Flour and Breads and Their Fortification in Health and Disease Prevention 0128146397, 9780128146392

Table of contents :
Content: Section 1: Introductory Chapters 1. Deamidation of Gluten Proteins as a Tool for Improving the Properties of Bread 2. Polycyclic Aromatic Hydrocarbons (PAHs) in Flour, Bread, and Breakfast Cereals 3. A Review of Adulteration Versus Authentication of Flour 4. The Fate of Alternaria Toxins in Wheat-Processing Chain 5. Organophosphorus pesticides (OPPs) in Bread and Flours Section 2: Flours and Breads Section 2.1: Monotypes 6. Flour and Bread From Black, Purple, and Blue-Colored Wheats 7. Emmer (Triticum turgidum ssp. dicoccum) Flour and Bread 8. Nutritional, Technological, and Health Aspects of Einkorn Flour and Bread 9. Maize: Composition, Bioactive Constituents, and Unleavened Bread 10. Amaranth: Potential Source for Flour Enrichment 11. Sorghum Flour and Flour Products: Production, Nutritional Quality, and Fortification 12. Banana and Mango Flours 13. Macadamia Flours: Nutritious Ingredients for Baked Goods Section 2.2: Bread Types 14. Sourdough Breads 15. Brewer's Spent Grain From By-Product to Health: A Rich Source of Functional Ingredients 16. Effect of Addition of Thermally Modified Cowpea Protein on Sensory Acceptability and Textural Properties of Wheat Bread 17. Bread Packaging: Features and Functions Section 2.3: Composite Flours and Breads 18. Nixtamalized Maize Flour By-product as a Source of Health-Promoting Ferulated Arabinoxylans (AX) 19. Chestnut and Breads: Nutritional, Functional, and Technological Qualities 20. Passiflora edulis Peel Flour and Health Effects Section 3: Fortification of Flours and Breads Section 3.1: Addition of Micronutrients 21. Micronutrient Fortification of Flours-Developing Countries' Perspective 22. Effects of Phytochemical Fortification of Flour and Bread on Human Health 23. Soybean-Fortified Wheat Flour Tortillas 24. Protein-Selenized Enriched Breads 25. Soybean-Fortified Nixtamalized Corn Tortillas and Related Products 26. Trends in Science of Doughs and Bread Quality Section 3.2: Addition of Macronutrients 27. Barley ss-Glucans and ss-Glucan-Enriched Fractions as Functional Ingredients in Flat and Pan Breads 28. Fortification of Bread With Soy Protein to Normalize Serum Cholesterol and Triacylglycerol 29. Resistant Starch (RS) in Breads: What It Is and What It Does 30. Flours Based on Exotic Fruits and Their Processing Residues-Features and Potential Applications to Health and Disease Prevention Section 4: Metabolic Responses to Flour and Bread Fortification 31. Dietary Breads and Impact on Postprandial Parameters 32. Folic Acid and Colon Cancer: Impact of Wheat Flour Fortification With Folic Acid 33. Effects of the Soybean Flour Diet on Insulin Secretion and Action 34. Flour Fortification and the Prevention of Neural Tube Defects (NTDs) 35. Minor and Ancient Cereals: Exploitation of the Nutritional Potential Through the Use of Selected Starters and Sourdough Fermentation 36. Quinoa Flour as an Ingredient to Enhance the Nutritional and Functional Features of Cereal-Based Foods 37. Faba Bean Flour to Improve Nutritional and Functional Features of Cereal-Based Foods: Perspectives and Future Strategies 38. The Glycemic Index: What It Is and How It Can Be Applied to Retinal Health 39. Wheat Flour Fortification to Prevent Iron-Deficiency Anemia

Citation preview

FLOUR AND BREADS AND THEIR FORTIFICATION IN HEALTH AND DISEASE PREVENTION

FLOUR AND BREADS AND THEIR FORTIFICATION IN HEALTH AND DISEASE PREVENTION Second edition Edited By

VICTOR R. PREEDY RONALD ROSS WATSON

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2019 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-814639-2 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Megan Ball Editorial Project Manager: Susan Ikeda Production Project Manager: Nilesh Kumar Shah Cover Designer: Mark Rogers Typeset by SPi Global, India

Contributors

Marijana Acˇanski

University of Novi Sad, Novi Sad, Serbia

Edith Agama-Acevedo

Instituto Polit
ecnico Nacional, Centro de Desarrollo de Productos Bióticos, Yautepec, Morelos, Mexico

Mohamed A. Ahmed School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland; Department of Food Technology, Faculty of Engineering and Technology, Sebha University, Sebha, Libya Saeed Akhtar Institute of Food Science & Nutrition, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan, Pakistan Rosario Alonso-Domı´nguez Biomedical Research Institute of Salamanca (IBSAL), Primary Care Research Unit, La Alamedilla Health Center, Castile and Leon Health Service (SACYL), Spanish Network for Preventive Activities and Health Promotion (redIAPP), Department of Nursing and Physiotherapy, University of Salamanca, Salamanca, Spain Joseph O. Anyango

Department of Dairy, Food Science and Technology, Egerton University, Egerton-Njoro, Kenya

Franklin B. Apea-Bah

Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada

Vanessa Cristina Arantes Ahmad Arzani Iran

Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil

Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan,

Mohamad F. Aslam Gladys Barrera

Department of Nutritional Sciences, King’s College London, London, United Kingdom

Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile

Emma Beckett School of Medicine & Public Health, The University of Newcastle and Hunter Medical Research Institute; School of Environmental & Life Sciences, The University of Newcastle, Newcastle, NSW, Australia Luis A. Bello-P
erez Instituto Polit
ecnico Nacional, Centro de Desarrollo de Productos Bióticos, Yautepec, Morelos, Mexico Sarah E. Berry Department of Nutritional Sciences, King’s College London, London, United Kingdom Trust Beta

Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada

Andrea Brandolini CREA—Research Centre for Animal Production and Aquaculture, S. Angelo Lodigiano, Italy Daniel Bunout Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Alma C. Campa-Mada Lydia Campbell

Research Center for Food and Development, CIAD, Sonora, Mexico

School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland

Elizabeth Carvajal-Millan

Research Center for Food and Development, CIAD, Sonora, Mexico

Pasquale Catzeddu Porto Conte Ricerche Srl, Alghero (SS), Italy Emma Chiavaro

Department of Food and Drug, University of Parma, Parma, Italy

Cristina Elizabeth Chuck Herna´ndez Sciences, Monterrey, M
exico Rossana Coda

Tecnológico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and

Department of Food and Environmental Science, University of Helsinki, Helsinki, Finland

Priscila da Costa Rodrigues Mestrado em Nutric¸ão, Alimentos e Metabolismo, Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil Chaiane Aline da Rosa Mestrado em Nutric¸ão, Alimentos e Metabolismo, Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil Marise Auxiliadora de Barros Reis Maria Pia de la Maza Peter R. Ellis

Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil

Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile

Department of Nutritional Sciences, King’s College London, London, United Kingdom

Ays¸e Naciye Erbakan Department of Internal Medicine, Nisa Hospital, Istanbul, Turkey Stephen R. Euston

School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland

Adriana S. Franca

Mechanical Engineering Department, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

xi

xii

CONTRIBUTORS

Javier Gonza´lez-Sa´lamo Departamento de Química, Unidad Departamental de Química Analítica, Facultad de Ciencias, Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Spain Daniela Guardado-F
elix Tecnológico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and Sciences, Monterrey; Biotechnology Postgraduate Regional Program, Faculty of Chemical and Biological Sciences, Autonomous University of Sinaloa, FCQB-UAS, Culiacan, Sinaloa, M
exico Elizabet Jani
c Hajnal

Institute of Food Technology, University of Novi Sad, Novi Sad, Serbia

Mehmet Hayta Erciyes University, Faculty of Engineering, Department of Food Engineering, Melikgazi, Kayseri, Turkey Erick Heredia-Olea M
exico

Tecnológico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and Sciences, Monterrey,

Javier Herna´ndez-Borges Departamento de Química, Unidad Departamental de Química Analítica, Facultad de Ciencias, Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Spain Antonio V. Herrera-Herrera Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Spain Alyssa Hidalgo

Department of Food Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy

Sandra Hirsch Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Majid Hussain Institute of Food Science & Nutrition, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan, Pakistan Elif Meltem I˙s¸c¸ imen Erciyes University, Faculty of Engineering, Department of Food Engineering, Melikgazi, Kayseri, Turkey Tariq Ismail Institute of Food Science & Nutrition, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan, Pakistan Marta S. Izydorczyk

Grain Research Laboratory, Canadian Grain Commission, Winnipeg, MB, Canada

Siwaporn Jitngarmkusol

Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

Sibel Kacmaz Faculty of Engineering, Department of Food Engineering, Giresun University, Giresun, Turkey Hitomi Kumagai Department of Chemistry and Life Science, Nihon University, Fujisawa-shi, Japan Ma´rcia Queiroz Latorraca Gladys O. Latunde-Dada

Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil Department of Nutritional Sciences, King’s College London, London, United Kingdom

Michele Cristiane Laux Mestrado em Nutric¸ão, Alimentos e Metabolismo, Faculdade de Nutric¸ão, Universidade Federal de Mato Grosso, Cuiabá, Brasil Marco A. Lazo-V
elez

University of Azuay, Food Engineering Program Research Strategic Groups (GEICA-UDA), Cuenca, Ecuador

Laura Leiva Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Wende Li Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada Glaucia Cariello Lima

Nutrition School-Federal University of Goias, Goi^ ania, Brazil

Mark Lucock School of Environmental & Life Sciences, The University of Newcastle, Newcastle, NSW, Australia Ma´rio Roberto Maro´stica Junior Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, São Paulo, Brazil Jorge Marquez-Escalante

Research Center for Food and Development, CIAD, Sonora, Mexico

Jasna Mastilovi
c Institute of Food Technology, University of Novi Sad, Novi Sad, Serbia Tricia McMillan Grain Research Laboratory, Canadian Grain Commission, Winnipeg, MB, Canada Ilkem Demirkesen Mert Ankara, Turkey Banu Mesci

Republic of Turkey Ministry of Food, Agriculture and Livestock, Food Enterprises and Codex Department,

Department of Internal Medicine, Goztepe Training and Research Hospital, Medeniyet University, Istanbul, Turkey

Marco Montemurro

Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

Dejan Orcˇi
c Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad, Novi Sad, Serbia Maria Paciulli

Faculty of Sciences, Department of Food and Drug, University of Parma, Parma, Italy

Antonella Pasqualone Kristian Pastor

Department of Soil, Plant and Food Sciences, University of Bari ‘Aldo Moro’, Bari, Italy

University of Novi Sad, Novi Sad, Serbia

Rita Paz-Samaniego

Research Center for Food and Development, CIAD, Sonora, Mexico

Esther Perez-Carrillo Tecnológico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and Sciences, Monterrey, M
exico Erica Pontonio Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

CONTRIBUTORS

Alessandro Pugliese

xiii

Department of Food and Drug, University of Parma, Parma, Italy

Agustı´n Rascon-Chu

Research Center for Food and Development, CIAD, Sonora, Mexico

Jos
e I. Recio-Rodrı´guez Biomedical Research Institute of Salamanca (IBSAL), Primary Care Research Unit, La Alamedilla Health Center, Castile and Leon Health Service (SACYL), Spanish Network for Preventive Activities and Health Promotion (redIAPP), Department of Nursing and Physiotherapy, University of Salamanca, Salamanca, Spain Laı´s M. Resende Food Science Graduate Program, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Massimiliano Rinaldi Department of Food and Drug, University of Parma, Parma, Italy Carlo Giuseppe Rizzello

Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

Cristina M. Rosell Department of Food Science, Institute of Agrochemistry and Food Technology, Spanish Scientific Research Council (CSIC), Valencia, Spain Natalia Sa´nchez-Aguadero Biomedical Research Institute of Salamanca (IBSAL), Primary Care Research Unit, La Alamedilla Health Center, Castile and Leon Health Service (SACYL), Spanish Network for Preventive Activities and Health Promotion (redIAPP), Department of Nursing and Physiotherapy, University of Salamanca, Spain Sergio O. Serna-Saldivar Monterrey, M
exico Paul A. Sharp

Tecnológico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and Sciences,

Department of Nutritional Sciences, King’s College London, London, United Kingdom

Khetan Shevkani Department of Applied Agriculture, Central University of Punjab, Bathinda, India Narpinder Singh

Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India

Sandeep Singh

Department of Food Science and Technology, Khalsa College, Amritsar, India

Prabhjeet Singh

Department of Biotechnology, Guru Nanak Dev University, Amritsar, India

Darryl M. Small Australia

School of Science, Applied Chemistry and Environmental Science Discipline, RMIT University, Melbourne, VIC,

Ba´rbara Socas-Rodrı´guez Departamento de Química, Unidad Departamental de Química Analítica, Facultad de Ciencias, Universidad de La Laguna (ULL), San Cristóbal de La Laguna, Spain Norberto Sotelo-Cruz

Department of Medicine, University of Sonora, Sonora, Mexico

Valentina Stojceska Centre for Sustainable Energy in Food Chains, Brunel University London, College of Engineering, Design and Physical Sciences, Uxbridge, United Kingdom William R. Sullivan VIC, Australia

School of Science, Applied Chemistry and Environmental Science Discipline, RMIT University, Melbourne,

Kanitha Tananuwong John R.N. Taylor

Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

Department of Consumer and Food Sciences, University of Pretoria, Pretoria, South Africa

Reiko Urade Institute for Integrated Radiation and Nuclear Science, Kyoto University, Osaka, Japan Michela Verni

Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

Amardeep Singh Virdi Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India Djura Vuji
c University of Novi Sad, Novi Sad, Serbia Milena Morandi Vuolo

Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, São Paulo, Brazil

K€ ubra Yıldız Department of Nutrition and Dietetics, Faculty of Health Sciences, Medeniyet University, Istanbul, Turkey

C H A P T E R

1 Deamidation of Gluten Proteins as a Tool for Improving the Properties of Bread Hitomi Kumagai*, and Reiko Urade† †

*Department of Chemistry and Life Science, Nihon University, Fujisawa-shi, Japan Institute for Integrated Radiation and Nuclear Science, Kyoto University, Osaka, Japan

O U T L I N E Introduction

3

Methods for Deamidation Acid/Alkali Enzyme Anion/Cation-Exchange Resin

4 4 5 6

Functions Changed by Deamidation Physicochemical Functions Physiological Functions

6 6 7

References

9

Abbreviations HMG glutenin high-molecular-weight glutenin subunit pI isoelectric point RAST radioallergosorbent test SDS sodium dodecyl sulphate TGase transglutaminase WDEIA wheat-dependent exercise-induced anaphylaxis

INTRODUCTION Wheat gluten formed from gliadin and glutenin, two major types of protein, plays a critical role in bread-making.1–3 Regarding the rheological properties of bread dough, gliadin contributes to its viscosity, whereas glutenin contributes to its elasticity and strength. Gliadin is a typical monomeric prolamin protein that is soluble in 60%–90% aqueous alcohol, whereas glutenin is a large polymer comprised of high- and low-molecular weight subunits cross-linked with disulfide bonds that is alcohol-insoluble but soluble in diluted acid or alkaline solutions. The physical and chemical properties of gluten proteins, including molecular weight, gliadin:glutenin ratio, and degree of primary amidation, are reportedly factors that can affect baking quality.4–9 Furthermore, the poor solubility and emulsification of gluten proteins under most food-processing conditions often preclude the addition of these proteins to a variety of foods. Physiologically, it is well known that gliadin is the principal allergen that induces serious allergic reactions in susceptible individuals. Allergic manifestations of reactions to gliadin include atopic eczema/dermatitis syndrome (AEDS), which often occurs in children,10–12 and wheat-dependent, exercise-induced anaphylaxis (WDEIA), which mainly occurs in adults.13,14 Certain tandem sequencing sites with glutamine residues of ω-5 gliadin and a high-molecular-weight glutenin subunit (HMW glutenin), which are the most potent antigens for WDEIA, have been identified as the primary structure of immunoglobulin E (IgE)-binding epitopes.15–17 One approach to mitigating the allergenicity of gluten proteins is to chemically modify the proteins by alkylation, acylation, or deamidation. Alkylation and acylation are sometimes used to change the functionality of proteins.18

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00001-0

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© 2019 Elsevier Inc. All rights reserved.

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1. DEAMIDATION OF GLUTEN PROTEINS AS A TOOL FOR IMPROVING THE PROPERTIES OF BREAD

FIG. 1 Deamidation of protein. An amide group in the side chain of glutamine or asparagine is converted to carboxyl group by typical deamidation. As a result, glutamine or asparagine is converted to glutamic acid or aspartic acid. TABLE 1 Percentages of Acid-Amidic and Acidic Amino Acids in Food Proteins Glutamine

Asparagine

Glutamic acid

Aspartic acid

Wheat gliadin

33.8

2.6

1.9

0.4

Soy 11S globulin

10.4

8.6

7.6

3.8

Milk αs1-casein

8.3

4.3

10.1

2.4

However, these modifications require chemicals that are not allowed in food intended for human consumption. Conversely, deamidation, a reaction that converts glutamine and asparagine residue to glutamic-acid and aspartic-acid residue, respectively (Fig. 1), can be achieved under mild conditions that are approved for food processing. Consequently, protein deamidation is recognized in the food industry as a method to improve the functionality of proteins in food systems.19–21 Deamidation is often applied to cereal and pulse proteins because the percentage of glutamine and asparagine residue in these proteins is higher than the corresponding levels in animal proteins. Notably, approximately one-third of the amino acids in wheat gliadin are comprised of glutamine residue (Table 1). There is ample evidence suggesting that deamidation improves solubility and surface properties. Also, physiological functions such as allergenicity and calcium bioavailability can be altered by deamidation. The popular methods for protein deamidation include acid, enzyme, and cation-exchange-resin treatment. A review of the current deamidation methods and the resulting functional improvements are presented in this chapter.

METHODS FOR DEAMIDATION Acid/Alkali Among the established chemical deamidation methods, treatment with an acid is the most popular.22–39 Deamidation under acidic conditions causes the direct hydrolysis of an amide to a carboxylate group.40 To this end, the amide in asparagine residues is much more amenable to deamidation than the amide in glutamine residues.41 Furthermore, conducting the reaction in an acid solution at a high temperature achieves a high degree of deamidation, but a certain level of peptide-bond hydrolysis inevitably occurs, which produces bitter-tasting peptides and reduces processing properties such as gelation and solubility. The degree of protein deamidation in an acid solution increases as the temperature increases. For example, up to 90% of wheat gluten was deamidated following the addition of 1.0 M hydrochloric acid (HCl) solution at 95°C for 30 min, whereas only up to 30% was deamidated in 1.0 M HCl solution at 50°C for 30 min.22 Although HCl is the most common acid used, organic acids such as acetic, malic, tartaric, and citric acid can also be used for deamidation. Wheat gluten was deamidated to approximately 60% in 0.034–0.133 M acetic acid at 121°C for 15 min, which was similar to the deamidation level observed in 0.055–0.137 M HCl at 121°C for 15 min.33 However, the degree of deamidation observed at the 0-min treatment time was already quite high (24%–52%), which might be explained by the 12-h reaction time, which is required to release ammonia from the deamidated samples during the determination of the deamidation level. Therefore, the actual degree of deamidation by acetic acid might be much lower. Although the degree of deamidation was not measured, wheat gluten was deamidated in 0.082 M acetic acid at 121°C for 1. INTRODUCTORY CHAPTERS

METHODS FOR DEAMIDATION

5

15 min, and various properties were compared with those of unmodified, succinylated, and deamidated and succinylated gluten.39 Wheat gluten was deamidated in 1.6 M malic acid, 0.8 M tartaric acid, or 1.0 M citric acid at up to 30% at 70°C for 20 h, which was equivalent to the deamidation level observed in 0.08 M HCl.38 Alkali deamidation is less popular than other methods,27,42,43 likely because a cross-linking reaction resulting in the formation of lysinoalanine occurs readily under alkaline conditions,44 reducing the nutritional value due to the loss of lysine and of protein-processing properties due to polymerization. Both lysinoalanine and lanthionine production increase with elevations in the reaction temperature from 50°C to 90°C, as well as with increases in the sodium hydroxide (NaOH) concentration from 0 to 1.5%. In addition, the formation of an intramolecular cyclic imide intermediate that produces β-carboxyl linkage occurs in alkali deamidation more predominantly than direct hydrolysis.40 The presence of anions enhances the deamidation rate of proteins. For example, the deamidation of soy protein at 100°C for 3 h in a pH 8.0 bicarbonate and phosphate solution enhanced deamidation levels up to 40% and 25%, respectively. However, the addition of acetate, sulfate, and chloride anions was ineffective.45 Sodium dodecyl sulfate (SDS), an anionic surfactant, also increases deamidation rates. The degree of cottonseed protein deamidation in 0.4 M HCl heated to 70°C for 5 h increased from 30% to 100% when SDS was increased from 0 to 0.08 M.46 These anions may promote the deamidation rate via a catalytic function induced by heating or HCl because proteins can be deamidated to a certain degree just by heating47–49 or by treatment with acid.

Enzyme As previously noted, chemical deamidation often causes undesired modifications, such as the cleavage of peptide bonds; however, deamidation by the addition of enzymes is an alternative method that does not cause peptide-bond hydrolysis. Enzymatic deamidation has reportedly been achieved using protease, transglutaminase (TGase), peptidoglutaminase, and protein glutaminase. Kato et al. developed a method to deamidate food proteins by treatment with proteases at pH 10.50,51 The authors noted that approximately 20% of the asparagine or glutamine residue in ovalbumin, lysozyme, and soy proteins (7S globulin and 11S globulin) were deamidated by treatment with papain, pronase, and chymotrypsin without (or with only slight) proteolysis. In addition, chymotrypsin immobilized on controlledpore glass was effective at deamidating ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten at pH 10 and 20°C. The percentages of deamidated ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten were 10.0, 8.4, 6.0, 5.0, and 8.0, respectively. In addition, the larger molecular-weight fractions of soy proteins and gluten dissociated into smaller molecular-weight fractions on SDS-polyacrylamide gels, suggesting the occurrence of partial peptidebond cleavage.52 TGase (glutaminyl-peptide: amine γ-glutamyltransferase, EC 2.3.2.13) is an enzyme that catalyzes amineincorporating and cross-linking reactions via the transfer of an acyl group between a γ-carboxyamide of the peptide-bound glutamine and a primary amine and/or lysyl side chain. In addition, TGase catalyzes the deamidation of glutamine residues. The TGases derived from blood plasma and erythrocytes and from Streptomyces mobaraensis are used in commercial products, as these enzymes can be produced in larger quantities. Generally, cross-linking will dominate the deamidation reaction. However, if the protein contains a large number of glutamine residues and only a few lysine residues, such as in gliadins and glutenins, water will react as a nucleophile, resulting in the deamidation of glutamines.53 In a structural analysis, Mazzeo et al. determined that 19 of 94 glutamine residues present in recombinant α-gliadin were converted to glutamic-acid residues by the addition of guinea-pig liver tissue TGase.54 The experiments were conducted on a recombinant α-gliadin, and on a panel of 26 synthetic peptides, overlapping the complete protein sequence by the use of advanced matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) and tandem MS methodologies. Bacillus circulans peptidoglutaminase (EC 3.5.1.43) is well-known enzyme that catalyzes the hydrolysis of glutamine residues in peptides.55 The isolated enzyme consists of two distinct isozymes: peptidoglutaminase I and II. Furthermore, the peptidoglutaminases are dimers comprised of subunits with molecular weights of 42 kDa and 51 kDa, and isoelectric point (pI) values of 4.1 and 4.0, respectively.56,57 Peptidoglutaminases are located intracellularly. Peptidoglutaminase I deamidates a C-terminal glutamine residue, and peptidoglutaminase II deamidates an internal glutamine residue, as well as a C-terminal residue. The peptidoglutaminases are inactive against higher-molecular-mass peptides and proteins.58, 59 Also, the activity of peptidoglutaminase is generally limited to the deamidation of glutamine residue on the short peptide chain; therefore, the deamidation of native casein and whey proteins by peptidoglutaminase is minimal.59 Hamada et al. investigated the deamidation of modified food proteins by peptidoglutaminase and found that prior proteolysis and disruption of the compact structure of intact proteins are required to increase protein deamidation.60 Therefore, a new enzyme that catalyzes the deamidation of native proteins without the requirement for pretreatment is desirable. 1. INTRODUCTORY CHAPTERS

6

1. DEAMIDATION OF GLUTEN PROTEINS AS A TOOL FOR IMPROVING THE PROPERTIES OF BREAD

Protein glutaminase (EC 3.5.1.44) is a monomer protein with a pI of 10.0 and a calculated molecular weight of 19,860, which catalyzes the deamidation of native high-molecular-weight proteins and lacks transglutaminase activity. This enzyme was first identified in the culture supernatant from Chryseobacterium proteolyticum, and the gene encoding it was subsequently cloned.61,62 Protein glutaminase was then de novo synthesized as a pre-pro-protein with a putative signal peptide of 21 amino acids and a pro-sequence of 114 amino acids. The amino-acid sequence had no apparent homology to that of peptidoglutaminase. The solution structure of protein glutaminase was determined by nuclear magnetic resonance (NMR).63 Hashizume et al. refined the crystal structures of the mature and pro forms of the enzyme expressed in Escherichia coli at 1.15 and 1.73 Å resolutions, respectively.64 The overall structure of the mature enzyme is homologous to the core domain of human transglutaminase-2 (factor XIII-like TG). Protein glutaminase contains a catalytic triad (Cys-His-Asp) located in the bottom of the active site pocket. The proposed catalytic mechanism of protein glutaminase is based on the structures of a pro-enzyme mutant containing two different reaction intermediates. Most important, the catalytic efficiency (kcat/Km) of protein glutaminase is higher during the deamidation of native proteins than of short peptides. The substrate specificity of protein glutaminase is restricted to glutamine residue in peptides. Consequently, asparagine residues, free glutamine, or other amides in peptides are not deamidated by the enzyme. It has been shown that the protein glutaminase can efficiently deamidate a variety of proteins, such as gluten, gliadin, α- and β-caseins, zein, and α- and β-lactalbumins without denaturing the proteins or proteolytic pretreatment.65–67 In the case of water-insoluble gluten, 72% of the total glutamine residue was deamidated at pH 7 and 40°C for 30 h. Changes in the secondary structure of gluten by deamidation has been revealed by Fourier transform infrared analysis, showing decreases in both the intermolecular and intramolecular β-sheets compared to nondeamidated gluten.

Anion/Cation-Exchange Resin Although enzymes such as protein glutaminase are available for protein deamidation, enzymes do not act on asparagine residue. Moreover, the enzymes cannot be used for gliadins and glutenin dissolved in aqueous alcohol because they are inactivated in an alcohol solution. Kumagai et al. developed a technique for the effective deamidation of proteins without causing any detectable peptide-bond hydrolysis using cation-exchange resins containing carboxylate.68–74 Cation-exchange resins with anionic groups such as sulfonate or carboxyl groups are effective for promoting deamidation. Heating soy protein to 85°C for 24 h in 0.05 M HCl enhanced the degree of deamidation from 30% to 70% by the addition of cation-exchange resins containing sulfonate anions.75 Cation-exchange resins are also efficient at deamidating proteins in a buffered solution at around neutral pH, as well as in an ethanol solution. Soy protein was deamidated up to 73% in 0.05 M Tris-HCl (pH 7.4) at 4°C by mixing with cation-exchange resins containing carboxyl anions for 6 h.70 No observable peptide-bond hydrolysis occurred under these conditions, and the degree of deamidation by the cation-exchange resin with carboxyl anions was approximately double the rate achieved using sulfonate anions. The deamidation of wheat gliadin was 28% in 60% ethanol at 4°C for 15 h in the presence of cationexchange resin containing carboxyl anions without any peptide-bond hydrolysis, which was likewise almost double the degree of deamidation achieved using sulfonate anions.68,69 Therefore, this method is applicable to ethanol-soluble proteins such as wheat gliadin and corn zein. To clarify the mechanism of action, the affinity of soy protein for the carboxyl group was analysed in various solutions using surface plasmon resonance (SPR) technology.76 The results indicated that the deamidation level and the amount of protein bound to the polymer-supported carboxylates were well correlated. Both the deamidation level and the affinity of soy protein for the carboxyl groups of the SPR chips were highest in 0.05 M phosphate at pH 6.0, but they decreased as the sodium concentration in a pH 10 solution increased. In addition, the deamidated soy protein did not bind to the carboxyl groups of the SPR chips. These results indicated that deamidation by cation-exchange resin occurs via the binding of the amide in the glutamine and asparagine residues in proteins with the carboxyl groups of the resin, and that the binding was affected by the sodium ion concentration in the solution.

FUNCTIONS CHANGED BY DEAMIDATION Physicochemical Functions Numerous studies have been conducted to evaluate physicochemical functions such as the solubility and surface properties of deamidated proteins. These functions are of great importance to the deamidation of proteins in processed foods. Wheat gliadin deamidated by acid,25, 26 transglutaminase,77 or carboxylated cation-exchange resins,69 as well as

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FUNCTIONS CHANGED BY DEAMIDATION

7

wheat gluten deamidated by acid,25 chymotrypsin,52 or protein glutaminase65 became highly soluble in water or a buffered solution at around neutral and alkaline pH compared with untreated proteins. However, although the dispersibility of wheat gluten deamidated by acid continued to increase up to the 10% deamidation level, it decreased when the deamidation level exceeded 10%.22 Further, the affinity of wheat gliadin and soy globulin for water was enhanced by deamidation with the use of carboxylated cation-exchange resins.68,78 The increase in the affinity for water could explain the improvement in the gelling properties and water-holding capacity of soy globulin deamidated without any detectable peptide-bond hydrolysis. The surface properties of a protein often change following deamidation because the carboxyl groups produced by deamidation affect the hydrophobic-hydrophilic balance of the protein. The emulsifying properties of wheat gliadin deamidated by transglutaminase77 and of wheat gluten deamidated by various acids (0.08 M HCl, 1.6 M malic acid, 0.8 M tartaric acid, or 1.0 M citric acid)38 or protein glutaminase65 were higher than the properties observed in undeamidated wheat gliadin and gluten, respectively. Acid deamidated wheat protein exhibited high emulsifying properties.30 However, the improved emulsifying properties might not be attributable to deamidation alone because in addition to deamidation, peptide-bond hydrolysis occurs following acid treatment. The foaming properties of wheat gliadin and gluten were enhanced by deamidation with carboxylated cationexchange resin.69 Both the emulsifying and foaming properties of wheat gliadin and gluten deamidated by 0.1 M HCl25 and wheat gluten deamidated by 0.1 N HCl or H2SO4,26 0.082 M acetic acid,33, 39 or chymotrypsin52 were higher than the corresponding properties observed in undeamidated proteins. The surface hydrophobicity of wheat gluten was enhanced by deamidation with 0.082 M acetic acid or 0.137 M HCl,33 which might be one of the reasons for the improvement in surface properties by deamidation. Deamidated gluten exhibited improved gelation properties,79 similar to deamidated soy globulin, which formed gel with no syneresis.78 The deamidation of wheat gluten by 0.08 M HCl or 0.8 M tartaric acid decreased the presence of β-sheets and slightly increased the appearance of random coils.38 These enhancements in physicochemical properties following deamidation could expand the usage of wheat proteins.

Physiological Functions Deamidation also affects the physiological functions of proteins. Soy globulin that has been deamidated by carboxylated cation-exchange resin after the removal of phytate, an inhibitor of calcium absorption, exhibited improved calcium-binding properties and enhanced calcium absorption when it was injected into the ligated loop of the small intestine with a calcium solution.72 Oral administration of phytate-depleted, deamidated soy globulin to young male rats lowered parathormone levels, indicating the promotion of calcium absorption from the small intestine and increased γ-carboxylated-type osteocalcin levels, which affect bone mineral density and mechanical bone strength and promote femur bone formation.74 Ovariectomy decreases the rate of calcium absorption from the small intestine and promotes bone resorption, which leads to reduced bone mineral density and induces osteoporosis. However, oral administration of the phytate-depleted, deamidated soy globulin restored the reduced calcium-absorption rate and enhanced bone mineral density and bone strength.80 The phytate-depleted, deamidated soy globulin was more effective at preventing osteoporosis than casein, which produced casein phosphopeptides (CPPs) that enhance calcium absorption in the small intestine during digestion. Wheat allergies account for approximately 30% of total food allergies81 and are serious because they cause severe anaphylactic reactions, and it is not likely that affected individuals will outgrow the allergy. The most common type of wheat allergy is WDEIA, an immediate hypersensitive reaction caused by wheat intake and physical exercise. The major WDEIA allergens are ω-5 gliadin and HMW glutenin, both of which have abundant repeat motifs containing glutamine residue, and some of these glutamine-rich tandem amino-acid sequences are the IgE-binding epitopes for WDEIA. The identified IgE-binding epitope sequences in gliadin include QQPFP, PQQPF,82 QQIPQQQ, QQLPQQQ, QQFPQQQ, QQSPEQQ, QQSPQQQ, QQYPQQQ, PYPP,15 QQFHQQQ, QSPEQQQ, YQQYPQQ, and QQPPQQ83 (Fig. 2), while those in HMW glutenin subunits are QQPGQ, QQPGQGQQ, and QQSGQGQ.83 Therefore, deamidation is effective at reducing allergenicity. Deamidation of gliadin by carboxylated cation-exchange resins effectively reduced its reactivity with the sera of patients that were radioallergosorbent test (RAST)-positive to wheat (Fig. 3).73 Although deamidation offers apparent benefits for overcoming wheat allergies, HCl-treated wheat protein could cause potential transcutaneous sensitization. Facial soap containing HCl-treated wheat protein-induced wheat allergies in individuals with no prior allergic symptoms. However, not all HCl-treated wheat proteins cause transcutaneous sensitization.36 Only HCl-treated wheat proteins in the 15,000–250,000 or higher-molecular-weight range reacted with the sera of patients, indicating that only the large polypeptide aggregates caused transcutaneous sensitization.

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1. DEAMIDATION OF GLUTEN PROTEINS AS A TOOL FOR IMPROVING THE PROPERTIES OF BREAD

IgE epitopes of wheat ω5 gliadin. (A) Amino-acid sequences of IgE epitopes found in ω-5 gliadin. (B) The primary structure of a representative ω-5 gliadin (accession number: BAE20328). IgE epitopes are underlined.

FIG. 2

FIG. 3 Reactivity of gliadin before and after deamidation with IgE in the sera of patients who test RAST-positive to wheat. The immunoreactivity of patient IgE to untreated or deamidated gliadin was evaluated. Briefly, each gliadin was fixed to a well in a microtiter plate, and serum from each patient RAST-positive to wheat was added and incubated. The bound IgE was quantified using a biotinylated goat antihuman IgE antibody.

Because the manufacturing process for the HCl-treated wheat protein used in the soap is confidential, the reason underlying why large polypeptide aggregates were produced by HCl treatment is unknown. In contrast to common wheat allergies in which the major allergens include ω-5 gliadin and HMW glutenin, IgE antibodies from these transcutaneous-sensitized patients bound to α/β-, γ-, and ω1,2-gliadin.84 Although the QPQQPFPQ sequence in γ-gliadin was identified as an IgE-binding epitope, deamidated peptides such as PEEPFP bound more strongly to the IgE antibodies from the patients.37, 84 However, when gliadin that was deamidated by carboxylated cationexchange resins, deamidated and hydrolyzed by HCl, or hydrolyzed by a protease was each applied to the skin of rats for several weeks, followed by the oral administration of gliadin, only the HCl-treated gliadin caused allergic reactions (unpublished data). Therefore, at least the combination of deamidation and hydrolysis is necessary to induce transcutaneous sensitization. The reason why HCl-treated wheat protein with large polypeptide aggregates caused transcutaneous sensitization requires clarification in future research. The improvement in the digestibility of wheat gliadin following deamidation is likely due to the increase in water solubility, which simplifies the hydrolysis of proteins in the stomach and small intestine via digestive enzymes.73 Enhanced digestibility is expected to promote a reduction in allergenicity due to the hydrolysis of the IgE-binding epitope structure by digestive enzymes. When sensitized to allergens, inflammation can promote the absorption of large molecules like proteins from the small intestine, which increases allergen levels in the blood. The absorption of allergens into the blood promotes the formation of allergen-specific IgE and the expression of FcεRI, a high-affinity IgE receptor, on the surface of mast cells. Allergic reactions subsequently occur, causing the release of histamines when the IgE epitopes of allergens are bound to IgE that is cross-linked to FcεRI. The oral administration of deamidated gliadin showed low allergenicity, suppressing the enhancement in intestinal permeability, serum allergen levels, allergen-specific IgE levels, mast-cell-surface expression of FcεRI, and serum and intestinal histamine levels.85

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In addition, deamidated gliadin-induced oral tolerance in a mouse model of wheat allergy,86 indicating the possibility of using deamidated gliadin to treat and potentially cure wheat allergies. Finally, the marked reduction in allergenicity following deamidation and the induction of oral tolerance might facilitate the ability to prepare bread and cake that patients allergic to wheat can consume.

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C H A P T E R

2 Polycyclic Aromatic Hydrocarbons (PAHs) in Flour, Bread, and Breakfast Cereals Sibel Kacmaz Faculty of Engineering, Department of Food Engineering, Giresun University, Giresun, Turkey

O U T L I N E Introduction

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Legislation on PAHs

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Analysis Methods

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Level of PAHs in Flour, Bread, and Breakfast Cereals

16

Conclusion

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References

19

INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are a complex group of chemical compounds present in the environment and foods. They are formed and released via the pyrolysis or incomplete combustion of organic materials (e.g., garbage, wood, gasoline, oil products and coal), and during industrial food processes such as smoking, frying, drying, baking, roasting, and charcoal barbecuing/grilling. Humans are exposed to PAHs mostly by food intake and environmental pollution.1–7 In the past decade, the occurrence, sources, toxicity, exposure, and carcinogenicity of PAHs have been extensively evaluated by major organizations, such as the European Food Safety Authority (EFSA),8 the Scientific Committee on Food (SCF),9 the International Programme on Chemical Safety (IPCS),10 the Joint FAO/WHO Expert Committee on Food Additives (JECFA),11 the International Agency for Research on Cancer (IARC),12 and the U.S. Environmental Protection Agency (EPA).13 The EPA13 selected 16 PAH compounds that are most commonly found in environmental samples.14–16 These 16 PAHs are identified in Table 1 as “EPA PAHs.” The EFSA8 evaluated data gathered by its member-states and then prioritized 15 PAH compounds as being toxicologically significant.17–19 The SCF,9 of the European Commission, finalized that these 15 PAH compounds are of serious concern for human health, and primarily suggested monitoring them in food.20 Moreover, the SCF proposed benzo[a]pyrene (BaP) as a suitable indicator for the occurrence and effect of carcinogenic PAHs in food, based on examinations of PAH profiles and evaluation of a carcinogenicity study.1,2 The IARC12 confirmed the toxicological importance of these PAH compounds. It also announced that these compounds can be regarded as possibly carcinogenic and genotoxic for humans, and thus they should be a priority in the assessment of the risk of long-term adverse health effects after PAH intake via eating food. The IARC has classified those PAH compounds as possible human carcinogens (group 2B), probable human carcinogens (group 2A), and carcinogenic to humans (group 1); it also said that other PAH compounds can behave as synergists21,22 (see Table 1).

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00002-2

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© 2019 Elsevier Inc. All rights reserved.

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2. POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN FLOUR, BREAD, AND BREAKFAST CEREALS

TABLE 1

PAHs Assessed by the Leading National Authorities and Their Carcinogenicity

Full name

Abbreviation

MW

PAH4b

PAH8c

EU priorityd PAHs

EPAe PAHs

IARC groupf

Benzo[a]pyrene

BaP

252

✓

✓

✓

✓

1

Chrysene

CHR

228

✓

✓

✓

✓

2B

Benz[a]anthracene

BaA

228

✓

✓

✓

✓

2B

Benzo[b]fluoranthene

BbF

252

✓

✓

✓

✓

2B

benzo[k)fluoranthene

BkF

252

✓

✓

✓

2B

indeno[1,2,3-cd]pyrene

IP

276

✓

✓

✓

2B

dibenz[ah]anthracene

DBahA

278

✓

✓

✓

2A

benzo[ghi]perylene

BghiP

276

✓

✓

✓

3

benzo[j]fluoranthene

BjFA

252

✓

2B

cyclopenta[c,d]pyrene

CPP

226

✓

3

dibenzo[al]pyrene

DBalP

302

✓

2A

dibenzo[ae]pyrene

DBaeP

302

✓

3

dibenzo[ai]pyrene

DBalP

302

✓

2B

dibenzo[ah]pyrene

DBahP

302

✓

2B

5-methylchrysene

MCH

242

✓

2B

benzo[c]fluorene

BcFL

216

✓

3

Pyrene

Pyr

202

✓

3

Phenanthrene

P

178

✓

3

Naphthalene

N

128

✓

2B

Fluorene

F

166

✓

3

Fluoranthene

Fl

202

✓

3

Anthracene

Ant

178

✓

3

Acenaphthylene

Acy

152

✓

Not assessed

Acenaphthene

Ace

154

✓

3

a

a b c d f e

International Union of Pure and Applied Chemistry (IUPAC) names. PAH4; the four EU marker PAHs proposed as the most suitable indicators of occurrence of PAHs in food by the European Union. PAH8; the eight EU marker PAHs proposed as the most suitable indicators of occurrence of PAHs in food by the European Union. 15 PAH compounds that the European Union suggested for monitoring. Classifications according to IARC (2010)21. Overall evaluation of carcinogenicity to humans: 1, carcinogenic; 2A, probably carcinogenic; 2B, possibly carcinogenic; 3, not classifiable. EPA PAHs; 16 PAHs that proposed to monitor by the EPA.

For BaP and other carcinogenic/genotoxic PAHs in foodstuffs, further monitoring has been requested by the European Commission (EC).23 Since then, the scientific opinion of the EFSA17 via Commission Regulation (EU) No. 835/201120,24 has underlined that BaP cannot only be a good marker for the occurrence and effect of the carcinogenicty of PAHs in foodstuffs, eight compounds (PAH8) were selected as the most appropriate indicators [PAH8; chrysene, benzo[k]fluoranthene, dibenz[a,h]anthracene, benz[a]anthracene (BaA), BaP, indeno[1,2,3-cd]pyrene (IP), benzo[b]fluoranthene (BbF), and benzo[ghi]perylene (BghiP)]. However, in comparison with the four PAH compounds [PAH4; i.e., chrysene (CHR), BaA, BaP, and BbF], PAH8 does not give much added value.17,18 Based on assessments conducted by the EFSA,8 IARC,12 SCF,9 and European Commission (EC),25 a set of the PAH4 compounds was identified as being markers for total PAH content in foodstuffs reported in Commission Regulation (EU) No. 835/2011.20,26 In general, they are known as “the four EU marker PAHs.” These prioritized PAHs are listed in Table 1.

1. INTRODUCTORY CHAPTERS

ANALYSIS METHODS

15

LEGISLATION ON PAHs The 16 EPA PAH compounds have been given a fairly high priority for environmental monitoring, but they have not been included in U.S. food legislation up to now, and as far as we know, no maximum limits have been set for PAHs in foods. However, there are only a limited number of regulations on the breathing, eating, or drinking of PAHs in the United States. But there are regulations specifying a valid maximum limit for BaP levels in drinking water (i.e., 0.2 μg/L) by the EPA13 in the Safe Drinking Water Act. Another limit was set on coal products (enforceable exposure limit of 0.2 mg/m3 air averaged over an 8-h period of work place exposure) based on evaluations of exposure by the Occupational Safety and Health Administration (OSHA) and the National Institute for Occupational Safety and Health (NIOSH).27, 28 The European Union (EU) shows too much concern and priority for the contamination of PAHs in foodstuffs. Thus, it has created legislation that sets maximum limits (MLs) for chemical contaminants in foodstuffs. The current maximum limits for PAHs in food are issued in Commission Regulation (EU) No. 835/2011,20 which amended Regulation (EC) No 1881/200624 for key foodstuffs (e.g., smoked meat and smoked meat products, smoked fish and fishery products, muscle meat of smoked fish, cocoa beans and derived products, infant formula, oils, fats, and processed, cerealbased food and baby food). According to this legislation, maximum limits established for these key food commodities include 35 μg/kg for cocoa beans and derived products; 30 μg/kg for smoked meat and other meat products; 10 μg/kg for oils and fats, fish, and fishery products; and 1 μg/kg for baby foods and dietary foods for the sum of four EU marker PAHs (ΣPAH4; PAH4; i.e., CHR, BaA, BaP, and BbF). For baked, packaged bread and breakfast cereals, although the consumption rate has gradually been increasing, the maximum limits of PAHs in these food products had not been established before this. Commission Regulation (EU) No. 835/2011 emphasizes that flour, bread, and cereals contain a pretty low level of PAHs. It also represented that current data for the levels of PAHs in flour, bread, and cereals are restricted and do not allow for the urgent setting of maximum levels to be determined with this data.20 In conclusion, Commission Regulation (EU) No. 835/2011 showed that bread and cereals have major effects on consumer health because of their high consumption, and recommended further monitoring of PAH levels in these product groups.17,18 However, on the basis of further reliable data about the level of PAHs in bread and cereals, the need for setting maximum levels will be evaluated by European Commission (EC).

