed by Spain (Lopez-Ortiz et al. 2008). Large kernel and thin or semihard shell thickness are among the desired nut characteristics in almond breeding. However, nutritional characteristics might be affected by kernel weights. In addition, almond kernels should have high contents of fatty acids, oil, and protein as nutritional values. Almonds can also be significant for their antioxidant properties. It is an excellent source of tocopherols; moreover, the polyphenols located in almond skin especially (Bolling et al. 2009) may also contribute to their health-promoting actions (Chen et al. 2005; Milbury et al. 2006; Chen and Blumberg 2008).

Phenolics The phenolics of almond are a mixture of flavonoids, phenolic acids, and tannins that contribute to their antioxidant capacity in an additive or even synergistic manner (Milbury et al. 2006; Chen and Blumberg 2008; Garrido et al. 2008). Almond and its by-products derived from industrial processing, such as hull, shell, and skin have been reported to have powerful free radical scavenging capacities (Pinelo et al.

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2004; Moure et al. 2007). Skin, while representing only approximately 4% of the total weight, contains 70–100% of the total phenolics present in the nut (Milbury et al. 2006). Almonds contain a variety of flavonoids including flavanols (catechin and epicatechin), flavonols (kaempferol, isorhamnetin, and quercetin), flavanones (naringenin and eriodictyol), anthocyanins ­(cyanidin and delphinidin), and proanthocyanidins (of both B- and A-type), as well as phenolic acids  (caffeic acid, chlorogenic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, protocatechuic acid, and vanillic acid) and some alcohols and benzoic aldehydes (eugenol, p-hydroxybenzaldehyde, and protocatechuic aldeide; Amarowicz et al. 2005; Monagas et al. 2007; Chen and Blumberg 2008; Garrido et al. 2008). The most abundant group of almond skin phenolics are flavanols (~50%), followed by flavonol glycosides (~25%), and phenolic acids (~20%). The remaining phenolic compounds (flavonol aglycones, flavanone glycosides, and aglycones) represent ~5% of total phenolic compounds (Monagas et al. 2007; Garrido et al. 2008). The total content of almond skin phenolics varies from about 160 to 800 μg/g, but this range is affected both by variety and processing (roasting and blanching). The mean total concentration of phenolic compounds has been found significantly higher in the skins of Spanish almonds (~410 μg/g) with respect to American almonds (~270 μg/g). The content of phenolic compounds in the American almonds has a high variability (Milbury et al. 2006; Garrido et al. 2008). Table 9.3 reports the content (μg/g) of phenolic compounds of almond skins from Spain and the US industrially processed (blanching + drying). The influence of industrial processing on almond skin polyphenols was studied by Garrido et al. (2008). In this study the phenolic composition of almond skins obtained from different processes (blanching, blanching + drying, and roasting) has been evaluated. The mean content of total polyphenols was higher (>twofold) in the roasted samples (28.4 mg/g) than in the blanched samples (13.3 mg/g). The drying of the blanched samples produced an increase ( + 34%) in the content of phenolic compounds of blanched samples. Moreover drying and roasting processes induce an increase of antioxidant activity, they can produce a series of transformations that can affect the concentration of some phenolic compounds (Piga et al. 2003), which could affect the antioxidant capacity of almond skins. The ORAC values of almond skins are within 0.331–3.0 mmol Trolox/g range (Monagas et al. 2007; Chen and Blumberg 2008; Garrido et al. 2008); these values are in the range found for some by-products derived from the winery industry, such as grape skins and seeds (0.428 and 2.11 mmol Trolox/g, respectively), using the same extraction procedure (Friedrich et al. 2000; Monagas et al. 2005). This suggests that almond skins could be considered as a value-added by-product to be used in the elaboration of antioxidant dietary ingredients.

Neutral Lipids Triacylglycerols (TGs) represent the major lipid class in tree nut oils (>95%). Among nuts, almonds have one of the lowest oil yield (on average ~50%), but its oil contains the highest TGs content (~98g/100 g oil; Miraliakbari and Shahidi 2007). Table 9.4 reports the TGs content (%) of almonds for different geographic origins. Five TGs are the major ones: OLL, OLO, PLO, OOO, and POO (P = palmitic, O = oleic, S = stearic, L = linoleic), together they represent ~80% of the total TGs content; four are minor: LLL, PLL, PLP, and SOO, representing ~20% of the total TGs content. POP and LnOO have also been found in small amounts (<1% and <3%, respectively; Cherif et al. 2004). The TGs composition of almonds for different origins is qualitative similar, but marked differences have been found in the TGs amount. The quantitative differences in the TGs composition can be useful for distinguishing almonds of different cultivars (Martin-Carratala et al. 1999; Cherif et al. 2004). The advantage of using TGs analysis compared to fatty acid profiles is that the stereospecific distribution of fatty acids on glycerol molecule is genetically controlled and, thus, the information content of intact TGs is usually higher (Aparicio and Aparicio-Ruiz 2000; Ulberth and Buchgraber 2000). The composition (%) of the major fatty acids (FAs) of some almond cultivars is reported in Table 9.5. The major fatty acid in almonds is oleic (C 18:1), representing ~60–70% of the total FAs content, followed by linoleic (C 18:2), ~10–20% and palmitic 5–8% (García-López et al. 1996; Askin et al. 2007; Miraliakbari and Shahidi (2007); little amounts (below 1%) of myristic (C 14:0) and linolenic acid

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Table 9.3 Content (μg/g) of Phenolic Compounds in Almond Skins from Spain and the United States. Compound Hydroxybenzoic acids and aldehydes p-hydroxybenzoic acid Vanillic acid Protocatechuic acid Protocatechuic aldehyde Hydroxycinnamic acids trans-p-coumaric Chlorogenic acid Flavan-3-ols ( + )-catechin (−)-epicatechin B-type procyanidins A-type procyanidins Flavonol glycosides Kaempferol-3-O-rutinoside Kaempferol-3-O-glucoside Kaempferol-3-O-galactoside Isorhamnetin-3-O-rutinoside and glucoside Isorhamnetin-3-O-galactoside Quercetin-3-O-glucoside Quercetin-3-O-galactoside Quercetin-3-O-rutinoside Flavanone glycosides Naringenin-7-O-glucoside Eriodictyol-7-O-glucoside Flavonol aglycones Kaempferol Quercetin Isorhamnetin Dihydroflavonol aglycones Dihydroquercetin Dihydrokaempferol Flavanone aglycones Naringenin Eriodictyol Total

Spaina

United Statesb

6.9 14.5 32.0 20.1

3.1 6.0 9.7 9.9

— 10.6

0.7 12.1

90.1 36.6 77.7 28.9

37.5 18.7 36.2 11.7

12.8 — — 43.2 — 2.4 — —

29.2 5.0 0.1 79.1 5.7 1.6 7.9 2.6

22.1 1.6

9.8 1.1

1.7 1.8 4.9

2.0 1.7 4.0

— —

9.0 1.1

2.8 2.4 413.0

4.2 2.7 274.8

Data reported by: Monagas, M., Garrido, I., Lebron-Aguilar, R., Bartolome, B., and Gomez-Cordoves, C., J. Agric. Food Chem., 55, 8498–507, 2007. a,b Garrido, I., Monagas, M., Gomez-Cordoves, C., and Bartolome, B., J. Food Sci., 73, C106–15, 2008. b Milbury, P. E., Chen, C. Y., Dolnikowski, G. G., and Blumberg, J. B., J. Agric. Food Chem., 54, 5027–33, 2006. a,b

(C 18:3) were also detected (Martin-Carratala et al. 1999; Kris-Etherton et al. 1999; Zacheo et al. 2000). Askin et al. (2007), have observed that the content of the major FAs is influenced by kernel weight. The contents of palmitic, stearic, and oleic acids, are positively correlated with kernel weight, contrary to the linoleic acid content. In addition, shell thickness is negatively correlated with contents of palmitic and stearic acids, but positively correlated with oleic acid.

191

Nut Bioactives Table 9.4 Mean Content (%) of Major Triacylglycerols of Almonds of Different Geographic Origins Triacylglycerols LLL OLL PLL OLO PLO PLP OOO POO SOO

Italya

Spainb

Francec

Tunisiad

United Statese

Australiaf

1.21 ± 0.27 6.59 ± 0.33 1.08 ± 0.10 17.72 ± 1.28 5.37 ± 0.37 0.35 ± 0.02 52.06 ± 2.79 12.14 ± 0.72 3.47 ± 0.61

1.87 ± 0.37 10.95 ± 1.96 1.76 ± 0.29 22.65 ± 2.61 8.31 ± 1.13 0.45 ± 0.05 39.27 ± 5.64 11.88 ± 0.32 2.82 ± 0.50

1.78 ± 0.02 9.46 ± 0.05 1.53 ± 0.02 22.99 ± 0.13 7.27 ± 0.06 0.40 ± 0.01 41.68 ± 0.15 11.35 ± 0.11 3.53 ± 0.04

1.96 ± 0.02 11.34 ± 0.09 1.73 ± 0.02 25.68 ± 0.16 9.54 ± 0.09 0.44 ± 0.01 34.27 ± 0.17 12.61 ± 0.14 2.43 ± 0.05

2.51 ± 0.77 11.67 ± 3.70 1.83 ± 0.65 24.14 ± 3.18 8.28 ± 1.92 0.39 ± 0.03 37.71 ± 9.49 10.94 ± 0.48 2.54 ± 0.48

2.24 ± 0.03 12.68 ± 0.08 2.08 ± 0.02 26.25 ± 0.16 10.69 ± 0.05 0.49 ± 0.01 30.29 ± 0.21 12.28 ± 0.07 2.99 ± 0.05

Data reported by:  Martin-Carratala, M. L., Llorens-Jordá, C., Berenguer-Navarro, V., and Grané-Teruel, N., J. Agric. Food Chem., 47, 3688–92, 1999. Cultivars examined:  a  Genco, Tuono, Cristomorto; b  Malaguena, Peraleja, Atocha, Del Cid, Desmayo Largueta, Ramillete, Marcona; c  Ferragnes; d  Achaak; e  Texas, Nonpareil, Titan, Wawona; f  Chellaston.

Table 9.5 Mean Content (%) of Major Fatty Acid of Almond Cultivars from Different Origins Fatty Acids Origin American cultivarsa Italian cultivarsa Spanish cultivarsb Tunisian cultivarsa Turkish cultivarsc

Palmitic C 16:0

Palmitoleic C 16:1

Stearic C 18:0

Oleic C 18:1

Linoleic C 18:2

5.76 5.03 7.42 7.50 7.50

0.369 0.401 0.632 0.368 0.724

1.72 1.77 2.03 1.70 1.83

62.75 64.53 68.46 73.10 70.84

20.42 11.87 20.45 19.50 18.49

Data reported by: Garcia-Lopez, C., Grané-Teruel, N., Berenguer-Navarro, V., Garcia, J., and Martin Carratalá, M. L., Journal of Agricultural and Food Chemistry, 44, 1751–5, 1996. b Miraliakbari, H., and Shahidi, F., Journal of Food Lipids, 15, 81–96, 2007. c Askin, M., Balta, M., Tekintas, F., Kazankaya, A., and Balta, F., Journal of Food Composition and Analysis, 20, 7–12, 2007. a,b

Polar Lipids Among polar lipids phosphatidylserine, phosphatidylinositol, and phosphatidylcholine are present at 0.13, 0.09, and 0.14 g/100 g of almond oil, respectively. Sphingolipids are also present (0.37 g/100 g of oil; Miraliakbari and Shahidi 2007, 2008).

Phytosterols and Tocols Almond oil contains between 2.2 and 2.8 g/kg of phytosterols, mainly β-sitosterol (~2.0 g/kg), with trace amounts of stigmasterol, Δ5-avenasterol, and campesterol (Maguire et al. 2004; Miraliakbari and Shahidi 2007). Almonds stand out for being one of the nuts with the highest α-tocopherol content. It ranges from about 8.0 to 25.0 mg/100 g of oil (Kornsteiner et al. 2006; Miraliakbari and Shahidi 2007; Lopez-Ortiz et al. 2008). Almond oil contains smaller amounts of β + γ-tocopherol, ranging from 0.1 to 3–0 mg/100 g of oil, and δ-tocopherol present in traces. Therefore, almonds are a dietary sources of α-, β-, and γ-tocopherol and can contribute to a balanced intake of vitamin E. This explains why almonds have been included in

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

the recommendations of The Dietary Guidelines for Americans (USDA 2005) in the context of enhancing the intake of this vitamin.

Hazelnut The hazelnut (Corylus avellana L.) belongs to the Betulacee family and is a popular tree nut worldwide, mainly distributed along the coasts of the Black Sea region of Turkey, southern Europe (Italy, Spain, Portugal, and France), and in some areas of the United States (Oregon and Washington). Turkey is the world’s largest producer of hazelnuts contributing ~74% to the total global production, followed by Italy (~16%), the United States (~4%), and Spain (~3%; Alasalvar et al. 2009a). Besides its economic value, hazelnuts provide a unique and distinctive flavor as an ingredient in a variety of food products for taste; active components such as free amino acids, sugars, and organic acids improve the sensory characteristics of products. Moreover, hazelnuts play a major role in human nutrition and health for its special composition of fat, which are highly rich in monounsaturated fatty acids, protein, carbohydrate, dietary fiber, vitamins, minerals, phytosterols, squalene, and antioxidant phenolics (Alphan et al. 1997; Richardson 1997; Ackurt et al. 1999; Yurttas et al. 2000).

Phenolics Hazelnuts have a hard, smooth shell. The seed is covered by a dark brown pellicular pericarp (skin or testa), which is typically removed before consumption after roasting of the kernel. The hazelnut native phenolics are almost exclusively located in the perisperm of the seed (Contini et al. 2008). Recent studies (Shahidi et al. 2007; Alasalvar et al. 2009b) have established that hazelnut wastes, especially the skin and hard shell, are a reliable source of natural antioxidants. Hazelnut skin shows superior antioxidative efficacy and higher phenolic content, compared to the hazelnut kernel and other by-products (Table 9.6). By comparison with almonds and peanut skins, the content obtained from the hazelnut has the highest level of phenolics. Five phenolic acids have been identified and quantified (both free and esterified forms) by Alasalvar et al. (2006) and Shahidi et al. (2007) in the hazelnut kernel and by-products. One of which is a hydroxylated derivative of benzoic acid (gallic acid) and four of which were cinnaminc acid derivatives (caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid). The p-coumaric acid is the most abundant phenolic acid in the hazelnut kernel with a mean content of ~2.5 μg/g. Whereas gallic acid is the most abundant with a mean content of ~39.1 and ~ 81.0 μg/g of skin and hard shell, respectively, implying the presence and the dominance of tannins, ranging nearly 60–65% of the total phenols (Contini et al. 2008). Tannins are much more powerful antioxidants than simple monomeric phenols, and may have unique roles in the human digestive metabolism as both savers of other biological antioxidants and protectors of nutrients

Table 9.6 Yield, Content of Phenolics and TAA in Extracts of Hazelnut Kernel and Hazelnut By-Products Extract Hazelnut kernel (with skin) Hazelnut skin Hazelnut hard shell Hazelnut green leafy cover Hazelnut tree leaf

Yielda

Phenolicsb

TAAc

2.26 ± 1.11 e 10.28 ± 1.02 f 2.53 ± 0.33 e 3.59 ± 0.85 e 1.64 ± 1.87 e

13.7 ± 0.5 e 577.7 ± 1.1 f 214.1 ± 0.3 g 127.3 ± 0.7 h 134.7 ± 1.0 i

29.0 ± 3.5 e 132.0 ± 4.0 f 120.0 ± 3.0 g 117.0 ± 2.5 g 148.0 ± 2.1 h

Source: Data reported by Shahidi, F., Alasalvar, C., and Liyana-Pathirana, C. M., J. Agric. Food Chem., 55, 1212–20, 2007. Notes: Data are expressed as means ± SD (n = 3) on an extract. Means ± SD followed by the same letter, within a column, are not significantly different (p >.05). a  Expressed as grams per 100 g of defatted samples. b  Expressed as milligrams of catechin equivalents per gram of extract. c  Expressed as micromoles of trolox equivalents per gram of extract.

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(lipid, proteins, and carbohydrates) from oxidative damages (Chung et al. 1998; Hagerman et al. 1998). By comparison with almonds and walnuts, hazelnuts have the highest content of condensed tannins (proanthocyanidins; Karamac et al. 2007). Yurttas et al. (2000) have identified six phenolic aglycones in Turkish and American hazelnuts: gallic acid, p-hydroxybenzoic acid, epicatechin and/or caffeic acid, sinapic acid, and quercetin. Protocatechuic acid has been reported to be the predominate phenolic acid in the skin of an American hazelnut (0.36 μg/g) by Senter et al. (1983). These differences could be ascribed to varieties and extraction procedures that exerted a great influence on the concentration and variability of the phenolic acids present. The phenolic content of hazelnuts could be used as an important criteria in evaluating hazelnut quality (Contini et al. 2008; Alasalvar et al. 2009b).

Neutral Lipids The lipid fraction is the major component of hazelnuts (~60%), and is composed of nonpolar (98.8%) and polar (1.2%) constituents (Amaral et al. 2006a; Alasalvar et al. 2009a). Triacylglycerols (TGs) are the major nonpolar lipid class, representing nearly 100% of the total nonpolar lipids in hazelnut oil (Alasalvar et al. 2003). The TGs are increasingly used in the food industry as a tool to assess the quality and authenticity of vegetable oils (Aparicio and Aparicio-Ruiz 2000), particularly the adulteration of olive oil with hazelnut oil (Parcerisa et al. 2000). Amaral et al. (2006a) identified and quantified 11 TGs for 19 cultivars coming from six different countries (United States, Italy, Spain, France, Germany, and England) during three consecutive years; 12 TGs (including one unknown) have been determined by Alasalvar et al. (2009a) in hazelnut oils of five native hazelnut varieties from Turkey (Table 9.7). The predominant TGs are OOO (65.3–70.8%), followed by OOL (13.6–15.4%), POO (8.9–11.4%), SOO (3.5–3.7%), and OLL (2.0%). They account for more than 95% of total TGs content; the remaining seven TGs contribute only about 2% to the total amount. TGs are usually determined by HPLC/ELSD, studies conducted with different RI detectors (Parcerisa et al. 1995) and MS detector (Parcerisa et al. 2000) have rendered higher values for LLL, OLL, POL, and PPO and much lower values for OOO. Trace amounts of PPP in hazelnut oil have been reported by Parcerisa et al. (1995). In contrast, Ayorinde et al. (1999) have analyzed hazelnut oil using MALDITOF-MS and pointed out that minor quantities of SLL and SOL could be ascribed to the coelution with Table 9.7 Mean Triacylglycerol Content (Relative %) of Hazelnut Oil Triacylglycerols LLL OLL PLL OOL POL PPL OOO POO PPO Unknown SOO PSO

Amaral et al. (2006a)a

Alasalvar et al. (2009a)b

0.23 ± 0.06 2.00 ± 0.27 0.09 ± 0.03 15.36 ± 1.23 1.57 ± 0.21 0.03 ± 0.01 65.32 ± 1.22 11.43 ± 0.56

0.25 ± 0.28 2.02 ± 1.99 0.05 ± 0.07 13.56 ± 5.28 0.77 ± 0.49 0.01 ± 0.01 70.82 ± 6.08 8.89 ± 1.93

0.10 ± 0.02 —

0.03 ± 0.01 0.02 ± 0.02 3.54 ± 1.51 0.05 ± 0.04

3.73 ± 0.76 0.13 ± 0.03

Cultivars and varieties examined:  a  Butler, Campanica, Cosford, Couplat, Daviana, Ennis, Fertille de Coutard, Grossal, Gunslebert, Lansing, Longa d’Espanha, Merveille de Bollwiller, Morell, Negreta, Pauetet, Round du Piemont, Santa Maria de Jesus, Segorbe, Tonda de Giffoni; b  Tombul, Yassi Badem, Sivri, Karafindik, Ham.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 9.8 Percentages of Fatty Acids of Lipid Fractions Extracted from Hazelnuts of Different Origins Fatty Acids (%) Origin Italy

Spain

Turkey United States

Cultivar T. Romanaa T.g.d.l.b Mortarella T. Giffonic Casina Segorbe Negret Ribet Tomboul Imperial Barcelona Daviana Montebello Butler Ennis Halls giant Willamette

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

5.78 6.04 4.98 5.38 5.70 4.66 5.04 5.52 5.48 5.07 5.20 5.19 5.10 5.77 5.41 4.72 5.20

0.22 0.30 0.15 0.19 0.21 0.17 0.19 0.19 0.21 0.18 0.17 0.19 0.21 0.24 0.23 0.17 0.18

2.50 2.81 2.72 2.86 2.96 2.58 2.49 2.68 2.04 3.36 1.93 2.29 3.34 2.68 1.38 2.19 2.43

82.55 82.29 78.90 82.83 78.58 79.85 79.06 76.45 74.13 76.42 78.72 79.19 80.31 79.55 77.08 79.13 80.76

8.59 8.17 12.83 8.37 12.19 12.34 12.82 14.69 17.78 14.52 13.58 12.75 10.46 11.26 15.55 13.23 11.04

0.10 0.10 0.11 0.10 0.10 0.12 0.13 0.15 0.11 0.13 0.14 0.11 0.17 0.11 0.11 0.14 0.11

Source: Data reported by Parcerisa, J., Richardson, D., Rafecas, M., Codony, R., and Boatella, J., J. Chromatogr.,º A, 805, 259–68, 1998. Note: Data are means of triplicate results. a Tonda Romana; b  Tonda gentile delle langhe; c  Tonda giffoni.

OLL and OOO. Therefore, the type of detector used has a significant influence on TGs profile (Alasalvar et al. 2009a). Amaral et al. (2006b) have also studied the effect of roasting on hazelnut lipids. They observed a decrease of TGs containing linoleic acid moieties and an increase of TGs containing oleic, palmitic, and stearic acids, with the increase of temperature and roasting time. In general, as roasting time increased, losses of TGs species are more pronounced in those containing more than four double bonds, probably because the rate of fatty acids breakdown is related to the increasing rate of oxidation with increasing unsaturation (Yoshida et al. 2003). Among nuts, hazelnuts have the highest oleic acid content (~80%; Miraliakbari and Shahidi 2007). The other major fatty acids are linoleic (~13%), palmitic (~5%), and stearic (~2%; Parcerisa et al. 1998; Koksal et al. 2006). The fatty acid composition of the hazelnut is influenced by variety and geographical origin (Table 9.8). It has been reported that the ratio of oleic to linoleic acid varies among hazelnut cultivars, and that their contents are inversely related (Parcerisa et al. 1995; Amaral et al. 2006c). The ratio oleic to linoleic, for cultivars reported in Table 9.8, varies from 4.1 to 9.8; this can really point to different behaviors for the several cultivars there were studied. In general, with roasting (Amaral et al. 2006a) and during fruit development (Seyhan et al. 2007), the relative levels of monounsaturated fatty acids increase while that of the polyunsaturated fatty acids decrease.

Polar Lipids Hazelnut oil components have been separated and quantified using column chromatography by Parcerisa et al. (1997), revealing that oil contains less than 0.2% of phospholipids (phosphotidylcholine and phosphatidylinositol). The lipid class composition of hazelnut oil has also been studied using Iatroscan by Alasalvar et al. (2003), revealing 1.2% of polar lipids in oil. Among polar lipids, phosphotidylcholine,

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Nut Bioactives Table 9.9 Mean Tocopherol and Tocotrienol Composition of Hazelnut Oils (mg/100 g) Tocols

Crews et al. (2005)a

α-Tocopherol β-Tocopherol γ-Tocopherol δ-Tocopherol α-Tocotrienol β-Tocotrienol γ-Tocotrienol Total

35.40 1.10 2.10 — — — — 38.60

Country of origin of hazelnuts: 

a 

Amaral et al. (2006b)b

Alasalvar et al. (2009a)c

24.47 0.84 0.97 0.01 0.19 0.03 0.12 26.63

30.50 1.07 10.15 0.43 0.17 0.09 0.22 42.63

Italy; b  Portugal; c  Turkey.

phosphotidylethanolamine, and phosphatidylinositol were present at 56.4, 30.8, and 11.7%, respectively. Traces of phosphatidic acid have also been detected (Miraliakbari and Shahidi 2008).

Phytosterols and Tocols Total sterols content varies from about 120 to 250 mg/100 g of hazelnut oil (Crews et al. 2005; Amaral et al. 2006c; Alasalvar et al. 2009a). There are differences among cultivars, though β-sitosterol is the major sterol in all cultivars (84–219 mg/100 g oil), followed by Δ5-avenasterol (2.2–18.2 mg/100 g oil), and campesterol (5.1–16.4 mg/100 g oil). Besides these compounds, cholesterol, stigmasterol, clerosterol, Δ7-avenasterol, Δ7-stigmastenol, cholestanol, brassicasterol, 24-methylenecholesterol, campestanol, Δ7-campesterol, Δ5,23-stigmastadienol, sitostanol, and Δ5,24-stigmastadienol have also been found, but they contribute less than 4% to the total (Crews et al. 2005; Amaral et al. 2006c; Alasalvar et al. 2009a). Hazelnut varieties are an excellent source of tocols ranging from 11 to 45 mg/100 g oil (Crews et al. 2005; Amaral et al. 2006b; Alasalvar et al. 2009a). In the majority of hazelnuts, seven tocol isoforms have been detected (α-, β-, γ-, and δ-tocopherols and α-, β-, and γ-tocotrienols; Table 9.9). The major tocopherol present is α-tocopherol, followed by γ- and β-tocopherol. Hazelnut oil has been reported to have the highest α-tocopherol level among tree nut oils (Kornsteiner et al. 2006). Differences in tocols composition could be ascribed to the geographical origin of hazelnuts (Crews et al. 2005), and the type of detector (UV, FL, or FL/DAD) and methodology used that exert a significant effect on the tocol profiles (Alasalvar et al., 2009a). Roasting of hazelnuts causes a modest decrease of vitamin E homologues (maximum 10%; Amaral et al. 2006b).

Peanut Peanut or groundnut (Arachis hypogaea L.) is universally popular and is used as a snack food or as an ingredient in the manufacture of a variety of food products such as peanut butter and peanut brittle (Venkatachalam and Sathe 2006). Peanuts may be consumed raw, roasted, pureed, or in a variety of other processed forms, and constitute a multimillion-dollar crop worldwide (Yu et al. 2005) with numerous potential dietary benefits. Recently several peanut cultivars were developed with elevated concentrations of the monounsaturated oleic acid, in relation to other highly oxidizable polyunsaturated fatty acids. The high oleic trait provides peanuts with potentially greater health benefits and serves to prolong shelf-life characteristics. Numerous phytochemical compounds with potential antioxidant capacity are present in peanuts including polyphenolics (Talcott et al. 2005), tocopherols (Hashim et al. 1993), and proteins (Bland and Lax 2000). Moreover, the peanut is a good source of folate and resveratrol. Regular peanut consumption lowers serum TG and increases dietary folate intake, thereby lowering plasma homocysteine concentration. Higher peanut butter consumption was associated with a decreased risk of type 2 diabetes in women (Yang et al. 2009).

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Phenolics Numerous phenolics have been identified in peanut skins, such as phenolic acids, flavonoids and stilbenes (Yu et al. 2005; Francisco and Resurreccion 2009). Yu et al. (2005) identified three classes of phenolic compounds in raw peanut skins: (1) phenolic acids including chlorogenic acid, caffeic acid, and ferulic acid; (2) flavonoids including epigallocatechin, epicatechin, catechin gallate, epicatechin gallate; and (3) stilbenes (resveratrol). Caffeic acid, chlorogenic acid, ellagic acid, resveratrol, and its glycoside were identified but not quantified, due to a very low concentration and peaks were suppressed by major procyanidins (Yu et al. 2006). By weight, 17% of peanut skins are procyanidins consisting of low and high molecular weight oligomers (Karchesy and Hemingway 1986). Table 9.10 shows the mean values of peanut skin phenolics of the major cultivar groups (Runner, Virginia, and Spanish). Ethyl protocatechuate, a protocatechuic acid ethyl ester, also has been identified in Spanish peanut skins (Huang et al. 2003; Yen et al. 2005), but no amount was provided by the authors. Caffeic acid has been detected only in Spanish skins. This compound was also detected by Yu et al. (2005) in peanut skins (peanut type not specified). The p-coumaric acid is predominantly present in peanut kernels (~60 μg/g; Talcott et al. 2005; Duncan et al. 2006) with respect to skins (~13 μg/g). Monomeric and oligomeric flavan-3-ol has been detected in peanuts. Monomeric (+)-catechin is more abundant than (−)-epicatechin in peanut skins (849 and 196 μg of catechin equivalents/g of peanut skin, respectively; Monagas et al. 2009). Total monomers account for only 19% in peanut skins, while procyanidins (both A- and B-type, including B-type dimers, and A-type dimers, trimers, and tetramers) represent ~80% of total flavan-3-ol content (~4600 μg of catechin equivalents/g of peanut skin). Peanut skins are characterized by a high proportion of A-type procyanindins, total dimers + trimers accounting for 40% and tetramers for 37% of the total monomers + oligomers content (Yu et al. 2006; Monagas et al. 2009). Moreover the presence of isoflavones (~90 μg/100 g of wet weight), mainly genistein, glycitein, and biochanin A, as well as lignans (~75 μg/100 g of wet weight; secoisolariciresinol) has been revealed in fresh, dry, and roasted peanuts (Kuhnle et al. 2008). Among nuts, only peanuts and pistachios are sources of stilbenes (Baur and Sinclair 2006). The trans-resveratrol is the main resveratrol isomer that occur in peanuts, with a mean content of ~8.5 μg/g (skin + kernel; Tokuşoğlu et al. 2005; Francisco and Resurreccion 2009). Tokuşoğlu et al. (2005), after exposure of peanuts to UV light, have observed the conversion of trans-resveratrol into cis-form with a range value of 0.04–0.50 μg/g. Roasted peanuts have a minor amount of resveratrol (~four-fold less) than raw peanuts (Sanders et al. 2000; Hurst et al. 2008). Even in pistachios, the thermal process induces a profound loss (>80%) of this compound (Ballistreri et al. 2009).

Table 9.10 Mean Values of Peanut Skins Phenolics of Different Cultivar Groups Compound Protocatechuic acid Caffeic acid p-Coumaric acid Epigallocatechin Catechin Procyanidin B2 Epicatechin Quercetin Resveratrol

Runner

Virginia

Spanish

7.62 ± 1.34 nd

34.03 ± 1.95 nd

23.35 ± 0.91 440.05 ± 16.70 74.35 ± 13.14

4.98 ± 0.63 1275.92 ± 77.10 535.03  ± 41.72

15.45 ± 0.87 3.49 ± 0.36 12.31 ± 1.42 1274.72 ± 67.50 448.30 ± 36.47

20.67 ± 5.63 60.06 ± 11.44 20.14 ± 1.49 4.30 ± 0.10

17.69 ± 1.61 144.75  ± 1.42 22.88 ± 2.92 3.66 ± 0.44

107.00 ± 18.99 238.55 ± 9.20 27.99 ± 2.10 15.04 ± 1.57

Source: Data reported by Francisco, M. L. L. and Resurreccion, A. V. A., Food Chem., 117, 356–63, 2009. Note: Data are expressed as μg/g of dry skin. nd = not detected.

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Nut Bioactives Table 9.11 Mean Content (%) of the Major Triacylglycerols of Peanut Oil Triacylglycerols OOL LLO POL OOO POO

19.3 ± 3.2 19.1 ± 10.0 13.2 ± 0.3 7.2 ± 3.9 6.8 ± 1.7

Source: Data reported by Sempore, G. and Bezard J., Journal of Chromatography A, 366, 261–82, 1986; Singleton, J. A. and Pattee, H. E., J. Am. Oil Chem. Soc., 64, 534–8, 1987.

Neutral Lipids Peanuts have the lowest lipid content among nuts (<50 mg/100 g; Maguire et al. 2004; Venkatachalam and Sathe 2006). The major fraction of peanut lipids are TGs (>95% of total lipid content; Singleton et al. 1999; Yoshida et al. 2005). Together, OOL, LLO, POL, OOO, and POO represent more than 60% of the total triaclyglycerols content (Table 9.11; Sempore and Bezard 1986; Singleton and Pattee 1987). Peanut oil contains predominantly unsaturated fatty acids (~81% of total fatty acids); the content of SFAs is ~13% of the total fatty acids (Venkatachalam and Sathe 2006). The main fatty acids are oleic followed by linoleic and palmitic, together they account for more than 80% of total fatty acids content. Stearic, arachidic, behenic, lignoceric, and linolenic acids account for ~8% of total fatty acids content. Moreover trace amounts of margaric, palmitoleic, gadoleic, and erucic acids have also been found (<0.5%) (Golombek et al. 1995; Venkatachalam and Sathe 2006).

Polar Lipids Polar lipids represent ~1.5% of total lipids content in peanut oil (Yoshida et al. 2005). Among polar lipids, phosphotidylcholine, phosphatidylinositol, and phosphotidylethanolamine are present at ~58, 20, and 19%, respectively (Yoshida et al. 2005).

Phytosterols and Tocols The total phytosterols content in peanuts range from about 135 to 220 mg/100 of the dry nut (Phillips et al. 2005; Kocygit et al. 2006). The predominant phytosterols are β-sitosterol (~77 mg/100 g of a dry nut), Δ5-avenasterol (~18 mg/100 g), campesterol (~13 mg/100 g), and stigmasterol (~12 mg/100 g). Together they account for more than 85% of total phytosterols content. Other sterols (poriferasta-7,25-dienol, campestanol, and sitostanol) are present in a minor amount (~10% of total sterols content; Maguire et al. 2004; Phillips et al. 2005). The total tocopherols content of peanut oil is the lowest among nuts (~4.8–16 mg /100 g oil; Kocygit et al. 2006; Kornsteiner et al. 2006; Arranz et al. 2008). The α-tocopherol is the dominant tocopherol (6.1–8.3 mg/100 g oil); β- and γ-tocopherols have been detected together with a mean content of about 8 mg/100 g oil, moreover traces of δ-tocopherol has been found (<2 mg/100 g oil; Kocygit et al. 2006; Kornsteiner et al. 2006). Low levels of vitamin E could be associated with a potentially higher risk of atherosclerosis or other degenerative diseases and increased intakes appear to be protective against these diseases (Elmadfa and Wagner 2003). On the other hand, Miller et al. (2005) have shown that the high dosage (>400 IU/d) in vitamin E supplements may increase all-cause mortality. However, low

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

quantities, which can be obtained from average nut consumption, have been shown to be beneficial for cardiovascular heart disease (Bramley et al. 2000).

Pistachio Pistachio (Pistacia vera L.) belongs to the Anacardiaceae family. It is the only species of the Pistacia genus that produces edible nuts. It is mainly cultivated in the United States, western Asia, and some Mediterranean countries (Italy, Turkey, Greece, and Tunisia). The pistachio nut is widely appreciated all over the world for its peculiar organoleptic characteristics; it is consumed raw, sun-dried, or roasted, as snack food and/or as ingredients in the confectionery industry, while the shells have been proposed as a raw material to prepare activated carbons (Galvano et al. 1996). Pistachio seeds are of high economic value due to their balanced composition, characterized by a low carbohydrate content of about 10%, a protein content of more than 20%, and a lipid content of about ~50%, all on a dry weight basis (Chahed et al. 2006). Pistachio kernels contain a remarkable amount of phenolic compounds such as anthocyanins, flavonoids, stilbenes, and a low amount of antioxidant vitamins, but the quantity of such constituents is deeply influenced by the processing employed (Seeram et al. 2006; Gentile et al. 2007). For this reason, Pistacia species have attracted the attention of researchers for their antioxidant potential, besides their antimicrobial, anti-inflammatory, and cytotoxic activities, particularly due to their phenolic constituents. The essential oils of the fruits and the leaves of the pistachio also have antibacterial and antifungal properties (Tsokou et al. 2007). Recently, the edible pistachio nut has been ranked among the top 50 food products with antioxidant potential (Halvorsen et al. 2006).

Phenolics The main phenolic compounds in the pistachio are anthocyanins (cyanidin-3-galactoside and cyanidin3-glucoside) ranging from about 24 to 43 mg/100 g dry matter (Table 9.12) (Bellomo and Fallico 2007; Ballistreri et al. 2009). These pigments are localized exclusively in the skin of pistachios (Seeram et al. 2006) and are synthesized during ripening, but cyanidin-3-galactoside (the major anthocyanin in pistachio skin) increased its concentration, with ripening, faster than cyanidin-3-glucoside (Ballistreri et al. 2009). The sun-drying process causes the loss of about 60% of anthocyanins. Cyanidin-3-glucoside seems to be more sensitive to the drying process than cyanidin-3-galactoside. In fact, the ratio between cyanidin-3-galactoside and cyanidin-3-glucoside is 3.22 in raw ripe pistachios and 5.23 after the sundrying process, respectively (Ballistreri et al. 2009). A similar ratio value (5.31) has been found for roasted pistachio (Seeram et al. 2006). Such a ratio (cy-3-gal/cy-3-glu) could be a useful index to evaluate thermal treatments of pistachios. The major isoflavones present are daidzein and genistein (Table 9.12), moreover, other isoflavones such as biochanin A, glycitein, and formononetin have also been detected in pistachios, although at very low levels (<0.04 mg/100 g; Thompson et al. 2006; Kuhnle et al. 2008). Small amounts of rutin and apigenin (0.2 and 0.02 mg/100 g, respectively; Seeram et al. 2006) such as the complex phenolics 3-alkylphenols or cardanols (~44 mg/100 g), mainly 3-(8-pentadecenyl)-phenol, 3-(10-pentadecenyl)-phenol, 3-pentadecenylphenol, and 3-(10-eptadecenyl)-phenol (Saitta et al. 2009), lignans (matairesinol, lariciresinol, pinoresinol, and secoisolariciresinol; <0.3 mg/100 g) and coumestan (coumestrol; <0.01 mg/100 g) were also found (Thompson et al. 2006; Kuhnle et al. 2008). The high variability of phenolics has been reported both in pistachios (Gentile et al. 2007; Kuhnle et al. 2008; Seeram et al. 2006; Thompson et al. 2006) and in other nuts, depending on variety, environmental factors, growth, harvesting, ripening, processing, sampling, and analytical methods (Gentile et al. 2007; Kuhnle et al. 2008, 2009; Ballistreri et al. 2009). The polyphenolic phytoalexin resveratrol (3,5,4΄-trihydroxystilbene) is an important bioactive polyphenol (Bowers et al. 2000). Stilbenes act against biotic and abiotic factors, defending plants from cold, heat, fungal infections, and the growth of molds. Resveratrol (trans-reveratrol, trans-3,5,4΄­trihydroxystilbene) has been detected in pistachio cultivars grown in Turkey and Italy, with a mean

199

Nut Bioactives Table 9.12 Mean Content of the Major Phenolic Compounds in Pistachios (mg/100 g Dry Matter) Phenolics Anthocyanins cyanidin-3-galactoside1 cyanidin-3-glucoside2 Total Flavonoids Proanthocyanidins3 Daidzein Genistein4 4

26.8 ± 9.0 3.9 ± 2.9 30.7

268.1 ± 8.31a 2.9 ± 1.1

Quercetin5 Daidzin5 Genistin5 Eriodictyol5 Luteolin5 Naringenin5 Total

2.7 ± 1.0 1.4 ± 0.1 1.2 ± 0.1 1.1 ± 0.3 0.9 ± 0.2 0.9 ± 0.2 0.1 ± 0.0 279.3

Stilbenes trans-resveratrol6

0.1 ± 0.1

Data reported by: 1  Bellomo, M. and Fallico, B., Journal of Food Composition and Analysis, 20, 352–9, 2007. 1,2,4,5,6  Ballistreri, G., Arena, E., and Fallico, B., Molecules, 14, 4358–69, 2009 3,4 Gentile, C., Tesoriere, L., Butera, D., Fazzari, M., Monastero, M., Allegra, M., and Livrea, M. A., Journal of Agricultural and Food Chemistry, 55, 643–8, 2007. 6  Tokuşog ˘ lu, O., Unal, M. K., and Yemis, F., Journal of Agricultural and Food Chemistry, 53, 5003–9, 2005; Grippi, F., Crosta, L., Aiello, G., Tolomeo, M., Oliveri, F., Gebbia, N., and Curione, A., Food Chemistry, 107, 483–8, 2008. a  Expressed as mg of cyanidine chloride equivalents/100 g dry matter.

content of about 0.1 mg/100 g in both origins (Tokuşoğlu et al. 2005; Gentile et al. 2007; Grippi et al. 2008; Ballistreri et al. 2009).

Neutral Lipids The pistachio contains about 50–60% of oil (Ruggeri et al. 1998; Satil et al. 2003; Arena et al. 2007) and the prevailing lipid constituents are TGs more than 90% of the total glycerolipids content; Miraliakbari and Shahidi 2007; Chahed et al. 2008). Percentages of the different TG types in the oils of the pistachio from seven different geographic origins are reported in Table 9.13. The main TGs in pistachio oil are: OLO, OLL, OOO, LLL, OLP, LLP, and OOP; together they account for more than 85% of the total TGs (Dyszel and Pettit 1990; Holcapek et al. 2003; Ballistreri et al. 2010b). Both OLnO and SLO have been identified in pistachio oil in small amounts (~1% each; Holcapek et al. 2003). The principal component analysis and linear discriminant analysis (PCA and LDA) of TG types were successfully used to distinguish the geographic origin of pistachios. They indicate that the TG characteristics studied are suitable for authenticating the geographical origin of pistachio seeds (Ballistreri et al. 2010b). Table 9.14 reports the composition (%) of the major fatty acids of pistachio oils of different geographic origins. Oleic, linoleic, and palmitic acids are predominant with respect to the others fatty acids, in fact they account for more than 95% of the total fatty acids content (Arena et al. 2007; Miraliakbari and Shahidi 2007). Myristic acid, pentadecenoic acid, Z-(7)-hexadecenoic acid, vaccenic (Z-(7)-octadecenoic

200

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 9.13 Content (%) of the Most Relevant Triacylglycerol Types in Pistachio Oils

Triacylglycerols

Italy var. Bianca

LLLn LLL OLLn LnLP OLL LLP OLnP OLMo OLO OLP PLP GLO OOO OOP POP GOO SOO SOP

1.4 8.9 2.7 1.1 14.3 7.6 1.6 0.6 18.5 10.6 2.1 0.6 16.8 8.4 1.6 0.7 2.0 0.5

Turkey var. Siirt 1.2 10.1 2.7 0.8 15.3 7.9 1.3 0.4 19.2 10.5 1.6 0.3 16.3 7.8 1.0 0.9 1.8 0.2

Iran var. Akbari

United States var. Kerman

Syria var. Jalab

Tunisia var. Mateur

Greece var. Kerman

1.9 14.2 3.3 1.3 18.9 10.5 1.1 0.4 18.2 11.6 2.1 0.4 8.9 5.1 1.0 0.5 0.7 0.3

1.0 13.9 3.6 1.3 18.1 11.1 1.3 0.5 17.4 11.8 2.8 0.3 9.2 5.7 1.1 0.2 0.5 0.3

1.6 10.3 2.4 0.7 16.0 7.4 1.4 0.2 20.6 10.2 1.7 0.3 16.3 6.7 1.2 0.9 1.4 0.6

1.2 11.2 2.8 1.2 16.9 8.3 1.4 0.7 19.6 10.6 2.1 0.6 13.9 6.3 1.1 1.1 0.9 0.0

0.7 9.8 2.2 0.6 15.1 8.0 1.3 0.4 19.4 11.0 2.1 0.4 17.1 8.1 1.4 0.9 1.4 0.2

Data reported by Ballistreri, G., Arena, E., and Fallico, B., Italian J. Food Sci., 22, in press, 2010b.

Table 9.14 Mean Content (%) of the Major Fatty Acid of Pistachio Oils of Different Geographic Originsa Samples Italy Turkeyb Greece Iran Mean value

Palmitic (C16:0)

Palmitoleic (C16:1)

Stearic (C18:0)

Oleic (C18:1)

Linoleic (C18:2)

Linolenic (C18:3)

9.7 11.6 10.8 10.8 10.7

0.9 0.8 0.9 0.9 0.9

1.6 2.0 2.1 1.1 1.7

71.1 67.4 68.3 55.1 65.5

14.4 16.2 15.4 28.9 18.7

0.4 0.4 0.5 0.6 0.5

Data reported by  a,b  Arena, E., Campisi, S., Fallico, B., and Maccarone, E., Food Chem., 104, 403–8, 2007. Satil, F., Azcan, N., and Baser, K. H. C., Chem. Nat. Comp., 39, 322–4, 2003; Acar, I., Kafkas, E., Ozogul, Y., Dogan, Y., and Kafkas, S., Italian J. Food Sci., 20, 273–9, 2008; Chahed, T., Bellila, A., Dhifi, W., Hamrouni, I., M’hamdi, B., Kchouk, M., and Marzouk, B., Grasas y Aceites, 59, 51–6, 2008.

b

acid), and arachidic acid are also present in small amounts. Moreover, traces of margaric, behenic, and gadoleic acids have also been found in some pistachio genotypes (Acar et al. 2008). According to Satil et al. (2003), these minor acids do not exceed 0.5%. The variations of fatty acid composition may be due to cultivar. In addition, the other main factors that are known to affect total fatty acid composition, and especially oleic acid content, are latitude, climatic conditions, and the ripening stage of the fruit when harvested (Ranalli et al. 1997; Aparicio and Luna 2002; Chahed et al. 2008). The ratio between unsaturated and saturated fatty acids is about 6.5 and the ratio between oleic and linoleic acid is about 4.5. These data, as well as the distribution of fatty acids, show that pistachio oil is very similar to olive oil (Parcerisa et al. 1998; Belitz et al. 2004).

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Nut Bioactives

Polar Lipids Among tree nut oils, the pistachio oil possesses the highest amount of polar lipids (~17 g/kg) (Miraliakbari and Shahidi, 2007, 2008). Phospholipids are present in a major quantity (~11 g/kg) with respect to sphingolipids (~6 g/kg). The main phospholipid is phosphotidylcholine, followed by phosphotidylserine and phosphatidylinositol at 45, 40, and 20%, respectively (Miraliakbari and Shahidi 2007, 2008). Although a contribution of phospholipids to antioxidant capacity, and therefore to oxidative stability, has been found (Lee et al. 2002; Arranz et al. 2008), the biological relevance of it would be uncertain.