ANALYSIS METHODS A number of analysis techniques have been described in the literature for some of the suitable PAHs in food commodities (e.g. flour, bread, and cereals), and they can be used through the combination of various experimental approaches.29 However, studies for new and more sensitive techniques for the determination of PAHs in these food commodities are ongoing. Basically, there are two main steps for PAH analysis in food samples: sample preparation, which comprises homogenization, extraction, and cleanup; and instrumentation, which comprises the separation, identification, and quantification of PAHs. Sample preparation is important and necessary prior to instrumental analysis for the isolation of target PAHs from solid food samples such as flour, bread, and cereals. The first stage of sample preparation is homogenization, where solid food samples are ground into a fine powder for better and easier extraction. It also can be useful for the solid samples that have been deep-frozen by milling under cooling with liquid nitrogen. Homogenization can be done by putting these food commodities into a grinding mortar and/or blender. Following homogenization of solid foods such as flour, bread, and cereals, PAHs can be extracted using a number of techniques and solvents before cleanup and measurement are done.30,31 The extraction of PAHs from these foodstuffs can be performed via an optimal extraction process with an appropriate solvent, such as pressurized liquid extraction (PLE) with n-hexane32, ultrasonic bath extraction with n-hexane33,34 or (hexane/acetone, 60:40, v/v),35 liquid-liquid extraction (LLE) with ethanolic solution of potassium hydroxide (KOH) (2 mol/l),36 ultrasound-assisted solvent extraction with n-hexane37, soxhlet extraction with hexane/dichloromethane (DCM)38 or n-hexane,39 reflux saponification extraction with KOH in a water-ethanol mixture (1:9, v/v),40 focused-microwave-assisted extraction utilizing aggregates of the IL 1-hexadecyl-3-butylimidazolium bromide (HDBIm-Br),41 and sonication extraction by ethyl ether–methylene chloride (1 ∶ 1).42 After the extraction stage, the cleanup process should be carried out to isolate PAHs from the extraction solution. A convenient cleanup process for PAH isolation prior to measurement is solid-phase extraction (SPE) on silica,32–34 gel

1. INTRODUCTORY CHAPTERS

16

2. POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN FLOUR, BREAD, AND BREAKFAST CEREALS

permeation chromatography (GPC),35 column chromatography on aluminum oxide (Al2O3),36 sep-pack silica cartridges,37 soaked silica,38 saponification,39 and silica-gel column chromatography.40 The second main step (and the most important one) is instrumentation. There are two main analytical techniques available for identification and quantification of PAH compounds: gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with ultraviolet (UV)/fluorescence detection (FLD). Gas chromatography (GC) can be used with flame ionization detection (FID),43 or it can be coupled to mass spectrometry (MS).32,34,36,38,39,44,45 GC-MS has its advantages because of its sensitivity and selectivity. However, the application of FID is not very useful anymore due to its restriction of selectivity and the fact that it does not allow the use of isotope-labeled internal standards. HPLC can be used with UV deduction,46 or fluorescence detection (FLD)33/diode array detection (DAD),35,37,42 or it can be coupled to MS.30,33 In conclusion, it can be said that the most-often-used and appropriate device is GC/MS for food commodities such as flour, bread, and cereals because of its advantages.29,30,32,34,45

LEVEL OF PAHs IN FLOUR, BREAD, AND BREAKFAST CEREALS According to the EFSA, food is the main route of human exposure to PAHs, and bread, cereals, and cereal-based products constitute one of the major contributing sources, due to the high amount of consumption.1,2,17,18 Furthermore, PAH contamination is inevitable in bread and cereal products due to the high-temperature nature of the baking process. Research on the level of PAHs in flour, bread, and cereals is limited. However, when examining the existing studies, some conclusions can be reached about the occurrence and level of PAHs in bread and breakfast cereals. Ciecierska and Obiedzi nski (2013)35 have studied the occurrence and possibility of PAH formation in the bakery chain, from raw resources to the final products. According to this study, PAHs can contaminate bread, not only from the raw materials such as flour, but also from the baking process. The raw materials and various kinds and parts (crumbs, crusts) of bread have been analyzed to determine the level of their contamination. It can be seen that the total of 19 PAHs in raw materials such as bran, flour, and grain have ranged from 1.07 to 3.65 μg/kg and, in bread, from 1.59 to 13.6 μg/kg depending on the part of bread and the baking temperature (between 230°C and 260°C) (see Table 2). Basically, similar profiles of PAHs were observed in all analyzed kinds of bread. Four low-molecular-weight (LMW) PAHs [namely, fluoranthene (Fl), phenanthrene, pyrene (Pyr), and anthracene (Ant)] are found at low levels. If the various kinds of bread (e.g., rye bread, wheat-rye bread, and whole-meal rye bread) are analyzed, it also can be seen that four low molecular weight PAHs around 90% of all PAHs. However, it was also reported that the four EU marker PAHs are detected at low levels. Moreover, when whole-meal rye bread is baked at the highest temperature (260°C), the BaP is detected only in the crust. In general, when all kinds of bread are examined, the highest total PAH content is found in the crust (up to 13 μg/kg for the sum of 19 PAHs), and the lowest is found in crumbs.35 When some bread doughs are examined, it can be seen that in the dough of wheat-rye bread, rye bread, and wholemeal rye bread, four low-molecular-weight (LMW) PAHs [namely, fluoranthene (Fl), phenanthrene, pyrene (Pyr), and anthracene (Ant)] constituted 100%, 100%, and 83% of the total content of 19 PAHs, respectively. Looking at the doughs’ contamination levels and the influence of the baking temperature on bread’s PAH content confirms that PAHs are formed during baking35. The effect of gas oven toasting on bread has been studied with respect to the formation of PAHs in bread baked from white flour, bread baked from brown flour, and sandwich bread baked from white flour. BaP is not detected in 10 of 18 samples, while in the remaining samples, the BaP levels vary from 2.83 to 16.54 μg/kg. However, BaP is not detected in original white and brown flour. The total PAHs vary from 1.06 to 149.3 μg/kg for bread baked from white flour.38 It is also found that the PAH levels in toasted bread from white flour ranged between 7.38 and 18.0 μg/kg.42 The PAH levels of dough and bread are compared in Table 3. Alomirah et al. (2011) have reported on the occurrence and levels of genotoxic PAHs in pita bread (see Table 3). Although there is no BAP in pita bread, it have found that genotoxic PAHs (PAH8) (0.94 μg/kg) and total PAHs (between 7.50 and 32.1 μg/kg) are at low levels. On the other hand, pita bread with meat fat drippings shows the highest amount of BAP (4.88 μg/kg) and PAH8 (14.0 μg/kg), indicating the probable role that meat fat plays in the enhancement of PAH formation.40 As such, the role of pyrolysis is significant as meat fat is dripped onto the heated surface (leading to incomplete combustion). Kacmaz et al. (2016) studied the level of the four EU marker PAHs in bread and breakfast cereal samples. Breakfast cereal samples showed a higher mean sum for ΣPAH4 than for the samples of bread. It has reported that BaP is the most prevalent PAH in these kinds of samples. On average, all analyzed samples showed low amounts of ΣPAH4,

1. INTRODUCTORY CHAPTERS

17

LEVEL OF PAHs IN FLOUR, BREAD, AND BREAKFAST CEREALS

TABLE 2

Mean and Range Content of PAHs With Standard Deviation in Selected Raw Materials (μg/kg)

Product

Mean
SDa or rangeb

BaA (μg/kg)

CHR (μg/kg)

BbF (μg/kg)

BaP (μg/kg)

Wheat

Mean
SD

0.05
0.00

nd

nd

Rye

Mean
SD

0.06
0.00

nd

White flour

Mean
SD

na

Mean
SD Whole wheat flour (brown flour)

P

19 PAHs (μg/kg)

Reference

nd

2.35
0.15

35

nd

nd

2.93
0.22

35

na

na

0.02–0.09

na

47

nd

nd

nd

nd

43.1

38

Mean
SD

nd

nd

nd

nd

56.4

38

Wheat flour

Mean
SD

nd

nd

nd

nd

1.07
0.14

35

Rye flour

Mean
SD

nd

nd

nd

nd

1.25
0.15

35

Whole-meal rye flour

Mean
SD

nd

nd

nd

nd

1.35
0.20

35

Wheat bran

Mean
SD

0.05
0.01

nd

0.11
0.01

nd

1.87
0.18

35

Rye bran

Mean
SD

0.05
0.01

nd

nd

nd

2.65
0.17

35

Bran from rye grinding to whole-meal rye flour

Mean
SD

0.08
0.02

0.16
0.03

nd

nd

3.65
0.23

35

a a a a a a a a a a a

a

Range: maximum-minimum value. SD, standard deviation. nd ¼ not detected; na ¼ not available.

b

TABLE 3

Mean and Range Content of PAHs With Standard Deviation in Dough and Bread Samples (μg/kg) P

Product

Mean
SD or rangeb

Dough of Baltonowski bread

BaA (μg/kg)

CHR (μg/kg)

BbF (μg/kg)

BaP (μg/kg)

SUM of 4PAHs

19 PAHs (μg/kg)

Reference

Mean
SD

nd

nd

nd

nd

na

0.57
0.05

35

Dough of rye bread

Mean
SD

nd

nd

nd

nd

na

0.66
0.07

35

Dough of whole-meal rye bread

Mean
SD

nd

nd

nd

nd

na

0.58
0.06

35

Baltonowski bread baked at a temperature between 230–250°C

Rangeb

0.05–0.09

nd–0.23

nd–0.15

nd

na

1.59–7.37

35

Rye bread baked at temperature between 235–255°C

Rangeb

0.07–0.18

0.15–0.27

nd

nd

na

5.62–13.04

35

Whole-meal rye bread baked at temperature between 240–260°C

Rangeb

0.06–0.29

0.15-0.53

nd–0.23

nd–0.24

na

2.67–13.55

35

Commercial bread (wheat bread, whole grain bread, and brown bread)

Rangeb

na

na

na

< LOQ– 0.20

0.11–0.22

na

32

Bread (whole wheat bread, whole-meal bread, rye bread, and white wheat bread)

Mean
SDa

0.03
0.02

0.05
0.03

0.05
0.02

0.17
0.05

0.28
0.09

na

34

Bread (whole wheat bread, whole-meal bread, rye bread, and white wheat bread)

Rangeb

black-grained wheat (223.76 g) > Klasic (186.01 g) > Glenlea (182.67 g).23 A high dough-stickiness value indicates stickier dough. Bakery characteristics are poor if the dough is too sticky. The flour from black-grained wheat had better baking properties than that obtained from Taifeng and Yecora Rojo, but its dough was somewhat stickier than flours from Klasic and Glenlea. The baking quality of wheat cultivars can be predicted according to their high-molecular-weight glutenin (HMWglu) subunits. For example, Taifeng has HMW-glu subunits similar to Anza (2 + 12 and 7 + 8 subunits), and Klasic has HMW-glu subunits similar to Yecora Rojo (1, 17 + 18, and 5 + 10 subunits), while black-grained wheat has HMW-glu subunits similar to Glenlea (2*, 7 + 8, and 5 + 10 subunits).23 Because good baking quality is strongly correlated with the presence of 1 and 5 + 10 or 2* and 5 + 10 HMW-glu subunits, while poor baking quality is usually associated with 2 +12 HMW-glu subunits, HMW-glu subunits (2* and 5 + 10) in black-grained wheat predict that black-grained wheat can be classified as bread wheat.23 On studying the effect of adding purple wheat (Konini) bran on the sensory quality of bread, Janeckova et al.27 observed that a 10%–30% addition of purple wheat bran affected the loaf volume, crust color and integrity, crumb structure, and taste of the resulting bread. When 20% of the baking flour was replaced with 10% purple wheat bran and 10% semolina, the loaf volume slightly decreased, with an accompanying change in shape from regular to slightly arched. Although the crust appeared integral and rather dull, the crumb porosity of the bread containing 10% bran and 10% semolina was comparable to the control bread, which had no flour replacement. Increasing the flour replacement to 20% finely milled bran and 10% semolina resulted in bread with the most preferred crust integrity and crumb porosity, but with less arching and less loaf volume compared to the control and 10% bran-10% semolina-containing bread. The 20% milled bran-bread was judged as the second-worst bread in terms of taste. A replacement of flour with 20% unmilled bran and 10% semolina produced bread with lower height and corresponding lower volume than the 20% milled bran-bread due to disruption of the gluten structure by the large particle size of the bran.27 Although the crust integrity, crumb porosity, color, and product gloss of the 20% unmilled bran-bread were less preferred to the 20% unmilled bran-bread, its aroma and taste were the most preferred among all the test samples. A 30% unmilled bran and 10% semolina replacement of flour resulted in the least-preferred bread in terms of all the sensory attributes (namely, loaf volume and shape, crust integrity and color, gloss, crumb porosity, aroma, and taste). Instrumental color analysis revealed the bread samples with 20% milled bran-10% semolina and 30% unmilled bran-10% semolina replacement of flour to be the most different from the control sample in terms of color.27 Milling of the bran (for the 20% milled bran sample) increased its surface area, probably resulting in more dietary fiber-bound phenolic compounds being exposed and participating in complex reactions that led to Millard Reaction Products.28 The bread sample with 30% unmilled bran-10% semolina had the darkest crust color, due to its highest bran content, compared to all the other bread samples.27 Texture analysis showed that increasing flour replacement with bran decreased the crust integrity in a dose-dependent manner up to 20% bran replacement of flour,27 due to a reduction in gluten that forms and strengthens the dough structure of the bread.29

TOTAL PHENOLIC AND ANTHOCYANIN CONTENT, PHENOLIC ACID COMPOSITION AND ANTIOXIDANT PROPERTIES OF COLORED WHEATS AND THEIR BREADS Total Phenolic Content Purple wheat bread (PWB) and two bread controls, whole wheat bread (WWB) and white flour bread (WFB), were prepared according to the method described by G
elinas and McKinnon,30 and their phenolic content and antioxidant properties were evaluated. The total phenolic contents of PWB, WWB, and WFB are shown in Table 1 (unpublished

2. FLOURS AND BREADS

TOTAL PHENOLIC AND ANTHOCYANIN CONTENT

79

TABLE 1 Total Phenolic Content of WWB, Wheat Flour Bread and PWB Bread name

Equivalent of ferulic acid (mg/kg)

WWB

1005

Wheat flour bread

515

PWB

1111

data). PWB showed the highest total phenolic content and their total phenolic content decreased in the same order [PWB (1111 mg/kg) > WWB (1005 mg/kg) > WFB (515 mg/kg)] as that of 2,2-diphenyl-1-picryhydrazyl free radical (DPPH) scavenging activity and oxygen radical absorbance capacity (ORAC). G
elinas and McKinnon30 reported that total phenolic contents (gallic acid equivalent), which ranged from 522 to 866 mg/kg for the whole meal of organic white wheat varieties, were up to above 1000 mg/kg for WWB, and about 400 mg/kg for WFB. Baking slightly increased the level of total phenolic content in bread crust, likely due to the Maillard reaction.30 Total phenolic contents (ferulic acid equivalent, or FAE) were 1973.5 and 811.6 mg/kg in the whole meal and flour of purple-grained wheat, and 7616.4 and 646.5 mg/kg in the bran and flour of blue-grained wheat, respectively.31 The total phenolic contents (FAE) of bran and whole meal were 2415 and 1108 mg/kg in black-grained wheat, 2,290 and 929 mg/kg in purplegrained wheat, 1416 and 706 mg/kg in blue-grained wheat, and 2215 and 817 mg/kg in white-grained wheat, respectively.32 Because there are differences in phenolic content among wheat genotypes, the level of phenolics in bread will be affected by raw wheat material and ingredients used. Yu and Beta33 studied the effects of various unit operations, during the bread-making process, on the soluble-free total phenolic content (SF-TPC), insoluble-bound total phenolic content (IB-TPC), and overall total phenolic content (the € sum of SF-TPC and IB-TPC), as well as the total anthocyanin content and antioxidant properties of one yellow (Oelands hvede) and two purple (Indigo comprising 97% purple, and Konini comprising 84% purple) wheat kernels. They also identified the soluble-free phenolic acids and insoluble-bound phenolic acids present in the samples. The unit operations studied included dough mixing, dough fermentation for 30 min and 65 min, and baking. The baking-quality indicators studied were the bread loaf, bread crust, and bread crumbs. Antioxidant properties studied included DPPH• and 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical scavenging activities, as well as ORAC. They observed that generally, processing increased (65%–68%) the SF-TPC for all three wheat varieties, moving from flour through dough mixing and fermentation to baking (Table 2). Although IB-TPC increased (10%–11%) on dough mixing, it decreased (7%–9%) on 30-min fermentation, but increased after 65-min fermentation (13%–14%), and subsequently during baking (26%–27%).33 The decrease in IB-TPC during 30-min fermentation is due to degradation of the fiber components in the wheat by hydrolytic enzymes, such as xylanases and glucanases, which are expressed by the fermenting organisms, thereby releasing bound phenolic acids that contributed to the SF-TPC. The complex baking process that involves starch gelatinization, protein denaturation, and production of Maillard reaction products (MRPs), increased both SF-TPC and IB-TPC. MRPs reportedly interfere with the Folin Ciocalteu assay used to determine total phenolic content, and they also exhibit antioxidant properties.34,35 It is worth noting that bread crust had higher SF-TPC and IB-TPC than bread crumbs, and this may be related to the MRPs that were produced during the baking process.33 The overall total phenolic content obtained by combining SF-TPC and IB-TPC yielded a trend similar to that of IB-TPC, which was observed for both yellow and purple kernels. There appeared not to be any distinct difference in the total phenolic content between the yellow and purple wheat kernels and bread (Table 2). When bread from the three varieties were subjected to simulated in vitro gastrointestinal digestion, their SF-TPCs € decreased by 35.5%, 5.8%, and 31.2% for Oelands hvede, Indigo, and Konini respectively (Table 3).36 This indicates that Indigo has the most stable phenolic content to digestion compared to the other two, and suggests that phenolic compounds in bread from Indigo are more bioaccessible than bread from the other two varieties. To date, there is no information on the effect of simulated in vitro gastrointestinal digestion on the insoluble-bound phenolic acids of bread from colored wheat varieties and their antioxidant properties. There is a need for such research in order to ascertain the amount of phenolic acid that passes through the upper gut unchanged and will be released by the colonic microbiota through anaerobic fermentation. In a similar study, Guo and Beta37 compared the total phenolic content (TPC), phenolic acid composition, and antioxidant capacity (using DPPH• radical scavenging activity) of whole grain, insoluble dietary fiber, and soluble dietary fiber from soft white wheat (MSU D8006) flour and purple wheat flour. The researchers reported that purple wheat flour had 20.5% higher TPC than soft white wheat flour (Table 4). Among the soft white wheat components, TPC of

2. FLOURS AND BREADS

80

FAE Total free phenolic content (mg FAE/100 g)

2. FLOURS AND BREADS

Treatment

€ Oelands hvedeA

Flour

113.23  1.97

105.44  0.82

Mixing

129.05  1.64

30-min fermentation

Total bound phenolic content (mg FAE/100 g)

Overall total phenolic content (mg FAE/100 g) € Oelands hvede

Indigo

Konini

123.68  5.53

238.59  7.24

227.26  4.77

234.94  6.02

135.96  2.37

137.08  1.32

267.62  4.01

261.72  7.30

265.78  2.14

129.64  2.37

125.73  5.79

127.96  2.63

265.78  7.14

253.73  8.26

263.63  4.11

137.53  6.74ef

141.73  5.26b

138.20  5.53bc

140.61  4.74b

284.03  5.92

268.41  9.80

278.14  11.48

176.72  0.99d

183.53  2.96cd

158.10  5.79a

154.75  5.79a

156.43 3.95a

344.01  9.57

331.47  6.78

339.96  6.91

228.81  5.92

219.16  6.08

229.86  1.81

122.57  5.00

116.80  3.16

119.59  6.05

351.38  10.92

335.96  9.24

349.45  7.86

123.81  6.08

114.28  3.78

119.05  5.26

163.13  0.26

159.22  4.74

161.64  8.16

286.94  6.35

273.50  8.52

280.69  13.42

€ Oelands hvedeB

IndigoB

111.26  0.49

125.36  5.26

121.82  3.95

125.76  4.93

128.70  0.82

138.57  2.37

136.14  4.77

128.00  2.47

135.67  1.48

65-min fermentation

142.3  0.66e

130.21  4.28fghi

Bread loaf

185.91  3.78c

Bread crust Bread crumbs

IndigoA kl ghi efg

a ij

KoniniA kl ij hi

b k

kl ghi efgh

a jk

efg bc cde

efg a

KoniniB efg bcd efg

g a

efg bcd def

gf a

A–B ¼ Columns labeled with the same capital superscript were considered as a group. a–l ¼ Significant difference was defined with different letters in each group. nd, not determined. Overall total phenolic content was calculated as the sum of free and bound phenolic content for each raw. Modified from Yu and Beta (2015) under Creative Commons Attribution License.

6. FLOUR AND BREAD FROM BLACK, PURPLE, AND BLUE-COLORED WHEATS

TABLE 2 Effect of the Bread-Making Process on Total Soluble-Free, Total Insoluble-Bound, and Overall Total Phenolic Content of Yellow and Purple Wheat Grains

81

TOTAL PHENOLIC AND ANTHOCYANIN CONTENT

TABLE 3 Effect of Simulated In Vitro Gastrointestinal Digestion on Total Phenolic Content and Antioxidant Properties of Yellow Wheat Bread and PWB Bread-making stages

TPC (mg FAE/100 g DW)

DPPH (μmol TE/100 g DW)

ABTS (μmol TE/100 g DW)

ORAC (μmol TE/100 g DW)

€ Oelands hvede

119.95  5.92

125.13  4.55

a

419.63  21.43

4367.99  311.44b

Indigo

166.47  6.58a

207.08  11.36a

461.31  12.50a

5040.53  326.25a

Konini

126.23  4.28b

173.34  9.09b

438.57  19.65a

4712.02  197.27ab

b

c

Results are expressed as mean  standard deviation (SD). Means in a column with different superscript letters are significantly (P < .05) different from each other. TPC, total phenolic content; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid); ORAC, oxygen radical absorbance capacity; FAE, ferulic acid equivalent; TE, Trolox equivalent; DW, dry weight basis. Data from Yu (2014) with permission from Lilei Yu, Canada.

TABLE 4

Total Phenolic Content and Antioxidant Capacity of Whole-Grain Soft White Wheat and Purple Wheat Floursa

Sample

Whole grain

Insoluble dietary fiber

Soluble dietary fiber

Soft white wheat

1.55  0.05b,B

8.81  0.02a,A

0.88  0.03b,C

Purple wheat

1.95  0.06a,B

9.09  0.05a,A

1.05  0.04a,C

Soft white wheat

2.08  0.07a,B

11.13  0.59b,A

0.88  0.06a,C

Purple wheat

2.18  0.03a,B

12.47  0.31a,A

0.95  0.04a,C

B

TPC (MG FAE /G SAMPLE DW)

DPPH (μMOL TEC/G DW)

TPC, total phenolic content; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight basis. a ¼ Values are expressed as mean  standard deviation of two measurements. b ¼ FAE, ferulic acid equivalent. c ¼ Trolox equivalent. Wheat varieties are compared by small letters (a–b): means in a column with different superscript letters are significantly (P < .05) different from each other. Wheat fractions are compared by capital letters (A–C): means in a row with different superscript letters are significantly (P < .05) different from each other. Data from Guo and Beta (2013) with permission from Elsevier Ltd.

whole grain was 43% higher than that of soluble dietary fiber, but 82% lower than that of insoluble dietary fiber extracted from whole grain. The TPC of insoluble dietary fiber was 90% higher than that of soluble dietary fiber from soft white wheat. This implies that insoluble dietary fiber has more phenolic compounds bound to it than the corresponding soluble dietary fiber. Similarly, among the purple wheat components, TPC of whole grain was 46% higher than that of soluble dietary fiber, but 78% lower than that of insoluble dietary fiber extracted from whole grain. The TPC of insoluble dietary fiber was 91% higher than that of soluble dietary fiber from purple wheat (Table 4).

Phenolic Acid Composition The phenolic acid composition of WWB, WFB, and PWB after hydrolysis is shown in Table 5 (unpublished data). A total of 10 types of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, syringic, m-coumaric, caffeic, p-coumaric, ferulic, and sinapinic acids) were detected in PWB, nine types of phenolic acids (gallic, p-hydroxybenzoic, vanillic, syringic, m-coumaric, caffeic, p-coumaric, ferulic, and sinapinic acids) in WWB, and eight types of phenolic acids (gallic, p-hydroxybenzoic, vanillic, syringic, caffeic, p-coumaric, ferulic, and sinapinic acids) in WFB. The major phenolic acids (>50 mg/kg) were ferulic acid (228 mg/kg) and p-coumaric acid (84 mg/kg) in PWB, ferulic acid (250 mg/kg) in WWB, and ferulic acid (65 mg/kg) in WFB. The total phenolic acids decreased as the same order [PWB (403 mg/kg) > WWB (313 mg/kg) > WFB (111 mg/kg)] as that of total phenolic content. PWB had 3.63 and 1.29 times higher total phenolic acids than WFB and WWB, respectively. Siebenhandl et al.31 reported that ferulic acid was 851.7 and 180.1 mg/kg in the whole meal and flour of purple-grained wheat and 3503.3 and 151.1 mg/kg in the bran and flour of blue-grained wheat; vanillic acid 35.1 mg/kg and 9.9 mg/kg in the whole meal and flour of purplegrained wheat and 99.8 mg/kg and 10.1 mg/kg in the bran and flour of blue-grained wheat; and p-coumaric acid 24.3 mg/kg and 4.6 mg/mg in the whole meal and flour of purple-grained wheat and 456.6 mg/kg and 6.3 mg/kg in the bran and flour of blue-grained wheat, respectively. The level of phenolic acids in the whole meal of soft wheat cultivars ranged from 455.92 to 621.47 mg/kg for ferulic acid, 8.44 to 12.68 mg/kg for vanillic acid,

2. FLOURS AND BREADS

82

6. FLOUR AND BREAD FROM BLACK, PURPLE, AND BLUE-COLORED WHEATS

TABLE 5 Phenolic Acid Composition of WWB, Wheat Flour Bread, and PWB (mg/kg) Phenolic acids

WWB

Wheat flour bread

PWB

Gallic acid

12

12

13

Protocatechuic acid

Not detectable

Not detectable

20

p-hydroxybenzoic acid

5

4

7

Vanillic acid

12

8

18

Syringic acid

4

8

7

m-coumaric acid

2

Not detectable

2

Caffeic acid

8

3

15

p-coumaric acid

12

9

84

Ferulic acid

250

65

228

Sinapinic acid

8

2

9

Total phenolic acids

313

111

403

8.86 to 17.77 mg/kg for syringic acid, and 10.40 to 14.10 mg/kg for p-coumaric acid.38 Potential health benefits have been demonstrated for phenolic compounds because of their ability to act as antioxidants.31 € With respect to the effect of bread-making-unit operations on the phenolic acid composition of yellow (Oelands hvede) 33 and purple (Indigo and Konini) wheat grains, Yu and Beta identified p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids in soluble-free phenolic extracts (Table 5), while in insoluble-bound phenolic extracts, these phenolic acids were identified, in addition to protocatechuic, caffeic, syringic, and sinapic acids (Table 6). Also, p-hydroxybenzoic, vanillic, and p-coumaric acids were absent from the soluble-free extracts of the flours and their mixed dough. However, fermentation to some extent, and largely baking, caused their release and detection in the extracts. As expected, ferulic acid was identified in the soluble-free extract of all the flours, and its level increased upon processing, from mixing through fermentation and baking. The levels of insoluble-bound phenolic acids, however, appeared to decrease upon mixing and fermentation for 30 min. However, 65-min fermentation generally increased their levels, while baking appeared not to affect their levels for all the wheat varieties (Table 7).33 It was observed that the concentrations of p-hydroxybenzoic, vanillic, and caffeic acids in the insoluble-bound phenolic fraction of the yellow variety were lower than that of the purple varieties. On the contrary, the concentrations of p-coumaric, ferulic, and sinapic acids in the insoluble-bound phenolic fraction of the yellow variety were higher than that of the purple varieties.33 In comparing the phenolic acid composition of whole grain, soluble dietary fiber, and insoluble dietary fiber from soft white wheat (MSU D8006) flour and purple wheat flour, Guo and Beta37 reported the major phenolic acids that were identified to include protocatechuic, vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acids, as well as the 8-50 , 5-50 , and 8-O-40 dimers of ferulic acid (Table 8). The ferulic acid dimers were absent from the whole grain of soft white wheat, as well as the soluble dietary fiber components of both varieties. Ferulic acid was the predominant phenolic acid identified in the samples. Generally, the purple wheat variety had higher levels of total quantified phenolic acids in its whole grain flour and soluble dietary fiber component, but lower levels of total quantified phenolic acids in its insoluble dietary fiber extract than the corresponding soft white wheat components (Table 8).

Total Anthocyanin Content Total anthocyanin content in PWB was 78 mg/kg (Table 9, unpublished data). Anthocyanin was not detectable in WWB and WFB. Anthocyanins are members of the bioflavonoid phytochemical class, which have been recognized to have health-enhancing benefits due to their antioxidant activity, antiinflammatory, and anticancer effects.17 The total anthocyanin content of bran, whole meal, and flour ranged from 415.9 to 479.7 mg/kg, 139.3 to 163.9 mg/kg, and 18.5 to 23.1 mg/kg in blue-grained wheat; from 156.7 to 383.2 mg/kg, 61.3 to 153.3 mg/kg, and 3.1 to 14.3 mg/kg in purple-grained wheat; and from 9.9 to 10.3 mg/kg, 4.9 to 5.3 mg/kg, and 1.5 to 1.7 mg/kg in red-grained wheat, respectively.16 Siebenhandl et al.31 reported total anthocyanin content of 225.8 and 17.0 mg/kg in the bran and flour of blue-grained wheat, and 34.0 and 8.2 mg/kg in the whole meal and flour of purple-grained wheat, respectively.

2. FLOURS AND BREADS

83

TOTAL PHENOLIC AND ANTHOCYANIN CONTENT

TABLE 6

Effect of the Bread-Making Process on Composition of Soluble-Free Phenolic Acids in Yellow and Purple Wheat Grains

Steps

Sample name

Flour

€ Oelands hvede Indigo Konini

Mixing

€ Oelands hvede Indigo Konini

30-min fermentation

€ Oelands hvede Indigo

65-min fermentation

Bread loaf

Bread crust

nd

Vanillic acid nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

nd

2.44  0.53

d

nd

nd

Konini

nd

€ Oelands hvede

4.19  0.71

nd ef

3.49  0.09

c

Indigo

nd

Konini

2.66  0.37

€ Oelands hvede

11.07  2.42

4.37  0.29

Indigo

7.39  2.41

Konini

nd f

nd cd

dc ab

5.20  0.83

11.87  1.93

c

3.44  0.32

nd

3.73  0.27

nd

Konini

nd

€ Oelands hvede

16.29  1.68

Indigo

8.64  1.87

Konini

15.24  1.37

€ Oelands hvede Indigo

Bread crumb

p-Hydroxybenzoic acid

de bc

ab

Total phenolic acid

nd

j

2.50  0.20

2.50  0.20

nd

j

2.02  0.07

2.02  0.07

nd

j

2.25  0.01

2.25  0.01

nd

10.01  1.23

10.01  1.23

nd

9.04  0.24

9.04  0.24

nd

10.58  0.69

10.58  0.69

1.31  0.17

11.25  0.67

15.01  1.37

nd

10.57  1.55

10.57  1.55

nd

12.55  1.13

12.55  1.13

nd

13.34  1.68

21.03  2.49

nd

13.25  2.06

13.25  2.06

nd

15.01  0.69

17.67  0.44

hi i ghi

ab

egh ghi ef cde de bcd

1.51  0.07

a

1.15  0.01

15.53  0.98

32.00  2.53

nd

12.41  0.90

16.15  1.17

5.19  0.04

nd

12.01  1.01

17.21  1.05

6.00  0.28

0.70  0.02

15.11  0.26

21.82  0.55

nd

14.76  0.74

31.05  1.82

nd

14.65  0.03

23.29  2.01

nd

16.44  0.57

31.68  1.54

nd nd

b

30.47  4.27 28.25  3.66

nd

1.26  0.06

13.53  1.49

ab

c

b

cde

14.40  0.42

a

cd

Ferulic acid

bcd

ab

a

p-Coumaric acid

ef efg

c

abc abcd abcd a

Content of phenolic acid was expressed as μg/g of dry weight. Values in each column with different letters are significantly different (P < .05). Total phenolic acid was calculated as the sum of each row. a–j ¼ Significant difference was defined with different letters in each group. nd, not detected. Modified from Yu and Beta (2015) under Creative Commons Attribution License.

Hosseinian et al.39 reported total anthocyanin content of 500.6 mg/kg in normal purple-grained wheat and 526.0 mg/kg in heat-stressed purple-grained wheat, and confirmed that cyanidin 3-glucoside was the predominant anthocyanin (103.0 mg/kg) in normal purple-grained wheat. The major anthocyanins were delphinidin 3-glucoside (56.5 mg/kg), delphinidin 3-rutinoside (49.6 mg/kg), cyanidin 3-glucoside (20.3 mg/kg), and cyanidin 3-rutinoside (16.8 mg/kg) in blue-grained wheat,17 and cyanidin 3-glucoside (19.73–46.44 mg/kg) in purple-grained wheat.16 On studying the effects of various unit operations during bread-making on the total anthocyanin content of purplegrained wheat, Yu and Beta33 reported that total anthocyanin content decreased upon mixing of the flour, but increased slightly during the fermentation process, and then decreased again on baking (Table 10). The decrease upon flour mixing was attributed to dilution of the constituent anthocyanins by other components in the flour while the increase upon fermentation was due to tissue degradation in the flour, promoting anthocyanin release. Baking decreased the total anthocyanin content because anthocyanins are heat labile. Complete thermal degradation of purple wheat anthocyanins have been reported during the preparation of muffins.4 The mechanisms and kinetics of thermal degradation of anthocyanins have been amply reviewed40 and therefore will not be treated in this chapter. It is, however, noteworthy that because anthocyanins are natural plant colorants, their thermal degradation will affect the color of products developed from their plant sources. There were € no anthocyanins detected in the yellow (Oelands hvede) variety. Among the purple wheat varieties, total anthocyanin content of Indigo was 46%–55% higher than that of Konini, both in their flour and bread (Table 10). Anthocyanins make important contribution to the antioxidant capacity of black, purple, and blue wheats in comparison with that of red and white wheats, which contain only very low anthocyanin levels.16

2. FLOURS AND BREADS

Breadmaking stages Flour

Mixing

2. FLOURS AND BREADS

65-min fermentation

Bread loaf

Bread crust

Bread crumbs

Sample name

Protocatechuic acid

p-Hydroxy benzoic acid

Vanillic acid

Caffeic acid

Syringic acid

p-Coumaric acid

Ferulic acid

Sinapic acid

Total phenolic acid

€ Oelands hvede

nd

22.73  1.29b

9.29  0.54f

4.54  0.18k

9.03  0.58b

26.13  1.19a

532.15  30.00defgh

69.36  2.69cd

673.25  36.47

Indigo

nd

25.69  1.17a

24.82  0.01a

6.53  0.15ef

9.95  0.23a

19.14  1.50def

447.02  24.14kl

35.97  2.80k

569.12  29.99

Konini

nd

24.60  1.24

19.20  0.97

7.10  0.30

9.05  0.09

21.64  0.68

466.66  0.28 jk

52.48  2.24

600.74  5.81

€ Oelands hvede

20.67  1.00

10.63  0.73

7.44  0.49

4.85  0.02

9.46  0.57

26.50  0.39

573.64  11.84

72.37  0.88

725.56  15.92

Indigo

19.37  0.60ab

24.48  1.68a

9.48  0.32f

4.52  0.00k

5.97  0.17f

19.63  1.25de

482.89  9.02ijk

51.74  2.88ij

618.07  15.94

Konini

14.37  0.84e

20.59  1.19c

13.58  0.18de

5.70  0.28g

7.24  0.36de

22.57  1.16bc

514.29  4.29hi

60.38  0.28ef

658.73  8.57

€ Oelands hvede

15.42  0.43d

7.83  0.39h

6.17  0.17h

nd

nd

21.02  0.96cd

411.57  13.46lm

64.66  2.88de

526.66  18.23

Indigo

15.46  0.51d

11.63  0.31f

8.68  0.73fg

nd

nd

14.56  0.54ij

363.99  18.02n

40.82  3.24k

455.14  23.35

Konini

11.13  0.68

13.79  0.42

12.76  0.51

nd

nd

j

13.57  0.03

386.39  11.13

j

49.10  2.84

486.74  15.60

€ Oelands hvede

17.56  0.04

11.70  0.61

12.86  0.92

5.20  0.08

9.40  0.46

26.57  0.56

526.64  31.76

71.83  1.05

681.75  35.47

Indigo

19.97  0.23ab

12.19  0.24f

19.18  0.69b

6.21  0.28f

6.83  0.32e

16.90  0.25gh

488.22  2.63hijk

54.86  0.48ghi

624.36  5.12

Konini

14.99  0.22

15.77  0.91

18.17  0.98

6.86  0.05

7.82  0.27

18.61  0.19

523.25  7.22

60.34  2.47

665.81  12.31

€ Oelands hvede

3.75  0.12

10.94  0.27

14.68  0.30

5.30  0.22

9.63  0.16

26.64  0.30

586.92  30.32

75.51  2.09

733.38  33.79

Indigo

4.12  0.08gh

9.99  0.32g

19.32  0.59b

6.62  0.16de

6.97  0.27e

17.31  0.33fgh

517.14  1.40hi

56.12  1.48fghi

637.57  5.16

Konini

a

a

f c

de

ghi

fg

e f

d fg

b

h

bc jk

e e

b cd

ij

cde hi

3.10  0.08

5.29  0.15

18.65  0.62

7.33  0.26

€ Oelands hvede

nd

4.80  0.21

Indigo

nd

Konini

nd

€ Oelands hvede

b ab

ab

cd ab

c a

a

efg a

bcd

mn efghi

fghi bc

hij bc

bc

ef b

8.12  0.09

19.39  1.47

564.64  37.89

65.31  1.81

691.84  43.37

15.45  0.21

gh

5.66  0.19

nd

27.87  0.79

631.54  42.35

80.92  3.40

766.23  47.15

4.44  0.10j

19.49  0.66b

7.17  0.24bc

nd

19.30  1.07de

551.35  6.76cdefg

57.45  4.15fgh

659.20  12.98

j

4.25  0.27

18.77  0.63

7.63  0.04

nd

20.70  4.12

616.95  21.59

71.57  3.64

739.88  26.59

3.95  0.14

6.17  0.59

14.51  0.11

5.19  0.05

9.80  0.16

23.93  1.64

569.32  26.73

70.96  4.66

703.82  34.08

Indigo

4.33  0.17g

5.62  0.21ij

18.33  1.00b

6.56  0.15ef

6.97  0.38e

17.10  0.63gh

498.80  7.71hij

51.14  0.89ij

608.85  11.08

Konini

3.25  0.13

4.32  0.27

18.65  1.21

6.96  1.64

8.18  0.13

16.05  0.98

554.64  24.93

58.22  0.79

670.28  28.61

i

ij ij

ghi

hi

i

j

b c

b cd

b

ab

a ij

bcd

c

de a

cd

a

c

b

hi

cdef a

ab cde

cdefg

de a

bc bc

fg

Content of phenolic acid was expressed as μg/g of flour, dry weight. Values in each column with different letters are significantly different (P < .05). Total phenolic acid was calculated as the sum of each row. a j ¼ Significant difference was defined with different letters in each group. nd, not detected. Modified from Yu and Beta (2015) under Creative Commons Attribution License.

6. FLOUR AND BREAD FROM BLACK, PURPLE, AND BLUE-COLORED WHEATS

30-min fermentation

84

TABLE 7 Effect of the Bread-Making Process on Composition of Insoluble-Bound Phenolic Acids in Yellow and Purple Wheat Grains

85

TOTAL PHENOLIC AND ANTHOCYANIN CONTENT

TABLE 8

Phenolic Acid Composition of Whole-Grain Soft White and Purple Wheat Varieties and Their Insoluble Dietary Fiber and Soluble Dietary Fiber Monomeric phenolic acid

Dimeric ferulic acid

PCA

VNA

CFA

SYA

p-COA

FA

SIA

iso-FA

8-50

5-50

8-O-40

Total

Short white wheat

nd

6.05

Nd

4.21

18.89

565.12

22.36

36.95

nd

nd

nd

653.58

Purple wheat

nd

34.02

nd

7.12

17.78

562.96

42.00

18.39

3.95

1.29

1.32

688.83

Sample WHOLE GRAIN

INSOLUBLE DIETARY FIBER Short white wheat

nd

13.91

nd

6.07

101.07

3543.82

32.71

155.00

43.35

27.90

55.96

3979.79

Purple wheat

34.63

72.75

20.14

10.21

81.66

3081.57

66.14

75.54

28.94

13.41

37.75

3522.74

SOLUBLE DIETARY FIBER Short white wheat

nd

nd

nd

nd

15.03

94.87

5.90

9.65

nd

nd

nd

125.45

Purple wheat

nd

nd

nd

nd

15.43

99.46

3.99

13.07

nd

nd

nd

131.95

Concentration of phenolic acids were expressed as μg/g sample, dry weight. PCA, protocatechuic acid; VNA, vanillic acid; CFA, caffeic acid; SYA, syringic acid; p-COA, p-coumaric acid; FA, ferulic acid; SIA, sinapic acid; iso-FA, iso-ferulic acid. Values are means of two measurements (standard deviations not shown); nd, not detected. Data from Guo and Beta (2013) with permission from Elsevier Ltd.

TABLE 9 Total Anthocyanin Content of WWB, Wheat Flour Bread, and PWB Bread name

Cyanidin 3-glucoside equivalent (mg/kg)

WWB

Not detectable

Wheat flour bread

Not detectable

PWB

78

TABLE 10

Effect of the Bread-Making Process on Total Anthocyanin Content of Yellow and Purple Wheat Grains € Oelands hvede

Indigo

Flour*

nd

10.41  0.39

Mixing*

nd

8.09  0.37c

4.22  0.07f

30-min fermenting*

nd

8.60  0.47bc

4.65  0.25ef

65-min fermenting*

nd

8.97  0.26b

4.80  0.08de

Bread loaf*

nd

4.64  0.24ef

2.15  0.04g

Bread crust*

nd

4.16  0.21f

1.99  0.07g

Bread crumb*

nd

4.81  0.26de

2.18  0.08g

Bread-making stages

Konini a

5.31  0.02d

* Results were expressed as mg cyanidin-3-glucoside equivalents/100 g. Mean values in both rows and columns with different superscript letters are significantly (P < .05) different from each other. Modified from Yu and Beta (2015) under Creative Commons Attribution License.

Antioxidant Properties The DPPH• free radical scavenging activity and kinetics of PWB, WWB, and WFB are shown in Table 11 and Fig. 1, respectively. PWB had the highest DPPH• scavenging activity (47.58%), at 60 min, when compared to WWB (34.06%) and WFB (32.20%). The DPPH• radical scavenging activity from kinetics curve was also in the following order: PWB > WWB > WFB (Fig. 1). DPPH•-scavenging activity of whole meal in several wheat genotypes were reported to be 33.51% for black-grained wheat, 25.57% for purple-grained wheat, 23.66% for blue-grained wheat, and 25.40% for white-grained wheat.32 The ORAC of three bread extracts decreased in the same order as that of DPPH• radical 2. FLOURS AND BREADS

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TABLE 11 DPPH• Radical Scavenging Activity and ORAC of WWB, White Wheat Bread, and PWB Sample

DPPH• scavenging (%)

ORAC (g Trolox equivalent/kg)

WWB

34.06

10.64

Wheat white bread

32.20

8.88

PWB

47.58

12.09

FIG. 1 DPPH• radical scavenging activity kinetics for whole wheat bread, white wheat bread, and purple wheat bread. DPPH radical scavenging (%)

50

40

30

20

Whole wheat meal bread Wheat white flour bread Purple wheat flour bread

10

0 0

10

20

30

40

50

60

Reaction time (min)

scavenging activity. The values are as follows: PWB (12.09 g/kg) > WWB (10.64 g/kg) > WFB (8.88 g/kg) (Table 11). High DPPH• and ORAC values are an indication of the high antioxidant capacity of the sample extract. A high correlation (r ¼ .96) was found between DPPH• scavenging activity and total phenolic content for whole wheat meals.32 This implies that high total phenolic content results in high antioxidant capacity. On studying the effects of various unit operations during the bread-making process on the antioxidant capacities of € phenolic extracts from one yellow (Oelands hvede) and two purple (Indigo and Konini) wheat kernels, Yu and Beta33 observed trends similar to that of their total phenolic content. While there were little differences in trends between the DPPH• and ABTS•+ radical scavenging activities of the extracts, the overall outcomes were similar. Mixing of the flour decreased the DPPH• and ABTS•+ radical scavenging activities of soluble-free extract but increased that of the insoluble-bound extract. Fermentation of the mixed dough for 30 min increased the DPPH• radical scavenging activity of the soluble-free extract, but had no effect on the insoluble-bound extract. The 30-min fermentation showed no effect on the ABTS•+ radical scavenging activity of the soluble-free extract but decreased the value for the insolublebound extract. Increasing the fermentation time to 65 min did not affect the DPPH• radical scavenging activity of either the soluble or the insoluble extract, as well as the ABTS•+ radical scavenging activity of the soluble extract, but increased the ABTS•+ value for the insoluble extract. Baking of these doughs into bread increased both the DPPH• and ABTS•+ radical scavenging activities of all the extracts for all the wheat varieties.33 As observed for the SF-TPC, when bread from the three varieties were subjected to simulated in vitro gastrointestinal digestion, their antioxidant capacity, measured using the DPPH• and ABTS•+ radical scavenging activities, were lower than that of undigested bread. Notwithstanding, they all demonstrated antioxidant capacity. Among the varieties, Indigo showed the highest antioxidant capacity, in agreement with the observation for the SF-TPC.36 € With respect to the antioxidant properties of the anthocyanin extracts from the yellow (Oelands hvede) and two purple (Indigo and Konini) wheat kernels, Yu and Beta33 observed a trend similar to that of the total anthocyanin content during the bread-making process. Mixing of the flour into dough had no effect on the DPPH• radical scavenging activity, but it decreased the ABTS•+ radical scavenging activity of the anthocyanin extracts. Fermentation and baking both increased the DPPH• and ABTS•+ radical scavenging activities of all the extracts. As with the total anthocyanin content, the antioxidant capacity of the variety Indigo was higher than that of Konini.33 In the study comparing the antioxidant capacity (DPPH• radical scavenging activity) of whole grain, soluble dietary fiber, and insoluble dietary fiber from soft white wheat (MSU D8006) flour and purple wheat flour, Guo and Beta37 2. FLOURS AND BREADS

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reported that purple wheat flour had 4.6% higher antioxidant capacity than that of soft white wheat flour (Table 4). The trend of the antioxidant capacity among the grain components (whole grain, soluble dietary fiber, and insoluble dietary fiber) were similar to that of their TPC. With regard to the soft white wheat components, antioxidant capacity of the whole grain was 58% higher than that of its soluble dietary fiber, but 81% lower than that of the insoluble dietary fiber extracted from the whole grain. The antioxidant capacity of the insoluble dietary fiber was 92% higher than that of the soluble dietary fiber from soft white wheat. Also, among the purple wheat components, antioxidant capacity of the whole grain was 56% higher than that of the soluble dietary fiber, but 82% lower than that of the insoluble dietary fiber extracted from whole grain. The antioxidant capacity of the insoluble dietary fiber was 92% higher than that of the soluble dietary fiber from purple wheat (Table 4). As explained earlier, the higher TPC of the insoluble dietary fiber resulted in a correspondingly higher antioxidant capacity in the insoluble fraction.