Phytosterols and Tocols Pistachio oil has ~100–280 mg/100 g phytosterols (Phillips et al. 2005; Kocygit et al. 2006; Miraliakbari and Shahidi 2007), of which ~85% has been reported to be β-sitosterol (Yousfi et al. 2002; Phillips et al. 2005; Arena et al. 2007). The Δ5-avenasterol and campesterol together account for ~12% of total sterols content. Other minor phytosterols have been found such as stigmasterol, clerosterol, campestanol, sitostanol, Δ5,24-stigmastadienol, Δ7-stigmastenol, Δ7-avenasterol, poriferasta-7,25-dienol, and β-sitostanol (Phillips et al. 2005; Arena et al. 2007; Miraliakbari and Shahidi 2007). Cumulatively, these minor sterols vary from 1 to 3%. The differences in phytosterols content could be due to a difference in varieties and thermal processes (Phillipis et al. 2005). Although sterols can undergo oxidation altering the sensorial properties of pistachio, their presence is important for the healthy properties: phytosterols are known for the capacity of lowering cholesterol both in plasma and in low density lipoprotein and may also exhibit antioxidant activity (Wang et al. 2002; Belitz et al. 2004; Kocygit et al. 2006). The total tocopherols concentration range from about 8 to 50 mg/100 g (Kocygit et al. 2006; Kornsteiner et al. 2006; Miraliakbari and Shahidi 2007; Arranz et al. 2008; Ballistreri et al. 2009). The γ-tocopherol is the major vitamin E isomer found in pistachios (>90% of total tocopherols content), followed by α-tocopherol (<5% of total tocopherols) and δ-tocopherol (~2% of total; Kocygit et al. 2006; Kornsteiner et al. 2006; Gentile et al. 2007). The γ-tocopherol has higher antioxidant activity compared to α-tocopherol, better preserving the lipidic fraction of the pistachio from oxidation (Belitz et al. 2004). A high loss of vitamin E has been observed in pistachios during sun-drying, in fact this thermal process causes a loss of about 38% of this vitamin (Ballistreri et al. 2009).

Chlorophylls and Xanthophylls The pistachio is the only nut containing chlorophyll. Both C-10 chlorophyll epimers (a and b) were found, with a prevalence of the a form (Bellomo and Fallico 2007). The highest chlorophyll content is in the unripe green pistachio while, with ripening, the level diminishes with the exception of the Italian pistachio that retained its green color also at maturity (Table 9.15). Lutein is the main carotenoids in the pistachio, also β-carotene was found but in a small amount (<2 mg/kg DM; Kornsteiner et al. 2006; Bellomo and Fallico 2007). As for chlorophylls, the lutein level Table 9.15 Mean Content of Chlorophylls and Xanthophylls in Pistachios (mg/kg Dry Matter) Chl a

Chl b

Chl tot

Lutein

Unripe

Iran Turkey

Sample

111.0 ± 5.66 150.6 ± 4.38

40.8 ± 3.00 49.7 ± 2.66

151.8 ± 8.05 200.3 ± 6.75

41.3 ± 1.64 52.1 ± 2.94

Intermediate

Turkey Turkey Greece Turkey Italy

89.4 ± 2.11 75.9 ± 4.07 27.8 ± 1.87 18.3 ± 1.69 113.3 ± 8.98

28.8 ± 0.85 28.3 ± 3.02 11.9 ± 1.98 7.1 ± 0.43 38.1 ± 2.62

118.2 ± 1.78 104.2 ± 7.01 39.7 ± 3.85 25.4 ± 1.58 151.3 ± 11.60

34.7 ± 2.58 31.8 ± 1.61 17.9 ± 2.71 18.1 ± 0.68 36.6 ± 1.63

Ripe

Source:  Data reported by Bellomo, M. and Fallico, B., J. Food Comp. Anal., 20, 352–9, 2007.

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depends upon the degree of ripeness and the origin of the pistachio (Table 9.15; Bellomo and Fallico 2007). The high level of chlorophylls and the presence of lutein contribute to the antioxidant activity of this nut.

Walnut Walnut, the seed of Juglans regia L. (Juglandaceae), is a traditional food in the Mediterranean, South America, and Asia, and is an ingredient in sauces, stuffing, snacks, appetizers, and dessert. Walnut trees are cultivated throughout southern Europe, northern Africa, eastern Asia, the United States, and western South America. China and the United States are the major producers, with about 25 and 20%, respectively, of the total world production (Crews et al. 2005). The walnut seed (kernel) represents from 40 to 60% of the nut weight, depending mainly on the variety (Labuckas et al. 2008). The seed has a good level of oil (~60%) in which polyunsaturated fatty acids predominate (Savage et al. 1999; Martinez et al. 2006), and contains a lower amount of α-tocopherol compared with almonds, hazelnuts, and pistachios (Miraliakbari and Shahidi 2007). In addition to oil, walnuts provide appreciable amounts of proteins (up to 24% of the walnut seed weight), carbohydrates (12–16%), fiber (1.5–2%), and minerals (1.7–2%; Sze-Tao and Sathe 2000). Among nuts, walnuts have the highest total phenolic and flavonoid contents and also possess the highest total antioxidant activity (Kornsteiner et al. 2006; Yang et al. 2009). Most phenolic compounds commonly identified in walnuts are phenolic acids, condensed tannins, and flavonoids, having serum cholesterol-modulating effects and strong antioxidant properties. These bioactive compounds play an important role in the prevention of chronic diseases.

Phenolics Walnut phenolics, similar to other nuts, reside in the highest concentration of their skin or kernel pellicle (~5% of the total weight), however the kernel may also contain antioxidative compounds (Colaric et al. 2005; Labuckas et al. 2008; Samaranayaka et al. 2008). Different phenolic acids, mainly ellagic and chlorogenic acids and dicarboxylic acid derivatives (glansreginins A and B), have been identified in walnuts, as well as flavonoids (catechin) and hydrolyzable tannins such as ellagitannins (glansrins A, B, and C; Fukuda et al. 2003; Ito et al. 2007; Gómez-Caravaca et al. 2008). These polyphenolic compounds are likely to protect the fatty acids from oxidation in walnuts (Shimoda et al. 2008). The most abundant polyphenolic compounds in the Chandler, Howard, and Hartley varieties result in glansreginins A and B, with a range of 76.3–335.6 and 35.5–99.7 mg of chlorogenic acid/kg of dry weight walnut, respectively. Aglycone and glycosilated ellagic acid account for 64–75% of total phenols (Gómez-Caravaca et al. 2008). These differences could be ascribed to agronomic and environmental factors that play important roles in the phenolic composition (Tomas-Barberan and Espin 2001). The total phenolic content of walnuts is ~1500 mg of gallic acid equivalents/100 g of nut and about 7.5-, 5-, 3-, and 2.5-fold higher than the total phenolic content of almonds, hazelnuts, pistachios, and peanuts, respectively (Anderson et al. 2001; Kornsteiner et al. 2006; Yang et al. 2009). The total flavonoids and bound flavonoids content is ~700 and ~200 mg of catechin equivalents/100 g of nut, respectively (Yang et al. 2009). The total flavonoids content of walnuts is about eight-, seven-, five-, and fourfold higher than the one in almonds, hazelnuts, pistachios, and peanuts, respectively. Bound phytochemicals cannot be digested by human enzymes and could survive stomach and small intestine digestion to reach the colon (Sosulski et al. 1982). It may be hypothesized that nuts with bound phytochemicals can be digested and absorbed at different sites of the gastrointestinal tract and play their unique health benefits (Yang et al. 2009).

Neutral Lipids Such as other tree nut oils, TGs are the major lipid class in walnut oil (>95 g/100 g oil; Miraliakbari and Shahidi 2007). There have been 13 TGs identified in walnut oil: LLL, OLL, LLLn, PLL, OLLn, OOL, POL, SLL, PLLn, LLnLn, OOO, SOL, and PLnLn (Oliveira et al. 2002; Amaral et al. 2004). The LLL

203

Nut Bioactives Table 9.16 Triacylglycerols Composition (%) of Walnut Oil Triacylglycerols LLL OLL LLLn PLL OLLn OOL POL POL + SLL PLLn LLnLn SLL OOO SOL PLnLn

Oliveira et al. (2002) 23.7 19.3 18.1 7.7 7.2 7.3 4.4 — 3.3 4.4 1.4 1.3 0.2 —

Amaral et al. (2004) 37.7 18.5 18.4 8.7 5.0 4.0 — 3.6 2.1 1.7 — traces — traces

is the major TG (23.7–37.7%), followed by OLL (18.5–19.3%), and LLLn (18.1–18.4%). Together they account for more than 60–75% of the total TGs content (Table 9.16). Among nut oils, walnut oil contains the lowest amount of monounsaturated fatty acids (MUFA; ~15–20 g/100 g of oil), and the highest level of polyunsaturated fatty acids (PUFA; >50 g/100 g of oil; Oliveira et al. 2002; Amaral et al. 2003; Crews et al. 2005; Miraliakbari and Shahidi 2007) which improve serum lipid profiles and reduce coronary heart disease (Zambon et al. 2000; Albert et al. 2002). But, for its high content of PUFA, walnut oil is relatively unstable when compared to other common nut oils (Savage et al. 1999). Table 9.17, gives the fatty acid composition of walnut oils from seven different countries. Fatty acid composition has been reported to differ significantly with variety and origin (Garcia et al. 1994; Crews et al. 2005). Independently from cultivars, varieties and geographical origins, the major fatty acids in walnut oil are linoleic acid followed by oleic, linolenic, palmitic, and stearic acids (Oliveira et al. 2002; Amaral et al. 2003; Crews et al. 2005; Martinez et al. 2006; Miraliakbari and Shahidi 2007). Besides these five main fatty acids, myristic, palmitoleic, margaric, arachidic, and eicosenoic acids have been detected in small amounts (<0.1%). Trans isomers of unsaturated fatty acids as well as γ-linolenic (C18:3ω6), C20:2ω6, C22:2ω6, and C22:5ω3 have also been detected in trace amounts (Amaral et al. 2003).

Polar Lipids Walnut oil contains ~2% of polar lipids (Miraliakbari and Shahidi 2007). Among polar lipids, phospholipids (phosphotidylserine, phosphatidylinositol and phosphotidylcholine) are the main class (~65% of total polar lipids content). Phosphotidylcholine is present in a major amount, more than phosphotidylserine and phosphatidylinositol, with a mean content of 0.38, 0.36, and 0.22 g/100 g of oil, respectively. Sphingolipids have a mean content of ~0.48 g/100 g of oil (Miraliakbari and Shahidi 2007, 2008).

Phytosterols and Tocols Walnut oil contains the highest amount of total sterols among nut oils (150–290 mg/100 g of oil; Miraliakbari and Shahidi, 2007, 2008). Table 9.18 lists the main sterols identified in the oils of six walnut cultivars. The main phytosterols are β-sitosterol >Δ5-avenasterol > campesterol. The β-sitosterol comprises ~85% of total sterol, followed by Δ5-avenasterol (~8%), campesterol (~4%), clerosterol (~1.5%), and finally cholesterol (~1%; Amaral et al. 2003; Crews et al. 2005; Martinez et al. 2006; Miraliakbari and

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Table 9.17 Fatty Acid Composition (%) of Walnut Oils Fatty acids Myristic (C14:0) Pentadecylic (C15:0) Palmitic (C16:0) Palmitoleic (C16:1) Margaric (C17:0) Heptadecenoic (C17:1) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Linolenic (C18:3) Arachidic (C20:0) Eicosenoic (C20:1) Behenic (C22:0) Docosenoic (C22:1) Lignoceric (C24:0)

China

France

Hungary

India

Italy

Spain

United States

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

5.1–5.4

6.5–7.3

5.8–7.7

5.7–5.8

7.3–8.1

7.1–7.5

6.3–8.0

0.1

0.1

0.1

nd–0.1

nd–0.1

nd–0.1

0.1

0.1

nd–0.1

nd

nd

nd–0.1

nd–0.1

nd–0.1

nd

nd

nd

nd

nd

nd

nd–0.1

2.7–2.9

1.7–2.9

2.1–2.2

2.5–2.6

2.2–2.9

1.9–2.8

2.3–3.1

16.9–21.0

15.1–18.9

17.4–22.2

19.8–20.6

14.5–15.3

14.3–19.2

13.4–16.7

60.1–64.1

57.4–64.3

58.3–60.8

57.3–58.1

60.2–63.1

57.6–62.5

59.6–63.1

10.3–10.4

11.3–15.4

10.8–11.6

12.6–13.5

11.8–14.3

12.4–13.2

12.5–15.5

0.1

nd–0.1

nd–0.1

0.1

0.1

0.1

0.1

0.1

0.2–0.3

0.2

0.2

0.2

0.2

0.2

nd

nd

nd

nd–0.1

nd

nd

nd–0.1

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd–0.1

Source: Data reported by Crews, C., Hough, P., Godward, J., Brereton, P., Lees, M., Guiet, S., and Winkelmann, W., J. Agric. Food Chem., 53, 4843–52, 2005. Note:  nd < 0.1.

Table 9.18 Sterol Content (mg/100 g of Oil) of Oil Extracted from Six Walnut Cultivars Cultivar cholesterol campesterol stigmasterol clerosterol β-sitosterol Δ5-avenasterolb Δ7-stigmasterol Δ7-avenasterol total

Franquette

Marbot

Mayette

Mellanaise

Lara

Parisienne

1.4 ± 0.03 8.6 ± 0.20 0.5 ± 0.07 1.5 ± 0.11 138.3 ± 1.80 7.3 ± 0.11 0.7 ± 0.05 1.4 ± 0.29 159.0 ± 5.00

2.0 ± 0.04 9.6 ± 0.30 0.8 ± 0.03 2.0 ± 0.06 175.7 ± 0.24 11.4 ± 0.41 nd

3.8 ± 0.12 8.1 ± 0.17 0.6 ± 0.07 1.5 ± 0.08 151.4 ± 0.49 13.3 ± 0.01 0.9 ± 0.01 nd

1.4 ± 0.03 6.5 ± 0.03 0.5 ± 0.03 1.1 ± 0.06 109.8 ± 0.06 7.3 ± 0.03 nd

0.9 ± 0.09 10.8 ± 0.18 nda

0.8 ± 0.04 6.1 ± 0.02 nd

1.2 ± 0.09 170.6  ± 0.21 9.8 ± 0.09 nd

0.5 ± 0.02 127.1 ± 0.15

2.8 ± 0.06 196.1 ± 0.36

2.0 ± 0.04 109.3 ± 0.04 2.5 ± 0.02 nd nd

1.1 ± 0.06 202.6 ± 1.67

179.6 ± 0.73

120.7 ± 0.06

Source: Data reported by Amaral, J., Casal, S., Pereira, J., Seabra, R., and Oliveira, B., J. Agric. Food Chem., 51, 7698–7702, 2003. a Not detected. b  β-sitostanol + Δ5-avenasterol.

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Shahidi 2007). Moreover, minor amounts (<1%) of stigmasterol, Δ5,24-stigmastadienol, Δ7-stigmasterol, and Δ7-avenasterol have been detected (Oliveira et al. 2002; Amaral et al. 2003; Crews et al. 2005). Besides sterols, methylsterols have also been found by Martinez et al. (2006) in walnut oil of three different varieties (Criolla, Chandler, and Franquette). Cycloartenol, cycloeucalenol, and 24-methylenecycloartanol result in the major components of the methylsterol fraction of walnut oil with ranges of 63–86%, 8–11%, and 5–27%, respectively. Trace amounts of cyclolaudenol have also been detected. Unlike sterol composition, methylsterols have greater differences among varieties due to the genotype as well as the crop year. The fact that the walnut oil contains high amounts of cycloartenol reaffirms the pathway model of sterol biosynthesis postulated by Guo et al. (1995): the cycloartenol transformation to sitosterol (Martinez et al. 2006). The γ-tocopherol is the predominant vitamin E homologue present in walnut oil (~205–525 mg/kg), followed by δ- (~12–34 mg/kg), and α-tocopherol (~3–10 mg/kg); β-tocopherol has been detected in traces (Oliveira et al. 2002; Crews et al. 2005; Li et al. 2007). The γ-tocopherol is considered to be the main contributor to the total antioxidant activity of walnut oil (Li et al. 2007). The total tocopherols content range from 149 to 632 mg/kg (Oliveira et al. 2002; Crews et al. 2005; Kocygit et al. 2006; Li et al. 2007; Miraliakbari and Shahidi 2007, 2008; Arranz et al. 2008). Even though there are marked differences in the content of total tocopherols in different walnut varieties, the relative proportions of the major tocopherols are rather similar. The antioxidative properties of tocopherols have been shown to prevent the oxidation of polyunsaturated fatty acids and stabilize the free fatty acids of walnut oil (Savage et al. 1999).

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10 Nut Bioactives: Phytochemicals and LipidBased Components of Brazil Nuts, Cashews, Macadamias, Pecans, and Pine Nuts Biagio Fallico, Gabriele Ballistreri, Elena Arena, and Özlem Tokus¸ og˘lu Contents Brazil Nut.................................................................................................................................................213 Phenolics.............................................................................................................................................214 Neutral Lipids.....................................................................................................................................214 Polar Lipids.........................................................................................................................................214 Phytosterols and Tocols......................................................................................................................215 Cashew.....................................................................................................................................................216 Phenolics.............................................................................................................................................216 Neutral Lipids.....................................................................................................................................216 Polar Lipids.........................................................................................................................................217 Phytosterols and Tocols......................................................................................................................217 Macadamia...............................................................................................................................................217 Phenolics.............................................................................................................................................218 Neutral Lipids.....................................................................................................................................218 Polar Lipids.........................................................................................................................................219 Phytosterols and Tocols......................................................................................................................219 Pecan........................................................................................................................................................219 Phenolics.............................................................................................................................................219 Neutral Lipids.................................................................................................................................... 220 Polar Lipids........................................................................................................................................ 220 Phytosterols and Tocols..................................................................................................................... 220 Pinenut.................................................................................................................................................... 221 Phenolics............................................................................................................................................ 221 Neutral Lipids.................................................................................................................................... 221 Polar Lipids........................................................................................................................................ 223 Phytosterols and Tocols..................................................................................................................... 224 References............................................................................................................................................... 224

Brazil Nut The Brazil nut (Bertholletia excelsa H.B.K.) (Figure 10.1) is a native plant from the Amazonian region (Brazil, Peru, Colombia, Venezuela, and Ecuador). Its fruit called ouriço, a capsule of a spherical format with a hard shell that contains around 15–24 seeds, is processed by breaking the pod and shell of the individual nut before exportation, usually to North America and Europe where seeds are used

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Brazil nut (Bertholletia excelsa H.B.K.) Figure 10.1  (See color insert) Brazil nut.

most extensively in confections (Sun et al. 1987). The Brazil nut is also used to produce oil, soaps, and shampoos and for the cosmetic industries (Bonelli et al. 2001). The Brazil nut has a well-known nutritional value due to its high content of lipids (50–70%), proteins (10–20%), and carbohydrates (10–20%; Elias and Bressani 1961; Gonçalves et al. 2009); it is high in essential fatty acids (mainly oleic and linoleic acids), amino acids containing sulfur (particularly methionine and cysteine), vitamins (A and E), fibers and minerals such as zinc and especially selenium. In fact, the Brazil nut is the best source of selenium from plant-based foods; selenium is a mineral needed for proper thyroid and immune function and it is known for its antioxidant activity (Nwanna et al. 2008). Brazil nuts promote diverse benefits to the health of humans owing to its anticarcinogenic properties (Ip and Lisk 1994). This effect seems to be related to the high content of selenium, mainly present in the form of Se-methionine, which is in a bioavailable form, and is also associated with compounds of low molecular weight (Kannamkumarath et al., 2002).

Phenolics There is a lack of information regarding the phenolics profile in a Brazil nut. However, the total phenolic content has been determined with the Folin-Ciocalteu method (112 mg/100 g of gallic acid equivalents) and summing the free and the bound phenolic contents (169 mg/100 g of the nut; Kornsteiner et al. 2006; Yang et al. 2009). The total flavonoid content was found to be ~108 mg/100 g (Yang et al. 2009). The content of phytoestrogens (isoflavones + lignans, mainly genistein and secoisolariciresinol) has been reported to be ~0.9 mg/100 g of the dry nut (Kuhnle et al. 2008).

Neutral Lipids Brazil nuts have a high oil content (>65%; Miraliakbari and Shahidi, 2007, 2008). Triacylglycerols (TGs) are the major lipid class (>95 g/100 g oil; 2007). Over 20 TGs have been identified in Brazil nut oil, which include: POL, SOL, OOO, PLP, POO, PoOO, PPoO, PoPoO, and SOO, together they account for ~80% of the total TGs content (Rodrigues et al. 2005). The mean fatty acid composition (%) of a Brazil nut is reported in Table 10.1. The Brazil nut contains an especially high concentration of palmitic and stearic acids (13.8 and 10.9%, respectively). The saturated fat content of a Brazil nut is the highest among all nuts (~20% of total fat). Oleic and linoleic acids together account for more than 70% of the total fatty acids content (Miraliakbari and Shahidi 2007; Chunhieng et al. 2008; Yang et al. 2009). The content of the ω-3 fatty acid (α-linolenic acid) is near 7% of total fat (Yang 2009).

Polar Lipids Brazil nut oil contains ~2% of polar lipids (Miraliakbari and Shahidi 2007). Among polar lipids, phospholipids (phosphatidylinositol, phosphotidylcholine, phosphatidylethanoalmine, phosphotidylserine and phosphatidic acid) are the main class. Phosphatidylinositol and phosphotidylcholine range from 16 to 31% and 24 to 53% of total phospholipids content, respectively. Phosphotidylserine, phosphatidic acid, and phosphatidylethanoalmine account for about 30, 24, and 21% of total phospholipids content,

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Nut Bioactives Table 10.1 Mean Content (%) of the Major Fatty Acid of Brazil Nut Oil Fatty Acids

%

C 12:01 C 14:02 C 16:02 C 16:12 C 17:03 C 18:04 C 18:14 C 18:24

0.2 0.1 13.8 0.3 0.2 10.9 35.4 38.3

C 18:35 C 20:06 C 20:16 C 22:07 C 22:17

0.2 0.5 0.2 0.1 0.3

Source: Data reported by: 1,2,4  Chunhieng, T., Hafidi, A., Pioch, D., Brochier, J., and Montet, D., J. Braz. Chem. Soc., 19, 1374–80, 2008; 2,4,6  Miraliakbari, H. and Shahidi, F., J. Food Lipids, 15, 81–96, 2007; 2–7  Yang, J., Liu, R. H., and Halim, L., LWT-Food Sci. Technol., 42, 1–8, 2009.

Table 10.2 Content (mg/ g of Oil) of Major Sterols of the Brazil Nut Oil Sterols

Phillips et al. (2005)

Miraliakbari and Shahidi (2007)

Yang (2009)

nd 0.65 0.06 0.02 0.13 0.86

0.12 1.11 0.22 0.12 nd 1.57

nd 1.32 0.58 0.03 nd 1.93

Cholesterol β-sitosterol Stigmasterol Campesterol Δ5-avenasterol Total Note: nd: not detected.

respectively (Miraliakbari and Shahidi 2007; Chunhieng et al. 2008). Sphingolipids are present in small amounts (<1% of total lipid content).

Phytosterols and Tocols The total sterols content of Brazil nut oil ranges from 0.9 to 1.9 mg/g (Phillips et al. 2005; Miraliakbari and Shahidi 2007; Yang 2009). The main phytosterol is β-sitosterol, followed by stigmasterol, campesterol, and Δ5-avenasterol (Table 10.2). Moreover small amounts of 22-nordehydrocholesterol, 24-methylenecholesterol, and Δ7-stigmasterol have also been detected (Miraliakbari and Shahidi 2007; Chunhieng et al. 2008). The total tocopherols content is ~20 mg/100 g of the nut (Kornsteiner et al. 2006; Miraliakbari and Shahidi 2007; Yang 2009). The γ-tocopherol is the major tocopherol (~15 mg/100 g of nut) followed by α- and δ-tocopherol (~2 and 1 mg/100 g of the nut, respectively). Synergistic effects of Se and vitamin E as well as other antioxidants play an important role in the prevention of prostate cancer cells (Zu and Ip 2003).

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Cashew (Anacardium occidentale L.) Figure 10.2  (See color insert) Cashew nut.

Cashew The cashew (Anacardium occidentale L.) (Figure 10.2) is one of the nut crops cultivated in the tropical regions of India, Brazil, and Africa. India is the largest producer and exporter of the cashew kernel, accounting for almost 50% of the world export (Paramashivappa et al. 2001). The kernel of a cashew nut valued in trade is covered with a thin reddish-brown skin or testa. The testa has been reported to be a good source of hydrolysable tannins (Pillai et al. 1963) with catechin and epicatechin as the major polyphenols (Mathew and Parpia 1970). There are many reports describing the radical scavenging activities of the by-products of a cashew nut (Amorati et al. 2001; Trevisan et al. 2006; Kamath and Rajini 2007). The cashew nut is a good source of proteins (20%), carbohydrates (23%), and fats (45%; Bhattacharjee et al. 2003). Of the fat, 56% is oleic acid (ω-9) and 18% is linoleic acid (ω-6; Venkatachalam and Sathe 2006; Mexis and Kontominas 2009; Yang 2009). The consumption of cashew nuts may prevent cardiovascular diseases and lower low density lipoprotein (LDL) without affecting high density lipoprotein (HDL; Baggio et al. 1988).

Phenolics The cashew nut shell liquid is a viscous liquid that surrounds the edible kernel of a cashew nut. It is an important by-product of cashew nut production, which is used in many industrial applications such as paints, foundry chemicals, and special coatings (Menon et al. 1985) and even for gasoline stabilization (Castro Dantas et al. 2003). The major phenolic constituents of the cashew nut shell liquid are anacardic acids that have antioxidant and antitumor activities and cardols and cardanols that have uses in polymer formulations and resins (Kubo et al. 1993; Ikeda et al. 2002; Kubo et al. 2006). Cashew nut shell liquid contains ~350 g/kg of alkyl phenols, anacardic acids while the cashew nut has only 0.6 g/kg of hydroxy alkyl phenols (Trevisan et al. 2006). The total phenolic content of the cashew skin and nut is ~240 mg GAE/g of skin and ~137 mg GAE/100 g of the nut, respectively (Kornsteiner et al. 2006; Kamath and Rajini 2007); more than 40% of the total polyphenol in the skin is reported to be constituted by (+) catechin and (−) epicatechin (Mathew and Parpia 1970). The total flavonoid content of the cashew nut is ~64 mg/100 g (~42 and ~22 mg/100 g of free and bound flavonoids, respectively; Yang et al. 2009). Isoflavones and lignans are also present in small amounts (0.012 and 0.170 mg/100 g of the dry nut; Kuhnle et al. 2008).

Neutral Lipids The lipid content of a cashew nut ranges from 40 to 45% (Bhattacharjee et al. 2003; Venkatachalam and Sathe 2006). Oleic and linoleic acids are the major fatty acids and in fact together account for ~75% of total fatty acid content (Table 10.3). Palmitic and stearic acids represent, together, ~23% of total fatty acids content. Palmitoleic and linolenic are present in small amounts (0.5 and 0.2%, respectively). Traces

217

Nut Bioactives Table 10.3 Mean Content (%) of the Major Fatty Acids of Cashew Oil Authors Venkatachalam and Sathe (2006) Mexis and Kontominas (2009) Yang (2009) Mean value

Palmitic (C16:0)

Palmitoleic (C16:1)

Stearic (C18:0)

Oleic (C18:1)

Linoleic (C18:2)

Linolenic (C18:3)

10.7 13.0 9.9 11.2

0.5 0.6 0.4 0.5

9.3 17.8 8.7 11.9

61.1 49.1 57.2 55.8

16.8 17.0 20.8 18.2

0.3 0.2 0.2 0.2

Macadamia (Macadamia tetraphylla) Figure 10.3  (See color insert) Macadamia.

of myristic, pentadecanoic, heptadecanoic, elaidic, arachidic, behenic, and lignoceric acids have also been detected (Mexis and Kontominas 2009; Yang 2009).

Polar Lipids The cashew nut contains ~3% of polar lipids (Koaze et al. 2001). The classes of polar lipids found in cashew nuts are cerebrosides and ceramides. The cerebroside concentration is ~0.04 mg/g of nut (Fang et al. 2005). Sphingolipids are important in biological systems and have preventive effects on colon ­carcinogenesis (Merill et al. 1997).

Phytosterols and Tocols The total phytosterols content of cashew nuts range from 1 to 2 mg/g of oil (Phillips et al. 2005; Yang 2009). The β-sitosterol represents ~89% of total sterols content, followed by stigmasterol and campesterol that together represent ~10% of total sterols content (Yang 2009). The Δ5-avenasterol, sitostanol, and campestanol have also been detected (Phillips et al. 2005). Phillips et al. (2005) have found higher levels of Δ5-avenasterol (~10% of total sterols content) and smaller amounts of stigmasterol (<1% of total sterols content). The mean content of total tocopherols is ~6 mg/100 g of oil. The γ-tocopherol account for more than 90% of total tocopherols content; α-, β-, and δ-tocopherol have also been detected (Kornsteiner et al. 2006; Yang 2009).

Macadamia The macadamia is a large evergreen tree indigenous to the coastal rainforests of Australia that belongs to the botanical family of Proteaceae (Figure 10.3). Australia is the largest producer of macadamia nuts followed by the United States and South Africa (Borompichaichartkul et al. 2009). There are two species, Macadamia tetraphylla and integrifolia, which produce edible nuts; these species are also known as a rough-shell and a smooth-shell type, respectively, for their characteristic surface texture of the shells (Koaze et al. 2002).

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The macadamia nut is rich in monounsaturated fatty acids, with oleic acid, which is claimed to be a potent inhibitor of fatty acid and cholesterol synthesis (Natali et al. 2007), contributing to more than 70% of the total fatty acids. This unsaturated fatty acid can help the decrease of cholesterol and triglyceride levels, thus lowering the risk of heart disease (Grag et al. 2003).

Phenolics The total phenolics content of a macadamia is about 498 mg/100 g of the nut (Yang et al. 2009). This value is higher with respect to almonds, Brazil nuts, hazelnuts, and pistachio nuts. Moreover macadamias have the highest bound phenolics content among nuts (~460 mg/100 g of the nut; Yang et al. 2009).

Neutral Lipids The macadamia has a high oil content of about 75 mg/100 g of the nut (Kaijser et al. 2000; Yang 2009). The major fraction of macadamia oil is represented by TGs accounting for about 80% of total lipids (Koaze et al. 2001). Even if 20 TGs have been detected in macadamia oil, the 10 TGs reported in Table 10.4 accounted for over 75% of the total TGs content (Holcapek et al. 2003; Lee et al. 2005; Rodrigues et al. 2005). The fatty acids composition (%) of macadamia oil is reported in Table 10.5. The main fatty acid is oleic ranging from 49.6 to 65.1%, followed by palmitoleic (17.3–30.8%) and palmitic (8.4–9.4%); together they account for about 90% of total fatty acids content (Gummeson et al. 2000; Rodrigues et al. 2005; Yang 2009). Traces of eicosatrienoic acid (C20:3) and behenic acid (C22:0) have also been detected (Yang 2009). Table 10.4 Content (%) of the Major Triacylglycerols of Macadamia Oil Triacylglycerols OOO SOL + OOO GOM + OOO OPoO OPO PoOPo GPoPo + POPo OOS POP

Holcapek et al. (2003)

Lee et al. (2003)

Rodrigues et al. (2005)

19.4 nd 19.4 16.1 9.9 8.2 6.1 nd 1.2

23.3 nd nd 20.8 13.6 8.8 nd 6.5 5.8

nd 24.0 nd 21.6 14.4 6.7 nd 7.2 1.9

Notes: nd: not detected. M: myristic, Po: palmitoleic, P: palmitic, L: linoleic, O: oleic, S: stearic, G: gadoleic.

Table 10.5 Fatty Acids Composition (%) of Macadamia Oil Fatty acids Myristic (C14:0) Palmitic (C16:0) Palmitoleic (C16:1) Stearic (C18:0) Oleic (C18:1) Linoleic (C18:2) Linolenic (C18:3) Arachidic (C20:0) Gadoleic (C20:1) Note: nd: not detected.

Gummeson et al. (2000) 1.8 9.4 30.8 2.2 49.6 2.5 0.1 1.6 1.8

Rodrigues et al. (2005) 1.0 9.4 19.3 3.4 59.8 2.0 0.1 2.6 2.5

Yang (2009) 1.0 8.4 17.3 3.2 65.1 2.3 0.1 2.3 nd

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Polar Lipids At maturity the Macadamia nut contains ~3% of polar lipids (Koaze et al. 2002). During drying and roasting, polar lipids increase from 2 to 7% (Koaze et al. 2001). Thermal treatments are essential for the preservation of the nuts and the development of their good flavor and taste. Among polar lipids, phospholipids (phosphatidylethanoalmine and phosphotidylcholine) are the main class accounting for ~85% of total polar lipids, followed by cerebrosides (~7%), acylsteryl-glucosides (~6%) and steryl-glucosides (~1%). Monoglycosyl-diglycerides have also been found in trace amounts (Koaze et al. 2002).

Phytosterols and Tocols The total phytosterols content of cashew nut ranges from 1 to 1.9 mg/g of oil (Kaijser et al. 2000; Phillips et al. 2005; Yang 2009). The β-sitosterol represents ~85% of total sterols content, followed by Δ5-avenasterol (~10%), stigmasterol (~5%), and campesterol (~1%) (Kaijser et al. 2000; Yang 2009). The mean content of total tocopherols is ~5 mg/100 g of oil. The α- and δ-tocopherol are the principal vitamin E isomer found in the macadamia nut (Kaijser et al. 2000; Yang 2009).

Pecan Pecans (Carya illinoinensis) (Figure 10.4), belonging to the Juglandaceae family, are an indigenous crop of the United States. Pecans are distributed over an area of geographic and climatic variation extending from northern Illinois and southeastern Iowa to the gulf coast of the United States (Villareal-Lozoya et al. 2007). The United States produces more than 80% of the world’s pecans. A pecan nut is a high-energy food (~690 kcal/100 g) as lipids [up to 75% (w/w)] and carbohydrates [up to 18% (w/w)] make up the bulk of the seed kernel weight (Venkatachalam et al. 2007). Pecan nuts are rich in proteins, vitamins, especially vitamin E, calcium, magnesium, potassium, zinc, fibers, and antioxidants (Taipina et al. 2009). Pecan oil is very low in saturated fatty acids (<9%), only high-oleic safflower (4–8%) and canola (<7.0%) oils are lower (Toro-Vazquez et al. 1999). Pecan kernels may improve human serum lipid profile and lower LDL levels, due to their high monounsaturated fatty acid content (Rajaram et al. 2001). The presence of phenolic compounds with high antioxidant capacity in the kernel and shell indicates that pecan can be considered an important dietary source of antioxidants, in fact the pecan has been ranked among foods with the highest phenolic content (Wu et al. 2004).

Phenolics The total phenolic content of pecans range from 1284 to 2016 mg of gallic acid equivalents/g of kernel, depending on cultivar, grown location, and storage conditions (Wu et al. 2004; Kornsteiner et al. 2006; Villareal-Lozoya et al. 2007). Proanthocyanidins or condensed tannins have been reported in pecan kernels with a mean content of 34 mg of catechin equivalents/g of a defatted kernel (Villareal-Lozoya et al. 2007). These types of compounds have biological activities such as antioxidant and antimutagenic properties, which are affected by

Pecan (Carya illinoinensis) Figure 10.4  (See color insert) Pecan.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

the degree of polymerization, the monomer structure, and the bond type between monomers (Grimmer et al. 1992). The main phenolic compounds are gallic and ellagic acids, ranging from 0.6 to 1.3 mg/g and 2.5 to 4.7  mg/g of a defatted kernel, respectively. Catechin and epicatechin are present in trace amounts (Villareal-Lozoya et al. 2007).

Neutral Lipids Pecan nuts contain 70–75% of oil, depending on cultivars, growing conditions, maturity, variety, and past productivity of the tree (Toro-Vazquez et al. 1999; Wakeling et al. 2001; Venkatachalam et al. 2007). Triacylglycerols (TGs) represent the major lipid class (~96% of total lipid content; Miraliakbari and Shahidi 2007). In general, pecan oil is mainly composed of oleic acid (~65%), linoleic acid (~25%), and palmitic acid (~6%) with small concentrations of palmitoleic, stearic, linolenic, and arachidic acids, which together account for ~4% of total fatty acids content (Table 10.6). Margaric, gadoleic, behenic, and eicosatrienoic acids have also been found in small amounts (<2%; Yang 2009).

Polar Lipids Phospholipids are the main polar lipids class with a level of about 1 g/100 g of pecan oil. The sphingolipids content is ~0.4 mg/100 g of pecan oil. Phosphatidylcholine and phosphatidylserine are the most abundant phospholipids (0.23–0.52 and 0.24–0.47 g/100 g of oil, respectively), followed by phosphatidylinositol (0.2–0.7 g/100 g of oil; Miraliakbari and Shahidi 2007, 2008).

Phytosterols and Tocols The total phytosterols content of the pecan nut ranges from 1.5 to 2.0 mg/g of oil (Ryan et al. 2006; Miraliakbari and Shahidi 2008; Yang 2009). The β-sitosterol represents ~80% of total sterols content, followed by stigmasterol (~17%) and campesterol (~3%; Yang 2009). The total tocopherols content range from 10 to 27 mg/100 g of pecan oil (Villareal-Lozoya et al. 2007; Miraliakbari and Shahidi 2008; Taipina et al. 2009). Variables such as genetics, environment, maturity and storage conditions may affect tocopherol content of pecans (Rudolph et al. 1992).

Table 10.6 Mean Content (%) of the Major Fatty Acids of Pecan Oil of Different Cultivars Cultivars Desirablea Kiowaa Pawneea Shawneea Western Schleyb Wichitab Mean value a

Palmitic (C16:0)

Palmitoleic (C16:1)

Stearic (C18:0)

Oleic (C18:1)

Linoleic (C18:2)

Linolenic (C18:3)

Arachidic (C20:0)

5.65 5.48 5.34 5.53 5.65 5.90 5.59

0.03 0.03 0.03 0.06 0.05 0.03 0.04

1.72 2.30 2.49 1.93 2.51 2.29 2.21

61.63 66.36 68.18 72.43 61.80 65.68 66.01

29.21 24.69 22.38 18.55 27.98 24.59 24.56

1.23 1.12 1.03 0.98 1.23 1.19 1.13

0.06 0.07 0.10 0.09 0.13 0.09 0.09

Venkatachalam, M., Kshirsagar, H. H., Seeram, N., Heber, D., Thompson, T. E., Roux, K. H., and Sathe, S. K., Journal of Agricultural and Food Chemistry, 55, 9899–907, 2007; Villareal-Lozoya, J. E., Lombardini, L., and CisnerosZevallos, L., Food Chemistry, 102, 1241–9, 2007. b Wakeling, L. T., Mason, R. L., D’Arcy, B. R., and Caffin, N. A., Journal of Agricultural and Food Chemistry, 49, 1277–81, 2001; Venkatachalam, M., Kshirsagar, H. H., Seeram, N., Heber, D., Thompson, T. E., Roux, K. H., and Sathe, S. K., Journal of Agricultural and Food Chemistry, 55, 9899–907, 2007.

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Nut Bioactives

Pinenut The species of genus Pinus, largely diffused both in Europe and North America, produce seeds edible with a good level of healthy substances. The most common pine seeds consumed by humans come from five pine tree species: the European stone pine, Pinus pinea, the Mexican Pinus cembroides, the Asiatic Pinus koraiensis and Pinus sibirica, and the Californian Pinus monophylla (Wolff and Bayard 1995; Wolff and Marpeau 1997). Pinus pinea is the specie principally widespread in the Mediterranean area (Spain, Portugal, Italy, Greece, Albania, and Turkey), where there is its highest production and consumption. It is called the “Stone Pine” and the “Umbrella Nut” as the tree grows in stony ground and shaped like an umbrella. Pine nuts (Figure 10.5), are consumed raw and roasted and as ingredients in many traditional dishes. They contain adequate amounts of all of the essential amino acids as recommended by FAO/WHO for an adult (Venkatachalam and Sathe 2006), and are a source of several nutrients, such as vitamins (B1, B2, and K) and minerals especially potassium and phosphorus (Dinsmore et al. 2003; Nergiz and Dönmez 2004). The composition of the pine nut shows variation among the species and even some subspecies, depending on geographical and climatic conditions (Sagrero-Nieves 1992).

Phenolics Total phenolics contents of the chloroform/methanol extract, expressed as equivalents of gallic acid, are about 423 mg/kg of oil equivalents (Miraliakbari and Shahidi 2008). This value is comparable with those of Brazil nuts and higher with respect to almond, hazelnut, and pistachio oils (Miraliakbari and Shahidi 2008).

Neutral Lipids The pine nut oil content varies from 31 to 75%, depending on the species (Wolff and Bayard 1995; Ruggeri et al. 1998; Nergiz and Dönmez 2004; Ryan et al. 2006; Miraliakbari and Shahidi 2008, Nasri et al. 2009). Triacylglycerols (TGs) represent the major lipid class 96%, while diacylglycerol, polar lipids, and free fatty acids are in lesser proportions (1.95%, 0.84%, and 0.93%, respectively; Nasri and Triki 2004; Miraliakbari and Shahidi 2007). The trioctadecylglycerols are the main TGs (65.8–77.2%) represented by LLO/LOL, OLnO, LLL, LOO, and LLPi, LOPi when the unusual polymethylene-interrupted unsaturated fatty acids with a cis-5 ethylenic bond were identified (Table 10.7). The high differences in the distribution of TGs, indicates that the TG content can vary among populations of the same species—even if they come from neighboring geographic areas—of different species, and overall is influenced by the experimental conditions (Imbs and Long 1996; Nergiz and Dönmez 2004, Nasri et al. 2009; Adhikari et al. 2010). Concerning the fatty acids composition, the presence of an uncommon series of polyunsaturated fatty acids has been highlighted. Conifer nuts contain a series of C18 and C20 polyunsaturated fatty acids in which the first double bond is in the Δ5 position, and the successive double bond is in the Δ9 or Δ11 position (Wolff and Bayard 1995). Pinolenic (cis-5, cis-9, cis12 18:3) acids exerting diverse physiological functions are used for the prevention or amelioration of hypercholesterolemia, thrombosis, and

Pine nut (Pinus pinea) Figure 10.5  (See color insert) Pine nut.

222

Table 10.7 TGs Distribution (% of Total TGs Content) in Pine Nuts of Different Origin and Species Turkey

TGsb

Tunisia

Spain

France

Greece

Italy

Turkey

TGsc

Commercial Pine Nut Oil

OLnO LOL LLL PLO OOO POO PLL SLO OLnL SOO PLnO LPP PLnL OPP POS OSS SLS PSS

23.50 18.60 10.80 10.60 10.30 5.38 5.32 3.39 2.23 1.87 1.23 0.87 0.83 0.77 0.46 0.28 0.25 0.14

LLO LOO LLL PLO OLnO POO OOO SOO OLnL LPP OPP LLLn PLL PLnLn PLnL POS LLS SLS

24.38 17.82 14.80 11.31 7.93 7.53 6.74 2.13 1.79 1.06 0.93 0.66 0.63 0.61 0.59 0.48 0.34 0.21

24.63 19.20 13.30 10.86 7.11 7.61 7.12 2.55 1.79 1.12 0.92 0.40 0.72 0.57 0.59 0.65 0.30 0.30

23.50 19.40 11.13 11.45 7.49 8.04 8.03 2.68 2.34 0.89 0.95 0.41 0.89 0.49 0.73 0.77 0.34 0.38

23.63 19.38 11.42 10.84 7.33 7.74 8.29 2.45 2.60 1.15 0.95 0.55 0.87 0.54 0.74 0.69 0.38 0.37

24.22 18.32 13.56 10.75 7.40 7.70 7.59 2.31 2.16 1.11 0.99 0.55 0.72 0.57 0.69 0.64 0.35 0.28

24.05 18.39 13.58 11.22 7.51 7.80 7.31 2.27 1.93 1.22 1.05 0.56 0.68 0.51 0.59 0.61 0.28 0.24

LLPi OLL/LOL LOPi OOPi/OPiO LLL OLO/LOO POL/PLO/OPL PLL/LPL OOO PLPi/LPiP/LPPi others              

24.70 17.00 17.00 8.80 8.40 7.20 3.60 3.50 3.20 2.10 3.00              

Nergiz, C. and Dönmez, I.. Food Chem., 86. 365–8. 2004. Nasri, N., Tlili, N., Ammar, K. B., Khaldi. A., Fady. B., and Triki. S., International Journal of Food Sciences and Nutrition. 60 (S1), 161–9, 2009. c Adhikari, P.. Zhu. XM., Gautam, A.. Shin. J. A., and Hu. J. N.. Food Chem., 119, 1332–8, 2010. a

b

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

TGsa

223

Nut Bioactives Table 10.8 Principal Fatty Acids of Pine Nut Oil and Related Rangea Fatty Acids C14 C16 C16:1 C17 C18 C18:1 9 C18:1 11 C18:2 5,9 C18:2 9,12 C18:3 5,9,12 C18:3 9,12,15 C20 C20:1 11 C20:2 5,11 C20:2 11,14 C 20:3 5,11,14 C22 C24 Total Sum of Δ5 TSb TMb TPb

% 0.05 4.59 0.22 0.05 2.34 24.28 0.90 2.05 48.52 14.80 0.57 0.76 0.95 0.36 0.75 3.22 0.12 1.51 106.01 20.43 9.41 26.34 70.26

Range 0.00–0.05 3.27–6.49 0.09–0.35 0.03–0.05 1.60–3.47 14.37–38.60 0.22–2.03 0.14–3.62 44.84–59.60 0.35–21.78 0.17–1.51 0.40–1.33 0.74–1.37 0.10–0.76 0.49–0.99 0.90–7.09 0.11–0.13 0.00–3.02

 

Data reported by Wolff, R., and Bayard, C. C., J. Am. Oil Chem. Soc., 72, 1043–6, 1995; Nergiz, C. and Dönmez, I., Food Chem., 86, 365–8, 2004; Venkatachalam, M. and Sathe, S. K., J. Agric. Food Chem., 54, 4705–14, 2006; Miraliakbari, H. and Shahidi, F., J. Food Lipids, 15, 81–96, 2007; Adhikari, P., Zhu, XM., Gautam, A., Shin, J. A., and Hu, J. N., Food Chem., 119, 1332–8, 2010. b TS: total saturated fatty acids; TM: total monounsaturated fatty acids; TP: total polyunsaturated fatty acids. a

hypertension (Sugano et al. 1994). The distribution of fatty acids, as well as for TGs, is influenced by the species. Table 10.8 reports the distribution of the principal fatty acids of pine nut oil belonging to different species. In most species oleic and linoleic acids are the principal acids accounting for about 70% followed by the pinoleic acid (about 17%). In Pinus pinea oleic, linoleic acids account for about 81%, followed by palmitic acid (about 6%). Moreover, in all species, the sum of all Δ5 unsaturated fatty acids is high (about 23%) and the polyunsaturated fatty acids content is more than threefold higher with respect to monounsaturated fatty acids, while in Pinus pinea the sum of all Δ5 unsaturated fatty acids is about 3% and the ratio between polyunsaturated/ monounsaturated fatty acids is about 1.4 (Wolff and Bayard 1995; Nergiz and Dönmez 2004, Venkatachalam and Sathe 2006; Miraliakbari and Shahidi 2007; Adhikari et al. 2010).