FUTURE PERSPECTIVES The protein, flour, and dough quality of purple and blue wheat varieties need further study in order to predict their potential for making bread and other baked products. It is important to breed black, purple, and blue wheat varieties as bread wheat in the future. For black, purple, and blue wheats, the effect of their phenolic compounds and anthocyanins on bread quality and sensory properties needs to be studied further. It is valuable to investigate the chemical transformations of phenolic acids and anthocyanins during baking, in order to provide better understanding of their effect on the functional and sensorial qualities, as well as their health-promoting properties. There is a need to study the effect of gastrointestinal digestion on the stability and release of bound phenolic acids and anthocyanins in the colored wheat breads and other baked products, to provide an understanding about their fate (whether they are more biologically active intact or as metabolites) and health-promoting effects in the colon. Black, purple, and blue wheat varieties are being suggested as potential candidates for the development of specialty foods, including naturally colored foods and functional foods, as well as natural colorants, nutraceuticals, and pharmaceuticals, due to their natural anthocyanin pigments with high antioxidant capacity and other health benefits.

SUMMARY POINTS • Black-grained wheat contains higher crude protein content compared with common white and red bread wheat varieties such as Klasic, Yecora Rojo, and Glenlea. • Black-grained wheat can be used as bread wheat because its HMW-glu subunits (2*, 7 + 8, and 5 + 10 subunits) are similar to those found in Glenlea. • The protein quality of black-grained wheat needs to be improved further through breeding technology to improve its gluten strength, which is weak when compared with the common bread wheat varieties Klasic, Yecora Rojo, and Glenlea. • In addition to phenolic acids, black, purple, and blue wheat varieties contain anthocyanins with proven antioxidant and other potential health benefits. • Anthocyanins and phenolic acids are the major antioxidant compounds in black, purple, and blue wheat varieties. • Although thermal processing decreases the anthocyanin content and corresponding antioxidant capacity of colored wheat varieties, unit operations such as fermentation for an hour and baking during the bread-making process increase the antioxidant capacity of the resulting bread through release of bound phenolic compounds and formation of Maillard reaction products. • PWB shows higher antioxidant capacity than WWB and WFB types, due to their anthocyanin content. • There is utilization potential for black, purple, and blue wheat varieties as novel food ingredients and for the development of other high-value products.

References 1. Yu LL, Zhou K, Parry JW. Inhibitory effects of wheat bran extracts on human LDL oxidation and free radicals. LWT-Food Sci Technol 2005;38 (5):463–70. 2. Regina A, Bird A, Topping D, et al. High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats. Proc Natl Acad Sci U S A 2006;103(10):3546–51. 3. Lila MA. Anthocyanins and human health: an in vitro investigative approach. J Biomed Biotechnol 2004;2004(5):306–13.

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4. Li W, Pickard MD, Beta T. Effect of thermal processing on antioxidant properties of purple wheat bran. Food Chem 2007;104(3):1080–6. 5. Li W, Pickard MD, Beta T. Evaluation of antioxidant activity and electronic taste and aroma properties of antho-beers from purple wheat grain. J Agric Food Chem 2007;55(22):8958–66. 6. Li W, Sun C, Ren G. Characteristic of black-grained wheat and its potential for utilization. China Condiment 2004;1(26):9–11. 7. Pei C, Sun Y, Sun C, Yan G, Ren Y. Research and utilization of black-grained wheat. Seed 2002;4:42–4. 8. Knievel DC, Abdel-Aal E-SM, Rabalski I, Nakamura T, Hucl P. Grain color development and the inheritance of high anthocyanin blue aleurone and purple pericarp in spring wheat (Triticum aestivum L.). J Cereal Sci 2009;50(1):113–20. 9. Knott DR. The inheritance in wheat of a blue endosperm color derived from Agropyron elongatum. Can J Bot 1958;36(5):571–4. 10. Zeven AC. Wheats with purple and blue grains: a review. Euphytica 1991;56(3):243–58. 11. Copp LGL. Purple grains in hexaploid wheat. Wheat Inf Serv 1965;18:19–20. 12. Sharman BC. ‘Purple pericarp’: a monofactorial dominant in tetraploid wheats. Nature 1958;181:929. 13. Sun S, Sun Y, Yuan W, et al. Breeding and qualitative analysis for black grain wheat 76 of superior quality. Acta Agron Sin 1999;25:50–4. 14. Dedio W, Hill RD, Evans LE. Anthocyanins in the pericarp and coleoptiles of purple wheat. Can J Plant Sci 1972;52(6):977–80. 15. Piech J, Evans LE. Monosomic analysis of purple grain color in hexaploid wheat. Zeitschrift f€ ur Pflanzenz€ uchtung 1979;82:212–7. 16. Abdel-Aal E-SM, Hucl P. Composition and stability of anthocyanins in blue-grained wheat. J Agric Food Chem 2003;51(8):2174–80. 17. Abdel-Aal E-SM, Young JC, Rabalski I. Anthocyanin composition in black, blue, pink, purple, and red cereal grains. J Agric Food Chem 2006; 54(13):4696–704. 18. Bean MM, Huang DS, Miller RE. Some wheat and flour properties of Klasic-a hard white wheat. Cereal Chem 1990;67:307–9. 19. Al-Mashhadi A, Naeem M, Bashour I, Rogers DE, Hoseney RC. Effect of fertilization on yield and quality of irrigated Yecora Rojo wheat grown in Saudi Arabia. Cereal Chem 1989;66(1):1–3. 20. Campbell AR. Inheritance of crude protein and seed traits in interspecific oat crosses [PhD Thesis]. Ames, Iowa, USA: Agronomy, Iowa State University; 1970. 21. Wall IS. The role of wheat proteins in determining baking quality. In: Laidman DL, Wyn Jones RO, editors. Recent advances in the biochemistry of cereals. London, UK: Academic Press; 1979. p. 275–311. 22. Payne PI, Seekings JA, Worland AJ, Jarvis MG, Holt LM. Allelic variation of glutenin subunits and gliadins and its effect on breadmaking quality in wheat: analysis of F5 progeny from Chinese spring  Chinese spring (Hope lA). J Cereal Sci 1987;6:103–18. 23. Li W, Beta T, Sun S, Corke H. Protein characteristics of Chinese black-grained wheat. Food Chem 2006;98(3):463–72. 24. Dick JW, Quick JS. A modified screening test for rapid estimation of gluten strength in early-generation durum wheat breeding lines. Cereal Chem 1983;60:315–8. 25. Li WD, Corke H, Sun SC. Some characteristics of Chinese black-grained wheat flour. In: Corke H, Lin R, editors. Asian food product development. Beijing, China: Science Press; 1998. p. 76–82. 26. Perten H. Rapid measurement of wet gluten quality by the gluten index. Cereal Foods World 1990;35:401–2. 27. Janeckova M, Hrivna L, Juzl M, et al. Possibilities of using purple wheat in producing bakery products. MendelNet 2014;412–6. 28. P
erez-Jim
enez J, Díaz-Rubio ME, Mesías M, Morales FJ, Saura-Calixto F. Evidence for the formation of maillardized insoluble dietary fiber in bread: a specific kind of dietary fiber in thermally processed food. Food Res Int 2014;55:391–6. 29. Noort MWJ, van Haaster D, Hemery Y, Schols HA, Hamer RJ. The effect of particle size of wheat bran fractions on bread quality-evidence for fibre-protein interactions. J Cereal Sci 2010;52(1):59–64. 30. G
elinas P, McKinnon CM. Effect of wheat variety, farming site, and bread-baking on total phenolics. Int J Food Sci Technol 2006;41:329–32. 31. Siebenhandl S, Grausgruber H, Pellegrini N, et al. Phytochemical profile of main antioxidants in different fractions of purple and blue wheat, and black barley. J Agric Food Chem 2007;55:8541–7. 32. Li W, Shan F, Sun S, Corke H, Beta T. Free radical scavenging properties and phenolic content of Chinese black-grained wheat. J Agric Food Chem 2005;53(22):8533–6. 33. Yu L, Beta T. Identification and antioxidant properties of phenolic compounds during production of bread from purple wheat grains. Molecules 2015;20:15525–49. 34. Chandrasekara N, Shahidi F. Effect of roasting on phenolic content and antioxidant activities of whole cashew nuts, kernels, and testa. J Agric Food Chem 2011;59(9):5006–14. 35. Lee K-G, Shibamoto T. Toxicology and antioxidant activities of non-enzymatic browning reaction products: review. Food Rev Int 2002;18 (2-3):151–75. 36. Yu L. Identification and antioxidant properties of phenolic compounds during production of bread from purple wheat grains and investigation of bread extracts after simulated gastrointestinal digestion [MSc Thesis]. Winnipeg, Manitoba, Canada: Department of Food Science, University of Manitoba; 2014. 37. Guo W, Beta T. Phenolic acid composition and antioxidant potential of insoluble and soluble dietary fibre extracts derived from select wholegrain cereals. Food Res Int 2013;51(2):518–25. 38. Moore J, Hao Z, Zhou K, Luther M, Costa J, Yu L. Carotenoid, tocopherol, phenolic acid, and antioxidant properties of Maryland-grown soft wheat. J Agric Food Chem 2005;53:6649–57. 39. Hosseinian FS, Li W, Beta T. Measurement of anthocyanins and other phytochemicals in purple wheat. Food Chem 2008;109(4):916–24. 40. Patras A, Brunton NP, O’Donnell C, Tiwari BK. Effect of thermal processing on anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends Food Sci Technol 2010;21(1):3–11.

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C H A P T E R

7 Emmer (Triticum turgidum ssp. dicoccum) Flour and Bread Ahmad Arzani Department of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, Iran

O U T L I N E Introduction

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Emmer Impact on Combating Malnutrition

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Type of Utilization

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Future Direction of Research

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Bread-Making History

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Summary Points

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Flour and Bread Fortification With Emmer

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References

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INTRODUCTION Wheat is one of the most abundant sources of protein and energy in the world, and it is consumed as bread—a major staple food—in most countries.1 The importance of wheat originates from the properties of wheat gluten, a cohesive network of strong endosperm proteins that stretch with the expansion of fermenting dough, and yet congeal and hold together when heated to produce a “risen” loaf of bread. The stretchable mass of gluten, with its ability to deform, expand, recover its shape, and trap gases, is critical to the production of bread and all fermented products. Of all the cereals, wheat is unique in this respect. Rye grain has this property to a lesser extent as well. Throughout the centuries, traditional bread varieties have been developed using the accumulated knowledge of craft bakers regarding how to make the best use of available raw materials to achieve the bread quality they desire. In some countries, the nature of bread-making has been preserved in its traditional form, whereas in others, it has changed considerably. The flatbreads of the Middle East and the steamed breads of China are examples of traditional bread varieties that are still a vital part of the culture of the countries in which they were originally produced and are still baked in large quantities. The number of people suffering from micronutrient malnutrition worldwide has grown rapidly during the past several decades. It can be claimed, therefore, that the current estimates of one-third to one-half of the world’s population suffering from micronutrient malnutrition2 reflect its global proliferation, particularly in developing countries. This alarming trend is thought to be caused primarily by the replacement of ancient crop varieties.1 Modern plant breeding has been historically directed toward high agronomic yield rather than nutritional quality. Increased grain yield may have resulted in lower density of minerals in grain, although evidence for the traditional bread varieties that still exist is contradictory.3 Biofortification, aimed at enhancement of micronutrient concentrations and its bioavailability in plant foods through genetic improvement, is a rational approach for diminishing the micronutrient malnutrition problems. The requisite conditions for diversification of crop species and the growing demand for nutritionally healthy food products and the therapeutic properties of foodstuff have resulted in a renewed interest in ancient wheats such

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as emmer (Triticum turgidum L. ssp. dicoccum Schrank ex Sch€ ubler) and einkorn (Triticum monococcum L.). Wheat varieties (Triticum spp.), the staple crops of early civilization, are complex, living, and dynamic species, with many mysteries yet to be discovered. The manufacture of flour for a variety of products, such as leavened and unleavened breads, cakes, biscuits, pasta, and noodles, is the most common food use of wheat grains. The physicochemical attributes of the gluten protein constitute of wheat are the primary factors contributing to the unique potential of its flour dough in bread-making.1 This unique potential in dough rheological properties and the bread-baking quality of wheat flour lead to its global distribution and utilization. The required elasticity of the dough is provided by the physicochemical properties of gluten, which makes risen dough rise to produce a loaf of baked bread. The gluten viscoelastic networks retain the carbon dioxide (CO2) gas bubbles trapped inside the fermenting dough, which is critical in the production of various fermented products and breads. Hulled wheats include species that bridge between cultivated (bread and durum) and wild wheats, and they have hulled kernels and nonfragile spikes. In addition, these ancient wheats were the earliest to be domesticated (almost 10,000 years ago) and contributed significantly to the phylogenesis of modern wheats.4 The hulled or ancient wheats, like modern wheats, exist at all three polyploidy levels: diploid (2x), tetraploid (4x), and hexaploid (6x). Emmer (T. turgidum ssp. dicoccum, 2n ¼ 4x ¼ 28), einkorn (T. monococcum L., 2n ¼ 2x ¼ 14), and spelt (Triticum spelta L., 2n ¼ 6x ¼ 42) comprise the three wheat species belonging to the group of cultivated ancient wheats. At the tetraploid level, T. turgidum L. ssp. dicoccum (Schrank ex Sch€ ubler) Thell (emmer wheat) is the domesticated form of T. turgidum L. ssp. dicoccoides (K€ orn. ex Asch. and Graebner) Thell (wild emmer), and, in turn, durum wheat (T. turgidum ssp. durum (Desf) Husn.) originated from the cultivated emmer (Fig. 1). The free-threshing hexaploid bread wheat (Triticum aestivum L., AABBDD) in common use today was most likely derived from a hulled hexaploid progenitor that originated from hybridization between the tetraploid emmer wheat (T. turgidum ssp. dicoccum, AABB) and the diploid goat grass (Aegilops tauschii, DD).5 The loss of strong glumes, which convert hulled wheat into free-threshing wheat, is considered to be an important trait for wheat domestication. The major genetic determinants of the free-threshing habit are recessive mutations at the Tg (tenacious glume) locus, accompanied by modifying effects of the dominant mutation at the q (speltoid) locus and mutations at several other loci.6 This characteristic is considered as the fundamental morphological difference between durum and emmer wheats. Emmer wheat originated in a more eastward location, in the mountains of the Fertile Crescent, an area in the Middle East stretching from Palestine, Jordan, and Lebanon to Syria, Iraq, and Iran, where its wild progenitor (T. turgidum ssp. dicoccoides) still thrives.7 As the main wheat in the Old World during the Neolithic and Bronze ages, it played a strategically important role as part of the human diet in the ancient civilizations, including those of the Assyrians, the Babylonians, and the Egyptians (Fig. 2). Later, it also spread to Ethiopia on the Abyssinian plateau, where it is still being cultivated and appreciated today. Emmer is still grown in some areas of the Balkans, Turkey, Italy, Iran, Caucasia, Ethiopia, and India. However, emmer has never been subjected to breeding programs, and only its landraces and wild forms are currently available.

FIG. 1 Spikes, spikelets, and grains of emmer wheat.

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FIG. 2 The Fertile Crescent (the area in light gray on the map) is known as the domestication site of wheat. It includes the Levant and Mesopotamia, as well as Sumer, the birthplace of the first civilization, and Mesopotamia in the Bronze Age, containing the Sumerian, Akkadian, Babylonian and Assyrian empires.

Consumers are becoming increasingly aware of the benefits of including a variety of cereal grains in their diets. Increased consumption of cereals should spawn consumer interest in seeking out breads and products made from cereal grains other than common bread wheat cultivars. The key factor in producing light-texture breads is the gluten quality of the flour. The dough properties and baking performance of wheat are determined by the structure and quantity of gluten proteins, which strongly depend on genotype. Whereas desirable gluten traits have been successfully obtained in common bread wheat, little effort has been applied in this area to other cereal crops. Increasing interest in natural and organic products has led to the rediscovery of emmer on the following grounds: • Its food characteristics, which make it especially suitable for preparing many different dishes using whole, pearled, and broken kernels and using flour and semolina to make bread, biscuits, and pasta • Its highly starch-resistant content and its nutritional and healing effects, especially in the treatment of such diseases as high blood cholesterol, colitis, and allergies • Its ability to grow in soils with conventional, low input and organic crop systems • Its superior tolerance to both abiotic and biotic stresses, such as pest, cold, heat, drought, and salinity • Its use as a potential source of genes for economically important traits in wheat breeding programs Its cultivation is especially significant in marginal areas of high altitude, where its low input requirements and cold resistance make the crop economically viable.

TYPE OF UTILIZATION Like einkorn and spelt, emmer is a hulled wheat. In other words, it has tough glumes (husks) that enclose the grains, as well as a semibrittle rachis. Upon threshing, a hulled wheat spike breaks up into spikelets. Thus, hulled wheats are

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mainly characterized by keeping their glumes adhered to the grain after threshing, as well as their semibrittle rachis. Milling or pounding is required to release the grains from glumes. Cultivation of the crop is associated with complications and challenges related to the harvesting techniques used and the need to dehisce the spikelets to obtain the grain for human consumption. Emmer’s main use is for human food, though it is also used for animal feed. While it is principally used in breadmaking, it is also used to create a number of other dishes, particularly in rural areas. It may be ground into flour and baked into unfermented bread (pancake). Some people crush it and cook it with milk or water to make a soft porridge. Ethnographic analyses judged by the taste and texture standards of traditional bread in some emmer-growing areas suggest that emmer makes good bread, and this is supported by evidence of its widespread consumption as bread in ancient civilizations. Emmer bread is widely available in Switzerland and found as pane di farro in bakeries in some parts of Italy. Its use for making pasta is a recent response to the health food market, though some believe that emmer pasta has an unattractive texture.8 Bulgur (made of cracked grains of emmer) is also mixed with boiling water and butter to produce a harder porridge. Emmer has been traditionally consumed as bulgur whole grains in different kinds of soups worldwide. In some rural places in Iran and Italy, emmer is also used like rice. Being rich in fiber, protein, minerals, carotenoids, antioxidant compounds, and vitamins, emmer becomes a complete protein source when combined with legumes, making emmer bread and pasta ideal for vegetarians or for anyone simply looking for a plant-based, high-quality protein source.

BREAD-MAKING HISTORY Bread has a long history and, indeed, will have a long future. It is a nourishing food that can be stored to be eaten at later time, which is a desirable attribute that enabled civilizations throughout history to survive. After ancient citizens initiated farming, they strived to develop tools to process the harvested crops, as well as procedures to cook the grains. The first bread was a type of flatbread dating to Neolithic times (New Stone Age), which began in approximately 8000 to 10,000 BC. At that time, bread was produced from emmer and einkorn wheat grains. It consisted of hand-crushed grain mixed with water, which was then laid on a heated stone and covered with hot ashes. People in Sumeria, in southern Mesopotamia, were the first to bake leavened bread. At approximately 6000 BC, they started to mix sourdough with unleavened dough. Sourdough is generated during the natural yeasting process of flour and water, during which CO2 is formed, which in turn causes the dough to rise. The Sumerians passed on their style of preparing bread to the Egyptians in approximately 3000 BC. The Egyptians refined the system and added yeast to the flour. Moreover, they developed a baking oven in which it was possible to bake several bread loaves simultaneously. The Egyptians experimented with adding yeast, which made the dough rise, and the leavened dough created bread that was lighter, yet bigger. The successful achievement of wheat bread loaf production by the Egyptians, the Greeks, and the Romans was considered by them as a sign of the high degree of their civilizations. Nowadays, many forms of bread are produced throughout the world. Hence, the term bread is used to describe a wide range of products with various shapes, sizes, textures, crusts, colors, elasticity, eating qualities, and flavors.

FLOUR AND BREAD FORTIFICATION WITH EMMER Some ancient wheats have a unique composition in secondary components, such as carotenoids and starch, which may play a role as functional food ingredients. Emmer is particularly appreciated for its content of resistant starch, fiber, carotenoids, and antioxidant compounds.9–12 Recently, Christopher et al.12 compared local emmer wheat with two commercial wheat cultivars for protein content, phenolic acid profile, total soluble phenolic content, type 2 diabetes relevant α-amylase, antioxidant activity, and α-glucosidase enzyme inhibitory activities under in vitro conditions. They observed the superiority of emmer wheat with its hull for its antihyperglycemic properties, total soluble phenolic content, and associated antioxidants. Although emmer flour does produce a satisfactory loaf of bread, the quality is not as good as bread made with common wheat. This may have led to emmer and spelt being attributed a higher protein quality than modern wheats, simply because they are mainly used as whole-meal or low-refined flours. However, it should be noted that lysine content and the chemical score are higher in whole grain than in white flour. White flour almost exclusively comprises the endosperm portion of the grains and excludes the bran (aleurone and pericarp layer), which is rich in globulins and albumins as well as essential amino acids such as lysine. Galterio and

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coworkers13 reported high lysine content (3.1%) for emmer grains. In contrast, it has been known that common wheat protein is deficient in lysine. The poor gluten quality of emmer is confirmed by its low gluten index value.13,14 In addition, Konvalina and coworkers15 stated that emmer has a high grain protein content, whereas the quality of its gluten is inferior to that of bread wheat, as determined by the gluten index and the Zeleny test. It is assumed that the poor gluten quality of emmer is due to its storage protein composition, which is dominated by high concentrations of the intermediate-molecular-weight glutenin (IMWG) group (50–78 KDa), poor synthesis of low-molecular-weight glutenin (LMWG) subunits (30–45 KDa), and the absence of gliadin fractions γ-42 and γ-45.13,16 The pH values of emmer sourdoughs were slightly greater than common wheat sourdoughs, but the concentration of free amino acids, phytase activity, and titratable acidity were higher than in wheat sourdoughs.17 Sensory analysis indicated that emmer flour can be made into fair products of bread.17 The potential use of emmer flour in bread-making could be more promising if it is blended with bread wheat flour. Consequently, the high lysine and low gluten content of emmer wheat could complement those of wheat flour, which is poor in lysine but rich in gluten content. During the last few decades, increasing attention has been paid to phytonutrients, which have significant effects on the reduction of the incidence of aging-related and chronic human diseases. Among the numerous antioxidant compounds present in foods, lipid-soluble antioxidants play a vital role in disease prevention. Interestingly, the natural antioxidant activity of these compounds might complement their positive functional characteristics in maintaining the freshness and shelf-life of food products. Wheat, as the major staple food for humans, is not only a source of energy and protein, but also of such antioxidant compounds. In bread wheat, however, the concentration of carotenoids and other antioxidants is low; these substances are more abundant in emmer wheat. In wheat, whole-grain flour and its bran fraction are a reliable source of fiber, especially the water-insoluble type. In contrast, white flour is not rich in total fiber but is relatively rich in soluble fiber. Epidemiological studies reveal a strong relationship between low fiber intake and many disease conditions, particularly those of the gastrointestinal tract.17 In developing countries, it is believed that a large amount of plant fibers consumed by people in rural areas protected them against many diseases common to people in urban areas, such as cardiovascular diseases, colon cancer, diverticulae, appendicitis, hemorrhoids, and varicose veins of the legs. Research relates high-fiber diets to decreased blood pressure in normal as well as in hypertensive subjects.18 For elevated blood serum lipids, dietary recommendations include increasing carbohydrate consumption to make up 65% of total daily calories, emphasizing complex carbohydrates from natural sources because they influence the absorption of fat-soluble substances from the digestive tract and the reabsorption of bile acids and neutral steroils. These recommendations are given to diabetics as well because cardiovascular disease is the most likely cause of death in these people.19 Therefore, there is growing evidence that high-fiber diets, especially those containing cereal fibers, have definite health benefits in reducing the risk of chronic diseases such as diabetes, cancer, and coronary heart disease. A diet rich in complex carbohydrates improves glucose metabolism in diabetic subjects by increasing their sensitivity to insulin, resulting in reduced dosage requirements.18 Moreover, a high-fiber diet is positively associated with the control of obesity and physical gastrointestinal tract disorders. Accordingly, consumers will be interested in utilizing functional cereal products, which will enhance their health and help them to avoid being overweight. Thus, high-fiber cereal products will be in demand and undoubtedly consumed in great quantities. However, as with most food items, the major criteria for consumer acceptability of cereal products are good flavor and texture. In other words, consumers expect functional cereal products, such as high-fiber breads, to have at least similar good-quality features as standard wheat bread. Hence, emmer flour can fully or partially replace wheat flour in bread products, in order to exploit the advantages of the higher fiber content of emmer wheat.

EMMER IMPACT ON COMBATING MALNUTRITION Considering the growing requirements for richness, diversity, and good quality of food products, the interest in emmer wheat has been greater than ever.20 Perrino et al.21 found high mean values of protein (17.1%) in the grains of 50 emmer accessions. There is also a belief that the gluten structure of emmer differs from that of modern wheat, so people with gluten allergies can safely use it without any adverse effects. Improvement in dietary quality may be the ultimate solution to micronutrient malnutrition in billions of people living in developing countries, which is basically the consequence of their extensive consumption of staple cereals with low quantities of available micronutrients.22,23 Therefore, micronutrient malnutrition is considered a great concern worldwide. Currently, enrichment of staple food crops with mineral nutrients is a high-priority research area as a temporary solution to this problem; however, the major strategy to improve the level of mineral nutrients is to exploit the natural genetic variation in grain concentrations of micronutrients in food crops. With the exception of Ca2+, modern

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wheat cultivars with a greater yield potential possess lower grain concentrations of mineral nutrients than the previous cultivars with a lower yield.24 Hence, modern wheat germplasm cannot contribute genetic potential to the development of new genotypes with higher levels of mineral nutrients. On the other hand, current research indicates that primitive wheats, such as T. monococum (einkorn wheat) and T. turgidum ssp. dicoccum (emmer wheat), contain a promising germplasm for improving grain micronutrient concentrations.25,26 To combat global micronutrient malnutrition, therefore, it is essential to approach fortifying and enriching staple food crops with mineral elements as an immediate but only temporary solution. International breeding efforts exploit biodiversity to improve the mineral nutrient levels in food crops. It is well established that modern, high-yielding wheat cultivars have lower concentrations of mineral nutrients than low-yielding older ones do.3,24 Alone, the germplasm of modern wheat cannot provide the genetic diversity required to develop new wheat cultivars with sufficiently high-grain mineral nutrient concentrations. On the other hand, ancient wheats, such as einkorn, emmer, and spelt, contain the germplasm resources that can be exploited to improve the micronutrient value of wheat grains.25,27

FUTURE DIRECTION OF RESEARCH The danger of genetic erosion of crop plants and the potential consequences for agriculture will be evident when their wild and primitive progenitors are considered throughout plant domestication and subsequent breeding. The challenge is to exploit the mostly unrealized potential of ancestral species as a component of sustainable crop production, particularly under less favorable environmental conditions. Therefore, the major task of modern breeders is not only to identify the valuable and outstanding traits of the primitive ancestors of crop plants and introduce them into cultivated crops, but also to undertake genetic improvement projects that address the crops themselves, particularly the domesticated ones. Lage et al.28 demonstrated that genetic variation for quality in tetraploid emmer wheat could be transferred to synthetic hexaploid wheats and combined with plump grains and high grain weight for the purpose of bread wheat improvement. Like other ancient kinds of wheat, emmer is high in protein, fiber, minerals, and phytochemicals. It is also considered to be very valuable in breeding programs for improving wheat cultivars for higher concentration and better composition of health-beneficial phytochemicals. In particular, studies on the genetic diversity of the nutritional and health-beneficial properties of emmer should be carried out in order to explore its potential in breeding programs and for improving the quality of both emmer and bread wheats. By collaborating with the private sector (i.e., millers), modern emmer products that suit the tastes of urban consumers can be developed either by blending the flours of emmer and common wheats or by utilizing the flour of emmer by itself. Diversification in emmer products in the flour industry can be promoted through models coming from countries like Italy that successfully produce emmer products. Emmer (ancient hulled wheat) was one of the first cereals ever domesticated in the Fertile Crescent. Emmer grain has the characteristics of two wild wheats (including wild einkorn) and is known to have been the primary wheat grown in Asia, Africa, and Europe through the first 5000 years of recorded agriculture. But over the centuries, emmer was gradually abandoned in favor of varieties of durum and bread wheats without hulls. By the beginning of the 20th century, higher-yielding wheat cultivars had replaced emmer almost everywhere except in small parts of the world (for the list of regions see Introduction). Wheat is the most widely grown crop and has traditionally been selected for its technological functionality, resulting in the selection of hard bread wheat (Triticum aestivum L.) cultivars with a high level of strong gluten proteins, or durum wheat (Triticum tugidum ssp. durum) making yellow-colored pasta products. However, little interest has been shown in the nutritional and healthy properties of grains and its improvement through breeding programs.29 Table 1 provides a rough comparison of the whole-grain flour compositions of four groups of wheat, including emmer, durum, and bread (hard and soft) wheat based on the means of the tested genotypes within each group. In addition, in spite of the great interspecific variations observed for the nutritional values of the grain in Triticum spp., large variations between emmer wheat and common wheat are also observed for the traits. Table 2 summarizes the available data on the compositions of whole-grain flour in emmer wheat and bread wheat and compares the nutritional status of these cultivated wheats. Current evidence from clinical and epidemiological studies implies that a diet high in whole grains may have a protective role against coronary heart disease,30,31 type 2 diabetes,32,33 age-related eye diseases, and certain types of cancer.34,35 The health-advantageous properties of whole-grain wheat flour have been attributed to the levels of natural antioxidants, including flavonoids, phenolic acids, phytic acids, tocopherols, and carotenoids.36,37 Due to lack of the D genome, the inferior gluten quality of emmer wheat (AABB) compared with bread wheat (AABBDD) is not surprising. Bread-making with emmer wheat flour could be more fruitful if a blend of flours from bread and emmer wheat was used. In this way, the health-promoting compounds, including high lysine content, of 2. FLOURS AND BREADS

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TABLE 1

Whole-Grain Flour Compositions of Emmer, Durum, and Hard and Soft Common Wheats Emmer (T. turgidum ssp. dicoccum)

Durum (T. turgidum ssp. durum)

Hard common wheat (T. aestivum)

Soft common wheat (T. aestivum)

Protein (g)

12.5

12.8

14.8

13.9

Fat (g)

2.4

1.6

1.7

1.6

Total carbohydrate (including fiber) (g)

71

69.5

69.7

70.7

Total fiber (g)

2.7

2.4

2.6

2.5

Ash (g)

1.8

2.1

1.6

1.5

Calcium (g)

38

48

55

54

Phosphorus (g)

360

300

317

275

Iron (mg)

4.7

na

8.2

6.5

Moisture (g)

12.3

14

12.2

12.3

All values expressed as a dry weight percentage. na, not applicable. Source: FAO Corporate Document Repository (http://www.fao.org/documents).

TABLE 2

Mean or Range of Compositions of the Whole-Grain Flours of Emmer and Bread Wheat

Component (dry matter)

Emmer (T. turgidum ssp. dicoccum)

Reference

Bread wheat (T. aestivum)

Reference

Digestible carbohydrate (%, or g per 100 g)

71

38

73a

39

Starch (%)

65

38

68.5

40

Amylose (% starch)

25.1

41

28.4a

42

Dietary fiber (%)

9.8

43

13.4

44

Protein (%)

13.5–19.05

45,46

12.9–19.9

47

Lipid (%)

2.16a

46,48

2.8

49

Ash (%)

2.3

45

1.9

45

Phosphorus (g/kg)

5.12

50

4.18

50

Potassium (g/kg)

4.39

50

5

50

Sulfur (g/kg)

1.88

50

1.4

50

Magnesium (g/kg)

1.67

50

1.44

50

Calcium (g/kg)

0.36

50

0.43

50

Iron (mg/kg)

49

50

38

50,51

Zinc (mg/kg)

54

50

35.0

50

Manganese (mg/kg)

24

50

26

50

Copper (mg/kg)

4.1

50

3.9

50

Sodium (mg/kg)

12

50

10

50

a

Determined by either taking an average value from the genotypes reported in a reference or taking an average from the listed references.

emmer flour could supplement those of bread wheat to attain a better dietary balance.1 However, limited information is currently available on the use of emmer flour as a partial substitute for wheat flour in bread production based on physicochemical and rheological properties of dough and bread, and further research is required to address this. At present, therefore, insufficient data are available on the use of emmer wheat flour as a full or partial substitute for wheat flour in making breads, pasta, and cookies. A number of research studies have investigated the health-related and nutritional properties of emmer wheat, while comparisons between emmer and common wheat have been shown 2. FLOURS AND BREADS

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to be associated with not only genetic loci affecting these traits, but also environment and genotype by environment interaction effects. This challenge is further complicated by the epigenetic modification—that is, histone modification, noncoding ribonucleic acid (RNA) molecules, and deoxyribonucleic acid (DNA) methylation, which can be altered by environmental stimuli and affects gene and regulation expression. However, the measures that need to be taken into account in any comparative study will have to include field conditions (such as water availability and plant density) and agronomic practices that are favorable to each of these groups, as well as the requirement for reliable phenotypic assessments in the multiyear and multiple-location trials.52 This has been even more challenging in the context of the interactions of genotypes by milling method and genotypes by baking approach, as well as genotypes by environment interaction.53

SUMMARY POINTS • The danger of genetic erosion of crop plants and the potential consequences for agriculture reinforce the need for further exploitation of the unrealized potential of ancestral species like T. turgidum ssp. dicoccum (emmer wheat), the domesticated ancestor of durum and bread wheat, essentially for the improvement of grain protein, fiber, minerals, and phytochemicals. • The recently growing interest in natural and organic products has led to an emmer rediscovery, not only due to its nutritional and healthy properties, but also because it is amenable to low-input and organic farming system. • Emmer flour can fully or partially replace wheat flour in most bakery products, such as bread and pasta. Modern cooks are discovering the full flavors, textures, and nutrition of whole-grain emmer pasta and bread, while they are also exploring new ideas, such as adding emmer grains to dishes such as soups. • Further research is needed to elucidate the physical, chemical, and nutritional properties of emmer grain and to address its beneficial health effects. • The superior tolerance of emmer wheat to environmental and biotic stresses such as pests, pollution, cold, heat, drought, and salinity could help farmers to sustainably manage harsh environments and to meet their subsistence needs without depending on mechanization, chemical fertilizers, pesticides, or modern agricultural processes.

Acknowledgments I would like to thank Dr. S.A.M. Mirmohammadi Maibody for depicting the map of Fertile Crescent and granting the permission to publish this image.

References 1. Arzani A, Ashraf M. Cultivated ancient wheats (Triticum spp.): a potential source of health-beneficial food products. Compr Rev Food Sci Food Saf 2017;16:477–88. 2. Miller BDD, Welch RM. Food system strategies for preventing micronutrient malnutrition. Food Policy 2013;42:115–28. 3. Shewry PR, Pelln TK, Lovegrove A. Is modern wheat bad for health? Nat Plant 2016;2:16097. https://doi.org/10.1038/nplants.2016.97. 4. Nesbitt M, Samuel D. From staple crop to extinction? The archaeology and history of the hulled wheats, In: Padulosi S, Hammer K, Heller J, editors. Hulled Wheats: Proceedings of the 1st International Workshop on Hulled Wheats. Castelvecchio Pacoli, Italy, 21 and 22 July 1995, IPGRI, Rome; 1996. 5. Arzani A, Khalighi MR, Shiran B, Kharazian N. Evaluation of diversity in wild relatives of wheat. Czech J Genet Plant Breed 2005;41:112–7. 6. Salamini F, Ozkan H, Brandolini A, Schafer-Pregl R, Martin W. Genetics and geography of wild cereal domestication in the near east. Nat Rev Genet 2002;3:429–41. 7. Luo MC, Yang ZL, You FM, Kawahara T, Waines JG, Dvorak J. The structure of wild and domesticated emmer wheat populations, gene flow between them, and the site of emmer domestication. Theor Appl Genet 2007;114:947–59. 8. D’Antuono LF. Traditional foods and food systems: a revision of concepts emerging from qualitative surveys on-site in the Black Sea area and Italy. J Sci Food Agric 2013;93:3443–54. 9. D’Antuono LF, Galletti GC, Bocchini P. Fiber quality of emmer (Triticum dicoccum Schubler) and einkorn wheat (T. monococcum L.) landraces as determined by analytical pyroly. J Sci Food Agric 1998;78:213–9. 10. Galterio G, Codianni P, Giusti AM, Pezzarossa B, Cannella C. Assessment of the agronomical and technological characteristics of Triticum turgidum ssp. dicoccum Schrank and T. spelta L. Nahrung/Food 2003;47:54–9. 11. Serpen A, G€ okmen V, Karag€ oz A, K€ oksel H. Phytochemical quantification and total antioxidant capacities of emmer (Triticum dicoccon Schrank) and einkorn (Triticum monococcum L.) wheat landraces. J Agric Food Chem 2008;56:7285–92. 12. Christopher A, Sarkar D, Zwinge S, Shetty K. Ethnic food perspective of North Dakota common emmer wheat and relevance for health benefits targeting type 2 diabetes. J Ethnic Food 2018;5:66–74. https://doi.org/10.1016/j.jef.2018.01.002. 13. Galterio G, Cappelloni M, Desiderio E, Pogna NE. Genetic, technological and nutritional characteristics of three Italian populations of ‘farrum’ (Triticum turgidum ssp. dicoccon). J Genet Breed 1994;48:391–8.

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In: Abdel-Aal ESM, Wood P, editors. Speciality grains for food and feed. St. Paul, MN: American Association of Cereal Chemists Inc.; 2005. p. 63–8. 21. Perrino P, Infantino S, Basso P, Di Marzio A, Volpe N, Laghetti G. Valutazione e selezione di farro in ambienti marginali dell’appennino molisano. L’Informatore Agrario 1993;43:41–4. 22. Bouis HE. Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proc Nutr Soc 2003;62:403–11. 23. Cakmak I. Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant Soil 2008;302:1–17. 24. Murphy KM, Reeves PG, Jones SS. Relationship between yield and mineral nutrient concentrations in historical and modern spring wheat cultivars. Euphytica 2008;163:381–90. 25. Genc Y, McDonald GK. Domesticated emmer wheat [T. turgidum L. subsp. dicoccon (Schrank) Thell.] as a source for improvement of zinc efficiency in durum wheat. Plant Soil 2008;310:67–75. 26. Ortiz-Monasterio I, Graham RD. Breeding for trace minerals in wheat. Food Nutr Bull 2000;21:392–6. 27. Hidalgo A, Brandolini A. Nutritional properties of einkorn wheat (Triticum monococcum L.). J Sci Food Agric 2014;94:601–12. 28. Lage J, Skovmand B, Pena RJ, Andersen SB. Grain quality of emmer wheat derived synthetic hexaploid wheats. Genet Resour Crop Evol 2006;53:955–62. 29. Leenhardt F, Lyana B, Rocka E, Boussard A, Potus J, Chanliaud E, Remesya C. Genetic variability of carotenoid concentration, and lipoxygenase and peroxidase activities among cultivated wheat species and bread wheat varieties. Eur J Agron 2006;25:170–6. 30. Behall KM, Scholfield DJ, Hallfrisch J. Whole-grain diets reduce blood pressure in mildly hypercholesterolemic men and women. J Am Diet Assoc 2006;106:1445–9. 31. Jacobs DR, Pereira MA, Stumpf K, Pins JJ, Adlercreutz H. Whole grain food intake elevates serum enterolactone. Br J Nutr 2002;88:111–6. 32. Fung TT, Hu FB, Pereira MA, Liu S, Stampfer MJ, Colditz GA, Willett WC. Whole-grain intake and the risk of type 2 diabetes: a prospective study in men. Am J Clin Nutr 2002;76:535–40. 33. Montonen J, Knekt P, Jarvinen R, Aromaa A, Reunanen A. Whole-grain and fiber intake and the incidence of type 2 diabetes. Am J Clin Nutr 2003;77:622–9. 34. Chatenoud L, Tavani A, La Vecchia C, Jacobs DR, Negri E, Levi F, Franceschi S. Whole grain food intake and cancer risk. Int J Cancer 1998;77:24–8. 35. Kasum CM, Nicodemus K, Harnack LJ, Folsom AR. Whole grain intake and incident endometrial cancer: the Iowa women’s health study. Nutr Cancer 2001;39:180–6. 36. Moore J, Hao Z, Zhou K, Luther M, Costa J, Yu LL. Carotenoid, tocopherol, phenolic acid, and antioxidant properties of Maryland-grown soft wheat. J Agric Food Chem 2005;53:6649–57. 37. Mpofu A, Sapirstein HD, Beta T. Genotype and environmental variation in phenolic content, phenolic acid composition, and antioxidant activity of hard spring wheat. J Agric Food Chem 2006;54:1265–70. 38. Lacko-Bartosova M, Curna V. Nutritional characteristics of emmer wheat varieties. J Microbiol Biotechnol Food Sci 2015;4:95–8. 39. Davis KR, Cain RF, Peters LJ, LeTourneau D, McGinnis J. Evaluation of the nutrient composition of wheat. II. Proximate analysis, thiamine, riboflavin, niacin and pyridoxine. Cereal Chem 1981;58:116–20. 40. Brandolini A, Hidalgo A, Moscaritolo S. Chemical composition and pasting properties of einkorn (Triticum monococcum L. subsp. monococcum) whole meal flour. J Cereal Sci 2008;47:599–609. 41. Mohammadkhani A, Stoddard FL, Marshall DR. Survey of amylose content in Secale cereale, Triticum monococcum, T. turgidum and T. tauschii. J Cereal Sci 1998;28:273–80. 42. Regina A, Berbezy P, Kosar-Hashemi B, Li S, Cmiel M, Larroque O, Bird AR, Swain SM, Cavanagh C, Jobling SA, Li Z, Morell M. A genetic strategy generating wheat with very high amylose content. Plant Biotechnol J 2015;13:1276–86. 43. Gebruers K, Dornez E, Boros D, Fra A, Dynkowska W, Bedo Z, Rakszegi M, Delcour JA, Courtin CM. Variation in the content of dietary fiber and components thereof in wheats in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9740–9. 44. Andersson A, Andersson R, Piironen V, Lampi AM, Nystrom L, Boros D, Fras A, Gebruers K, Courtin CM, Delcour JA, Raskzegi M, Bedo Z, Ward JL, Shewry PR, Aman P. Contents of dietary fibre components and their relation to associated bioactive components in whole grain wheat samples from the HEALTHGRAIN diversity screen. Food Chem 2013;136:1243–8. 45. Loje H, Moller B, Laustsen AM, Hansen A. Chemical composition, functional properties and sensory profiling of einkorn (Triticum monococcum L.). J Cereal Sci 2003;37:231–40. 46. Grausgruber H, Sailer C, Ghambashidze G, Bolyos L, Ruckenbauer P. Genetic variation in agronomic and quantitative traits of ancient wheats. In: Grausgruber H, Ruckenbauer P, editors. Genetic variation for plant breeding: Proceedings of the 17th EUCARPIA General Congress. Vollman J. Vienna: BOKU–University of Natural Resources and Applied Life Sciences; 2004. p. 19–22. 47. Shewry PR, Hawkesford MJ, Piironen V, Lampi A, Gebruers K, Boros D, Andersson AAM, Aman P, Rakszegi M, Bedo Z, Ward JL. Natural variation in grain composition of wheat and related cereals. J Agric Food Chem 2013;61:8295–303. 48. Suchowilska E, Wiwart M, Borejszo Z, Packa D, Kandler W, Krska R. Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions. J Cereal Sci 2009;49:310–5. 49. Hidalgo A, Brandolini A, Ratti S. Influence of genetic and environmental factors on selected nutritional traits of Triticum monococcum. J Agric Food Chem 2009;57:6342–8. 50. Suchowilska E, Wiwart M, Kandler W, Krska R. A comparison of macro- and microelement concentrations in the whole grain of four Triticum species. Plant Soil Environ 2012;58:141–7.

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51. Zhao FJ, Su YH, Dunham SJ, Rakszegi M, Bedo Z, McGrath SP, Shewry PR. Variation in mineral micronutrient concentrations in grain of wheat lines of diverse origin. J Cereal Sci 2009;49:290–5. 52. Shewry PR, Hey S. Do “ancient” wheat species differ from modern bread wheat in their contents of bioactive components? J Cereal Sci 2015;65:236–43. 53. Kucek LK, Dyck E, Russell J, Clark L, Hamelman J, Leader SB, Senders S, Jones J, Benscher D, Davis M, Roth G, Zwinger S, Sorrells ME, Dawson JC. Evaluation of wheat and emmer varieties for artisanal baking, pasta making, and sensory quality. J Cereal Sci 2017;74:19–27.

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C H A P T E R

8 Nutritional, Technological, and Health Aspects of Einkorn Flour and Bread Alyssa Hidalgo*, and Andrea Brandolini† *Department of Food Environmental and Nutritional Sciences (DeFENS), University of Milan, Milan, Italy † CREA—Research Centre for Animal Production and Aquaculture, S. Angelo Lodigiano, Italy

O U T L I N E Introduction

99

Changes During Storage

104

Einkorn Kernel

100

Technological Issues

105

Flour Composition Starch and Dietary Fiber Proteins Lipids Vitamins and Antioxidants Microelements and Ash Enzymatic Activities

100 100 101 102 102 103 104

Adverse Reactions

107

Summary Points

107

References

108

Further Reading

110

Abbreviations DM dry matter SDS sodium dodecyl sulfate

INTRODUCTION Einkorn (Triticum monococcum L. subsp. monococcum), a close relative of durum and bread wheats, is a diploid (2n ¼ 2x ¼ 14) hulled wheat domesticated approximately 10,000 years ago in the Karacada
g region of Turkey. One of the founder crops of agriculture along with barley and emmer, einkorn spread to Europe during the Neolithic Revolution. For several thousands of years, it was the staple food of European farmers, as confirmed € by archaeological remains and by the colon content analysis of Otzi, a frozen Copper Age man found in the Alps 1 in 1991. From the Bronze Age, einkorn cultivation lost momentum because of the availability of higher-yielding, free-threshing tetraploid and hexaploid wheats. However, it was still consumed in Roman times, as well as in the ensuing Dark Ages. Nowadays, traditional einkorn crops may be found in marginal areas of the Mediterranean region, Turkey, the Balkan countries, southern Italy, southern France, Spain, and Morocco, whereas its wild progenitor, T. monococcum ssp. boeoticum, still thrives in the central and eastern parts of the Fertile Crescent. The plant is often used only for animal feeding, and the straw is employed in the construction of thatched roofs. In recent years, the trend toward low-impact and sustainable agriculture,2 coupled with a stronger interest in the nutritional aspects of food, led to the rediscovery of

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00008-3

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8. NUTRITIONAL, TECHNOLOGICAL, AND HEALTH ASPECTS OF EINKORN FLOUR AND BREAD

FIG. 1 Einkorn spikes, hulled seeds (bottom), and threshed kernels (top).

several ancient cereals, including einkorn. New research projects are assessing T. monococcum potential for human consumption in terms of both nutritional and technological quality; meanwhile, breeding programs for the constitution of high-yielding, free-threshing einkorn lines suitable for modern cropping practices are ongoing in several countries, including Italy, Germany, and Canada.