Polar Lipids Sphingolipids are the main polar lipids with a level of about 0.28–0.57 g/100 g of pine nut oil. Phosphatidylcholine and phosphatidylserine are the most abundant phospholipids (0.14–0.37 and

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

0.15–0.33 g/100 g of oil, respectively), followed by phosphatidylinositol (0.07–0.19 g/100 g of oil; Miraliakbari and Shahidi 2007, 2008).

Phytosterols and Tocols Sterols in pine nut oil range from 0.14 to 0.43 g/100 g of oil (Phillips et al. 2005; Miraliakbari and Shahidi 2007; Nasri et al. 2007; Adhikari et al. 2010). The principal phythosterols are β-sitosterol (mean content 0.172 g/100 g of oil, range 0.112–0.320), campesterol (mean content 0.031 g/100 g of oil, range 0.017–0.660), and Δ5-avenasterol (mean content 0.026 g/100 g of oil, range 0.007–0.040), followed by stigmasterol, sitostanol. The presence of 22-nordehydrocholesterol, campestanol, Δ7-campesterol, clerosterol, Δ5,24 stigmastadienol, Δ7-avenasterol, cholesterol and others were reported also but in a minor amount. The high level of Δ7-avenasterol in pine nuts from Greece, Italy, and Turkey is useful to discriminate the nuts from this origin (Miraliakbari and Shahidi 2007; Nasri et al. 2007). Pine nut oil contains high levels of tocopherols, with a wide range of concentration from about 30 to 170 mg/100 g of oil. Seed coming from Spain has the highest level of tocopherols, while the ones from Turkey have the lowest content The most frequent form of tocopherol is the γ-tocopherol (range 250–1680 mg/kg of oil), followed by the α-form (range 9–166 mg/kg), the β-form (about 23 mg/kg) and δ-forms (range 22–59 mg/kg) (Miraliakbari and Shahidi 2007, 2008; Nasri et al. 2009; Adhikari et al. 2010).

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Gummeson, P. O., Lenman, M., Lee, M., Singh, S., and Stymne, S. 2000. Characterisation of acyl-ACP desaturases from Macadamia integrifolia Maiden & Betche and Nerium oleander L. Plant Science 154:53–60. Holcapek, M., Jandera, P., Zderadicka, P., and Hruba, L. 2003. Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Journal of Chromatography A 1010:195–215. Ikeda, R., Tanaka, H., Uyama, H., and Kobayashi, S. 2002. Synthesis and curing behaviors of a crosslinkable polymer from cashew nut shell liquid. Polymer 43:3475–81. Imbs, A. B., and Long, Q. P. 1996. Fatty acids and triacylglycerols in seeds of pinaceae species. Phytochemistry 42:1051–3. Ip, C., and Lisk, D. J. 1994. Bioactivity of selenium from Brazil nut for cancer prevention and selenoenzyme maintenance. Nutrition and Cancer 21:203–12. Kaijser, A., Dutta, P., and Savage, G. 2000. Oxidative stability and lipid composition of macadamia nuts grown in New Zealand. Food Chemistry 71:67–70. Kamath, V., and Rajini, P. S. 2007. The efficacy of cashew nut (Anacardium occidentale L.) skin extract as a free radical scavenger. Food Chemistry 103:428–33. Kannamkumarath, S. S., Wrobel, K., Wrobel, K., Vonderheide, A., and Caruso, J. A. 2002. HPLC-ICP-MS determination of selenium distribution and speciation in different types of nut. Analytical and Bioanalytical Chemistry 373:454–60. Koaze, H., Karanja, P. N., Onyango, A. N., Ishibashi, K., and Baba, N. 2001. Changes in the amount of lipid classes of macadamia nuts and cashew nuts by drying and roasting. Food Preservation Science 27:11–4. Koaze, H., Karanja, P. N., Kojima, M., Baba, N., and Ishibashi, K. 2002. Lipid accumulation of macadamia nuts during kernel development. Food Preservation Science 28:67–73. Kornsteiner, M., Wagner, K., and Elmadfa, I. 2006. Tocopherols and total phenolics in 10 different nut types. Food Chemistry 98:381–7. Kubo, I., Masuoka, N., Ha, T. J., and Tsujimoto, K. 2006. Antioxidant activity of anacardic acids. Food Chemistry 99:555–62. Kubo, I., Ochi, M., Viera, P. C., and Komatsu, S. 1993. Antitumor agent from the cashew (Anacardium occidentale) apple juice. Journal of Agricultural and Food Chemistry 41:1012–5. Kuhnle, G. G. C., Dell’Aquila, C., Aspinall, S. M., Runswick, S. A., Mulligan, A. A., and Bingham, S. A. 2008. Phytoestrogen content of beverages, nuts, seeds, and oils. Journal of Agricultural and Food Chemistry 56:7311–5. Lee, J. H., Jones, K. C., Lee, K. T., Kim, M. R., and Foglia, T. A. 2005. High-performance liquid chromatographic separation of structured lipids produced by interesterification of macadamia oil with tributyrin and tricaprylin. Chromatographia 58:653–8. Mathew, A. G., and Parpia, H. A. B. 1970. Polyphenols of cashew skin. Journal of Food Science 35:140–3. Menon, A. R. R., Pillai, C. K. S., Sudha, J. D., and Mattew, A. G. 1985. Cashew nut shell liquid: Its polymeric and other industrial products. Journal Scientific Indian Research 44:324–8. Merill, A. H., Schmelz, Jr., E. M., Dillehay, D. L., Spiegel, S., Shayman, J. A., and Schroeder, J. J. 1997. Sphingolipids—The enigmatic lipid class: Biochemistry, physiology, and pathophysiology. Toxicology and Applied Pharmacology 142:208–25. Mexis, S. F., and Kontominas, M. G. 2009. Effect of g-irradiation on the physicochemical and sensory properties of cashew nuts (Anacardium occidentale L.). LWT—Food Science and Technology 42:1501–7. Miraliakbari, H., and Shahidi, F. 2007. Lipid class compositions, tocopherols and sterols of tree nut oils extracted with different solvents. Journal of Food Lipids 15:81–96. Miraliakbari, H., and Shahidi, F. 2008. Antioxidant activity of minor components of tree nut oils. Food Chemistry 111:421–7. Nasri, N., Fady, B., and Triki, S. 2007. Quantification of sterols and aliphatic alcohols in Mediterranean stone pine (Pinus pinea L.) population. Journal of Agricultural and Food Chemistry 55:2251–5. Nasri, N., and Triki, S. 2004. Analyse des lipides des graines de pins de Tunisie: Pinus halepensis Mill. et Pinus pinea L. Rivista Italiana delle Sostanze Grasse, LXXXI, 244–7. Nasri, N., Tlili, N., Ammar, K. B., Khaldi, A., Fady, B., and Triki, S. 2009. High tocopherol and triacylglycerols contents in Pinus pinea L. seeds. International Journal of Food Sciences and Nutrition 60 (S1): 161–9.

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Natali, F., Siculella, L., Salvati, S., and Gnoni, G. V. 2007. Oleic acid is a potent inhibitor of fatty acid and cholesterol synthesis in C6 glioma cells. Journal of Lipid Research 48:1966–75. Nergiz, C., and Dönmez, I. 2004. Chemical composition and nutritive value of Pinus pinea L: Seeds. Food Chemistry 86:365–8. Nwanna, L. C., Oishi, C. A., and Pereira-Filho, M. 2008. Use of phytase to improve the digestibility of alternative feed ingredients by Amazon tambaqui, Colossoma macropomum. ScienceAsia 34:353–60. Paramashivappa, R., Kumar, P. P., Vithayathil, P. J., and Rao, A. S. 2001. Novel method for isolation of major phenolic constituents from cashew (Anacardium occidentale L.) nut shell liquid. Journal of Agricultural and Food Chemistry 49:2548–51. Phillips, K., Ruggio, D., and Ashraf-Khorassani, M. 2005. Phytosterol composition of nuts and seeds commonly consumed in the United States. Journal of Agricultural and Food Chemistry 53:9436–45. Pillai, M. K. S., Kedlaya, K. J., and Selvarangan, R. 1963. Cashew seed skin as a tanning material. Leather Science 10:317. Rajaram, S., Burke, K., Connell, B., Myint, T., and Sabate, J. 2001. A monounsaturated fatty acid-rich pecanenriched diet favorably alters the serum lipid profile of healthy men and women. Journal of Nutrition 131:2275–9. Rodrigues, C. E. C., Silva, F. A., Marsaioli, Jr., A., and Meirelles, A. J. A. 2005. Deacidification of Brazil nut and macadamia nut oils by solvent extraction: Liquid-liquid equilibrium data at 298.2 K. Journal of Chemical & Engineering Data 50:517–23. Rudolph, C. J., Odell, G. V., Hinrichs, H. A., Hopfer, D. A., and Kays, S. J. 1992. Genetic, environmental, and maturity effects on pecan kernel lipid, fatty-acid, tocopherol, and protein-composition. Journal of Food Quality 15:263–78. Ruggeri, S., Cappelloni, M., Gambelli, L., Nicoli, S., and Carnovale, E. 1998. Chemical composition and nutritive value of nuts grown in Italy. Italian Journal of Food Science 10:243–52. Ryan, E., Galvin, K., O’Connor, T. P., Maguire, R., and O’Brien, N. M. 2006. Fatty acid profile, tocopherol, squalene and phytosterol content of brazil, pecan, pine, pistachio and cashew nuts. International Journal of Food Sciences and Nutrition 57:219–8. Sagrero-Nieves, L. 1992. Fatty acid composition of Mexican pine nut (Pinus cembroides) oil from three seed coat phenotypes. Journal of the Sciences of Food Agriculture 59:413–4. Sugano, M., Ikeda, I., Wakamatsu, K., and Oka, T. 1994. Influence of Korean pine (Pinus koraiensis)-seed oil containing cis-5, cis-9, cis-12-octadecatrienoic acid on polyunsaturated fatty acid metabolism, eicosanoid production and blood pressure of rats. Journal of British Nutrition 72:775–83. Sun, S. S., Leung, F. W., and Tomic, J. C. 1987. Brazil nut (Bertholletia excelsa H. B. K.) proteins: Fractionation, composition, and identification of a sulfur-rich protein. Journal of Agricultural and Food Chemistry 35:232–5. Taipina, M. S., Lamardo, L. C. A., Rodas, M. A. B., and del Mastro, N. L. 2009. The effects of gamma irradiation on the vitamin E content and sensory qualities of pecan nuts (Carya illinoensis). Radiation Physics and Chemistry 78:611–3. Toro-Vazquez, J. F., Charo-Alonso, M. A., and Perez-Briceno, F. 1999. Fatty acid composition and its relationship with physicochemical properties of pecan (Carya illinoensis) oil. Journal of the American Oil Chemists’ Society 76:957–65. Trevisan, M. T. S., Pfundstein, B., Haubner, R., Wurtele, G., Spiegelhalder, B., Bartsch, H., and Owen, R. W. 2006. Characterization of alkyl phenols in cashew (Anacardium occidentale) products and assay of their antioxidant capacity. Food and Chemical Toxicology 44:188–97. Venkatachalam, M., and Sathe, S. K. 2006. Chemical composition of selected edible nut seeds. Journal of Agricultural and Food Chemistry 54:4705–14. Venkatachalam, M., Kshirsagar, H. H., Seeram, N., Heber, D., Thompson, T. E., Roux, K. H., and Sathe, S. K. 2007. Biochemical composition and immunological comparison of select pecan [Carya illinoinensis (Wangenh.) K. Koch] cultivars. Journal of Agricultural and Food Chemistry 55:9899–907. Villareal-Lozoya, J. E., Lombardini, L., and Cisneros-Zevallos, L. 2007. Phytochemical constituents and antioxidant capacity of different pecan [Carya illinoinensis (Wangenh.) K. Koch] cultivars. Food Chemistry 102:1241–9. Wakeling, L. T., Mason, R. L., D’Arcy, B. R., and Caffin, N. A. 2001. Composition of pecan cultivars Wichita and Western Schley [Carya illinoinensis (Wangenh.) K. Koch] grown in Australia. Journal of Agricultural and Food Chemistry 49:1277–81.

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11 Bioactive Lipids in Cereals and Cereal Products Ali A. Moazzami, Anna-Maija Lampi, and Afaf Kamal-Eldin Contents Introduction............................................................................................................................................. 229 Cereal Lipids and their Importance......................................................................................................... 230 Extraction and Analysis of Cereal Lipids............................................................................................... 234 Bioactive Lipids in Cereal Grains........................................................................................................... 235 Wheat (Triticum aestivum L.)............................................................................................................ 235 Rye (Secale cereale, L.)..................................................................................................................... 237 Barley (Hordeum vulgare L.)............................................................................................................. 237 Oats (Avena sativa, L.)....................................................................................................................... 238 Corn/Maize (Zea mays, L.)................................................................................................................ 238 Rice (Oryza sativa L.)........................................................................................................................ 239 Sorghum (Sorghum bicolor [Monech] L.)......................................................................................... 240 Pearl millet (Pennisetum glaucum, L.)............................................................................................... 242 Concluding Remarks............................................................................................................................... 242 References............................................................................................................................................... 242

Introduction Cereal grains, belonging to the plant family Poaceae, are mainly composed of starch and contain much smaller amounts of lipids compared to the oilseeds. The seven major cereals are common wheat, rye, barley, oats, rice, maize, sorghum, and pearl millet (Figure 11.1). Cereal lipids are mainly located in the germ, the bran, and/or the endosperm, depending on the cereal. Lipids associated with the starch can be divided into surface lipids and internal lipids according to their extractability in cold or hot n-propanol (Morrison 1981). Surface lipids, which exist on the surface of starch granules (Galliard and Bowler 1987), resembles the rest of kernel lipids while internal lipids consist mainly of monoacyl lipids (lysophospholipids) and free fatty acids that form inclusion complexes with amylase (Morrison and Coventry 1985). In these amylolipid inclusion complexes, the hydrocarbon chain of the lipid lies within the hydrophobic amylose tube while the polar ends stay outside the helix (Morrison 1988; Morrison et al. 1993). Although the amount of starch for internal lipids is limited, for example, about 1% of the starch in barley (Becker and Acker 1971; Morrison 1995), they influence the swelling and gelatinization of these starched affecting properties such as the baking quality (Morrison et al. 1993). In oats, a considerable amount of lipids exist in the starchy endosperm as surface lipids. Cereal grains and their products differ in their lipid contents and composition. Cereal lipids have greater variability in composition compared to oils from oilseeds as they are composed not only of neutral lipids but also contain considerable amounts of polar lipids. The neutral lipids are composed mainly of triacylglycerols (TAG) and plant sterols, which can be free or sterol esters (SE) (to fatty acids or phenolic acids) while the polar lipids include a wide range of glycerolipids including diacylglycerols (GAG), monoacylglycerols (MAG), phospholipids (PL), and glycolipids (GL). Plant sterols (originally free or freed from ester combinations) are the major fraction in the unsaponifiable fractions of cereal 229

230

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Poaceae (=Gramineae)

Panicoideae

Paniceae

Pennisetum (millet)

Andropogoneae

Sorghum (sorghum)

Oryzeae

Oryza (rice)

Aveneae

Avena (oats)

Triticum (wheat)

Secale (rye)

Triticeae

Tribe

Genus

Festucoideae

Hordeum (barley)

Sub-family

Zea (maize/corn)

Family

Figure 11.1  Genetic relationships between cereal grains.

lipids, where they exist with tocopherols, hydrocarbons, waxy alcohols, and lipophilic compounds other than fatty acids. Corn oil (or corn germ oil), wheat germ oil, and rice bran oil (RBO) are produced in considerable amounts and are commercialized. While corn oil is commonly sold as salad oil, wheat germ oil and RBO are mainly used as nutraceuticals and for cosmetics.

Cereal Lipids and their Importance The main fatty acids in cereals are the saturated fatty acids, palmitic (16:0) and stearic (18:0), the monounsaturated fatty acid oleic acid (18:1Δ9), and the diunsaturated fatty acid linoleic acid (18:2, Δ9,12) existing with smaller amounts of other fatty acids. These fatty acids are mainly assembled in glycerolipids (i.e., triacylglycerols [TAG] and variable amounts of phospholipids [PL], and glycolipids [GL]), and in SE and waxes (or policosanols) in the different cereal grains. The compositional properties of oils and lipid extracts depend not only on the cereal gain of origin but very much on the extraction method used (vide infra). Phospholipids (Figure 11.2) are useful for their emulsifying properties and are used in several food applications and might have beneficial nutritional effects (Canty and Zeisel 1994; Ishinaga et al. 2005). Glycolipids (Figure 11.3), the other polar lipid class, contribute polarity and viscosity to cereal lipids. Fatty acids are also components of cereal wax esters (Figure 11.4), known as policosanols, in which they are esterified to high molecular weight aliphatic alcohols having chain lengths varying from 24 to 34 carbon atoms. Policosanols are known to lower blood cholesterol (Canetti et al. 1995; Torres et al. 1995; Carbajal et al. 1998; Gouni-Berthold et al. 2002; Janikula 2002). Plant sterols in cereal lipids mainly belong to the 4,4-desmethyl sterol family with β-sitosterol, campesterol, and stigmasterol being the major ones but sterols from the 4-monomethylsterols, dimethylsterols, and triterpene alcohols (Figure 11.5), which are biosynthetic intermediates are also encountered. Cereal grains also contain saturated counterparts of plant sterols—the stanols. Plant sterols can be present in free form or as SE with fatty acids or phenolic acids. Plant sterols are appreciated for a number of bioactivities including cholesterol-lowering (Andersson et al. 2004), antiatherogenic (Chan et al. 2006), and anticarcinogenic effects (Awad and Fink 2000; Bradford and Awad 2007; Jones and AbuMweis 2009). Single meal intervention studies with sterol-rich corn oil and wheat germ muffins caused a 40% reduction in cholesterol absorption (Ostlund et al. 2002, 2003). The γ-oryzanol, which is a mixture of ferulic acid esters of triterpene alcohols and phytosterols, are effective inter alias for their cholesterol-lowering (Sugano et al.

231

Bioactive Lipids in Cereals and Cereal Products O

O

O

Fatty acyl

Fatty acyl

P

O O

OR –

O

O

Phosphatidic acid

R=H

Phosphatidylglycerol

R = Glycerol CH2OH-CH(OH)-CH2OH

Phosphatidylcholine

R = Choline CH2-CH2-N(CH3)2-CH3

Phosphatidylethanolamine

R = Ethanolamine CH2-CH2-NH2

Phosphatidylserine

R = Serine CH2-CH(NH3+)-COO–

Phosphatidylinositol

R = Inositol OH HO

OH OH OH

Figure 11.2  Structures of phospholipids encountered in cereal grains.

1997; Wilson et al. 2007), antioxidant (Juliano et al. 2005), anti-inflammatory (Akihisa et al. 2000), and anticarcinogenic effects (Yasukawa et al. 1998). In addition, γ-oryzanol was found to suppress the activation of the transcription factor NF-κB (Nagasaka et al. 2007), which is associated with inflammation and insulin resistance (Shoelson et al. 2003; Ruan et al. 2002), immunity (Hayden et al. 2004), and carcinogenesis (Pikarsky et al. 2004; Dolcet et al. 2005). Cereal lipids are distinct from oils from oilseeds in their high levels of tocotrienols that coexist with tocopherols, which are the biologically most active antioxidants (Figure 11.6). In cereal grains, the tocopherols are mainly concentrated in the germ while the tocotrienols are predominant in the hulls and endosperm (Peterson 1994). Tocotrienols, which are unsaturated counterparts of tocopherols, were found to lower low-density lipoprotein (LDL) and total cholesterol in chickens, pigs, and humans by inhibiting the activity of HMG-CoA reductase, the rate limiting enzyme in cholesterol biosynthesis (Qureshi et al. 1986; Weber et al. 1990; Qureshi et al. 1991a, 1991b). It seems that a higher dietary ratio of tocotrienols to tocopherols is necessary for the effect, which is greater for γ- and δ-tocotrienols than for α-tocotrienol (Qureshi 1989). In addition, palm oil tocotrienols were shown to have antiproliferative effect on human cancer cells (Guthrie et al. 1997; Nesaretnam 1998). Besides the above-mentioned components, cereal lipids may contain variable levels of other compounds. For example, lipids extracted from wheat, rye, and probably barley may contain small amounts of alkylresorcinols (Figure 11.7). Alkylresorcinols are 1,3-dihydroxybenzene derivatives with an odd numbered alkyl chain at position 5 of the benzene ring and therefore possess amphiphilic properties. The length of the saturate alkyl tail varies between 15 and 27. Different alkylresorcinols are referred to by their chain length and the degree of unsaturation. In rye, there are five homologues of alkylresorcinol including C17:0, C21:0, C23:0, and C25:0 among them C17:0 (25%) and C21:0 (32%) are the major homologues (Evans et al. 1973; Gohil et al. 1988; Ross et al. 2001; Nystrom et al. 2008). Alkylresorcinols have been shown to have bioactivities in vitro and in vivo experiments. They increase

232

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

O

OH

OH

Fatty acyl

O

O

HO

OH

HO

OH O

O

OH

O

OH Monogalactosyl monoacylglycerol (MGMG) O

OH OH

HO Fatty acyl

O

O

O

OH

OH

Digalactosyl monoacylglycerol (DGMG) O

OH OH

HO Fatty acyl

O Fatty acyl

O

O

O

OH

O Monogalactosyl diacylglycerol (MGDG) O OH

HO Fatty acyl

O Fatty acyl

OH

OH O

HO

O

O

O

OH

O

OH

O Digalactosyl diacylglycerol (DGDG) Figure 11.3  Structures of major glycolipids in cereal grains.

Fatty acyl

O n

O

Figure 11.4  Structure of policosanols, wax esters of fatty acids, and long-chain alcohols in cereal grains.

the γ-tocopherol level in rat liver and the lung by possibly inhibiting γ-tocopherol metabolism (Ross et  al. 2004a, 2004b). In vitro, alkylresorcinols have been reported to have anticancer, enzyme binding, and DNA-cleaving properties. Because of their amphiphilic properties, they can make monolayers and insert them into phospholipid membranes (Kozubek 1999). They can also interfere with TAG synthesis and inhibit the accumulation of TAG in cultured adipocytes (Tsuge et al. 1992; Rejman and Kozubek 1997, 2004). Since alkylresorcinols intake is mainly associated with rye and wheat, they have been suggested as a biomarker of whole-grain wheat and rye intake (Chen et al. 2004; Ross et al. 2004; Soderholm et al. 2009).

233

Bioactive Lipids in Cereals and Cereal Products

Campesterol

HO

Sitosterol

HO

HO

Sitosteryl ferulate

CH=CHCOO

O

OMe

O

OH

Sitosteryl stearate

CH2OH

Stigmasterol

OH O O

Sitosteryl β-D-glycoside

O

O

H2C OH

OH OH

O O Sitosteryl (6'-O-acyl)-β-D-glycoside

OH

OH

Figure 11.5  Structures of the major 4,4-desmethylsterols and examples of esterified and glycosilated combinations of sitosterol.

R1 HO R2

O Tocopherols R1

HO R2

O Tocotrienols

Figure 11.6  Basic structures of tocopherols and tocotrienols in cereals. α-Tocols (R1 = R2 = methyl), β-tocols (R1 = methyl, R2 = hydrogen), γ-tocols (R1 = hydrogen, R2 = methyl), and δ-tocols (R1 = R2 = hydrogen).

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

HO n = 13–23 Figure 11.7  Structures of alkylresorcinols, amphiphilic lipids in wheat, rye, and barley.

Extraction and Analysis of Cereal Lipids Cereal lipids consist of a variety of lipid classes, and lipid profiles of different grain components may vary considerably (Chung and Ohm 2000, 2009). Moreover, some lipids are easily extracted while others are effectively protected by complex carbohydrates and proteins of the grains. Thus analysis of cereal lipids is a challenging task, and various approaches have been taken to extract them. The choice of the extraction method depends mainly on whether the task is to determine total lipids or to focus on an individual lipid class. Cereal lipids may be classified by the extractability. Most lipids belong to free lipids that are easily extracted with relatively nonpolar solvents (such as hydrocarbons) by using shaking or a Soxhlet extractor. Another category of lipids are bound lipids that interact with other grain constituents and need more polar solvents to be released from them. The solvents used include e.g., water-saturated n-butanol and mixtures of chloroform and methanol. The most tightly bound lipids are starch lipids, which can only be extracted by hot aqueous alcohol mixtures that are able to penetrate into starch granules (Dimberg et al. 2001). There are also differences in the chemical properties of cereal lipids. While nonpolar lipids such as acyl glycerols are dominant in seed oils, free fatty acids, glycolipids, and other polar lipids are also important in cereal lipids, which has to be taken into account when choosing an extraction method. The solvent has a vital effect on lipid yields. As reviewed by Zhou et al. (1999) total lipid amounts of oat groats vary a lot and were 5.6% and 8.8% when lipid were extracted with n-hexane and ethanol, respectively (Zhou et al. 1999. The amounts of glycolipids, phospholipids and mono- and diacylglycerols increased as the polarity of the solvent increased. When pressurized liquid extraction was used to extract oil from corn and oats, the yields were the greatest when methylene chloride was used as the solvent. This indicates that in addition to the polarity of the solvent, other characteristics of the solvent may also influence extraction efficiency (Moreau 2003). Oil yields were always higher when extraction was performed at 100°C than at 40°C, and by increasing the polarity of the solvent, the amount of polar lipids was increased. The most generally accepted method to measure lipid contents includes acid hydrolysis of the cereal product, solvent extraction of lipids, and gas chromatographic analysis of fatty acids, where total lipids are calculated from individual fatty acids. This procedure is used in the AOAC method for total fats in cereal products (Horwitz 2000). Due to the hydrolysis step, lipid molecules may partly be decomposed, and thus the extract should not be used for characterization of lipid classes. Lipid class profiles have been analyzed by several chromatographic methods after crude lipid extraction and possible purification steps. The most commonly used technique to fractionate lipid classes is normal phase liquid chromatography (LC). Since most lipids do not have chromophores that would absorb UV or VIS light, evaporative light-scattering detectors and other universal detectors are being used. More recently, mass spectrometry has also been applied to resolve individual lipids and lipid profiles and also used for quantification (Byrdwell 2001). As an example, an HPLC method with a diol column and a gradient elution has been used to separate nonpolar lipid classes (Moreau et al. 1996), and applied further to separate free sterols and steryl conjugates (Nystrom et al. 2007). Analytical methods used for analyzing three bioactive lipid classes in cereals, namely sterols, tocols, and alkylresorcinols were recently reported in a series of papers (Andersson et al. 2008; Lampi et al. 2008; Nurmi et al. 2008). Methods to analyze cereal lipids in more general were also shortly presented and evaluated [66]. To analyze sterols, wholemeal cereal samples were subjected to acid and alkaline

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hydrolyses to liberate sterols from conjugates (i.e., esters and glycosides). After hydrolyses, nonsaponifiable lipids were purified by liquid partitioning, and sterols were enriched by solid-phase extraction and derivatized to trimethyl silyl ethers. Finally sterols were analyzed by gas chromatography (Nurmi et al. 2008). Hydrolysis of glycosides by either acid or enzymatic hydrolysis is needed to include these conjugates in total sterol analysis (Moreau et al. 2002). For analysis of tocopherols and tocotrienols, wholemeal samples were subjected to hot alkaline hydrolysis after which the nonsaponifiable lipids could be concentrated. Separation of individual compounds was performed by normal-phase HPLC and detection by fluorescence (Lampi et al. 2008). Although alkaline hydrolysis as such is not needed because tocopherols and tocotrienols are not esterified, it has been shown that higher yields are obtained with hydrolysis than with solvent extraction, for example, from oats (Peterson 2007). Since cereals contain both tocopherols and tocotrienols, specific analysis of the eight vitamers is needed. This can be achieved by using silica columns (Kamal-Eldin 2000), and more recently also by some reversed-phase HPLC columns (Abidi 2003). Alkylresorcinols and alkenylresorcinols were solvents extracted from whole grains, and after evaporation of the solvents were analyzed either in underivatized form or as trimethyl silyl ethers by gas chromatography (Anderson 2008). When analyzing alkylresorcinols, extraction may be performed on intact grains, because they are specifically located in one of the outermost layers of the grains. In analyses of these bioactive lipids, mass spectrometry was used to verify the identity of the molecules (Anderson 2008; Nurmi et al. 2008).

Bioactive Lipids in Cereal Grains Wheat (Triticum aestivum L.) Lipids constitute 2.5–3.3% of the wheat kernel, of which 30–36% are located in the germ, 25–29% in the aleurone layer, and 35–45% in the endosperm (Hargin and Morrison 1980; Chung and Pomeranz 1981; Morrison 1988). The germ represents 2–3% of the wheat grain and it contains about 10% of lipids depending on the milling and extraction methods (Dunford and Zhang 2003). The germ and aleurone lipids are predominantly nonpolar (72–85%) with small amounts of polar lipids (13–23%) (Morrison 1988), and are distinctly different from the endosperm lipids that are especially rich in phospholipids and glycolipids. The fatty acid composition of wheat germ oil is constituted by palmitic acid (17%), stearic acid (1%), oleic acid (17%), linoleic acid (59%), and α-linolenic acid (6%; Eisenmenger and Dunford 2008). The major TAG species in wheat germ oil are PLP (2%), POO (2%), PLO (5%), OOO (6%), PLL (16%), PLLn (8%), OOL (6%), LLO (12%), LLL (30%), LLLn (13%; Jakab and Forgacs 2002). Depending on the grain quality, storage and milling, wheat germ oil contains 5–25% FFA and needs to be refined before use in edible applications. The relative proportions of wheat flour lipids are approximately shown in Table 11.1 with nonpolar lipids (51%), phospholipids (23%), and glycolipids (26%; Macmurra and Morrison 1970). The major phospholipids of wheat include lysophosphatidylcholine (LPC), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), and N-acylphosphatidyl ethanolamine (APE), and N-acyl lysophosphatidyl ethanolamine (ALPE) while the main galactolipids are digalactosyldiglycerides (DGDG), monogalactosyldiglycerides (MGDG), and monogalactosyldiglycerides (AMGDG). The four main phospholipids in wheat germ oil are PC (28.4%), LPC (30.4%), PE (5.2%), and PS (4.6%; Colborne and Laidman 1975). The total sterol contents range from 670 to 960 mg/Kg dm in different winter and spring wheat varieties and other wheat genotypes (Nurmi 2008). Spelt, durum wheat, and einkorn wheat contain the highest sterol contents with considerable variation in the relative distribution of the different phytosterols (Pomeranz et al. 1966). The most abundant phytosterol in all wheat genotypes is sitosterol (40–61% of total) followed by campesterol, sitostanol, and campestanol. Wheat germ oil also contains phytosterols (8000 mg/Kg oil) of which β-sitosterol (63%) and campesterol (27%) are the major phytosterols (Hassanein and Abedel-Razek 2009). Wheat bran, like rye and rice brans, contains steryl ferulates, which possesses antioxidant activity especially in the bulk lipid systems (Nystrom 2005). The content of steryl ferulate in wheat ranges from trace amounts in flours with low-ash content to 340 mg/Kg in wheat

236

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 11.1 Relative Distribution of Major Wheat Flour Lipids Lipid Class Nonpolar lipids Acylglycerols

Free fatty acids (FFA) Sterols

Polar lipids Phospholipids (PL)

Glycolipids (GL)

Species

Relative (%)

Triacylglycerols (TAG) 1,2-diacylglycerols (1,2-DAG) 1,3-diacylglycerols (1,3-DAG) Monoacylglycerols (MAG) Free sterols Sterol esters

20.8 6.2 6.0 1.3 7.0 2.1 7.5

Phosphatidyl choline (PC) Lysophospatidyl choline (LPC) Phosphatidyl ethanolamine (PE) Lysophosphatidyl ethanolamine (LPE) N-acylphosphatidyl ethanolamine (APE) N-acyllysophosphatidyl ethanolamine (ALPE) Phosphatidyl serine (PS) Phosphatidyl inositol (PI) Monogalactosyl monoacylglycerols (MGMG) Monogalactosyl diacylglycerols (MGDG) Digalactosyl monoacylglycerols (DGMG) Digalactosyl diacylglycerols (DGDG) Sterolglucosides (SG) + ceramide monoacylglycerols (CMG) Ceramide dglycosides (CDG) 6-O-Acyl sterol glucosides (ASG) 6-O-Acyl monogalactosyl diacylglycerol (AMGDG)

5.8 7.1 0.8 0.9 4.9 2.9 0.2 0.1 0.4 4.9 0.6 13.5 1.8 0.03 1.6 3.6

Source: Modified from Macmurray, T., and Morrison, W. R., J. Sci. Food Agr., 21(10), 520–8, 1970.

bran. Campestanyl ferulate and sitostanyl ferulate are the main compounds, followed by campesteryl ferulate and sitosteryl ferulate (Hakala et al. 2002). Wheat germ oil is also rich in tocopherols (0.1–0.15%) mainly α-tocopherol (70%), β-tocopherols (19%), and γ-tocopherol (7%) and sterols (0.8%) mainly β-sitosterol (63%) and campesterol (27%) existing with smaller amounts of sterol glucosides and acylated sterol glucosides (530 ppm; Hassanein and Abedel-Razek 2009). Although wheat, like other cereals, is a moderate source of sterols, ­tocopherols, and tocotrienols in comparison with vegetable oils, the amount of these bioactives obtained via consumption of wheat and other cereals is important considering their relatively high daily consumption (Morton et al. 1995; Normen et al. 2001; Valsta et al. 2004; Jimenez-Escrig et al. 2006). The beneficial health promoting effects of wheat germ oil are basically due to their high contents of vitamin E and unsaturated fatty acids mainly linoleic acid. Wheat flour includes small amounts of carotenoids (0.8 mg/Kg), mainly lutein (85%) and lutein esters (15%; Howitt 2009). Depending on variety, wheat grains contain policosanols (3–56 mg/Kg) of high molecular weight; C20–C36, mainly in the bran fraction (75%) compared to the germ (25%; Irmak and Dunford 2005). Wheat also contains alkylresorcinols, which mainly occur (99%) in an intermediate layer of caryopsis, including the hyaline layer, inner pricarp, and testa, and are not detectable in other parts of the kernel including the endosperm and germ (Teuscik 1978; Landberg et al. 2008). Alkylresorcinols range widely from 200 to 1480 mg/Kg in whole wheat from North America and Europe (Hengtrakul et al. 1990; Ross et al. 2001, 2003; Chen et al. 2004; Andersson et al. 2008; Kulawinek et al. 2008). The biosynthesis of alkylresorcinols can be affected by biotic and abiotic factors within different cultivars

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237

and also within the same ecological niche (Kulawinek et al. 2008; Magnucka et al. 2007a, 2007b). The ratio of C17:0 to C21:0 in wheat is generally about 0.1 and it is much lower in durum (0.01), which can be used and has the characteristics of wheat alkylresorcinols (Ross et al. 2001, 2003; Landberg et al. 2006; Andersson et al. 2008; Kulawinek et al. 2008; Kulawinek and Kozubek 2008).

Rye (Secale cereale, L.) Whole grain rye flour contains 1.3–2.8% lipids (Ryan et al. 2007; Nystrom et al. 2008) rich in linoleic acid (59%), oleic acid (17%), palmitic acid (15%), and α-linolenic acid (9%; Ryan et al. 2007). Phytosterols, steryl ferulates, tocopherols, tocotrienols, alkylresorcinols, and squalene possess structural association with lipids and may be coextracted with the lipid phase of rye (Hakala et al. 2002; Liukkonen et al. 2003; Kariluoto et al. 2006; Nystrom et al. 2008; Kamal-Eldin et al. 2009). The total content of plant sterol in whole meal rye of different varieties ranges between 77.4 and 142 mg/100g dm and sitosterol contributes (45–55%) of the total sterols in rye followed by other sterols and stanols; campesterol (15–20%), sitostanol (8–12%), campestanol (6–9%), stigmasterol (3–4%), and other minor sterols (14%; Piironen et al. 2002; Zangenberg et al. 2004; Nystrom et al. 2008). Rye bran, similar to wheat and rice brans, contains steryl ferulates at about 6 mg/Kg. Uneven distribution of steryl ferulates in the grains leads to considerable differences in the milling products (depending on extraction). In rye, steryl ferulate content ranged from trace amounts in flours with low-ash content to 200 mg/Kg in the bran. Campestanyl ferulate and sitostanyl ferulate are the main compounds, followed by campesteryl ferulate and sitosteryl ferulate (Nystrom et al. 2005; Hakala et al. 2002). Total tocols in the whole meal rye range from 18 to 67 mg/Kg dry matter (DM) and includes α/βtocopherols and α/β tocotrienols in which α-tocopherol (30–35%) and α-tocotrienol (35–40%) are the major isoforms (Piironen et al. 1986; Balz et al. 1992; Ryynanen et al. 2004; Zielinski et al. 2007; Nystrom et al. 2008). Squalene has also been reported to be present in rye kernels (3 mg/Kg; Ryan et al. 2007. The total content of alkylresorcinols in rye kernels varies widely between 568 and 3220 mg/Kg dry matter (Evans et al. 1973; Gohil et al. 1988; Ross et al. 2001; Nystrom et al. 2008), while the relative proportion of homologous varies less than the total content of alkylresorcinol and are characteristic for rye and wheat (Ross et al. 2001; Chen et al. 2004; Landberg et al. 2006; Nystrom et al. 2008; KamalEldin 2009). These differences may relate to the fact that rye samples in these studies came from different varieties and were grown under different agroclimatic conditions.

Barley (Hordeum vulgare L.) The amount of lipids in the barley kernel was estimated at 1.8–4.7% (Parsons and Price 1974; Price and Parsons 1974; Moreau et al. 2007). These lipids are classified by thin layer chromatography into neutral lipids (71%), phospholipids (20%), and glycolipids (9%). The neutral lipids consist of palmitic acid (∼27%), stearic acid (∼1%), oleic acid (∼18%), linoleic acid (∼50%), and α-linolenic acid (5%) as the major fatty acids. The fatty acid compositions of the phospholipid and glycolipid fractions were comparable with higher percentages of palmitic acid (31–37%) and lower percentages of α-linolenic acid (2–4%) in the phospholipid fraction and higher levels of linoleic acid (59%–66%) at the expense of oleic acid (5–8%) in glycolipids (Parsons and Price 1974; Price and Parsons 1974). The neutral lipids of barley consisted of TAG (52%), sterols (20%), SE (3%), DAG (11%), FFA (11%), and hydrocarbons (4%). The phospholipids of barley grains were composed of phosphatidyl choline (PC, 44.4%), lysophosphatidyl choline (LPC, 37.3%), phosphatidyl ethanolamine (8.8%), phosphatidyl serine (4.8%), phosphatidyl inositol (PI, 1.3%), diphosphatidyl glycerol (DPG, 1.5%), and small amounts of phosphatidyl glycerol, two unknowns, phosphatidic acid, where linoleic acid was the predominant fatty acid in all (Parsons and Price 1979). Barley grains contain 990–1150 mg/Kg DM of sterols composed mainly of β-sitosterol (53–61%), campesterol (14–20%), stigmasterol (∼2.8–4.7%), Δ5-avenasterol (∼3.2–7.5%; Andersson et al. 2008). The sterols content in barley oils ranged from 1.2 to 5.6% of which about 25–33% were free sterols and the rest were sterol esterified to fatty acids (Moreau et al. 2008). Barley kernels were reported to contain 42–80 mg/Kg of tocopherols and tocotrienols depending on variety and agroclimatic conditions (Peterson and Qureshi 1993). Hull-less barley is characterized by a lower level of total tocopherols and tocotrienols

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but higher levels of γ- and δ-tocopherols (Cavallero et al. 2004). Barley oil contains about 420–900 ppm of tocopherols and tocotrienols (Wang et al. 1993) being 29% tocopherols (α-tocopherol [142 ppm], γ-tocopherol [104 ppm], and β- and δ-tocopherols [7 ppm]) and 71% tocotrienols (α-tocotrienol [558 ppm], γ-tocotrienol [85 ppm], and δ-tocotrienol [5 ppm]; Wang et al. 1993). Barley grains contain low levels of alkylresorcinols (32–103 mg/Kg DM) compared to rye (360–3200 mg/Kg) and wheat (300–1000 mg/Kg) and unlike rye and wheat, barley grain AR contains very high percentages of C25:0 (15–48% of total), C23:0 (8–19%; Andersson et al. 2008).

Oats (Avena sativa, L.) Compared to other cereals, oat contains higher amounts of lipids (4.5–18%) located throughout the kernel (Peterson and Wood 1997). Unlike other cereal grains, oat contains a high level of lipids as surface lipids in the endosperm, being most frequent in the subaleurone cells and in endosperm cells in the vicinity of the embryonic axis and less frequently in cells of the middle and inner endosperm. During development of oat kernels, the distinct oil bodies initially in the embryonic axis and the aleurone layer lose their integrity and fuse together into large masses in the rest of the endosperm. This fusion is probably related to an insufficient amount of the protecting oil-body membrane proteins (oleosins; Heneen et al. 2008, 2009). Oat lipids are a heterogeneous mixture of acyl lipids and unsaponifiable components. The neutral lipids are mainly TAG and account for 50–60% of total oat lipids (Sahasrabudhe 1979a). The major fatty acids in acyl lipids are oleic acid (29–52%), linoleic acid (26–48%), palmitic acid (14–23%), stearic acid (0.5–3.9%), and linolenic acid (1–3.5%; Frey and Hammond 1975; Youngs and Puskulcu 1976; Roche et al. 1977; Sahasrabudhe 1979b; Zhou et al. 1999). Oat oil is also rich in polar lipids; that is, phospholipids (6–26%) and glycolipids (6–17%; Bedford and Joslyn 1937; Youngs et al. 1977; Sahasrabudhe 1979b; Alkio et al. 1991; Forssell et al. 1992; Zhou et al. 1999). The main phospholipid of oats is lecithin or PC (50% of total; Youngs et al. 1977) followed by phosphatidylethanolamine and phosphatidylglycerol (Sahasrabudhe 1979b). Oats also contain glycolipids where diacylglycerol is associated with the sugars glucose and galactose (Hauksson et al. 1995). The major glycolipid in oats is digalactosyldiacylglycerol (DGDG; Hauksson et al. 1995; Andersson et al. 1997; Aro et al. 2007; Moreau et al. 2008) and is present in the oat kernel at almost 0.5 g/Kg (Hamberg 1998). Avenoleic acid (Figure 11.8), which is a oxylipin from oat seeds is mainly found (65%) in the structure of glycolipids (Hamberg 1997). Oats also contain unsaponifiable lipid fraction including phytosterols, tocopherols, and tocotrienols. Oat phytosterols are present in free, esterified, glycosylated, and acyglycosylated forms. The total sterols in oats reported in five major cultivars studied in HEALTHGRAIN projects ranged from 620  to 680 mg/Kg in which almost 2% are stanols (Shewry et al. 2008). The β-sitosterol is the major sterol (360–440 mg/Kg) followed by campesterol (50–60 mg/Kg), stigmasterol (20–40 mg/ Kg), Δ5-avenasterol (20–30 mg/Kg), and Δ7-avenasterol (almost 10 mg/Kg). The total tocopherols and tocotrienols in oat grains range from 20–40 mg/Kg (Peterson and Qureshi 1993; Peterson 1995; Emmons et al. 1999; Handelman et al. 1999; Shewry et al. 2008). The α-tocotrienol is generally the main vitamer (almost 65%) followed by α-tocopherol (20%–25%) and small amounts of β-tocopherol and β-tocotrienol (Bryngelsson et al. 2002; Shewry et al. 2008).

Corn/Maize (Zea mays, L.) The kernel of Zea mays, L., contains about 4% oil concentrated in the germ. A by-product of the wet milling, corn germ contains 30–50% oil, which is a third of the commercialized oils. As with other cereal lipids, the composition of the oil depends very much on the milling process and the polarity of HO O

OH

Figure 11.8  15(R)-Hydroxy-9(Z),12(Z)-octadecadienoic acid (Avenoleic acid), a major component in the glycolipids of oats.

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Bioactive Lipids in Cereals and Cereal Products

the extraction solvent. Corn germ oil, extracted with hexane, is about 98% TAGs, 1% FFA, 0.5% SE, and 0.5% free sterols (Moreau and Hicks 2005). Corn germ oil is mainly composed of linoleic acid (57.3%), oleic acid (27.7%), palmitic acid (10.7%), and stearic acid (1.9%; Moreau et al. 2009). The major TAGs are LLL (29%), OLL (25%), and LLP (17%), which are present with other species containing the four major fatty acids (Holcapek 2003). Authors have reported 4–9% phospholipids in corn kernel lipids (Tan and Morrison 1979; Harrabi et al. 2009) and about 1.5% in crude germ oil (Blanchard 1992). The relative proportions of the phospholipid molecular species were approximately: PC (57%), phosphatidyl inisitolamine (20%), phosphatidylethanolamine (14%), phosphatidic acid (7%), and phosphatidylglycerol (2%) (Harrabi et al. 2009). Crude corn oil contains ∼1.5% unsaponifiable materials (Kuksis 1964). A sample of corn germ oil contained 0.84% total sterols, mainly β-sitosterol (60%), campesterol (18%), stigmasterol (6.5%), together with smaller amounts of campestanol, sitostanol, Δ5-avenasterol, and smaller amounts of monomethyl and dimethyl sterols (Moreau et al. 2009). The major SE were linoleate and oleate esters of sitosterol, campesterol, and stigmasterol (Billheimer et al. 1983). Cold-pressed corn oil was found to contain a total of ∼1135 ppm of tocochromanols: α-tocopherol (170 ppm), γ-tocopherol (650 ppm), δ-tocopherol (65 ppm), a-tocotrienol (115 ppm), γ-tocotrienol (117), δ-tocotrienol (∼1 ppm), and plastochromanol-8 (17 ppm; Gruszka and Kruk 2007). Crude corn oil contains ∼100 ppm of waxes extending from C44 to C58 with characteristic bimodal distribution around C48 and C54 (Henon et al. 2001). Hexane-extracted corn germ oil contained lutein (1.4 ppm) and zeaxanthin (0.9 ppm), while whole kernel oil and corn fiber oil contained significantly higher levels (Moreau et al. 2007). Corn oil contains ∼0.05% total hydrocarbons, mainly composed of odd- and even-chain normal paraffins extending from C13 to C34 (Kuksis 1964). Corn grains contain the highest level of carotenoids (11.1 mg/Kg DM), especially xanthophylls, among cereals (0.4–3 mg/Kg) with zeaxanthin (6.4 mg/Kg), β-cryptoxanthin (2.4 mg/Kg), α- and β-carotene (1.4 mg/Kg), and lutein (0.9 mg/Kg; Panfili et al. 2004). As special components, the corn kernel was shown to contain two polyamine conjugates, diferuloylputrescine and p-coumaryl-feruloylputrescine (Figure 11.9), which are present at low levels with corn germ oil extracted with hexane and at slightly higher levels in oils extracted with ethanol or isopropanol (Moreau et al. 2001).