EINKORN KERNEL As mentioned in the previous section, einkorn is a hulled wheat (i.e., its kernels are tightly protected by the glumes, which must be removed before milling); a few free-threshing genotypes are nevertheless reported.3,4,5 Einkorn spikes, hulled seeds, and threshed seeds are depicted in Fig. 1. The seeds are small,6,7 with an average weight of 25–28 g/1000 kernels; however, some genotypes can reach 35 g/1000 kernels,8 a value in the lower range for bread wheat.9 Seed size has a marked influence on many compositional and qualitative traits because big, heavy kernels have a higher proportion of starchy endosperm and smaller amounts of the external pericarp and aleurone layers. The einkorn germ proportion is only marginally superior to that of bread wheat (3.1 versus 2.9%, respectively); instead, sharp differences are observed for bran (22.9 versus 16%) and endosperm (74.0 versus 81%).10 The distribution of nutrients varies considerably across the kernel. The bran has high levels of minerals, proteins, enzymes, and some antioxidants such as tocotrienols and phenolics; the germ is rich in proteins, lipids, enzymes, and tocopherols; in the starchy endosperm, there are many storage proteins (gliadins and glutenins) and carbohydrates.

FLOUR COMPOSITION Starch and Dietary Fiber Einkorn endosperm, which accumulates storage products, is mostly starch. Two polymers compose starch: the linear amylose and the branched amylopectin. Starch molecules are organized in semicrystalline granules of varying dimensions. In Triticum ssp., two types are found: large, A-type granules (12–24 μm diameter), which form shortly after anthesis and may continue to grow throughout grain filling; and small, B-type granules (5 μm), that arise some days after anthesis.11 The A-granules, which are less numerous than B-granules, constitute most of the starch mass. Einkorn A-granules (with mode diameter of 13.2 μm) are smaller than those of bread wheat (mode: 23.8 μm),12 and this difference influences several flour characteristics, including gelatinization, pasting and swelling properties, enzyme susceptibility, and solubility. 2. FLOURS AND BREADS

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TABLE 1 Einkorn Compositiona Compound

Mean

Protein (g/100 g)b

Range

18.2

15.5–22.8

Lipid (g/100 g)c

4.2

4.0–4.4

Starch (g/100 g)b

65.5

60.6–71.4

Amylose (g/100 g starch)b

25.7

23.2–28.6

8.4

5.3–13.6

78.0

61.5–115.9

Ash (g/100 g)b

2.3

2.1–2.8

Zinc (mg/kg)e

54.8

42.7–71.1

47.0

37.2–62.6

49.3

34.4–68.2

6.4

4.9–8.3

Carotenoids (mg/kg)d Tocols (mg/kg)d

Iron (mg/kg)

e e

Manganese (mg/kg) e

Copper (mg/kg) a b c d e

Principal nutritional characteristics of einkorn whole-meal flour. Data from8; 65 einkorn accessions tested. Data from28; 5 einkorn accessions tested. Data from36; 57 einkorn accessions tested. € Data from56 and Ozkan (personal communication); 54 einkorn accessions tested.

The total starch content of T. monococcum is 65.5% (range: 60.6%–71.4%; see Table 1), whereas in Triticum aestivum, the value is higher (68.5%; range: 63.0%–75.0%).8,13 The amylose fraction, which has a special role on the pasting properties of the flour and the shelf life of bread, is approximately 26%–30% of total starch (range: 15%–35%),8,14,15,16 which is similar to bread wheat. Not all starch is rapidly assimilated during digestion; the fraction that resists digestion and absorption in the human small intestine, reaching the colon, where it undergoes fermentation to yield small fatty acids, is defined as resistant starch and has physiological functions similar to those of dietary fiber. Triticum species in general have low resistant starch content (9.2%–15.0%), and einkorn has approximately 10.8%,17 albeit lower values (2.56%) were reported.14 The dietary fiber, not digestedin the small intestine, is partially or totally fermented in the large intestine, and it has beneficial effects on the regulation of several physiological functions of the human body. Dietary fiber includes all nondigestible carbohydrates (i.e., nonstarch polysaccharides, resistant starch, resistant oligosaccharides, and other nondigestible minor components). Water-insoluble compounds (cellulose, fractions of hemicellulose, and lignin) act mainly on gastrointestinal functions, easing the transit of food through the bowel; water-soluble components (β-glucan, fructan, pectin, gums, and mucilages) are more easily fermentable in the colon than insoluble dietary fiber, and reduce the colon pH, increase the number and different profiles of intestinal microorganisms, regulate the absorption of some nutrients (e.g., sugars and lipids), as well as other beneficial effects. The total dietary fiber content of T. monococcum is low (Table 1): Values below7,18,19 or around9,20 10% are reported (as opposed to 11.5%–18.3% in bread wheat). Arabinoxylans, β-glucans, and lignin, which all are components of dietary fiber, are found in limited quantities in einkorn. Total arabinoxylan content of 1.45%–2.35% and β-glucan content of 0.25%–0.35% are reported in einkorn9; similar results for β-glucan have been observed by other researchers as well.7, 14,19 Lignin concentrations are similar among various wheat species, and in einkorn, they have an average content of 2.6%, with a range between 2.25% and 3.05%.9 On the other hand, fructan (another dietary fiber component) is more abundant in T. monococcum (1.74%–1.90%)14,21 than in other wheats (1.57%, 1.26%, 1.17%, and 0.95% for bread wheat, spelt, durum wheat, and emmer, respectively).21 Although such values might seem quite low, in fact wheat already provides approximately 70% of fructans in the American diet,22 indicating that even minimal changes could bring about noticeable improvement in wheat-consuming countries.

Proteins T. monococcum kernels show a superior protein content compared to that of bread wheat,6 averaging 18 g/100 g of dry matter (DM) and often exceeding 20 g/100 g DM8,19 (Table 1). Although part of this superiority is linked to its reduced seed size (and the related higher proportion of aleurone), the endosperm is also a good source of protein: for example, concentrations ranging from 15.4% to 25.2% in refined flour of 25 accessions cropped in three locations are reported.23 2. FLOURS AND BREADS

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Genes play a significant role, as evidenced by the broad within-einkorn variation (15.5–22.8 g/100 g8), as well as by the identification of two quantitative trait loci controlling the total protein content.24 The amino acid composition of T. monococcum seed proteins and the nutritional adequacy are very similar to those of polyploid wheats. Nevertheless, when adjusted to a common protein level (16.7%), the essential amino acid content is slightly superior in einkorn than in bread wheat cultivar (cv.) Centauro (on average, 32.2% as opposed to 29.1% of total protein, respectively).20,25,26 Additionally, some einkorn accessions with a lysine content higher than bread wheat are described.7,27

Lipids Although the relative proportions of einkorn and common wheat germs are similar, einkorn has 50% greater lipid content than that of bread wheat (4.2 versus 2.8 g/100 g DM).28 The analysis of fatty acid composition identifies up to 14 different compounds (Table 2). Linoleic (C18:2n6), oleic (C18:1n9 + C18:1n7), and palmitic (C16:0) acids are by far the most abundant of these compounds, ranging between 50.9%–54.0%, 24.8%–26.4%, and 13.9%–16.7%, respectively.20,28,29 In bread wheat, linoleic acid is also the prevalent fatty acid, but palmitic acid is more abundant than oleic acid, whereas the levels of other fatty acids are similar between species. Consequently, einkorn has higher monounsaturated fatty acids (MUFAs) and lower polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) than bread wheat.28 Low SFA concentrations along with high MUFA and PUFA concentrations in food play a role in the prevention of cardiovascular diseases, as MUFA and PUFA influence lipids and cholesterol synthesis, reducing thrombosis and atherosclerosis risks. From a technological perspective, high-MUFA and low-PUFA content provides better stability to oxidation and longer shelf life of the products.28

Vitamins and Antioxidants Vitamins are organic compounds needed by organisms in limited amounts. The concentration of folate, a watersoluble form of vitamin B9 important in the prevention of neural tube defects in the fetus, was 429–678 ng/g DM in a group of five einkorns—that is, in the same range of 150 bread wheat varieties (323–774 ng/g DM).30 TABLE 2 Fatty Acids of Einkorn Wheat Meana

Range

C14:0

0.53

0.49–0.61

C15:0

0.12

0.11–0.13

C16:0

16.65

16.07–17.73

C16:1n9

0.09

0.09–0.10

Palmitoleic

C16:1n7

0.18

0.16–0.20

Margaric

C17:0

0.11

0.10–0.13

Stearic

C18:0

1.18

1.10–1.26

Fatty acid Myristic

Palmitic

b

Oleic

C18:1n9

24.77

23.23–26.51

Linoleic

C18:2n6

50.86

49.89–51.47

Arachidic

C20:0

0.18

0.15–0.19

Gadoleic

C20:1n11

2.75

2.47–3.04

Linolenic

C18:3n3

1.95

1.82–2.08

Behenic

C22:0

0.23

0.19–0.25

Lignoceric

C24:0

0.41

0.37–0.45

SFA

19.4

18.7–20.6

MUFA

27.8

26.3–29.2

PUFA

52.8

51.9–53.3

a Mean (and range) relative percentages of fatty acids in einkorn whole meal; data from28; 5 einkorn accessions tested. b (C18:1n9 + C18:1n7).

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Carotenoids are lipid-soluble antioxidants: Some carotenoids (α- and β-carotenes) are involved in the biosynthesis of vitamin A, which is essential for many biological functions,31 while others (e.g., lutein and zeaxanthin) protect cells and tissues from free radicals, contributing to eyesight maintenance, inhibition of some cancers, and prevention of degenerative and cardiovascular diseases.32,33 Carotenoid concentration in einkorn whole meal is far superior to those of the polyploid wheats.6,23,34 Detailed studies, performed by high-performance liquid chromatography (HPLC), found that in all Triticum species, the yellow pigment consist mostly of lutein (>90%).35,36,37 While the highest concentration is in the germ (32.1 mg/kg), lutein is also detected in the endosperm (6.3 mg/kg) and bran (4.3 mg/kg).10 In einkorn whole meal, lutein content averages 8.4–8.5 mg/kg,35,36 but values greater than 10 mg/kg, up to a maximum of 13.64 mg/kg DM (see Table 1), were observed.36 Furthermore, several accessions showed significant amounts of carotenes (>25% of total carotenoids), sometimes together with high lutein content. Therefore, einkorn carotenoid concentration is four to eight times higher than that of bread wheat and twice that of durum wheat, in which the yellow color of the semolina and the derived pasta is perceived as an important quality. Interestingly, color perception in refined flour is not only related to carotenoid content, but also to flour granulometry.5 Tocols (vitamin E) consist of tocopherols and tocotrienols, each including four derivatives (α, β, γ, and δ). They protect against chronic pathologies such as cardiovascular and neurological disorders, cancer, cataracts, and inflammatory diseases. The total tocol content of einkorn is higher than that of bread and durum wheats,36,38,39 with an average concentration of 77.96 mg/kg DM and a maximum value of 115.85 mg/kg DM (see Table 1); as a comparison, the tocol content of T. aestivum and Triticum turgidum is approximately 62.75 and 52.91 mg/kg DM, respectively.36 In T. monococcum, the most abundant compound is β-tocotrienol (61.9% of the total), followed by α-tocotrienol (16.4%), α-tocopherol (15.6%), and β-tocopherol (6.1%). While tocopherols are almost exclusively present in the germ, tocotrienols are more abundant in the bran fraction, although significant quantities are present in the germ and in the flour.10 The mean tocotrienol:tocopherol ratio (3.68) is higher than those of T. aestivum and T. turgidum (2.97 and 1.79, respectively). Notwithstanding the lower concentrations, the endosperm contributes the majority of lutein (76.6%), as well as one-fourth of total tocols,10 indicating that the refined flour still retains good nutritional value. Polyphenols (phenolic acids, flavonoids, alkylresorcinols, etc.), mainly localized in the external layers of the kernel, are plant metabolites essential for plant growth and reproduction, and they are produced as a response against plant pathogens. In humans, they exert a protective role against oxidative damage diseases such as coronary heart disease, stroke, and cancers.40 The phenolic acids are present in soluble-free, soluble-bound, and insoluble-bound forms. Insoluble-bound phenolics, linked to cell-wall structural components such as cellulose, lignin, and proteins, are more abundant than the soluble forms. In einkorn and in bread wheat, ferulic acid is the main phenolic component of both the soluble and the insoluble fractions. Einkorn and bread wheat have similar free total polyphenols41,42,43 and total phenolic acid content,44,45 but T. monococcum has a higher concentration of conjugated phenolic acids, along with a lower content of bound phenolic acids.46 Overall, the total phenolic acid content of einkorn is in the range 449–816 mg/kg DM.44,46 Flavonoid content in six einkorn accessions, instead, averaged 1.13  0.28 μmol/g (range, 0.80–1.59 μmol/g) versus 1.32 0.04 μmol/g for bread wheat.45 Alkylresorcinols, molecules with antimicrobial activity and have effects on biological membranes, are more abundant in einkorn whole-meal flours (595 mg/kg) than bread wheat (410–416 mg/kg) and durum wheat (399 mg/kg); the relative homologue composition in einkorn was C17:0 (2%), C19:0 (14%), C21:0 (47%), C23:0 (27%), and C25:0 (10%).47 Phytosterols reduce serum and/or plasma total cholesterol and low-density lipoprotein (LDL) cholesterol levels, have anti-inflammatory, anti-atherogenicity, and antioxidation activities and may offer protection against some cancers, such as colon, breast, and prostate cancer.48,49,50 Cereals have moderate sterol content but, as the staple food for humans, they provide relevant amounts to the human diet. Phytosterol content is highest in the germ and bran fraction.51 Einkorn is the Triticum species with the highest concentration, averaging 1054 mg/kg (range: 976–1187 mg/kg), which is 25% higher than winter wheat. In T. monococcum. The main phytosterol is sitosterol (500 mg/kg DM; 47% of all sterols), followed by total stanols (229 mg/kg; 22%), campesterol (195 mg/kg; 19%) and other compounds (130 mg/kg; 12%).52 As a result of its peculiar antioxidant content and composition, radical scavenging activity of einkorn whole-meal flour is superior to that of bread wheat whole-meal flour.43,45,53,54,55

Microelements and Ash Cereals and cereal-based foods are extensively consumed worldwide, and as such, they represent the primary source of iron and zinc for humans in many developing countries. However, cereals are inherently poor in their 2. FLOURS AND BREADS

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concentration and bioavailability, leading to a low intake of these micronutrients, and approximately half of the world population suffers from micronutrient deficiencies. Iron and zinc deficiencies are responsible for health problems such as impairment of the immune system, disturbed physical growth and mental and cognitive development, and increased rates of anemia, morbidity, and mortality. Some researchers have evaluated microelement variation in the cultivated wheat gene pool as a way to improve the nutritional value of cereals. A broad genotypic variation for micronutrient content was observed in the seeds of 54 einkorn accessions, with mean values of 47.04 mg/kg for iron, 54.81 mg/kg for zinc, 49.29 mg/kg for manganese, and 6.40 mg/kg for copper.56 In general, T. monococcum averages higher concentrations than T. aestivum for most microelements, including iron (45.9–52 versus 36–38.2 mg/kg, respectively)57,58,59 zinc (53–72 versus 35 mg/kg),58,59 manganese (28–46 versus 26–30 mg/kg),58,59 copper (9 versus 6 mg/kg),58 strontium (5.4 versus 3.0 mg/kg),59 molybdenum (1.2 versus 0.65 mg/kg),59 magnesium (1.5–1.6 versus 1.1–1.4 g/kg),58,59 phosphorus (5.2–5.4 versus 3.1–4.7 g/kg)58,59 and selenium (50.0–54.8 versus 29.8–39.9 μg/kg42; 178.5–440 versus 32.9–237.9 μg/kg57). On the other hand, potassium and calcium content did not differ between the two species. However, whole-meal flour contains phytic acid, which is a strong chelator of positively charged mineral cations such as calcium, iron, and zinc. Phytic acid may bind minerals, and the resulting salts are excreted, thus limiting the absorption of several micronutrients. Not surprisingly, the total ash content of einkorn whole meal is high, ranging from 2.1 to 2.8%7,8,34; in comparison, the mean ash content of bread wheat is mostly less than 2.0%.

Enzymatic Activities Whole-meal flour contains many enzymes, some of which (e.g., amylases, proteases, oxidases, and lipases) are active during food processing. Starch degradation is catalyzed by alpha- and beta-amylases. Alpha-amylases randomly hydrolyze α1–4 bonds, but not α1–6 bonds, thus determining the formation of linear and branched oligosaccharides, maltotriose, glucose, and maltose. These simple reducing sugars are metabolized by yeasts during leavening, and also are substrates of the Maillard reaction. Alpha-amylase activity may be enhanced by late rainfall on the ripe kernels, but decreases during flour aging.60 Genetic differences among wheat species exist: Lower alpha-amylase activity was observed61 in wholemeal flours of einkorn than in those of T. turgidum and T. aestivum (0.20  0.006, 0.24  0.030 and 0.29  0.032 Ceralpha U/g DM, respectively). Beta-amylases lead to the complete hydrolysis of amylose to maltose, while amylopectin gives maltose (60%) and dextrines (40%). T. monococcum displays significantly lower β-amylase activity than T. turgidum and T. aestivum (12.0  0.36, 48.6  1.44 and 58.5  2.29 Betamyl-3 U/g DM, respectively),61 and this influences the quality of the end product, as high beta-amylase activity leads to excessive heat damage (Maillard reaction) in baking products because of greater maltose formation during the mixing step.3,4 Lipoxygenase (LOX) catalyzes the oxidation of PUFAs (mainly linoleic and linolenic acids) to fatty acid radicals, which are responsible for the oxidative degradation of some antioxidant compounds, such as carotenoids and tocols: Therefore, high LOX activity is detrimental to the nutritional value of the end products. A study performed on 57 accessions from different Triticum species showed that einkorn exhibited lower LOX activity (0.12–0.91 μmol/min g DM) than both durum and bread wheat (3.48  0.701 and 8.02  0.492 μmol/min g DM, respectively),62 confirming preliminary observations.63 Polyphenol oxidase (PPO) catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones, which react with amines and thiol groups or polymerize nonenzymatically into dark or brown products,64 causing undesirable darkening of products and polyphenol degradation. PPO is abundant in the bran and in the germ. In a trial performed on 59 accessions from different wheat species cropped over two years, T. monoccocum whole-meal flours showed a higher polyphenol oxidase than T. turgidum and T. aestivum (362.1  9.46, 340.4  9.34, and 307.2  6.16 U/g DM).61 Peroxidases are enzymes that utilize peroxide to oxidize a wide range of hydrogen donors, including phenolics, ascorbic acid, amines, and nitrite. No significant genetic differences among einkorn, durum, and bread wheat were found63 in a study considering one sample from each species.

Changes During Storage Several factors influence the postmilling nutritional value of whole-meal and refined flours, including storage conditions and technological transformations. Low conservation temperatures preserve over long periods of time the antioxidant properties of freshly milled flours, which are instead quickly lost under more exacting conditions, such as storage at temperatures higher than 20°C.65,66

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TECHNOLOGICAL ISSUES During its heyday, T. monococcum was mainly eaten as porridge or plainly cooked; this use did not require leavening, a process discovered and employed for bread-making by the Egyptians. In more recent times, einkorn was preferentially fed to animals, keeping for humans the threshable durum and bread wheats. Therefore, no selection whatsoever favoring bread-making attitude was exerted on einkorn during the early history, and a similar trend was probably maintained ever after. As a result, until recently, einkorn was deemed unsuitable for the manufacture of bakery products because of its sticky dough and inadequate rheological properties. In fact, Brabender farinograph stability and degree of softening, as well as Chopin alveograph gluten strength, are generally poor.23,67 Nevertheless, breads with loaf volumes and characteristics similar to those of bread wheat are sometimes obtained.68 The screening of a broad collection (>1000 accessions) of T. monococcum ssp. monococcum and ssp. boeoticum accessions led to the identification of several genotypes having sodium dodecyl sulfate (SDS) sedimentation (a small-scale test for evaluating the baking attitude of wheat flours) values greater than 60 mL,6 which is a threshold for breadmaking potential. Further analysis of the most promising genotypes showed alveograph values and farinograph stability indices similar to those of bread wheat,6,23 thus opening new horizons for the use of this ancient crop. The breads prepared from einkorn refined flour following standard microbaking tests showed a broad volume variation, ranging from very poor to outstanding (Fig. 2); the finest samples compared favorably with the best bread wheats.6,23 The characteristics that differentiate breads prepared with the refined flour of T. monococcum from those prepared with T. turgidum or T. aestivum are the appealing, deep-yellow color of the crumbs and the intense flavor.67 Whereas only some einkorn genotypes are suitable for bread-making, all the accessions show excellent attitude for the preparation of other bakery products, such as cookies and pastry (Fig. 3). Bread-making quality is strictly related to the composition of storage protein. Compared to bread wheat, einkorn flour is characterized by a very high gliadin/glutenin ratio and low amounts of high-molecular-weight glutenins.69 Electrophoretic analysis of 668 T. monococcum ssp. monococcum accessions detected 39 glutenin bands (six x subunits and eight y subunits in the high-molecular-weight fraction, and 25 in the low -molecular-weight region) and 44 gliadin bands (20 in the ω-region, 10 in the γ-region, and 14 in the α/β-region).70 Eight glutenin and eight gliadin bands correlated significantly with an increase in bread-making quality, as assessed by the SDS sedimentation test. Particularly relevant was the effect of three linked glutenin fragments that, coupled with reduced or absent ω-gliadins (Fig. 4), improved the SDS sedimentation volume from 24 to 45 mL. A molecular map, integrating restriction fragment length polymorphism (RFLP), acid polyacrylamide gel electrophoresis, and storage protein information, positioned the loci for the three glutenin and the ω-bands on the short arm of chromosome 1, a region rich in glutenin and gliadin coding genes.24 Dough stickiness and leavening, as well as end-product and staling kinetics, are also influenced by the pasting properties of the flour. Einkorn amylographic viscosity values are superior to those of spelt and common wheat and, in most instances, emmer as well.7 A Rapid Viscosity Analyzer (RVA) study of the pasting properties of 65 einkorn samples showed that einkorn has higher peak viscosity and final viscosity than T. turgidum and T. aestivum,8 a difference probably related to the smaller size and different gradings of einkorn starch granules. Some external factors show a relevant influence on bread-making. Parboilization leads to the gelatinization of starch granules and denaturation of proteins, thus inducing major changes in the technological properties of the flour. The limited information available for einkorn describes a steep decline, after low-moisture parboilization, in SDS

FIG. 2 Einkorn breads. Bread loaves prepared from refined flour of einkorn (from left, cv. Monlis, accessions ID1395 and ID331, and advanced lines SAL 98-38 and SAL 98-32) and bread wheat (cv. Blasco, right).

2. FLOURS AND BREADS

FIG. 3 Einkorn bakery products. Bakery products prepared from einkorn flour. From left to right: einkorn flour and seeds (foreground), bread and cake (center), and cookies and pasta (background).

FIG. 4

Fingerprinting of einkorn storage proteins. (Left) SDS polyacrylamide gel electrophoresis of the glutenin fraction: the asterisks indicate the three bands that correlate to bread-making quality. (Right) Acid polyacrylamide gel electrophoresis of the gliadin fraction: the asterisks indicate the scarce or absent ω-gliadin bands correlated to breadmaking quality.

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107

sedimentation values (and thus in bread-making quality), as well as a decrease in viscosity; the changes are stronger under more drastic steaming conditions.71 Storage conditions also modify the technological characteristics of the flours: SDS sedimentation values and viscosity of einkorn flour change during conservation, particularly at high temperatures (30°C and 38°C).60

ADVERSE REACTIONS Wheat is a major diet constituent for much of humankind, but wheat consumption often leads to fastidious or even life-threatening health problems. Hypersensitivity reactions to wheat flour occur both by inhalation (baker’s asthma) and ingestion (food allergy and celiac disease), but sometimes they also may arise by contact. Baker’s asthma and food allergy are abnormal immune system reactions to one or more wheat proteins. They may result in a wide range of symptoms, including rash, difficult breathing, and nausea, and sometimes might cause anaphylaxis as well. Although the immunoglobulin E (IgE)-binding action of einkorn salt extracts is similar to that of T. aestivum and T. turgidum, and many potential allergens are present in this diploid wheat,72 their inhibitory activity toward human α-amylase is minimal or nonexistent.72,73,74 Similar results are described for wheat-dependent, exerciseinduced anaphylaxis (WDEIA), an allergic reaction prompted by ω-5-gliadins.76 Celiac disease is an inflammatory immunomediated condition triggered by the gluten prolamins of several cereals (including Triticum spp.) in predisposed individuals. The main causal agent is the gliadin fraction of gluten. All three structural types of gliadins (α/β, γ, and ω) are active; nevertheless, glutenin components can exacerbate celiac disease. Two pathological effects can be distinguished: a rapid cytotoxic effect on the intestinal epithelium and an immune response involving T-cells that recognize specific prolamin epitopes. Up to 35 immunologically active sequences have been detected. The majority of these epitopes are immunogenic, stimulating specific T-cell lines and clones derived from jejunal mucosa or peripheral blood of celiac patients; however, a few of them are toxic and induce mucosal damage. Some studies suggest reduced or absent toxicity of T. monococcum,77,78 which lacks a highly immunoreactive α/βgliadins peptide (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) encoded by genes located on chromosome 6D79; genome D is absent in einkorn. Furthermore, in vitro studies show that einkorn prolamines do not agglutinate human myelogenous leukemia K562(S) cells and do not induce lesions in intestinal cell cultures of celiac patients, properties attributed to the reaction between a toxic peptide and a “protective” peptide that inhibits the agglutinating activity in K562(S) cells.78 In particular, einkorn accession ID331 could be less effective in inducing celiac disease due to its inability to activate the innate immune pathways, possibly because of a protective action of its ω-gliadins.80 However, the T. monococcum genotypes Monlis and ID331 activate the celiac disease T-cell response,81 suggesting that both einkorns are toxic for celiac patients. Moreover, the sequencing of einkorn complementary deoxyribonucleic acid (cDNA) clones related to the storage protein genes of T. monococcum revealed the presence of 4 toxic peptides and 13 immunogenic peptides82 belonging to all the storage protein classes, therefore indicating that einkorn has the full potential to induce the celiac disease syndrome. In vivo tests proved that einkorn, apparently well tolerated by the majority of celiac disease patients, is nevertheless toxic on the basis of histological and serological criteria.83,84 In conclusion, no wheat species or cultivar, including einkorn, is safe for patients with a diagnosis of celiac disease,85,86 but einkorn may be of value for patients with gluten sensitivity or for prevention of celiac disease.83,84 Another adverse reaction to cereal ingestion is irritable bowel syndrome, a chronic functional gastrointestinal disorder favored by the dietary intake of fermentable oligosaccharides, disaccharides, and monosaccharides, and polyols (FODMAP). Its symptoms are alleviated by a diet low in specific short-chain carbohydrates,87 and so wheats with low FODMAP concentrations should be prioritized. Unfortunately, einkorn has a higher fructans content than most other wheats,88,21 albeit still within the bread wheat range.21 However, long dough proofing times (>4 h) effectively diminish FODMAPs in the final product by up to 90%,21 leading to wheat bakery products suitable for consumption by patients with irritable bowel syndrome.

SUMMARY POINTS • Einkorn is a hulled wheat—that is, after harvesting, its seeds are tightly enclosed by glumes. • Domesticated in the Fertile Crescent approximately 12,000 years ago, it was instrumental in the diffusion of agriculture. • Broadly cropped and eaten in Europe and the Near East for several thousand years, it was replaced by the more productive durum and bread wheats during the Bronze Age.

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• Einkorn kernels have higher protein and antioxidant content (i.e., carotenoids and tocols) than other wheats. • The lipidic fraction is rich in MUFAs. • Some genotypes show very good bread-making attitude, producing outstanding bread loaves with appealing deep yellow crumbs. • All einkorns are suitable for bakery product preparation. • Einkorn flour apparently elicits weaker reactions than other wheats, but it is toxic for celiac patients.

References 1. Oeggl K. The diet of the Iceman. In: Bortenschlager S, Oeggl K, editors. The Iceman and his natural environment: palaeobotanical results. Wien-New York: Springer; 2000. p. 89–116. 2. Hidalgo A, Brandolini A. Nitrogen fertilisation effects on technological parameters and carotenoid, tocol and phenolic acid content of einkorn (Triticum monococcum L. subsp. monococcum): a two-year evaluation. J Cereal Sci 2017;73:18–24. 3. Hidalgo A, Brandolini A. Evaluation of heat damage, sugars, amylases and colour in breads from einkorn, durum and bread wheat flour. J Cereal Sci 2011;54:90–7. 4. Hidalgo A, Brandolini A. Heat damage of water biscuits from einkorn, durum and bread wheat flours. Food Chem 2011;128:471–8. 5. Hidalgo A, Fongaro L, Brandolini A. Wheat flour granulometry determines colour perception. Food Res Int 2014;64:363–70. 6. Borghi B, Castagna R, Corbellini M, Heun M, Salamini F. Breadmaking quality of einkorn wheat (Triticum monococcum ssp. monococcum). Cereal Chem 1996;73:208–14. 7. Løje H, Møller B, Laustsen AM, Hansen Å. Chemical composition, functional properties and sensory profiling of einkorn (Triticum monococcum L.). J Cereal Sci 2003;37:231–40. 8. Brandolini A, Hidalgo A, Moscaritolo S. Chemical composition and pasting properties of einkorn (Triticum monococcum L. subsp. monococcum) whole meal flour. J Cereal Sci 2008;47:599–609. 9. Gebruers K, Dornez E, Boros D, Fra A, Dynkowska W, Bedo Z, Rakszegl M, Delcour JA, Courtin CM. Variation in the content of dietary fiber and components thereof in wheats in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9740–9. 10. Hidalgo A, Brandolini A. Protein, ash, lutein and tocols distribution in einkorn (Triticum monococcum spp. monococcum) seed fractions. Food Chem 2008;107:444–8. 11. Morrison WR, Gadan H. The amylose and lipid contents of starch granules in developing wheat endosperm. J Cereal Sci 1987;5:263–75. 12. Stoddard FL. Survey of starch particle-size distribution in wheat and related species. Cereal Chem 1999;76:145–9. 13. Lineback DR, Rasper VF. Wheat carbohydrates. In: Pomeranz Y, editor. Wheat chemistry and technology. St. Paul, Minnesota, USA: American Association of Cereal Chemists, Inc; 1998. p. 277–372. 14. Brandolini A, Hidalgo A, Plizzari L, Erba D. Impact of genetic and environmental factors on einkorn wheat (Triticum monococcum L. subsp. monococcum) polysaccharides. J Cereal Sci 2011;53:65–72. 15. Mohammadkhani A, Stoddard FL, Marshall DR. Survey of amylose content in Secale cereale, Triticum monococcum, T. turgidum and T. tauschii. J Cereal Sci 1998;28:273–80. 16. Rodríguez-Quijano M, Vásquez JF, Carrillo JM. Waxy proteins and amylose content in diploid Triticeae species with genomes A, S and D. Plant Breed 2004;123:294–6. 17. Shewry PR, Hey S. Do “ancient” wheat species differ from modern bread wheat in their contents of bioactive components? J Cereal Sci 2015;65:236–43. 18. Abdel-Aal E-SM, Hucl P, Sosulski FW. Compositional and nutritional characteristics of spring einkorn and spelt wheats. Cereal Chem 1995;72:621–4. 19. Grausgruber H, Scheiblauer J, Sch€ onlecher R, Ruckenbauer P, Berghofer E. Variability in chemical composition and biologically active constituents of cereals. In: Vollman J, Grausgruber H, Ruckenbauer P, editors. Genetic variation for plant breeding: Proceedings 17th EUCARPIA eneral Congress. Tulln, Austria, 8–11 September; 2004. p. 23–6. 20. Gabrovská D, Fiedlerová V, Holasová M, Mascková E, Smrcinov H, Winterová R, Michalová A, Hutar M. The nutritional evaluation of underutilized cereals and buckwheat. Food Nutr Bull 2002;23:246–53. urschum T, Schweiggert RM, Carle R. Wheat and the irritable bowel syndrome-FODMAP levels of modern 21. Ziegler JU, Steiner D, Longin CFH, W€ and ancient species and their retention during bread making. J Funct Foods 2016;25:257–66. 22. Moshfegh AJ, Friday JE, Goldman JP, Chug Ahuja JK. 1999. Presence of inulin and oligofructose in the diets of the Americans. J Nutr 1999;129:1407–11. 23. Corbellini M, Empilli S, Vaccino P, Brandolini A, Borghi B, Heun M, Salamini F. Einkorn characterization for bread and cookie production in relation to protein subunit composition. Cereal Chem 1999;76:727–33. 24. Taenzler B, Esposti RF, Vaccino P, Brandolini A, Effgen S, Heun M, Sch€afer-Pregl R, Borghi B, Salamini F. A molecular linkage map of einkorn wheat: mapping of storage-protein and soft-glume genes and bread making QTLs. Genet Res Camb 2002;80:131–43. 25. Abdel-Aal E-SM, Hucl P. Amino acid composition and in vitro protein digestibility of selected ancient wheats and their end products. J Food Compos Anal 2002;15:737–47. 26. Acquistucci R, D’Egidio MG, Vallega V. Amino acid composition of selected strains of diploid wheat Triticum monococcum L. Cereal Chem 1995;72:213–6. 27. Matuz J, Bartók T, Mórocz-Salamon K, Bóna L. Structure and potential allergenic character of cereal proteins. I. Protein content and amino acid composition. Cereal Res Commun 2000;28:263–70. 28. Hidalgo A, Brandolini A, Ratti S. Influence of genetic and environmental factors on selected nutritional traits of Triticum monococcum. J Agric Food Chem 2009;57:6342–8. 29. Suchowilska E, Wiwart M, Borejszo Z, Packa D, Kandler W, Krska R. Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions. J Cereal Sci 2009;49:310–5.

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30. Piironen V, Edelmann M, Kariluoto S, Bedo Z. Folate in wheat genotypes in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9726–31. 31. Zile MH. Vitamin A and embryonic development: an overview. J Nutr 1998;128:455S–458S. 32. Krinsky NI. The biological properties of carotenoids. Pure Appl Chem 1994;66:1003–10. 33. Rao AV, Rao LG. Carotenoids and human health. Pharmacol Res 2007;55:207–16. 34. D’Egidio MG, Nardi S, Vallega V. Grain, flour and dough characteristics of selected strains of diploid wheat Triticum monococcum L. Cereal Chem 1993;70:298–303. 35. Abdel-Aal E-SM, Young JC, Wood PJ, Rabalski I, Hucl P, Falk D, Fregeau-Reid J. Einkorn: a potential candidate for developing high lutein wheat. Cereal Chem 2002;79:455–7. 36. Hidalgo A, Brandolini A, Pompei C, Piscozzi R. Carotenoids and tocols of einkorn wheat (Triticum monococcum ssp. monococcum L.). J Cereal Sci 2006;44:182–93. 37. Abdel-Aal E-SM, Rabalski I. Bioactive compounds and their antioxidant capacity in selected primitive and modern wheat species. Open Agric J 2008;2:7–14. 38. Lachman J, Hejtmánková K, Kotíková Z. Tocols and carotenoids of einkorn, emmer and spring wheat varieties: selection for breeding and production. J Cereal Sci 2013;57:207–14. 39. Lampi A-M, Nurmi T, Ollilainen V, Piironen V. Tocopherols and tocotrienols in wheat genotypes in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9716–21. 40. Quiñones M, Miguel M, Aleixandre A. Beneficial effects of polyphenols on cardiovascular disease. Pharmacol Res 2013;68:125–31. 41. Brandolini A, Hidalgo A, Gabriele S, Heun M. Chemical composition of wild and feral diploid wheats and their bearing on domesticated wheats. J Cereal Sci 2015;63:122–7. 42. Lachman J, Miholová D, Pivec V, Jíru K, Janovská D. Content of phenolic antioxidants and selenium in grain of einkorn (Triticum monococcum) emmer (Triticum dicoccum) and spring wheat (Triticum aestivum) varieties. Plant Soil Environ 2011;57:2235–43. 43. Lavelli V, Hidalgo A, Pompei C, Brandolini A. Radical scavenging activity of einkorn (Triticum monococcum L. subsp. monococcum) wholemeal flour and its relationship to soluble phenolic and lipophilic antioxidant content. J Cereal Sci 2009;49:319–21. 44. Li L, Shewry R, Ward JL. Phenolic acids in wheat varieties in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9732–9. 45. Serpen A, G€ okmen V, Karag€ oz A, K€ oksel H. Phytochemical quantification and total antioxidant capacities of emmer (Triticum dicoccum Schrank) and einkorn (Triticum monococcum L.) wheat landraces. J Agric Food Chem 2008;567285–92. 46. Brandolini A, Castoldi P, Plizzari L, Hidalgo A. Phenolic acids composition, total polyphenols content and antioxidant activity of Triticum monococcum, Triticum turgidum and Triticum aestivum: a two-years evaluation. J Cereal Sci 2013;58:123–31. 47. Andersson AM, Kamal-Eldin A, Fras A, Boros D, Åman P. Alkylresorcinols in wheat varieties in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9722–5. 48. Awad AB, Fink CS. Phytosterols as anticancer dietary components: evidence and mechanism of action. J Nutr 2000;130:2127–30. 49. Berger A, Jones PJ, Abumweis SS. Plant sterols: factors affecting their efficacy and safety as functional food ingredients. Lipids Health Dis 2004;3:5. 50. Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R. Efficacy and safety of plant stanols and sterols in the management of blood cholesterol levels. Mayo Clin Proc 2003;78:965–78. 51. Nystr€ om L, Paasonen A, Lampi AM, Piironen V. Total plant sterols, steryl ferulates and steryl glycosides in milling fractions of wheat and rye. J Cereal Sci 2007;45:106–15. 52. Nurmi T, Nystr€ om L, Edelmann M, Lampi AM, Piironen V. Phytosterols in wheat genotypes in the HEALTHGRAIN diversity screen. J Agric Food Chem 2008;56:9710–5. 53. Lachman J, Orsák M, Pivec V, Jírů K. Antioxidant activity of grain of einkorn (Triticum mono-coccum L.), emmer (Triticum dicoccum Schuebl [Schrank]) and spring wheat (Triticum aestivum L.) varieties. Plant Soil Environ 2012;58:15–21. 54. Fogarasi A-L, Kun S, Tanko G, Stefanovits-Banyai E, Hegyesne-Vecseri B. A comparative assessment of antioxidant properties, total phenolic content of einkorn, wheat, barley and their malts. Food Chem 2015;167:1–6. 55. Yilmaz VA, Brandolini A, Hidalgo A. Phenolic acids and antioxidant activity of wild, feral and domesticated wheats. J Cereal Sci 2015;64:168–75. € 56. Ozkan H, Brandolini A, Torun A, Altintas S, Kilian B, Braun HJ, Salamini F, Cakmac I. Natural variation and QTL identification of microelements content in seeds of einkorn wheat (Triticum monococcum), In: Buck HT, Nisi JE, Salomón N, editors. Wheat production in stressed environment, Series developments in plant breeding, Mar del Plata, Argentina12:; 2007. p. 455–62. 57. Zhao FJ, Su YH, Dunham SJ, Rakszegi M, Bedo Z, McGrath SP, Shewry PR. Variation in mineral micronutrient concentrations in grain of wheat lines of diverse origin. J Cereal Sci 2009;49:290–5. 58. Erba D, Hidalgo A, Bresciani J, Brandolini A. Environmental and genotypic influence on trace element and mineral concentrations in whole meal flour of einkorn (Triticum monococcum L. subsp. monococcum). J Cereal Sci 2011;54:250–4. 59. Suchowilska E, Wiwart M, Kandler W, Krska R. A comparison of macro-and microelement concentrations in the whole grain of four Triticum species. Plant Soil Environ 2012;58:141–7. 60. Brandolini A, Hidalgo A, Plizzari L. Storage-induced changes in einkorn (Triticum monococcum L.) and breadwheat (Triticum aestivum L. ssp. aestivum) flours. J Cereal Sci 2010;51:205–12. 61. Hidalgo A, Brusco M, Plizzari L, Brandolini A. Polyphenol oxidase, alpha-amylase and beta-amylase activities of T. monococcum, T. turgidum and T. aestivum: a two-year study. J Cereal Sci 2013;58:51–8. 62. Hidalgo A, Brandolini A. Lipoxygenase activity in whole meal flours from Triticum monococcum, Triticum turgidum and Triticum aestivum. Food Chem 2012;131:1499–503. 63. Leenhardt F, Lyan B, Rock E, Boussard A, Potus J, Chanliaud E, Remesy C. Genetic variability of carotenoid concentration, and lipoxygenase and peroxidise activities among cultivated wheat species and bread wheat varieties. Eur J Agron 2006;25:170–6. 64. Okot-Kotber M, Liavoga A, Yong K, Bagorogoza K. Activation of polyphenol oxidase in extracts of bran from several wheat (Triticum aestivum) cultivars using organic solvents, detergents, and chaotropes. J Agric Food Chem 2002;502410–7. 65. Hidalgo A, Brandolini A. Kinetics of carotenoids degradation during the storage of einkorn (Triticum monococcum L. ssp. monococcum) and breadwheat (Triticum aestivum L. ssp. aestivum) flours. J Agric Food Chem 2008;56:11300–5.

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66. Hidalgo A, Brandolini A, Pompei C. Kinetics of tocols degradation during the storage of einkorn (Triticum monococcum L. ssp. monococcum) and breadwheat (Triticum aestivum L. ssp. aestivum) flours. Food Chem 2009;116:821–7. 67. Lomolino G, Morari F, Dal Ferro N, Vincenzi S, Pasini G. Investigating the einkorn (Triticum monococcum) and common wheat (Triticum aestivum) bread crumb structure with X-ray microtomography: effects on rheological and sensory properties. Int J Food Sci Technol 2017;52:1498–507. 68. D’Egidio MG, Vallega V. Bread baking and dough mixing quality of diploid wheat Triticum monococcum L. Ital Food Beverage Technol 1994;4:6–9. 69. Wieser H, Mueller KJ, Koehler P. Studies on the protein composition and baking quality of einkorn lines. Eur Food Res Technol 2009;229:523–32. 70. Brandolini A, Vaccino P, Bruschi G. Technological properties of einkorn flour: The role of storage proteins and starch. In: Proceedings of the Tenth International Wheat Genetic Symposium, September 1–6, Paestum, Italy; 2003. p. 427–30. 71. Hidalgo A, Brandolini A, Gazza L. Influence of steaming treatment on chemical and technological characteristics of einkorn (Triticum monococcum L. ssp. monococcum) wholemeal flour. Food Chem 2008;111:549–55. 72. Larre C, Lupi R, Gombaud G, Brossard C, Branlard G, Moneret-Vautrin DA, Rogniaux H, Denery-Papini S. Assessment of allergenicity of diploid and hexaploid wheat genotypes: identification of allergens in the albumin/globulin fraction. J Proteome 2011;74:1279–89. 73. Sánchez-Monge R, García-Casado G, Malpica JM, Salcedo G. Inhibitory activities against heterologous α-amylases and in vitro allergenic reactivity of einkorn wheats. Theor Appl Genet 1996;93745–50. 74. Zevallos VF, Raker V, Tenzer S, Jimenez-Calvente C, Ashfaq-Khan M, R€ ussel N, Pickert G, Schild H, Steinbrink K, Schuppan D. Nutritional wheat amylase-trypsin inhibitors promote intestinal inflammation via activation of myeloid cells. Gastroenterology 2017;152:1100–13. 76. Lombardo C, Bolla M, Chignola R, Senna G, Rossin G, Caruso B, Tomelleri C, Cecconi D, Brandolini A, Zoccatelli G. Study on the immunoreactivity of Triticum monococcum (Einkorn) wheat in patients with wheat-dependent exercise-induced anaphylaxis for the production of hypoallergenic foods. J Agric Food Chem 2015;63:8299–306. 77. Pizzuti D, Buda A, D’Odorico A, D’Incà R, Chiarelli S, Curioni A, Martines D. Lack of intestinal mucosal toxicity of Triticum monococcum in celiac disease patients. Scand J Gastroenterol 2006;41:1305–11. 78. Vincentini O, Maialetti F, Gazza L, Silano M, Dessi M, De Vincenzi M, Pogna NE. Environmental factors of celiac disease: cytotoxicity of hulled wheat species Triticum monococcum, T. turgidum ssp. dicoccum and T. aestivum ssp spelta. J Gastroenterol Hepatol 2007;22:1816–22. 79. Mølberg O, Ulhen AK, Jensen T, Flaete NS, Fleckenstein B, Arentz-Hansen H, Raki M, Lundin KE, Sollid LM. Mapping of gluten T-cell epitopes in the bread wheat ancestors: implications for celiac disease. Gastroenterology 2005;128:393–401. 80. Iacomino G, Di Stasio L, Fierro O, Picariello G, Venezia A, Gazza L, Ferranti P, Mamone G. Protective effects of ID331 Triticum monococcum gliadin on in vitro models of the intestinal epithelium. Food Chem 2016;212:537–42. 81. Gianfrani C, Maglio M, Aufiero VR, Camarca A, Vocca I, Iaquinto G, Giardullo N, Pogna N, Troncone R, Auricchio S, Mazzarella G. Immunogenicity of monococcum wheat in celiac patients. Am J Clin Nutr 2012;96:1339–45. 82. Vaccino P, Becker HA, Brandolini A, Salamini F, Kilian B. A catalogue of Triticum monococcum genes encoding toxic and immunogenic peptides for celiac disease patients. Mol Gen Genomics 2009;281:289–300. 83. Zanini B, Basche R, Ferraresi A, Ricci C, Lanzarotto F, Marullo M, Villanacci V, Hidalgo A, Lanzini A. Randomised clinical study: gluten challenge induces symptom recurrence in only a minority of patients who meet clinical criteria for non-coeliac gluten sensitivity. Aliment Pharmacol Ther 2015;42:968–76. 84. Zanini B, Villanacci V, De Leo L, Lanzini A. Triticum monococcum in patients with celiac disease: a phase II open study on safety of prolonged daily administration. Eur J Nutr 2015;54:1027–9. 85. Prandi B, Tedeschi T, Folloni S, Galaverna G, Sforza S. Peptides from gluten digestion: a comparison between old and modern wheat varieties. Food Res Int 2017;91:92–102. 86. Šuligoj T, Gregorini A, Colomba M, Ellis HJ, Ciclitira PJ. Evaluation of the safety of ancient strains of wheat in coeliac disease reveals heterogeneous small intestinal T cell responses suggestive of coeliac toxicity. Clin Nutr 2013;32:1043–9. 87. Rao SSC, Yu S, Fedewa A. Systematic review: dietary fibre and FODMAP-restricted diet in the management of constipation and irritable bowel syndrome. Aliment Pharmacol Ther 2015;41:1256–70. 88. Brandolini A, Hidalgo A, Plizzari L, Erba D. Impact of genetic and environmental factors on einkorn wheat (Triticum monococcum L. subsp. monococcum) polysaccharides. J Cereal Sci 2011;53:65–72.