Rice (Oryza sativa L.) The bran of rice is a part of the rice grain, including the testa and pericarp that exists between the paddy husk and endosperm. Rice bran, which represents ∼10% of kernel by weight, is produced as a byproduct of the rice milling and contains 15–20% oil called RBO. The RBO is composed of triacylglycerols (TAG, 68–84%), diacylglycerols (DAG, 2–3%), monoacylglycerols (MAG, 1–6%), free fatty acids (FFA, 2–6%), wax esters (2–4%), phospholipids (PL, 1–4%), glycolipids (GL, 1–7%), and unsaponifiables (3–4%; Ghosh 2007). The fatty acids of RBO are composed of palmitic acid (P, 17–22%), stearic acid (S, 1–2%), oleic acid (O, 37–44%), linoleic acid (L, 34–38.5%), and α-linolenic acid (1–2%) with no significant differences between lipid classes (Rukmini 1988; Denev et al. 2009; Yoshie et al. 2009). Reena et al. (2009) found these fatty acids to be organized in the TAG molecular species of RBO as follows: PPP (0.9%), POP (4.9%), POS (0.5%), PLP (5.9%), SOO (1.1%), PLO (18.1%), SLO (12.9%), PLL (11.2%), OOO (10.2%), LOO (15.2%), LLO (12.8%), LLL (5.5%). The level of FFA in RBO varies widely O R

OH

H N H

HO

N

OMe O

Diferuloylputrescine

R = OCH3

p-coumaryl-feruloylputrescine

R=H

Figure 11.9  Putrescines in maize.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

depending on the activity of a 30 kDa rice bran lipase (Rajeshwara and Prakash 1995; Gopalakrishna et al. 2002). The phospholipids are mainly composed of phosphatidyl cholines (PC, ∼32%), phosphatidyl inositols (PI, 23%), phosphatidyletanolamines (PE, 21%), phosphatidyl serines (PS, 2.6%), lyso-PCs (6.8%), N-acyl-PEs (6.9%), N-acyl-lyso-PEs (6.9%), as well as calcium and magnesium salts of phosphatidic acid (PA; Glushenkova et al. 1998). Rice bran oil is characterized by higher levels of unsaponifiable materials (2–4%) compared to other cereals and oilseeds (1–2%). The constituents of RBO unsaponifiables include phytosterols and the main constituent γ-oryzanols, tocopherols/tocotrienols, squalene, and other hydrocarbons. According to Deepman et al. (2007), crude RBO contains 1.1% free sterols, 1.9% oryzanols, 0.7% SE, 0.2% tocols, 1.3% wax esters, and 0.04% squalene, which are reduced in refined RBO. Phytosterols are present in RBO as free sterols, γ-oryzanols, and sterol-fatty acid esters. The total phytosterol content in RBO is ca 2 g/100 g oil (of which ∼38% existed in free form) and is composed of campesterol (12.8%), campestanol (1.8%), stigmasterol (8.8%), sitosterol (24.3%), sitostanol (4.1%), cycloartenol (16.4%), and 24-methylene cycloartanol (25.8%; Jiang and Wang 2005). The presence of γ-oryzanols (1.53 g/100 g oil, m.p. ∼138°C) in RBO was first realized via UV absorption at 230, 290, and 315 nm in heptane, thus their total content in the oils can be roughly estimated by UV spectroscopy (Kaneko and Tsuchiya 1954). These components were later identified as mixed ferulic acid esters of triterpene alcohols and sterols (Xu and Godber 1999). About 80% of RBO oryzanols are composed of cycloartenyl, 24-methylene cycloartanyl, and campesteryl ferulates (Xu and Godber 2001). Later, 23 different compounds including 24-methylene cholesterol, Δ5 and Δ7-campesterols, campestanol, Δ5 and Δ7-sitosterol, stigmasterol, cycloartenol, 24-methylene cycloartenol, 24- and 25-hydroxy-24-­methylcycloartanol, and hydroxydehydrocycloartenol esterified to trans-ferulate and to a lesser extent to cis-ferulate and caffeate (Fang et al. 2003). The SE and policosanols (wax esters), representing ∼4.5% of RBO are widely used in cosmetic, nutraceuticals, and pharmaceutical preparations (Buffa 1976; Ito et al. 1983). The policosanols (∼1.3% of RBO), esters of long-chain fatty alcohols (C24 –C40) with long-chain fatty acids (C22–C24), include even- and oddnumbered carbon species, ranging from C46 to C64, with central predominance around C54 (ester of tricontanol [C30:0] with lignoceric acid [C24:0]). The SE fraction (∼3.5% of RBO) mainly includes oleic and linoleic acid esters of the 4-desmethylsterols (campesterol, sitosterol, stigmasterol, and Δ7-stigmasterol, 76%), the 4-monomethyl sterol (citrostadienol, 9%), and the 4,4-dimethylsterols (cycloartenol, and 24-methylenecycloartanol, 15%; Gunawan et al. 2006). The RBO contains ∼0.1% of vitamin E compounds (tocopherols and tocotrienols) distributed as follows: α-tocopherol (118 ppm), γ-tocopherol (5 ppm), α-tocotrienol (148 ppm), and γ-tocotrienol (814 ppm; Abidi and Rennick 2003) (Figure 11.10).

Sorghum (Sorghum bicolor [Monech] L.) The kernels of sorghum contain 5.0–8.2% lipids, mainly located in the germ. The fatty acids composition of the total sorghum kernel lipids is dominated by oleic (31.1–49.0%) and linoleic (27.59–50.73%) acids, which exist with small amounts of palmitoleic acid (0.4–0.6%), palmitic acid (11.7–20.2%), stearic CH3O

O O

OH

O

O OH

O

Oryzadine

HO

Figure 11.10  Chemical structure of oryzadine in rice bran.

N H

O

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acid (1.1–2.6%), and α-linolenic acid (1.7–3.9%; Mehmood et al. 2008). Sorghum kernel lipids are composed of ∼94% neutral lipids (nonpolar), ∼5% glycolipids, and ∼1% phospholipids. The neutral lipids are dominated by TAG (85%), which exist together with DAG (4%), MAG (2.5%), free sterols (4%), SE (2.8%), and FFA (2.8%). The major glycolipids are sterol glycosides (10%), esterified sterol glycoside (38%), cerebrosides (9%), monogalactosylmonoacylglycerol (4%), digalactosylmonoacylglycerol (5%), monogalactosyldiacylglycerol (9%), and DGDG (21%). The major phospholipids are LPC (42%), LPE (14%), PC (36%), phosphatidylethanolamine (5.5%), phosphatidylglycerol (1%), and phosphatidic acid (1.5%; Osagie 1987). Sorghum kernel lipids contain about 3300 ppm of total desmethylsterols (Avato et al. 1990) composed of cholesterol (0.7–2.1%), campesterol (18.7–29.1%), stigmasterol (12.4–20.5%), β-sitosterol (43.8–57.9%), Δ5-avenasterol (4.1–7.4%), and Δ7-stigmasterol (trace to 2.5%; Maestri et al. 1996). In addition, sorghum lipids were reported to contain two ketosteroids (stigmasta-3,5-diene-7-one and ergosta-3,5-diene-7-one) as well as α-amyrin and β-amyrin, and the hopanoids simiarenol, fernenol, trematol, isoarborinol, and sorghumol (Avato et al. 1990), which are biosynthesized from 2,3-oxidosqualene in the leaves (Heupel 1985) (Figure 11.11). Sorghum lipids contain about 340 ppm of tocopherols: approximately α-tocopherol (0.5%), γ-tocopherol (95%), and γ-tocopherol (0.5%: Mariod 2004). Sorghum lipids contain about 4750 ppm of fatty alcohols: C26:0 (13%), C28:0 (52%), C30:0 (25%), and C32:0 (10%; Leguizamon et al. 2009). Avato et al. (1990) studied sorghum wax and found the following species: C42 (1%), C44 (2%), C46 (3%), C48 (1%), C50 (1%), C52 (1%),

H

HO

HO H

Trematol

H

HO

H

H

Fernenol

H

H

HO H

H

H

Isoarborinol

H

H

Sorghumol R2 R1

H

H

H

H

H

H

HO

HO Simiarenol

α-Amyrin R1 = methyl, R2 = hydrogen β-Amyrin R1 = hydrogen, R2 = methyl Figure 11.11  Structures of triterpene alcohols in sorghum.

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C54 (1%), C55 (1%), C56 (19%), C57 (1%), C58 (37%), C59 (1%), C60 (25%), and C62 (2%). These included alkanes, aldehydes, alcohols, and acids in the carbon range C21–35 with acids and alcohols of C28 and C30 predominating. It was reported that sorghum grain wax is composed of hydrocarbons (1–7%), wax esters (4–13%), fatty alcohols (32–34%), fatty acids (25–27%), and aldehydes (21–32%; Bianchi et al. 1979). The alcohols, acids, and aldehydes were mainly C28:0 and C30:0 while the hydrocarbons were mainly C27:0 and C29:0 (Hwang et al. 2002). The major wax lipids determined by HPLC were policosanols (37–44%), aldehydes (44–55%), and acids (4–5%; Hwang et al. 2004).

Pearl millet (Pennisetum glaucum, L.) Pearl millet typically contains about 1–5% lipids, which are concentrated in the germ. About 10% of the total lipids are bound to the starch. The fatty acid components of the total lipids are palmitic (19%), stearic acid (5%), palmitoleic (0.6%), oleic (25%), linoleic (46%), α-linolenic (3.2%), and arachidic acid (0.5%; Rooney 1978). About 85% of the lipids are neutral (nonpolar), 12% phospholipids, and 3% glycolipids (Osagie and Kates 1984)[185]. According to these authors, the neutral lipids of millet are mainly TAG (86%) existing together with free sterols (5.4%), SE (3.5%), DAG and MAG (3.2%), and FFA (2.1%). The major phospholipids are LPC (42%), LPE (21%), PC (24%), phosphatidylethanolamine (6.4%), phosphatidylglycerol (1.1%), phosphatidic acid (1.2%), and biphosphatidic acid (1.52). The major glycolipids are acyl-monogalactosyldiacylglycerol (2%), sterol glycosides (14.2%), esterified sterol glycoside (37%), cerebrosides (11.1%), monogalactosylmonoacylglycerol (1.4%), digalactosylmonoacylglycerol (2.3%), and DGDG (15.4%). These results are comparable to those reported for sorghum (vide supra). Pearl millet contains—in the pericarp, aleurone, and germ—lipases that have higher activities than those in other cereal grains but no lipoxygenase enzymes (Lai and Varrianomarston 1979, 1980; Galliard 1999).

Concluding Remarks Although cereal grains contain considerably less lipid compared to oilseeds, cereal lipids have ­several advantages with references to their composition. While oils from seeds are mainly triacylglycerols, cereal lipids contain relatively much higher levels of polar lipids (phospho- and glycol-lipid) and other bioactive compounds including phytosterols and tocols. Apart from palm oil and a few other oils, cereal grains provide the most important dietary source of tocotrienols for most people in the world. Although the contents of sterols and tocopherols in cereal kernels can be modest, cereals still contribute significantly to their intake because of the amounts consumed. The research and the utilization of cereal lipids in functional foods are limited and deserve special attention.

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Part III

Mycotoxic Bioactives of Fruits and Cereals

12 Mycotoxic Bioactives in Cereals and Cereal-Based Foods Anuradha Vegi Contents Introduction............................................................................................................................................. 253 Mycotoxigenic Fungi in Cereals and Cereal-Based Foods..................................................................... 254 Mycotoxigenic Fusarium Species in Cereals.................................................................................... 254 Mycotoxigenic Aspergillus in Cereals............................................................................................... 254 Penicillium Species in Cereals........................................................................................................... 255 Mycotoxins in Cereals and Cereal-Based Foods.................................................................................... 255 Aflatoxins........................................................................................................................................... 256 Ergot Alkaloids.................................................................................................................................. 257 Fumonisins......................................................................................................................................... 257 Ochrotoxins........................................................................................................................................ 257 Trichothecenes................................................................................................................................... 258 Zearalenone........................................................................................................................................ 259 Occurrence of Mycotoxic Bioactives during Cereal-Based Food Processing........................................ 259 Barley Malting................................................................................................................................... 260 Baking and Extrusion......................................................................................................................... 260 Corn Milling...................................................................................................................................... 260 Worldwide Distribution of Mycotoxic Bioactives...................................................................................261 Analytical Methods to Detect and Quantify Mycotoxins in Cereals.......................................................261 Microbiological (Culture) Methods....................................................................................................261 Gaseous Chromatography Methods to Detect Aspergillus, Fusarium, and Penicillium Volatiles............................................................................................................262 Polymerase Chain Reaction (PCR) Based Methods.......................................................................... 262 Immunological Detection Methods................................................................................................... 263 Summary................................................................................................................................................. 265 References............................................................................................................................................... 265

Introduction Mycotoxic bioactives 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 the mycotoxic bioactive under optimal conditions. These fungi and their mycotoxins pose a serious threat to not only the plant species such as cereals on which they survive and grow, but also are toxic to animal and human health who consume mycotoxin-contaminated cereal-based foods. The detrimental effects of these toxic bioactives to plants and animals have been researched extensively. Some of the toxigenic fungal species and a variety of mycotoxins that they produce in some cereals and cereal-based foods will be discussed in this chapter. The mycotoxin presence during some ­cereal-based 253

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food processing will also be included in the chapter. Occurrence, worldwide distribution, and ­toxicities of important mycotoxic bioactives will also be highlighted. The chapter will also focus on some of the detection and quantification methods for mycotoxigenic fungal species and their toxins in cereals and cereal-based foods. A myriad of toxigenic fungal species and mycotoxins have been studied and even as you read this chapter, there are many more mycotoxic bioactives being discovered and studied worldwide. The author of this chapter would strongly encourage the readers to find literature that focus on new fungal bioactives and updated information on known mycotoxins.

Mycotoxigenic Fungi in Cereals and Cereal-Based Foods Cereal grains are the staple food sources worldwide for both animal and human consumption. Cerealbased foods have high nutritional value including carbohydrates (45–76%), proteins (6–17%), and fat (1–7%), depending on the type of the cereal grains such as wheat, barley, oat, corn, and rice (Butt et al. 2008; Shewry 2007; Sugiyama et al. 2003; Thondre and Henry 2009; Zhao et al. 2009). Many filamentous plant pathogenic fungi are known to attack cereal crops in the farm (field fungi) as well as during storage (storage fungi). These fungi belong to many genera including Aspergillus, Fusarium, Penicillium (Abarca et al. 2001; Burgess et al. 1987; Castella et al. 2002; Muller et al. 1997; Pitt 2000). More information of these fungi is given in the following sections.

Mycotoxigenic Fusarium Species in Cereals Fusarium species belong to the field fungi group that attack cereal grains in the field. Fusarium head blight (FHB) is a disease of cereal grains like wheat, corn, oats, rye, and barley (Burgess et al. 1987; Muller et al. 1997). In the United States, FHB is mainly caused by F. graminearum (Stack 2003). The yield and quality of grain are reduced due to FHB, and grain that is harvested is usually contaminated with trichothecene mycotoxins such as deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON; Cook 1981a; Kommedahl and Windels 1981). The trichothecene mycotoxins produced by F. graminearum affect grain safety and are also hazardous to human and animal health (Prelusky et al. 1994). Trichothecenes are also potent phytotoxins (Desjardins and Hohn 1997). Warm, wet weather conditions (like continuous wetness at 25°C) at the time of wheat anthesis were important factors for severe FHB in wheat (Bai and Shaner 1994; McMullen et al. 1997a). Mutants of F. graminearum that do not produce DON were studied to determine the role of DON in plant pathogenesis (Proctor et al. 1995). Additional studies confirmed that production of DON plays an important role in the spread of FHB within a spike of wheat (Bai 2001). Barley is also affected by FHB, which is endemic in Northeast Asia (Cook 1981b) and has affected the Red River Valley region of the United States causing major losses (McMullen et al. 1997b). Fusarium adversely affects malting barley. Fusarium reduces the kernel plumpness of infected malting barley as well as wort color during brewing process (Flannigan 1996; Schwarz et al. 2001). Corn, a staple food source for people worldwide including Mexico where human consumption of corn products is as high as 60% of the total corn produced, is also susceptible to Fusarium species (Garcia and Heredia 2006; Sydenham et al. 1990). Fumonisins are a group of mycotoxins that are produced by F. verticillioides in corn, and they are highly toxic to both animals and human (Sydenham et al. 1990). Rice, an important component of the diet in many Asian countries, is also plagued by the Fusarium species. In Nepal, the common species found on rice were F. verticillioides and F. graminearum producing various mycotoxins such as beauvericin, moniliformin, gibberellic acid, nivalenol (NIV) and DON (Desjardins et al. 2000).

Mycotoxigenic Aspergillus in Cereals Aspergillus species infect and grow in stored cereal grains and produce mycotoxins such as aflatoxins and ochratoxin A (OTA; Madhyastha et al. 1993; Pitt 1987). Species known to produce OTA in

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cereals include A. auricomus, A. melleus, A. ochraceus, A. ostianus, A. petrakii, A. sclerotiorum, and A. ­sulfureus (in the A. ochraceus group); A. alliaceus and A. albertensis (in section Flavi); A. carbonarius and A. niger (in section Nigri); A. glaucus (in section Aspergillus), and P. nordicum (Abarca et al. 2001; Bayman et al. 2002; Castella et al. 2002; Dalcero et al. 2002). The chemical composition of cereal grains is reported to be changed due to colonization by ochratoxin A-producing fungi. When autoclaved barley and wheat samples were separately inoculated with A. alutaceus and P. verrucosum and incubated at 28°C for 7, 15, and 30 days, the OTA production of P. verrucosum on both barley and wheat increased significantly over time (Madhyastha et al. 1993). Higher OTA levels were produced by Aspergillus alutaceus on wheat than on barley. The lipid contents of both barley and wheat decreased due to colonization by A. alutaceus and P. verrucosum, and a small decrease in their starch content was observed. Also, a higher concentration of protein in wheat was observed (Madhyastha et al. 1993). Ochratoxin A has been reported to affect cereal grains and to be nephrotoxic in animals that consume infected grain (Keblys et al. 2004; Madhyastha et al. 1993; Pitt 1987). Aflatoxins are another set of mycotoxins that are produced by A. flavus and A. parasiticus in corn and corn-based food products (Garcia and Heredia 2006). These mycotoxins are highly carcinogenic, cause liver disease, and in extreme cases of exposure even cause death (Azziz-Baumgartner et al. 2005, Bandyopadhyay et al. 2007, Williams et al. 2004). These aflatoxigenic fungal species also affect other crops such as peanuts and cottonseed (Horn 2007; Payne et al. 1998). Aspergillus species also produce other bioactive toxins such as citrinin in cereal grains such as wheat (Abramson et al. 1990; Betina 1984).

Penicillium Species in Cereals Many stored cereals and cereal-based food products are contaminated with mycotoxins produced by Penicillium species. Various factors are involved in the contamination of stored cereal grains by mycotoxigenic Penicillium. Mechanical damage can occur at harvesting and also by rodents, birds, and insects that facilitate infection by fungi. Increasing moisture content, grain temperature, fungal spore content, and free fatty acids are the main factors involved in fungal spoilage of cereal grains (Abramson 1991). The food substrate plays an important role on the type of mycotoxin produced in Penicillium species. Penicillium verrucosum produced OTA and citrinin on bread, but the same strain produced only citrinin when yeast extract agar (YES) was used as a substrate, and produced no mycotoxins when cheese was used as a substrate (Kokkonen et al. 2005). Penicillium nordicum, when tested on the same substrates, produced OTA on all three substrates used. Penicillium crustosum produced another mycotoxin named roquefortin C on all three substrates, and produced a mycotoxin, penitrem A, only on cheese (Kokkonen et al. 2005). Citrinin and OTA had nephrotoxic effects, whereas penitrem A and roquefortin C were neurotoxic to mammals such as swine (Keblys et al. 2004). Corn or maize silage, which is an important animal feed, is also reported to be contaminated with the mycotoxigenic Penicillium species. The researchers have reported that not only the ensiled corn but also freshly harvested silage was contaminated with mycotoxins (patulin, mycophenolic acid, cyclopiazonic acid, and roquefortine C) produced by Penicillium (Mansfield et al. 2008). In a study conducted during 2003–2005 among bakery mills of Lithuania, freshly milled rye flour were mostly contaminated with Penicillium species including P. biforme, P. brevicompactum, P. chrysogenum, P. cyclopium, P. expansum, P. roqueforti, and P. velutinum. These Penicillium species have the capability to produce mycotoxins such as citrinin, cyclopiazonic acid, OTA, patulin, and roquefortin C (Lugauskas et al. 2006).

Mycotoxins in Cereals and Cereal-Based Foods Bioactive mycotoxins are secondary metabolites produced by fungal pathogens in cereal grains and cereal-based foods under optimal conditions in the field, during storage, or in processing. There are

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several types of mycotoxins that plague the cereal food industry. Some of these mycotoxins are described in the following sections.

Aflatoxins Cereals are commonly contaminated with aflatoxins. A wide variety of aflatoxins are produced by mycotoxigenic Aspergillus flavus and A. parasiticus in cereals and cereal-based foods (Figure 12.1A; Brase et al. 2009; Garcia and Heredia 2006). Structurally aflatoxins are polyketide-derived furanocoumarins and they are mainly divided into six types: aflatoxin B1, B2, G1, G2, M1, and M2. Aflatoxin B1 is the most common form as well as very carcinogenic (Brase et al. 2009; IARC 1993).

(a)

O

O

O

O O

CH3

O

O

(b)

N H .O H

N O

O

H3C

N

.H

CH3 O

N

CH3 H

N. H (c)

OH

O OH

H3C

O

OH

CH3

O

OH CH3

O O

HO O

CH3

OH

NH2

O HO

O

Figure 12.1  Structure of (A) aflatoxin B1, (B) ergotoxine, and (C) fumonisin B1. (From ChemID, Aflatoxin B1, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; ChemID, Ergotoxine, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009; Chem ID, Fumonisin B1, Chem ID Plus Advanced, National Library of Medicine, Betheseda, MD, 2009.)

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When cereal-based food products contaminated with aflatoxins are consumed, they cause cancer, liver disease, and other health problems in animals and humans. West African countries such as Nigeria are plagued by aflatoxin contamination where corn is an important part of the diet (Azziz-Baumgartner et al. 2005; Bandyopadhyay et al. 2007). Because of health concerns caused by aflatoxin containing foods, the United States and a few other countries have limited the dietary intake of aflatoxin to 20 ng/g and in Europe it is 4 ng/g (FAO 2004).

Ergot Alkaloids Structurally ergot alkaloids belong to indole alkaloids derived from a tetracyclic ergoline ring system (Figure 12.1B; Brase et al. 2009). Ergot disease caused by Claviceps purpurea can have up to 10% yield loss in wheat and almost 5% in rye crops, as reported by McMullen and Stoltenow (2002). Barley is also reported to be affected by ergot alkaloids (Schwarz et al. 2006). One of the first known diseases caused by a mycotoxin in human populations and animals is ergotism. Loss of limbs, gangrene, and an effect on the central nervous system are attributed to ergot alkaloids in animals and humans (De Costa 2002; van Dongen and de Groot 1995). Even though ergot alkaloids are known to cause damage to both cereal crops and to animals and humans who consume cereal-based foods, no regulatory limits are set as yet for ergot alkaloids in cereals.

Fumonisins Fusarium group of fungi, especially F. verticillioides and F. proliferatum produce another set of mycotoxins known as fumonisins in cereal grains such as corn, sorghum, and rice (CAST 2003). Their structure is very similar to that of the backbone of sphingolipids (Figure 1C; ApSimon 2001; Brase et al. 2009). The fumonisin group consists of fumonisins A1-A4, B1-B4, C1-C4, and P1-P4; however, fumonisins B1, B2, and B3 are of major concern in cereals and cereal-based foods. Fumonisins are the causative agents in the brain damage of horses known as leukoencephalomalacia (Marasas et al. 1988). The fumonisins in corn also affect human populations as they are reported to be involved in esophageal cancer (Rheeder et al. 1992). In experimental animals, such as rodents, fumonisin B1 is considered to be hepatotoxic (Domijan et al. 2008). Because of their prevalence in corn and cornbased foods, the maximum levels for total fumonisins in those foods were set by the FDA at 2–4 µg/g (depending on the type of corn product) and European countries adopted a stricter limit of 0.2 µg/g of fumonisins in processed corn baby food (FAO 2004).

Ochrotoxins Two main types of ochratoxins are present including ochratoxin A (OTA) and B (OTB). Ochratoxin A is a fungal secondary metabolite that is a chlorinated isocoumarin derivative linked to L-phenylalanine and the most important mycotoxin in cereals (Figure 12.2A). Ochratoxin A is produced by two main fungal species, A. ochraceus and P. verrucosum in stored cereal grains (Pitt 2000). A liquid chromatographic method was used to test for the presence of OTA in wheat, barley, green coffee, and roasted coffee in the United States. Ochratoxin A contamination (>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).

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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

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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.

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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 <.05) higher level of DON in germinated barley than during steeping. Steeping decreased the preharvest formed DON concentration, as DON is water soluble. The DON levels increased by 18%–114% after five days of germination (Schwarz et al. 1995). The kilning process did not affect the DON concentration, as DON is heat stable (Niessen et al. 1991; Schwarz et al. 1995). Deoxynivalenol and diacetoxyscirpenol produced by Fusarium affected the malting process, and FHB infected barley resulted in gushing of beer (Munar and Sebree 1997; Schapira et al. 1989). Mycotoxins were found in the beer after contaminated barley was used for brewing (Munar and Sebree 1997; Schwarz et al. 1995).

Baking and Extrusion High temperature processing steps such as baking seem to show a varied affect on different mycotoxins. Some mycotoxins such as OTA were not destroyed during bread making (Scudamore 2003; Subirade 1996). However, DON—which is supposed to be a heat-resistant mycotoxin—decreased in concentration during baking of bread, biscuits, and cookies (Scott et al. 1983). In a study on the effect of wheat bread making on aflatoxins, the total aflatoxins concentration were reduced by 41% after baking (El-Tawila et al. 2003). Jackson et al. (1997) have shown that the interior regions of baked corn muffins had increased levels of fumonisin B1 when compared to the outer layers of the muffin, indicating destruction of fumonisins under high temperature processing. Breakfast cereals and similar foods are produced using extrusion processing. Higher temperatures (>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.

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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

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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

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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

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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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McDevitt, J. J., P. S. Lees, W. G. Merz, and K. J. Schwab. 2004. Development of a method to detect and ­quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples. Mycopathol. 158 (3): 325–35. McMullen, M. P., J. Enz, J. Lukach, and R. Stover. 1997a. Environmental conditions associated with Fusarium head blight epidemics of wheat and barley in the Northern Great Plains, North America. Cereal Res Commun. 25 (3/2): 777–8. McMullen, M. P., R. Jones, and D. Gallenberg. 1997b. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Dis. 81:1340–8. McMullen, M., and C. Stoltenow. 2002. Ergot. In Publication PP551. Fargo, ND: NDSU Agriculture and University Extension. Mirocha, C. J., H. K. Abbas, C. E. Windels, and W. Xie. 1989. Variation in deoxynivalenol, 15-acetyldeoxynivalenol, and zearalenone production by Fusarium graminearum isolates. Appl Environ Microbiol. 55 (5): 1315–6. Moss, M. O., and U. Thrane. 2004. Fusarium taxonomy with relation to trichothecene formation. Toxicol Lett. 153: 23–8. Muller, H. M., J. Reimann, U. Schumacher, and K. Schwadorf. 1997. Fusarium toxins in wheat harvested ­during six years in an area of Southwest Germany. Nat Toxins 5:24–30. Muller, G., H. Rosner, B. Rohrmann, W. Erler, G. Geschwend, U. Grafe, B. Burkert, et al. 2003. Effects of the mycotoxin ochratoxin A and some of its metabolites on the human cell line THP-1. Toxicol. 184:69–82. Munar, M. J., and B. Sebree. 1997. Gushing—A malster’s view. J Am Soc Brew Chem. 55:119–22. Ng, W., M. Mankotia, P. Pantazopoulos, R. J. Neil, P. M. Scott, and B. P. Lau. 2009. Survey of dry pasta for ochratoxin A in Canada. J Food Prot. 72 (4): 890–3. Nicholson, P., D. R. Simpson, G. Weston, H. N. Rezaroor, A. K. Lees, D. W. Parry, and D. Joyce. 1998. Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol. 53:17–37. Niessen, L., S. Donhauser, and H. Vogel. 1991. Zur problematic von Mycotoxinen in der Brauerei. Brauwelt. 131:1510. Niessen, L., and R. F. Vogel. 1997. Specific identification of Fusarium graminearum by PCR with gaoA ­targeted primers. System Appl Microbiol. 20:111–3. Niessen, L., and R. F. Vogel. 1998. Quantitative estimation of Fusarium graminearum DNA using a solid phase PCR assay (DIAPOPS). J Food Mycol. 1:73–84. Nilsson, T., T. O. Larsen, L. Montanarella, and J. O. Madsen. 1996. Application of solid-phase microextraction for analysis of volatile fungal metabolites. J Microbiol Methods. 25:245–55. Noots, I., J. A. Delcour, and C. W. Michiels. 1998. From field barley to malt: Detection and specification of microbial activity for quality aspects. Crit Rev Microbiol. 25:121–53. Olsson, J., T. Borjesson, T. Lundstedt, and J. Schnurer. 2002. Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose. Int J Food Microbiol. 72: 203–14. Pasanen, A. L., S. Lappalainen, and P. Pasanen. 1996. Volatile organic metabolites associated with some toxic fungi and their mycotoxins. Analyst. 121:1949–53. Payne, G. A., K. K. Sinha, and D. Bhatnagar. 1998. Process of contamination by aflatoxin-producing fungi and their impact on crops. In Mycotoxins in Agriculture and Food Safety, 279–306. New York: Marcel Dekker. Petziner, E., and K. Ziegler. 2000. Ochratoxin A from a toxicological perspective. J Vet Pharmacol Ther. 23:91–8. Pitt, J. I. 1987. Penicillium viridicatum, Penicillium verrucosum, and production of ochratoxin A. Appl Environ Microbiol. 53:266–9. Pitt, J. I. 2000. Toxigenic fungi and mycotoxins. Br Med Bull. 56 (1): 184–92. Prelusky, D. B. 1997. Effect of intraperitoneal infusion of deoxynivalenol on feed consumption and weight gain in the pig. Nat Toxins. 5:121–5. Prelusky, D. B., B. A. Rotter, and E. G. Rotter. 1994. Toxicology of mycotoxins. In Mycotoxins in Grains: Compounds Other Than Aflatoxin, ed. H. L. Trendholm, 359–403. St Paul, MN: Eagan Press. Proctor, R. H., T. M. Hohn, and S. P. McCormick. 1995. Reduced virulence of Gibberella zeae caused by ­disruption of a trichothecene toxin biosynthetic gene. Mol Plant-Microbe Interact. 8 (4): 593–601.

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Reischer, G. H., M. Lemmens, A. Farnleitner, A. Adler, and R. L. Mach. 2004. Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan probe. J Microbiol Methods. 59:141–6. Rheeder, J. P., W. F. O. Marasas, P. G. Thiel, E. W. Sydenham, G. S. Shepherd, and D. J. van Schalkwyk. 1992. Fusarium moniliforme and fumonisins in corn in relation to esophageal cancer in Transkei. Phytopathol. 82:353–7. Salas, B., B. J. Steffenson, H. H. Casper, B. Tacke, L. K. Prom, T. G. Fetch, and P. B. Schwarz. 1999. Fusarium species pathogenic to barley and their associated mycotoxins. Plant Dis. 83:667–74. Samson, R. A., E. S. Hoekstra, F. Lund, O. Filtenborg, and J. C. Frisvad. 2000. Methods for the detection, isolation and characterization of food-borne fungi. In Introduction to Food- and Airborne Fungi. 6th edition, eds. R. A. Samson, E. S. Hoekstra, J. C. Frisvad, and O. Filtenborg, 283–97. The Netherlands: Centraalbureau voor Schimmelcultures. Schapira, F. D., M. P. Whitehead, and B. Flannigan. 1989. Effects of mycotoxins diacetoxyscirpenol and deoxynivalenol on malting characteristics of barley. J Ins. Brew 95: 415–7. Schilling, A. G., E. M. Moller, and H. H. Geiger. 1996. Polymerase chain reaction-based assays for speciesspecific detection of Fusarium culmorum, F. graminearum and F. avenaceum. Phytopathol. 86: 515–22. Schnerr, H., L. Niessen, and R. F. Vogel. 2001. Real-time detection of the Tri5 gene in Fusarium species by Light Cycler-PCR using SYBR Green I for continuous fluorescence monitoring. Int J Food Micro. 71:53–61. Schwarz, P. B., H. H. Casper, and S. Beattie. 1995. Fate and development of naturally occurring Fusarium mycotoxins during malting and brewing. J Am Soc Brew Chem. 53:121–7. Schwarz, P. B., J. G. Schwarz, A. Zhou, L. K. Prom, and B. J. Steffenson. 2001. Effect of Fusarium graminearum and F. poae infection on barley and malt quality. Monatsschrift for Brauwissenschaft. 54: 55–63. Schwarz, P. B., S. M. Neate, and G. E. Rottinghaus. 2006. Widespread occurrence of ergot in upper midwestern U.S. Barley, 2005. Plant Dis. 90:527. Scott, P. M., S. R. Kanhere, P.-Y. Lau, J. E. Dexter, and R. Greenhalgh. 1983. Effects of experimental flour milling and breadbaking on retention of deoxynivalenol (vomitoxin) in hard red spring wheat. Cereal Chem. 60: 421–4. Scudamore, K. A., J. Banks and S. J. MacDonald. 2003. Fate of ochratoxin A in the processing of whole wheat grains during milling and bread production. Food Addit Contam. 20 (12): 1153–63. Scudamore, K. A., and S. Patel. 2009. Fusarium mycotoxins in milling streams from the commercial milling of maize imported to the UK, and relevance to current legislation. Food Addit Contam. 26 (5): 744–53. Scudamore, K. A., S. Patel, and V. Breeze. 1999. Surveillance of stored grain from the 1997 harvest in the United Kingdom for ochratoxin A. Food Addit Contam. 16 (7): 281–90. Seo, J. A., J. C. Kim, D. H. Lee, and Y. W. Lee. 1996. Variation in 8-keto trichothecene and zearalenone production by F. graminearum isolates from corn and barley in Korea. Mycopathol. 134:31–7. Sharma, R. P., and Y. W. Kim. 1991. Trichothecene. In Mycotoxins and Phytoalexins, eds. R. P. Sharma and D. K. Salunche, 339–59. Boca Raton, FL: CRC Press. Shewry, P. R. 2007. Improving the protein content and composition of cereal grain. J Cereal Sci. 46 (3): 239–50. Shier, W. T., A. C. Shier, W. Xie, and C. J. Mirocha. 2001. Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins. Toxicon. 39:145–38. Skaug, M. A. 2003. Levels of ochratoxin A and IgG against conidia of Penicillium verrucosum in blood ­samples from healthy farm workers. Ann Agric Environ Med. 10 (1): 73–7. Subirade, I. 1996. Fate of ochratoxin A during breadmaking. Food Addit Contam. 13 (Suppl.): 25–6. Sugiyama, M., A. C. Tang, Y. Wakaki, and W. Koyama. 2003. Glycemic index of single and mixed meal foods among common Japanese foods with white rice as a reference food. Eur J Clin Nutr. 57 (6): 743–52. Stack, R. W. 2003. History of fusarium head blight with emphasis on North America. In Fusarium Head Blight of Wheat and Barley, eds. K. J. Leonard and W. R. Bushnell, 14. St. Paul, MN: APS Press. Sydenham, E. W., P. G. Thiel, W. F. O. Marasas, G. S. Shephard, D. J. Van Schalkwyk, and K. R. Koch. 1990. Natural occurrence of some Fusarium mycotoxins in corn from low and high esophageal cancer prevalence areas of the Transkei, southern Africa. J Agric Food Chem. 38:1900–3. Thondre, P. S., and C. J. K. Henry. 2009. High-molecular-weight barley β-glucan in chapatis (unleavened Indian flatbread) lowers glycemic index. Nutr Res. 29 (7): 480–6.

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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). 273

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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

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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).

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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

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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

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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.

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.

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.

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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.

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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 CH3 HO

CH3

CH3

O

OH

O O O H3C O CH3

O

CH3

Figure 13.11  T-2 toxin.

H H3C

H

OH

O O

O OH

CH2 OH

Figure 13.12  Deoxynivalenol.

CH3

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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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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

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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

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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,

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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.

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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.

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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.

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 291

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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

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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.

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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

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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

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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.

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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).

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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

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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

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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 <16°C during reproduction and maturation (November to December). Gardening and soil fertilization during fall and winter seasons, spreading of plant litter (especially rubbish and other plant materials remaining from pistachio nut processing), and animal manures were shown to marginally increase spore density in pistachio orchards. Aspergillus species are soil borne in pistachio growing regions and their spore density is greatly affected by the fungal population in the soil, and any gardening and soil practices would result in increased density in pistachio orchards during the nut growing season (Mojtahedi et al. 1978; Moradi et al. 2004). The results indicated that the population density of Aspergillus species varied in different years and regions. Variations in spore density among different agro-ecological zones may be related to the variation in prevailing climatic conditions and cultural practices in the different zones. Setamou et al. (1997) pointed out that there was a trend of the higher rate of aflatoxin contamination and A. flavus infection from south to north, which may affect infection and contamination incidences of Mussidia nigrivenella cultivars grown in each zone. The effects of temperature on contamination for peanuts to A. flavus and aflatoxin indicated that the infection and aflatoxin production in peanuts can be related to the occurrence of soil moisture stress during pod-filling when soil temperatures are near optimal for A. flavus infection and growth (Craufurd et al. 2006).

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Infection of pistachios by A. flavus is associated with early splits, a condition in which the hull splits before the nut is mature (Doster and Michailides 1994a, 1994b). In both pistachios and almonds, high aflatoxin contamination is associated with damage by the navel orange worm larvae (Doster and Michailides 1994a, 1994b; Thomson and Mehdy 1978). The two overriding conditions that influence aflatoxin contamination are temperature and moisture (Widstrom 1996; Payne and Brown 1998). In corn and peanuts, high temperatures and drought stress lead to high levels of aflatoxin contamination (Payne and Brown 1998). The researchers found that peanuts grown with adequate moisture had no aflatoxin. Similarly, peanuts grown under prolonged drought with temperatures less than 25°C or greater than 32°C were free of aflatoxin. Colonization by A. flavus and aflatoxin contamination were maximized at 30.5°C. High temperature and drought conditions increase the air-borne inoculum of the fungus (Jones et al. 1981; McGee et al. 1996). The increased growth and reproduction of the fungus at higher temperatures is presumably related to its relatively high optimum growth temperature. The fungus can grow over a wide range of temperatures (12–48°C), but its optimum for growth is 37°C (Klich et al. 1992). The higher temperatures and drought conditions also may favor A. flavus over other fungi because of its ability to grow on substrates with low water activity. The fungus can grow at an Aw as low as –35 megapascals (Mpa; Klich et al. 1992). Interestingly, the optimum temperature or aflatoxin production is 25–30°C (Ashworth et al. 1969; Maggon et al. 1977). Temperature and drought stress also likely predispose the plant to increased infection; however, little is known about the mechanisms. High day and night temperatures also have been associated with higher aflatoxin levels in almonds (Doster and Michailides 1995a, 1995b). The source of inoculum for A. flavus is in the soil but the predominant survival structure is not known. Soil temperature (McGee et al. 1996) and moisture (Jones et al. 1981) greatly influence the number of conidia in the soil and the air.

Harvest Conditions Many efforts have been done to reduce and prevent the contamination of pistachios to mycotoxins because of their hazardous affects on human and animal health. Harvesting of nuts should begin as soon as possible to prevent fungal attack and insect damage. In most cases, the infection of nuts by mycotoxigenic fungi as well as their mycotoxin occurs before harvesting under field conditions and affected by the factors influencing the contamination (Denizel et al. 1976; Emami et al. 1977; Danesh et al. 1979; Mojtahedi et al. 1979; Heperkan et al. 1994). The major strategy to reduce the kernel aflatoxin contamination in pistachios is early peaking, which has been shown to be critical. In harvest delay, the frequency of contaminated nuts as well as the key factors in infection will be highlighted (Crane 1978; Kader et al. 1982; Panahi et al. 2005). For example, the frequency of air-borne Aspergillus species, opportunity, and time for frequency and intensity of kernel colonization, the number of cracked pistachios and as a consequence the amount of aflatoxin greatly increased in delay of harvest (Esmaeilpour 2004; Panahi et al. 2005). Therefore, it is critical to harvest the nuts in a timely manner or as early as possible—the first week after the physiological maturation of pistachio nuts. The application of this strategy in Iran has led to exporting pistachios and a significant reduction in rejected pistachios from EU countries. The results of the research indicated that the infection of nuts to A. flavus occur under field conditions as well as aflatoxin production, which may be developed during the next stages. The key factors in infection are cracking and early splitting pistachios, which expose the kernels to the air bore spores of Aspergillus species. The spores of Aspergillus on the kernels can grow and compete with other fungal species and produce aflatoxins. This indicates the hull of pistachio nuts physically protects the kernels against air-borne fungal species and several insects. If the pistachio nuts aren’t harvested timely according to the environmental conditions and cultivars, it increases over-maturation that causes an increment in the frequency of early splitting and cracking pistachios. This implies the possibility of health risk to the consumer in late harvested nuts. In the Ohadi cultivar, the results regarding the best harvest time in Kerman showed that the highest dry weight of fruit is in the last week of September, the highest weight of the nuts is in the first week of October, and the maximum maturation percentage of fruit is in the second week of October. The highest rate of over-maturated fruits is in the third week of October and the lowest rate in the last week of August and first week of September (Esmaeilpour 2004; Panahi et al. 2005).

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The most suitable harvest time can be estimated by quality indices such as percentages of shelling, fruit maturation, and shell-coloration and kernel dry weight, usually as 1–3 weeks after easy peeling. Due to the high moisture of pistachio nuts (40–50%) and shell dehiscent (fruit maturation) during harvest, the nuts are very sensitive to physical and mechanical damages and their quality is influenced by different peeling methods (Crane 1978; Kader et al. 1982; Esmaeilpour 2004; Panahi et al. 2005). On the other hand, any delay in harvest can effectively influence microbial charge as well as the external quality of pistachios. Dry kernel weight, the amount of pure fat including maturation and change in the shell color represent that the pistachio is physiologically matured and the hull is easily separated. The nuts harvested before the time of maturation resulted in failure of kernel development. On the other hand, harvesting past the time leads to staining on the surface of the shell decreasing marketing value. This staining does not deeply penetrate into the shell, which may result in different compounds when cell disruption occurs. When an early split occurs, the split will normally start at 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. 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 1993). Another kind of split that can occur on a pistachio hull shortly (less than 15 days) before harvest is called a growth split. 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). The results indicated that less than 2% of the total pistachios are early splitting, which may or may not contain aflatoxin; however, there is no aflatoxin that could be detected in pistachios without any cracking in the hull. About 6–8 weeks after beginning the harvest the pistachio hulls are being tattering, which may subject nuts to insect contamination as well as an increase in the amount of aflatoxin kernel content especially in those pistachios that contained Apomyelois ceratoniae (Crane 1978; Esmaeilpour 2004;Panahi et al. 2005). A positive correlation was observed among increasing spore density and the reproduction and maturation period of pistachio nuts in the spring and summer seasons. The results indicated that the peak of spore density coincides with cracking and harvesting of pistachio nuts in the orchards (Moradi et al. 2009). Therefore, late harvesting (after the second half of September) not only causes an increase in the cracking of pistachio nuts but also provides further exposure to high spore density of A. flavus, resulting in more aflatoxin production and contamination of kernels in the orchards. Damage from birds, pests, shell discoloration, and falling pistachio nuts from trees on contaminated soil surface are other risk factors associated with late harvesting (Sommer et al. 1986; Moradi 2005; Moradi and Javanshah 2006; Moradi and Mirabolfathy 2007). Several reasons may explain the increased spores of the Aspergillus species in pistachio orchards such as cracking of pistachio nuts (especially early splitting pistachios), environmental factors, contaminated nuts in processing units, cultural practices, frequency and time of irrigation, plant litter, animal manures, and pistachio nuts dropping to the ground (Thomson and Mehdy 1978; Mojtahedi et al. 1979, 1980; Sommer et al. 1986; Doster and Michailides 1995, 1999; Mirabolfathy et al. 2006; Moradi 2005; Moradi and Javanshah 2006; Moradi and Mirabolfathy 2007). Other parameters that will be seriously affected with a delay in harvest are over-maturing pistachios, which has shown significant differences in different times of harvest. For example, the frequency of over-mature pistachios on September 6 and October 22 were not detectable to 17.2%, respectively (Esmaeilpour 2004; Panahi et al. 2005). The results of a research indicated that the contamination of pistachios to aflatoxin was 9.4 ppb on October 22, while it was not detectable in September.. This implies that the harvest time has important effects on the risk of the contamination of pistachios to the toxigenic fungal species and accumulation of

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aflatoxin in the kernels. Delay in peeling pistachios also caused a considerable increase in the contamination of pistachios to aflatoxin, for example, 48 hours delay increased the aflatoxin concentration up to 15.6 ppb (Esmaeilpour 2004; Panahi et al. 2005). The delay in harvest will influence the appearance quality and staining of a pistachio shell. The shell staining has a direct correlation with aflatoxin kernel content. On the other hand, appearance quality decreases the marketing of pistachios, which may have high risk of aflatoxin contamination (Doster and Michailides 1999). The delay in harvest causes significant reduction in the frequency of intact pistachios, as the hull protects the kernel against air-borne fungal species, dust, loss of kernel moisture, and insect damages. These parameters are critical in infection of pistachio kernels to Aspergillus species and aflatoxin production that has been reported by Sommer et al. (1986). In a delay of harvest, the frequency of over-matured pistachios that provides opportunity for high early splitting formation and the high amount of aflatoxin in the kernels increase (Shatzki and Pan 1997). Kader et al. (1982) reported that the kernel moisture during the harvest is between 30 and 45%, which favors infection by Aspergillus species as well as competing with other microflora of the kernels. This implies that the pistachio kernels at harvest time are sensitive to physical and mechanical damages and their quality is influenced by the different methods of processing. On the other hand, any delay in this process may have a considerable effect on increasing microbial charge and external quality of products. Fluctuations of spore density in the processing plants during the peeling stage were influenced by the spores released during processing. Early splitting, cracking, shrunk and dried hull nuts, damage by insects and birds, and the collection of fallen pistachio nuts from the ground have been found to increase spore density throughout the peeling and bleaching stages (Moradi 2005), which cause high surface infection of healthy pistachio nuts during processing. The harvest time is of great importance on the contamination of pistachio nuts with fungal species and mycotoxins. Regarding the cultivar, soil texture, and weather conditions of the region, it is recommended that the yields of each cultivar should be harvested at physiological maturation (Doster and Michailides 1995). For determining the physiological maturation time, the fruit’s physical properties, cultivar, the soil texture, environmental conditions, processing facilities, and kernel composition should be considered. For example, the Ouhadi cultivar should be harvested when the kernel moisture, fats, s­ ugars, and physical properties are between 40–42%, 54–56%, 14–16%, and 70–75%, respectively (Panahi et al. 2005). It has been demonstrated that during the harvesting time, the density and frequency of the Aspergillus species as a group increase (Figure 14.3). This implies a positive relation between physiological maturation and the population density of Aspergillus species in pistachio orchards. In all studies carried out on the population density of Aspergillus species in the pistachio orchards, A. flavus was detected in low frequency. This species, however, is a main aflatoxin producer (Michailides and Morgan 1990; Mirabolfathy et al. 2006). The increase in the density of Aspergillus species in the pistachio orchards during harvest can be explained due to an increase in cracked pistachios, pistachios that fell to the ground, processing pistachio nuts, and pistachios in contact with the orchard surface. On the other hand, the longer the time that nuts are exposed to the air-borne spores can increase the chance of infection as well as accumulation of mycotoxins in kernels. Sampling during pistachio nut maturation in California showed that A. flavus was not isolated from nuts in July and from only 0.3% of the nuts in August. However, 11.8% of nuts were infected at harvest (September 15). There was a positive correlation between the level of A. flavus infection and the time that pistachio kernels exposure to the air-borne spores. At harvest, 28% of the insect-damaged nuts contained A. flavus compared to only 6% of the undamaged ones. When tested for A. flavus, 70% of the A. flavus isolates produced AFB(1). However, A. flavus was not detected in 29 nut samples (Thomson and Mehdy 1978).

Processing and Storage Freshly harvested pistachio nuts arrive at the processing plant and undergo the following fully mechanized processing steps in the sequence given in Figure 14.4, before being ready for storage and the subsequent shipment for exporting.

304

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Weighing and unloading Hulling (removal of hull) Cleaning & washing Water floatation tank (for the seperation of blank and/or underweight nuts) Washing Heated, forced air continuous pre-drying “Stick-tight” seperation and removal (to remove from the main product stream, the nuts with the skin still adhering to the shell) Picking conveyer belt (optional) (to remove from the main product stream, the nuts with visible defects) Main drying stage, using batch or continuous dryers (uses high volume forced air, heated and then ambient air flow, to reduce the water activity of the product) Sun drying or aeration silos (to further reduce water activity of the product) Mechanical sizing Mechanical seperation of open Vs closed-shell nuts Picking conveyer belt (to remove from the main product stream, the nuts with visible defects) Packaging

Figure 14.4  Fully mechanized processing steps in the sequence given.

Although the application of methyl bromide is proven to control Aspergillus species, several factors are affecting the contamination of pistachios in pre- and postharvest as well as low relative humidity and the fact that the spores of these species could be found everywhere, it is not necessary to decontaminate storage in the main pistachio producing dry areas such as Iran. The study of relative humidity of the storage in Rafsanjan and analyzing the possibility of postharvest contamination of pistachio nuts with aflatoxin has shown that, due to the low relative humidity during the storage of pistachio nuts, the spores of fungi A. flavus, which are present on most of the stored pistachios, will not have the opportunity for growth and production of toxin (Ershad 1973; Emami et al. 1977). The population density of Aspergillus species in different processing plants showed that several factors such as processing plant type (traditional or mechanized), different parts of a processing plant, cultivar, frequency of cracked pistachios (particularly, early splitting and irregular cracking pistachios with dry and shriveled hull), and pistachios that fell from the tree are the main factors affecting frequency and density of kernel contamination (Moradi and Mirabolfathy 2007). The comparison of traditional and mechanized processing plants showed that processed pistachios from the traditional processing plants have the higher density of A. flavus and A. niger groups than mechanized ones (Moradi and Mirabolfathy 2007). Therefore, the mechanized processing plants are preferred to traditional. On the other hand, some of processing plants may increase the density of Aspergillus species in processed pistachios compared to the density of these species before processing (Figure 14.5). In

Control of Mycotoxin Bioactives in Nuts: Farm to Fork

Population density (CFU/nut)

1400 1200 1000 800

305

Prepeeling Peeling After washing Predryer Sun drying Storage

600 400 200 0

Figure 14.5  Population density of Aspergillus species during different stages of pistachio nut processing (CFU/nut). (From Moradi, M. and Mirabolfathy, M. Iranian Journal of Pajouhesh and Sazandeghi (In Persian), 77, 105–10, 2007.)

Figure 14.6  Schematic of floating tank using in pistachio processing terminals. (From Momtazan Ind. Co. at http:// www.momtazan.com/images/FLOATING TANK.jpg)

most cases, while in processing plants the population density is significantly less than before processing (Moradi and Mirabolfathy 2007; Hokmabadi 2008). Comparison of different washing systems showed that water shower baths are more efficient than current or noncurrent pools to reduce population densities of molds (Figures 14.6 and 14.7). However, this system is not capable of separating the pistachios contaminated with aflatoxin. The system of a flowing water basin, due to the relative functionality for separating light and aflatoxin-contaminated pistachios from the healthy pistachios, is preferred to the above-mentioned system. However, this system causes the cross-contamination of the infection during the washing and mixing of pistachios. Combining the two mentioned systems might be effective for separating the contaminated pistachios from the healthy pistachios (Moradi and Mirabolfathy 2007). The population density of Aspergillus species in three successive days (1st, 2nd, and 3rd days) during sun-drying, pistachios were not significantly different (Figures 14.5 and 14.8; Hokmabadi et al. 2007; Moradi and Mirabolfathy 2007). Drying the nuts in a dryer caused significant reduction in the population density of Aspergillus even several hours after drying (Figure 14.9; Kashani-Nejad et  al. 2003; Hokmabadi et al. 2007). It seems to be, therefore, the weather conditions (especially temperature and relative humidity) in the Kerman province that has no favored effects to increase growth Aspergillus species and contamination of pistachios under sun-drying conditions. This is also true for the storage conditions in Kerman province during the autumn and winter seasons. Overall, the infection and aflatoxin production take place in preharvest. During the processing of nuts, a large amount of spores will be released from the cracked pistachios and pistachios fallen to the ground, which will

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Figure 14.7  Designed and tested rewashing system after floating tank. (From Hokmabadi, H., Tajabadi Pour, A., Moradi, M., et al., Acta Hort. (ISHS), 741, 259–64, 2007.)

No . spores in 100 nuts

60 3rd terminal

50

1st terminal

2nd terminal

40 30 20 10 0

0

8

12

24

32

40

48

Hours of pistachio exposure in sun field

Figure 14.8  Fluctuation in the colony of aflatoxin producer fungus in three sun fields. (From Hokmabadi, H., Tajabadi Pour, A., Moradi, M., et al., Acta Hort. (ISHS), 741, 259–64, 2007.)

No . spores in 100 nuts

60

Second mechanical dryer

50 40 30 20 10 0

0 4 8 Hours of pistachio exposure in mechanical dryer

No . spores in 100 nuts

60 50

First mechanical dryer

40 30 20 10 0

0

8

16

24

Hours of pistachio exposure in mechanical dryer

Figure 14.9  Fluctuation in the colony of aflatoxin producer fungus in two mechanical dryers. (From Hokmabadi, H., Tajabadi Pour, A., Moradi, M., et al., Acta Hort. (ISHS), 741, 259–64, 2007.)

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Control of Mycotoxin Bioactives in Nuts: Farm to Fork

cause ­cross-contamination of healthy and noncracked hulled pistachios, and thus problems will appear through the further stages of storage and transportation—if the conditions are favored (Moradi and Mirabolfathy 2007).

Sorting Out Contaminated Pistachios

Con. (ng/g)

The studies on the processed pistachios showed that there are positive relations between contaminated pistachios and the shell characteristics, which can be used as an advantage to sorting them out (Figure 14.10; Farsaie et al. 1978, 1981; Moradi and Javanshah 2006). The intensity of a shell staining pistachio has a positive correlation with early splitting, content of kernel aflatoxin, quality, damaged by pests especially Apomyelois ceratoniae, and the density of Aspergillus species as well as in a negative way with kernel weight. Sommer et al. (1976) 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. Doster and Michailides (1993) observed that early split pistachio nuts tend to have shell stains near the perimeter of the shell suture split. Furthermore, they also reported that early split nuts with shell staining had a higher incidence of Aspergillus molds than nonstained, early split nuts. The highest population density of Aspergillus species belong to the stained, yellow, small, deformed, and floated pistachios (Moradi and Javanshah 2006). However, stained and deformed pistachios have the highest content of kernel aflatoxins. These pistachios are usually sorted out from pistachio bulks during processing. Based on the time of early splitting formation, the physical appearance and their quality are significantly different. The old early splitting has a dry and shriveled hull with a high content of aflatoxin, while the new ones have a semidry or soft hull with a low amount of contamination. This indicated that the temperature, kernel moisture, and time to colonize the kernels in the pistachio orchards favor the infection, frequency, and density of kernel colonization and aflatoxin production as well as competing with other microflora to occupy the ecological niches. The contamination of different parts of the pistachio fruit, involving the kernel, shell, and the hull with aflatoxin shows that the average of total aflatoxin decreases in a kernel, shell, and hull, respectively (Moradi 1998). The results indicated that the concentration of aflatoxin B1 increased in small, stained, and yellow shell discoloration pistachios and decreased in yellow shell discoloration, stained, and deformed pistachios, respectively. The percentage or existence of yellow shell discoloration, stained, and deformed pistachios can be used to determine the contamination of pistachio bulks. The least amount was attributed to semimechanized terminal and pistachio sample in Yazd province (Moradi and Javanshah 2006). 900 800 700 600 500 400 300 200 100 0

A B1

GG F F F F Nss D S S1

A

Total

FF EE Nss

D S2

CD C D CD S

Y

Cd C

CB

CB

D

S

EE D S3

S

Y

S4

Figure 14.10  Distribution of aflatoxin in processed pistachios in processing plants S1: pre-sun drying; S3: yellow discoloration in sinker pistachios; S2: small; S4: yellow discoloration in floater pistachios; Nss: nonshell discoloration; S: shell discoloration; D: deformed; Y: yellow shell discoloration.

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For small pistachios, results showed that there was no correlation between the nut size and contamination by aflatoxin, because the aflatoxin concentration was high in some and low in other nonstained small pistachios. Perhaps one of the reasons is the size of the nut; that is, small pistachios are collected in one group in processing plants, which cause contamination of those genetically or by factors such as looking small and they are not contaminated under normal conditions. However, this needs more investigation classifying them based on weight and appearance as well as establishing their correlation to aflatoxins. Schatzki and Pan (1997) reported that the average aflatoxin level appears to fall when the nut size gets very small (below 0.5 g/nut). It seems that pistachio nuts in very small sizes reduce the ability and possibility of fungi entrance. The results indicated that stained, yellow shell discoloration, and deformed pistachios in sinking and floating pistachios are the main source of aflatoxin in pistachio processing terminals. Normally, these pistachios are sorted out during processing in terminals.

Ecology of Aspergillus Species in Pistachio Orchards Aspergillus species have been frequently isolated from the soil, pistachio litter, and animal manures in orchards. Aflatoxin-producing strains of A. flavus and A. parasiticus distributed worldwide in the soil and air, are classified as both field and storage fungi. In most regions, Aspergillus species are widely distributed, which implies their high tolerance in different geographical climates. The conidia for this fungus could be found in the air and soil of most regions, which cause serious problems in the process of producing agricultural yields. The rubbish remaining from nut processing had been seriously colonized by Aspergillus species and spore bulks of Aspergillus species observed with a disarmed eye, simply (Moradi et al. 2004). Similarly, Doster and Michialides (1994) reported that various Aspergillus molds heavily infest, infect, and sporulate on litter from pistachio trees, such as the fallen pistachio fruit and male inflorescence in commercial orchards, and Aspergillus spp. is commonly associated with litter from pistachio trees on the orchard floor. Sheep manure had the highest population density among other kinds of manures such as cow and poultry manure commonly used in Kerman pistachio orchards. On the other hands, the density of the Aspergillus species is related to the stage and percentage of animal manure colonization (Moradi et al. 2004). When animal manures are applied on the soil surface, they can act as a suitable substrate for increasing populations of Aspergillus species. Therefore, animal manures should be applied as fertilizer canals in pistachio orchard. A. niger population density was more than that of A. flavus; however, A. flavus has greater importance due to its aflatoxin production. During the peeling and bleaching of pistachio nuts, a large number of spores are released in the processing plants. Spores can infect the rubbish remaining from pistachio nut processing and develop when they are transported to orchards during the fall and winter seasons. Colonization of a rubbish bulk showed that Aspergillus species could grow around it but no observed colonization at its center. The Aspergillus species locally grows on a rubbish bulk. The differences in local colonization in a rubbish bulk by Aspergillus species may be due to higher moisture content, temperature, and combination microorganisms that cause a growth inhibition for Aspergillus species in the center of a rubbish bulk (Moradi et al. 2004). However this subject isn’t well known and needs more research. It has been documented that Aspergillus species can grow in lower moisture contents than most other fungi (Purcell et al. 1980; Jones et al. 1981; McGee et al. 1996; Rodriguez-del-Bosque 1996). Spreading of the litter increases spore density of the Aspergillus species in orchard spaces and if they mix with soil during fall and winter seasons, can increase their populations. They have a role as the source of primary inoculum in pistachio orchards for infection of pistachio nuts to the Aspergillus species (Mirabolfathy 1981; Moradi et al. 2004). Gardening processes cause the scattering of a large number of Aspergillus spores in dust to aerial parts of trees, especially when pistachio nuts are sensitive to infection. Wicklow et al. (1993) reported that most of the Aspergillus flavus conidial inoculum survived the first winter after burial in a cornfield in Illinois and Georgia. Aspergillus flavus has frequently been detected in bird and insect damaged nuts. Doster and Michailides (1993) 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

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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. Fallen leaves on the orchard floor are a suitable substrate for sporulation of the Aspergillus species as well as potential sources of primary inoculum for over-wintering of propagules of the Aspergillus species in pistachio orchards in later season, especially when mixed or buried in the soil during gardening processes. The role of plant debris in over-wintering and increments of the Aspergillus species populations were studied in different crops by some researchers. For example, increasing primary and secondary inoculum and their effects on population of the Aspergillus species were reviewed in some of the crop by Diener et al. (1987), development of Aspergillus species in plant litter, manures, pistachio fruit fallen on the ground, and male inflorescence of pistachio trees (Mirabolfathy 1981; Doster and Michailides 1994; Moradi and Ershad 2000); populations of A. flavus in the soil associated with rye green manure and peanut fruit debris (Griffin and Garren 1976); and a comprehensive review concerning ecology of the Aspergilli of soils and litter by Klich et al. (1992). The observations show that Aspergillus species can colonize and sporulate on pistachio litter at an early stage of decomposition. It has a relation to time and frequency of irrigation in pistachio orchards in the fall season, although the role of irrigation and its effects on populations of Aspergillus species is not clear in pistachio orchards. Aspergillus species have been isolated from the soil with different frequencies in pistachio producing areas. The results indicated that Aspergillus species are soil-borne in pistachio orchards. Their populations are affected by gardening processes, time, and frequency of irrigation, existence, and no existence of plant debris and animal manures in the soil. For example, any kind of gardening processes that cause dust can disperse the spores in the space of an orchard and colonize kernels of damaged nuts. Similarly, Doster and Michailides (1994b) reported that although the importance of Aspergillus propagules in the soil of pistachio orchards is not clear, the spores in the soil could disperse in dust to aerial parts of trees by such practices as disking. The passage of sheep and cattle in Iranian pistachio orchards cause dust, increasing the number of spores in the air. The relation between time and frequency of irrigation and population density of A. flavus showed a positive correlation between them. Irrigation could periodically increase propagules in soil. There is positive correlation between soil moisture and A. flavus populations. Regarding the little amount of rain in pistachio orchards in Iran and the United States in a fall season, irrigation may play an important role in colonization of pistachio litters by Aspergillus species. The observation indicates a positive relation between time of irrigation and density fluctuations in some of the leaf samples during the fall season (Moradi et al. 2004). Similarly, Doster and Michailides (1994) reported that rains usually do not fall in California pistachio orchards during summer; irrigation may play an important role in wetting pistachio litter enough for colonization by Aspergillus molds. Gardening processes such as burying or removing all kinds of plant debris, avoidance of transporting the rubbish to pistachio orchards, applying animal manures as fertilizer canals, avoidance of the manipulation of soil in harvest or in the little time remaining to harvest, and no irrigation of orchards in harvest time if it is possible may all be useful to reduce primary and secondary inoculums of Aspergillus species that can infect the pistachio nut.

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Mojtahedi, H., Rabie, C. J., Lubben, A. M. S., and Danesh, D. 1979. Toxic aspergilli from pistachio nuts. Mycopahtologia 31:123–7. Moradi, M. 1998. Distribution of aflatoxin B1 in the pistachio nuts as measured by Enzyme-Linked immonosorbent assay (ELISA) at preharvest and postharvest. Extension booklet of IPRI (In Farsi and English), 13. Moradi, M. 2005. Ecology and biology of Aspergillus flavus and Aspergillus niger groups in pistachio orchards in Kerman Province, Iran. IV International Symposium on Pistachios and Almonds, May 20–26, 2005, Tehran (Iran), Abstract Book. Moradi, M., and Ershad, J. 2000. Determination of density of the molds Aspergillus species in the Kerman pistachio orchards in different months of years. Proceeding of the 14th Iranian Plant Protection Congress, Isfahan, Iran, 128. Moradi, M., Ershad, D., Mirabolfathy, M., and Panahi, B. 2004. The role of plant debris, soil and manure on population density of Aspergillus flavus and Aspegillus niger groups in pistachio orchards of Kerman Province (in Persian). Iran. J. Plant Pathol. 40:221–34. Moradi, M., Hokmabadi, H., and Mirabolfathy, M. 2009. Incidence of Aspergillus species airborne spores in pistachio growing regions of Iran. International Journal of Nut research and related science. 1:15–25. Moradi, M., and Javanshah, A. 2006. Distribution of aflatoxin in processed pistachio nut terminals. Acta Hort. (ISHS) 726:431–6. Moradi. M., and Mirabolfathy, M. 2007a. Critical points in pistachio nut contamination by Aspergillus species. In Modern Fungicides and Antifungal Compounds V, 353–4. 15th International Reinhardsbrunn Symposium, May 06–10, 2007. Friedrichroda, Germany. Moradi, M., and Mirabolfathy, M. 2007b. Changing of Aspergillus species populations in pistachio processing terminals. Ir. J. Pajouhesh and Sazandeghi (In Persian) 77:105–10. Morton, S. G., Eadie, T., and Llewellyn, G. C. 1979. Aflatoxigenic potential of dried figs, apricots, pineapples and raisins. J. AOAC 62:958–62. Moss, M. O. 1996. Centenary review. Mycotoxins. Mycol. Res. 100:513–23. Nesci, A. V., Bluma, R. V., and Etcheverry, M. G. 2005. In vitro selection of maize rhizobacteria to study potential biological control of Aspergillus section Flavi and aflatoxin production. Europ. J. Plant Pathology 113 (2): 159–71. Palumbo, J. D., Baker, J. L., and Mahoney, N. E. 2006. Isolation of bacterial antagonists of Aspergillus flavus from almonds. Microbiol. Ecol. 52 (1): 45–52. Panahi, B., Mirdamadiha, F., and Talaie, A. 2005. Determination of the best time of harvest in different commercial Iranian pistachio nuts. In XIII GREMPA Meeting on almonds and pistachios = XIIIème Réunion du GREMPA sur l’amandier et le pistachier, ed. M. M. Oliveira, 215–19. Zaragoza: CIHEAMIAMZ. Payne, G. A., and Brown, M. P. 1998. Genetics and physiology of aflatoxin biosynthesis. Ann. Rev. Phytopathol. 36:329–62. Pearson, T. 1996. Machine vision system for automated detection of stained pistachio nuts. Lebensmitelw. U. Technol. 29:203–9. Pearson, T. C., Slaughter, D. C., and Studer, H. E. 1994. Physical properties of pistachio nuts. Trans. ASAE 37:913–18. Phillips, D. J., Mackey, B., Ellis, W. R., and Hansen, T. N. 1979. Occurrence and interaction of Aspergillus flavus other fungi in almonds. Phytopathology 69:829–31. Phillips, D. J., Purcell, S. L., and Stanley, G. I. 1980. Aflatoxins in almonds. Agricultural Reviews and Manuals ARM-W-20. Washington, DC: U.S. Department of Agriculture. Phillips, D. J., Uota, M., Monticelli, D., and Curtis, C. 1976. Colonization of almond by Aspergillus flavus. J. Am. Soc. Hort. Sci. 101:19–23. Placinta, C. M., J. P. F. D’Mello, and A. M. C. Macdonald. 1999. A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Anim. Feed Sci. Technol. 78:21–37. Purcell, S. L., Phillips, D. J., and Mackey, B. E. 1980. Distribution of Aspergillus flavus and other fungi in several almond-growing areas of California. Phytopathology 70:926–29. Rodriguez-del-Bosque, L. A. 1996. Impact of agronomic factors on aflatoxin contamination in pre-harvest field corn in northeastern Mexico. Plant Dis. 80:988–93. Rotter, B. A., Thompson, B. K., Lessard, M., Trenholm, H. L., and Tryphonas, H. 1994. Influence of low-level exposure to Fusarium mycotoxins on selected immunological and hematological parameters in young Swine. Fundam. Appl. Toxicol. 23:117.

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Samarajeewa, U., Sen, A. C., Cohen, M. D., and Wei, C. L. 1990. Detoxification of aflatoxins in food and feeds by physical and chemical methods. J. Food Protec. 53:489–501. Sauphanor, B., and Bouvier, J. C. 1995. Cross-resistance between benzoylureas and benzoylhydrazines in the codling moth, Cydia pomonella L. Pest. Sci. 45:369–75. Schade, J. E., McGreevy, K., King, A. D. J., Mackey, B., and Fuller, G. 1975. Incidence of aflatoxin in California almonds. Appl. Microbiol. 29:48–53. Schatzki, T. F. 1995a. Distribution of aflatoxin in pistachios. 1. Lot distributions. J. Agric. Food Chem. 43:1561–5. Schatzki, T. F. 1995b. Distribution of aflatoxin in pistachios. 2. Distribution in freshly harvested pistachios. J. Agric. Food Chem. 43:1566–9. Schatzki, T. F. 1998. Distribution of aflatoxin in pistachios. 5. Sampling and testing U.S. pistachios for ­aflatoxin. J. Agric. Food Chem. 46:2–4. Schatzki, T. F., and De Koe, W. J. 1999. Distribution of aflatoxin in pistachios. 6. Buyer’s and seller’s risk. J. Agric. Food Chem. 47:3771–5. Schatzki, T. F., and Ong, M. S. 2000. Distribution of aflatoxin in almonds. 2. Distribution in almonds with heavy insect damage. J. Agric. Food Chem. 48:489–92. Schatzki, T. F., and Ong, M. S. 2001. Dependence of aflatoxin in almonds on the type and amount of insect damage. J. Agric. Food Chem. 49:4513–9. Schatzki, T. F., and Pan, J. L. 1996. Distribution of aflatoxin in pistachios. 3. Distribution in pistachio process streams. J. Agric. Food Chem. 44:1076–84. Schatzki, T. F., and Pan, J. 1997. Distribution of aflatoxin in pistachios. 4. Distribution in small pistachios. J. Agric. Food Chem. 45:205–7. Scholten, J. M., and Spanjer, M. C. 1996. Determination of aflatoxin in pistachio and shells. J. Assoc. Off. Anal. Chem. Int. 79:1360–4. Scudamore, K. A. 2008. Mycotoxins. In Bioactive Compounds in Foods, eds. J. Gilbert and H. Senyuva. 134–72. New York: Wiley-Blackwell. Sedaghati, N., and Alipour, H. 2006. The effect of different time of irrigation on occurrence of early split (ES) of pistachio nuts. Acta Hort. (ISHS) 726:582–6. Setamou, M., Cordwell, K. F., and Hell, K. 1997. Aspergillus flavus infection and aflatoxin contamination of preharvest maize-in Benin. Plant Dis. 81:1323–7. Sibbett, G. S., and Van Steenwyk, R. A. 1993. Shedding “mummy” walnuts is key to destroying navel orangeworm in winter. Calif. Agric. 47:26–8. Sommer, N. F., Buchanan, J. R., and Fortlage, R. J. 1976. Aflatoxin and sterigmatocystin contamination of pistachio nuts in orchards. Appl. Environ. Microbiol. 32 (1): 64–7. Sommer, N. F., Buchanan, J. R., and Fortlage, R. J. 1986. Relation of early splitting and tattering of pistachio nuts to aflatoxin in the orchard. Phytopathology 76:692–4. Sweeney, M. J., and Dobson, A. D. W. 1998. Review: Mycotoxin production by Aspergillus, Fusarium and Penicillium species. Int. J. Food Microbiol. 43:141–58. Tajabadipour, A., Panahi, B., and Zadehparizi, R. 2006. The effects of rootstock and scion on early splitting and cracked hull of pistachio. Acta Hort. (ISHS) 726:193–8. Thomson, S. V., and Mehdy, M. C. 1978. Occurrence of Aspergillus flavus in pistachio nuts prior to harvest. Phytopathology 68 (8): 1112–4. Torabi, M., and Malakouti, J. 1998. Effects of nutrition on contamination of pistachio to Aflatoxin. First Gathering on Aflatoxin in Pistachio, by IPRI, Kerman, Iran (In Persian). UCD. 2009. University of California Davis, Fruit and nut website. Available at http://fruitsand nuts. ucdavis. edu/crops/pistachio.shtml#tools, accessed on 1998. United States Department of Agriculture Research Service. 2007. Available at http://www.ars.usda.govresearch/projects/projects.htm/accn_no = 412279, accessed on 2007. Varela, L. G., Welter, S. C., Jones, V. P., Brunner, J. F., and Riedl, H. 1993. Monitoring and characterization of insecticide resistance in codling moth (Lepidoptera: Tortricidae) in four western states. J. Econ. Entomol. 86:1–10. Varga, J., Kevei, E., Rinyu, E., Teren, J., and Kozakiewicz, Z. 1996. Ochratoxin production by Aspergillus species. Appl. Environ. Microbiol. 62:4461–64. Wicklow, D. T., Bobell, J. R., and Palmquist, D. E. 2003. Effect of intraspecific competition by Aspergillus flavus on aflatoxin formation in suspended disc culture. Mycol. Res. 107:617–23.

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Part IV

Functionality, Processing, Characterization, and Applications of Fruit and Cereal Bioactives

15 Isolation Characterization of Bioactive Compounds in Fruits and Cereals Xiaoke Hu and Zhimin Xu Contents Introduction..............................................................................................................................................319 Isoflavones...............................................................................................................................................319 Extraction, Purification, and Identification Methods of Isoflavones.................................................. 320 Carotenoids..............................................................................................................................................321 Extraction, Purification, and Identification Methods of Carotenoids................................................ 322 Anthocyanins.......................................................................................................................................... 324 Extraction, Purification, and Identification Methods of Anthocyanins.............................................. 324 Tocopherols and γ-Oryzanol................................................................................................................... 326 Extraction, Purification, and Identification Methods of Tocopherols and γ-Oryzanol...................... 327 Summary................................................................................................................................................. 329 References............................................................................................................................................... 329

Introduction Most phytochemical bioactive compounds in fruits and cereals have been recognized as having healthpromoting capability. They are being widely used in various functional foods to provide health benefits and prevent chronic diseases, such as cardiovascular diseases (CVD), obesity, diabetes, and certain cancers. These bioactive compounds are in a range with diverse chemical structures such as plant sterols, carotenoids, flavones, polyphenols, tocols, and so on. Consumers are concerned about maintaining health and preventing diseases through healthy diets. The functional foods with the bioactive compounds are fast becoming one of the most profitable sections in food markets. The purpose of this chapter is to introduce isolation and analysis method of the major bioactive compounds in common fruits and cereals. They include isoflavones, anthocyanins, carotenoids, tocopherols, and γ-oryzanol. The information will be helpful for food and nutrition scientists to indentify and quantify those bioactives in their original sources and in various food products. It is also valuable for the food industry in developing, monitoring, and quality controlling the functional foods made from those bioactive compounds.

Isoflavones Soybeans have been well-recognized as an excellent source of high-quality dietary protein and lipids. Soybeans contain important biological active substances, such as saponin and isoflavones (Ohsawa 1998; Watanabe 2001). Soy foods have received considerable attention for their potential role in reducing the formation and progression of certain types of cancers and some chronic ailments such as cardiovascular

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disease, Alzheimer’s disease and osteoporosis (Messina 1999; Zhao et al. 2002). Soy isoflavones are the most important antioxidants in soybeans because of their phenol structure and redox potential, which provide excellent antioxidant activity (Meng et al. 1999). Numerous mechanisms of soybean isoflavones have been suggested for the inhibition of carcinogenesis. Because of the classically competitive activity to estrogen, isoflavones were considered in the estrogen-related cancer prevention (Adlercreutz et al. 1995). Watanabe and Koessel (1993) and Hirayama (1986) found that the mortality of breast, endometrial, ovarian, and colon cancers in Japan was inversely associated with dietary consumption of soy and soy products. Lee et al. (1991) also observed an inverse correlation between breast cancer risk and the consumption of soy products. Three out of four case-controlled studies since 1991 have suggested that soy intake reduces breast cancer risk and one case-control study showed a decreased risk in women with high amounts of urinary phytoestrogens (Barnes et al., 1995; Ingram et al. 1997). According to the studies from Watanabe et al. (2000), isoflavones can reduce the risk of endometrial and ovarian cancer by reducing the plasma E2 level and competitively binding to ER in the target organ. Estrogenic effect of isoflavones in the low estrogen environment of males may help to inhibit the growth of prostatic cells (Modugno et al. 2001). Cardiovascular disease (CVD) is the single leading cause of death for both males and females in the United States and other developed countries in the world. In the developing countries, CVD generally ranks among the top five causes of death as well. The World Health Organization estimates that by 2020, heart disease and stroke will surpass infectious diseases to become the leading cause of death and disability worldwide (Lopez and Murray 1998). Soybeans have been recommended as a health food, as they not only manifest antioxidant capacities (Di Giacomo et al. 2009, Hsieh et al. 2009) but also reduce the risk of CVD (Martin et al. 2008; Retelny et al. 2008).

Extraction, Purification, and Identification Methods of Isoflavones Isoflavones have two phenol structures linked by a carbon chain. The hydroxyl group(s) on the phenol structure contributes to the antioxidant activity of isoflavone. There are several different types of isoflavones based on different substitute groups and sugar moieties on the two phenol rings (Figure 15.1). Soybean is the richest source of isoflavones among beans. The total level of isoflavones in soybeans is up to 0.5% (Liu 1997). Their concentration in the sample depends on several variables—such as variety and the type of food product—to obtain a concentrated extract with all isoflavones and free of interfering compounds from the matrix for a challenging project (Luthria 2006; Rostagno et al. 2009). Solid soy samples, such as soybeans and soy protein, require grinding into homogenous powder before extraction. Optimal liquid extraction includes the intimate contact between a solid material and a solvent that has a maximal solubility for the isoflavones and minimal solubility for the matrix and other compounds. Common organic solvents for liquid extraction are pure aqueous methanol (MeOH), ethanol (EtOH), acetonitrile, or acetone with or without the addition of small amounts of acids. The mixing and extracting procedure is soaking, mixing, shaking, or soxhlet extraction (Murphy 1981). Incubation at a warm temperature can speed up the extraction process without degradation of the isoflavones. Also, the extraction could be assisted with some modern techniques, such as ultrasonically assisted extraction (UAE), pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), and microwave-assisted extraction (MAE). These techniques combined with similar solvents have been used for accelerating the extraction of soy isoflavones. Evaporation techniques and solid phase extraction (SPE) are commonly used for concentration and further purification of isoflavones fraction (Rostagno et al. 2009). Usually, a series of purification steps before analysis will be applied to the extract from the sample matrix. After solvent extraction, the insoluble materials are usually removed by filtration or centrifugation. If an extract is obtained using PLE or SPE filtration the centrifugation is not required. The lipid extraction with polar solvent removes undesired sample components such as the lipophilic components to preserve reversed-phase chromatographic columns. The SFE have been used by some studies to provide a clean concentrated isoflavone extract prior to the chromatographic analysis (Wang and Sporns 2000; Bajer et al. 2007). The increased trend for the use of SPE contributes to its easy automation and coupling online with a further analysis method. Compared with manual methods, an automated SPE is less labor intensive and requires less sample handing time and provides better recovery. The method is

321

Isolation Characterization of Bioactive Compounds in Fruits and Cereals R3O

O

8 2

R2

5

Name Daidzein Glycitein Genistein Daidzin Glycitin Genistin Acetyldaidzin Acetylglycitin Acetylgenistin Malonyldaidzin Malonylglycitin Malonylgenistin

4′

O

R1 R1

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

OH R2

H OCH3 H H OCH3 H H OCH3 H H OCH3 H

R3 H H H Glu Glu Glu Glu–COCH3 Glu–COCH3 Glu–COCH3 Glu–COCH2COOH Glu–COCH2COOH Glu–COCH2COOH

Figure 15.1  Chemical structures of 12 isoflavones isolated from soybeans. (Adapted from Luthria, D. L., Biswas, R., and Natarajan, S., Food Chem., 105, 325–33, 2007.)

more reproducible and performed in a closed system. Rostagno et al. (2005) developed an automated SPE method for soy isoflavones achieving very high recoveries (99.37%) and reproducibility ( > 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

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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.)

Isolation Characterization of Bioactive Compounds in Fruits and Cereals

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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.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

325

Isolation Characterization of Bioactive Compounds in Fruits and Cereals

(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

7 6

8

1 + O

A

C

5 R2

4

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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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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,

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

328

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

(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

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. 337

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

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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

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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

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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

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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

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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

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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.

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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.

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

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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.

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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

352

Table 17.2 Simple Phenolic Compounds in Olive Flesh of the Seven Types of Tunisian Table Olives Expressed in mg per 100 g of Dry Weight Phenolic Compound Gallic acid Protocatechuic acid

Meski M1

Meski M2

Meski M3

Meski M4

Chemlali CH

Besbessi B

Tounsi T

ND

ND

ND

ND

ND

ND

ND

ND

34.6 ± 1.6

29.45 ± 3.8

ND

ND

13.79 ± 6.6

219.8 ± 3.2

35.55 ± 1.3

274.8 ± 9.7

283.2 ± 9.8

83.34 ± 5.8

ND

85.78 ± 9.7

Tyrosol

16.9 ± 2.4

24.99 ± 0.9

11.25 ± 1.1

10.35 ± 1.9

56.60 ± 2.7

28.52 ± 4.7

24.62 ± 6.9

p-Hydroxy-benzoic acid

5.9 ± 0.1

14.4 ± 0.02

20.21 ± 5.2

30.71 ± 4.6

10.61 ± 1.9

ND

ND

ND

ND

ND

25.04 ± 5.2

ND

11.5 ± 0.9

ND

Vanillic acid

4.41 ± 0.2

6.5 ± 1.1

7.98 ± 1.3

6.05 ± 1.2

2.33 ± 0.6

3.27 ± 0.7

5.56 ± 1.7

Caffeic acid

1.35 ± 0.01

ND

ND

ND

2.32 ± 0.01

ND

1.21 ± 0.6

Vanillin

ND

1.69 ± 0.03

3.27 ± 0.4

2.48 ± 0.8

ND

ND

ND

p-Coumaric acid

ND

2.08 ± 0.02

1.86 ± 0.02

ND

11.02 ± 3.8

3.91 ± 0.04

1.17 ± 0.09

Ferulic acid

4.1 ± 0.35

ND

ND

ND

2.12 ± 0.25

1.43 ± 0.06

ND

m-Coumaric acid

1.35 ± 0.02

ND

ND

ND

1.04 ± 0.05

ND

ND

Benzoic acid

213 ± 25.2

ND

ND

ND

ND

ND

ND

o-Coumaric acid

ND

2.99 ± 0.2

1.29 ± 0.07

ND

ND

ND

ND

Oleuropein

ND

ND

ND

ND

ND

ND

ND

466.81

88.2

355.25

387.29

169.38

48.65

132.13

p-Hydroxy-phenyl-acetic acid

Total

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.

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

ND

Hydroxytyrosol

Impacts of Food and Microbial Processing on the Bioactive Phenolics of Olive Fruit Products

353

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.

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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).

References Adams, M. R., and Moss, M. O. 1995. Food Microbiology. North Yorkshire: The Royal Society of Chemistry. Allalout, A., Krichène, D., Methenni, K., Taamalli, A., Oueslati, I., Daoud, D., and M. Zarrouk. 2009. Characterization of virgin olive oil from super intensive Spanish and Greek varieties grown in northern Tunisia. Scientia Horticulturae 120:77–83. Ayed, L., and Hamdi, M. 2003. Fermentative decolorization of olive mill wastewater by Lactobacillus plantarum. Process Biochem. 39:59–65. Ben Othman, N., Roblain, D., Chammem, N., Thonart, P., and Hamdi, M. 2009. Antioxidant phenolic compounds loss during the fermentation of Chetoui olives. Food Chem. 116:662–9. Ben Othman, N., Roblain, D. Thonart, P., and Hamdi, M. 2008. Tunisian table olive phenolic compounds and their antioxydant capacity. J. Food Sci. 73 (4): 235–40.

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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 361

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

363

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): (18.4) Fe2+  + H2O2 → Fe3+  + •OH + OH– • • •– 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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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

365

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

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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

367

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

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

N

N

Folin reagent

SO3H

N

Et Et ABTS•+ radical cation

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

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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).

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

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

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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.

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

372

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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).

Comparison of Methods for Assessing Antioxidant Capacity Antioxidant Assay

Simplicity

ORAC

Difficult and expensive

FRAP

Simple

CUPRAC

Simple

ABTS/TEAC

Rather difficult and expensive

DPPH

Rather difficult

Folin

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

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.)

Mechanism

End-Point

Property Measured

Lipophilic and Hydrophilic AOC

HAT

Fixed time

Decrease in florescence integrated value lag time

+

ET

Fixed time

Increase in absorbance

–

ET

Fixed time

Increase in absorbance

+

ET (or mixed HAT–ET)

Fixed time

Decrease in absorbance

+

ET (or mixed HAT–ET)

Fixed time

Decrease in absorbance

+

ET

Fixed time

Increase in absorbance

–

Antioxidant Activity/Capacity Assay Methods Applied to Fruit and Cereals

Table 18.4

373

374

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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

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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

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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  <  .05) than in mature and overripe fruits. Free polyphenols and flavonoids were at lower levels than hydrolyzed ones. Caffeic acid and quercetin were the dominant antioxidant substances in ripe durian. In these fruits, methanol extracts contained a relatively high capacity of 74.9 ± 7.1% inhibition using β-carotene-linoleic acid assay. FRAP and CUPRAC assays supported this finding. The correlation coefficients between polyphenols and antioxidant capacities of durian samples with all applied assays were about 0.98. The results showed that the bioactivity of ripe durian was high and the total polyphenols were the main contributors to the overall antioxidant capacity.

Antioxidant Capacities of Regularly Consumed Cereals The Basic Difficulties Encountered in the TAC Assays of Cereals The Contribution of Antioxidants Bound to insoluble Fractions and of High Molecular-Weight Polyphenols to the Measured TAC Grain phytochemicals may exist in free, soluble conjugates, and insoluble forms, the latter fraction being exceptionally rich in antioxidants. For example, bound phytochemicals are the major contributors to the TAC of cereals with the percentages: 90% in wheat, 87% in corn, 71% in rice, and 58% in oats (Adom and Liu 2002). Most reports on cereal antioxidant capacity have presented the limitation of incomplete extraction from insoluble fractions. For example, Pérez-Jiménez and Sauro-Calixto (2005) have emphasized that the cereal antioxidant capacities in methanol-water (50:50, v/v) and acetone–water (70:30, v/v) extracts ranged from 1.1. to 4.4 μmol TE/g dry weight (dw), and a significant amount of hydrolyzable phenolics with a high antioxidant capacity (5–108 μmol TE/g dw) was found in the residues of these aqueous-organic extracts. Nonextractable polyphenols are either high molecular weight compounds (e.g., condensed tannins and hydrolyzable phenolics) or polyphenols bound to dietary fiber and protein, which may be found in the residues of aqueous-organic extracts. As a result, the true TAC of cereals considering full contribution of bound phytochemicals to the overall antioxidant activity has been underestimated in literature (Pellegrini et al. 2006) because of the insufficiency of extractability of antioxidants by common organic solvents from insoluble fractions (as many antioxidants are covalently bound to the cell walls of these fractions), and due to the nonextractability of certain polyphenolics (Pérez-Jiménez and Sauro-Calixto 2005). However, it should also be noted that insoluble fractions of cereals are not necessarily chemically inert, since functional groups of antioxidants bound to the surfaces of these insoluble fractions may exert their antioxidant effects by scavenging free radicals (and also quenching synthetic radicals like ABTS•+ and DPPH• used as reagents in TAC assays) that are present in the solvent matrix (Serpen et al. 2007b). Thus TAC assays may be partly carried out in aqueous-organic solvent media with the aid of interfacial interactions between two heterogeneous phases without a strict requirement for complete solubilization of these fractions prior to assay.

Possible Losses of Antioxidants During Various Treatments of Cereals such as Heat- and Physical-Processing and Alkaline Hydrolysis Losses in measured TAC during heat processing of cereals may be partly counterbalanced by the contribution of the Maillard reaction products to TAC (Pérez-Jiménez and Saura-Calixto 2005). For example, extractable polyphenols in bread decreased by 78% with respect to the raw flour possibly as a result of the thermal effect (250°C), but despite the reduction in phenolics content, TAC of bread was higher than that of the wheat flour (Pérez-Jiménez and Saura-Calixto 2005). The high molecular-weight

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polymers (mainly insoluble), melanoidins and melanoproteins, formed in heat-processed foods through the Maillard reaction, possess a relevant antioxidant activity (Serpen et al. 2007a). However, while evaluating this contribution to the observed TAC, it should also be kept in mind that some reducing sugars and amino acids that are not classified as true antioxidants may also show interference to TAC measurement of these Maillard reaction products (Serpen et al. 2007a). Industrial (basically physical) processing can also inactivate 20–50% of phenolic compounds at 65°C, producing a loss of tocotrienols, tocopherols, and other antioxidants by disintegration of the membranous structures; however, avenanthramides and lignans seem to be unaffected by these treatments (MartinezTome et al. 2004). It is possible that phenolic acids may be partially destroyed during prolonged alkaline hydrolysis of grains (i.e., through alkaline digestion followed by subsequent ethyl acetate extractions). Adom and Liu (2002) found that about 10% ferulic acid was lost during alkaline hydrolysis carried out for 1–4 hours. Alkaline hydrolysis has been shown to reduce up to 90% of the antioxidant activity of cereal-based insoluble matters (Serpen et al. 2007b). During oxidation, an antioxidant (A–H) may lose protons together with electrons (e.g., A–H + O2 ↔ AOO – + H + ), and this redox equilibrium easily shifts to the right in alkaline medium. Therefore, the oxidative loss of antioxidants is facilitated during alkaline hydrolysis, which may give rise to serious negative errors in TAC measurements. For this reason, such hydrolysis operations should be carried out by a strict exclusion of oxygen (e.g., under controlled argon or nitrogen atmosphere throughout the operation), and preferably in the absence of transition metal salts that may catalyze such undesired oxidation reactions.

Summary of TAC Measurements in Individual Cereal Samples Gorinstein et al. (2008) studied polyphenols, phenolic acids, fibers, and determined their antioxidant capacities in water, acetone, and methanol extracts of buckwheat, rice, soybean, quinoa, and three amaranth cultivars by using TRAP, FRAP, CUPRAC, and NO • assays, which comprised of contributions from polyphenols and phenolic acids (especially from the most abundant ferulic acid). The correlation coefficients between total phenolics and antioxidant activities of cereal and pseudocereal methanol extracts with FRAP, NO•, CUPRAC, and TRAP were 0.99, 0.97, 0.96, and 0.77, respectively. The weakest correlation was with dietary fibers, an average one exhibited with tannins and marked correlation was shown with phenolics. All the applied methods have shown that pseudocereals have higher antioxidant activity than some cereals (rice and buckwheat) and can successfully replace cereals in case of allergy. Stratil and coworkers used FCM (with Folin–Ciocalteu reagent), PBM (Price and Butler) and AAPM (with 4-aminoantipyrine) methods for assessment of phenolic compounds and three commonly used methods, TEAC, DPPH, and FRAP for the evaluation of antioxidant capacity of six kinds of cereal (Stratil et al. 2007). The total content of phenolic compounds determined by FCM decreased (mostly with PBM) in sequence from buckwheat hulled > 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

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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 < .001 (Velioğlu et al. 1998). The purpose of another study was to examine the antioxidant properties of water and 80% methanolic extracts of cereal grains and their different morphological fractions by using the ABTS method. Wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), rye (Secale cereale L.), oat (Avena sativa L.), and buckwheat (Fagopyrum esculentum Moench) were sampled (Zieliński and Kozłowska 2000). Among the water extracts, only the one prepared from buckwheat exhibited antioxidant activity at the concentration analyzed. The following hierarchy of antioxidant activity was provided for 80% methanolic extracts that originated from whole grain: buckwheat > 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

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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

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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).

REFERENCES Adom, K. K., and Liu, R. H. 2002. Antioxidant activity of grains. J. Agric. Food Chem. 50:6182–7. Ames, B. N., Shigenaga, M. K., and Hagen, T. M. 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. 90:7915–22. Apak, R., Güçlü, K., Özyürek, M., and Karademir, S. E. 2004. Novel total antioxidant capacity index for dietary polyphenols, vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC Method. J. Agric. Food Chem. 52:7970–81. Apak, R., Güçlü, K., Özyürek, M., Karademir, S. E., and Altun, M. 2005. Total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: The CUPRAC Method. Free Radic. Res. 39:949–61.