Further Reading 89. Zoccatelli G, Sega M, Bolla M, Cecconi D, Vaccino P, Rizzi C, Chignola R, Brandolini A. Expression of α-amylase inhibitors in diploid Triticum species. Food Chem 2012;135:2643–9.

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C H A P T E R

9 Maize: Composition, Bioactive Constituents, and Unleavened Bread Narpinder Singh*, Sandeep Singh†, and Khetan Shevkani‡ *Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India † Department of Food Science and Technology, Khalsa College, Amritsar, India ‡ Department of Applied Agriculture, Central University of Punjab, Bathinda, India

O U T L I N E Introduction

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Structure and Composition

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Making Unleavened Bread

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Dry and Wet Milling Dry Milling Wet Milling

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Technological Issues

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Summary Points

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Bioactive Compounds and Resistant Starch Bioactive Compounds

115 115

References

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Resistant Starch

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Abbreviations G0 G00 GI RDS RS SDS

elastic modulus viscous modulus glycemic index rapidly digestible starch resistant starch slowly digestible starch

INTRODUCTION Maize (Zea mays), also known as corn, is grown throughout the world, and >1000 million tons of corn is cultivated on approximately 185 million ha of land worldwide. The United States, China, Brazil, Argentina, India, France, and Indonesia are the main corn-producing countries. Corn of various types (flour corn, flint corn, dent corn, sweet corn, popcorn, waxy corn, and amylomaize) and color (ranging from white and yellow and red to purple) is grown. Floury corn (Zea mays var. amylacea.) also known as soft corn, has mainly white grains with rounded or flat crowns, consisting almost entirely of soft starch and a small portion of hard starch. Flint corn (Zea mays var. indurata) also known as Indian corn, has soft starch in the middle, surrounded by a hard shell, and its color ranges from white to red. Dent corn (Zea mays var. indentata) is either yellow or white in color, with a depressed crown. Sweet corn (Zea mays var. saccharata and Zea mays var. rugosa) has a higher sugar content than other corn types, and it is consumed in different forms (boiled/ roasted/frozen/canned). Popcorn (Zea mays var. everta) is mainly used for popping and has a greater ability to pop, which is linked to dense starch filling in the endosperm. Waxy corn (Zea mays var. ceratina) has starch mainly consisting Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00009-5

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of amylopectin (99%), and amylose is present in very small amounts. Waxy cornstarch produces paste that has low tendency toward retrogradation with high transmittance and resembles potato starch. Waxy cornstarch is used in many foods (e.g., fruit pies, canned foods, frozen foods, and dairy products) and nonfood applications (gummed tapes). White corn has a white endosperm containing higher amounts of vitreous endosperm relative to floury endosperm and is preferred for nixtamalized products such as tortillas. Blue, purple, and red corn kernels are rich in anthocyanins with well-established antioxidant and bioactive properties.1 Interest in researching and using pigmented corn rich in anthocyanins or carotenoids and with phenolic compounds having antioxidant and bioactive properties has increased due to their health benefits.

STRUCTURE AND COMPOSITION Corn grain is composed of endosperm (82%–83%), germ (10%–11%), pericarp (5%–6%), and tip cap (0.8%–1.0%). The pericarp is the outermost layer, characterized by high crude fiber content, mainly consisting of hemicellulose, cellulose, and lignin. Hemicellulose is present in the highest concentration in the crude fiber. The pericarp thickness varies in different corn types and extends to the base of the kernel joining the tip cap. The pericarp and tip cap contribute only a negligible amount to the total kernel lipids. The endosperm is composed of a large number of cells, each packed with starch granules embedded in a continuous matrix of protein. The cell walls consist of nonstarch polysaccharides (β-glucan and arabinoxylan), proteins, and phenolic acids. Endosperm is rich in starch and proteins. Corn grain has two types of endosperm: floury and horny endosperm. Floury endosperm contains loosely packed starch granules surrounding the central fissure, whereas horny endosperm has tightly packed, smaller starch granules toward periphery. The grains of different varieties of corn may contain lipids up to 5.91%2; however, the crude fat content of the endosperm is relatively low (about 1%). The lipids present in endosperm contain more saturated fatty acids (SFAs) than do germ lipids. The germ is composed of the embryo, the living organ of the grain, and the scutellum, which nourishes the embryo. The germ is characterized by high fat and protein content (approximately 33% and 18%, respectively) and low starch content (approximately 8%).3 Germ oil is low in SFAs and high in polyunsaturated fatty acids (PUFAs). Germ oil is relatively stable due to its high levels of natural antioxidants (tocols) and is considered healthy because of its high concentration of oleic and linoleic acids. However, the composition and nutritional value of corn types vary with seed maturity, harvesting date, variety, storage, and drying conditions applied after harvesting.4, 5 Starch is the most abundant component, constituting 74.4% and 76.8% of corn grain on a dry basis.5 The starch is mainly concentrated in the endosperm and constitutes about 82%–83% of grain. Amylose and amylopectin are the major constituents of cornstarch. Amylose is essentially a linear polymer of glucopyranose units linked through α-D-1–4 glycosidic linkages, while the amylopectin is a highly branched polymer. The amylose may contain 1500 glucopyranose units, with an average total molecular weight of approximately 2.5  105, whereas amylopectin has an average molecular weight of approximately 108. Normal corn generally contains 25% amylose and 75% amylopectin. However, corn varieties with amylose content as high as 85% (amylomaize) and as low as 1% or less (waxy) have also been reported. The amylose and amylopectin are packed within the starch granules, and their packing arrangement corresponds to crystallinity, which varies among different corn types. In normal and waxy starches, the branched molecule of amylopectin constitutes the crystallites. The branches of the amylopectin molecule form double helices arranged in crystalline regions, while amylose, less-ordered amylopectin, and branch points connecting the double helices are attributable to the amorphous regions within the granules.6 X-ray diffraction (XRD) is used to reveal the presence and characteristics of the crystalline structure of the starch granules. The A-, B-, and C-type patterns are different polymeric forms of the starch, which vary in the packing of their amylopectin double helices. Corn and other cereal starches exhibit a typical A-type pattern, in which double helices comprising the crystallites are densely packed, with low water content. Tuber starches show the B-type, in which crystallites are less densely packed and have a more open structure containing a hydrated helical core.7 The C-type pattern is an intermediate/mixture of A and B-types, which is a characteristic of pulse starches. The A-, B-, and C-type patterns of starches from a number of sources are shown in Fig. 1. Waxy cornstarch exhibits a higher crystallinity than normal, and sugary cornstarch, while starch from sugary corn has a larger amount of amylose-lipid complex.8 The differences in crystallinity in various cereal starches also have been attributed to the difference in proportion of short-, medium-, and long-branch amylopectin chains, wherein long amylopectin chains with a degree of polymerization (DP) > 13 contribute to crystallinity, owing to the formation of double helices compared to that with DP 6–12.9 In corn grains, starch is present in granular form that differs in size and shape among botanical sources, as well as other types/varieties of corn. Normal cornstarch shows a bimodal particle size distribution, with granules of size 10 μm with relative proportions of 10.2% and 89.8%, respectively.10 The average size of small and large granules is reported to range from 1 to 7 μm and 15–20 μm, respectively.3 Scanning electron micrograms of normal 2. FLOURS AND BREADS

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Relative intensity

STRUCTURE AND COMPOSITION

0

5

10

15

20

25

Corn

A-type

Potato

B-type

Mungbean

C-type

30

35

2q

FIG. 1

XRD patterns of corn, potato, and mungbean starch.

corn, waxy corn, and sugary cornstarch are shown in Fig. 2. Normal corn and waxy cornstarch granules displayed spherical or angular shapes, whereas sugary cornstarch displayed irregular-shaped granules consisting of lobes.11 The irregular-shaped granules with average size of 36 μm for white cornstarch, and a defined round shape with average sizes of 20 μm and 40 μm for black and blue cornstarch, respectively, have been reported.12 When starch is heated in excess water, the granules swell to several times their original size. This results from the absorption of water and loss of crystalline order. The changes in starch slurries during heating and cooling are measured with a Rapid Visco Analyzer and a Brabender Visco-Amylo-Graph. These instruments measure changes in the viscosity of starch pastes during heating and cooling with continuous stirring. During heating, the viscosity increases with increase in temperature due to swelling of the starch granules. This is followed by a decrease in viscosity caused

FIG. 2 Scanning electron micrograms of (A) normal corn, (B) waxy corn, and (C) sugary cornstarch granules. Reprinted from Sandhu KS, Singh N, Lim ST. Functional properties of normal, waxy and sugary corn starches. J Food Sci Technol 2007;44:565–571, with permission. 2. FLOURS AND BREADS

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by rupturing and fragmentation of granules. During cooling, the starch molecules reassociate to form a gel, wherein amylose molecules aggregate into a network, embedding remnants of starch granules. The pasting properties of starch are influenced by the constituents that leached out from the granules during heating and the interactions between the chains. Sugary cornstarch gave pasting curves with flatter peaks, while normal and waxy corn show sharper peaks. Waxy corn starch has a higher peak viscosity and a lower breakdown viscosity than normal and sugary cornstarches. The higher peak viscosity of waxy cornstarch has been related to the absence of amylose-lipid complex, as these starches had negligible amylose.11 The lipids and phospholipids form a complex with the amylose and long branch-chains of amylopectin in the cereal starches, which results in reduction in swelling and inhibited amylose leaching.13 Waxy starches have negligible amounts of lipids and create pastes with higher peak viscosity. Sugary and normal cornstarches have higher amylose content, which restricts swelling and limits the increase in viscosity by developing aggregated structures. Waxy cornstarch has a lower pasting temperature than normal cornstarch.11 Black cornstarch showed higher peak viscosity, followed by white and blue cornstarches; this finding was attributed to the difference in starch organization and damaged starch content.12 Protein is the second-most-abundant constituent of corn grains. Corn varieties differ in protein content. The grains of waxy corn contained higher (11.03%) protein content than normal corn (8.05%–8.62%) and flint-dent (8.5%–8.7%) corn.2, 5 The storage proteins of endosperm are located within subcellular bodies, simply known as protein bodies, and comprise the protein matrix. Protein bodies consist almost entirely of a prolamine-rich protein fraction known as zein, which accounts for about 60% of the total grain proteins.14 Zein is deficient in lysine and tryptophan and contains 21.4% glutamine, 19.3% leucine, 9.0% proline, 8.3% alanine, 6.8% phenylalanine, 6.2% isoleucine, 5.7% serine, and 5.1% tyrosine.15. At least four major fractions have been identified within the zein storage protein: α-zein (21–25 kDa), β-zein (17–18 kDa), γ-zein (27 kDa), and δ-zein (9–10 kDa). Of these, α-zein is the most abundant fraction, contributing about 35% of the total zein protein, with large amounts of hydrophobic amino acids such as leucine, proline, alanine, and phenylalanine.14, 16 The β- and δ-zeins are soluble in aqueous alcohols in the presence of a reducing agent, and γ-zeins are soluble in both aqueous and alcoholic solvents in the presence of salt and reducing agents.17 However, the composition and concentration of zein proteins vary with corn genotypes, location of protein bodies in the grain, and maturity.14

DRY AND WET MILLING Dry Milling Dry milling of corn is done to produce grits of different particle sizes, which are used to make a number of products, like breakfast cereals and snacks, and grains with higher proportions of harder endosperm are preferred for dry milling.3 The main objective of dry milling is to get the maximum amount of grit with minimum contamination of the hull, germ, and tip cap. The grains are cleaned to remove impurities, conditioned with cold or hot water or steam, and then tempered for varying amounts of time depending upon the product required. The conditioning makes the hull germ tough and makes endosperm mellow. The grains are then passed through either a Beall Degerminator or fluted roller mills to separate the germ and hull. After degermination, the material is dried to around 15% moisture content, cooled, and graded to obtain fractions of different particle sizes, ranging from large hominy grits to fine flour. The products obtained from dry milling include flaking grits; coarse, medium, and fine grits; coarse or granulated meal; and fine meal. The fractions of corn dry milling differ in composition and end-use suitability.18 In India, the corn traditionally is milled in stone mills to get whole meal (or atta) that is used in the preparation of unleavened flat bread (or roti).

Wet Milling All over the world, large quantities of corn is wet-milled to produce starch and other valuable by-products such as gluten, germ, and bran. The first step in the wet-milling of corn is steeping in water (30–40 h at 50°C) in the presence of sulfur dioxide (SO2; 0.02%) under carefully controlled conditions to soften the kernels. SO2 prevents fermentation and facilitates the separation of starch from protein. After steeping, the water is drained and grains are coarsely ground to free the germ from endosperm and hull. Then the germ portion is separated, dried, and expelled for oil extraction. The fiber and starch suspension is then fine-milled and separated by screening, centrifugation, and washing. Centrifugation with a battery of hydrocyclones successively purifies cornstarch (reduces protein and lipid content) and affects physicochemical and functional properties of the starch.19 The starch is dried and converted to a number of valuable products, such as dextrose, fructose syrups, dextrins, modified starches, and sorbitol, by chemical processes,

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TABLE 1

Phenolic and Anthocyanin Content in Various Corn Types Total phenolicsa (mg/100 g of dry weight)

Anthocyanin contentb (mg/100 g of dry weight)

Corn types

Free

Bound

Total

Whitec

34.7  0.4

226.0  6.3

260.7  6.1

1.33  0.02

Yellowc

43.6  1.8

242.2  13.1

285.8  14.0

0.57  0.01

Redc

38.2  0.4

205.6  4.5

243.8  4.6

9.75  0.44

Bluec

45.5  0.5

220.7  0.5

266.2  0.7

36.87  0.71

High-carotenoidc

50.0  2.5

270.1  9.4

320.1  7.6

4.63  0.06

Blackd

103  2.6

354  3.1

457  7.4

76.2  2.2

Purpled

83.7  2.5

381  4.4

465  9.8

93.2  1.1

82.3  0.8

384  7.1

465  4.4

85.2  2.2

d

Red

73.1  1.4

271  4.5

343  8.6

99.5  1.8

d

Orange

40.3  1.4

175  2.3

215  5.1

30.6  0.9

d

Yellow

104  2.2

447  4.3

551  3.8

70.2  0.9

d

33.4  1.5

136  3.2

170  1.1

1.54  0.9

d

Blue

White a b c d

Expressed as gallic acid. Expressed as cyanidin-3-glucoside. de la Parra et al.20 Lopez-Martinez et al.21

enzymatic processes, or a combination. Here, α-amylase, β-amylase, glucoamylase, and pullulanase enzymes from bacterial and fungal sources are used for starch hydrolysis.

BIOACTIVE COMPOUNDS AND RESISTANT STARCH Bioactive Compounds Various bioactive constituents, such as carotenoids, anthocyanins, and phenolic compounds, which are associated with health-promotion and disease-prevention properties present in fruits and vegetables, have also been reported in corn. These compounds are present mainly in whole grains, the outer bran layers and germ being rich in these compounds. Total phenolic and anthocyanin content of various corn types are shown in Table 1. The anthocyanins present in blue corn come from cyanidin and malvidin (mainly from derivatives of the former), whereas in red corn, they come from pelargonidin, cyanidin, and malvidin. The carotenoids with molecules containing oxygen are also known as xanthophylls, and they are the source of the yellow color in corn. The carotenoids vary in corn according to type and genotype. Yellow corn has more carotenoids than floury corn. Lutein and zeaxanthin are the major carotenoids in corn, and to a lesser extent, α-cryptoxanthin, β-cryptoxanthin, α-carotene, and β-carotene are present. Blue and white corn is low in lutein and zeaxanthin content, whereas yellow and high-carotenoid corn varieties have higher content of these elements.20 Several desirable health-related properties of lutein and zeaxanthin have been identified; for example, lutein has been shown to have antitumor-promoting activity and suppression of tumor growth in mice.22 Lutein and zeaxanthin also have been associated with the prevention of age-related macular degeneration, a human disorder similar to cataracts that causes early blindness. Bioactivities of purple-, blue-, and red-pigmented corn has been associated to the presence of anthocyanins. The antimutagenic and radical scavenging activities of anthocyanins are well documented. The carotenoids content of raw corn and its processed products is always different because certain amounts of carotenoids are lost during processing. The losses in carotenoid content depend upon the processing conditions, such as canning, freezing, and extrusion (Table 2). Carotenoids are sensitive to heat, light, air, and pH; however, lutein has better heat stability. High-carotenoid corn genotypes have the best overall phytochemical profile, followed by yellow, blue, and red corn. Purple, black, and red corn phenotypes contain higher amounts of anthocyanins than yellow, orange, and white phenotypes.21 White corn genotypes have lower antioxidant activity due to the lower amounts of

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Carotenoid Content of Various Corn Types and Processed Corn

TABLE 2

Carotenoid content (μg/100 g of dry weight) Corn types

Lutein 5.73  0.18

Whitea

Zeaxanthin

β-Cryptoxanthin

β-Carotene

6.01  0.06

1.27  0.06

4.92  0.18

Yellowa

406.2  4.9

353.2  23.1

19.1  1.2

33.6  1.2

Reda

121.7  12.1

111.9  9.2

13.1  1.8

20.2  1.9

Bluea

5.17  0.49

14.3  1.0

3.41  0.39

23.1  2.1

322.3  10.7

23.1  1.0

45.8  3.9

245.6  9.4

High-carotenoida White cornb (fresh)

5.5  1.2

28.5  5.2

0.4  0.1

0.82  0.08

White cornb (canned)c

6.6  0.5

30.5  3.4

0.5  0.2

0.68  0.17

6.3  1.1

47.7  10.2

0.9  0.2

2.37  0.42

330  19.8

209.0  12.0

31.6  15.5

15.69  0.60

336.4  67.5

215.9  42.2

42.8  10.0

11.66  2.47

361.6  34.2

212.3  36.0

33.1  3.9

16.68  1.83

10.4  0.4

16.2 0.7

15.5  0.3

18.6  0.8

80.4  5.5

140.5  11.7

68.2  2.2

35.4  1.3

b

White corn (frozen) b

Golden corn (fresh) b

c

Golden corn (canned) b

Golden corn (frozen) d,e

Yellow corn oil Yellow corn oil

d,f

Yellow corn germ oil

d,e

1.4  0.2

0.9  0.1

Nil

Nil

Yellow corn germ oil

d,f

2.1  0.3

2.9  0.4

Nil

Nil

Yellow corn fiber oil

d,e

6.3  0.6

5.7  0.4

6.8  1.2

5.6  0.2

Yellow corn fiber oil

d,f

25.1  1.4

26.4  1.9

17.9  0.4

10.7  0.2

a b c d e f

de la Parra et al.20 Scott and Elridge.23 Corn and brine were analyzed in all canned samples, with brine content factored out of the final calculations. Moreau et al.24 Hexane extracted. Ethanol extracted.

anthocyanins and carotenoids. The utilization of pigmented corn instead of white corn can create nutritionally better products. However, in many industrially manufactured products (e.g., nixtamalized products, such as tortillas and tacos), white corn is preferred. The phenolic compounds exhibit antioxidant and a number of other bioactive properties, such as cell differentiation, pro-carcinogen deactivation, deoxyribonucleic acid (DNA) repair, inhibition of N-nitrosamine formation, and change of estrogen metabolism.25 Grains of different corn types (normal corn, waxy corn, baby corn, popcorn, and sweet corn) contained a number of phenolic compounds (namely, protocatechuic, vanillic, sinapic, syringic, p-hydroxybenzoic, caffeic, p-coumaric, ferulic and isoferulic acids, cyanidin-3-O-glucoside, kaempferol, and quercetin).26–28 These compounds were mainly concentrated in the pericarp and embryo/germ of grains, wherein pericarp contained the highest content of total phenolics.27 The phenolics also had been related to color characteristics of grains. Madhujith and Shahidi29 reported a greater contribution of quercetin and catechin to grain color. Ferulic acid is an important phenolic compound in corn and other cereal grains. It was the most prominent phenolic compound in purple corn varieties.30 It is present in different forms (free, conjugated, and bound) and the concentration of each form varies in the different corn types (Table 3). The high-carotenoid corn contains a higher amount of total ferulic acid compared to white, yellow, red, and blue corn. Furthermore, among 18 Mexican corn phenotypes and strains, the orange phenotype contained the highest amount of total ferulic acid, while the white phenotype had the least-bound ferulic acid content.21 Ferulic acid acts as an antioxidant and is used as an ingredient in various supplements that claim to slow down the aging process. It has been approved in certain countries as a food additive to prevent lipid oxidation. It can readily form a resonance-stabilized phenoxyl radical, which accounts for its potent antioxidant potential. Corn has a higher antioxidant capacity compared to wheat, oat, and rice.1 The antioxidant activity of bound phytochemicals in corn grains has been reported to be 157.68 μmol/g against 68.74, 43.60, and 39.76 μmol/g in the grains of wheat, oats, and rice, respectively.1 2. FLOURS AND BREADS

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TABLE 3

Ferulic Acid Content in Various Corn Types Ferulic acida (mg/100 g of dry weight)

Corn types

Free

Soluble conjugated

Bound

Whiteb

0.50  0.02

0.76  73

119.20  11.2

120

Yellowb

0.65  0.01

1.47  102

100.84  5.0

102

Redb

0.58  0.02

1.26  48

128.45  11.5

130

Blueb

0.68  0.05

1.45  27

127.85  8.1

130

High-carotenoidb

0.97  0.08

1.96  33

150.08  12.4

153

Blackc

1.87  0.3

–

150  1.4

151

Purplec

1.97  0.1

–

152  1.5

154

2.02  0.5

–

151  2.9

153

c

Red

Total

2.02  0.4

–

149  2.2

152

c

Orange

2.42  0.2

–

161  3.3

164

c

Yellow

2.01  0.1

–

138  1.1

140

c

1.57  0.6

–

146  2.3

148

c

Blue

White a b c

Expressed as ferulic acid. de la Parra et al.20 Lopez-Martinez et al.21

Ferulic acid is suggested to be useful in alleviating oxidative stress and attenuating the hyperglycemic response associated with diabetes. A wide range of therapeutic properties of ferulic acid, such as antiflammatory, antiatherogenic, antidiabetic, antiaging, neuroprotective, radioprotective, and hepatoprotective, have been reported.31 However, most of the ferulic acid (approx. 94%–98%) is present in bound form.1, 21 which results in its reduced bioavailability. Processing has a profound effect on the bioavailability of the phenolic compounds. Processing techniques such milling or grinding, microfluidization, and bioprocessing (germination and fermentation) enhanced the bioavailability of the bound phenolics by increasing their accessibility through particle size reduction, structural breakdown of cereal matrices, and their liberation from grain components.32 However, thermal-processing techniques had variable effects. Lime cooking, tortilla baking, and tortilla chip frying increased the amount of free and soluble-conjugated ferulic acid,20 while extrusion cooking reduced the bioavailability of phenolic compounds of cereal grains due to the decomposition of heat-labile phenolic compounds, as well as the polymerization of some phenolic compounds under high pressure.32 Efforts are being made in a number of countries to develop varieties of corn that are high in carotenoid content to derive the health benefits that come from its dietary consumption. New varieties of corn with high β-carotene content have been bred to combat vitamin A deficiency in African countries33. Knowledge of the carotenoid synthesis pathway, which involves certain enzymes regulating the proportion of various carotenoids, has been utilized to develop new corn lines with increased concentrations of specific carotenoids33. The health implications of yellow and pigmented corns in processed products need to be studied in depth.

Resistant Starch During heating, starch is gelatinized and the semicrystalline structure of starch disintegrates. The cooling of the gelatinized starch pastes lead to recrystallization of the starch chains, known as retrogradation. The retrogradation rate and its extent vary with the starch properties (molecular and crystalline structure) and storage conditions (temperature, duration, water content). Cereal starches with higher amylose content and longer amylopectin chains show higher retrogradation rates.8, 34 The changes in G0 among the cooked pastes of cornstarches with different amylose contents measured using a Haake dynamic rheometer at 10°C during a 10-h period are compared in Fig. 3. Sugary cornstarch with higher amylose had a higher retrogradation rate than normal cornstarch.8 The retrogradation makes the starch resistant to breakdown by digestive enzymes, which consequently reduces the glycemic index (GI)35. The GI characterizes the carbohydrates consumed in the form of different foods on the basis of the postprandial level of blood glucose36. Carbohydrates that break down quickly during digestion and release glucose rapidly into the bloodstream are considered to have high GI. Starch is classified into three groups according to the rate of glucose release 2. FLOURS AND BREADS

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FIG. 3 Changes in G0 at 10°C during 10 h holding

300

of cooked paste from cornstarches varying in amylose content (measured using a dynamic rheometer, Haake, RheoStress 6000).

Amylose 42.3%

G' (Pa)

200

Amylose 32.7%

100

Amylose 27.6%

0

0

100

200

300

400

500

600

Time (min)

and the absorption into the gastrointestinal tract: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistance starch (RS).37 RDS is the portion of starches that can be rapidly hydrolyzed by digestive enzymes, SDS is digested at a relatively slow rate, and RS is not digested by digestive enzymes and consequently transferred into the colon, where it performs physiological functions similar to dietary fibers as prebiotics and promotes the growth of probiotics in the human gut. It is fermented in the large intestine by colonic microflora, resulting in the production of gases, organic acids, and short-chain fatty acids having a number of beneficial effects not only on gastric health and postprandial blood glucose levels, but also on cardiovascular health and the management of hyperlipidemia and obesity.9 Waxy cornstarch is more rapidly digested than high-amylose starch, perhaps due to a greater surface area per molecule of the amylopectin than amylose. Normal cornstarch is more susceptible to amylolysis than high-amylose cornstarch, which may be due to the presence of surface pores and channels that facilitate enzymatic diffusion38. The association between amylose chains and the potential for amylose-lipid complex formation39, higher crystalline lamella thickness, and a thicker peripheral layer40 are the factors that make the high-amylose cornstarch granules resistant to amylolysis. High-amylose cornstarch granules are hydrolyzed predominantly by exocorrosion, whereas normal cornstarch is internally hydrolyzed in an inside-out pattern.38 Furthermore, normal starches with short double helices are more susceptible to enzymatic hydrolysis and exhibit high RDS and SDS content, whereas long amylopectin chains form more stable helices, which contribute to enhanced resistance to digestibility41. Mechanical and thermal treatments change the structure and digestibility of starch. Waxy and low-amylose starches are readily damaged by milling, which renders them more vulnerable to amylolysis.42 Thermal treatments such as autoclaving, baking, steam cooking, and parboiling affect the gelatinization and retrogradation processes, and consequently the formation of RS in foods. Thakur et al.28 reported that waxy corn resulted in extrudates that have better digestibility, but poor expansion and textural properties that limited its use in the extrusion-processing industry. Extruded starches from corn, kidney beans, and field peas were reported to have lower retrogradation (syneresis) and paste viscosities, but a higher water solubility index compared to their native starches.43 Cornstarch was observed to have a lower retrogradation tendency than kidney bean and field pea starches. The tendency of retrogradation of extruded starches depended upon conditions during extrusion processing. The retrogradation tendency was reported to be higher in starches extruded at lower feed moistures than those at higher moistures. Extruded starches showed higher RDS and lower RS content. All starches extruded at higher feed moisture showed significantly lower RDS than those extruded at lower feed moisture. Cornstarch contained lower RS content than kidney bean and field pea starch. Starches extruded at higher feed moistures showed higher RS content than those extruded at lower feed moistures.43 Hi-MAIZE, containing >80% amylose and 42% RS, is a commercial source of RS that is widely used in various baked goods. Starches are chemically modified to change the functional properties, which also change the susceptibility to the action of enzymes. Esterification, etherification, and cross-linking of starch make it resistant to α-amylase.44

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Chung et al.45 reported that the chemical substitution of corn starch, such as hydroxypropylation and acetylation and oxidation of cornstarch increase RS, whereas cross-linking does not affect starch digestibility considerably. Chung et al.,45 studied RDS, SDS, and RS of the modified cornstarch in the prime and gelatinized states using enzymatic hydrolysis. These authors reported that the oxidized starch had a higher percentage of RDS (33.5%) compared to hydroxypropylated starch (27.9%), cross-linked and acetylated starches (approximately 24%), and unmodified starch (25.6%). They observed that the oxidized starch was rapidly hydrolyzed during the early stage of digestion (up to 60 min) because the chain degradation of starch occurred during the oxidation. Therefore, the fast rate of hydrolysis resulted in the greatest amount of RDS. These authors reported that the exceptionally high swelling ability of hydroxypropylated starch enhanced the access of digestive enzymes inside the granules, thereby increasing the RDS content. Although the acetylated starch swelled more readily than unmodified starch, the acetyl groups could hinder the enzymatic action during the hydrolysis.

UNLEAVENED BREAD MAKING PROPERTIES The different sizes of the fractions of grit are produced during dry milling of corn, and they vary in composition and end-use suitability. Flaking grits are used for making corn flake cereal. Coarse and medium grits are used in the processing of breakfast cereals and snacks. Fine grits are preferred in porridge-making and is used as a brewing adjunct, often to reduce the cost of beer. Coarse or granulated meal is used in pancakes, muffin mixes, and various bakery products. Finely ground corn is used in the production of tortillas, a type of unleavened bread. Tortilla is an important product for the populations of suburban and rural areas of meso-American countries, such as Mexico and Guatemala. Tortillas are prepared from white, yellow, and blue corn; and white corn is preferred in commercial processing. Similar bread made from corn in South America is called arepa, and it is thicker than tortillas. Wheat meal is generally used for making unleavened bread called chapatis (roti) in India and Pakistan. However, cornmeal also is used to prepare roti in winter and is very popular in North Indian states. Yellow cornmeal is preferred for the preparation of roti. Roti made from cornmeal is less pliable than that made from wheat meal, due to the differences in the composition and rheological properties. Dough is generally characterized by empirical (farinograph and mixograph) and fundamental rheological measurements. Farinographs and mixographs are extensively used to characterize dough-mixing properties, such as the water absorption, development time, stability, and mixing tolerance of flour and meal. In a recent study involving mixographic analysis of dough from different corn types, it was reported that water absorption of 40% was appropriate for the formation of consistent and stable dough, and there was an increase in the proportion of intermolecular and antiparallel β-sheets, as well as α-helices and β-sheets, with an increase in the hydration of dough46. Further, it was shown that red and yellow corn varieties resulted in stronger and more stable dough than do white and waxy corn. In addition, the dough from waxy corn suffered greater breakdown during mixing than that from normal corn.46 Dynamic oscillation measurement involves small deformation and is a fundamental approach to study dough rheology. Dynamic oscillation technique is being preferred for studying the structure and fundamental properties of cereal flour dough. Mechanical spectra of corn- and wheat-meal dough obtained using the Haake dynamic rheometer is shown in Fig. 4. G0 of cornmeal and wheat-meal dough was greater than G00 , indicating a predominance of elastic character. Higher G0 with lower G00 and tan δ of cornmeal dough as compared to wheat-meal dough reflect its higher rigidity and stiffness. Wheat-meal dough shows lower G0 and G00 as compared to cornmeal dough. In addition, the difference between the moduli was lesser, showing good balance between these. The “roti” prepared from wheat meal has better textural qualities than cornmeal because of good balance between the moduli. Cornmeal is kneaded into soft, pliable dough with water for roti-making. Cornmeal dough lacks the viscoelasticity of wheat dough; therefore, warm water is used during kneading. Warm water helps with the aggregation of particles due to the partial gelatinization of starch. After mixing, dough is divided into small balls, rounded, flattened, and sheeted into circular disks (about 5 in. in diameter and 0.25 in. in thickness) with a rolling pin or by hand. The disks are roasted on a heavy iron or earthen griddle until crisp. They are turned several times during the roasting process. After roasting, roti is coated with butter oil or butter. In the Punjab states of India and Pakistan, roti is traditionally served hot with Sarson ka saag (mustard leaf gravy) and salad consisting of onions, radishes, and lemons. Corn varieties differ in terms of the chapati-making properties. Yellow-orange corn is generally preferred for chapati-making because of its superior sensory properties. On the other hand, waxy corn was not observed to be suitable for the preparation of good-quality chapati because of the presence of a negligible amount of amylose and high protein content, which restricted the development of a starch-protein complex during dough formation, resulting in the preparation of hard/tough (i.e., high rupture force and low extensibility) chapatis.28

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Corn meal

Wheat meal

100

0.7

100

0.7

0.6

0.6

60

0.4

40

0.3 0.2

20 0.1 0

0 0

1

2 Time (min)

3

4

0.5

60

0.4

40

0.3

Tan d

0.5

G', G" (x1000 Pa)

80

Tan d

G', G" (x1000 Pa)

80

0.2

20 0.1

0

0 0

1

2 Time (min)

3

4

FIG. 4 Mechanical spectra of dough from cornmeal and wheat meal. ■—G0 , ▲—G00 , ●—tan δ.

TECHNOLOGICAL ISSUES Knowledge of the carotenoid synthesis pathway33 could help in developing new varieties of corn with higher concentrations of specific carotenoids. These varieties could be useful in combating the problems related to vitamin A deficiency in developing countries. The processing conditions have variable effects on the bioactive constituents in corn. Therefore, processing methods with minimum losses of bioactive constituents for different corn products need to be developed. The utilization of pigmented corn varieties instead of white corn could provide nutritionally better products; however, in many industrially manufactured products, white corn is preferred.

SUMMARY POINTS • High-amylose corn is a good source of RS, which can be used in various food products for its health benefits. RS has been associated with improved cholesterol metabolism and reduced risk of type II diabetes and colon cancer. • Various bioactive compounds with health-promoting and disease-preventing properties in fruits and vegetables have also been reported in corn. • Ferulic acid is an important phytochemical present in higher amounts in high-carotenoid corn, as compared to white, yellow, red, and blue corn. • Purple, blue, and red corn inhibit colorectal carcinogenesis in male rats and possess antimutagenic and radical scavenging activities. These bioactivities have been associated with the presence of anthocyanins. • The health implications of pigmented corn in various processed products need to be studied in additional depth. • Corn varieties with high β-carotene content can combat vitamin A deficiency in developing countries. Knowledge of the carotenoid synthesis pathway can be utilized to develop such varieties.

References 1. Adom KK, Liu RH. Antioxidant activity of grains. J Agric Food Chem 2002;50:6182–7. 2. Thakur S, Kaur A, Singh N, Virdi AS. Successive reduction dry milling of normal and waxy corn: grain, grit, and flour properties. J Food Sci 2015;80:C1144–55. 3. Singh N, Kaur A, Shevkani K. Maize: grain structure, composition, milling, and starch characteristics. In: Chaudhary DP, Kumar S, Singh S, editors. Maize: nutrition dynamics and novel uses. New Delhi: Springer; 2014. p. 65–76. 4. Gehring CK, Cowieson AJ, Bedford MR, Dozier III WA. Identifying variation in the nutritional value of corn based on chemical kernel characteristics. Worlds Poult Sci J 2013;69:299–312. 5. Odjo S, Bera F, Beckers Y, Foucart G, Malumba P. Influence of variety, harvesting date and drying temperature on the composition and the in vitro digestibility of corn grain. J Cereal Sci 2018;79:218–25. 6. Waterschoot J, Gomand SV, Fierens E, Delcour JA. Starch blends and their physicochemical properties. Starch 2015;67:1–13. 7. Tester RF, Karkalas J, Qi X. Starch structure and digestibility enzyme-substrate relationship. Worlds Poult Sci J 2004;60:186–95. 8. Singh N, Inouchi N, Nishinari K. Structural, thermal and viscoelastic characteristics of starches separated from normal, sugary and waxy maize. Food Hydrocoll 2006;20:923–35. 9. Shevkani K, Singh N, Bajaj R, Kaur A. Wheat starch production, structure, functionality and applications—a review. Int J Food Sci Technol 2017;52:38–58. 2. FLOURS AND BREADS

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10. Kaur A, Shevkani K, Singh N, Sharma P, Kaur S. Effect of guar gum and xanthan gum on pasting and noodle-making properties of potato, corn and mung bean starches. J Food Sci Technol 2015;52:8113–21. 11. Sandhu KS, Singh N, Lim ST. Functional properties of normal, waxy and sugary corn starches. J Food Sci Technol 2007;44:565–71. 12. Agama-Acevedo E, Barba de la Rosa AP, Mendez-Montealvo G, Bello-Perez LA. Physiochemical and biochemical characterization of starch granule isolated from pigmented maize hybrids. Starch 2008;60:433–41. 13. Singh N, Singh J, Kaur L, Sodhi NS, Gill BS. Morphological, thermal and rheological properties of starches from different botanical sources. Food Chem 2003;81:219–31. 14. Singh N, Singh S, Kaur A, Bakshi MS. Zein: structure, production, film properties and applications. In: John MJ, Thomas S, editors. Natural polymers. Vol. 1. London: RSC; 2012. p. 204–18. 15. Pomes AF. Zein. In: Encyclopedia of polymer science and technology: plastics, resins, rubbers, fibers. Vol. 15. InterScience Publishers; 1971. p. 125–32. 16. Gianazza E, Viglienghi V, Righetti PG, Salamini F, Soave C. Amino acid composition of zein molecular components. Phytochemistry 1977;16:315–7. 17. Wilson CM. Multiple zeins from maize endosperms characterized by reversed-phase high performance liquid chromatography. Plant Physiol 1991;195:777–86. 18. Shevkani K, Kaur A, Singh G, Singh B, Singh N. Composition, rheological and extrusion behaviour of fractions produced by three successive reduction dry milling of corn. Food Bioprocess Technol 2014;7:1414–23. 19. Singh N, Shevkani K, Kaur A, Thakur S, Parmar N, Virdi AS. Characteristics of starch obtained at different stages of purification during commercial wet milling of maize. Starch 2014;66:668–77. 20. de la Parra C, Serna SO, Liu RH. Effect of processing on the phytochemical profiles and antioxidant activity of corn for production of masa, tortilla and tortilla chips. J Agric Food Chem 2007;55:4177–83. 21. Lopez-Martinez LX, Oliart-Ros RM, Valerio-Alfaro G, Lee CH, Parkin KL, Garcia HS. Antioxidant activity, phenolic compounds and anthocyanins content of eighteen strains of Mexican maize. LWT- Food Sci Technol 2009;42:1187–92. 22. Park JS, Chew BP, Wong TS. Dietary lutein from marigold extract inhibits mammary tumor development in BALB/c mice. J Nutr 1998;128:1650–6. 23. Scott CE, Eldridge AL. Comparison of carotenoid content in fresh, frozen and canned corn. J Food Compos Anal 2005;18:551–9. 24. Moreau RA, Johnston DB, Hicks KB. A comparison of the levels of lutein and zeaxanthin in corn germ oil, corn Fiber oil and corn kernel oil. J Am Oil Chem Soc 2007;84:1039–44. 25. Shahidi F. Functional foods: their role in health promotion and disease prevention. J Food Sci 2004;69:R146–9. 26. Kandil A, Li J, Vasanthan T, Bressler DC. Phenolic acids in some cereal grains and their inhibitory effect on starch liquefaction and saccharification. J Agric Food Chem 2012;60:8444–9. 27. Das AK, Singh V. Antioxidative free and bound phenolic constituents in botanical fractions of Indian specialty maize (Zea mays L.) genotypes. Food Chem 2016;201:298–306. 28. Thakur S, Singh N, Kaur A, Singh B. Effect of extrusion on physicochemical properties, digestibility, and phenolic profiles of grit fractions obtained from dry milling of normal and waxy corn. J Food Sci 2017;82:1101–9. 29. Madhujith T, Shahidi F. Antioxidant potential of pea beans (Phaseolus vulgaris L.). J Food Sci 2005;70:S85–90. 30. Cuevas Montilla E, Hillebrand S, Antezana A, Winterhalter P. Soluble and bound phenolic compounds in different Bolivian purple corn (Zea mays L.) cultivars. J Agric Food Chem 2011;59:7068–74. 31. Srinivasan M, Sudheer AR, Menon VP. Ferulic acid: therapeutic potential through its antioxidant properties. J Clin Biochem Nutr 2007;40:92–100. 32. Wang T, He F, Chen G. Improving bioaccessibility and bioavailability of phenolic compounds in cereal grains through processing technologies: a concise review. J Funct Foods 2014;7:101–11. 33. Harjes CE, Rocheford TR, Bai L, Brutnell TP, Kandianis CB, Sowinski SG, Stapleton AE, Vallabhaneni R, Williams M, Wurtzel ET, Yan J, Buckler ES. Natural genetic variation in lycopene epsilon cyclase tapped for maize biofortification. Science 2008;319:330–3. 34. Shevkani K, Singh N, Singh S, Ahlawat AK, Singh AM. Relationship between physicochemical and rheological properties of starches from Indian wheat lines. Int J Food Sci Technol 2011;46:2584–90. 35. Fredriksson H, Bjorck I, Andersson R, Liljeberg H, Silverio J, Elliasson AC, Aman P. Studies on -amylase degradation of retrograded starch gels from waxy maize and high-amylopectin potato. Carbohydr Polym 2000;43:81–7. 36. Jenkins AL. The glycemic index: looking back 25 years. Cereal Foods World 2007;52:50–3. 37. Englyst HN, Kingman SM, Cummings JH. Classification and measurement of nutritionally important starch fractions. Eur J Clin Nutr 1992;46: S33–50. 38. Zhang GY, Ao ZH, Hamaker BR. Slow digestion property of native cereal starches. Biomacromolecules 2006;7:3252–8. 39. Morita T, Ito Y, Brown IL, Ando R, Kiriyama S. In vitro and in vivo digestibility of native maize starch granules varying in amylose contents. J AOAC Int 2007;90:1628–34. 40. Jenkins PJ, Donald AM. The influence of amylose on starch granule structure. Int J Biol Macromol 1995;17:315–21. 41. Lehmann U, Robin F. Slowly digestible starch-its structure and health implications: a review. Trends Food Sci Technol 2007;18:346–55. 42. Tester RF, Morrison WR. Properties of damaged starch granules. Composition and swelling of fractions of wheat-starch in water at various temperatures. J Cereal Sci 1994;20:175–81. 43. Sharma S, Singh N, Singh B. Effect of extrusion on morphology, structural, functional properties and in vitro digestibility of corn, field pea and kidney bean starches. Starch 2015;67:721–8. 44. Hood LF, Arneson VG. In vitro digestibility of hydroxypropyl distarch phosphate and unmodified tapioca starch. Cereal Chem 1976;53:282–90. 45. Chung HJ, Shin DH, Lim ST. In vitro starch digestibility and estimated glycemic index of chemically modified corn starches. Food Res Int 2008;41:579–85. 46. Thakur S, Singh N, Kaur A. Characteristics of normal and waxy corn: physicochemical, protein secondary structure, dough rheology and chapatti making properties. J Food Sci Technol 2017;54:3285–96.

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C H A P T E R

10 Amaranth: Potential Source for Flour Enrichment Narpinder Singh*, Prabhjeet Singh†, Khetan Shevkani‡, and Amardeep Singh Virdi* *Department of Food Science and Technology, Guru Nanak Dev University, Amritsar, India † Department of Biotechnology, Guru Nanak Dev University, Amritsar, India ‡ Department of Applied Agriculture, Central University of Punjab, Bathinda, India

O U T L I N E Introduction

123

Transgenic Applications

132

Grain Characteristics

124

Technological Issues

132

Grain Composition

124

Summary Points

133

Grain Protein Characteristics

127

Acknowledgments

133

Nutraceutical Properties of Amaranth Proteins

130

References

133

Food and Nonfood Applications

130

Further Reading

135

List of Abbreviations AmA API CaMV cDNA GI kDa PAGE SDS

amaranth albumin amaranth protein isolate cauliflower mosaic virus complementary DNA glycemic index kilo Dalton polyacrylamide gel electrophoresis sodium dodecyl sulfate

INTRODUCTION Amaranth (Amaranthus), a traditional Mexican plant is a cosmopolitan genus of herbs with approximately 60 plant species, the majority of which are wild.1 It was cultivated 5000–7000 years ago in areas of Mexico, but the cultivation was discontinued in the early 15th century until the late 1970s, when it was rediscovered and utilized as a new economic crop.2,3 Amaranthus plants have inflorescences and foliage in colors ranging from purple to red to gold. It is a dicotyledonous plant and is also considered as a pseudocereal because of its properties and characteristics.4 Amaranth is generally cultivated in arid zones, where commercial crops cannot be grown. Furthermore, amaranth is also known for its high nutritive value and several nutraceutical health-benefiting effects.5–7

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© 2019 Elsevier Inc. All rights reserved.

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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT

The Amaranthus plant has a good capacity to produce a high biomass and is used to produce grains, leafy vegetables, and ornamentals. Several species of amaranth are often considered as weeds. Amaranthus cruentus and Amaranthus hypochondriacus are the two species that are primarily cultivated for grain, whereas Amaranthus blitum, Amaranthus dubius, Amaranthus tricolor, Amaranthus lividus, and Amaranthus spinosus are used as vegetables. Amaranthus tricolor and Amaranthus caudatus are also grown for ornamental and decorative purposes. Amaranthus viridis, Amaranthus retroflexus, Amaranthus hybridus, Amaranthus gracilis, Amaranthus gangeticus, Amaranthus paniculatus, and Amaranthus graecizans are wild types of the plant. Its leaves are a potential alternative source of betalains because of betacyanin pigments, and they also show anticancer activity.

GRAIN CHARACTERISTICS Amaranth grains are nearly spherical and about 1 mm in diameter, and they vary in color from creamish yellow to reddish and have unique compositions of protein, carbohydrates, and lipids. A. hypochondriacus produces creamish yellow grains, while A. caudatus has red grains (Fig. 1). Hunter color L*, a*, and b* values are around 62–68, 5.5–6.7, and 21.2–23.7, respectively, for grains of A. hypochondriacus, versus 49–51, 13–13.8, and 10.6–13.2, respectively, for A. caudatus. These two species also differ in grain size. A. hypochondriacus grains were larger and showed greater thousand kernel weight, varying between 0.62 and 0.88 g, as compared to A. caudatus grains, with thousand grain weight ranging between 0.46 and 0.70 g.8 Amaranth seeds have circular embryos or germ, which surrounds the starch-rich perisperm and, together with the seed coat, represent the bran fraction, which is relatively rich in fat and protein.9 The bran fraction is proportionally higher in amaranth seeds in comparison to common cereals, such as maize and wheat, which explains the higher levels of protein and fat present in these seeds.9

GRAIN COMPOSITION Amaranth is a good source of starch, proteins, lipids, dietary fiber, and minerals. On a dry basis, amaranth grains contain 12.5%–15.5% proteins, 73.7%–77.0% carbohydrates, 7.1%–8.0% lipids and 3.0%–3.5% minerals,10 while the dietary fiber content ranges from 19.5%–49.3%.11 Starch is the most abundant carbohydrate in amaranth grain. Amaranth grain has approximately 62%–65% starch, which is made up of amylose and amylopectin. Amylose is essentially a linear polymer of glucose, while amylopectin is highly branched, consisting of a main chain of (1–4)-linked α-D-glucose, along with short chains of (1–6)-α-D-glucose-linked branches. Amaranth starch has low amylose content, ranging from 2% to 12% depending on the genotype. Amylopectin is the most abundant component of amaranth starch. Amaranth starch may contain 90%–98% amylopectin, which is composed of 1700 amylopectin molecules on average,12 which in turn exhibit smooth polymodal chain length distribution, with the peak maxima at degree of polymerization (DP) around 11–12.13 In addition to amylose and amylopectin, amaranth starch granules contain 0.16%–0.28% bound lipids.14 Although starch granule lipids are present as minor components, they have a great effect on starch properties and functionality.15 Amaranth starch granules have diameters ranging between 0.5 and 2.5 μm, which is similar to rice but smaller than that found in the starches of other cereal grains. A comparison of amaranth starch granules with those of wheat, rice, and potato is illustrated in Fig. 2. Amaranth starch has polygonal-shaped granules and displays an A-type X-ray pattern (Fig. 2), which is similar to wheat, rice, and maize starches. Amaranth starch granules are mostly small and show unimodal size distribution; however, the granular size and distribution vary widely among cultivars and species.13 FIG. 1 Grains of two different amaranth species.

A. hypocondriacus

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A. caudatus

125

GRAIN COMPOSITION

FIG. 2 Scanning electron micrographs of different starches.

FIG. 3 X-ray diffractions (XRDs) of wheat and amaranth starch. From Singh, N. Unpublished data.

Relative intensity

Wheat

Amaranth

5

10

15

20

25

30

2␪(°)

Amaranth starch shows greater crystallinity than wheat starch, with strong reflections at 2°θ ¼ 15.1 degrees, 17.2 degrees, 18.1 degrees, and 23.2 degrees (Fig. 3). An additional peak at 2°θ ¼ 20.0 degrees is usually present, indicating the presence of amylose-lipid complexes. Amaranth starch shows intercultivar variability in crystallinity. Its starch has pasting temperature and gelatinization temperature ranges (To–Tp) between 69°C–72°C and 60°C–77°C, respectively. The difference in pasting behavior among starches from various genotypes has been observed due to differences in amylose content and crystallinity, as well as the presence or absence of amylose-lipid complexes. The pasting curve of starch separated from two genotypes of A. hypocondriacus is illustrated in Fig. 4. In comparison to cereal, pulse, tuber, and root starches, amaranth starch generally produces more stable pastes (low breakdown) owing to its small size and low amylose content.3

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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT

100

3000

80

2000 60 1500 40

IC-540860

1000

Temperature (°C)

Viscosity (cP)

2500

RMA-22

20

500 0

0 0

3

6

9

12

Time (min)

FIG. 4 Pasting curves of starch separated from different amaranth genotypes. From Singh N. Unpublished data. GIs of Various Foods

TABLE 1 Food

GI

Amaranth grain (raw)a

87

a

94

White bread

a

Amaranth grain (popped)

101

a

Amaranth grain (roasted)

106

a

Amaranth grain (flaked)

106

Amaranth grain (extruded)a

91

Amaranth grain (popped)b

97

Pearl barleyb

25

Sweet cornb

53

White riceb

64

Brown riceb

55

Parboiled riceb

47

b

48

Bulgur wheat Corn flakes

b

84

Puffed wheat

b

74

b

70

Wheat bread

b

69

Whole-meal bread b

28

Lentils

b

18

Soybean

Baked beans (canned)

b

48

a Capriles et al.18 [GI (predicted), determined using the equation 39.71 + 0.549 (hydrolysis index) of Goni et al.58]. b Foster-Powell et al.59; the reference food is glucose.

Amaranth grain is a high-glycemic food. Amaranth starch granules digested faster within the first hour than normal cornstarch.16 The rapid digestion of amaranth starch was attributed to its small size, low amylose content, and high tendency to lose its crystalline and granular starch structure completely during heating. The glycemic index (GI) of amaranth is compared with that of other cereal grains and foods in Table 1. The GI defines the carbohydrates present 2. FLOURS AND BREADS

GRAIN PROTEIN CHARACTERISTICS

127

in various foods on the basis of the postprandial level of blood glucose.17 The relationship between the rate of in vitro digestion and the glycemic response to food is well known. Raw amaranth seeds had rapidly digestible starch (RDS) content of 30.7% (dry weight basis) and a predicted GI of 87.2.18 Amaranth grains are enriched in various minerals, such as calcium, phosphorus, iron, potassium, and zinc, and vitamins E and B complexes, all of which are concentrated in the bran and germ portions of the grain. Amaranth contains higher amounts of riboflavin and ascorbic acid than cereal and is a good source of vitamin E.7 It also is a rich source of polyphenols (flavonoids), with relatively high antioxidant activity. Caffeic acid, p-hydroxybenzoic acid, and ferulic acid are the main phenolic compounds found in amaranth grains.19 The presence of polyphenols such as rutin (4.0–10.2 mg/g flour) and nicotiflorin (7.2–4.8 mg/g flour) in A. hypochondriacus varieties grown in the Mexican Highlands also has been reported.20 The calcium, magnesium, and oxalate concentrations in the grains of 30 amaranth genotypes of A. cruentus, A. hybridus, and A. hypochondriacus have been studied.21 Calcium and magnesium concentrations in the grains ranged between 134 and 370 mg/100 g and 230 and 387 mg/100 g, respectively, whereas the oxalate content ranged between 178 and 278 mg/100 g. Although dietary oxalate is a potential risk factor for kidney stone development and reduces the availability of calcium and magnesium, most of the oxalates in the amaranth grains are in insoluble form; as a result, the absorption may be low. However, this needs to be confirmed by bioavailability investigation. The dietary fiber and lipid content in the amaranth grain ranged between 8% and 17% and 3.0% and 10.5%, respectively. Although amaranth grain contains higher lipids than most of the cereals, the composition of its oil is quite similar to that of cereals, being high in unsaturated fatty acids (approximately 77%). Amaranth oil contains mainly linoleic acid and also contains tocotrienols that are associated with cholesterol-lowering activity in mammalian systems.22 Amaranth grain oil contains a significant amount (up to 8%) of squalene, exhibiting anticarcinogenic and hypocholesterolemic effects.23,24 Therefore, amaranth oil has the potential of replacing other squalene sources (e.g., shark and whale, which are endangered species).

GRAIN PROTEIN CHARACTERISTICS Cereals are normally deficient in lysine and tryptophan, whereas legume proteins show a deficiency of sulfurcontaining amino acids (namely, cysteine and methionine). Amaranth proteins, on the contrary, contain significant amounts of both sulfur-containing amino acids and lysine. Amaranth grains have higher protein (11%–17%) than most cereals. Amaranth is an appropriate grain for people who are allergic to gluten. The germ and endosperm of amaranth grain contain 65% and 35% of protein against an average of 15% and 85% in most cereals, respectively. The amino acid composition of various amaranth protein fractions is given in Table 2. Albumins and globulins are relatively rich in lysine and valine, while glutenins are high in leucine, threonine, and histidine. Aside from the amino acid composition, the protein quality also depends on bioavailability or digestibility. Protein digestibility, available lysine, net protein utilization, and protein efficiency ratio, which are indicators of protein nutritional quality, are substantially higher for amaranth protein than for cereal grains.25 Therefore, amaranth proteins are a promising food ingredient, capable of complementing and supplementing cereal or legume proteins.25 The protein digestibility corrected amino acid score of amaranth whole-meal flour is higher (0.64) than those of wheat (0.40) and oats (0.57).26 An average protein digestibility of 74.2% for raw amaranth whole-meal flour was reported.26 Thermal processing improves protein digestibility due to the opening of carbohydrate-protein complexes, the inactivation of antinutritional factors such as trypsin inhibitors, or both.26 Apart from their nutritional properties, amaranth proteins also possess functional properties that play important roles in formulation and processing.27 Amaranth protein isolate (API) improved the quality attributes (i.e., crust color, appearance, volume, height, springiness, and cohesiveness) and produced gluten-free muffins comparable to those with gluten.28 Contrary to legumes and cereals, where grain proteins generally serve as storage molecules for the growing plantlets, amaranth grain consists of the highest amount of albumins, which are usually biologically active. According to the Osborne classification, amaranth grain consists of three major fractions [namely, albumins (51%), globulins (16%), and glutelins (24%)] and a minor fraction (i.e., alcohol-soluble fraction or prolamine between 1.4% and 2.0%),29,30 whereas legume grain contains salt-soluble globulins as the major storage protein fraction. Based on size exclusion chromatographic analysis, it was reported that globulins were prominent proteins in kidney beans and field peas, while amaranth proteins contained both globulins and albumins as major fractions.31 Cereals such as maize and wheat, on the contrary, contain alcohol-soluble prolamins as the major storage proteins.32 The characterization of grain proteins of amaranth has been carried out using different techniques of extraction and electrophoresis.29,32–34 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of amaranth proteins 2. FLOURS AND BREADS

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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT

TABLE 2 Amino Acid Composition of Amaranth (A. hypochondriacus L.) Protein Fractions Protein Fractions Amino Acid

Meal

Albumins

Globulins

Prolamins

Glutelins

Isoleucinea

–

3.7

4.2

6.2

5.8

Leucinea

–

5.7

5.7

5.7

10.5

Lysinea

–

7.6

6.7

4.2

4.6

Methioninea

–

4.1

3.4

7.4

3.1

Cystinea

–

5.9

3.9

6.5

6.2

Phenylalaninea

–

5.1

5.0

9.0

6.8

Tyrosinea

–

3.3

4.3

4.0

3.8

–

3.9

4.1

3.2

8.6

–

4.5

4.7

2.7

3.8

–

2.5

1.1

1.1

4.7

–

5.1

4.0

4.7

3.6

–

8.1

9.5

9.4

2.7

–

6.2

8.7

6.2

6.1

–

17.5

17.3

13.4

13.2

Glycine

–

6.2

6.6

4.4

4.9

a

Proline

–

3.7

3.9

4.7

4.6

Serine

a

–

4.8

4.9

5.1

5.3

Serine

b

7.3

6.4

7.7

8.0

9.0

10.7

10.5

13.9

10.7

10.3

3.0

2.3

2.3

1.8

2.4

7.3

8.9

9.3

6.8

8.5

5.1

3.4

4.0

7.2

5.4

6.6

6.2

5.4

8.6

6.3

5.7

5.0

4.0

4.5

5.9

1.9

2.9

2.8

3.0

3.0

5.9

4.0

5.0

4.5

5.0

3.9

3.5

4.0

4.5

5.0

6.2

5.5

6.0

10.0

8.0

3.4

3.0

2.0

3.9

4.3

5.7

6.6

7.0

6.7

4.2

a

Threonine a

Valine

a

Histidine a

Alanine

a

Arginine

a

Aspartic acid

a

Glutamic acid a

b

Glycine

b

Histidine

b

Arginine

b

Threonine b

Alanine

b

Proline

b

Tyrosine b

Valine

b

Isoleucine b

Leucine

Phenylalanine b

Lysine

b

a

Expressed as g of amino acids/100 g of crude protein. Expressed in percent in moles. From Barba de la Rosa AP, Gueguen J, Paredes-Lopez O, Viroben G. Fractionation procedures, electrophoretic characterization and amino acid composition of amaranth seed protein. J Agric Food Chem 1992;40:931–936; Segura-Nieto M, Vaazquez-sanchez N, Rubio-Velazqez H, Olguin-Martin LE, Rodriguez-Nester CE, HerreraEstrella L. Characterization of amaranth (Amaranthus hypocondriacus) seed proteins. J Agric Food Chem 1992;40:1553–1558.

b

reveals a wide range in the molecular weight of polypeptide subunits among varieties and species (Fig. 5). On the basis of differential extraction, the amaranth albumin (AmA) was classified as albumin 1 and albumin 2.35 Albumin 1 is extractable with water, saline solution, or both, whereas albumin 2 is extractable with water after the removal of albumin 1 and globulin with saline solution. Albumin 2 has amarantin as the major protein component.30 The subunit size of albumin proteins varied from 10 to 37 kDa,29,33 with low molecular subunits being more abundant.36 Barbara de la

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GRAIN PROTEIN CHARACTERISTICS

97.4 66.0

43.0

110 96 91 73 61 56 53 43

(A)

IC540862

IC540860

IC540839

IC467901

IC042311–7

IC042254–5

IC042265–2

IC095341

52 43.0

42 40 38 33

29.0

28

26 24

26 23

23

22

20

20.0

18 17 16 14.3

IC38312

66.0

34 31

20.0

VL–0344

97.4

38

29.0

RMA–30

RMA–22

PRA–3

PRA–2

Annapurna

Marker

IC540869

IC540828

Amaranthus hypochondriacus

IC467902

IC423448

IC423393

IC363742

IC258399

IC38181

IC38165

Marker

Amaranthus caudatus

10

20 16

14.3

15 14

(B)

FIG. 5 SDS-PAGE (12% resolving gel) analysis (under reducing conditions) of (A) A. caudatus and (B) A. hypochondriacus protein isolates. From Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. Thesis submitted to Guru Nanak Dev University, India, 2014.

Rosa et al.,20 however, differentiated the albumin fraction into two groups of proteins corresponding to around 18 kDa and between 40 and 80 kDa. The proteins of around 18 kDa were termed methionine-rich proteins due to their high methionine content (between 16% and 18%).37 Determination of sedimentation coefficient by centrifugation has also been widely used to characterize the proteins. On the basis of sedimentation coefficients, the amaranth seed globulins are categorized into 10S and 12.7S, as compared to 7/8S and 11/12S for the legume seed globulins. The electrophoretic behavior of 10S and 12.7S amaranth globulin fractions on denaturing gel was observed to be similar to that of 7S and 11S storage proteins of legumes; hence, they are referred to as 7S and 11S, respectively.34 The higher sedimentation coefficients of amaranth globulins, as observed on linear sucrose gradients, suggested that these proteins contain polypeptides with higher molecular weight than those present in 7S and 11S from pea globulins.36 The 7S and 11S amaranth seed globulins also differed in their solubility in salt solution, with the former being extractable with 0.1 M and the latter with 0.8 M of sodium chloride (NaCl).20 The 7S globulin fraction of amaranth grain was characterized by the presence of a main band of 38 kDa and lacked disulfide bridges, whereas the 11S-like globulins consisted of both acidic (35–38 kDa) and basic polypeptides (22–25 kDa). These results were in agreement with earlier studies finding that globulins consisted of polypeptides of heterogenous sizes, as demonstrated by SDS-PAGE analysis.36 However, these observations contradicted the findings of Gorinstein et al.,29 which reported that the globulin was composed of polypeptides of only 14–18 kDa. Martinez et al.,30 proposed that both 7S and 11S globulins correspond to one type of globulin, whereas polymerized globulins (albumin 2) and glutelins correspond to two other types of globulins. This notion, however, needs to be supported by establishing sequence homology and by proving a common genetic origin of globulin, albumin 2, and glutelin. The most important component of globulins is amarantin, which alone constitutes 90% of the total globulins and about 19% of the total grain protein.38 Amarantin is a homohexameric molecule of approximately 300–400 kDa, comprised of subunits of about 59 kDa. Each of these subunits consists of an acidic polypeptide of 34–36 kDa and a basic polypeptide of 22–24 kDa linked by disulfide bonds. The additional subunit of 54 kDa present in amarantin has been proposed to act as an inducer of polymerization.30

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10. AMARANTH: POTENTIAL SOURCE FOR FLOUR ENRICHMENT

The amaranth glutelin showed high similarity with 11S globulins and was comprised of three major polypeptide groups of 22–25 kDa, 35–38 kDa, and around 55 kDa.39 It is likely that both glutelins and 11S globulins may belong to the same structural gene family. The differences in composition of alcohol-soluble proteins have been reported in various studies. Gorinstein et al.29 observed only low-molecular-weight subunits of 10–20 kDa via SDS-PAGE analysis of the alcohol-soluble proteins, whereas Barba de la Rosa et al.33 reported the presence of subunits with both low and high molecular mass. Furthermore, great similarity between the electrophoretic patterns of reduced prolamine and glutelins was also observed in the latter study. The lack of consistency in the composition of various protein fractions of amaranth grain, which is evident from the literature, may be due to the various procedures used in the extraction and analyses. In view of the nutritional importance of the amaranth, it is imperative that a systematic study should be undertaken to analyze the proteome of amaranth leaves and grains by employing the latest techniques of proteomics. This will enable the identification and characterization of nutritionally important proteins, the genes for which can then be cloned and expressed heterologously in other crops for enhancing their nutritive value.

NUTRACEUTICAL PROPERTIES OF AMARANTH PROTEINS The albumins and globulins are rich in lysine and valine, whereas prolamins have comparatively higher content of methionine and cystine. Glutelins, on the other hand, contain higher levels of leucine, threonine, and histidine. Compared to legume grain albumins, which contain several antinutritional factors, the AmA fraction is considered safe. The AmA fraction is comparable with egg-white proteins and can be used as an egg substitute in various products. The 11S globulin fraction is rich in peptides of angiotensin-converting enzyme (ACE) inhibitor, whereas the glutelin fraction contains antihypertensive activity, as well as the anticarcinogenic lunasinlike peptide,40 thus signifying its nutraceutical properties. Amaranth proteins also exhibit antioxidant activity, which increases after gastrointestinal digestion41 and potential antitumor properties through putative mechanism of action.42 In addition, they affect the metabolism of liver lipids and have a hypotriglyceridemic effect in rats.24 Simulated gastrointestinal digestion of APIs resulted in the identification of several bioactive peptides, which showed very high antithrombotic activity. These bioactive peptides belong to 11S globulin and agglutinin fractions of amaranth.43 An amaranth peptide (SSEDIKE) attenuated the expression of the CCL20 (Caco-2 cells transfected with a luciferase reporter under the control of the CCL20 promoter) gene at the transcript level in colonic epithelial cells. Thus, the antiinflammatory effect of amaranth protein hydrolysate was demonstrated at molecular levels.44 Milk allergens are a major cause of intestinal inflammation and are considered as allergens. Oral administration of the amaranth-charged peptide (SSEDIKE) resulted in the inhibition of the allergen response in a mouse model system.44 Delgado et al.45 demonstrated that the simulated gastrointestinal digestion of Amaranthus mantegazzianus proteins yielded bioactive peptides (i.e., AWEEREQGSR, YLAGKPQQEH, IYIEQGNGITGM, and TEVWDSNEQ), which showed potential antioxidant activity and belongs to the 11S globulin protein family. Furthermore, amaranth bioactive cationic (HVIKPPSRA and KFNRPETT) and neutral (GDRFQDQHQ) peptides were also shown to exhibit the in vivo inhibition of Cu+2/H2O2-induced LDL oxidation.46

FOOD AND NONFOOD APPLICATIONS The unique small starch granule size and composition has been suggested to be responsible for unique gelatinization and freeze/thaw characteristics, which could be exploited in the development of products by the food industry.47 Amaranth starch can be used in many food preparations, such as custards, pastes, and salad, and nonfood applications such as cosmetics, biodegradable films, paper coatings, and laundry starch. The modified A. viridis starches also have the potential to partially replace fat in fat-rich foods such as mayonnaise and salad cream.48 Amaranth flour is used as a thickener in gravies, soups, and stews. Sprouted amaranth is used in salads. Defatted amaranth flours may be preferred over full-fat flours, as they have better flavor stability and can be marketed as low-fat or low-calorie products. In addition, extracted oil can be used for other purposes and these flours have improved functionality.10 However, the improvement in the functional properties of flours depends on protein characteristics, which in turn vary based on the cultivars.10,27 The cooking of amaranth improves its digestibility and absorption of nutrients. Amaranth flour lacks the gluten proteins present in wheat, and hence it is not suitable for bread-making. A blend of amaranth flour with wheat meal or flour is used in the preparation of an unleavened flatbread known as chapatis in India and tortillas in Latin America. Amaranth flour is also used to prepare biscuits, muffins, pancakes, pastas, flatbreads, extruded products, and other foodstuffs. In India, the grains are most commonly used in the form of candy known as laddoos. 2. FLOURS AND BREADS

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Waxy and normal maize starch granules were transformed into starch nanocrystals, and protein films were prepared after blending with amaranth protein formulations.49 One study demonstrated that nanocomposite films exhibited improved water vapor permeability, water uptake, surface hydrophobicity, mechanical behavior, and delayed weight loss in soil than that of normal protein films. It was proposed that the good dispersion of the nanoreinforcements in the protein matrix is due to the formation of disulfide bonds among waxy maize nanocrystals, as well as hydrogen bonding between normal maize nanocrystals and protein matrix.49 Amaranth proteins compared well with pulse (kidney bean and field pea) proteins in terms of edible/biodegradable film-forming31 and gluten-free muffinmaking (Fig. 6) properties.28 Furthermore, API edible films prepared from different ultra-high-pressure treatments demonstrated uniform surface and more opaque characteristics, along with improved mechanical properties, lower water solubility, and water vapor permeability, as compared to nontreated protein dispersions.50,51 Encapsulation of folic acid into API-pullulan electrospun fibers resulted in photoprotection of folic acid.52 It has been reported that APIs at acidic pH (2.0) show improved foam stability over those with alkaline pH.53 Increased unfolding, greater flexibility, and the net charge on amaranth proteins under acidic conditions were attributed to

FIG. 6 Crusts (I) and crumb cross sections (II) of muffins prepared from (A) cornstarch; (B) cornstarch with wheat gluten; (C) cornstarch with kidney bean protein isolate; (D) cornstarch with field pea protein isolate; and (E) cornstarch with API. The starch was replaced with calculated amount of proteins in order to get a protein content of 10%. From Shevkani K, Singh N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Int J Food Sci Tech 2014;49:2237–2244.

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the higher foam stability of proteins. These findings thus demonstrated that the viscoelasticity and flexibility of foams were influenced by the solubility of protein isolates at different ionic strengths and pHs of solubilizing solvents.53 Suarez and Añón54 also demonstrated that APIs solubilized at low pH (2.0) adsorbed in an oil/water interface twice as quickly as APIs prepared at pH 6.3 and allowed in the formation of oil-in-water emulsions. These findings indicated the treatment of APIs at various ionic and pH conditions may be helpful in the improvement of texture and sensory properties of food products with improved nutraceutical values. In comparison to amaranth grain, vegetable amaranth has received less attention. Vegetable amaranth is used as a delicacy or a food staple in many parts of the world. Amaranthus leaves are used as a vegetable in the northern states of India. However, its use is limited to saag, a dish prepared by cooking mustard leaves with garlic, ginger, green chilies, and salt. Vegetable amaranth tastes better than spinach and is substantially higher in calcium, iron, and phosphorous.

TRANSGENIC APPLICATIONS Improving the balance of essential amino acids in important crop plants remains one of the major objectives of plant breeders. Transgenic technology presents an attractive alternative for improving the nutritional quality of grain proteins. Heterologous transgenic expression of storage protein genes with higher levels of limiting amino acids has been reported. Transgenic expression of high levels of a particular amino acid may affect the normal physiology of seed development adversely or produce seeds with a biased amino acid composition. Therefore, expressing a gene for a heterologous protein with a balanced amino acid composition is a better alternative. A gene of a 35-kDa albumin protein (AmA1), which is expressed during early to mid-maturation stages of embryogenesis in the amaranth seed was cloned.55 The amino acid composition of this protein conforms to the World Health Organization (WHO)–recommended values for a highly nutritional protein because it is rich in various essential amino acids. Potato is the most important noncereal crop in terms of total global food production; therefore, transgenic expression of this gene in the tubers of this crop has been achieved. Heterologous expression of AmA1 under constitutive [Cauliflower mosaic virus (CaMV) 35S promoter] and tuber-specific promoter (granule-bound starch synthase) in potato resulted in significant enhancement in total protein content, with an increase in essential amino acids.56 Furthermore, the growth and production of tubers in transgenic plants were also higher than for control plants. The maize, which is a staple food in many countries but lacks essential amino acid in the grains, has also been targeted for heterologous expression of amaranth proteins to enhance the nutritional quality of its protein. Complementary DNA (cDNA) of an 11S globulin storage protein, amarantin, which has a high content of essential amino acids, was expressed in maize under the CaMV 35S promoter and an endosperm-specific promoter (rice glutelin-1).57 Heterologous expression of this gene resulted in an increase of 18% in lysine, 28% in sulfur-containing amino acids, and 36% in isoleucine, as well as a 32% increase in total seed protein. Furthermore, the heterologously expressed protein was digested by simulated gastric and intestinal fluids, thus confirming the biodigestibility of the transgenic protein. These studies, therefore, validate the potential of using various amaranth genes for supplementing and complementing the proteins in both cereal and noncereal staple crops. However, detailed studies on the analysis of proteomes of amaranth species needs to be carried out to identify the candidate genes, which can be employed for transgenic improvement. Furthermore, the generation of mutants in amaranth is also required to determine the role of specific proteins in the growth and development of the plant so that appropriate improvement of the germplasm can be undertaken through conventional breeding strategies.

TECHNOLOGICAL ISSUES The presence of diversity in composition among amaranth genotypes necessitates in-depth characterization of biochemical constituents for its specific applications in the food industry. The smaller granules in amaranth starch, which resemble the size of fat globules in cow’s milk, can be exploited to mimic fat in a number of food products. Some of the genotypes have higher polyphenols with higher antioxidant activity, which also could be utilized in the development of new products. Amaranth grain has the potential to develop various food products for people suffering from celiac disease, a disorder that makes the body intolerant to gluten proteins.

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SUMMARY POINTS • Amaranth grain is a good source of dietary fiber and has high GI. It is low in resistant starch (RS), and its starch has uniquely small granules with low tendency toward retrogradation. • Grain amaranth has higher protein than most of the cereal grains and is an appropriate food for people who are allergic to gluten. • Amaranth grain oil is quite similar to that of cereals, being high in unsaturated fatty acids, and it contains mainly linoleic acid. Its oil also contains tocotrienols, which are associated with cholesterol-lowering activity in mammalian systems. Amaranthus grain oil also contains a significant amount of squalene, which has anticarcinogenic and hypocholesterolemic effects. • Amaranth grain proteins are mainly composed of three major fractions (namely, albumins, globulins, and glutelins) with little or no storage of prolamin. Amarantin is the most important component of globulins and constitutes 90% of the total globulins and about 19% of the total grain protein. • Heterologous expression of the amarantin gene (AmA1) in potato results in significant enhancement in total protein content, with an increase in essential amino acids as well. • Amaranth is also a good source of minerals such as iron, magnesium, phosphorus, copper, and manganese. Its unique composition also makes it an attractive food complement and supplement.

Acknowledgments The financial assistance to NS from the Department of Science and Technology, Ministry of Science and Technology, Government of India, is acknowledged.

References 1. Stallknecht GE, Schulz-Schaeffer JR. Amaranth rediscovered. In: Janick J, Simon JE, editors. New crops. New York: John Wiley & Sons; 1993. p. 211–8. 2. Cai YZ, Corke H, Wu HX. Amaranth. In: Wrigley CW, Corke H, Walker CE, editors. Encyclopedia of grain science. Vol. 1. Oxford: Elsevier; 2004. p. 1–10. 3. Zhu F. Structures, physicochemical properties, and applications of amaranth starch. Crit Rev Food Sci Nutr 2017;57:313–25. 4. Breene WM. Food uses of grain amaranth. Cereal Foods World 1991;36:426–30. 5. Plate AY, Ar^eas JA. Cholesterol-lowering effect of extruded amaranth (Amaranthus caudatus L.) in hypercholesterolemic rabbits. Food Chem 2002;76:1–6. 6. Mendonca S, Saldiva PH, Cruz RJ, Ar^eas JA. Amaranth protein presents cholesterol-lowering effect. Food Chem 2009;116:738–42. 7. Rastogi A, Shukla S. Amaranth: a new millennium crop of nutraceutical values. Crit Rev Food Sci Nutr 2013;53:109–25. 8. Kaur S, Singh N, Rana JC. Amaranthus hypochondriacus and Amaranthus caudatus germplasm: characteristics of plants, grain and flours. Food Chem 2010;123:1227–34. 9. Bressani R. Composition and nutritional properties of amaranth. In: Peredes-Lopez O, editor. Amaranth biology, chemistry and technology. Boca Raton: CRC Press; 1994. p. 185–205. 10. Shevkani K, Singh N, Kaur A, Rana JC. Physicochemical, pasting, and functional properties of amaranth seed flours: effects of lipids removal. J Food Sci 2014;79:C1271–7. 11. Pedersen B, Knudsen KB, Eggum BO. The nutritive value of amaranth grain (Amaranthus caudatus). Plant Foods Hum Nutr 1990;40:61–71. 12. Wilhelm E, Aberle T, Burchard W, Landers R. Peculiarities of aqueous amaranth starch suspensions. Biomacromolecules 2002;3:17–26. 13. Singh N, Kaur S, Kaur A, Isono N, Ichihashi Y, Noda T, Rana JC. Structural, thermal, and rheological properties of Amaranthus hypochondriacus and Amaranthus caudatus starches. Starch 2014;66:457–67. 14. Hoover R, Sinnott AW, Perera C. Physicochemical characterization of starches from Amaranthus cruentus grains. Starch-St€ arke 1998;50:456–63. 15. Shevkani K, Singh N, Bajaj R, Kaur A. Wheat starch production, structure, functionality and applications—a review. Int J Food Sci Technol 2017;52:38–58. 16. Konishi Y, Nojima H, Okuno K, Asaoka M, Fuwa H. Characterization of starch granules from waxy, nonwaxy, and hybrid seeds of Amaranthus hypochondriacus L. Agric Biol Chem 1985;49:1965–71. 17. Jenkins AL. The glycemic index: looking back 25 years. Cereal Foods World 2007;52:50–3. 18. Capriles VD, Coelho KD, Guerra-Matias AC, Areas JA. Effects of processing methods on amaranth starch digestibility and predicted glycemic index. J Food Sci 2008;73:H160–4. 19. Klimczak I, Malecka M, Pacholek B. Antioxidant activity of ethanolic extracts of amaranth seeds. Nahrung 2002;46:184–6. 20. Barba de la Rosa AP, Fomsgaard IS, Laursen B, Mortensen AG, Olvera-Martınez L, Silva-Sanchez C, Mendoza-Herrera A, Gonzalez-Castaneda J, De Leon-Rodrıguez A. Amaranth (Amaranthus hypochondriacus) as an alternative crop for sustainable food production: phenolic acids and flavonoids with potential impact on its nutraceutical quality. J Cereal Sci 2009;49:117–21. 21. Gelinas B, Seguin P. Oxalate in grain amaranth. J Agric Food Chem 2007;55:4789–94. 22. Becker R. Preparation, composition and nutritional implications of amaranth seed oil. Cereal Foods World 1989;36:426–9.

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23. Sun H, Wiesenborn D, Tostenson K, Gillespie J, Rayas-Duarte P. Fractionation of squalene from amaranth seed oil. J Am Oil Chem Soc 1997;74:413–8. 24. Escudero NL, Zirulnik F, Gomez NN, Mucciarelli SI, Mucciarelli SI, Gimenez MS. Influence of a protein concentrate from Amaranthus cruentus seeds on lipid metabolism. Exp Biol Med 2006;231:50–9. 25. Guzman-Maldonado SH, Paredes-Lopez O. Biotechnology for the improvement of nutritional quality of food crop plants. In: Paredes-Lo’pez O, editor. Molecular biotechnology for plant food production. Lancaster: Technomic Publishing; 1999. p. 553–620. 26. Bejosano FP, Corke H. Protein quality evaluation of Amaranthus wholemeal flours and protein concentrates. J Sci Food Agric 1998;76:100–6. 27. Shevkani K, Singh N, Rana JC, Kaur A. Relationship between physicochemical and functional properties of amaranth (Amaranthus hypochondriacus) protein isolates. Int J Food Sci Technol 2014;49:541–50. 28. Shevkani K, Singh N. Influence of kidney bean, field pea and amaranth protein isolates on the characteristics of starch-based gluten-free muffins. Int J Food Sci Technol 2014;49:2237–44. 29. Gorinstein S, Moshe R, Greene LJ, Arruda P. Evaluation of four amaranthus species through protein electrophoretical patterns and their amino acid composition. J Agric Food Chem 1991;51:851–4. 30. Martinez EN, Castellani OF, Anon MC. Common molecular features among amaranth storage proteins. J Agric Food Chem 1997;46:4849–53. 31. Shevkani K, Singh N. Relationship between protein characteristics and film-forming properties of kidney bean, field pea and amaranth protein isolates. Int J Food Sci Technol 2014;50:1033–43. 32. Gorinstein S, Denue IA, Arruda P. Alcohol soluble and total proteins from amaranth seeds and their comparison with other cereals. J Agric Food Chem 1991;39:848–50. 33. Barba de la Rosa AP, Gueguen J, Paredes-Lopez O, Viroben G. Fractionation procedures, electrophoretic characterization and amino acid composition of amaranth seed protein. J Agric Food Chem 1992;40:931–6. 34. Barba de la Rosa AP, Paredes-Lopez O, Gueguen J. Characterization of amaranth globulins by ultracentrifugation and chromatographic techniques. J Agric Food Chem 1992;40:937–40. 35. Konishi Y, Horikawa K, Oku Y, Azumaya J, Nakatani N. Extraction of two albumin fractions from amaranth grains: comparison of some physicochemical properties and putative localization in the grains. J Agric Biol Chem 1991;55:1745–50. 36. Segura-Nieto M, Vaazquez-sanchez N, Rubio-Velazqez H, Olguin-Martin LE, Rodriguez-Nester CE, Herrera-Estrella L. Characterization of amaranth (Amaranthus hypocondriacus) seed proteins. J Agric Food Chem 1992;40:1553–8. 37. Segura-Nieto M, Barba-de-la-Rosa AP, Paredes-Lopez O. Biochemistry of amaranth proteins. In: Paredes-Lopez O, editor. Amaranth biology, chemistry and technology. Boca Raton: CRC Press; 1994. p. 75–106. 38. Romero-Zepada H, Paredes-Lopez O. Isolation and characterization of amarantin, the 11S amaranth seed globulin. J Agric Food Chem 1996;19:329–39. 39. Abugoch LE, Martinez EN, Anon MC. Influence of the extracting solvent upon the structural properties of amaranth (Amaranthus hypochondriacus) glutelin. J Agric Food Chem 2003;51:460–5. 40. Silva-Sanchez C, Barba de la Rosa AP, Leon-Galvan F, de Lumen BO, De Leon-Rodrıguez A, Gonzalez de Mejıa E. Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed storage proteins. J Agric Chem 2008;56:1233–40. 41. Delgado MCO, Tironi VA, Añón MC. Antioxidant activity of amaranth protein or their hydrolysates under simulated gastrointestinal digestion. LWT Food Sci Technol 2011;44:1752–60. 42. Barrio DA, Anon MC. Potential antitumor properties of a protein isolate obtained from the seeds of Amaranthus mantegazzianus. Eur J Nutr 2010;49:73–82. 43. Sabbione AC, Nardo AE, Añón MC, Scilingo A. Amaranth peptides with antithrombotic activity released by simulated gastrointestinal digestion. J Funct Foods 2016;20:204–14. 44. Moronta J, Smaldini PL, Fossati CA, Añon MC, Docena GH. The anti-inflammatory SSEDIKE peptide from Amaranth seeds modulates IgEmediated food allergy. J Funct Foods 2016;25:579–87. 45. Delgado MCO, Nardo A, Pavlovic M, Rogniaux H, Añón MC, Tironi VA. Identification and characterization of antioxidant peptides obtained by gastrointestinal digestion of amaranth proteins. Food Chem 2016;197:1160–7. 46. Fillería SFG, Tironi VA. Prevention of in vitro oxidation of low density lipoproteins (LDL) by amaranth peptides released by gastrointestinal digestion. J Funct Foods 2017;34:197–206. 47. Becker R, Wheeler EL, Lorenz K, Stafford AE, Grosjean OK, Betschart AA. A compositional study of amaranth grain. J Food Sci 1981;46:1175–8. 48. Fasuan TO, Gbadamosi SO, Akanbi CT. Modification of amaranth (Amaranthus viridis) starch, identification of functional groups, and its potentials as fat replacer. J Food Biochem 2018; https://doi.org/10.1111/jfbc.12537. In press. 49. Condes MC, Añón MC, Mauri AN, Dufresne A. Amaranth protein films reinforced with maize starch nanocrystals. Food Hydrocoll 2015;47:146–57. 50. Condes MC, Añón MC, Mauri AN. Amaranth protein films prepared with high-pressure treated proteins. J Food Eng 2015;166:38–44. 51. Condes MC, Añón MC, Dufresne A, Mauri AN. Composite and nanocomposite films based on amaranth biopolymers. Food Hydrocoll 2018;74:159–67. 52. Aceituno-Medina M, Mendoza S, Lagaron JM, López-Rubio A. Photoprotection of folic acid upon encapsulation in food-grade amaranth (Amaranthus hypochondriacus L.) protein isolate—pullulan electrospun fibers. LWT Food Sci Technol 2015;62:970–5. 53. Bolontrade AJ, Scilingo AA, Añón MC. Amaranth proteins foaming properties: film rheology and foam stability-part 2. Colloids Surf B: Biointerfaces 2016;141:643–50. 54. Suarez SE, Añón MC. Comparative behaviour of solutions and dispersions of amaranth proteins on their emulsifying properties. Food Hydrocoll 2018;74:115–23. 55. Raina A, Datta A. Molecular-cloning of a gene encoding a seed-specific protein with nutritionally balanced amino-acid-composition from Amaranthus. Proc Natl Acad Sci U S A 1992;89:1774–8. 56. Chakraborty S, Chakraborty N, Datta A. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. Proc Natl Acad Sci U S A 2000;97:3724–9.

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57. Rascón-Cruz Q, Sinagawa-García S, Osuna-Castro JA, Paredes-López O. Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize. Theo App Gene 2004;108:335–42. 58. Goni I, Garcia-Alonso A, Saura-Calixto F. A starch hydrolysis procedure to estimate glycemic index. Nutr Res 1997;17:427–37. 59. Foster-Powell K, Holts S, Brand-Miller JC. International table of glycemic index and glycemic load values. Am J Clin Nutr 2002;76:5–56.

Further Reading 60. Kaur S. Studies on functional properties and utilization of leaves and grains of amaranthus. Ph.D. thesis submitted to Guru Nanak Dev University, India; 2014.

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C H A P T E R

11 Sorghum Flour and Flour Products: Production, Nutritional Quality, and Fortification Joseph O. Anyango*, and John R.N. Taylor† *Department of Dairy, Food Science and Technology, Egerton University, Egerton-Njoro, Kenya † Department of Consumer and Food Sciences, University of Pretoria, Pretoria, South Africa

O U T L I N E Introduction

137

Flour Milling

144

Grain Structure With Respect to Milling Pericarp Endosperm Germ

138 138 138 138

Products Made From Sorghum Flour Traditional Products Modern Products

144 144 145

Grain Chemical Components: Functional and Nutritional Attributes Starch Proteins Lipids Nonstarch Polysaccharides (NSPs) Phytochemicals Phenolic Compounds

139 139 139 141 141 142 142

New Developments in Sorghum Flours Bread-Making Technology Biofortification

146 146 147

Technological Issues

149

Summary Points

149

References

150

Abbreviations Approx. GAX HMW LMW NSP RDA

approximately glucuronoarabinoxylans high molecular weight low molecular weight nonstarch polysaccharides recommended dietary allowance

INTRODUCTION Sorghum [Sorghum bicolor (L.) Moench] is a tropical cereal. The sorghum grain is naked grain—that is, during threshing, the glumes are removed from the grain. Thus, there is no husk to remove during milling. The grain is approximately 4 mm in length, more or less spherical in shape, but somewhat flattened at the germ end.1

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The 1000-kernel weight ranges from approximately 25 to 35 g. Sorghum grain color varies from almost white to almost black, with shades of red and brown being common as well.2 The grain color strongly affects the flour color. Sorghum is uniquely adapted to arid and semiarid conditions, and also can survive periods of waterlogging.3 It is the fifth-most-important cereal crop worldwide, with the production of approximately 63.8 million tons in 2016.4 The 10 leading sorghum-producing countries are the United States, Nigeria, India, Mexico, Sudan, Argentina, China, Ethiopia, Burkina Faso, and Egypt. In the United States, Mexico, and Argentina, sorghum is mainly used for animal feed. However, in the other countries, particularly India and the African countries, it is mostly used for human food and for brewing beer. Sorghum is a gluten-free cereal5 that contains various phenolic compounds that appear to have health benefits,6 which makes the grain suitable for developing functional foods and nutraceuticals.

GRAIN STRUCTURE WITH RESPECT TO MILLING The sorghum grain (caryopsis), like all other cereal grains, consists of three distinct parts: the pericarp (outer layer), endosperm (storage tissue), and germ (embryo). It should be noted that the aim of milling is generally not only to reduce the grain to small particles, but to also separate the pericarp and germ from the endosperm, with the resulting flour being endosperm of varying purities. The pericarp and germ are removed, as most people find that the color, texture, and taste imparted by them to food products adversely affects their appeal.

Pericarp The pericarp, which is rich in insoluble dietary fiber, accounts for about 4.3%–8.7% of sorghum grain.7 It is subdivided into three tissues—namely, the epicarp, mesocarp, and endocarp (listed from the outside in). The epicarp is covered with a thin layer of wax and contains most of the sorghum grain pigments; hence, it has a great influence on grain color. The mesocarp contains starch granules, which is a feature unique to sorghum and pearl millet.1 It has been suggested that the presence of starch granules in the mesocarp could account for the high friability of the sorghum pericarp.8 Friability is a negative attribute of the pericarp for dry milling, as it causes fragmentation into fine pieces, escaping separation and thereby contaminating the flour. Some sorghum cultivars have a pigmented subcoat (known as testa) between the pericarp and the endosperm. The pigmented testa contains condensed tannins,7 which protect these sorghums against insects, birds, and fungal attack. Until recently, sorghum tannins were viewed as undesirable due to their antinutritive properties. For example, they complex with food macromolecules such as proteins, reducing their digestibility. Likewise, these polyphenolic compounds inhibit the absorption of essential minerals, such as iron.9, 10 However, current research indicates that tannins have health benefits, which are discussed later in this chapter.

Endosperm The endosperm is the largest part, constituting 82%–87% of the sorghum grain7 and containing mainly starch and protein. It is made up of the aleurone layer, the peripheral area, and corneous (hard) and floury (soft) areas. The latter two account for the largest portion of the endosperm. In sorghum, the aleurone layer is just one cell layer thick. It contains protein bodies, phytin bodies, and oil bodies (spherosomes). The peripheral endosperm region comprises several layers of dense cells containing essentially just protein bodies. The corneous endosperm cells contain a continuous matrix of kafirin protein-containing bodies, glutelin matrix protein, and starch granules. In the floury endosperm, starch granules, matrix protein, and protein bodies are discontinuous, with air spaces in the cells. The proportion of starch relative to protein is also higher. The relative proportion of corneous and floury endosperm is largely genetically controlled. When milled, sorghums with a higher proportion of corneous endosperm yield a grittier flour, due to stronger adhesion between the starch granules and surrounding protein. The tannin sorghums invariably have a high proportion of the softer, floury endosperm.

Germ The germ is the living part of the sorghum grain, and it consists of two main parts: the embryonic axis and scutellum.1 The germ is very rich in lipids (approximately 28% by weight, or 76% of total grain lipids). It is also

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relatively protein-rich (approximately 18% by weight, or 15% of total grain proteins). The germ proteins are mainly albumins and globulins, which are rich in lysine and other essential amino acids.11

GRAIN CHEMICAL COMPONENTS: FUNCTIONAL AND NUTRITIONAL ATTRIBUTES Starch Starch constitutes approximately 71% of sorghum grain.1 Sorghum starch gelatinization temperature, in the range 66°C–81°C, is high in comparison to wheat, and possibly slightly higher than maize.2 It also seems to be quite variable between sorghum cultivars. Generally, sorghum has a lower starch digestibility than maize.2 On this basis, it has been suggested that sorghum may be a particularly suitable food for diabetic and obese persons.12 However, there is little if any direct evidence to support this contention. It appears that the lower starch digestibility is not an intrinsic property of sorghum starch, but rather primarily a result of the endosperm protein matrix, cell wall material, and tannins (if present) inhibiting enzymatic hydrolysis of the starch.2 Protein disulfide bond cross-linking involving kafirin proteins in the protein matrix around the starch granules seems to be of major importance in reducing starch digestibility.13

Proteins Sorghum grain protein content varies quite a bit, ranging from approximately 7%–16% with an average of about 11%1 (see Table 1). The major sorghum grain proteins are prolamin storage proteins, as in virtually all other cereal TABLE 1 Nutrient Composition per 100 g of Whole Sorghum and Morvite (Fortified, Precooked Sorghum Flour) Morvite (% RDA for persons 10 years or older)b

Nutrient

Sorghuma

Morviteb

Energy (kJ)

1374

1506

Protein (g)

11.6

7.1

Lipid (g)

3.4

3.8

Carbohydrate (g)

77.0

77.8

Dietary fiber (g)

6.3–11.5

2.7

Sodium (Na) (mg)

6

208

Calcium (Ca) (mg)

29

120

15

Iron (Fe) (mg)

4.5

2.1 (electrolytic)

15

Zinc (Zn) (mg)

1.4

2.25

15

I (μg)

No data

23

15

Selenium (Se) (μg)

No data

8.25

15

Vitamin A (μg retinol equiv.)