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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.

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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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387

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

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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

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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

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications To separation/fractionation of soluble compounds

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.

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

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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

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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 (< 850 µm). As expected, the extraction yield increased by increasing the operating pressure at a constant temperature. Regarding the temperature effect, the crossover effect takes place at 30 MPa; that is, at pressures ranging from 15 to 25 MPa the oil extraction yield decreases by increasing temperature. However, at 30 MPa the oil extraction yield increases by increasing the temperature. The highest oil extraction yield was obtained

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at 30 MPa and 70°C (5.25%). But at 25 MPa and 40°C slightly lower oil extraction yield was obtained (4.77%). On the other hand, the highest squalene extraction yield was obtained at 20 MPa and 50°C. Due to the low solubility of oil at those conditions, the high concentration of squalene (15.3%) was obtained. This compares favorably with hexane extraction, which results in a squalene concentration of only 6.01%. A similar crossover effect in the pressure and temperature range was reported by Westerman et al. (2006). In this study they investigated the effect of pressure (10–30 MPa), temperature (30–50°C), solvent flow rate (0.2–1 kg/h), scale of the extraction, and sample pretreatment on the oil extraction rate and yield. Additionally, they carried out solubility studies on squalene and amaranth oil in the pressure and temperature range of 10–30 MPa and 35–60°C, respectively. The highest extraction yield (7.95%, which corresponds to 98% of the yield extracted with hexane) was obtained at 30 MPa and 50°C and 2 hours. Based on the data reported, by selecting the proper extraction conditions, supercritical CO2 can ­selectively extract squalene at higher concentrations than can be reached with traditional organic solvents.

Barley Barley has been extensively used as animal feed and in alcoholic beverages. However its interest in human consumption has increased mainly due to the high content of dietary fiber. Barley mainly consists of starch (50–60%), protein (9–15%), fiber (15–20%), lipids (2–5%), and ash (2–3%; Oscarsson et al. 1996). Barley is low in lipid content but is a good source of tocols (46–68 mg/kg), phytosterols (899–1153 mg/kg), and alkylresorcinols (33–103 mg/kg; Andersson et al. 2008). Colombo et al. (1998) reported the extraction of barley lipids using supercritical CO2. Their objective was to optimize the tocols extraction. Extractions were carried out on 50 g of barley (Hordeum vulgare L) at a constant temperature (40°C) and CO2 flow rate of (0.88 g/s) for 60 minutes in the pressure range of 7.9–23.7 MPa. As expected the highest yield of 4.38% was obtained at the highest pressure (23.7 MPa). At a low pressure (7.9–9.9 MPa), the extract mainly consists of triglycerides and free fatty acid. The higher pressure is required to extract tocols. The tocols yield was 30–50% higher as compared to hexane extraction. Concentration of α-tocopherol and α-tocotrienol were 1.67% and 0.64% in the extract, respectively. Fratianni et al. (2002) carried out a comparison between traditional and supercritical methods for the extraction of tocols from barley. Traditional methods include Soxhlet and Folch methods, in which tocols are extracted using organic solvent. Fractional extraction was carried out at a constant temperature (40°C) on a 2 g sample in the pressure range of 20–35 MPa. Total lipids extracted by Soxhlet and Folch were 4.4% and 4.7%, respectively. Total lipids extracted using supercritical CO2 in one step (4.0%) were slightly lower as compared to a sequential extraction (4.3%). During sequential extraction, at a low pressure (20 MPa) of the overall extraction about 70% of the lipids, as well as 96% of the total tocols were extracted. Total tocols recovery in multistep (83%) was comparable with single step (82%). However, it was lower when compared to a solvent extraction method like Soxhlet (92%) and Folch (94%). Supercritical fluid extraction using CO2 could be effectively used as a good alternative to extract tocols.

Corn Corn is the most widely grown crop in the United States and is primarily used for human consumption. It mainly consists of starch and is low in protein, fiber, and lipids. Due to the low content of lipids, it is not economical to extract oil from the corn itself. Corn is mainly processed by a wet- or dry-milling process. Wet-milling is geared toward the production of starch that can be used for corn syrup or ethanol production. Corn germ from a wet-milling process could contain about 40–45% lipids. The drymilling process is used to produce ethanol. In this process corn distillers grain (CDG) is obtained as a

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by-product. It is very low in starch and sugar but rich in fiber, protein, and lipid content. Lipid content in CDG varies anywhere between 5 and 20% depending on the variety of corn. Primarily it is used as animal feed due to its unstable nature and poor flavor quality. The main applications of supercritical fluids to corn is: removal of off-flavors, production of food grade defatted flour, and oil extraction from different corn fractions. Defatted dry corn germ flour obtained by supercritical CO2 extraction has better nutritive and physicochemical properties compared with defatted flour obtained with hexane (Christianson et al. 1984). The CO2 defatted germ flour has a good flavor quality. It contains 20% protein with a good amino acid balance making it a good food protein supplement. Additionally the high pressure extraction (34.5–55 MPa) reduced the peroxide enzymes tenfold when compared with hexane extracted flour, and the flavor quality of the flour was maintained for two months in storage tests. Wu et al. (1990) studied the effect of supercritical extraction on the improvement of CDG flavor. They compared CO2, binary mixtures (CO2 + ethanol, CO2 + water) and ternary mixtures (CO2 + ethanol + water). Extraction yields > 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.

396

Table 19.3 Supercritical Extraction of Corn Oil

Sample Size (g) 2.5 60 1000 1000 1000

Bioactive Compounds

Particle Size (mm)

Moisture (%)

Vessel Volume (ml)

Pressure (MPa)

Temperature (°C)

CO2 Flow Rate

Run Time (min)

(%, w/w)

Tocols (mg/g oil)

Sterol (mg/g oil)

— 420 — — 10–48

8 — 13 8 —

— 90 2000 2000 —

55 63, 68, 81 55, 82 55 30

80 85, 100 50 50 42

2 L/min 45.75 g/min 33 g/min 18 L/min —

60 30 — — —

12.5 99%a 21, 43 24.00 95%a

1.71 — 0.8–1.8 — —

15.8 — — — —

Percentage as compare to hexane extratable oil.

a

Extraction Yield

Process Parameters Studied

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

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Com

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

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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

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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

Supercritical Extraction of Rice Bran Oil Ricebran Sample Size (g) 20 20 150 300 5 10, 35 35

Particle Size (mm) — — — — — — —

Extraction Yield

Process Parameters Studied Moisture (%)

Vessel Volume (ml)

Pressure (MPa)

Temperature (°C)

CO2 Flow Rate

Run Time (min)

— 3.10 — 8.48 6.00 10.00 —

50 250 1000 1000 — 250 —

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

40, 60, 80 40 35 40 70–100 40, 50 40

1.08 g/min 6–10 L/min 20.5 g/min 3.5 kg/h — 10–14 g/min —

60, 120, 240 20, 80 60 240 90 — —

Bioactive Compounds

(%, w/w)

Tocols (mg/g oil)

Oryzanol (mg/kg oil)

Sterol (mg/ kg oil)

Reference

24.65 22 17.98 96a 19–20.4 16–19 16, 17

1050–1228 284 — 420 — — —

18 11 — 18.08 — 10, 13 15

— — 7.25 27.53 — — —

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

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

Table 19.4

Percentage as compare to hexane extratable oil.

a

399

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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.

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

401

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

402

Table 19.5 Supercritical Extraction of Wheat Germ Oil Wheat Germ

Extraction Yield

Process Parameters Studied

Particle Size (mm)

Moisture (%)

Vessel Volume (ml)

Run Time (min)

25

0.3, 075

9.36

75

5

0.505

5.1

0.35, 0.5

35

(%, w/w)

Tocols (mg/g oil)

Phenolics (mg/g oil)

Sterol (mg/g oil)

180, 300

55

0.5, 1, 1.5, 2.0 L/min 1.5, 2, 2.5 L/min 1.5 L/min

8

244.3–416.7

—

—

90

—

6.6–23.1

—

—

180

6.4–11.7

1.1–2.9

—

9.3–96

10, 25, 40, 55 69

40, 60, 80

—

60

2–20 a

2.6

—

—

80

0.18 kg/ min —

45, 60

11

26.9

—

—

10, 60, 80

2300

10, 25, 40, 55 20, 40

60

2–20 a

—

—

—

0.13 kg/ min 0.11 kg/ min —

60–120

8.5–9.2

2.1

—

—

—

1300

20, 40

40, 60

60

9

—

—

—

400

20, 25, 30

40

480

6–9

α-T:0.9

—

—

<1

10

15, 24, 38, 51, 60

40, 44, 50, 56, 60

2 g/min

10–60

276–8.96

2.7–7.1

2.62–6.63

—

Pressure (MPa)

Temperature (°C)

Co2 Flow Rate

10

5, 10, 15, 20, 25, 30 14, 28, 41

10, 20, 30, 40, 50, 60 30, 40, 50

—

1000

25, 38

—

—

100

4500

—

—

35

—

—

100, 3×1500 100

450

1.9–2.3

—

Small flakes Small flakes 0.18–0.25

2

0.75

600–700

220

40, 60

based on weight difference in the extraction vessel before and after extraction

a

Reference Molero and Martinez 2000 Ge et al. 2002 Panfili et al. 2003 Dunford and Martinez 2003 Esienmenger et al. 2006

Zacchi et al. 2006

Piras et al. 2009 Gelmez et al. 2009

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Sample Size (g)

Bioactive Compounds

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

403

place at lower pressures, where the overall extraction yield is low (Gelmez et al. 2009; Zacchi et al. 2006). Similarly the fractions collected at a lower solvent/feed ratio show higher tocol content (Dunford and Martinez 2003; Panfili et al. 2003). Yields of tocols increase by increasing the pressure at a constant temperature. Based on the data reported, the total tocol recovery yield obtained was higher than 87% to almost complete extraction. The tocol content of the wheat extracts is highly dependent on the tocol content in the starting material. However, there is a discrepancy of 1 or even 2 orders of magnitude on the tocols concentration of supercritical extracts reported. Considering only the data in the order of magnitude of 1000 mg of tocols/kg of oil extracted, the tocol content of the extract ranged from 2131 to 7100 mg tocols/kg of oil (Gelmez et al. 2009; Panfili et al. 2003; Zacchi et al. 2006). Any results falling outside of this range were not considered. Phenolic acid. Phenolic acid is reported as mg gallic acid equivalent/g extract (Gelmez et al. 2009). It shows a similar behavior to that of tocols. By increasing pressure, the total phenolic yield increases, but the total phenolic content decreases due to the diluting effect of the oil in the extracts. The highest phenolic content of the extracts were 6.63 mg GAE/g of extract. Carotenoids. Panfili et al. (2003) reported that the most representative carotenoid in wheat germ oil is lutein, followed by zeaxanthin and β-carotene. The concentration of total carotenoids, as well as that of the individual carotenoids, in extracted oil at 38 MPa and 55°C, increased as the extraction time increased. The highest extraction yield was obtained at the end of the extraction as 45% of the wheat germ content. Sterols. The total content of phystosterols (3.7 mg/g oil) was similar in hexane and supercritical carbon dioxide extracted oils. The β-sitosterol (78%) was the predominant phytosterol followed by campesterol (16%; Eisenmenger and Dunford 2008). In addition to producing a high quality of wheat germ oil, the supercritical extraction process produces food grade defatted wheat germ flour. This by-product could be used for further processing in the food industry. Additionally the low residual oil content improves the shelf life of the by-product.

Supercritical Fluid Extraction: Industrial Process Implementation for Cereal Lipids Industrial process implementation generally responds to a market demand, the replacing of a weak process, or even as a solution to a specific problem that traditional technologies are not able to properly solve. The steps for an industrial process implementation are listed in Table 19.6. The first step is to prove that the technology is capable of meeting the product specifications and process requirements defined by the customer or the market. In order to do that solubility data, partition coefficients, and separation factors of the compounds to be extract are required. Then the extraction of the compound(s) from the original feed material is evaluated in a bench scale supercritical extraction system. In this step the following process parameters must be optimized: conditioning of the raw material, extraction kinetics and separation conditions. Once the process parameters have been optimized, an initial cost estimate is calculated based on production requirements (i.e., amount of feed to process per year, working days per year, and number of hours per day) and the optimized process parameters. This cost estimation provides information related to operating costs ($/kg of feed or $/kg of final product), plant size, and plant configuration. If the investment and operating costs are satisfactory, the next step is to scale up the process to pilot plant and/or semi-industrial scale. The objectives in this step are to confirm that the process is scalable using the process parameters selected, optimize utility requirements, and recirculation parameters of the solvent as well as address any issues related to the material handling. The final step is to provide the final cost calculation. The capital cost of the supercritical extraction plant will depend on multiple factors, such as the number and size of the vessels, design pressure, flow rate, automation, specialized materials of construction,

404

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications Table 19.6 Industrial Process Implementation Steps for Industrial Process Implementation 1) Process parameters optimization: Lab scale • Conditioning the raw material • Extraction kinetics • Separation conditions 2) Initial cost estimate 3) Scale up to pilot plant scale/semi-industrial scale • Verification and tuning the optimum lab scale process parameters • Optimization of utility requirements • Optimization of recirculation parameters of the solvent • Material handling 4) Final cost calculation

Table 19.7 Estimated Operating Costs for a Supercritical Extraction Plant Used in the Cereal Oil Extraction Amaranth Amount to be processed/year, t/year Concentration of product in the feed stock,%, w/w Working days/year Number of hours/day Estimated operating costs, $/kg feedstock

200 8 330 24 1.31

Wheat 1000 10 330 24 0.32

Rice Bran 2000 20 330 24 0.19

Note: Includes power consumption, CO2 losses, maintenance and labor.

required hazard classification area, cleaning in place requirements, or the Good Manufacturing Practice (GMP) compliance. Generally, the capital cost of the supercritical fluid extraction plant is higher than the conventional extraction plant, while the operating costs are significantly lower. However, to compare properly the capital costs of a supercritical fluid extraction plant versus a traditional extraction process, it is necessary to take into consideration all the associate equipment used in the conventional extraction process, such as distillation or evaporation systems for solvent recovery or solvent removal from meal, as well as the associated costs of building requirements, instrumentation, and electrical connections to meet explosion proof requirements. The operating costs will depend on the location of the plant, as it will have a direct impact on the labor, feed stock, electrical, and CO2 costs. The plant size is determined by the amount to be processed per year, concentration of the product to extract in the feedstock, bulk density of the feed stock, as well as the process parameters, such as linear velocity, residence time and solvent/feed. Table 19.7 illustrates the estimated operating costs for a supercritical extraction plant used in the cereal oil extraction, specifically in amaranth, wheat germ, and rice bran. The operating costs were calculated assuming the installation of a supercritical processing plant in the United States. As discussed previously in this chapter, the cereal oils obtained required less refining as compared to cereals processed with hexane. Additionally the supercritical defatted meals could target the organic and/or food market as a final product or for further processing.

Conclusions Cereal oils are conventionally processed by mechanical pressing and/or extraction using organic ­solvents. The main drawbacks of these technologies are the high residual oil content in the case of ­mechanical pressing, and solvent residues in the meal and the extraction with traditional solvent extraction. Solvent removal implies high energy costs, as well as degradation and losses of bioactive components.

Supercritical Fluid Extraction of Bioactive Compounds from Cereals

405

Additionally, the traditional solvent extraction uses large quantities of toxic solvents that have adverse health effects. Supercritical fluid technology overcomes these limitations. Additional advantages offered by supercritical fluid technology are: supercritical extracted oils required fewer refining steps; manipulation of solubility and selectivity of the solvent by tuning process parameters to allow selective extraction of specific compounds; higher concentration of bioactive compounds are generally obtained; and the meals obtained could target organic and food markets in the form of light cereals, protein concentrates, and so on. Supercritical fluid technology is a viable technology in the nutraceutical and pharmaceutical sectors replacing conventional technologies and providing solutions that traditional technologies cannot provide. In order to successfully implement this technology it is necessary to fully understand it, focusing on the optimization of the plant design and components as well as the process parameters that provide minimum operating costs. This technology can become a part of an integrated process line, combining with traditional processes such as a supercritical fluid extraction + conventional extraction, a supercritical fluid extraction + supercritical fluid chromatography, or a conventional process + supercritical drying.

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20 Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives Marina Stefova and Violeta Ivanova Contents Introduction............................................................................................................................................. 409 Sample Preparation Procedures...............................................................................................................410 Spectrophotometry...................................................................................................................................410 High Performance Liquid Chromatography (HPLC)..............................................................................411 Matrix-Assisted Laser Desorption/Ionization (MALDI).........................................................................417 References............................................................................................................................................... 423

Introduction Phenolic compounds, as very important constituents of grape and essential components for wine ­quality, have been extensively studied in recent years. Scientists became very interested in the nutritional and beneficial health effects of polyphenolic compounds. Red and white grapes and wines have different phenolic compositions and characteristics for each variety. The polyphenolic content of a finished wine depends on grape variety, but also on different wine-making procedures (Nagel and Wulf 1979; Gil-Muñoz et al. 1999; Gómez-Plaza et al. 2000; Monagas et al. 2003; Bautista-Ortín et al. 2007; Ivanova et al. 2009). Red wine production includes the procedure of maceration, which is not applied in white wine production (i.e., white wines are produced without grape mash, having no contact with the grape skins or maceration is kept to a minimum). Various assay methods for analysis of phenolic compounds have been developed in order to study the concentration of total phenolics, specific subgroups or specific phenolic compounds. Among the analytical techniques, reversed phase high-performance liquid chromatography (RP-HPLC) is commonly employed for separation of the complex mixtures of phenolic compounds (McMurrough and McDowell 1978; Wulf and Nagel 1978; Betés-Saura et al. 1996; Palomino et al. 2000; Viñas et al. 2000; Careri et al. 2003; Monagas et al. 2003; Tsao and Yang 2003; Castillo-Muñoz et al. 2007; Gómez-Alonso et al. 2007). HPLC requires costly equipment and consumables and is not available in the wineries and spectrophotometric determinations, as easier and faster assays, could be used for routine quality control during the grape ripening and wine production. Thus, a number of chromatographic and spectrophotometric methods have been developed and applied for analysis of phenolic compounds in grapes and wine. Since different parameters influence the extraction of phenolic compounds from grapes and wine—such as their chemical nature, extraction method, sample storage time, and conditions—different previous purification steps and extraction procedures have been proposed as suitable for the extraction of phenolics from grapes and wine.

409

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Sample Preparation Procedures The separation methods have to be capable of determining most of the compounds of interest. The complexity of grapes and wine requires the use of sample preparation and/or clean-up procedures. When analyzing phenolic compounds from grape seeds and skins, an initial extraction step must be performed, including milling of grape solids in the presence of a suitable extraction solvent. Most of the procedures for extraction of polyphenols from grapes use aqueous methanol, acetone, or ethanol as a solvent for extraction, mostly acidified with HCl or with formic acid. Thus, Marinova et al. (2005) used 80% aqueous methanol (v/v) for phenolic and flavonoid compounds extraction. Montealegre et al. (2006) used methanol, water, and formic acid (50:48.5:1.5, v/v/v) for extraction of phenolic compounds from lyophilized grape skins and seeds. Kennedy et al. (2002) extracted the phenolics from grape skins with 66% aqueous acetone during 24 hours at 20°C and evaporated the solvent after filtration of the extract. Ivanova et al. (2009) tested the efficiency of methanol and acetone (80% aqueous solutions, v/v, containing 0.1% HCl that do not cause degradation as 1% HCl may induce hydrolysis of acetylated anthocyanins [Revilla et al. 1998]) observing that an overall slightly better extraction efficiency of the phenolics was achieved using acetone, mainly evident for extraction of flavan-3-ols and flavonols from grape skin and seed. This can be attributed to the more efficient dissolution of seed’s lipidic external layer caused by acetone, which is less polar and thus a better solvent for lipids than methanol, yielding the largest amounts of polyphenols. Also, one step is not enough for total extraction of the analyzed components from the skins, seeds, and pulp and therefore, two subsequent extractions are usually performed. Separation and fractionation of phenolic compounds from wine and grapes can be performed by ­solid-phase extraction (SPE) as an efficient method for separation of different fractions of phenolics that offers the advantages of reduction of solvents and time, increased speed and selectivity, and improved recoveries. Among the numerous applications of SPE, C18 Sep-Pak cartridges are mostly used for ­fractionation of phenolics prior to HPLC analysis. Elution with ethyl acetate or diethyl ether allows isolation of flavan-3-ol monomers and oligomers, followed by elution with methanol to isolate polymeric proanthocyanidins and anthocyanins. Successful fractionation of phenolic compounds from red wine belonging to different groups: phenolic acids, flavonols, anthocyanin monomers and polymers, procyanidins and catechins has been performed by Oszmianski et al. (1998) in four fractions using C18 cartridges and elution with methanol, 16% acetonitrile and ethyl acetate. In addition, the sample purification has been performed using column chromatography on Sephadex LH-20 or on Toyopearl 40 (S) or 50 (F) eluting the components with methanol/water or ethanol/water that allows phenolic fractionation and analysis of wine pigment composition (Mateus et al. 2002).

Spectrophotometry Spectrophotometric methods, more affordable for routine analyses and particularly because of their speed and simplicity, are widely used for the determination of various subgroups belonging to polyphenols. Based on different principles, these assays have been used to quantify total polyphenols (Slinkard and Singleton 1977; Di Stefano and Cravero 1989; Ivanova et al. 2009, 2010), anthocyanins (Di Stefano and Cravero 1989; Burns et al. 2000; Ho et al. 2003), flavonoids (Mazza et al. 1999; Zhishen et al. 1999; Kim et al. 2003; Marinova et al. 2005), flavan-3-ols (Di Stefano and Cravero 1989) in wine and fruits, as well as color intensity and hues of wine (Glories 1984). The Folin–Chiocalteu method (Slinkard and Singleton 1977) is most widely used for determination of total phenolics in grapes and wine. It is based on a redox reaction of phosphomolybdic-phosphotungstic acid (Folin–Chiocalteu reagent) to a blue-colored complex in an alkaline solution in the presence of phenolic compounds and shows maximum absorbance at a wavelength of 765 nm. For determination of total flavonoids, the colorimetric method with AlCl3 is usually applied to analyze wines (Zhishen et al. 1999). This method is based on the formation of stable complexes with the C-4 carboxyl group and either the C-3 or C-5 hydroxyl group of flavones and flavonols, exhibiting maximum absorbance at 510 nm. Determination of flavan-3-ols can be performed using the p-DMACA (p-dimethylaminocinnamaldehyde)

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives

411

method that was first reported by Thies and Fisher in 1971. This method is based on formation of a colored product from the reaction between tannins and the aldehyde reagent (Thies and Fischer 1971). Applying this method, monomeric procyanidins ((+)-catechin and (–)-epicatehicn)) are determined, reacting with p-DMACA reagent and measuring the absorbance of the formed adducts at 640 nm. An important feature of this method is that it theoretically responds only to the units that are not substituted in the A-ring, and thus only one unit per chain, regardless of its length unless there is some branching. In addition, the vanilin assay, which is more sensitive to polymeric tannins than to momoneric flavan-3ols, is also a widely used method for quantification of proanthocyanins in grapes and wine, as well as in other food samples (Goldstein and Swain 1963; Burns 1971; Deshpande and Cheryan 1987). Simple and fast anthocyanin analysis of wine and grape extracts can be performed by the method proposed by Di Stefano and Cravero (1989) based on dillution of the sample in a mixture of ethanol, water, and HCl in appropriate amounts (ethanol/water/HCl = 70/30/1) and measuring the absorbance at 540 nm. The color intensity depends on the content and structure of the anthocyanins present in wine and it is usually determined as sum of the absorbances at 420 nm, 520 nm, and 620 nm (Glories 1984). The ratio A420/A520 defines the hue (tint) of the wine and gives a measure of the wine redness. A direct measurement of undiluted wine at 420, 520, and 620 nm can be carried out using 1 or 2 mm optical path and the color intensity (CI), hue (H), proportion of red color (% Rd), proportion of blue color (% Bl), and proportion of yellow color (% Ye) are calculated using the following equations:

CI = A420 + A520 + A620 % Ye = A420/CI · 100

% Rd = A520/CI · 100

H = A420/A520 % Bl = A620/CI · 100,

where % Ye is the percentage of yellow color in the overall color, % Rd is the percentage of red color, and % Bl is the percentage of blue color in the overall wine color. The values of the color intensity vary between 0.3 and 1.8 depending on the variety, while the hue values of young wines are 0.5–0.7 and increase throughout wine aging. The results for the color of wine, measured at these three wavelengths, are easy to interpret, which is of great importance for the winemakers to control the wine making and wine aging stages.

High Performance Liquid Chromatography (HPLC) Reverse phase liquid chromatography (HPLC) coupled to UV-Vis (ultraviolet-visible) detection is the standard method for analysis of various classes of polyphenolic compounds (Wulf and Nagel 1978) using C18 column, a binary solvent system with a polar acidified solvent, such as aqueous formic acid, acetic acid, phosphoric acid, or perchloric acid solution (solvent A) and an organic modifier such as methanol or acetonitrile, possibly acidified (solvent B), (McMurrough and McDowell 1978; Wulf and Nagel 1978; Betés-Saura et al. 1996; Palomino et al. 2000; Viñas et al. 2000; Monagas et al. 2003; Careri et al. 2003; Tsao and Yang 2003; Castillo-Muñoz et al. 2007; Gómez-Alonso et al. 2007). Phenolic compounds exhibit characteristic absorbtion in the UV-Vis region enabling the distinction of the various classes: anthocyanins have an absorbance maximum around 520 nm, as well as in the UV range around 280 nm, flavonols at around 360 nm, and hydroxycinnamic acids can be detected at their absorption maximum at 320 nm. Flavan-3-ols exhibit the maximum absorbance around 280 nm and these substances possess fluorescence properties that the other wine polyphenols do not have that enable their more specific detection and determination. Figure 20.1 shows the UV-Vis spectra of anthocyanin monoglucosides. A typical chromatogram obtained for a wine sample, recorded at 520 nm is presented in Figure 20.2. Liquid chromatography coupled to mass spectrometry (LC/MS) is applicable to a wide range of ­compounds and, in recent years, it has become the most sophisticated technique for analysis of phenolic compounds in wine and grapes and for studying the structure of compounds formed by the reaction of anthocyanins with other compounds. Especially, this technique is very effective for studies of glycoside compounds, allowing characterization of the aglycone and sugar moiety (Pérez-Magariño et al. 1999; de Villiers et al. 2004; Monagas et al. 2005).

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications 528

0.20 0.18 0.16 0.14 276

AU

0.12 0.10 0.08 0.06 0.04

348

276 290

0.02 0.00 250

300

515

357

525

525

350 400 450 Wavelength/nm

500

550

Mv-3-glc , UVmax = 528 nm Pt-3-glc , UVmax = 525 nm

Pn-3-glc , UVmax = 515 nm

Dp-3-glc , UVmax = 525 nm Figure 20.1  UV-Vis spectra of anthocyanin monoglucosides. (Mv-malvidin, Pt-petunidin, Pn-peonidin, Dp-delphinidin, and glc-glucoside)

Mv-3-Glc

0.20 0.18 0.16

520nm

0.02

Pt-3-AcGlc CoumVitisin A Mv-3-AcGlc cis-Mv-3coum-Glc Pt-3coum-Glc Mv-3caffeoylGlc Pn-3coum-Glc Mv-3coum-Glc

Dp-3-Glc

0.06 0.04

Cy-3-Glc

0.08

Vitisin A Vitisin B

0.12 0.10 Pt-3-Glc Pn-3-Glc

AU

0.14

0.00 0.00

5

10

15

20

25

30

35

40 45 tR/min

50

55

60

65

70

75

80

Figure  20.2  Chromatogram obtained for a wine sample monitored at 520 nm for separation of anthocyanins (Dp-delphinidine, Cy-cyanidine, Pt-petunidine, Pn-peonidine, Mv-malvidine, Glc-glucoside, Coum-coumaroyl, Ac-acetyl). Chromatographic conditions: C18 column (250 mm × 2.1 mm i.d., 5 µm packing, Waters, Milford, MA) protected by a guard column of the same material (20 × 2.1 mm i.d.; Waters, Milford, MA); mobile phase consisting of water/formic acid (95:5; solvent A), and acetonitrile/water/formic acid (80:15:5; solvent B) at a flow rate of 0.25 mL/min at 38°C. Gradient program of solvent B: isocratic for 2 min with 0%; 2–5 min, 0–2%; 5–12 min, isocratic with 2%; 12–15 min, 2–3%; 15–25 min, 3–8%; 25–40 min, 8–20%; 40–45 min, 20–25%; 45–55 isocratic with 25%, 55–70 min, 25–65% and 70–75 min, 65–0%. (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives

413

Coupling HPLC to mass spectrometry allows characterization and structure elucidation of the anthocyanin pigments present in trace amounts in grapes and wine (Baldi et al. 1995; Cameira-dos-Santos et al. 1996; Wang and Sporns 1999; Revilla and González-San José 2001; Núñez et al. 2004; La Torre et al. 2006). This especially counts for the ion trap mass spectrometers, which enable several subsequent fragmentation steps thus allowing identification of complex molecules in a step-by-step removal of the various groups. However, distinguishing between glucoside and galactoside in flavonoid glycosides, or catechin and epicatechin units is not possible with mass spectrometry (MS). In that case, enzymatic reactions, applying appropriate enzymes (e.g., ß-glucosidase) can be used in order to identify the sugars in glycosides. In addition, an acid-catalyzed cleavage of the glycosidic bond can be performed for sugar determination. The acid-catalyzed cleavage is also successfully applied for determination of proanthocyanidin composition in the presence of a nucleophilic agent. Examples of chromato­grams recorded with a diode array detector at 280 nm and fluorimetric detector (excitation wavelength of 275 nm and emission wavelength of 322 nm) are reported in Figure 20.3 (phloroglucinol used as a nucleophilic reagent for depolymerization). Analytical features of LC/MS, such as sensitivity, selectivity, speed of analysis, cost, and effectiveness have been continually improved and are focused on separation, detection, and structural characterization of novel compounds in wine. LC/MS provides the capability to analyze the phenolic compounds either in the positive or in the negative ion mode, generating cations ([M + H]+, M + Na]+) or anions ([M−H]–, [M−Cl]–. The positive ion mode has been proven as efficient for anthocyanin analysis, as well as for flavan-3-ols detection, while the negative ion mode is more suitable for phenolic acids, flavonoids, and can be successfully applied for flavan-3-ols identification. One of the first applications of mass spectrometry for grape polyphenols identification was published in 1990 by Lee and Jaworski where positive and negative ion modes were applied for analysis of extracts from grape samples and three compounds have been detected: catechin–gallate ([M–H]– at m/z 441, fragment ions at m/z 151 and 137), catechin–catechin–gallate ([M–H]– at m/z 729, fragment ion at m/z 577) and gallocatechin– gallate ([M–H]– at m/z 459). The LC/MS system equipped with an electrospray ionization (ESI), giving access to molecular weights of different species present in the samples, has been successfully applied for studying different groups of polyphenols in wine and grapes, such as nonflavonoids (phenolic acids and stilbenes) and flavonoids (flavonols, dihydroflavonols, anthocyanins, flavan-3-ols: oligomers and polymers). A list of nonanthocyanin compounds detected by LC/ESI-MS in red wine samples is reported in Table 20.1 also containing UV and MS data. Liquid chromatography/mass spectrometry analysis of phenolic acids is usually performed in negative ion mode. With regard to hydroxybenozoic acids, all of them are detected as [M–H]– showing a characteristic fragmentation [M–H-44]–, which corresponds to elimination of CO2 group from the carboxylic acid and production of the corresponding fragment ions (Monagas et al. 2005), as shown in Table 20.1. The common compounds present in grapes and wine belonging to the group of hydroxycinnamic acid derivatives are caffeoyltartaric (caftaric) acid at m/z 311 (fragment ions: m/z 179, 149), p-coumaroyltartaric (coutaric) acid at m/z 295 (fragment ion at m/z 163), and feruloyltartaric (fertaric) acid at m/z 325 (fragment ion at m/z 193), which have a characteristic fragmentation pattern [M–H-132]– that corresponds to a loss of tartaric acid residue (Baranowski and Nagel 1981; Baderschneider and Winterhalter 2001; Monagas et al. 2005). The phenolic acid GRP (2-S-glutathionylcaffeoyltartaric acid), detected in wine at m/z 616 (fragment ions at m/z 484, 440 and 272; Boselli et al. 2006) was also confirmed to be present in wine (Singleton et al. 1985, 1986). For analysis of flavan-3-ols, both positive and negative ion modes can be applied. Figures 20.4 and 20.5 present extracted ion chromatograms relative to the analysis of wine flavan-3-ols in negative-ion mode. Since MS does not distinguish flavan-3-ol monomers ( + )-catechin ([M–H]– = m/z 289) and (–)-­epicatechin ([M–H]– = m/z 289), further identification of these compounds can be achieved by coelution with the corresponding standard compounds. A characteristic feature of flavan-3-ols, compared to the other phenolic compounds, is the Retro-Diels-Alder (RDA) rearrangement on the C-ring of catechin and epicatechin derivatives followed by the elimination of 152 Da fragment (Table 20.1). The glucoside derivatives of flavonols (myricetin, quercetin, laricitrin, and syringetin) known to be the predominant flavonol hexosides in grapes and wine are identified on the basis of their [M–H]– signals

414

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

0.09

UV/Vis 280 nm

0.08 0.07

AU

0.06 0.05 3

0.04 0.03 0.02 1

0.01

4

2

5 6

0 0 (b)

10

20

30

40 tR/min

50

60

80

160 140

FLD Ex 275 nm Em 322 nm 280 nm

3

120 100 mV

70

80 60 4

40 20

6

2

0 0

5

10

15

20

25

30

35

40 45 tR/min

50

55

60 65

70

75

80

Figure 20.3  Chromatograms obtained for wine sample recorded with (a) diode array detector at 280 nm and (b) fluorimetric detector, Ex-275 nm, and Em-322 nm for analysis of flavan-3-ols (terminal units) and phloroglucinol adducts (extension subunits) after acid-catalyzed depolymerization with phloroglucinol. Peak identification: 1-Epigallocatechin(4α→2)-phloroglycinol, 18.8 min; 2-Catechin-(4α→2)-phloroglucinol, 29.9 min; 3-Epicatechin-(4α→2)-phloroglucinol, 30.7 min; 4-Catechin, 40.2 min; 5- Epicatechin-3-O-gallate-(4α→2)-phloroglucinol, 47.8 min; 6-Epicatechin, 52.1 min. Chromatographic conditions: C18 column (250 mm × 4.6 mm i.d., 5 µm packing Waters, Milford, MA) protected by a guard column of the same material (20 × 4.6 mm i.d.; Waters, Milford, MA). A binary solvent consisted of water/formic acid (98:2; solvent A) and acetonitrile/water/formic acid (80:18:2; solvent B) at a flow rate of 1 mL/min at 30°C: Gradient program of solvent B: isocratic for 5 min with 0%; 5–35 min, 0–10%; 35–70 min, 10–20%; 70–75 min, 20%–100%; and 75–80 min, 100–0%. (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

and formed fragment ions ([M–H–162]–), which correspond to the elimination of a glucose molecule ­(Ribereau-Gayon 1964; Mattivi et al. 2006; Castillo-Muñoz et al. 2007), while the glucuronide derivatives (e.g., myricetin-3-O-glucuronide, quercetin-3-O-glucuronide) are characterized by a loss of 176 Da ([M–H–176]), corresponding to the elimination of a glucuronide group (Cheynier and Rigaud 1986). Previous studies have shown the presence of astilbin (dihydroquercetin-3-O-rhamnoside, [M–H]– = m/z 449) and engeletin (dihydrokaempferol-3-O-rhamnoside, [M–H]– = m/z 433) in skins of white grapes and

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives

415

Table 20.1 UV and MS Spectral Data for Nonanthocyanin Phenolics Detected in Vraneca Wines λ max/nm

[M-H]–

Fragments (m/z)

Gallic acid Protocatechuic acid Caftaric acid cis-Coutaric acid trans-Coutaric acid GRP Fertaric acid Syringic acid

272.3 293.7, 255.8 328 310.4 310

125 109 179, 149 163 163

312.8 272.3

169 153 311 295 295 616 325 197

Flavan-3-ols Procyanidin B3 Catechin Procyanidin B1 Procyanidin B4 Epicatechin Procyanidin B2

λ max/nm 284.2 276.5 264.7 264.7 276.5 264.7

[M–H]– 577 289 577 577 289 577

Fragments (m/z) 559, 451, 425, 407, 289, 245 245, 205, 179 559, 451, 425, 407, 289, 245 559, 451, 425, 407, 289, 245 245, 205, 179 559, 451, 425, 407, 289, 245

Flavan-3-ols Catechin Epicatechin

λ max/nm 276.5 276.5

[M+H]+ 291 291

Fragments (m/z) 273, 165, 139, 123 273, 165, 139, 123

Dihydroflavonols Dihydromyricetin-3-O-rha Astilbin Engeletin

λ max/nm

[M–H]– 465 449 433

Fragments (m/z) 339, 301 303, 285 287, 269

Flavonols Myricetin-3-glcA Myricetin-3-glc Quercetin-3-glcA Quercetin-3-glc Laricitrin-3-glc Syringetin-3-glc Quercetin

λ max/nm 343.5 343.5 353.4 353.4 364.2 358.2 368.7

[M+H]+ 495 481 479 465 495 509 303

Fragments (m/z) 319 319 303 303 333 481, 392, 347  

Phenolic Acids

193 153

Source: Adapted from Monagas, M., Suárez, R., Gómez-Cordovés, C., and Bartolomé, B., Am. J. Enol. Vitic., 56, 139–47, 2005. Labels: GRP = grape reaction product, which is 2-S-glutathionylcaffeoyltartaric acid, glc: glucoside, glcA: glucuronide, rha: rhamnoside. a Vranec is a red grape variety used for production of quality wines typical for the Balkan Peninsula.

in white wines (Trousdale and Singleton 1983) showing characteristic fragmentation that corresponds to the elimination of the rhamnoside group (–164 amu; Souquet et al. 2000). Another compound, dihydromyricetin-3-O-rhamnoside at m/z 465, shows the same fragmentation pattern (Vitrac et al. 2001). The characteristic MS fragmentations of astilbin and engelitin detected in wine samples are given in Figure 20.6. Structural identification and characterization of grape anthocyanins: glucosides, acetylglucosides, and p-coumaroylglucoside derivatives of delphinidin, cyanidin, petunidin, peonidin, and malvidin as the main colored compounds are achieved by electrospray ionization in the positive mode and MSn analyses, as a highly effective tool for compound differentiation. They have similar fragmentation patterns: their mass spectra contain two signals, the molecular ion, M + and the fragment ions [M-162]+, [M-204] + , or [M-308]+ due to elimination of glucose, acetylglucose, or p-coumaroylglucose groups, respectively (Baldi et al. 1995; Vivar-Quintana et al. 2002; Salas et al. 2004; Kelebek et al. 2007). The λmax values for cyanidin-3-glucoside and peonidin-3-glucoside are lower than λmax for the other three glucosides and the acylated derivatives with either p-coumaric acid or caffeic acid absorb in the 300–340 nm region, as

416

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

B1

Intens. x107

2.5 2.0 1.5

B2

1.0

B3

0.5 0.0

(–)-Epicatechin

(+)-Catechin B4

12.5

15.0

17.5

20.0

22.5

25.0 Time [min]

27.5

30.0

32.5

Figure  20.4  Negative ion-mode extracted ESI-MS chromatograms obtained from wine analysis in the m/z range 200–1200. Extracted m/z values correspond to the ions of (+)-catechin (m/z 289), (–)-epi­ca­techin (m/z 289), and procyanidins: B1 (m/z 577), B2 (m/z 577), B3 (m/z 577), and B4 (m/z 577). (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

(–)-Epicatechin-3-O-gallate

Intens. x106

1.0 0.8

(–)-Gallocatehin

0.6 0.4

(–)-Epigallocatechin

0.2 0.0

0

10

20

30 Time [min]

40

50

Figure  20.5  Negative ion-mode extracted ESI-MS chromatograms obtained from wine analysis in m/z range 200– 1200. Extracted m/z values correspond to the ions of ion chromatograms extracted at m/z values corresponding to the negative ions of (–)-gallocatechin (m/z 305), (–)-epigallo­ca­techin (m/z 305), and (–)-epicatechin-3-gallate (m/z 441). (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

previously described by Wulf and Nagel (1978) and in agreement with other published data (Piovan et al. 1998; de Villiers et al. 2004). Typically for reversed-phase liquid chromatography, the components elute in order of their polarity: (1) delphinidin-3-glucoside, (2) cyanidin-3-glucoside, (3) petunidin-3-glucoside, (4) peonidin-3-glucoside, and (5) malvidin-3-glucoside. Acetyl and p-coumaroyl derivatives elute in the same order as anthocyanin-3-monoglucosides. In this way, the order of elution of the anthocyanins is monoglucoside < acetylmonoglucoside < coumaroylmonoglucoside (Vivar-Quintana et al. 2002; Kelebek et al. 2007). Fragmentations and λmax values of different groups of pigments are listed in Table 20.2. The MS/MS analyses are successfully used for structural characterization of anthocyanin derivatives formed during maceration and wine aging, in particular the pyranoanthocyanins, which arise from reactions of anthocyanins with pyruvic acid (Bakker et al. 1997; Revilla et al. 1999; Hayasaka and Asenstorfer 2002; Heier et al. 2002; Morata et al. 2003; Alcalde-Eon et al. 2004, 2006; Chinnici et al. 2009). All these molecules have the same main fragment ion that corresponds to carboxy-pyrano-

417

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives

Intensity

Intensity

303

100 95 (M–H)– = 449 90 Astilbin 85 80 285 OH 75 70 OH 65 60 O HO 55 50 H 45 40 O 35 OH O 30 O 25 CH 20 151 OH OH OH 3 15 323 177 10 431 5 0 200 300 400 500 600 700 800 900 m/z

100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0

269 (M–H)– = 433 Engelitin 287

OH O

HO

H O OH

O

259 243 339 415 200

300

400

O

CH3 OH

700

800

OH OH

500

600

m/z

Figure 20.6  MS/MS fragmentations of (a) astilbin and (b) engelitin detected in wine under the negative ESI mode. (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

anthocyanin aglycone, released after loss of 162, 204, and 308 mass units (Table 20.2; Fulcrand et al. 1998; Mateus et al. 2003). A series of pyranoanthocyanins formed by reactions of anthocyanins with acetaldehyde (pyrano-anthocyanins) have also been shown to be present in wines (Table 20.2). Other pyranoanthocyanins, formed by a reaction of acetaldehyde, flavanols and anthocyanins have been detected in wine samples (He et al. 2006) and have been identified as flavanyl-pyranoanthocyanins. An example of the fragmentation of catechin-pyrano-malvidin-3-glucoside [M + H]+ at m/z 805 is reported in Figure  20.7. As shown in Table  20.2, the compound detected with a molecular mass of 805 unites corresponds to (epi)catechin-pyrano-malvidin-3-glucoside (fragment ions: 643, 491) and those with m/z 1093 (fragment ions: m/z 931, 803) and 1135 (fragment ions: m/z 931, 845) to procyanidin dimer-pyrano-malvidin-3-glucoside and procyanidin dimer-pyrano-malvidin-3-acetylglucoside (Francia-Aricha et al. 1997). Anthocyanins that can condense either directly or by mediation of acetaldehyde with flavan-3-ols, forming anthocyanin–flavanol direct condensation pigments or ethyl-bridged pigments as a result of reactions that take place spontaneously during the maceration and aging of wine, have been detected in wines (Table 20.2). Thus, the compounds with m/z 779, 925, and 795 have been identified as (epi) catechin-ethyl-peonidin-3-glucoside, (epi)catechin-ethyl-peonidin-3-p-coumaroylglucoside and (epi) catechin-ethyl-petunidin-3-glucoside, respectively. An example of fragmentation of (epi)catechin)(epi)catechin-malvidin-3-glucoside (m/z 1069) is shown in Figure 20.8.