10–20

200

20

Vitamin B1 (mg)

0.24

0.35

25

Vitamin B2 (mg)

0.15

0.4

25

Niacin (mg)

3.0

4.5

25

Vitamin B6 (mg)

0.48

0.5

25

Folic acid (μg)

84

50

25

Pantothenic acid (mg)

No data

1.5

25

Vitamin C (mg)

0

39

65

Vitamin E (α-tocopherol) (mg)

1.2

1.5

15

a b

13

Data from USDA Nutrient Database (www.nal.usda.gov/fnic/foodcomp/search) and Serna-Saldivar and Rooney.1 Manufacturer’s data.

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grains, and are known as kafirins.14 Kafirins are classified into four major species based on the differences in molecular weight, solubility, structure, and amino acid composition and sequence. These are the α-, β-, γ-, and δ-kafirins (Table 2). Our recent research15 has shown that the γ-kafirin class has the highest glass transition temperature (Tg) (Fig. 1) among the four classes, and it is needed to form and stabilize spherical kafirin microstructures (microparticles) due to its ability to form disulfide cross-links. Perhaps γ-kafirin encases the other kafirin classes in a similar manner as reported for prolamin protein bodies.16 This knowledge of the thermal properties of isolated γ-kafirin will be useful in the development of stable, gluten-free dough. Kafirins have low nutritional quality because they are poor in essential amino acids, particularly lysine.11 They are also poorly digestible, especially when cooked in water, as occurs during most food preparation processes.17 This may constitute a major nutrition problem for children in developing countries where sorghum is a major staple.18 However, an important positive health impact with respect to the kafirins is that, because they are so different in structure from the wheat gliadin and glutenin storage proteins (Table 2), sorghum does not elicit morphometric or immunomediated alteration of duodenal explants from patients suffering from celiac disease.5 Celiac disease, a syndrome characterized by damage to the mucosa of the small intestine, is caused by the ingestion of wheat gluten and similar proteins.19 Celiac disease is becoming recognized as a major nutritional problem in Western countries, affecting at least 1 in 150 people. The only treatment is lifelong avoidance of foods containing wheat and similar cereals such as rye, triticale, and barley. Hence, sorghum is a viable and important alternative for making baked products such as bread. However, sorghum flour dough does not have the viscoelastic, gas-holding property of wheat flour dough. This is because when mixed with water, gluten proteins become hydrated and form a three-dimensional (3D) network, which is responsible for the unique viscoelastic property of wheat dough20, 21). Because kafirins are more hydrophobic than gluten proteins, related to the high levels of leucine (Table 2), kafirins are difficult to hydrate. It has been suggested that this poor hydration of kafirins also may be linked to their mainly α-helical structure, in contrast to the high-molecularweight (HMW) glutenin subunits of wheat, which have a high level of β-sheets and β-turn structures.14 As Table 2 demonstrates, kafirins are much smaller proteins than HMW glutenins, which probably also has a bearing on their lack of elasticity. Additionally, as kafirins are encapsulated in protein bodies,17 this probably makes them unavailable

TABLE 2

Classification and Properties of Sorghum Kafirins and Wheat Gliadins and Glutenins Molecular mass (kDA)

Total fraction (%)

Protein

Group

Subunit structure

Kafirins

α-

Monomeric, oligomeric, and polymerica

22–27a

66–84a

22 Gln, 9 Pro, 0.7 Gly, 15Ala, 15 Leu, 1 Cys, 0.6 Meta

β-

Monomeric and polymeric

16–20

7–13

18 Gln, 19 Pro, 13 Ala, 12 Leu, 5 Cys, 6 Met

γ-

Oligomeric and polymeric

28–29

9–16

14 Gln, 23 Pro, 9 Gly, 9 Leu, 7 Cys, 1 Met

δ-

Unknown

13–15

Unknown

16 Met, 1Trpa

γ-

Monomericb

26–36b

40–50b

32–36 Gln, 16–17 Pro,3 Gly, 3 Ala, 2–3 Cys, 1.5 Metb

α-

Monomeric

30–34

35 Gln, 15–18 Pro, 2.3 Gly, 2–3 Ala, 2 Cys, < 1 Met

ω-

Monomeric

30–50

40–53 Gln, 20–29 Pro, 0.7–1 Gly, < 1 Ala, 0 Cys, 0 Met

Low-molecular-weight (LMW)–Gliadin

Monomeric

16–19

23–27 Gln, 9 Pro, 5 Gly, 7 Ala, 5 Leu, 6–9 Cys, 3–5 Met

HMW-Glutenin

Polymeric

45–106b

LMW-Glutenin

Polymeric

32–42

Gliadins

Glutenins

a b

Belton et al.14 Shewry et al.42

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30-40b

Partial amino acid composition (mol %)

33–36 Gln, 11–13 Pro, 18–20 Gly, 3–4 Ala, 4 Leu, 0.5–1 Cys, < 1 Met 33–35 Gln, 13–16 Pro,2–3 Gly, 2–3 Ala, 8 Leu, 2–3 Cys, 1.6–2 Met

GRAIN CHEMICAL COMPONENTS: FUNCTIONAL AND NUTRITIONAL ATTRIBUTES

FIG. 1

0

270.5°C

–5

Total kafirin Heat flow (mW)

141

Residual kafirin

–10

Typical DSC thermograms of kafirin proteins and kafirin microparticles. (A) Proteins. (B) Microparticles. Arrows mark melting temperatures, which are probably Tg. From Anyango JO, Taylor JRN, Taylor J. Role of γ-kafirin in the formation and organization of kafirin microstructures. J Agric Food Chem 2013;61:10757
65.

g- kafirin

246.2°C

–15 248.6°C

–20

–25 140

175

210

245

280

Temperature (°C)

(A) 0

–5

Total kafirin microparticles

Heat flow (mW)

256.9°C

Residual kafirin ⬙microparticles⬙

–10 253.7°C

–15

Residual +30% g- kafirin microparticles

–20

–25 140

(B)

Residual +15% g- kafirin microparticles

253.9°C

260.0°C

175

210

245

280

Temperature (°C)

for participation in dough fibril formation, unlike gluten proteins, which are present in the continuous matrix after seed desiccation.22

Lipids Sorghum contains around 3.4% lipids (Table 1), somewhat more than wheat but less than maize; the majority of these are neutral triglycerides (triacylglycerols). The triglycerides of sorghum are rich in unsaturated fatty acids. The predominant fatty acids are linoleic (C18:2), at 38%–49% of the total and oleic (C18:1), at 31%–38% of the total.1 Sorghum is also rich in tocopherols (vitamin E), at about 1.2 mg/100 g (Table 1).

Nonstarch Polysaccharides (NSPs) Sorghum contains some 6%–11% nonstarch polysaccharides (NSP) (dietary fiber), probably a slightly lower level than wheat. The major NSPs of the sorghum endosperm are water-unextractable (insoluble) glucuronoarabinoxylans (GAX).2, 23 Because they are water-insoluble, sorghum GAX are probably not functional in bread-making, unlike wheat arabinoxylans. Concerning their nutritional attributes, they probably have good laxation properties, but they do not have the cholesterol-lowering effects associated with soluble dietary fiber.

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Phytochemicals Sorghum grain contains several types of potentially health-promoting phytochemicals, including various phenolic compounds, plant sterols, and policosanols. Examples of the health benefits that have been indicated for sorghum phytochemicals include antioxidant, anti-inflammatory, cancer-preventive, and anti-arrhythmic activities associated with the phenolics; satiety-promoting activities specifically associated with the tannin-type phenolics6; and cholesterol-lowering activity associated with the policosanols.23 In addition, sorghum foods are antidiabetic and act to prevent cardiovascular disease, while lactic acid bacteria-fermented products have probiotic effects emanating from their unique microflora,24

Phenolic Compounds The quantity of phenolic compounds in sorghum grain can be substantial, particularly in red, tannin-type sorghums (Table 3). The main groups of phenolic compounds in sorghum grain are phenolic acids, flavonoid-type compounds, and tannins (proanthocyanidins)6 (Fig. 2). It should be noted that the tannins are present only in tannin (Type II and Type III) sorghums. Sorghum phenolic acids are mainly derivatives of benzoic acid (Fig. 2A) and cinnamic acid (Fig. 2B). They are concentrated in the pericarp and occur mostly in bound form (esterified to cell wall polymers). Ferulic acid is the most abundant bound phenolic acid in sorghum. Other phenolic acids abundant in sorghum include syringic, protocatechuic, caffeic, p-coumaric, and sinapic. Flavonoids form the largest group of phenolic compounds in sorghum. They are made up of a benzopyran nucleus, with an aromatic substituent at carbon 2 of the C ring (Fig. 2C). Many sorghum flavonoids have been isolated and identified, with anthocyanins being the major class found in sorghum.6 The anthocyanins contribute most of the color of sorghum. Sorghum anthocyanins are unique because they do not contain the hydroxyl group in the 3-position of the C-ring and thus are called 3-deoxyanthocyanins. The two common sorghum 3-deoxyanthocyanidins are yellow (apigeninidin) and orange (luteolinidin) (Fig. 3C). In sorghum, the 3-deoxyanthocyanins are concentrated in the pericarp, and the highest levels are found in sorghums with black pericarp. TABLE 3

Effect of Milling Extraction Rate on the Nutrient Content of Sorghum Flour

Flour

Extraction rate (%)

Protein (g/100 g)

Oil (g/100 g)

Ash (g/100 g)

(A) EFFECT ON PROTEIN, OIL, AND ASH (TOTAL MINERALS) Whole grain

100

11.59

3.60

1.90

Roller milled

83.6

14.12

2.79

1.34

Decorticated then hammer milled

75.7

13.73

2.61

1.22

Extraction rate (%)

Total phosphorus (mg/g)

Phytate phosphorus (mg/g)

Iron (ppm)

Zinc (ppm)

(B) EFFECT ON PHOSPHORUS, IRON AND ZINC 100

4.0

3.1

179

36

90

3.4

2.7

65

30

80

2.4

1.6

83

21

73

1.9

1.2

76

10

Extraction rate (%)

Total phenolics (g catechin equiv./ 100 g)

Condensed tannins (g catechin equiv./ 100 g)

Antioxidant activity (μMol Trolox equiv./g)

(C) EFFECT ON TOTAL PHENOLICS, CONDENSED TANNINS AND ANTIOXIDANT ACTIVITY IN A RED, TANNIN CULTIVAR 100

1.25

7.73

373

90

0.87

6.73

322

70

0.42

0

178

A: Data from Kebakile MM, Rooney LW, Taylor JRN. Effects of hand pounding, abrasive decortication-hammer milling, roller milling and sorghum type on sorghum meal extraction and quality. Cereal Foods World 2007;52:129–137; B: Data from Klopfenstein CF, Hoseney RC. Nutritional properties of sorghum and the millets. In: Dendy DAV, editor. Sorghum and millets: chemistry and technology. St. Paul, MN: American Association of Cereal Chemists; 1995. p. 125–168; C: Data from Chiremba C, Taylor JRN, Duodu KG. Phenolic content, antioxidant activity and consumer acceptability of sorghum cookies. Cereal Chem 2009;86:590–594.

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GRAIN CHEMICAL COMPONENTS: FUNCTIONAL AND NUTRITIONAL ATTRIBUTES

3 R3

R1

R1

R2 2

O

R2

143

O

2

3

OH

4 OH

5

R3

4

R4

6

5 R4

(B) (A)

OH OH

R1 OH 8

R3O

O +

6

HO

O OH OH

2

OH HO

3

OH

n = 1 - > 10

O

4

OH

OR2

OH OH HO

OH

O

OH

(C)

(D)

OH

FIG. 2 Basic structures of sorghum phenolic compounds: (A, B) phenolic acids, (C) flavonoids, (D) proanthocyanidins (condensed tannins). From Dykes L, Rooney LW. Sorghum and millet phenols and antioxidants. J Cereal Sci 2006;44:236–51.

FIG. 3 Traditional-type foods from sorghum flour. (A) Sudanese kisra flatbread being removed from a hotplate after baking; (B) instant sorghum porridge products from South Africa: (left and front) Morvite porridge, (center) fruit-enriched infant porridge, (right) protein-fortified porridge.

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Sorghum tannins are probably exclusively of the condensed type (proanthocyanidins), which are HMW polyphenols that consist of polymerized polymers of flavonoid subunits, mainly flavan-3-ols and/or flavan-3,4-diols (Fig. 3D). The proanthocyanidins in tannin sorghums are mainly the B-type, as they are linked mostly by C4 ! C8 interflavan bonds with (
)–epicatechin as extension units and catechin as terminal units.6 Of the phenolics, the tannins have the highest antioxidant activity when considered on a molar basis.

FLOUR MILLING The technology of milling sorghum grain into flour is not nearly so well developed as that of wheat milling.8 Mechanical milling of sorghum grain is normally done using either disc mills or hammer mills. This is often preceded by removal of the bran layers (pericarp and germ), a process referred to as decortication or dehulling. Decortication is generally achieved by using dehulling equipment to perform dry abrasion. These technologies are not particularly efficient, and the quality of the flour can be variable. Small roller mills (simple versions of the type used for wheat milling) are a recent development in sorghum milling. These small roller mills combine both decortication and reduction in particle size. Research indicates that small roller mills are advantageous over other sorghum-milling technologies in that they produce flour at higher extraction rates (flour yield) and much higher throughput.25 An important issue with respect to milling sorghum is that that its endosperm consists of two main components: a hard outer part, corneous (also referred to as vitreous) endosperm; and a softer, inner part, floury endosperm. The hard corneous endosperm resists reduction to a fine particle size.26 Hence, milling the corneous endosperm to flour can result in a high level of starch damage, which can adversely affect the bread-making quality of the flour. Removal of the bran strongly affects the flour composition. There is an increase in protein content due to removal of the dietary fiber-rich pericarp (Table 3). However, the protein quality is adversely affected with the amount of lysine, the first indispensable amino acid in sorghum, being reduced by about 20%11 due to removal of at least part of the germ, which is rich in high-quality protein. Germ removal also reduces the lipid content of the flour, including the tocopherols. Minerals and B-vitamins are also substantially reduced, as they are concentrated in the germ and aleurone layer. However, mineral bioavailability may be improved27 because the level of the antinutrient phytic acid, which binds divalent minerals such as iron, zinc, and calcium, is also reduced, as it is similarly located (Table 3). In addition, antioxidant activity of the flour is reduced when the bran is removed. This is because phenolics, primarily responsible for antioxidant activity in sorghum, and the nontannin anthocyanis pigments are concentrated in the pericarp, and the condensed tannins, if present, are concentrated in the testa layer, which is directly above the aleurone layer of the endosperm.6

PRODUCTS MADE FROM SORGHUM FLOUR Traditional Products Across Africa, the major sorghum food product is porridge. This is prepared by cooking sorghum flour with water. Porridges range in solid content, from about 10% for a thin gruel to 30% for a stiff porridge of mashed potato-like consistency. Depending on regional tastes, the sorghum porridges may be cooked to a neutral pH, or acidified to < pH 4.0 by lactic acid fermentation or acidification with fruit juice, or made alkaline (pH 8.2) due to cooking with wood ash. These treatments affect the nutritional value of sorghum porridge. As stated, wet cooking in general substantially reduces the protein digestibility of sorghum foods.17 This adverse effect is alleviated by lactic acid fermentation.28 Other nutritional benefits of lactic acid fermentation can include improving starch digestibility, increasing the levels of B-vitamins, reducing antinutrients such as tannins and phytic acid, and most important, rendering the porridge microbiologically safe.29 Alkaline cooking adversely affects the sorghum protein’s quality and availability.27 In North Africa and India, sorghum flour is widely used to make flatbreads. In the production of the major African flatbreads, which are kisra, produced in Sudan (Fig. 3A), and injera, produced in Ethiopia and Eritrea, a slurry of flour undergoes lactic acid fermentation. In injera-making, a portion of the fermented slurry of flour is cooked and then added back. The fermented flour is then diluted into a batter, which is poured onto a circular hot plate. The resulting flatbread is moist and flexible, and a cellular structure is formed by the fermentation gases. Kisra is about 3 mm thick, and injera is thicker (approximately 6 mm), probably because of the precooking of part of the flour. In contrast, the major Indian sorghum flatbread, known as roti or chapati, is a thin, dry, crisper product with a puffed texture, due to steam production during baking. 2. FLOURS AND BREADS

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It should be noted that these traditional products made from sorghum flour are almost exclusively made in the home, and that generally the sorghum used to produce this flour is home grown. Hence, the options for flour fortification are limited. An exception is instant acidified sorghum porridge powder, called Morvite, which is commercially manufactured in South Africa (Fig. 3B). To make porridge, one simply mixes boiling water or milk with the Morvite. This porridge powder is made by pregelatinizing the starch using technologies such as extrusion cooking or gun puffing. The product is fortified with a range of minerals and vitamins so that a 100-g flour serving generally meets 15%– 25% of an adult’s micronutrient recommended dietary allowance (RDA) (Table 1). Other similar vitamin- and mineralfortified sorghum porridge products are available (Fig. 3B). These are variously enriched with soya to provide 40% of an adult’s protein RDA, and with fruit, which when made with milk, can provide 50% and 25% of a 2–3-year-old’s protein and energy RDAs, respectively. The possibility of combining infrared (IR) treatment (micronization) with extrusion cooking to produce a ready-to-eat sorghum-and-cowpea–based porridge supplemented with cooked cowpea leaves has been investigated.30, 31 A daily serving of this cereal would provide about 40% of the protein and lysine requirements for a preschool child, with significant improvement of iron and zinc bioavailability.

Modern Products Because sorghum is considered a safe alternative to wheat for celiacs, and because of the need to find local alternatives to wheat for bread-making in tropical countries where wheat cannot be grown., the production of bread, cakes, and cookies from sorghum is being widely investigated.23, 32 The production of good-quality nonwheat bread with a light, airy texture is a skilled craft. A number of techniques are used to do this. However, the scientific reasons behind most of them remain a matter of conjecture. The general principle of successful nonwheat bread-making is that the solute molecules in the dough (mainly starch) have substituted for the gas-holding, viscoelastic properties of wheat gluten molecules. Specifically, the solute molecules have to interact with each other and with the water molecules to hold the gas produced during yeast fermentation, allowing the dough to expand during fermentation and to set into a firm cellular structure during baking. In this context, it should be noted that sorghum has no special characteristics compared to other gluten-free cereals such as maize or rice. However, the more bland taste of white, tan-plant sorghum cultivars compared to other cereals and other sorghum types seems to be preferred in bread and cakes. Nonwheat bread dough is prepared with a much higher proportion of water (80%–110% relative to flour, compared to around 65% for wheat dough).32 Thus, the dough is actually a stiff batter, similar in consistency to that used for cakemaking. The high amount of water presumably allows greater starch granule expansion during baking, perhaps enabling the formation of a stabler gas cell structure. Commonly, raw starch (normally maize or cassava starch) is also included in the recipe, at around 30% on a flour basis.32 This also is probably related to a requirement for greater starch granule expansion during baking. As indicated in Flour Milling Section earlier in this chapter, flour from the corneous endosperm of sorghum is subject to high levels of starch damage. Damaged starch granules have high water absorption, but at lower temperatures than intact granules. Hydrocolloids such as xanthan gum and hydroxypropyl methyl cellulose are commonly included in commercial gluten-free bread formulations.23, 32 The probable function of hydrocolloids is to increase the viscosity of the aqueous phase of the dough so as to reduce the rate of gas loss from the dough. Hydrocolloids add considerably to the ingredient cost of gluten-free breads. A much cheaper, but somewhat less effective, alternative is to precook a portion of the flour in order to produce pregelatinized starch23 as is done in injera-making. This porridge is then added to the flour, water, and other ingredients during dough-making. Notwithstanding the use of these techniques, gluten-free breads are invariably denser in texture than wheat breads. They have a coarser crumb structure, with proportionally larger gas cells and thicker cell walls (Fig. 4A). Gluten-free cakes and muffins more closely resemble their wheat-flour counterparts (Fig. 4B). This is probably because the role of gluten is less important in cake-making. The flour is a much lower proportion of the solutes, as there are high levels of sugar and fat, and eggs are generally included,23 all of which play a role in gas cell stabilization. Cookies are easily made from sorghum flour,23, 33 as unlike bread and cakes, they are not leavened. No special ingredients are required. However, sorghum cookies tend to be denser and have a harder texture than their wheat counterparts and can have a somewhat gritty mouthfeel.33 We have experimentally produced protein-fortified sorghum cookies with the addition of defatted soya flour, for use as a supplementary food to combat protein-energy malnutrition. Consumer sensory evaluation by primary-school children of these protein-fortified sorghum cookies showed them to be as acceptable as 100% wheat cookies.34 Commercialization of production of biscuits fortified with soy flour is underway (Fig. 5). 2. FLOURS AND BREADS

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FIG. 4 Modern-type foods made with sorghum flour. (A) Slice of gluten-free bread from Sweden (note the relatively coarse and open crumb structure); (B) a 100% sorghum-flour cake made by the Food Research Processing Centre, Khartoum, Sudan.

FIG. 5 Commercial sorghum-soy cookies being made.

NEW DEVELOPMENTS IN SORGHUM FLOURS Bread-Making Technology As previously explained, a problem with making good-quality gluten-free breads from sorghum and other grains is that expensive additives such as hydrocolloids are required. This is especially disadvantageous in countries with developing economies in the tropics, where sorghum is a major crop and should be utilized in bread-making. Kafirin proteins are not functional in terms of providing the required viscoelastic characteristics to the dough. Recent research,

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147

however, has shown that if kafirin or zein, the very similar prolamin protein of maize, is mixed with a solvent at elevated temperature (75°C), the resulting dough has viscoelastic properties.21 This occurs because the temperature of 75° C is above the glass transition temperature of the kafirin and zein proteins. In addition, recently we showed that viscoelastic masses could be formed from zein and kafirin preparations of varying subclass compositions by coacervation from glacial acetic acid (Fig. 6A).35 It is presumed that dissolving the prolamins in glacial acetic acid enables protonation and complete solvation. The γ-subclass is necessary for the retention of viscoelastic mass softness for both coacervated kafirin and zein, and the elastic recovery of coacervated kafirin (Fig. 6B). These findings offer the possibilities of developing processes to produce good-quality breads from sorghum or maize flour without using expensive additives.

Biofortification As indicated previously, as sorghum is primarily a food in developing tropical countries, the vast majority of sorghum flour food products are made in the home, and they are often produced from home-cultivated grain. Thus, flour fortification is problematical. A more viable option is biofortication, which can be defined as increasing the concentration of nutrients in crops using conventional plant breeding or recombinant deoxyribonucleic acid (DNA) technology (genetic engineering). Polleti et al.,36 reviewing the progress made in the nutritional fortification of cereals, stated that

FIG. 6 (A) Formation of a cohesive mass or hydrated aggregate on addition of acetic acid (5.4%) to kafirin or zein preparations at 50°C. (a) Commercial zein, (b) zein, (c) kafirin minus gamma-, (d) kafirin A-cohesive mass, (b–d) hydrated aggregate. (Continued)

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(B) FIG. 6, CONT’D (B) Stereo microscopy of hydrated aggregates or visco-elastic masses formed from different kafirin and zein preparations treated with either water or 33% acetic at 50°C or coacervated from glacial acetic acid. From Taylor J, Anyango JO, Muhiwa PJ, Oguntoyinbo SI, Taylor JRN. Comparison of formation of visco-elastic masses and their properties between zeins and kafirins. Food Chem 2018;245:178–188.

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SUMMARY POINTS

149

effective biofortification of cereal staples can reach the poor in rural areas, has low recurrent costs, is sustainable in the long term, and in the case of genetic improvement, it only requires an upfront investment. High iron- and zinc-biofortified sorghum lines have also been developed by conventional breeding by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).37 Biosorghum (www.biosorghum.org), a Bill and Melinda Gates Foundation Grand Challenges in Global Health project, has used recombinant DNA technology with the aim of developing biofortified sorghums specifically for small-holder farmers in Africa. Biosorghum lines have increased iron and zinc bioavailability by 6%–20% and 25%–39%, respectively, through reduction in phytate levels (by up to 86%).37–39 The essential amino acid composition (specifically, lysine content and protein digestibility of Biosorghum) have been increased by up to 97% and 59%, respectively, through the suppression of synthesis of specific kafirin proteins.16, 37, 40 Lines of Biosorghum can also contain substantial levels of pro-vitamin A (β-carotene) (up to about 12 μg/g), achieved through expression of β-carotene biosynthesis that enhances all-trans-β-carotene accumulation in sorghum endosperm.41 Breeding is ongoing for sorghum with an improved pro-vitamin A trait.

TECHNOLOGICAL ISSUES The technology of sorghum milling needs to be improved in terms of milling efficiency, flour functional quality, and retention of micronutrients and phytochemicals. To achieve this, sorghum roller-milling technology needs to be further developed. The technology of leavened bread production using sorghum also needs to be further developed so as to improve bread quality and avoid the use of additives that can increase the cost of the bread and may be unacceptable to consumers. Good progress is being made on in-depth research that will enable sorghum kafirin proteins to provide dough viscoelasticity. Biofortification has already provided some progress toward achieving a nutritious sorghum crop. However, this technology needs to be further developed in order to fill the gaps in knowledge about improving the amount of essential amino acids such as threonine and tryptophan and be implemented in countries where sorghum is a major staple so as to improve the nutritional status of rural people.

SUMMARY POINTS • Sorghum is a major staple crop and food in arid, tropical, and subtropical regions of Africa and India. • Sorghum has some valuable nutritional characteristics. It is gluten-free and generally contains high levels of phytochemicals, particularly antioxidant-rich phenolics. • Sorghum flour-milling technology has not been developed to the same level as that of wheat milling. The extraction rate of flour substantially affects its chemical composition; with decreasing extraction rates, considerable loss of micronutrients and phenolic phytochemicals occur. • Sorghum flour is widely used to produce traditional foods, including porridges of many types and flatbreads. • Micronutrient-fortified instant sorghum porridges are popular foods in South Africa. • Leavened breads can be produced from sorghum flour, although the quality is not particularly good, and additives such as hydrocolloids generally have to compensate for the absence of gluten. • Recent research has shown that wheat gluten-like viscoelastic masses can be formed from kafirin (sorghum prolamin protein) preparations of varying subclass compositions by coacervation from glacial acetic acid. This suggests that kafirin has the potential to replace wheat-gluten functionality in dough-based products like bread. • Good-quality cakes and cookies can be readily made with sorghum flour, as the absence of gluten is less important than in bread. Commercial production of cookies fortified with soy flour is in progress. • Progress has been made on the biofortification of sorghum through conventional plant breeding or recombinant DNA technology. Research shows that biofortified sorghum can have substantially improved iron and zinc bioavailability and protein quality, and contain substantial amounts of pro-vitamin A (β-carotene) achieved using recombinant DNA technology. Some of these varieties are already under field trial.

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References 1. Serna-Saldivar S, Rooney LW. Structure and chemistry of sorghum and millets. In: Dendy DAV, editor. Sorghum and millets: chemistry and technology. St. Paul, MN: American Association of Cereal Chemists; 1994. p. 69–124. 2. Taylor JRN, Emmambux MN. Developments in our understanding of sorghum polysaccharides and their health benefits. Cereal Chem 2010;87:263–71. 3. Doggett H. Sorghum. 2nd ed. Harlow: Longman Scientific and Technical; 1988. 4. FAO. Crop prospects and food situation. www.fao.org/3/a-i6558e.pdf; 2016. [Accessed January 2018]. 5. Ciacci C, Maiuri L, Caporaso N, Bucci C, Del Giudice L, Massardo DR, Pontieri P, Di Fonzo N, Bean SR, Ioeger B, Londei M. Celiac disease: in vitro and in vivo safety and palatability of wheat-free sorghum food products. Clin Nutr 2007;26:799–805. 6. Dykes L, Rooney LW. Sorghum and millet phenols and antioxidants. J Cereal Sci 2006;44:236–51. 7. Waniska RD, Rooney LW. Structure and chemistry of the sorghum caryopsis. In: Smith CW, Frederiksen RA, editors. Sorghum: origin, history, and production. New York: John Wiley & Sons; 2000. p. 649–88. 8. Taylor JRN, Dewar J. Developments in sorghum food technologies. In: Taylor SL, editor. Advances in food and nutrition research. vol. 43. San Diego: Academic Press; 2001. p. 218–64. 9. Yeung CK, Glahn RP, Wu X, Liu RH, Miller DD. In vitro iron bioavailability and antioxidant activity of raisins. J Food Sci 2003;68:701–5. 10. Graham RD, Welch RM, Bouis HE. Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: principles, perspectives, and knowledge gaps. Adv Agron 2001;70:77–142. 11. Taylor JRN, Sch€ ussler L. The protein compositions of the different anatomical parts of sorghum grain. J Cereal Sci 1986;4:361–9. 12. Dicko MH, Gruppen H, Traore AS, Voragen AGJ, Van Berkel WJH. Sorghum grain as human food in Africa: relevance of content of starch and amylase activities. Afr J Biotechnol 2006;5:384–96. 13. Ezeogu LI, Duodu KG, Emmambux MN, Taylor JRN. Influence of cooking conditions on the protein matrix of sorghum and maize endosperm flours. Cereal Chem 2008;85:397–402. 14. Belton PS, Delgadillo I, Halford NG, Shewry R. Kafirin structure and functionality. J Cereal Sci 2006;44:272–86. 15. Anyango JO, Taylor JRN, Taylor J. Role of γ-kafirin in the formation and organization of kafirin microstructures. J Agric Food Chem 2013;61:10757–65. 16. Da Silva LS, Jung R, Zhao Z, Glassman K, Taylor J, Taylor JRN. Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines. J Cereal Sci 2011;54:160–7. 17. Duodu KG, Taylor JRN, Belton PS, Hamaker BR. Factors affecting sorghum protein digestibility. J Cereal Sci 2003;38:117–31. 18. Millward DJ. The nutritional value of plant-based diets in relation to human amino acid and protein requirements. Proc Nutr Soc 1999;58:249–60. 19. Catassi C, Fasano A. Celiac disease. In: Arendt EK, Dal Bello F, editors. Gluten-free cereal products and beverages. Burlington: Academic Press; 2008. p. 1–28. 20. Belton PS. On the elasticity of gluten. J Cereal Sci 1999;29:103–7. 21. Oom A, Pettersson A, Taylor JRN, Stading M. Rheological properties of kafirin and zein prolamins. J Cereal Sci 2008;47:109–16. 22. Shewry PR. The synthesis, processing, and deposition of gluten proteins in the developing wheat grain. Cereal Foods World 1999;44:587–9. 23. Taylor JRN, Schober TJ, Bean SR. Novel food and non-food uses for sorghum and millets. J Cereal Sci 2006;44:252–71. 24. Taylor JRN, Duodu KG. Effects of processing sorghum and millets on their phenolic phytochemicals and the implications of this to the healthenhancing properties of sorghum and millet food and beverage products. J Sci Food Agric 2015;95:225–37. 25. Kebakile MM, Rooney LW, Taylor JRN. Effects of hand pounding, abrasive decortication-hammer milling, roller milling and sorghum type on sorghum meal extraction and quality. Cereal Foods World 2007;52:129–37. 26. Chandrashekar A, Mazhar H. The biochemical basis and implications of grain strength in sorghum and maize. J Cereal Sci 1999;30:193–207. 27. Klopfenstein CF, Hoseney RC. Nutritional properties of sorghum and the millets. In: Dendy DAV, editor. Sorghum and millets: chemistry and technology. St. Paul, MN: American Association of Cereal Chemists; 1995. p. 125–68. 28. Taylor J, Taylor JRN. Alleviation of the adverse effects of cooking on protein digestibility in sorghum through fermentation in traditional African porridges. Int J Food Sci Technol 2002;37:129–38. 29. Taylor JRN, Dewar J. Fermented products: beverages and porridges. In Smith CW, Frederiksen R. A., eds. Sorghum: origin, history, and production. New York: John Wiley & Sons; 2000, pp. 751–795. 30. Vilakati N, MacIntyre U, Oelofse A, Taylor JRN. Influence of micronization (infrared treatment) on the protein and functional quality of a readyto-eat sorghum-cowpea African porridge for young child-feeding. LWT Food Sci Technol 2015;63:1191–8. 31. Vilakati N, Taylor JRN, MacIntyre U, Kruger J. Effects of processing and addition of a cowpea leaf relish on the iron and zinc nutritive value of a ready-to-eat sorghum-cowpea porridge aimed at young children. LWT Food Sci Technol 2016;73:467–72. 32. Schober TJ, Bean SR. Sorghum and maize. In: Arendt EK, Dal Bello F, editors. Gluten-free cereal products and beverages. Burlington: Academic Press; 2008. p. 101–18. 33. Chiremba C, Taylor JRN, Duodu KG. Phenolic content, antioxidant activity and consumer acceptability of sorghum cookies. Cereal Chem 2009;86:590–4. 34. Serem CA. Development of soy fortified sorghum and bread wheat biscuits as a supplementary food to combat protein energy malnutrition in young children. PhD thesis, Pretoria, South Africa: University of Pretoria; 2010. 35. Taylor J, Anyango JO, Muhiwa PJ, Oguntoyinbo SI, Taylor JRN. Comparison of formation of visco-elastic masses and their properties between zeins and kafirins. Food Chem 2018;245:178–88. 36. Polleti S, Gruissem W, Sauter C. Nutritional fortification of cereals. Curr Opin Biotechnol 2004;15:162–5. 37. Taylor JRN, Belton PS, Beta T, Duodu KG. Increasing the utilisation of sorghum, millets and pseudocereals: developments in the science of their phenolic phytochemicals, biofortification and protein functionality. J Cereal Sci 2014;59:257–75. 38. Kruger J, Taylor JRN, Oelofse A. Effects of reducing phytate content in sorghum through genetic modification and fermentation on in vitro iron availability in whole grain porridges. Food Chem 2012;131:220–4.

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39. Kruger J, Taylor JRN, Du X, De Moura FF, L€ onnerdal B, Oelofse A. Effect of phytate reduction of sorghum, through genetic modification, on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay, Caco-2 cell uptake assay, and suckling rat pup absorption model. Food Chem 2013;141:1019–25. 40. Da Silva LS, Taylor J, Taylor JRN. Transgenic sorghum with altered kafirin synthesis: kafirin solubility, polymerization and protein digestion. J Agric Food Chem 2011;59:9265–70. 41. Che P, Zhao Z-Y, Glassman K, Dolde D, Hu TX, Jones TJ, Gruis DF, Obukosia S, Wambugu F, Albertsen MC. Elevated vitamin E content improves all-trans β-carotene accumulation and stability in biofortified sorghum. Proc Natl Acad Sci U S A 2016;113:11040–5. 42. Shewry PR, D’Ovidio R, Lafiandra D, Jenkins JA, Mills ENC, Bekes F. Wheat grain proteins. In: Khan K, Shewry PR, editors. Wheat chemistry and technology. 4th ed St. Paul, MN: AACC International, Inc; 2009. p. 223–98.

2. FLOURS AND BREADS

C H A P T E R

12 Banana and Mango Flours Luis A. Bello-P
erez, and Edith Agama-Acevedo Instituto Polit
ecnico Nacional, Centro de Desarrollo de Productos Bio´ticos, Yautepec, Morelos, Mexico

O U T L I N E Introduction

153

Agronomic Characteristics Banana Mango

154 154 155

Chemical Composition Banana Mango Metabolomic of Banana and Mango

155 155 157 157

Uses of the Fruits

157

Unripe Flour

158

Mango Flour (MF) Banana Flour

159 159

Use of Unripe Flour Mango Flour (MF) Banana Flour

161 161 161

Technological Issues

162

Summary Points

162

Acknowledgments

163

References

163

INTRODUCTION During the past decade, nutrition science has studied the relationship between dietary habits and disease risk, and the findings largely support the concept that diet plays a significant role in the modulation of various functions in the body. Nowadays, there is a growing interest in the consumption of foods and ingredients rich in dietary fiber (DF).1 This implies that the diet and its components could contribute to an improved state of well-being, a reduction of risks related to certain diseases, and even improvement in quality of life. It is generally accepted that plant-derived foods such as wine, fruits, nuts, vegetables, grains, legumes, and spices, have beneficial effects on human health, particularly on age-related diseases such as cardiovascular and neurodegenerative diseases, type 2 diabetes, and several types of cancer. As a result, there is a huge interest among consumers and the food industry in products that can promote health and well-being. These foods have been generically named functional foods (Table 1). Important items reviewed in this chapter include the nutraceutical ingredients of fruits, where polyphenols (e.g., flavonoids, anthocyanins, tannins) that show antioxidant capacity, and indigestible carbohydrates, present as the substance called DF, exist. DF includes polysaccharides, oligosaccharides, lignin, and other associated substances. DF has beneficial physiological effects, which could include laxation, blood cholesterol attenuation, and blood glucose attenuation.2 The presence of significant amounts of bioactive compounds, such as flavonoids and carotenoids, in DF in fruit imparts considerable nutritional value. The food industry is trying to find new sources of DF to use as ingredients, which has become a very useful trend within the industry. The most widespread consumed products with DF are those derived from whole grains. However, over the past decade, DF derived from fruits (citrus fruits, apples, and others) has been steadily introduced in the Eastern world markets.

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00012-5

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© 2019 Elsevier Inc. All rights reserved.

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12. BANANA AND MANGO FLOURS

Do not have FDA-approved health claimsb

FDA-approved health claimsa

TABLE 1 Examples of Functional Foods, With Their Possible Effects on Human Health Functional food

Functional ingredients or bioactive compounds

Potential health benefit

Whole oat products

β-glucans

Lower cholesterol levels

Psyllium

Soluble fiber

Specially fortified margarine or salad dressing

Plant stanol or sterol sters

Sugarless chewing gums and candies

Sugar alcohols

Does not promote tooth decay

Fatty fish

Ω-3 fatty acids

Reduce risk of heart diseases

Cranberry juices

Proanthocyanidins

Reduce urinary tract infections

Chocolate

Flavonoids

Reduce LDL cholesterol

Garlic

Organo-sulfur compounds

Lower cholesterol levels

Green tea

Catechins

Cruciferous

Glucosinolates, indoles

Reduce risk of some types cancer

Tomatoes and tomato products

Lycopene

Dark green leafy vegetables

Lutein

Reduce risk of age-related macular degeneration

Fermented dairy

Probiotics

Support gastrointestinal tract health boost immunity

a

The functional foods listed in this section have been tested in clinical trials, and all carry FDA-approved health claims. The foods described do not have FDA-approved health claims. However, their potential health benefits are currently being investigated. The first column shows some foods, the second column the active compunds and the third column the benefits due to the consumption. FDA ¼ Food and Drug Administration; LDL ¼ low-density lipoprotein.

b

The DF presented in some fruits, including unripe fruit, has better nutritional quality than those found in whole grain in most cases. Their significant contents of associated bioactive compounds (flavonoids, carotenoids, etc.) and more balanced composition (higher overall fiber content, greater soluble dietary fiber (SDF)/insoluble dietary fiber (IDF) ratio, water and fat holding capacities, lower metabolic energy value, and phytic acid content).3 Also, it was postulated the concept of antioxidant DF due to the possible interactions between the components of the indigestible fraction and polyphenols. The antioxidant DF reaches the colon, and compounds with antioxidant activity are released after fermentation of the components of DF by the microbiota with the concomitant production of an antioxidant environment. The interactions between DF components and polyphenols can be produced during gastrointestinal digestion by hydrogen bonding, hydrophobic interactions, and covalent linkages; also, the polyphenols can be trapped by entanglement of the polysaccharides. There is a high starch content in the flours of unripe banana and mango, which in the uncooked state show high resistance to hydrolysis by digestive enzymes. Also, during the use of those flours as ingredients in such cooked foods as bakery products, snacks, and pasta, an important decrease in the resistant starch (RS) content can be observed. However, RS is important as a component of DF because during its fermentation in the colon, it mainly produces butyric acid, which is associated with the prevention of colon cancer.

AGRONOMIC CHARACTERISTICS Banana Bananas (Musa paradisiaca spp.) are considered to be one of the first fruits harvested by primitive agricultures, and it has been presented in diverse cultures and civilizations from various centuries. Its origin is Southeast Asia, including the north of India, Cambodia, Sumatra, Java, the Philippines, and Taiwan. The introduction of the banana cultivar occurred in the 16th century, in the Santo Domingo and Cuba islands. At the end of the 19th century, the first

2. FLOURS AND BREADS

CHEMICAL COMPOSITION

155

commercial plantations were established in Jamaica, and thereafter in Mexico and in several countries in Central America. The banana is a climateric fruit cultivated in many countries, primarily those located in the tropical and subtropical regions (approximately between 120 and 130 countries), and it represents a major staple. The world’s production of bananas is approximately 104 million tons. The main producers of bananas are Brazil, China, Ecuador, the Philippines, and India. The countries leading in banana exports are Ecuador, Colombia, Costa Rica, and the Philippines. However, the Food and Agriculture Organization of the United Nations (FAO) calculated that bananas have a loss of 53.8% between the farm and the market.4 In this sense, it is important to search for alternatives for the use of unripe bananas as a starch source for diverse industrial applications or when made into flour. Edible Musa plants are classified in AA, AB, BB, AAA, AAB, ABB, AAAA, AAAB, and ABBB genomic groups. In general, dessert banana cultivars in the world are AA or AAA; this last group includes almost all bananas sold. Cooking bananas, also named plantains, are predominantly AAB, ABB, or BBB. The great biodiversity of banana plants provides the potential for diverse uses and applications.5 Unripe bananas contain large amounts of starch, cellulose, hemicellulose, and lignin in the pulp. The nutritional/nutraceutical potential of unripe banana flour (UBF) has been reported6; also, due to the high starch content, this flour and the peel of the fruit can be used for ethanol production,7, 8 but the use of whole UBF (using both pulp and peel) can be an alternative that merits more investigation.

Mango The mango (Mangifera indica L.) is the most important member of the Anarcadiaceae family. This fruit is native to southern Asia, especially Burma and eastern India. It spread early into Malaysia, eastern Asia, and eastern Africa. The genus Mangifera includes approximately 50 species, but only 3 or 4 produce edible fruits. The fruit that is typically referred to as mango has a weight between 150 g and 2 kg with an ovoid-oblong shape, with a size between 4 and 25 cm long and 1.5–10 cm thick. The mango tree has become adapted in many tropical and subtropical regions of the world. Conversely, its fruit is fragile, and significant postharvest wastage occurs in producing due to insufficiently established practices of handling, convoying, storage, and ripening. When the fruit is ripe, it is consumed raw as a dessert, while the rest of the processing is to make other products such as nectar, juice powder, canned mango slices in syrup chutney, etc.9 Mangoes are harvested at a mature green stage and stored for ripening. If the fruit is fully grown and ready for picking, the stem snaps easily. Depending on the variety of the fruit and environmental conditions, it takes 6–10 days to ripen under ambient conditions (temperature and relative humidity), and become overripe and spoiled within 15 days after harvest, provided that fruits are eliminated and not consumed. The climacteric characteristics of mango are associated with important postharvest loss; the FAO reported that 54.5% of the fruit is lost from farm to market.4

CHEMICAL COMPOSITION Banana The chemical composition of banana varieties depends on the ripening state; however, agronomic traits, the type of soil, and climatic conditions alter the major and minor components of the fruit. Lower moisture content was found in the unripe pulp of banana (69%) than in ripe pulp (74%), the carbohydrate content was higher in unripe pulp (28.7%) than in ripe banana (21.8%), but an inverse pattern was obtained for fiber (Tables 2 and 3). The unripe pulp had 2.0% and the unripe 0.5%; this pattern might be related to the higher pectin levels present in the ripe state of the pulp.5 Starch is the main carbohydrate in unripe bananas (73%–77%, on a dry basis), and is replaced by simple sugars such as sucrose, glucose, and fructose when the fruit starts maturing. The starch present in diverse food crops such as unripe bananas is gelatinized and consumed after the cooking and preparation of foods. It was demonstrated that a fraction of the starch consumed in the diet escapes digestion and absorption in the small intestine of healthy people, and so it is then fermented in the large intestine with the production of short-chain fatty acids. This fraction was called RS, and its consumption has been associated with reduction of the glycemic index (GI), low absorption of cholesterol, and prevention of colon cancer. RS was found in unripe banana; because of this fruit in this maturity stage is considered the natural product with the highest RS content.14

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12. BANANA AND MANGO FLOURS

TABLE 2

Chemical Composition of Bakery Products With Banana and Mango Flour (g/100 g)

Bread

Cookies

Chemical composition Sample

Moisture

Ash

Protein

Fat

References

Control

3.8  0.1

0.6  0.1

9.5  0.3

6.9  0.4

30-UPF

6.0  0.3

0.8  0.1

8.6  0.0

5.4  0.5

50- UPF

6.4  0.2

0.9  0.1

7.5  0.0

4.0  0.9

25- UPF

0.9  0.3

2.0  0.0

7.3  0.0

15.7  0.0

Utrilla-Coello et al.21

Control

2.8  0.0

1.5  0.1

7.6  0.2

18.8  0.2

Fasolin et al.46

44.6-UBF

2.8  0.0

1.7  0.2

6.9  0.1

19.0  1.7

66.9- UBF

2.6  0.1

1.9  0.0

7.8  0.2

19.7  0.4

Control

12.0  0.1

4.3  0.0

9.1  0.1

13.8  0.1

75-MDF

11.9  0.0

4.3  0.0

8.3  0.0

13.0  0.3

15.41-UPS

4.8  0.1

1.2  0.1

9.1  0.1

26.9  0.4

17.56- UPS

2.0  0.1

0.9  0.1

6.1  0.1

28.3  0.8

Control

4.9  0.0

2.4  0.0

8.9  0.1

13.2  0.0

7-UBF

2.7  0.6

2.7  0.0

9.6  0.0

13.2  0.1

Control

31.5  0.9

3.2  0.0

11.4  0.1

16.2  0.2

40-MDF

26.0  0.1

3.9  0.0

10.2  0.2

11.2  0.1

Control

13.7  0.0

2.0  0.0

4.1  0.0

18.4  0.0

100-UPF

26.6  0.8

3.3  0.0

9.8  0.0

13.2  0.9

Control

12.1

2.0  0.0

16.3  0.8

2.3  0.0

20-UBF

12.8

2.3  0.0

14.4  0.9

2.3  0.0

Agama-Acevedo et al.28

Vergara-Valencia et al.22

Bello-P
erez et al.47

Maldonado and Pacheco23

Vergara-Valencia et al.22

Juárez-García et al.24

Pacheco and Testa25

UPF, Unripe plantain flour; UPS, unripe plantain starch; MDF, mango DF. The number in the sample corresponds to the level of flour used in the product.