Matrix-Assisted Laser Desorption/Ionization (MALDI) The determination of molecular masses of polyphenols by mass spectrometry could be performed using the electrospray ionization (ESI; Cheynier et al. 1997; Hayasaka and Asenstorfer 2002), the atmospheric pressure chemical ionization (APCI) or the matrix-assisted laser desorption/ionization (MALDI; Sugui et al. 1998, 1999; Wang and Sporns 1999; Robards 2003; Reed et al. 2005; Es-Safi et al. 2006; Tholey and Heinzle 2006; Carpentieri et al. 2007). MALDI was first demonstrated by Karas et al. (1987) and originally developed for large biomolecules analyses. MALDI is usually coupled with a time of flight (TOF) analyzer, one of the oldest and simplest mass analyzers (Sporns and Wang 1998), since the timing of the ionization is very precise using a short nanosecond laser pulse and the initial ion velocities are remarkably consistent. The TOF gives access to a theoretically unlimited mass range and also

418

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 20.2 UV-Vis and MS Spectral Data of Anthocyanins and Their Derivatives Detected in Wine Samples Pigment Compounds Dp-3-glc Cy-3-glc Pt-3-glc Pn-3-glc Mv-3-glc Dp-3-acetylglc Cy-3-acetylglc Pt-3-acetylglc Pn-3-acetylglc Mv-3-acetylglc Dp-3-p-coumglc Cy-3-p-coumglc Pt-3-p-coumglc Pn-3-p-coumglc Mv-3-p-coumglc Dp-3-caffeoylglc Pt-3-caffeoylglc Pn-3-caffeoylglc Mv-3-caffeoylglc Dp-3,7-diglc Pt-3,7-diglc Pn-3,7-diglc Mv-3,7-diglc Dp-3-glc + L(+)lactic acid Pt-3-glc + D(-)lactic acid Pt-3-glc + L(+)lactic acid Pn-3-glc + D(-)lactic acid Pn-3-glc + L(+)lactic acid Mv-3-glc + D(-)lactic acid Mv-3-glc + L(+)lactic acid Carboxy-pyrano-Dp-3-glc Carboxy-pyrano-Pt-3-glc Carboxy-pyrano-Pn-3-glc Carboxy-pyrano-Mv-3-glc (Vitisin A) Carboxy-pyrano-Dp-3-acetylglc Carboxy-pyrano-Pt-3-acetylglc Carboxy-pyrano-Pn-3-acetylglc Carboxy-pyrano-Mv-3-acetylglc (acetylVitisin A) Carboxy-pyrano-Dp-3-p-coumglc Carboxy-pyrano-Pt-3-p-coumglc Carboxy-pyrano-Pn-3-p-coumglc Carboxy-pyrano-Mv-3-p-coumglc (p-coumVitisin A) Pyrano-Dp-3-glc Pyrano-Pt-3-glc Pyrano-Pn-3-glc Pyrano-Mv-3-glc (Vitisin B) Pyrano-Mv-3-glc-dimer

λmax/nm

M+/[M+H]+

Fragments (m/z)

References

525 516 525 516 528 527 523 529 522 530 530 520 532 526 532 532 531 525 534 523 522

465 449 479 463 493 507 491 521 505 535 611 595 625 609 639 627 641 625 655 627 641 625 655 537 551 551 535 535 565 565 533 547 531 561 575 589 573 603

303 287 317 301 331 303 287 317 301 331 303 287 317 301 331 303 317 301 331 303 317 301 331

*, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** *, **, ***, **** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** *, ** *, ** *, ** **** **** **** *, ****

679 693 677 707

371 369 399

**** **, **** **, **** *, ****

489 503 487 517 1093

327 341 325 355 931, 803

**, **** **, **** **, **** *, **** *

526

507 508 503 510

508 514

492 490

317 301 331 331 371 385 369 399 371 385 399

419

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives

Table 20.2 (Continued) UV-Vis and MS Spectral Data of Anthocyanins and Their Derivatives Detected in Wine Samples Pigment Compounds Pyrano-Pn-3-acetylglc Pyrano-Mv-3-acetylglc (acetylVitisin B) Pyrano-Mv-3-p-coumglc (p-coumVitisin B) Dp-3-glc-4-vinylphenol Cy-3-glc-4-vinylphenol Pt-3-glc-4-vinylphenol Pn-3-glc-4-vinylphenol Mv-3-glc-4-vinylphenol Mv-3-acetylglc-4-vinylphenol Dp-3-p-coumglc-4-vinylphenol Pt-3-p-coumglc-4-vinylphenol Pn-3-p-coumglc-4-vinylphenol Mv-3-p-coumglc-4-vinylphenol Dp-3-glc-4-vinylcatechol Pt-3-glc-4-vinylcatechol Pn-3-glc-4-vinylcatechol Mv-3-glc-4-vinylcatechol Mv-3-acetylglc-4-vinylcatechol Dp-3-p-coumglc-4-vinylcatechol Pt-3-p-coumglc-4-vinylcatechol Mv-3-p-coumglc-4-vinylcatechol Dp-3-glc-(epi)cat Cy-3-glc-(epi)cat Pt-3-glc-(epi)cat Pn-3-glc-(epi)cat Mv-3-glc-(epi)cat (epi)cat-(epi)cat-Mv-3-glc Mv-3-acetylglc-(epi)cat Mv-3-acetylglc-flavan-3-ol-dimer Dp-3-p-coumglc-(epi)cat Cy-3-p-coumglc-(epi)cat Pt-3-p-coumglc-(epi)cat Pn-3-p-coumglc-(epi)cat Mv-3-p-coumglc-(epi)cat (epi)cat-pyrano-Mv-3-glc Dp-3-glc-gallcat Cy-3-glc-gallcat Pt-3-glc-gallcat Pn-3-glc-gallcat Mv-3-glc-(epi)gallcat Dp-3-p-coumglc-gallcat Cy-3-p-coumglc-gallcat Pt-3-p-coumglc-gallcat Pn-3-p-coumglc-gallcat Mv-3-p-coumglc-(epi)gallcat Dp-3-glc-ethyl-(epi)cat

λmax/nm

M+/[M+H]+

Fragments (m/z)

References

494

529 559

325 355

** **

497

663

355

****

503

581 565 596 579 609 651 727 741 725 755 597 611 595 625 667 743 757 771 753 737 767 751 781 1069 823 1135 899 883 913 897 927 805 769 753 783 767 797 915 899 929 913 943 781

419

** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** * * * ** ** ** ** ** * ** ** ** ** ** ** ** ** ** ** **

502 500 504 505 504 501 505 509 510 506 510 513

531 282 279 281

433 417 447 447 419 433 417 447 435 449 433 463 463 435 449 463 591 575 605 589 619 907, 619 619 931, 845, 641, 435 591 605 589 619 607 591 621 605 635 607

605 635 329

(Continued)

420

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 20.2 (Continued) UV-Vis and MS Spectral Data of Anthocyanins and Their Derivatives Detected in Wine Samples λmax/nm

Pigment Compounds

M+/[M+H]+

Cy-3-glc-ethyl-(epi)cat Pt-3-glc-ethyl-(epi)cat Pn-3-glc-ethyl-(epi)cat Mv-3-glc-ethyl-(epi)cat Mv-3-glc-ethyl-flavan-3-ol dimer Mv-3-acetylglc-ethyl-(epi)cat Mv-3-p-coumglc-ethyl-(epi)cat Dp-3-glc-ethyl-(epi)gallcat Cy-3-glc-ethyl-(epi)gallcat Pt-3-glc-ethyl-(epi)gallcat Pn-3-glc-ethyl-(epi)gallcat Mv-3-glc-ethyl-(epi)gallcat Mv-3-acetylglc-ethyl-(epi)gallcat Mv-3-glc-ethyl-Mv-3-glc

Fragments (m/z)

765 795 779 809 1097 851 955 797 781 811 795 825 867 1012

References ** ** ** ** * ** ** ** ** ** ** ** ** *

343 327 357 357 357 329 343 357

Labels: Dp: delphinidin, Cy: cyanidin, Pt: petunidin, Pn: peonidin, Mv: malvidin, glc: glucoside, acetylglc: ­acetylglucoside, p-coumglc: p-coumaroylglucoside, (epi)cat: (epi)catechin, gallcat: gallocatechin. * Adapted from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD thesis, Ss Cyril and Methodius Univ ersity, Skopje, Republic of Macedonia, 2009. ** Adapted from Alcalde-Eon, C., Escribano-Bailón, M. T., Santos-Buelga, C., and Rivas-Gonzalo, J. C., Changes in the detailed pigment composition of red wine during maturity and ageing. A comprehensive study, Anal. Chim. Acta, 563, 238–54, 2006. *** Adapted from Alcalde-Eon, C., Escribano-Bailón, M. T., Santos-Buelga, C., and Rivas-Gonzalo, J. C., Separation of pyranoanthocyanins from red wine by column chromatography. Anal. Chim. Acta, 513, 305–18, 2004. **** Adapted from Boido, E., Alcalde-Eon, C., Carrau, F., Dellacassa, E., and Rivas-Gonzalo, J. C., Aging effect on the pigment composition and color of Vitis vinifera L. Cv. Tannat wines. Contribution of the main pigment ­families to wine color. J. Agric. Food Chem., 54, 6692–704, 2006.

O+

HO

OCH3 OGk OCH3 OH

O O

HO

OH

m/z 805

O+

HO

- Glucose

O

OCH3 OH

O

HO

OH

m/z 643

OCH3 OH

Retro-Diels-Alder

OCH3 OH

O+

HO

OCH3 OH

OCH3 OH

OCH3 OH

OCH3 OH

OCH3 OH

O

OH O

HO

CH2

OH

+

OH

OH

m/z 491

m/z 152

Figure  20.7  Fragmentation of catechin-pyrano-malvidin-3-glucoside with m/z 805 under positive ESI-MS mode. (Adapted from He, J., Santos-Buelga, C., Mateus, N., and De Freitas, V., J. Chromatogr. A., 1134, 215, 2006.)

provides high resolution giving access to accurate mass determination. A basic characteristic of MALDI is the application of the analyte with a suitable matrix substance and irradiation of this mixture by a pulsed laser, including ablation of the sample and matrix molecules accompanied by ionization of the analytes. MALDI can be operated in the positive or negative ion mode and it yields mostly singly charged ions, generating cations such as [M + H]+, [M + Na]+ and [M + K]+ or anions such as [M–H]– and ­[M + Cl]– (Fulcrand et al. 1998). Recent investigations demonstrated that MALDI as a sensitive and efficient technique could be used for characterization of different molecules in food science: anthocyanins in red wine and fruit juice (Wang and Sporns 1999), carotenoids in crop plants (Fraser et al. 2007),

421

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives OH

OH

OH

O

HO

OH OH

OH

OH

O

–162 OH

OH

OH + OH

OH

HO

O OH

OH OH

+

OCH3 O

O OH

HO

OCH3 OH

O

OH

OCH3

HO

O

HO

OCH3 OH

O+

OH

OH

OH

OH

HO

OH

O

HO

O OH

OH m/z 907

OH OH

m/z 1069 –288 OH

OH

OH

OH

O

HO

OH OH HO

+

+ OCH3 OH

O

O

HO

OH OH

OCH3 OH

OH m/z 619 Figure  20.8  Fragmentation of (epi)catechin-(epi)catechin-malvidin-3-glucoside (m/z 1069). (Adapted from FranciaAricha, E. M., Guerra, M. T., Rivas-Gonzalo, J. C., and Santos-Buelga, C., J. Agric. Food Chem., 45, 2262–66, 1997.)

plant oligosaccharides (Lerouxel et al. 2002), black tea stain consisting of polyphenols (Yamada et al. 2007), and polygalloyl polyflavan-3-ols in grape seed extracts (Krueger et al. 2000). This technique has become the routine method of choice for peptide and protein characterization (Marentes and Grusak 1998; Schiller et al. 2004; Joss et al. 2006; Piraino et al. 2007; Sheoran et al. 2007; Zhang et al. 2008). The advantages of MALDI-TOF-MS over the other methodologies include the ease of use, speed of analysis, high sensitivity, wide applicability combined with a good tolerance toward contaminants, as well as the ability to analyze complex mixtures (Karas 1996). The most widely used sample preparation methods for MALDI-TOF-MS analysis are: the dried droplet technique when a mixed solution of the sample with analyte(s) and matrix is deposited onto a sample plate and allowed to dry, and in the sandwich method the sample is placed “in a sandwich” between two matrix layers. Two critical concerns for successful MALDI analyses are the choice of the matrix and the sample preparation (Sugui et al. 1999). A number of substances have been tested as possible MALDI matrices and applied as well. In general, there are no rules for predicting the suitability of a substance as a matrix, with the exception of the absolute requirement for absorption of UV laser energy. The most frequently used MALDI matrices are derivatives of benzoic acid (e.g., 2,5-dihydroxybenzoic acid [2,5-DHB]), which is suitable for analysis of low molecular weight compounds, and analysis of proteins and glycoproteins (Juhasz et al. 1993) and derivatives of cinnamic acid (e.g., α-cyano-4-hydroxycinnamic acid [CHCA] or synapic acid [SA]). Carpentieri et al. (2007) used 2,5-DHB for fast fingerprinting of red wines. Fullerenes have also been used as matrices (Ugarov et al. 2004), but there are no published data for their application for anthocyanins analyses in wine and grape samples even though these compounds have been attracting scientists all over the world working extensively on their syntheses and properties studies. The first use of fullerenes for laser desorption of biomolecules involved the application of a protein sample solution directly onto the predeposited

422

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

fullerene film (Michalak et al. 1994). The fullerene matrix has then been used for MALDI-TOF analysis of small hydrophobic molecules (e.g., steroids, fatty acids; Mernyak et al. 2008). Figure 20.9 illustrates the positive-ion MALDI-TOF-MS spectra of different tested matrices: CHCA, SA, 2,5-DHB, and C70 fullerene, and it is shown that the obtained ­quasimolecular, fragment and adduct ion peaks for CHCA, SA and 2,5-DHB have relatively high intensity in the range of m/z 100–700 compared to the peaks of C70 fullerene matrix (Ivanova 2009). Taking into account the molecular mass of fullerene (m/z 840), it is expected that the obtained signal peaks would have very low intensity in the range of interest for the anthoycanin identification, from m/z 100 to 700 and they would not interfere the identification of the sample peaks in this low mass range. So, applying the fullerene as a matrix, all five anthocyanins (malvidin, peonidin, petunidin, delphinidin, and cyanidin) have been identified in their aglycone forms since applying higher laser energy, needed for ionization of fullerene molecules, causes fragmentation of the glucosides. In Figure 20.10, an example of application of different MALDI matrices, CHCA, SA, 2,5-DHB, and C70 fullerene, for MALDI-TOF-MS analysis of grape skin extract is presented (Ivanova 2009).

×104

×104

190.05 172.04

[M+H]+

1.5 [M+H-H2O]+ 1.0

379.09

Intens. [a.u.]

(c)

400

500

600

700

m/z

137.02

HO O

154.02

+

0.5

100

200

300

200

300

400

500

600

700

m/z

840.00

M

+

1.5

OH 2,5-dihydroxybenzoic acid

1.0

199.02 273.04

100

(d)

OH

[M+K]+

1.0

0.0

x104

[M+Na]+

M

0.0

237.07 387.15 449.14 431.14

177.02

1.5 102.06

[2M+H]+

0.5

[M+H-H2O]+

×104

sinapinic acid

OH

α-cyano-4-hydroxycinnamic acid

300

OH

HO H3CO

335.11

200

O

H3CO

1.0

O

HO

212.02

100

[M+H]+

1.5

NC

[M+Na]+

102.11

207.05 225.08

[M+H-H2O]+

146.05

0.0

[2M+H]+

Intens. [a.u.]

0.5

Intens. [a.u.]

(b)

Intens. [a.u.]

(a)

0.5 449.01

400

500

820.69 816.00

600

700

m/z

0.0

720.10

100 200 300 400 500 600 700 800 900 m/z

Figure  20.9  Positive-ion MALDI TOF mass spectra of the matrices (a) CHCA, (b) SA, (c) 2,5-DHB, and (d) C70 fullerene. (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

423

Analytical Methodology for Characterization of Grape and Wine Phenolic Bioactives (b)

×104

Intens. [a.u.]

Intens. [a.u.]

(a)

331.1

×104 207.1

3.0

1.5

1.0

2.0

104.1

1.0

0.5

0.0

317.1 369.1 138.1 228.0 301.1 358.1 493.1 190.1 266.0 535.1

100

200

300

400

500

600

700

m/z

×104

1.25 1.00

639.1

104.1

639.1

0.0

(d)

Intens. [a.u.]

Intens. [a.u.]

(c)

225.1

100

179.1

200

493.1 237.1 331.0 387.1 449.1 535.1 609.1 677.1 263.0

300

400

500

600

700

m/z

400

500

600

700

m/z

×104 104.1

331.1

2.0

104.1 493.1

639.1

1.5

0.75 1.0

0.50 0.25 0.00

331.1

138.1 176.0

100

200

0.5

535.1 317.1 479.1 531.1 625.1 677.1 358.1 609.1 301.1 369.1 463.1

300

184.1

86.0

400

500

600

700

m/z

0.0

147.0

100

317.1 301.1 219.01

200

300

Figure  20.10  Positive-ion MALDI TOF mass spectra of the skin extract of the Vranec grape (red grape variety) in the presence of different MALDI matrices: (a) CHCA, (b) SA, (c) 2,5-DHB, and (d) C70 fullerene “sandwich.” (Data from Ivanova, V., Development of methods for identification and quantification of phenolic compounds in wine and grape applying spectrophotometry, liquid chromatography and mass spectrometry, PhD Thesis, Ss Cyril and Methodius University, Skopje, Republic of Macedonia, 2009.)

For better identification of anthocyanin glycosides (acetyl and p-coumaroyl derivatives) in wine and grapes further investigations are needed to be performed, in order to test, for example, the effect of the derivatized and/or acidified fullerenes as possible matrices. The ability to use a lower laser power is thus one advantage of these matrices. There are a number of other advantages that this new class of matrices possesses: high analyte ionization efficiency, small molar ratios (less than 1) of matrix/analyte, and a broader optical absorption spectrum, which should obviate specific wavelength lasers for MALDI acquisitions (Uragov et al. 2004).

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424

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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21 High Pressure Processing Technology on Bioactives in Fruits and Cereals Özlem Tokus¸ og˘lu and Christopher Doona Contents Introduction............................................................................................................................................. 429 High Pressure Processing (HPP)............................................................................................................. 430 HPP: Principles of Operation..............................................................................................................431 Large-Scale and Research HPP Equipment........................................................................................431 HPP on Bioactive Components................................................................................................................431 HPP Effects on Antioxidant Phenolics and Antioxidant Activity.......................................................432 Acknowledgment.................................................................................................................................... 439 References............................................................................................................................................... 440

Introduction In recent years, there has been increasing interest in moving from conventional methods of processing for food preservation toward the use of novel and emerging nonthermal food processing technologies, to control or eliminate microbes, enzymes, or chemical reactions and deliver more fresh-like, nutritious, valueadded, and safe high-quality food products to satisfy consumer demand for less processed foods with an extended shelf-life and that are free from additives. High pressure processing (HPP), irradiation, pulsed electric field (PEF), ultraviolet light (UVL), and other nonthermal processing methods are becoming increasingly popular to treat foods, capable of eliminating harmful microorganisms in foods, while minimizing thermal degradation reactions in foods compared to thermal processing (Barbosa-Cánovas et al. 1998; Barbosa-Cánovas et al. 2005; Guerrero-Beltráni et al. 2005). More information on various nonthermal processing technologies is available from the Nonthermal Processing Division of the Institute of Food Technologists (http://www.ift.org/divisions/nonthermal/). The major aims of utilizing these ­methods are to improve food safety and food quality concomitantly, and thereby facilitate the development of innovative high value products and the creation of new opportunities for expanding markets. High hydrostatic pressure or ultrahigh-pressure processing or HPP is one technology that has begun to fulfill its potential to satisfy both consumer and scientific requirements, and it is a leading alternative in replacing thermal processing in some food applications in the drive to meet increasing consumer demand for foods featuring improved organoleptic qualities and higher acceptance (Patterson et al. 2008). The technology is especially beneficial for heat sensitive products (Barbosa-Cánovas et al. 2005). HPP can be conducted at ambient or moderate temperatures, thereby eliminating thermally induced cooked offflavors. Compared to thermal processing, the HPP of foods results in products with a fresher taste, better appearance, and texture. In the 1990s, the HPP technology began gaining prominence in the food industry because of its advantages for inactivating microorganisms and enzymes at ambient or relatively low temperatures with less adverse affect on the flavor, color, and nutritional constituents of foods compared to conventional ­thermal processes (Hoover et al. 1989; Mertens and Knorr 1992; Cheftel 1995; Cheftel and Culioli 1997). 429

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Companies began marketing commercial HPP-treated products, such as jam, fruit juice, sauces, rice wine, and rice cake (Hayashi 1997). In recent years, HPP has been successfully implemented in food industries worldwide (United States, Europe, and Japan) to extend shelf-life or improve safety of fruit products (avocado, guacamole, salsa, applesauce, fruits juices, etc.), ready-to-eat (RTE) meats, and fresh oysters. Among the most successfully commercialized HPP-treated food products are sliced, cooked ham and a range of tapas products in Spain (Gassiot and Masoliver 2010). Tapas products are convenient heat-and serve mini-pork sausages made with Spanish paprika and marinated diced pork. The benefits of HPP for increasing the retention of food organoleptic attributes and other more fresh-like characteristics combined with increased convenience and extended shelf-life will no doubt continue to increase for the market (Deliza et al. 2005). The HPP provides an alternative means of killing vegetative bacteria, spoilage organisms (yeasts and molds), viruses, and bacterial spores that can cause food spoilage or food-borne diseases without compromising food sensory quality attributes or food nutrients. In many cases with vegetative pathogens and ­bacterial spores, the survival curves for organisms subjected to HPP exhibit nonlinear inactivation kinetics “shoulders” (Doona et al. 2005; Feeherry et al. 2005) or “tailing” (Doona et al. 2008). Predictive microbiology models provide convenient tools to assess whether a process will ensure the safe preservation of foods. Two examples are the quasi-chemical model (Taub et al. 2003; Ross et al. 2005) and the Weibull distribution model, both of which are nonlinear models that can accurately describe the nonlinear inactivation kinetic models of vegetative pathogens (Escherichia coli, Listeria monocytogenes) in foods treated with HPP (Doona et al. 2008; Doona, Ross, and Feeherry 2008). An enhanced version of the quasi-chemical model is being developed to account for unique features of the inactivation kinetics of bacterial spores of Bacillus amyloliquefaciens by HPP, including the presence of a subpopulation of increased baro-resistance. As indicated above, the HPP pasteurization safely inactivates vegetative cells, and some enzymes, while retaining nutritive content, sensory attributes, and a fresh-like character of foods. HPP tends to affect cell membranes, enzymes, and large molecules. Macromolecules such as proteins and starches can undergo changes in their native structure during HPP treatments (and during thermal treatments) that can be used to influence texture. Doona et al. (2006) studied the retrogradation kinetics, water dynamics, and thermometric characteristics of HPP-treated wheat starch (Doona et al. 2008; Patterson et al. 2008; Ahmed and Ramaswamy, 2006; Feehery et al. 2005). In contradistinction, HPP generally has little effect on the primary structure of low molecular weight food individual components such as flavors, vitamins, pigments, peptides, lipids, and saccharides. In general, HPP tends not to destroy the covalent bonds between atoms of the constituent molecules. The energy used during HPP treatment is relatively low and covalent bonds tend to have low compressibility below 2000 MPa, whereas the process affects hydrogen bonds and ionic and hydrophobic interactions in macromolecules. HPP protects nutraceuticals, functional food ingredients, and so on, whose functionality can be compromised by the use of heat. HPP is an innovative, emerging technology with potential for optimizing intake of nutrient and nonnutrient phytochemicals in foods (Deliza et al. 2005).

High Pressure Processing (HPP) HPP conditions in the range of 300–700 MPa at moderate initial temperatures (around ambient) are generally sufficient to inactivate vegetative pathogens for pasteurization processes, some enzymes, or spoilage organisms to extend shelf-life. For example, HPP is used to inactivate spoilage organisms and extend shelf-life (and provide extra assurance against pathogens) at conditions of 400 MPa and 15°C (in a 320 L unit) for sliced cooked ham, and 600 MPa and 15°C (in a 218 L unit) for dry-cured ham and tapas products (Gassiot and Masoliver 2010). To inactivate bacterial spores such as Clostridum botulinum for the production of ambient shelf-stable, low-acid foods requires high pressure and high temperature combinations. Such processes typically involve high pressures in the range of 600–800 MPa and higher initial temperatures around 80−90°C. During pressurization, rapid adiabating heating generates temperatures above 121°C. This process, achieving commercial sterility in low-acid foods, is called “pressure-assisted thermal sterilization” and has several technical advantages over conventional thermal sterilization methods (shorter processing times, improved food quality, and increased energy efficiency).

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HPP: Principles of Operation A typical HPP process (Ohio State University 2009) involves treating packaged food products (usually in flexible plastic pouch material or plastic bottles) by loading them into a high pressure vessel filled with an incompressible transmission fluid (usually water) then closing the vessel. Using one or more pumps, fluid is pumped into the vessel to increase the pressure to the intended end-level and then the pumping is stopped. The packaged food products are subjected to these combinations of high hydrostatic pressure and temperature for a sufficient time to induce inactivation of the target organisms or enzymes and then the pressure is released. Since pressure is transmitted uniformly throughout the package and product, the food retains its original shape. This works particularly well for unstructured foods containing water, whereas foods with internal air pockets (strawberries, marshmallows, some bakery items) tend to collapse, and dry solids tend not to have enough moisture to allow efficient microbial destruction. When the product is removed from the high pressure vessel, the package is covered with water. In the case of RTE meats, for example, some companies use cold drying equipment to remove the water and prepare the package for labeling and packing. Cold drying helps maintain product quality by reducing the potential effects of using heat.

Large-Scale and Research HPP Equipment There are a number of high pressure equipment manufacturers worldwide making HPP equipment for food preservation (Ohio State University 2009). Systems cost in the range of $0.5–2.5 million, depending on the size of the vessel, extent of automation, and other design features. Units can range in sizes of 420 L, 350 L, or 150 L, and systems run in batch or semicontinuous modes of operation for food industry purposes. Figure 21.1 demonstrates large-scale HPP equipment, including a 420 liter unit (Figure 21.1a) and a 350 liter unit (Figure 21.1b). Both units are in a horizontal configuration. The illustration in Figure 21.1c depicts the semicontinuous mode of operation, in which carriers full of packaged food products enter on a conveyor belt from the left and are loaded into the pressure vessel. After the HPP treatment, the carriers are removed from the pressure vessel, and exit the area to the right on the conveyor belt. The treated products are removed from the carrier, dried, labeled, and packed for shipping and distribution. Figure 21.1d shows a smaller vertical configuration HPP unit used to process oysters. A 215 L batch ­system has the capacity to produce about 10 million pounds of food per year and products may cost about $0.03–0.10 more per pound than thermally processed counterparts. Significantly smaller laboratory-scale units are also manufactured to operate on the same general basic principles but for research purposes, and they are available at research facilities and universities worldwide. Figure 21.2 depicts one such unit that operates at pressures of 100,000 psi with sample sizes of 10−30 mL. Figure 21.2a shows the front view of the high pressure research unit with the accompanying workstation to the left and the bath cover emanating from the top. In addition to containing heat in the bath where the high pressure chamber vessel cylinder is located, the bath cover also acts as a potential safety shield. Figure 21.2b details the arrangement of the pressure unit’s components, with the chamber vessel cylinder located in the covered bath and connected to the pump and an assortment of valves for regulating and releasing high pressures. Figure 21.3a shows the actual interior of the high pressure unit, with the components labeled in accordance with the schematic of the interior side view in Figure 21.3b. Figure 21.4a shows the top view of the bath with the bath cover removed and the bath fluid drained to reveal the chamber vessel cylinder (with the cap removed). The corresponding cross-sectional side view of the bath (Figure 21.4b) shows the chamber vessel cylinder with the cap in place and a thermocouple inserted into the sample chamber.

HPP on Bioactive Components Consumer perception of food quality depends not only on microbial quality, but also on other food ­factors such as biochemical and enzymatic reactions and structural changes (Cheftel 1995; Patterson 2005). In this context, HPP can have an effect on food yield and on sensory qualities such as food color

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

(a)

(b)

(c)

(d)

Figure 21.1  (For Figure 21.1d, see color insert) Large-scale HPP equipment: horizontal configuration units, including (a) a 420 liter unit (Courtesy of NC Hyperbaric.), (b) a 350 liter unit (Courtesy of Avure Technologies Inc.), and (c) an illustration depicting the semicontinuous mode (Courtesy of Avure Technologies Inc.), in which carriers enter the pressure vessel area on a conveyor belt from the left. After HPP treatment, the carrier is removed from the vessel and exits to the right on the conveyor belt. For comparison, the vertical configuration unit shown in (d) is used to process oysters (Courtesy of Avure Technologies Inc.)

and texture (Hogan et al. 2005). High pressures can also be used to enhance extraction of compounds from foods. Recent studies have shown that high pressure extraction (HPE) can shorten processing times, and provide higher extraction yields while having less negative effects on the structure and antioxidant activity of bioactive constituents. The use of HPE enhances mass transfer rates, increases cell permeability, and increases diffusion of secondary metabolites (Richard 1992; Dornenburg and Knoor 1993). Also, HPP increased the capacity to extract phenolic constituents, and HPP-treated samples retain higher levels of bioactive compounds (Zhang et al. 2004, 2005ab; Ahmed and Ramaswamy 2006; Tokuşoğlu et al. 2010).

HPP Effects on Antioxidant Phenolics and Antioxidant Activity The study of Patras et al. (2009) was undertaken to assess the effect of HPP treatments and conventional thermal processing on antioxidant activity, levels of bioactive antioxidant compounds (polyphenols,

433

High Pressure Processing Technology on Bioactives in Fruits and Cereals (a)

(b)

Cover

Bath heater Chamber cylinder

Bath Air supply

Electric air pressure regulator Air/oil pump

Pressure transducer Dump valve

Shift valve Intensifier

Pump on–off

On–off valve

Solenoid

Solenoid

Drain

Control system

Figure 21.2  A smaller HPP unit for laboratory research that operates up to 100,000 psi with sample sizes of 10−30 mL: (a) front view of the HPP unit with the bath cover emanating from the top (Courtesy of Avure Technologies Inc.) and (b) schematic of the HPP unit locating the bath cover and HPP chamber inside the controlled temperature bath.

ascorbic acid, and anthocyanins), and the color of strawberry and blackberry purées (Patras et al. 2009). It was reported that key antioxidants (cyanidin-3-glycoside, pelargonidin-3-glucoside, and ascorbic acid) in strawberry and blackberry purées and the antioxidant activity of these purées were quantified after various HPP treatments (400, 500, 600 MPa/15 min/10–30°C) and thermal treatments (70°C/2 min). Table 21.1 shows the antioxidant indices of HPP-treated and thermally processed strawberry and blackberry purées (Patras et al. 2009). The three different pressure treatments did not cause any significant changes in ascorbic acid levels. Following thermal processing (P70 ≥ 2 min), the ascorbic acid content degraded by 21% compared to the unprocessed purée. Similarly, no significant changes in anthocyanin compounds were observed in HPP-treated and unprocessed purées, while conventional thermal

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

(b)

Bath/bath cover

Bath overflow hose Insulated bath tank

Bath drain valve

Bath TC I

Pressure fluid and bath reservoir

External bath chiller circulation

Intensifier valve assembly Spare intensifier side

Working intensifier side

Figure 21.3  (a) Actual and (b) schematic diagram of the interior of the HPP unit viewed from the side.

treatments significantly reduced the anthocyanin levels (Patras et al. 2009). Patras et al. (2009) reported that antioxidant activity of HPP-treated strawberry and blackberry purées were significantly higher than in thermally processed purées (Patras et al. 2009). Qui et al. (2006) studied the stability and isomerization of lycopene by HPP. Standard lycopene and tomato purée were pressurized at 100, 200, 300, 400, 500, or 600 MPa for 12 minutes and at controlled temperature (20 ± 1°C), then stored at refrigerator temperature (4 ± 1°C) and ambient laboratory temperature (24 ± 1°C) under lightproof conditions. Afterward, HPP-treated and controlled lycopene and

435

High Pressure Processing Technology on Bioactives in Fruits and Cereals (a)

(b)

TC fitting End closure

End cap UHP tubing coil

Chamber cylinder thermocouple Chamber cylinder

Bath thermocouple

Figure 21.4  Close-up of the top view reveals (a) the actual chamber vessel cylinder (with the cap removed) inside the thermal bath (with bath cover removed and the bath fluid drained) and (b) the corresponding cross-sectional side view of the bath shows the chamber vessel cylinder with the cap in place.

436

Table 21.1 The Antioxidant Indices of HPP-Treated and Thermally Processed Strawberry and Blackberry Purées Total Phenols, mg GAE/100g DWe

Anthocyanin, mg/100g DW

Ascorbic Acid, mg/100g DW

Treatment

Strawberry

Blackberry

Strawberry

Blackberry

Strawberry

Blackberry

Unprocessed Thermal HPP (400 MPa) HPP (500 MPa) HPP (600 MPa)

1.55 ± 0.07a 1.16 ± 0.01b 1.25 ± 0.05b 1.30 ± 0.02ab 1.33 ± 0.02a

2.86 ± 0.23a 2.78 ± 0.26a 3.87 ± 1.11a 3.70 ± 0.57a 4.80 ± 1.79b

855.02 ± 6.52a 817.01 ± 5.26b 859.03 ± 6.56a 926.00 ± 5.93a 939.01 ± 0.99c

1694.19 ± 3.0a 1633.62 ± 8.4a 1546.26 ± 8.0a 1724.65 ± 0.7b 1778.44 ± 6.0b

202.27 ± 0.50a 145.82 ± 6.40b 173.34 ± 6.51ab 202.53 ± 5.40a 204.30 ± 1.60a

1004.90 ± 8.60a 975.28 ± 7.90b 1039.21 ± 4.51a 1014.21 ± 0.10a 1014.47 ± 1.00a

f

g

Strawberry 633.10 ± 9.31a 496.11 ± 0.04b 574.30 ± 3.93c 577.10 ± 6.52c 599.11 ± 0.60c

Source: Adapted from Patras, A., Brunton, N. P., Pieve, S. D., and Butler, F., Innov. Food Sci. Emerg. Technol., 10, 308–13, 2009b. Notes: Values are mean ± standar deviation, n = 3, mean values in a column with different letters are significantly different at p < .05; nd = not detected. a Dry weight. b Expressed as mg/100g DW pelargonidin-3-glucoside. c Expressed as mg/100g DW cyanidin-3-glucoside.

Blackberry nd nd nd nd nd

Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Antiradical Power (g/L)–1

437

High Pressure Processing Technology on Bioactives in Fruits and Cereals

its cis-isomers in tomato purée samples were measured (Qui et al. 2006) by HPLC and IR spectral analysis after 2, 4, 8, and 16 days of storage (Table 21.2). It was found that 500 and 600 MPa led to the highest reduction of lycopene, while 400 MPa could retain the maximal stability of lycopene (Qui et al. 2006). The highest stability of lycopene in tomato purée was found when pressurized at 500 MPa and stored at 4 ± 1°C in the study of Qui et al. (2006), which retained most of the total lycopene content in tomato purée (6.25 ± 0.23 mg/100 g; see Table 21.2). It was established that HHP is an alternative preservation method for producing ambient-stable tomato products in terms of lycopene conservation (Qui et al. 2006). Prasad et al. (2009a) determined that HPE has tremendous potential for use in flavonoid extraction. After 30 minutes of HPE of Litchi (Litchi chinensis Sonn.) fruit pericarp (LFP), the extract yield, total phenolic level, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH), and superoxide anion scavenging ability were determined by Prasad et al. (2009a). The extraction yield by treatments of 400 MPa HPE for 30 minutes was 30%, while that by conventional extraction (CE, control) was 1.83%. There was no significant difference in the total phenolic content (as mg/g DW [dry weight]) among the two extraction methods (HPE and CE). It was found that the DPPH radical scavenging activity obtained by HPE (400 MPa) was the highest (74%), while that of ascorbic acid was the lowest (44%), when using a 10 mg/mL concentration. Additionally, HPE could provide a more effective alternative than CE, because HPE requires less organic solvents and a shorter extraction time (Prasad et al. 2009a). Table 21.3 describes the quantification of the individual flavonoids epicatechin (EC), epicatechin gallate (ECG), catechin (C), and procyanidin B2 and total flavonoids from LFP tissues by conventional extraction (CE), ultrasonic extraction (UE), and HPE. Both EC and ECG were identified and quantified as the major flavonoids, while C and procyanidin B2 were identified as the minor compounds (Prasad et al. 2009a). The total flavonoid content detected was 0.65, 0.75, 0.29, and 0.07 mg/g dry weight by HPE at 200 and 400 MPs, UE, and CE, respectively. The HPE increased the flavonoid extraction yield up to 2.6 times in comparison with UE, and up to 10 times compared with CE. Patras et al. (2009) reported of the effect of thermal and HPP on antioxidant activity and the color stability of tomato and carrot purées. High pressure processed purées had significantly higher antioxidant capacities when compared to thermally treated samples. High pressure treatments at 600 MPa retained more than 93% of ascorbic acid (vitamin C) as compared to thermally processed tomato purées (Table 21.4; see Patras et al. 2009). Table 21.2 Total Lycopene Losses in Lycopene Standard (as Percentage) and Total Lycopene Content in Tomato Puree (as mg/100g) as a Function of Storage Time at 4 ± 1°C, at Six Different HHP Conditions Storage Time (Days) LYCOPENE 0

Untreated (0MPa)

Pressure Applied (MPa) 100

200

300

400

500

600

2.10 ± 0.02

2.10 ± 0.02

2.11 ± 0.02

2.11 ± 0.02

2.13 ± 0.02

20.8 ± 1.12

56.3 ± 3.02

3.05 ± 0.23 5.22 ± 0.34 6.13 ± 0.40

2.10 ± 0.02 2.40 ± 0.05 2.49 ± 0.07

2.11 ± 0.02 2.52 ± 0.09 2.63 ± 0.09

2.11 ± 0.02 2.34 ± 0.07 2.45 ± 0.09

2.13 ± 0.02 2.29 ± 0.09 2.39 ± 0.11

20.8 ± 1.12 21.7 ± 1.19 22.7 ± 1.21

56.3 ± 3.02 57.4 ± 3.34 57.4 ± 3.34

7.89 ± 0.44

4.21 ± 0.23

3.29 ± 0.28

3.78 ± 0.22

2.70 ± 0.28

25.7 ± 1.41

60.4 ± 3.76

TOMATO PUREE 0 5.16 ± 0.12 2 5.18 ± 0.13 4 5.18 ± 0.13 5.17 ± 0.13 8

5.33 ± 0.13 5.39 ± 0.12 5.37 ± 0.12 5.37 ± 0.13

5.39 ± 0.11 5.42 ± 0.12 5.43 ± 0.12 5.40 ± 0.15

5.48 ± 0.12 5.50 ± 0.13 5.51 ± 0.13 5.51 ± 0.13

5.55 ± 0.12 5.50 ± 0.13 5.50 ± 0.13 5.48 ± 0.14

6.25 ± 0.23 6.20 ± 0.21 6.21 ± 0.20 6.19 ± 0.22

5.10 ± 0.10 5.11 ± 0.11 5.10 ± 0.12 5.08 ± 0.10

4.37 ± 0.10

5.17± 0.12

5.22 ± 0.16

5.26 ± 0.12

5.18 ± 0.13

6.11 ± 0.23

4.88 ± 0.12

2 4

8 16

16

Source: Adapted from Qiu, W., Jiang, H., Wang, H., and Gao, Y., Food Chem., 97, 516–23, 2006.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

Table 21.3 The Quantification of Individual Flavonoids from Litchi Fruit Pericarp Tissues by Conventional Extraction, Ultrasonic Extraction and High Pressured-Extraction Flavonoids (mg/g DW)∗ Epicatechin Epicatechin gallate Catechin Procyanidin B2 Total flavonoids

Extraction Methods CE

UE

HPE at 200 MPa

HPE at 400 MPa

0.0414 ± 0.001 0.0121 ± 0.003 0.0002 ± 0.0 0.0175 ± 0.0003 0.0712 ± 0.004

0.16 ± 0.04 0.06 ± 0.01 0.0020 ± 0.0005 0.0731 ± 0.0011 0.2951 ± 0.051

0.32 ± 0.002 0.019 ± 0.04 0.0016 ± 0.001 0.14 ± 0.03 0.6516 ± 0.07

0.348 ± 0.06 0.2527 ± 0.04 0.0160 ± 0.07 0.1346 ± 0.03 0.7513 ± 0.2

Source: Adapted from Prasad, K. N., Yang, B., Zhao, M., Ruenroengklin, N., and Jiang, Y., Journal of Food Process Engineering, 32, 828–43, 2009a. Notes: Values reported are means of triplicate determinations (n = 3) ± SD. DW∗ = dry weight; CE = conventional extraction; UE = ultrasonic extraction; HPE = high-pressure extraction.

Table 21.4 Effect of Thermal (TP) and High Pressure Treatments on Antiradical Power, Total Phenols, Ascorbic Acid and Total Carotenoid Content in Tomato Purées Samples Unprocessed TP HPT400 MPa HPT500 MPa HPT600 MPa LSD∗

Antiradical Power (g/l)−1

Total Phenols (mg GAE/100g)

Total Carotenoids (mg/100g βCE)

Ascorbic Acid (mg/100g)

0.37 ± 0.04 0.34 ± 0.03 0.43 ± 0.01 0.40 ± 0.02 0.47 ± 0.03 0.04

360.56 ± 9.89 341.13 ± 4.83 337.36 ± 15.31 367.50 ± 17.58 371.73 ± 15.15 24.35

37.02 ± 3.07 33.40 ± 1.55 28.42 ± 2.65 30.25 ± 7.17 100.85 ± 0.11 8.44

204.83 ± 4.88 125.14 ± 5.174 115.25 ± 5.54 95.67 ± 3.71 192.13 ± 4.83 9.05

Source: Adapted from Patras, A., Brunton, N., Da Pieve, S., Butler, F., and Downey, G., Innov. Food Sci. Emerg. Technol., 10, 16–22, 2009a. Notes: Values reported are means of triplicate determinations (n = 3) ± SD; expressed on dry weight basis. ∗Least significant difference (p = 5%).

Yen and Lin (1996) reported that the level of retention of ascorbic acid in guava purée proceeded according to the following decreasing order: (400 MPa for 15 min) > (88–90°C for 24 s) > (600 MPa for 15 min). In the study given by Patras et al. (2009), ascorbic acid levels were in the order (600 MPa) > (water immersed purées) > (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.

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

O

OH

CH2OR

R:

O

O C H 3C

HO p-coumaryl-

acetyl

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.

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Fruit and Cereal Bioactives: Sources, Chemistry, and Applications

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.

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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.

Index A 3-Acetyl deoxynivalenol (3-ADON), 258 4-Acetyl nivalenol (4-ANIV), 258 3-ADON, see 3-Acetyl deoxynivalenol (3-ADON) Aflatoxins cereals with aflatoxinB 1,256 mycotoxic bioactives in fruits and cereals structures and formation. 277-279 mycotoxigcnic fungi in cereals and cereal-based foods Aspergillus spp.. 254-255 research on biosynthesis, 289 Alkylresorcinols, 5-7 cereals grains, structures, 234 cereals lipids, 231 content barley, 33-34 alkylresorcinols. 34 in rye, 400-401 wheat, 236 Almond; see also Nut bioactives neutral lipids fatty acids (FAs), 189-190 triacylglycerols (TGs), 189, 191 phcnolics, 188 content, 190 flavonoids, 189 flavonol glycosides. 189 industrial processing. 189 ORAC values, 189 phenolic acids. 189 phytosterols and tocols. 191—192 polar lipids. 191 Amaranth grain study, 393 Anacardium occidentale L., see Cashew 4-ANIV, see 4-Acetyl nivalenol (4-ANIV) Anthocyanins, 85-86 content and composition, 324 extraction, purification and identification methods chromatographic and electrophoretic techniques, 324 MS hyphenated techniques. 325 stability of, 325 health function of, 324 roles, 324 Antioxidants defined, 4 Apple. 107 anthocyanins, 109 antioxidant activity of TAC values, 113 bioactives, 108 cultivars. 109 dihydrochalcones, 109 flavanols, 109

© 2011 by Taylor & Francis G r o u p , L L C

hydroxycinnamates. 109 phenolic compounds, 108 characteristics, 113 clarification treatments on, 112 constituents of. Ill polyphenols, 108 procyanidins and catechins, 109-111, 113 production and consumption in world, 108 total polyphenols, 108-109 Apricot fruit aesculetin and scopoletin, 88 bioactives in, 87-89 carotenoids. 88 chlorogenicacid, 88 flavonols in. 88 genotype, 87-88 HCA levels, 88 Arbequina cultivars. 354 Aspergillus spp. and aflatoxin production. 294 distribution in world, 293 mycotoxigenic effect on cereals, 254-255 nuts, 293 pistachio, 295, 297-309 Asyrian tablet. 280 Avena saliva, L., see Oats 1?

Barley alkylresorcinols. 33 influence on levels, 34 antioxidant activity discrepancies, 35 DPPH and ABTS radical scavenging activity, 35 hydroxyl and superoxide anion radical tests, 35 methanol extracts, 35 phenolic compounds and metal chelation, 35 radical scavenging activities, 35 solvent extraction method. 35 surface methodology, 35 synthetic antioxidants, 35 carotenoids and phytosterols, 33 fatty acid, 237 germination and kilning on total phenolic acids. 34 hull-less barley, 34 lipids, 237 phenolics aleurone, 32 anthocyanins, 32 Chinese varieties, 32 cultivars and cultivars, 33

443

444

Index

extraction behavior, 33 flavonols. 32 location, 33 phenolic content, 33 proanthocyanins, 32 whole barley flour. 32 phytochemicals, 31 sterols, 237 tocols, 31 rich fractions, 32 yield, 394 tocopherols, 32, 238 utilization, 34 Baybcrry. 154, 156 Chinese cultivars, 155 Dongkui, 154 hyphenated technique of HPLC, 154 phcnolics in, 155 Xiangshan and Biqi, 154 Berry fruits, phytochemical bioactives in, 144 bayberry, 154. 156 Chinese cultivars, 155 Dongkui, 154 hyphenated technique of I1PLC, 154 phenolics in, 155 Xiangshan and Biqi. 154 blackberry anthocyanin and phenolics in, 145 anlhocyanin-containing extract (ACE). 144 cyanidin-3-glucosidc. 144 ferric reducing antioxidant power (FRAP), 144 used, 144 yalova. 145 blueberry anthocyanins and total phenolics in. 147-148 benefits, 145 catechin and epicatechin, 147 in Eastern Black Sea Region, Turkey. 146 Ericaceae and Vaccinium (V), 145 glycosylated and acylated forms, 147 ovariectomized rat model, 146 profiles of. 147 published data on, 147 in United States, 146 V. Angustifolium, 146 V. ashei, 146 V. corymbosum, 145 V. Myrtilloides, 146 chokeberry

aronia berries, 156 contents of llavononcs. 157 dark color of, 157 extracts of, 156 raw fruits of, 156 cranberry benefits, 158 consumption, 159 Helicobacter pylori, 159 peptic ulcers, 159 percentage scavenging capacities of, 159 phenolic profiles of. 159 © 2011 by Taylor & Francis G r o u p , L L C

currant blackcurrant, 157 elderberry anthocyanidin in, 160 antioxidant effects of, 161 cyanidin-3-glucosidc, 161 cyanidin-3-sambubioside, 161 dark blue, 160 European elder. 160 Florida elder, 160 herbal medicine treatments, 161 gooseberry ascorbic acid, 162 phenolic acids, levels of, 161-162 red currant anthocyanins and flavonols in. 158 strawberry antineurodegenerative biological properties, 153 ellagic acid in, 153-154 in United States, 153 Bertholletia excelsa H.B.K., see Brazil nut Blackberry anthocyanin and phenolics in. 145 anthocyanin-containing extract (ACE), 144 cyanidin-3-glucoside, 144 ferric reducing antioxidant power (FRAP), 144

used, 144 yalova, 145 Blueberry anthocyanins and total phenolics in average values for, 147 carotenoids, 147 genotypes, 148 benefits, 145 blueberry bioactives, 146 catechin and epicatechin. 147 glycosylated and acylated forms, 147 ovariectomized rat model. 146 profiles of, 147 published data on, 147 in Eastern Black Sea Region. Turkey, 146 Ericaceae and Vaccinium (V), 145 in United States, 146 V. Angustifolium, 146 V. ashei, 146 V. corymbosum, 145 V. Myrtilloides, 146 Brazil nut, 213; see also Nut bioactives neutral lipids, 214 nutritional value, 214 phenolics, 214 phytosterols and tocols, 215 polar lipids, 214-215

c Cancer and cereal grains in chemoprevention. mechanisms anlioxidant related, 11-12 dietary fiber related, 11

445

Index

glucose response, mediation, 12 phytoestrogen related, 12 epidemiological and intervention studies, 10-11 studies. 10 Cardiovascular disease (CVD) cereals and cereals products cereal bran wax components, 13 coronary heart disease (CUD) risk, 12 soluble fibers, 12 studies, 12 Carotenes, 5 Carotenoids, 321 chemical structures, 322 citrus fruit, 128 levels and distributions, 129 extraction, purification and identification methods alkaline/enzymatic hydrolysis saponification procedure, 323 analysis. 323 butylated hydroxytoluene (BHT), 322 characteristics, 322 GC and GC-mass spectrometry (MS), 323 HPLC, advantages, 323 organic solvents uses, 323 photodiode array detector (DAD), 323 polyisoprenoid compounds. 322 problems, 323 SFE technique, 323 ultraviolet-visible (UV-Vis), 323 in fruit and cereals. 5 structures, 6 tropical fruit, 129-130 analytical methods. 130 a-and P-carotenes, 129 Ataulfo mango cultivar, 130 lutein levels. 129 uses, 322 Carya illinoinensis, see Pecans Cashew; see also Nut bioactives neutral lipids. 216-217 phenolics, 216 phytostcrols and tocols, 217 polar lipids. 217 CDG, see Corn distillers grain (CDG) Cereal bioactive compounds in extrusion, 342 effect of antioxidant activity. 343-344 experiment on, 343 phenolic content, 343-344

lipids, 343 socioeconomic factors, 343 Cereals antioxidant composition, 361 definitions, 362-363 barley, 31 antioxidant activity, 35 phenolics, 32-34 tocols, 32 bran bound phenolic content, 379 © 2011 by Taylor & Francis G r o u p , L L C

corn antioxidant activity. 40-41 carotenoids, 38-39 phenolics, 36—38 phytosterols, 3 9 - 4 0 tocols, 35-36 direct measurement procedure. 380 ferulic acid equivalents (FAE). 379 Folin-Ciocalteau procedure, 379 grains alkylrcsorcinols structures, 234 amphophilic lipids structures. 234 barley. 237-238 corn/maize, 238-239 4,4-desmethylsterols structures, 233 genetic relationships between, 230 glycolipids structures, 232 long-chain alcohols structures. 232 oats, 238 pearl millet. 242 phospholipids structures, 231 policosanols structures, 232 rice, 239-240 rye, 237 sorghum, 240-242 tocopherols and tocotricnols structures, 233 wax esters of fatty acids structures, 232 wheat, 235-237 lipids alkylrcsorcinols, 231 extraclion and analysis, 234-235 fatty acids, 230 7-oryzanoI, 230-231 plant sterols, 230 tocotrienols, 231 methanolic extracts antioxidant activity and total phenolics content, 378 methods for assessing antioxidant capacity. comparison, 373 millet luten free, 59 oats antioxidant activity, 45 avenanthramides, 4 2 - 4 4 phenolics, 41-42 tocols, 41 oxidative stress. 362-363 primary (chain breaking) antioxidants. 363 prooxidant terms, 362-363 rice antioxidant activity, 47-49 phenolics, 47 tocols and oryzanols, 45-47 rye alkylresorcinols. 53-54 antioxidant activity, 54-55 phenolic, 50-52 phytosterols. 52—53 tocols, 49-50 salicylic acid probe 11 PLC chromatogram. 371 hydroxylation products, 371

446

secondary (preventive) antioxidants Fenton-type reactions, 363-364 lower oxidation, 363 phenolic antioxidants, prooxidative effect, 363 sorghum gluten free, 59 total antioxidant capacity (TAC) ABTS-TEAC assay, 367-368 antioxidant activity tests, 372 chromophores used in, 367 cupricion reducing antioxidant capacity (CUPRAC) method, 368-372 DPPH method, 368 ET-based assays, 365—366 FRAP assay, 368 IlAT-based assays, 364-365 heat and physical processing and alkaline hydrolysis, 376-377 high molecular-weight polyphenols, 376 measurements in. 377-380 oxygen radical absorbance capacity (ORAC) assay, 365 Trolox-cquivalent antioxidant capacity (TEAC), 366-367 values, 380 total oxyradical scavenging capacity (TOSC) assay, 378 wheat antioxidant activity, 58-59 phenolics. 55-57 tocols, 55 wheat flour and rice, 379 Chanomeles japonica, see Japanese flowering quince Che'toui cultivar fruit, 354 Chinese quince, 116 Chokeberry aronia berries, 156 contents of flavonones, 157 dark color of. 157 extracts of, 156 raw fruits of, 156 CITcont, see Citrinin contamination (CITcont) Citrinin contamination (CITcont), 438 Citrus fruits antioxidant activity 2.2-diphenyl-l-picrylhydrazyl (DPPH), 130 ferric reducing antioxidant power (FRAP). 130 methoxylated flavones, 130 oxidation of LDL, 131 phenolic acids, 130 scavenging activity, 130 caffcic and chlorogenic, 124 carotenoids. 128 levels and distributions of, 129 fcrulic and sinapic, 124 hydroxycinnamic acids, 124 /i-coumaric acids. 124 phytochemicals monophcnols and phenolic acids, 124-125 polyphenolics flavonoids, 127 Codling moth (CM), 292 © 2011 by Taylor & Francis G r o u p . L L C

Index

Corn aflatoxin B, and aflatoxin G,, 278 antioxidant activity anthocyanins, 40 blue and white corn cultivars, 40 carotenoid corn cultivar, 40 dietary source, 41 DPPH radical scavenging activity, 40 ferulic acid, 40 HCL-methanol solvent extraction protocol, 40 radical scavenging activity, 40 singlet oxygen quenching activity, 41 soluble phenolic contents, 40 avcnolcic acid. 238 campesterol and stigmasterol, 239 carotenoids carotene, 38 colored cultivars, 38 content. 38 cryptoxanthin contents, 38 xanthophylls, 38 corn distillers grain (CDG), 394-395 corn oil. 239 ear with fusarium ear rot. 278 Fusarium spp. on, 254 Penicillium spp. on. 255 phenol ics anthocyanin content. 38 blue genotypes. 38 bound fcrulic acid. 36 carotenoid in cultivars, 38 commercial corn oil. 36 ferulic acid dehydrodimers hydrolysis, 36 ferulic acid distribution. 37 ferulic acids. 36 free and soluble conjugates, 36 nixtamalization, 38 phenolic content in fiber. 37 sinapic acid. 36 phytosterols content. 39 distribution, 39 extraction, 39 fatty ester, 39 ferulate, 39 free, 39 source, 39 polyamine conjugates, 239 putrescines in, 239

sterols, 239 studies extraction yield. 395 fatty acid composition, 395 ferulate phytosterol ester (FPE), 397 free fatty acid, 395 phospholipids. 395 phytosterols, 397 tocols, 397 tocols cultivar differences, 36 dry milling, 36

447

Index germ oil tocopherols. 36 processing/fractionation. 36 refining process, 36 tocopherols, 36 wet-or dry-milling, 394 Corn distillers grain (CDG), 394-395 Corylus avellana L., see Hazelnut Cottonseed, balancing livestock rations, 285 Cranberry benefits, 158 consumption, 159 Helicobacter pylori, 159 peptic ulcers, 159 percentage scavenging capacities of. 159 phenolic profiles of. 159 Crude oat oil, 397 CUPRAC method, see Cupricion reducing antioxidant capacity (CUPRAC) method Cupricion reducing antioxidant capacity (CUPRAC) method antioxidant capacities. 368-372 chromogenic oxidizing reagent. 368 flavonoid glycosides, 369 with flavonoids-La(III) complexes, extractive separation determination, 369-370 lipophilic and hydrophilic antioxidants. measurement, 369 measurement, 370 phcnolics and flavonoids. hydroxyl radical scavenging assay, 370 reaction and chromophore, 368 Currant blackcurrant, 157 red currant anthocyanins and flavonols in. 158 Cydonia oblonga Mill., see Quince fruit 1) Date fruit, 98 bioactives in tannins, 99 coumaric acid. 100 Tantbouchlc and Deglet-Nour varieties, 99 Deoxynivalenol (DON), 258-259 Diabetes, cereal and cereal products clinical and observational studies, 14 dietary fiber, 13 glycemic index (GI), 13 Iowa Women's Health Study, 14 Multi-Ethnic Study of Atherosclerosis (MESA), 14 satiety and satiation, 13 whole grain, 14 DON, see Deoxynivalenol (DON) Dough, bioactives effect on antioxidants baking effects, 342 food matrix. 342 redox status and rhcology. 340-341 sorghum brans. 342 whole grain antioxidants and rheology, 341-342 © 2011 by Taylor & Francis G r o u p , L L C

dietary fiber, 338 problems, 338 rheology and product texture insoluble fiber components, 338-339 resistant starch, 340 soluble fiber components, 339 whole grain fiber, effects of, 339—340 Drupe fruits phenolic and beneficial bioactives in, 83 cherries, 84 sweet cherries, 84-85 tart cherries, 85-87 Dry bean antioxidant activity anthocyanins. 66 cyanidin, 66 delphinidin, 66 dietary antioxidants, 66 DPPH assay, 66 hydrophilic ORAC, 67 LAP and ORAC values, legumes, 66 legumes, 66 lipophilic antioxidant capacity (LAP), 67 phenolic acids contribution, 66 polymerization of phenols, 67 radical scavenging activity. 66 superoxide anion scavenging activity, 66 mean carotenoid values. 66 phenolic compounds antioxidants, 65 crude extracts, 66 flavonoid, 66 free content, 65 luteolin. 66 phenolic acids contents, 65 tannin content. 65 p-sitosterol, 66 tocopherol vitamin E activity, 65 Dry peas antioxidant activity cpicatcchin and condensed tannins. 64 lentils seed. 64 phenolics contents and antioxidant activities, 64 mataircsinol. 64 phenolic compounds beans. 63 catechins. 63 composition, 64 cotyledon. 63 ^-coumaric acid. 6 3 - 6 4 daidzein. 64 flavonoids, 63 genistein, 64 green split, 64 hydroxybenzoic acids, 63 hydroxycinnamic acids, 63 lentil seed coat. 64 peas, 63 phenol content, 64 phenolic acids, 63

448 proanthocyanidins. 63 procyanidin polymers, 63 prodelphinidins, 64 protocatechuic, 63 solvent extraction technique, 64 trans-fcrulic. 64 trans-resveratrol, 63 trimer procyanidins, 64 vanillic acid, 63 secoisolariciresinol, 64 tocopherol and carotenoids P-carotene, 63 bengal gram, 63 black gram. 63 green gram. 63 Y-tocopherol, 63 horse gram, 63 vitamin C. 64

i: Edible fruits, classification, 84 Elderberry anthocyanidin in, 160 antioxidant effects of, 161 cyanidin-3-glucoside, 161 cyanidin-3-sambubioside, 161 dark blue, 160 European elder. 160 Florida elder. 160 herbal medicine treatments. 161 Enterococcus casseliflavsus, see Olive fruits Ergot alkaloids, cereals with, 257 Ergot on wheat spikes. 278 Ergotoxine, cereals with. 256 Eriobotryajaponica Lindl., see Loquat fruit European plums. 89. 94

F Fatty acids in cereals, 5 Ferulate phytosterol ester (FPE). 397 FHB, see Fusarium head blight (FHB) Flavonoids anthocyanudins, 26 anthocyanins. 26 isolated from. 27 antioxidant activity, 27-28 anthocyanidins, 28 anthocyanins. 26-28 flavones, 28 flavonols. 28 hydroxyl groups, 27 isoflavones, 28 metal chelating activity. 28 radical scavenging activity, 27 C 6 - C 3 - C 6 configuration, 26 chemical structure, 4 family in food plants, 5 flavones and flavonols. 26 food antioxidants. 27 © 2011 by Taylor & Francis G r o u p , L L C

Index health benefits, 28-29 acyl CoA-cholesterol acyltransferase activity, 29 antiaromatase activity, 28 anticarcinogenic activities, 28 anti-inflammatory activities, 28-29 antitumor activities, 28-29 cytotoxicity, 28 hesperetin. 29 human breast carcinoma cells. 28 MCF7 human breast cancer cells. 29 naringenin. 29 prostaglandin production. 29 structure-activity relationship, 28 thromboxane synthase. 29 as hydrogen donors, 27 isoflavones in edible legumes, 27 isolated from cereals, pseudocereals and legumes, 26 metal chelators, 27 radical scavengers, 27 Fruit and cereal mycotoxigenic bioactives, 7 phytochcmicals in carotenoids, 5 lipids, 5-7 phenolics, 3-5 Fruits Actinidia deliciosa, 375 antioxidant capacities, 372 assays, 375 caffcic acid and qucrcctin. 375 CUPRAC test, 374 phenolic components, 375 sun-dried apricot and sulfited-dried apricot, 374-375 total phenolic content, 375 total antioxidant capacity (TAC) ABTS-TEAC assay, 367-368 antioxidant activity tests, 372 chromophores used in, 367 cupricion reducing antioxidant capacity (CUPRAC) method, 368-372 DPPH method, 368 ET-based assays. 365-366 FRAP assay, 368 HAT-based assays, 364-365 heat and physical processing and alkaline hydrolysis, 376-377 high molecular-weight polyphenols, 376 measurements in, 377-380 oxygen radical absorbancc capacity (ORAC) assay, 365 Trolox-equivalent antioxidant capacity (TEAC), 366-367 values, 380 Fruits and cereals, bioactive isolation anthocyanins content and composition, 324 extraction, purification and identification methods, 324-326 health function, 324 roles, 324

449

Index carotenoids. 321 extraction, purification and identification methods. 322-323 uses, 322 isofiavones extraction, purification and identification methods. 320-321 soybeans. 319-320 tocopherols and 7-oryzanol corn, 326 extraction, purification and identification

negative ion-mode extracted ES1-MS chromatograms, 416 Retro-Diels-Alder (RDA) rearrangement, 413 reversed-phase liquid chromatography, 416 hydroxycinnamic acids and derivatives caffeic acid and p-coumaric acid. 178 ferulic and sinapic acid. 178 2-S-glutathionylcaffeoyltartaric acid. 178 structures. 180 hydrozybenzoic acids, 177-178 structures, 179 matrix-assisted laser desorption/ionization (M ALDI) atmospheric pressure chemical ionization (APCI),417 (epi)catechin-(epi)catechin-malvidin-3-glucoside. fragmentation, 421 fullerenes matrix, 421-422 positive ions, mass spectra. 422-423 time of flight (TOF) analyzer, 417,420 nonflavonoids. 177 phenolic bioactives in, 171 sample preparation procedures, 410 spectrophotometric methods flavan-3-ols, determination, 410-411 Folin—Chiocalteu method. 410 p-dimethylaminocinnamaldehyde (/>-DM ACA), 410-411 stilbenes, 178-179 structures. 180 wine making and aging polyphenols evolution during, 180-181

methods, 327-329 rice, 326 structure, 327 vitamin E, 326 Fumonisin, cereals with. 256 Fusarium head blight (FHB) caused by F. graminearum, 254 trichotheccne mycotoxins, 254 wet weather conditions, 254

c; Gooseberry ascorbic acid. 162 phenolic acids, levels of, 161-162 Grapes and grape-based products anthocyanins acylated monoglucoside, 172 color, 172 dclphinidin and cyanidin. 172 maceration period. 172 malvidin, 172 3-0-glucosides, 172 petunidin and peonidin, 172 structures. 174-175 transformations, 175 UV-Vis and MS spectral data, 418-420 assay methods. 409 carbinol and chalcone forms, 172 enological practices, 172 flavan-3-ols catechin-and epicatechin-based polymers, 174 structures, 175-176 tannins and proanthocyanidins, 174 flavonoids HCA derivatives, 172 structure and flavonoid groups. 173 flavonolsanddihydroflavonols. 177 structures, 178 high performance liquid chromatography (HPLC) anthocyanins. structural identification and characterization. 415-417 chromatogram, 412. 414 flavan-3-ols, 411 flavonol hexosides. 413-414 liquid chromatography/mass spectrometry (LC/MS). 411.413 monoglucosides, UV-Vis spectra, 412 MS/MS analyses, 416-417 © 2011 by Taylor & Francis G r o u p , L L C

II Hazelnut; see also Nut bioactives neutral lipids, 193-194 phenolics, 192-193 phytosterols and tocols, 195 polar lipids, 194-195 Health promoting effects of cereal and cereal products, 9 bran antioxidant activity and dietary fiber content. 10 and cancer cereal grains in chemoprcvention, possible mechanisms, 11—12 epidemiological and intervention studies, 10—11 studies, 10 and cardiovascular disease (CVD) cereal bran wax components. 13 coronary heart disease (CHD) risk. 12 soluble fibers. 12 studies. 12 in obesity and diabetes clinical and observational studies. 14 dietary fiber. 13 glycemic index (GI), 13 Iowa Women's Health Study, 14 Multi-Ethnic Study of Atherosclerosis (MESA), 14 satiety and satiation. 13 whole grain, 14

450 sorghum antioxidant activity and dietary fiber content. 10 wheat grain antioxidant activity and dietary fiber content, 10 High pressure extraction (HPE), 432 High pressure processing (IIPP), 429 anthocyanins in grape, 439 antioxidant indices, 436 bacterial spores, 430 bioactive components, 431 antioxidant phenolics and activity, effects, 432-434, 437-439 citrinin contamination (CITcont). 438 high pressure extraction (HPE), 432 chamber vessel cylinder, 435 fresh oysters, 430 interior, actual and schematic diagram, 434 large-scale equipment, 432 Litchi Fruit Pericarp Tissues, 437 macromolecules, 430 operation principles. 431 pressure-assisted thermal sterilization, 430 ready-to-eat (RTE) meats, 430 research equipment, 431 smaller unit for laboratory research, 432 pressure treatments, 433 thermal and high pressure treatments effect of, 438 tomato puree lycopene standard and total lycopene content, 437 thermal (TP) and high pressure treatments, 439 total carotenoid, effect, 439 total phenols, effect, 439 Hordeum vulgare L., see Barley HPE, see High pressure extraction (HPE) HPP, see High pressure processing (HPP) I Isofiavones chemical structures, 321 extraction, purification and identification methods evaporation techniques and SPE, 320 MAE, 320 mixing and extracting procedure, 320 optimal liquid extraction, 320 phenol structures, 320 PLE, 320 SFE,320 UAE, 320 soybeans, 319 cardiovascular disease (CVD), 320 estrogenic effect, 320 importance, 320 mechanisms, 320

J Japanese flowering quince, 116 Japanese plums, 89 © 2011 by Taylor & Francis G r o u p . L L C

Index L Lipid soluble constituents and cereal grains, 6 Loquat fruit phenolics antioxidant capacity, 118 carotenoid profiles, 117-118 cultivars, 118 cyanidine glycoside, 117 hydroxycinnamic and benzoic acid, 117 total phenolic content, 119 vitamin A, 117-118

M Macadamia. 217; see also Nut bioactives neutral lipids. 218 phenolics, 218 phytosterols and tocols, 219 polar lipids, 219 Maize aflatoxin B, and aflatoxin G,, 278 antioxidant activity anlhocyanins, 40 blue and white corn cultivars, 40 carotenoid corn cultivar, 40 dietary source, 41 DPPH radical scavenging activity, 40 ferulic acid, 40 HCL-mclhanol solvent extraction protocol. 40 radical scavenging activity. 40 singlet oxygen quenching activity, 41 soluble phenolic contents. 40 M ALDI, see Matrix-assisted laser desorption/ ionization (MALDI) Malus domestica Borkh., see Apple Matrix-assisted laser desorption/ionization (MALDI) atmospheric pressure chemical ionization (APCI), 417 (epi)catechin-(epi)catechin-malvidin-3-glucoside, fragmentation, 421 fullcrcnes matrix, 421-422 positive ions, mass spectra, 422-423 time of flight (TOF) analyzer, 417, 420 Monophenols hydrogen abstraction. 23 hydrogen donation and radical scavenging activity, 23 monophenols tocopherol and tocotrienols, 22 Montmorency and Balaton tart cherries. 85 Mycotoxic bioactives in fruits and cereals, 7 accumulation, 275 analytical methods, 261 baking and extrusion, 260 barley malting, 260 kilning process, 260 steeping step, 260 black and protruding sclerotia. 277 corn milling. 260 economic consequences. 274 ergot, 276 formation causes, 275-276 fusaria, 276

451

Index immunological detection enzyme-linked immunosorbent assays (ELISA), 263 immunoaffinity column (1 AC), 263-265 immunoblotting techniques, 263 TaqMan probe mechanism. 264 inactivation assessments, 283-285 AFLATEST assay. 287 application of ammonia, 286 biological detoxification, 287 chlorine dioxide, 287 citric acid, 287 enzymatic inactivation, 288 extrusion cooking. 285-286 extrusion process, 285-286 feed additives, 286-287 interpretations on, 288-289 intrinsic and extrinsic factors, interaction between, 284 sulfhydryl compounds. 287-288 microbiological (culture) methods, 261 gaseous chromatography, 262 polymerase chain reaction (PCR) based methods, 262-263 structures and formation, 273-276 aflatoxins, 277-279 citrinin, 279 deoxynivalenol, 282 crgotaminc, 280 lumonisins. 280-281 ochratoxin, 279-280 patulin, 280 trichothecenes, 281 T-2 toxin, 282 type A trichothecene, 282 zearalenone, 283 worldwide distribution, 261 Mycotoxigenic fungi in cereals and cereal-based foods Aspergillus spp. aflatoxins and ochratoxin A (OTA). 254-255 chemical composition, 255 citrinin, bioactive toxins. 255 Fusarium spp. FHB, 254 trichothecene mycotoxins, 254 wet weather conditions and, 254 Penicillium spp. mechanical damage, 255 OTA, 255 Mycotoxins impact on nuts anatoxin content, 292-293 Aspergillus spores population, 293 damage from insects, 292 deficit irrigation, 294 fungicides and chemicals. 293 pistachio aflatoxin, 295-297 botanical information, 294 contaminated, sorting, 307-308 ecology of Aspergillus spp.. 308-309 harvest conditions, 301-303 © 2011 by Taylor & Francis G r o u p , L L C

irrigation, 298-299 kernel moisture, 299-301 nutrition, 299 processing and storage. 303-307 pruning, 298 species, rootstock. 297-298 temperature and relative humidity, 299-301 tree distance. 297 weather conditions, 297 pre-and postharvest influencing, 292 processing, 293 research and development. 293 N Navel orangeworm (NOW), 292 Nectarine bioactives in, 97-98 cultivars, 97 RAC values, 98 Nut bioactives; see also Mycotoxins impact on nuts almond neutral lipids, 189-191 phcnolics, 188-189 phytosterols and tocols, 191-192 polar lipids, 191 bioactive substances. 187 Brazil nut, 213 neutral lipids, 214 nutritional value, 214 phcnolics, 214 phytosterols and tocols, 215 polar lipids, 214-215 cashew neutral lipids. 216-217 phenolics, 216 phytosterols and tocols, 217 polar lipids, 217 clinical trials and epidemiological studies. 185-186 dietary fiber, 188 folic acid, 188 hazelnut neutral lipids, 193-194 phcnolics, 192-193 phytosterols and tocols, 195 polar lipids, 194-195 macadamia, 217 neutral lipids. 218 phenolics, 218 phytosterols and tocols, 219 polar lipids, 219 peanut/groundnut. 195 neutral lipids. 197 phenolics. 196-197 phytosterols and tocols, 197-198 polar lipids. 197 pecans neutral lipids. 220 phcnolics, 219-220 phytosterols and tocols, 220 polar lipids, 220

452 phenolic compounds, 188 phytosterols. 188 pinenut neutral lipids, 221-223 phenolics, 221 phytostcrols and tocols, 224 polar lipids, 223-224 pistachio chlorophylls and xanthophylls. 201-202 neutral lipids, 199-200 phenolics. 198-199 phytosterols and tocols, 201 polar lipids. 201 vitamin E. 188 walnut neutral lipids, 202-203 phenolics, 202 phytosterols and tocols, 203-205 polar lipids. 203

() Oats antioxidant activity carotene-linoleic oxidation model, 45 cultivar, 45 DPPH radical scavenging activity, 45 epicatcchin concentration, 45 tocols, 45 avenanthramides antiatherogenic. 42 anti-inflammatory activities, 42 antioxidant activity, 42 bioavailability, 42 collins, 42 concentrations, 42 content. 44 health benefits, 42 impact on, 44 phenolic content, 43 P-glucan content, 398 components, 41,44 crude oat oil. 397 delivering phytosterols, 4 4 fatty acid compositions. 397 IIEALTI1GRAIN projects, 238 lipids, 238 Nutrim-OB, 398 oat hull oil, 44 phenolics alkali concentration, 42 autoclaving, 42 caffeic acid, 41-42 content. 43 contents, 42 cultivar, 42 phenolic acid, 41 presence. 41 soluble esters, 41 phytosterol contents, 44 and tocopherols, 238 © 2011 by Taylor & Francis G r o u p , L L C

Index supercritical fluid technology, application, 397 tocols composition, 41 concentration, 41 processing, 41 source, 41 storage, 41 tocotrienol and tocopherol, 41 tocotrienols, 238 Obesity, cereal and cereal products clinical and observational studies, 14 dietary fiber, 13 glycemic index (GI), 13 Iowa Women's Health Study, 14 Multi-Ethnic Study of Atherosclerosis (MESA). 14 satiety and satiation, 13 whole grain. 14 Ochratoxins. cereals with. 257-258 Olive fruits composition and bioactive phenolics content benefits, 349 consumption, 349 development. 348 flesh, 349 platelet-activating factor activity, 350 pulp, 348 texture, 348 oil processing impact on phenolics cultivars, 354 Lactobacillusplantarum, appliction of, 354-355 natural antioxidants, 353 nutritional, biological and organoleptic value, 354 phenols. 353 postharvest effects on phenolics features, 350 mature-green olives, 350 microbial invasion. 350 Spanish method. 350 storage of, 350 table processing effect on phenolics fermentation, 351 flesh. 352 Greek style method. 351 hydroxytyrosol concentration, 353 inoculation, 351 Spanish and Californian style, 351 survival studies, 353 y-Oryzanol chemical structures, 329

corn, 326 extraction, purification and identification methods ascorbic acid, 327 BHT, 327 Folch extraction. 327 gamma-oryzanol. 328 GC-flame ionization detection (FID), 328 lipophilic compounds, 327 normal-phase HPLC, 328 online L C - G C method, 329 pyrogallol, 327 soxhlet extraction. 327

Index SPE, uses, 328 Zorbax reversed-phase column, 328 rice, 326 structure of, 327 vitamin E, 326 Oryza saliva L., see Rice

P Peach bioactives in, 97-98 cultivars, 97 RAC values, 98 Peach twig borer (PTB), 292 Peanut/groundnut. 195; see also Nut bioactives neutral lipids, 197 phenolics, 196-197 phytosterols and tocols, 197-198 polar lipids, 197 Pear phenolics amount, 116 arbutin chemical structure, 114 contents, 115 flavonol glucosides, 116 hydroxycinnamic acid esters, 116 in juice. 116 procyanidins, 115 profiles, 117 Pearl millet, 242 Pecans; see also Nut bioactives neutral lipids, 220 phenolics, 219-220 phytosterols and tocols. 220 polar lipids, 220 Penicillium spp. in cereals, 255 Penicillum verrucosum, Ochratoxin A in, 7 Pennisetum glaucum, L., see Pearl millet Phenolic acids aleurone, 23 antioxidant activity, 23 P-coumaric acid. 23 in cereals, pscudoccrcals and legumes, 23-24 fcrulic acid. 23-24 intramolecular hydrogen bonding of ortho substituted phenols, 24 monohydroxy phenolic acids, 23 pericarp, 23 radical acceptor, 23 second hydroxyl group, 23 tocols, 23 Phenolic compounds family of, 4 Phoenix dactylifera, see Date fruit Phytochemical bioactives in berry fruits bay berry, 156 Chinese cultivars, 155 Dongkui, 154 hyphenated technique of HPLC. 154 phenolics in. 155 Xiangshan and Biqi, 154 © 2011 by Taylor & Francis G r o u p , L L C

453 blackberry anthocyanin and phenolics in, 145 anthocyanin-containing extract (ACE), 144 cyanidin-3-glucoside, 144 ferric reducing antioxidant power (FRAP), 144 used, 144 yalova, 145 blueberry anthocyanins and total phenolics in. 147 benefits, 145 blueberry bioactives, 146-147 in Eastern Black Sea Region, Turkey, 146 Ericaceae and Vaccinium (V), 145 in United States, 146 V. Angustifolium, 146 V. ashei, 146 V. corymbosum, 145 V. Mynilloides, 146 chokeberry aronia berries. 156 contents of flavonones, 157 dark color of. 157 extracts of, 156 raw fruits of, 156 cranberry benefits, 158 consumption, 159 Helicobacter pylori, 159 peptic ulcers. 159 phenolic profiles, 159 scavenging capacities, 159 currant. 158 blackcurrant. 157 red currant. 157 elderberry antioxidant effects of, 161 cyanidin-3-glucoside, 161 cyanidin-3-sambubioside. 161 dark blue, 160 European elder. 160 Florida elder, 160 herbal medicine treatments. 161 special anthocyanidin in. 160 gooseberry ascorbic acid. 162 phenolic acids, levels of, 161-162 raspberry black raspberry, 152-153 red raspberry, 147-152 Rosaceae family, 147 strawberry anti neurodegenerative biological properties, 153 ellagic acid in, 153-154 Phytochemicals, 62 alkylresorcinols and alkenylresorcinols, 25-26 anticarcinogenic, 25 antimicrobial. 25 1.3-dihydroxybcnzene. 25 metabolites, 25 peroxy radical scavenging. 25 avenanthramides, 29

454 carotenoids. 29-30 carotene, 29 cryptoxanthin, 29 lutein, 29 polyene system, 29 xanthophylls. 30 zeaxanthin, 29 citrus fruit, bioactivity 2,2-diphenyl-l-picrylhydrazyl (DPPH), 130 ferric reducing antioxidant power (FRAP), 130 methoxylated flavones, 130 oxidation of LDL, 131 phenolic acids, 130 scavenging activity, 130 DPPII radical scavenging, 134 essential oils, role of, 134 flavonoids, 26-27 antioxidant activity. 26-27 health benefits, 28-29 fruit and cereals carotenoids, 5 functional and lipid soluble constituents. 5-7 phenolics, 3-5 health benefits, 132-133 citrus flavonoids, 132 cyclooxygenase-2 (COX-2), inhibition of, 132 7,12-dimethylbenz[a]anththracene (DMBA), 133 Epstein-Barr virus early antigen (EBV-EA), 133 flavonoids, antiprolifcration activity, 132 health promoting properties. 132 and limonoids, 132 methoxylated flavones and limonoids of, 132 naringenin of, 132 scavenger receptors (SR-A), 132 LDL antioxidant testing. 134 monophenols and phenolic acids citrus fruits, 124-125 phenolic acids, 23-25 tocopherols and tocotrienols, 22-23 tropical fruits, 125 oryzanols, 29 phytostcrols, 30 antioxidant activity, 30 blood cholesterol, 30 fatty acid. 30 fcrulate esters, 30 free sterols, 30 oil polymerization prevention, 30 phenolic compounds. 30 phenolic esters. 30 phytosanols, 30 steryl glycosides, 30 policosanols, 29 pulses dry beans, 6 3 - 6 4 dry peas, 65-67 tropical fruit, bioactivity discrepancies, 131 ellagic acid, 132 nonionic resin, 131 ORAC values, 131 © 2011 by Taylor & Francis G r o u p , L L C

Index quercetin and gallic acid, 132 radical scavenging ABTS activity, 131 in vivo bioactivity, 134 Pinenut; see also Nut bioactives neutral lipids, 221-223 phenolics, 221 phytosterols and tocols, 224 polar lipids, 223-224 Pinus pinea, see Pinenut Pistachio; see also Nut bioactives chlorophylls and xanthophylls, 201-202 mycotoxins impact on aflatoxin, 295-297 botanical information, 294 contaminated, sorting. 307-308 ecology of Aspergillus spp., 308-309 harvest conditions. 301-303 irrigation, 298-299 kernel moisture, 299-301 nutrition, 299 processing and storage, 303-307 pruning, 298 species, rootstock, 297-298 temperature and relative humidity, 299-301 tree distance, 297 weather conditions, 297 neutral lipids. 199-200 phenolics, 198-199 phytostcrols and tocols, 201 polar lipids, 201 Plant polyphenols, 361 Plum and prune anthocyanins. 90 average individual amount. 94 cyanidin-3-glucosides and cyanidin 3-rutinosidcs. 93 cyanidin rutinoside and peonidin rutinoside, 94 pigmentation in, 93 antioxidant activity of, 96 bioactives in, 89 biological functions, 96 catechins and proantocyanidins, 93 chlorogcnic acid and derivatives, 90 cultivars, 89 dehydrating. 97 flavan-3-ol catcchin. 90, 92 flavonol glycosides. 92 flavonol rutin. 90 genotypes, 89 hydroxycinnamates. 90 ncochlorogcnic acid, 90 Prunus Breeding Program. 96 Prunus domestica and Prunus salicina, 91 quercetin glycosides in, 91-92 red-fleshed plum, 95 rutin. 91 total phenolic contents, 95 Polyphenolics flavanones capillary electrophoresis, 126 hesperidin, 126 narirutin levels, 127

455

Index flavonoids distribution, 125 anthocyanins, 126 in citrus fruits, 127 cyanidin glucosides, 126 flavones and flavonols, 126 hcspcridin. 126 limes and lemons, 126 narirutin. 126 in sweet oranges and tangerines, 126 tangerine flavanones profile. 126 flavonols polymethoxylated flavones nobiletin and tangeretin, 128 quercetin-3-rhamnoside, 127 phenolic compounds flavanols and xanthones, 128 procyanidin levels, 128 Polyphenols as plant secondary metabolites, 3 subgroups of, 4 Polyunsaturated fatty acids (PUFAs), 393 Pome fruits bioactive phytochemicals in apple, 107-114 loquat, 117-119 pear, 114-116 quince, 116-117 Processing effects bioactivcs recovery einnamic and benzoic acids, optimal extraction conditions, 134 flavanone glycosides, 134 molasses, 133 polymethoxylated flavones, 133 bitterness reduction in citrus, 133 adsorption, 133 deacidification. 133 enzymatic hydrolysis. 133 Provitamin A activity, 5 Prunus armeniaca L., see Apricot fruit Prunus cerasus, see Tart cherries Prunus domesticus L., see Plum and prune; plum and prune Prunus dulcis, see Almond Pscudocereals amaranth and quinoa P-carotene bleaching assays, 62 DPPH and ABTS radical scavenging, 62 grain, 62 health and antioxidant activities, 62 lipid lowering activities, 62 low gluten foods. 61 oil, 62 pscudocereals, 61 tannic. 62 tocopherols, 62 antioxidant activity DPPH radical scavenging activity, 61 LDL peroxidation, 61 radical scavenging activity in ORAC test, 61 © 2011 by Taylor & Francis G r o u p , L L C

buckwheat antioxidants, 60 cultivated species, 59 phytochemicals. 59 contents, 62 phenol ics acetone extract. 60 buckwheat, 60 contents, 60 dried buckwheat, 60 solvent extraction. 60 tartary buckwheat, 60 tocopherol, 61 Pseudocydonia sinensis, see Chinese quince PUFAs, see Polyunsaturated fatty acids (PUFAs) Pyrus communis L., see Pear

Q Quince fruit, 116 K Raspberry, 151 black raspberry, 153 chemopreventive potential, 152 cyanidin-3-glucoside, 152 cyanidin-3-rutinoside, 152 cyanidin-3-sambubioside, 152 cyanidin-3-xylosylrutinoside, 152 familial adenomatous polyposis (FAP). 152 frcczc-dricd powder. 152 pharmacokinetic studies, 152 in United States, 148 red raspberry, 147-148, 151-152 "ahududu" or "framboise," 149 anthocyanins in, 150 importantance, 149 in Turkey, 149 Rosaceae family, 147 yellow raspberry in Turkey, 149 raspberry, 147-153 Red-fleshed plum, 95 Red raspberry anthocyanins. 150 ellagitannins ellagic acid. 151 lambcrtianin C, 151 levels, 150-152 SanguiinH-6, 151 flavonols, 152 Rheology and product texture in dough antioxidants cysteine, effects of, 340 disulfide cross-linking. 340 redox status, 340 spring-back, 341 transglutaminase, uses of, 341-342 insoluble fiber components. 338-339 resistant starch advantages. 340

456 soluble fiber components, 339 whole grain and commercial hydrocolloids, 340 effect of, 339-340 sodium stearoyl lactylate (SSL), 340 Rice alkaloid antioxidant isolated from pigmented rice, 45 antioxidant activity crude methanol extract, 49 cultivars. 49 DPPH radical, 49 extracts of rice bran, 49 phenolic content, 48 phytostcrol content. 49 radical scavenging activity, 47 reducing antioxidant power, 49 sitostanyl ferulate, 49 superoxide radical scavenging activities, 49 tocopherol, 49 bran, 45 carotenoid levels, 47 chemical structure of oryzadinc, 240 F. verticillioides and F. graminearum, 254 health benefits, 45 monoacylglycerols and free fatty acids, 239 oleic acid and linoleic acid, 239 palmitic acid, 239 phenol ics black rice cultivars, 47 degradation, 47 level, 47 phenolic acid reduction, 47 pigmented rice cultivars, 47 xylanasc, 47 phospholipids and glycolipids, 239 phytosterols content, 47 stearic acid, 239 tocols and oryzanols alkaloid antioxidant isolated. 45 bran oil, 46 composition, 45-47 dcodorization. 47 y-oryzanol, 46 levels, 4 6 solvent extraction protocols, 46 stabilization of bran. 46 tocotrienols, 46 triacylglycerols and diacylglycerols. 239 unsaponifiable matter, 240 wax esters, 239 Rice bran extraction yield, 400 fatty acid composition. 400 oryzanols, 398, 400 tocols, 398,400 Rye alkylresorcinols, 53 acetone and methanol solvent, 54 alkylenylresorsinol content, 53 composition, 53-54 contents, 53-54 © 2011 by Taylor & Francis G r o u p , L L C

Index cultivar, 53-54 extraction. 53-54 fermentation, 53-54 level, 53 loss during fermentation, 54 alkylresorcinols content. 400-401 antioxidant activity ABTS radical scavenging activity, 54 DPPH radical scavenging activities, 54 extracts and effectiveness, 54-55 radical scavenging activity, 54 campestanyl ferulate and sitostanyl ferulate, 237 compounds, 49 consumption, 49 linoleic acid, 237 rx-linolenic acid, 237 lipids, 237 oleic acid and palmitic acid, 237 phenolic changes, 52 composition, 50, 52 contents, 50, 52 cultivars, 50-52 fermentation. 52 ferulic acid dehydrodimers (diFA), 50 flour extraction rate, 52 pericarp. 52 phenolic acids, 50 rye bran and whole rye comparison. 50 trends. 52 phytosterols, 237 alkylresorcinols. 52-53 content. 52-53 fractionation process, 53 pearling, 53 steryl ferulate levels, 53 plant sterol, 237 squalcne, 237 steryl ferulates and tocopherols. 237 tocols, 237 (3-tocotrienols. 50 cultivar, 50 ct-tocotricnols, 49 tocotrienols and alkylresorcinols, 237

s Secale cereale, L., see Rye Sorghum, 240 alcohols, acids and aldehydes. 242 lipids, 241 structures of triterpene alcohols, 241 tocopherols, 241 Sorghum bicolor [Monech] L., see Sorghum Strawberry antineurodegenerative biological properties, 153 ellagic acid in, 153-154 ellagitannin in, 154 in United States, 153 Supercritical fluid extraction (SFE) carbon dioxide and propane, 386

Index

cereal lipids, industrial process implementation plant, operating costs, 404 steps, 4 0 3 - 4 0 4 of cereals amaranth grain, 393-394 barley, 394 carotenoids, 393 corn, 394-395, 397 oats, 397-398 polyunsaturated fatty acids (PUFAs), 393 rice, 398, 400 rye, 400-401 sterol esters, 393 sterols, 393 tocols, 393 wheat, 401,403 of corn oil, 396 critical parameters, 386 density, 387 flow diagram, 392 liquid feed height equivalent to theoretical plate (11ETPS), 393 separation steps and regeneration of solvent, 392 process description, 387-388 processing parameters moisture, 392 particle size/shape, 391-392 pressure, 390 separation parameters. 390 solubility of compounds. 390 solvent/feed ratio. 391 solvent flow rate, 391 P-T diagram of pure substance. 386 separation of soluble compounds basic schemes for, 388 solid matrix, compounds cap automation mechanism, 389 cascade operation of multiple extraction, schematic diagram. 390 flow diagram, 388-389 vapor-liquid equilibrium line. 386 water, 386 of wheat germ oil, 402 Sweet cherries. 84-85 polyphenolic compounds in, 86

T TAC, see Total antioxidant capacity (TAC) Tart cherries anthocyanins in, 85 L-value of, 86-87 polyphenols. 85. 87 stone fruit family, 85 TEAC, see Trolox-equivalent antioxidant capacity (TEAC) Terpen alcohol, 5-6 Tocopherols. 5 chemical structures, 329 corn. 326 © 2011 by Taylor & Francis G r o u p , L L C

457

extraction, purification and identification methods ascorbic acid, 327 BHT, 327 Folch extraction. 327 gamma-oryzanol. 328 GC-flame ionization detection (FID), 328 lipophilic compounds. 327 normal-phase HPLC, 328 online L C - G C method, 329 pyrogallol, 327 soxhlet extraction, 327 SPE, uses, 328 Zorbax re versed-phase column, 328 rice, 326 structure of. 327 vitamin E, 326 Tocopherols and tocotricnols phenolic hydrogen, 22-23 radical scavenging. 22—23 tocols, 22 vitamin E and antioxidant activities. 22 Tocotrienols, 231 TOSC assay, see Total oxyradical scavenging capacity (TOSC) assay Total antioxidant capacity (TAC) in cereals ABTS-TEAC assay, 367-368 antioxidant activity tests, 372 chromophorcs used in, 367 cupricion reducing antioxidant capacity (CUPRAC) method, 368-372 DPPH method, 368 ET-bascd assays, 365-366 FRAP assay, 368 H AT-based assays, 364-365 heat and physical processing and alkaline hydrolysis, 376-377 high molecular-weight polyphenols, 376 measurements in, 377-380 oxygen radical absorbance capacity (ORAC) assay, 365 Trolox-cquivalcnt antioxidant capacity (TEAC), 366-367 values, 380 in fruits ABTS-TEAC assay, 367-368 antioxidant activity tests, 372 chromophores used in, 367 cupricion reducing antioxidant capacity (CUPRAC) method, 368-372 DPPH method, 368 ET-based assays, 365-366 FRAP assay, 368 HAT-based assays, 364-365 heat and physical processing and alkaline hydrolysis, 376-377 high molecular-weight polyphenols, 376 measurements in, 377-380 oxygen radical absorbance capacity (ORAC) assay, 365

458

Index

Trolox-equivalent antioxidant capacity (TEAC), 366-367 values, 380 Total oxyradical scavenging capacity (TOSC) assay, 378 Trichothecene mycotoxins, 254 cereals with. 258 structure, 259 diacetoxyscirpenol and IIT-2 toxin, 262 T-2 toxin and neosolaiol, 262 Trilicum aestivum L., see Wheat Trolox-equivalent antioxidant capacity (TEAC), 366-367 Tropical fruits antioxidant activity 2,2'-azino-bis-(3-cthylbcnzthiazolinc-6-sulfonic acid) radical scavenging (ABTS) activity, 131 discrepancies, 131 nonionic resin, 131 OR AC values, 131 carotenoids, 129-130 analytical methods, 130 a-and P-carotenes, 129 Ataulfo mango cultivar, 130 lutein levels, 129 chlorogenic acid analogues, 125 phenolic acids of, 125 phytochemicals bioactivity ellagic acid. 132 monophenols and phenolic acids, 125 qucrcctin and gallic acid. 132

V Volatile fungal metabolites, 262 Vranec wines UV and MS spectral data, 415 \V Walnut; see also Nut bioactives neutral lipids, 202-203 phenol ics, 202 phytosterols and tocols, 203-205 polar lipids, 203 Wheat alkylresorcinols, 236 antioxidant activity DPPI1 and ABTS radical scavenging activities, 58 fcrulated oligosaccharide, 59 LDL oxidation, 59 linoleate oxidation. 59 ORAC radical scavenging activities, 58 pearling percentage, 59 primarily radical scavenging, 58 separation of aleurone. 58 Ultraflo L, 59 white spring durum cultivar, 58 p-carotene, 57 P-cryptoxanthin, 57 bioactive compounds, 401 biotic and abiotic factors within. 236—237 eampestanyl fcrulale and sitostanyl fcrulatc, 236 © 2011 by Taylor & Francis G r o u p . L L C

carotenoids. 236. 403 concentrations, 57 extraction yield, 401 fatty acid composition. 401 genotypes, 235 germ oil, 401 grain quality, 235 lipids, 235 relative distribution, 236 lutein and lutein esters. 57, 236 milling. 57 phenolic acid, 403 phenol ics p-gluconasc. 55 composition. 55-57 content. 56 cultivar, 57 ferulic acid and/>-coumaric acid. 55 phospholipids, 401 phytosterol content, 57, 235 policosanols. 236 soft wheat cullivars. 57 stanols content, 57 sterol contents, 235 sterols content, 57, 403 studies. 401 tocols, 401, 403 p-tocopherols, 55 germ fraction, 55 y-tocophcrols, 55 milling fractions, 55 a-tocopherols. 55 tocopherols, 236 wheat bran oil, 57 zeaxanthin, 57 Wickson plums, 94 Wine phenolic bioactives anlhocyanins UV-Vis and MS spectral data. 418-420 assay methods for, 409 high performance liquid chromatography (HPLC) anthocyanin monoglucosidcs. UV-Vis spectra, 412 anthocyanins, structural identification and characterization, 415—417 chromatogram. 412. 414 flavan-3-ols. 411 flavonol hexosides, 413-414 liquid chromatography /mass spectrometry (LC/MS), 411.413 MS/MS analyses, 416-417 negative ion-mode extracted ESI-MS chromatograms, 416 Retro-Diels-Alder (RDA) rearrangement, 413 reversed-phase liquid chromatography. 416 making and aging polyphenols evolution during. 180-181 matrix-assisted laser desorption/ionization (M ALDI) atmospheric pressure chemical ionization (APCI), 417 (epi)catechin-(epi)catechin-malvidin-3-glucoside. fragmentation. 421 fullerenes matrix, 421-422

Index positive ions, mass spectra, 422-423 time of flight (TOF) analyzer, 417, 420 sample preparation procedures, 410 spectrophotometric methods flavan-3-ols. determination, 410-411 Folin-Chiocaltcu method, 410 /)-dimethylaminocinnamaldehyde (/i-E 410-411

© 2011 by Taylor & Francis G r o u p , L L C

459 X Xanthophylls, 5

z ),

Zea mays, L., see Corn/maize Zearalenone. cereals with. 259