TABLE 3 Chemical Composition of Spaghetti Products With Banana Flour (g/100 g) Chemical composition

Spaghetii

Sample

Moisture

Ash

Protein

Fat

References Ovando-Martínez et al.10

Control

9.6  0.1

0.9  0.0

12.5  0.0

0.54  0.0

10-HBV

12.7

2.2  0.0

15.8  0.7

2.4  0.0

20-HBV

12.8

2.3  0.0

14.4  0.9

2.3  0.0

Control

9.5  0.5

0.58  0.0

12.8  0.0

1.31  0.0

20-HPI

10.0  0.0

1.8  0.0

20.1  0.0

3.6  0.0

30-HPI

9.6  0.1

1.6  0.0

20.4  0.0

3.3  0.0

12.5-UPF

11.97  0.1

2.12  0.0

10.88  0.2

4.01  0.1

Patiño-Rodríguez et al.12

25-UPF

10.15  0.0

2.50  0.1

10.57  0.0

3.81  0.0

Camelo-M
endez et al.13

37.5-UPF

10.12  0.0

2.21  0.1

10.31  0.0

4.40  0.0

Flores-Silva et al.11

UPF, Unripe plantain flour. The number in the sample corresponds to the level of flour used in the product.

DF is a nutritional component also present in the unripe banana. From a strictly nutritional point of view, it is not a nutrient because it does not directly contribute to the basic metabolic processes of the body. The role of DF is more physiologic because it stimulates the intestinal peristalsis and intestinal evacuation. Due to that, DF is not digested in the small intestine; it is fermented in the large intestine and promotes the growth of the intestinal flora, which are beneficial, while also binding diverse substances such as cholesterol. The physiological properties of DF are important in the prevention and treatment of obesity, coronary heart disease, colon cancer, and diabetes. 2. FLOURS AND BREADS

157

USES OF THE FRUITS

Other compounds associated with the prevention of diverse health problems that are present in the banana are bioactives such as polyphenols, which show antioxidant properties due to the ability to trap free radicals, which damage the biomolecules and cause cellular aging. The bioactive compounds are joined to DF and can exert their function in the large intestine, where the level of free radicals is important.

Mango The main components of mango are water and carbohydrates, with small amounts of DF, proteins, lipids, and vitamins (Table 2). The level of these components depends on the variety (e.g., the carbohydrate amount ranges between 90.1% and 93.6%, and the DF content between 3.85% and 12.64%). Mango is a good source of vitamin C, with values between 27 and 80 mg/100 g of fresh pulp. In the unripe state, the mango’s main carbohydrate is starch; in the mature fruit, the starch is replaced by monosaccharides and disaccharides such as glucose, fructose, and sucrose.9 For this reason, the isolation and characterization of mango starch using pulp was reported as an alternative source with industrial potential.15 The parenchymatous tissues and cell walls supply the DF in fruits and vegetables. The DF of mango pulp depends on the variety and the ripening stage, ranging between 12.64% and 3.85%.16 Due to the high amount of peel produced in industry, mango peel was studied as a DF source. This DF is a rich source of indigestible polysaccharides, principally IDF. DF content in mango peel ranged between 65 and 71 g/100 g of dry sample, with the total soluble polyphenol level ranging between 44 and 70 mg/g of dry sample.17 Mango DF was obtained from unripe whole fruit (pulp and peel), presenting a DF content of 28.1 g/100 g of dry sample, with a good balance of SDF (14.25%) and IDF (13.8%), which is important from a nutritional point of view.18 A polysaccharide present in mango is pectin, which provides firmness; when the fruit is unripe, the pectin concentration is high and the level decreases during ripening of the tissue.

Metabolomic of Banana and Mango Diverse metabolites present in UBF has been determined to be naringin, myricetin, rutin, and hesperidin, and they can present nutraceutical characteristics due to antioxidant capacity.12 In the same sense, unripe mango flour showed diverse metabolites that have nutritional value as mangiferin, quercetin, and kaempferon.19, 20

USES OF THE FRUITS Table 4 shows the commercial uses of mango and banana. Both fruits are preferentially consumed as fresh products; however, due to their seasonality and commercialization in various regions of the world, they are processed in various places, with the objective to find availability during the entire year. Mango is consumed in industrialized products as juice, concentrate pulp, and nectar, but banana has minor use as industrialized products; it is found in the market as frozen pulp, snacks, and dried pulp. TABLE 4 Commercial Uses of Mango and Banana Product

Mango

Banana

Juice

X

–

Concentrate

X

–

Frozen pulp

X

X

Marmalade

X

–

Snacks

X

X

Dried pulp (powder)

X

X

Pieces in syrup

X

–

Some commercial products using ripe banana and mango fruits.

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12. BANANA AND MANGO FLOURS

UNRIPE FLOUR The agronomic problems of banana and mango are similar. An important amount of the cultivates are lost due to pests such as black sigatoga (banana), inflorescence, and vegetative malformation of mango, caused by the fungal pathogen Fusarium subglutinans. The latter is one of the most important diseases of this crop occurring in most mango-growing countries worldwide. Additionally, hurricanes, cyclones, and tropical storms damage these fruits during the growing of cultivars. When the fruits reach their full size and physiological maturity, they are cut. Due to insufficiently established practices of handling, convoying, storage, and ripening during the postharvest stage, important amounts of the fruits are lost. The ideas to diversify the final use of fruits such as banana and mango include the development of functional ingredients, including those with low amounts of glycemic carbohydrates and with slow digestible starches. The interest in foods with low amounts of glycemic carbohydrates increased in the recent decade. A high intake of food products rich in nondigestible carbohydrates has been related to several physiological and metabolic effects. In the digestive tract, nondigestible carbohydrates exert a buffering effect that treats excess acid in the stomach, increases fecal bulk, and stimulates intestinal evacuation; besides, it provides a favorable environment for the growth of the beneficial intestinal flora. Consumption of food products with high amounts of nondigestible carbohydrates such as DF (particularly highly viscous SDF), is usually associated with moderate postprandial glycemic response, an important property in the dietetic treatment of diabetes. Unripe fruits are a rich source of starch. It has been said that starch-rich products vary in digestibility (Table 5).26 The rate and extent of starch digestion are reflected in the magnitude and duration of the glycemic response. Most starchrich foods contain a portion of rapidly digestible starch (RDS), another portion of slowly digestible starch (SDS), and a third portion that is resistant to digestion in the small intestine. The nutritional quality of a food is related to its GI. Differences in glycemic and insulinemic responses to dietary starch are directly related to the rate of starch digestion (Table 6). Recently, the benefits of starchy foods that present high levels of SDS were reported, because a product that provides the nutritional benefits of starch (a supply of glucose), but does not produce the postprandial hyperglycemic and hyperinsulinemic spikes associated with RDS, is most desirable. For example, people with type 2 diabetes enjoy benefits from the consumption of foods with high amounts of SDS because doing so does not produce the hyperglycemia followed by hypoglycemia typical of the disease. In addition, SDS may prolong satiety. Although not all low-GI foods are rich in SDS, the opposite is usually true.26 TABLE 5

Nondigestible Carbohydrate Content of the Products Added With Plantain Starch or Plantain or Mango Flour (g/100 g) Indigestible carbohydrates

Spaghetii

Bread

Cookies

Sample

TDF

TIF

IIF

SIF

References

36.9  0.8

19.7  1.0

17.1  0.7

Utrilla-Coello et al.21 Vergara-Valencia et al.22

25-HPI

7.0  0.3

Control

13.3  0.1

–

–

–

75-FDM

17.4  0.1

–

–

–

Control

4.9  0.0

–

–

–

7-HBV

5.4  0.2

–

–

–

Control

14.2  0.1

–

–

–

40-FDM

16.6  0.1

–

–

–

Control

2.3  0.0

18.4  0.7

12.4  0.5

5.9  0.2

100-HPI

5.1  0.1

26.1  0.9

22.3  0.8

3.8  0.1

Control

3.8  0.0

–

–

–

10-HBV

4.3  0.0

–

–

–

20-HBV

4.7  0.0

–

–

–

Maldonado and Pacheco23

Vergara-Valencia et al.22

Juárez-García et al.24

Pacheco and Testa25

Control

–

–

10.7  0.5

5.1  0.3

30-HPI

–

–

17.5  0.5

3.8  0.4

45-HPI

–

–

26.1  0.2

3.3  0.2

Ovando-Martínez et al.10

TIF, Total indigestible fraction; IIF, insoluble indigestible fraction, SIF, soluble indigestible fraction; UPF, unripe plantain flour; UPS, unripe plantain starch; MDF, mango DF. The number in the sample corresponds to the level of flour used in the product.

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159

UNRIPE FLOUR

Spaghetii

Bread

Cookies

TABLE 6 Hydrolysis and GI of Products Added With Plantain or Mango Flours Sample

HI

GI

Method

References

Control

–

116.8

Holm et al.27

Agama-Acevedo et al.28

50-HPI

–

98.6

25-HPI

39.9  2.0

54.4

Grandfelt et al.29

Utrilla-Coello et al.*,21

Control

71.2  1.0

69.5

Grandfelt et al.29

Vergara-Valencia et al.*,22

40-FDM

54.9  0.8

55.5

Control

80.5  2.2

77.6

Grandfelt et al.29

Vergara-Valencia et al.*,22

40-FDM

54.9  0.8

55.5

Control

81.9  3.2

78.8

Grandfelt et al.29

Juárez-García et al.*,24

100-HPI

65.1  2.4

64.3

Control

–

85.1  0.4

Grandfelt et al.29

Flores-Silva et al.*,11

30-HPI

–

76.0  0.4

UPF, Unripe plantain flour; UPS, unripe plantain starch; MDF, mango DF. The number in the sample corresponds to the level of flour used in the product. The number in the sample corresponds to the level of flour used in the product. Predicted glycemic index+ (GI) ¼ Calculated by the equation proposed by Goñi et al. (1997)30 GI ¼ 39.21 + 0.803 (H90). Predicted glycemic index * ¼ 0.862 HI + 8.198 (Granfeldt29).

Mango Flour (MF) In this frame of reference, unripe MF was prepared with the objective to produce a DF-rich powder. The total starch content of DF-rich powder was 29.8%; this level is important during the processing of food products because adding this functional ingredient could contribute to the formation of RS, as has been shown with mango starch extrudates.31 Total dietary fiber (TDF) content in DF-rich powder was 28.1%,18 and in mango peels, the TDF ranges from 65%–71%.17 TDF in DF-rich powder could be considered low. This value might be related to its high starch content (29.9%). In some applications as an ingredient, DF-rich powder has starch levels that might be important, given the additional functional properties imparted by this polysaccharide. DF-rich mango powder presents a balance between SDF (14.3%) and IDF (13.8%). This characteristic also might be important from the nutritional point of view. Nowadays, the bioactive compounds have been important to human health; in this group, polyphenols are included. Extractable polyphenol (EPP) or total soluble polyphenol content in DF-rich mango powder was 16.1 mg/g. EPPs appear to be absorbed in the digestive tract, exerting systemic effects. The antiradical efficiency (AE) was tested in EPP, obtaining a 15  103 value, which is considered suitable for antioxidant DF production.3

Banana Flour Traditionally, bananas are used to prepare regional food products and dishes, and sometimes also to produce vinegar and spirits. Some varieties or cultivars are used in small rural communities as special diets and traditional medicine [e.g., for babies, elderly people, and patients with stomach problems (antidiarrheal and intestinal disorders), gout, and arthritis]. In some South American countries (e.g., Colombia, Ecuador), precooking banana flour is used to produce an elaborate regional dish named empanadas. The use of unripe banana to prepare flour might be advantageous because fruits with damaged peels or those that fall due to cyclones and hurricanes can be used for this purpose. This possibility to diversify the use of bananas can be economically beneficial for farmers. The idea to use unripe bananas to produce functional flour comes from the high starch and nonstarch polysaccharides (i.e., DF) present in the fruit in this stage. It was reported that unripe bananas have the highest RS content,14 and consequently, they are a good source of indigestible and functional carbohydrates. Additionally, the polyphenol content increases the functional character of unripe bananas. Table 7 shows several procedures to prepare banana flour using pulp. The various flours show a high starch content and an important amount of DF, but the prata variety had the lowest DF content (1.17%). The procedure and variety used in our laboratory produced UBF with a high DF level. Precooking of banana produced flour with higher starch content, although DF was not tested. In order to increase the indigestible carbohydrates of UBF and decrease the

2. FLOURS AND BREADS

160 TABLE 7

12. BANANA AND MANGO FLOURS

Chemical Composition of Unripe Banana and Mango Flours Chemical composition

Sample

Flour production

Protein

Fat

Fiber

Starch

References

Green banana (Prata)

Dehydrated

4.73  0.84

0.70  0.03

1.17  0.02

75.20  0.47

Borges et al.32

Unripe banana (M. paradisiaca)

Dehydration by freeze-drying

2.92  0.10

0.83  0.01

9.67  0.05

74.65  2.08

Dehydration by a double-drum dryer

3.30  0.25

0.5  0.05

9.01  0.19

63.50  0.55

Pacheco-Delahaye et al.33

Dehydration by an irradiation microwave

3.12  0.18

0.17  0.15

9.43  0.20

64.52  0.25

Conventional dehydration

3.08  0.08

0.31  0.01

9.37  0.45

74.30  2.32

Peeled and cut-sliced (1 cm), rinsed in citric acid solution (0.3%), dried (50°C), and ground

3.4  0.3

Nd

10.4  1.4

76.8  1.0

Liquefaction

5.3  0.6

Nd

31.8  2.1

52.4  1.1

Cut-sliced (1 cm), rinsed in citric acid solution (0.3%), dried (50°C), and ground.

4.03  0.06

3.24  0.03

17.14  0.19

73  0.06

Acid treated (1.6 M HCl, 38°C, 11 days)

Nd

Nd

60.75  0.25

Nd

Unripe banana (M. paradisiaca)

Peeled and cut-sliced (1 cm), rinsed in citric acid solution (0.3%), dried (50°C), and ground

3.3  0.4

2.7  0.38

14.5  0.46

73.4  0.92

Unripe cooking bananas: Dwarf Kalapua

Treatment in water (85°C, 5 min) and dried (65°C, 48 h)

Unripe banana (M. paradisiaca)

Unripe banana (M. paradisiaca)

3.49

Nd

87.07

Bluggoe

2.84

3.29

Nd

86.42

Plantain French somber

2.88

1.13

Nd

90.19

2.8  0.13

0.33  0.35

8.95

65.7  2.8

2.8  0.19

0.78  0.51

7.76

76.5  2.9

2.6  0.20

0.82  0.47

6.28

76.1  3.7

Prata ana

2.9  0.04

0.47  0.29

8.86

68.2  4.0

Prata comun

2.5  0.02

0.58  0.48

10.46

72.4  3.6

Mysore

2.6  0.06

0.42  0.24

15.56

61.3  6.9

Maca

3.3  0.10

0.52  0.28

11.28

64.9  2.5

3.1  0.3

1.4  0.2

11.4  1.2

71.3  2.8

3.3  0.9

1.3  0.9

12.6  1.4

68.4  4.2

Nanica Nanicao

Green cooking banana: “Alukehel”

Sliced pulp and extraction buffer (ascorbic acid and EDTA) were homogenized, freezedried, and sieved (60 mesh); the residue was washed with ethanol and acetone and dried at room temperature

Pressure-cooked (15 lb/in, 5 min), sliced (0.5 cm), dipped in sodium metabisulfite solution (1%, w/v, 5 min), and dried

“Monthan”

Aguirre-Cruz et al.35

Juárez-García et al.24

Yomeni et al.36 3.77

Green banana: Ouro da mata

Rodríguez-Ambriz et al.34

da Mota et al.37

Suntharalingam and Ravindran38

Unripe mango

Cut-sliced (1 cm) and dried (50°C)

4.2  0.11

2.4  0.02

28.0  0.2

29.8  0.06

Vergara-Valencia et al., 200718

Mango peel

Peels were blanched (3 min), wet-milled, washed at 95°C (5 min), pressed, dried, and milled (particle size 15 mm)

5.2

2.5

34.4

Nd

Larrauri et al.17

Diverse process for production of unripe banana and mango flours and the effect on its chemical composition. Nd, not determined.

2. FLOURS AND BREADS

USE OF UNRIPE FLOUR

161

starch content, enzymatic34 and chemical35 treatments were used (Table 7). The enzymatic treatment was produced with α-amylase for 3 h at 70°C after starch gelatinization. The TDF in this fiber-rich powder increased from 10.4% in UBF to 31.8%, and the total starch and available starch (AS) decreased. The total indigestible fraction shows an important level (approximately 70 g/100 g of the dry fiber-rich powder), showing that there is a high fraction that is not digestible, and that can be used as a functional ingredient in the preparation of foodstuffs.34 A fiber-rich powder was prepared with an acid treatment using UBF prepared with the pulp and peel. UBF presented a TDF content of 17.14% and a starch level of 73.01%. The diverse acid treatment increased the DF content in the unripe flour from 19.8% to 60%. The authors concluded that this fiber-rich powder could be used in food and medical applications because of the increase in consumption of fiber-rich products.35 Physical treatment of unripe banana (AAB) flour was carried out due to its being environmentally friendly and low cost. Whole fruits were immersed in boiling water for 5, 15, or 25 min; nonboiling fruits were used as the control. Flours of the pulp were prepared and conditioned at 30% moisture content for 24 h in a sealed glass container; after that, the container were placed in an oven at 120°C for 24 h. Some of the glass containers were stored at 20°C for 7 days after treatment in the oven. Scanning electron microscopy (SEM) showed intact starch granules, and with X-ray diffraction (XRD) study, some crystallinity peaks were observed. The flours stored at 20°C presented high RS content (15–18 g/ 100 g) even after cooking. This result suggests that the modified UBF can be used in foods where cooking is necessary as bakery products, pasta, and snacks.39 Other alternative is the use of this modified UBF in products that do not require thermal treatments as smoothies, yogurt, fruits, breakfast cereals, and other foods, but the RS content is higher (59 g/100 g). Another hydrothermal treatment is the annealing. UBF was conditioned at 70% moisture content and stored at 65° C (a temperature lower than the gelatinization temperature of starch) for 24 h. Some glass containers were stored at 4°C for 7 days. After annealing, the starch granules in the flour showed a decrease in birefringence with the aggregation of starch granules; the stored flour showed greater birefringence, which was attributed to the reorganization of starch chains during storage. Even when the treated UBF was cooked, they showed RS and SDS,40 where the consumption of both RS and SDS is associated with beneficial health effects.41 Esterification of UBF with citric acid showed high RS content (94 g/100 g) after cooking 20 min, which suggests its use in bakery products, snacks, pasta, and other foods, with high DF content.42

USE OF UNRIPE FLOUR Mango Flour (MF) Unripe MF has been tested in the preparation of cookies and bread (Table 2). In these formulations, MF was added to replace the wheat germ used in the control sample. In the cookies, the wheat flour:MF ratio was 25:75, and for bread, it was 60:40. Both products added with MF presented an increase in TDF level. Cookies with MF added presented a TDF amount of 17.4%, and the control was 13.3%, and bread with MF added showed a TDF of 16.6% and its control was 14.2%; however, depending on the type of product, the increase in soluble and insoluble DF were different. Bakery products with MF added presented similar AS content. A larger difference in AS was evident between the two breads because AS content in the control sample is approximately 50% more than that of the MF-added products, suggesting that it could be an alternative for products with reduced digestible starch content. The starch hydrolysis index (HI) of products “as eaten” showed that control bread exhibited a 34.7% hydrolysis at 180 min, which was higher than in cookies with MF added and the control sample. The HI and derived predicted glycemic index (pGI) for products containing MF were lower than those determined for their respective control samples, indicating that this fiber exerts a significant effect on the rates of digestion and absorption of the starch component of the meal. Bakery products with MF concentrate may be an alternative for people with special caloric requirements.22

Banana Flour UBF was added to bread, where the wheat flour has been totally replaced. The UBF-bread increased up to 100% of DF over its control sample. RS content also was higher (6.7%) in UBF-bread than control bread (1.0%). The insoluble indigestible fraction was higher in the bread with UBF added (22.3%) than in the control (12.4%). These results had a beneficial impact on the HI of starch and GI, because HI and GI were higher (65.1% and 64.3%, respectively) in the bread with UBF added than control bread (81.9% and 78.8%, respectively).24

2. FLOURS AND BREADS

162

12. BANANA AND MANGO FLOURS

Partial substitution of semolina spaghetti was achieved with 15%, 30%, and 45% of UBF, with the aim of increasing the indigestible carbohydrates (i.e., DF). The cooked spaghetti with the highest substitution level presented 12.4% of RS, with 30% of indigestible fraction (an alternative method to determine DF). The spaghetti with UBF added showed cooking loss between 5.3% and 6.2%, and they can be considered acceptable for good-quality pasta (8%). The spaghetti with UBF replacing semolina showed antioxidants that increased with the substitution level (Table 3).10 The texture and preference study of the spaghettis with UBF substituted showed that the hardness and elasticity of the spaghettis were similar to control sample (with 100% semolina). The darkness of the spaghetti increased with the concentration of UBF in the formulation. The preference study of the spaghettis with UBF and the control by consumers did not show any difference. The acceptability of the spaghettis analyzed increased when they were consumed with tomato sauce.43 Recently, diverse studies of the use of UBF in gluten-free products were reported. Spaghetti was prepared with blends of chickpea, maize, and UBF at different levels. The cooking loss ranged from 10%–11% for those spaghettis and was higher than the control (5.4%). The RS content in the gluten-free cooked spaghettis ranged between 2.7% and 4.1%, and the control sample showed RS content of 2%. The pGI was medium (75), and this value was associated with the indigestible carbohydrates present in the UBF.11 The overall acceptability in preference testing by consumers of those gluten-free spaghettis was an average of 5.5, as opposed to the control sample (100% semolina) of 7.2.44 A similar blend of chickpea, blue maize, and UBF was used to prepare gluten-free spaghetti, with the objective of increasing the antioxidant capacity due to the anthocyanins of blue maize. The antioxidant capacity of the spaghettis increased when blue maize increased in the blend with values of 118–145 mmol of trolox equivalents to 145; the DF content was an average of 12%, assessed by the AOAC method, in which RS is not included. The SDS content increased with higher maize flour (white and blue) in the spaghetti, with anthocyanins having a slight effect on the SDS content. However, no appreciable effect was found in the pGI; it ranged between 60 and 64, which is considered medium.13 Other types of gluten-free food matrixes (snacks) were prepared with blends of UBF, chickpea, and maize at different levels. The snacks showed DF content between 13.7% and 18.2%, with appreciable SDS content at the highest UBF level in the snacks (13.3%). The pGI of the gluten-free snacks was low (28.3–35.1). The chili-flavored snack presented an overall acceptability level that was similar to commercial snacks (7.5).44 Recently, UBF was used as a potential nutraceutical ingredient to increase DF and reduce starch digestibility in another food matrix with low water content (i.e., a cookie). The cookie with UBF showed DF content “as eaten” of 43.7% (16 h of hydrolysis) and 62.2% (4 h of hydrolysis). The cookie which had a commercial source of DF (Hi-MAIZE 260) added showed 19.1% and 21.6%, respectively. The pGI was lower in the cookie with UBF (34.7) than the cookie with the commercial ingredient (42.9). UBF can be used as an ingredient in gluten-free cookies with high DF content.45

TECHNOLOGICAL ISSUES Banana flour and MF can be prepared using single procedures in order to minimize production costs. The idea is to establish small factories close to plantations where the crops that did not meet sufficiently quality control for sale as fruits could be used to create flours. The use of the fruit flours in bakery products has been demonstrated, with functionality and nutritional characteristics in their carbohydrate content. The indigestible carbohydrate level increased in products that had fruit flour added, decreasing the glycemic response. Additionally, they showed antioxidant capacity. The results obtained from the application of unripe fruit flours in foodstuffs indicate that these kinds of products might be used as dietary aids by people with special caloric requirements, without lessening preference by consumers.

SUMMARY POINTS • Flours obtained from unripe fruits such as mangoes and bananas are an important source of indigestible carbohydrates and polyphenol compounds. • Mango and banana flours can reduce the glycemic response. • UBF can be modified by enzymatic and chemical treatments to increase the amount of indigestible carbohydrates. • Mango and banana flours can be added to diverse bakery products, and evaluation of their nutraceutical potential is necessary. • Research has showed that unripe bananas have the highest RS content. • Bioactive compounds, such as flavonoids and carotenoids, exist in the DF of fruits.

2. FLOURS AND BREADS

REFERENCES

163

Acknowledgments The authors acknowledge the economic support from Swine Innovation Porc-IPN, COFAA-IPN, EDI-IPN and CONACYT-Mexico.

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erez LA. Pasta with unripe banana flour: physical, texture, and preference study. J Food Sci 2009;74:S263–7. 44. Flores-Silva P, Rodríguez-Ambriz SL, Bello-P
erez LA. Gluten-free snacks using plantain–chickpea and maize blend: Chemical composition, starch digestibility, and predicted glycemic index. J Food Sci 2015;80:961–6. 45. García-Solís SE, Bello-P
erez LA, Agama-Acevedo E, Flores-Silva PC. Plantain flour: a potential nutraceutical ingredient to increase fiber and reduce starch digestibility of gluten-free cookies. Starch/Staerke 2018;70:1700107. 46. Fasolin LH, Almeida GC, Castanho PS, Netto-Oliveira ER. Cookies produced with banana meal: chemical, physical and sensorial evaluation. Ci^enc Tecnol 2007;27:524–9. 47. Bello-P
erez LA, Sáyago-Ayerdi SG, M
endez-Montealvo G, Tovar J. In vitro digestibility of banana starch cookies. Plant Foods Hum Nutr 2004;59:79–83.

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13 Macadamia Flours: Nutritious Ingredients for Baked Goods Kanitha Tananuwong, and Siwaporn Jitngarmkusol Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

O U T L I N E Introduction

165

Production of Macadamia Nuts, Flours, and Other Macadamia-Derived Products Overall Processing Steps Production of Macadamia Flours

166 166 167

Nutrient Composition of Macadamia Nuts and Flours Overall Nutrient Composition Proteins Carbohydrates Lipids Micronutrients

Possible Applications of Macadamia Flour in Baked Goods Nutritional Aspects Functional Aspects

172 172 172

167 167 167 168 169 170

Technological Issues

172

Adverse Reactions

172

Summary Points

173

References

173

Functional Properties of Macadamia Flours and Proteins

170

Macadamia Flours Macadamia Proteins

170 171

Abbreviations EAA Essential amino acid MUFA Monounsaturated fatty acid SFA Saturated fatty acid wb Wet basis

INTRODUCTION Macadamia is a nutritious tree nut indigenous to Australia, originating from the coastal rain forests in Queensland and New South Wales. Among the existing species, the smooth-shelled macadamia (Macadamia integrifolia) is commercially grown for harvesting edible nuts.1 As shown in Table 1, the major producers of macadamia nuts are Australia, the United States, and South Africa.2–5 The total production of these three countries accounts for more than 70% of the global production. The other macadamia producers are located in southern Africa, including Kenya and Malawi, as well as in South and Central America, such as Guatemala and Brazil.3 The United States is not only one of the world’s leading macadamia producers, but also the largest market for the nuts. The import volume of shelled macadamia nuts was 9332 metric tons in 2014. In comparison with other tree nuts, macadamia was ranked seventh in terms of global production volume during the 2015 and 2016 crop years (the top five were almond, cashew, walnut, pistachio, and hazelnut).6

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00013-7

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© 2019 Elsevier Inc. All rights reserved.

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13. MACADAMIA FLOURS: NUTRITIOUS INGREDIENTS FOR BAKED GOODS

TABLE 1 Annual Production of Macadamia Nut-in-Shell in Selected Countriesa Production of nut-in-shell, 10% moisture content (metric tons) Year

Australia

United States (Hawaii)

South Africa

2013

35,200

21,123

40,631

2014

43,600

19,682

48,638

2015

48,300

22,370

49,841

2016

52,000

22,889

41,075

a

From 2013 to 2016, annual production of macadamia tended to increase for Australia and the United States, whereas fluctuation is shown for South Africa. Data from Australian Macadamia Society, Australian_Macadamia_Society. Australian Production and Prices from 2013. 2017. http://australian-macadamias.org/ industry/site/industry/industry-page/about-aussie-macadamias/statistics/statistics/australian-production-and-prices-from-2013. Department of Agriculture, Forestry and Fisheries, Republic of South Africa. Department_of_Agriculture_Forestry_and_Fisheries_Republic_of_South_Africa. A Profile of the South African Macadamia Nut Market Value Chain. 2016. http://www.nda.agric.za/doaDev/sideMenu/Marketing/Annual%20Publications/Commodity%20Profiles/field% 20crops/MACADAMIA%20NUTS%20MARKET%20VALUE%20CHAIN%20%20PROFILE%202016.pdf. The Southern African Macadamia Growers’ Association, The_Southern_African_Macadamia_Growers’_Association. Industry Statistics on the Southern African Macadamia Industry. 2017. https://www.samac. org.za/industry-statistics-southern-african-macadamia-industry/ and U.S. Department of Agriculture, U.S._Department_of_Agriculture. Hawaii Macadamia Nuts, Final Season Estimates (July 2017). 2017. https://www.nass.usda.gov/Statistics_by_State/Hawaii/Publications/Fruits_and_Nuts/072017MacNutFinal.pdf.

Macadamia kernels provide a high number of calories because approximately 70% of the kernel weight is lipids. Nevertheless, the lipids are rich in monounsaturated fatty acids (MUFAs), which may help reduce serum cholesterol and thus lower the risk of cardiovascular disease.7 In addition, macadamia kernels are a good source of proteins, dietary fiber (DF), vitamins, and minerals. Defatted or reduced-fat macadamia flours obtained as by-products of macadamia oil production can be nutrient-rich ingredients for foods and beverages. This chapter provides a review of the production and nutritional quality of macadamia nuts and flours. Details on functional aspects, as well as possible applications of macadamia flours in food products, are also provided.

PRODUCTION OF MACADAMIA NUTS, FLOURS, AND OTHER MACADAMIA-DERIVED PRODUCTS Overall Processing Steps Mature macadamia nuts consist of 69% by weight of the outer green husk, or pericarp (2–4 mm thick), with an inner brown shell, or testa (2–3 mm thick), covering the remaining 31% of the round kernel.7,8 After harvesting, dehusking should be done within 24 h to retard respiratory heat generation, microbial growth, and other biochemical reactions related to quality deterioration.7 The husk waste can be processed to create mulch. The resulting nut-in-shells still have a high moisture content—up to 30% on a wet basis (wb). Therefore, drying is the next crucial step for extending shelf life and increasing kernel yield after cracking. To avoid browning of the kernel core, especially after roasting, incremental drying has been commercially adopted. Wet-in-shell nuts are air-dried in the shade, bins, or aerated silos for up to 4 weeks to reduce the moisture content to 10%–15% wb. The nuts are then moved to bin- or silo-type hot air dryers operated under multiple drying temperatures (40°C–60°C). This process may require up to 6 days in order to acquire approximately 3.0% wb moisture of the nut-in-shells, or 1.5% wb moisture of the kernels.7,9,10 A major disadvantage of this drying process is the long operating time required for it. Alternative drying methods have thus been developed to shorten the drying time. These methods include combined hot-air and microwave or radiofrequency drying,9,11,12 hybrid heat pump–hot-air drying.13,14 Multistage heat-pump drying under nitrogen gas and normal air was also introduced not only to reduce drying time, but also to help maintain the quality of macadamia nuts.15 The dry-in-shell nuts are further cracked manually or by using machines with rotating rubber or steel rollers.8 The shell may be used as a fuel source, as a filler in the plastic industry, or processed to activated carbon. Whole kernels are graded via sink–float separation technique based on their specific gravity. Examples of the grading procedure and product characteristics are listed in Table 2. Grade I and II kernels may be roasted and seasoned to produce snacks. Lower-grade or broken kernels may be used as an ingredient in confectionery, desserts, and baked goods, or even used for oil and paste production. Due to the kernels’ high lipid content, mechanical extraction of the oil using an expeller or screw press can be commercially performed. The refined, cold-pressed oil can be used for food and cosmetic purposes. The pressed cake is normally ground into meal and used as a protein supplement in animal feed.7,8 Pulverization of full-fat macadamia

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TABLE 2

Grading and Application of Macadamia Kernela

Grading of kernel

Lipid content (% wb)

Criteria

Application

Grade I

>75

Float in water bath (specific gravity 1.00)

Edible nuts

Grade II

65–75

Float in saline bath (specific gravity 1.024)

Edible nuts

Grade III

45–65

Float in saline bath (specific gravity 1.150)

Raw material for oil extraction

a

Grading is based on specific gravities of macadamia kernels. Higher-grade kernels have lower specific gravity due to greater oil content. Data from Axtell and Fairman Axtell BL, Fairman RM. Minor Oil Crops. FAO agricultural services bulletin no. 94. Rome: Food and Agriculture Organization of the United Nations; 1992. and Macfarlane and Harris. Macfarlane N, Harris RV. Macadamia nuts as an edible oil source. In: Pryde EH, Princen LH, Mukherjee KD, eds. New Sources of Fats and Oils. Illinois: American Oil Chemists’ Society; 1981:103–108.

kernels results in nut paste, which can be used as an ingredient in nut spreads, desserts, baked goods, or even savory sauces. The flavor and aroma of the paste can be enhanced by using the roasted kernels as raw materials. The overall processing of macadamia is depicted in Fig. 1.

Production of Macadamia Flours Studies have shown that macadamia oil cake can be an efficient protein source in feed for cattle, lamb, broiler chicken, and fish without adverse effects.16–19 This macadamia by-product may also be a source of nutrients for humans, with insignificant amounts of antinutrients. The production of flour from macadamia oil cake or meal is simple. Further drying and defatting may not be required because the oil cake has less than 10% moisture and approximately 13% lipid content.20 The remaining lipid content in this low-fat macadamia flour is comparable with that found in full-fat soy flour (at approximately 20% lipid content).21 However, removal of lipids from the flour may provide several benefits, including improvement of some functional properties of the flour22 and alleviation of rancid flavor in the flour during storage.

NUTRIENT COMPOSITION OF MACADAMIA NUTS AND FLOURS Overall Nutrient Composition The amount of nutrients in macadamia kernels may vary according to their cultivars, level of maturity upon harvesting, and cultivating location and conditions.7 Sathe et al.23 performed an extensive review of the chemical composition of macadamia kernels. According to the review, the moisture, lipid, protein, ash, and sugar content in the edible nuts are 1.4%–2.1%, 66.2%–75.8%, 7.9%–8.4%, 1.1%–1.2%, and 1.4%–4.6%, respectively. Munro and Garg stated that the DF content of macadamia kernels cultivated in Australia (6.4%) is slightly less than that of kernels cultivated in the United States (8.6%). Nevertheless, the nutrient composition of US macadamia kernels and other nuts, as reported by the US Department of Agriculture,21 is used as the representative data set for the following discussion. The nutrient content of whole and defatted macadamia nuts, in comparison with those of the top three nuts consumed on a global basis, is shown in Table 3. Whole macadamia kernel has the highest lipid content, and it also provides the lowest amount of proteins and carbohydrates. However, the protein and carbohydrate content of defatted macadamias are comparable to those of the other defatted nuts. Note that the proximate composition of the defatted nuts in Table 3 is estimated from the nutrient content of the whole nuts on a fat-free basis. These values also represent the estimated chemical composition of the flours from defatted nuts. In the case of macadamia, the speculated data for the defatted nuts are in accordance with the values determined from the defatted flours in a previous study. Jitngarmkusol et al.22 reported the proximate composition of totally defatted flours (0.5%–0.9% fat, wb) from three macadamia cultivars grown in northern Thailand. The protein, carbohydrate, and ash content of those defatted flours are 30.00%–32.85%, 45.66%–51.84%, and 4.38%–5.53%, wb, respectively.

Proteins Defatted macadamia nuts and flours can be a potential source of proteins in the human diet. The quality of the macadamia proteins, in terms of essential amino acid (EAA) content, is also comparable to that of other nut proteins

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13. MACADAMIA FLOURS: NUTRITIOUS INGREDIENTS FOR BAKED GOODS

FIG. 1 Overall processing of macadamia. Critical steps of macadamia processing include incremental drying of the nut-in-shell, cracking, and grading. Premium-grade macadamia kernels are the main products, whereas macadamia flours are by-products of macadamia oil production.

Mature macadamia (up to 45% moisture, wb)

Dehusking

Husk waste

Wet-in-shell macadamia (up to 30% moisture, wb)

Incremental drying

Dry-in-shell macadamia (3% moisture, wb)

Cracking

Broken kernels

Whole kernels

Shell waste

Grading Edible chips or halves Lower-grade kernels

Premium-grade kernels

Edible nuts Cold press

Grinding

Paste Crude oil

Refining

Oil cake

Grinding/sieving

Refined oil

Meal or flour

(see Table 3). EAAs, including histidine and arginine, account for 50% of the total macadamia proteins, which is a similar percentage to that found in cashew nut proteins (50%), but slightly higher than those in almond (41%) and hazelnut proteins (46%). The main EAA present in all four nuts is arginine (23%–35% of total EAAs). However, tree nut proteins, similar to other plant proteins, are inferior to animal proteins because they are incomplete. Lysine is the first limiting amino acid in macadamias, whereas the first limiting amino acid in almonds, hazelnuts, cashew nuts, and most of the tree nuts is tryptophan.21,23

Carbohydrates According to the carbohydrate composition, DF is the major form of carbohydrate found in whole and defatted macadamias, almonds, and hazelnuts, whereas starch is the main carbohydrate in both forms of cashew nuts (see Table 3). Therefore, defatted macadamia flours are also good sources of DF. Because this type of macadamia flour contains approximately 3.4% wb of crude fiber,22 soluble fiber is possibly present as the major DF in the flours. Sugar is another category of carbohydrate found in tree nuts. More than 90% of the total sugars in macadamias, as well as the other three nuts, is sucrose. The sugar content of macadamia kernels may affect the quality of the kernels 2. FLOURS AND BREADS

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NUTRIENT COMPOSITION OF MACADAMIA NUTS AND FLOURS

TABLE 3

Nutrient Composition of Full-Fat and Defatted Nutsa Macadamia

Almond

Hazelnut

Cashew

Composition (g/100 g)

Full-fat

Defatted

Full-fat

Defatted

Full-fat

Defatted

Full-fat

Defatted

Moisture

1.36

5.61

4.41

8.81

5.31

13.53

5.20

9.26

Proteins

7.91

32.65

21.15

42.23

14.95

38.09

18.22

32.45

EAAs

4.02

16.59

8.75

17.47

6.95

17.71

9.15

16.30

Lipids

75.77

–

49.93

–

60.75

–

43.85

–

Saturated fatty acids

12.06

–

3.80

–

4.46

–

7.78

–

MUFAs

58.88

–

31.55

–

45.65

–

23.80

–

PUFAs

1.50

–

12.33

–

7.92

–

7.85

–

Carbohydrates

13.82

57.04

21.55

43.03

16.70

42.55

30.19

53.77

Sugars

4.57

18.86

4.35

8.69

4.34

11.06

5.91

10.53

DF

8.60

35.49

12.5

24.96

9.70

24.71

3.30

5.88

Starches

1.05

4.33

0.72

1.44

0.48

1.22

23.49

41.83

Ash

1.14

4.70

2.97

5.93

2.29

5.83

2.54

4.52

Calcium

0.09

0.37

0.27

0.54

0.11

0.28

0.04

0.07

Magnesium

0.13

0.54

0.27

0.54

0.16

0.41

0.29

0.52

Phosphorus

0.19

0.78

0.48

0.96

0.29

0.74

0.59

1.05

Potassium

0.37

1.53

0.73

1.46

0.68

1.73

0.66

1.18

a

All data are for raw (unroasted) nuts. Data of defatted nuts are estimated from those of the full-fat nuts, recalculated on a defatted basis. EAAs are tryptophan, threonine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, arginine, and histidine. Carbohydrate is calculated by subtraction of the sum of the proteins, total lipids, moisture, and ash from the total weight of the samples. Data from the U.S. Department of Agriculture. U.S._Department_of_Agriculture. USDA national nutrient database for standard reference, Rrelease 28. 2016; http://www.nal.usda. gov/fnic/foodcomp/search/.

(and thus the flours made from them) in terms of color development during the drying and roasting processes. Wall and Gentry24 indicated that macadamia kernels with higher sucrose and reducing sugar content are prone to greater browning development, particularly after roasting. Hydrolysis of sucrose can occur during thermal processing, resulting in an increased amount of reducing sugars available for the Maillard reaction. Variations in the sugar content of macadamia kernels may be caused by the differences in cultivars and levels of maturity. For instance, immature kernels have higher sucrose and reducing sugar content; thus, they are more likely to become defected brown kernels after roasting.

Lipids If macadamia flours are partially defatted, residual lipids also enhance the nutritional qualities of the resulting flours. Macadamia oil contains the greatest amount of MUFAs (81%) compared to the MUFAs in almond oil (66%), hazelnut oil (79%), and cashew nut oil (60%). The major MUFA in all the nuts listed in Table 3 is oleic acid, accounting for 99% of the total MUFA content in almonds, hazelnuts, and cashew nuts. The composition of the MUFAs in macadamias, however, is slightly different. The nuts contain 74% oleic acid and 22% palmitoleic acid in the MUFA fraction. Regular consumption of MUFA-rich macadamia kernels or its oil has been shown to reduce total cholesterol and low-density lipoprotein (LDL) cholesterol in the blood; however, its effects on serum triglycerides and high-density lipoprotein (HDL) cholesterol (the so-called good cholesterol) are still inconclusive in clinical studies.7 Palmitic acid and linoleic acid are the major saturated fatty acids and polyunsaturated fatty acids (PUFAs), respectively, in the four types of tree nuts. The saturated fatty acid fraction in macadamia oil (17%) is higher than that found in almond and hazelnut oil (8%), but slightly lower than the corresponding fraction in cashew nut oil (20%). On the contrary, macadamia oil contains the smallest proportion of PUFAs (2%), in comparison to those in the other three nuts (14%–26%).21 2. FLOURS AND BREADS

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Micronutrients Significant amounts of micronutrients are also present in defatted macadamia nuts and flours. The ash and total mineral contents in all the defatted nuts shown in Table 3 are similar. Potassium, phosphorus, magnesium, and calcium are major minerals found in tree nuts. Defatted macadamia nuts and flours contain moderate amounts of these four minerals, in comparison with the other three nuts. As for vitamins, macadamia nuts, almonds, hazelnuts, and cashews are good sources of thiamine, tocopherols, folate, and phylloquinone, respectively.21 Phenolic compounds are also present in macadamias and other tree nuts. Wu et al.25 reported the total phenolic content of macadamia nuts, almonds, hazelnuts, and cashews as 1.56, 4.18, 8.53, and 2.74 mg of gallic acid equivalent per gram of the whole nut, respectively. The authors also determined the oxygen radical scavenging capacity of lipophilic and hydrophilic extracts of the tree nut samples. Greater antioxidant capacity has been reported for the hydrophilic extracts of the nuts. Defatted macadamia nuts and flours thus may contain significant amount of hydrophilic antioxidants.

FUNCTIONAL PROPERTIES OF MACADAMIA FLOURS AND PROTEINS Macadamia Flours Functional properties of flours, including water and oil absorption capacities, emulsification/stabilization, and foam formation, arise from interactions between their chemical composition and other food components. Proteins and carbohydrates are key substances because they contain both hydrophilic and hydrophobic portions, as well as charged functional groups. Variations in the qualitative and quantitative aspects of these macromolecules may contribute to different functional properties of the flours. Because functional properties of food ingredients greatly influence the overall qualities of the food products, data on these aspects are required in order to use the flours effectively. Jitngarmkusol et al.22 determined the functional properties of partially and totally defatted macadamia flours prepared from three macadamia cultivars grown in northern Thailand. The overall results from this study are shown in Table 4. Greater removal of lipids from the flours results in enhanced water and oil absorption capacities, as well as foaming capacity. After extraction of the total lipids, the proteins and carbohydrates of the macadamia flours may become more soluble and may interact more with the surrounding water or oil. The foaming stability of all macadamia flours is inversely related to the foaming capacity of the flours. However, alteration in chemical composition after the complete removal of lipids apparently does not affect emulsion activity and emulsion stability of the flours.

TABLE 4

Chemical Composition and Functional Properties of Partially and Totally Defatted Macadamia Floura Macadamia flour Partially defatted

Totally defatted

Chemical composition/functional property

HAES 741

HAES 344

HAES 800

HAES 741

HAES 344

HAES 800

Lipids (%)

12.15  0.22

12.80  0.29

14.90  0.33

1.03  0.01

0.58  0.01

0.61  0.01

Proteins (%)

30.96  0.12

30.40  0.21

31.92  0.17

36.45  0.29

35.32  0.78

33.12  0.22

Carbohydrates (%)

49.29  0.33

49.94  0.79

46.49  0.68

52.23  0.21

55.74  0.80

57.09  0.18

Water-absorption capacity (g water/g dry flour)

5.59  0.35

5.40  0.07

3.68  0.16

6.72  0.14

4.71  0.17

4.48  0.09

Oil-absorption capacity (g oil/g dry flour)

3.39  0.04

3.16  0.06

3.05  0.09

4.40  0.79

4.93  0.06

4.65  0.17

Emulsion activity (%)

56.21  1.08

51.94  0.60

50.99  1.53

50.81  0.71

50.47  0.23

49.05  2.79

Emulsion stability (%)

51.68  1.10

53.22  0.59

50.44  2.44

54.20  2.03

53.52  0.32

54.26  0.73

Foaming capacity (%)

22.67  0.58

31.00  1.00

33.67  2.52

126.00  3.46

62.33  3.06

65.67  3.21

Foaming stability (%)

91.85  0.78

86.52  1.11

84.07  2.76

56.27  2.00

73.53  1.77

75.07  1.28

Lipid removal improves the water- and oil-absorption capacities and foaming capacity of macadamia flour. Data are shown as mean  SD of triplicate analyses and calculated on a dry basis. HAES, Hawaii Agricultural Experiment Station, a systematic nomenclature of macadamia. Data from Jitngarmkusol, S., Hongsuwankul, J., Tananuwong, K. Chemical compositions, functional properties, and microstructure of defatted macadamia flours. Food Chem 2008; 110 (1):23–30.

a

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FUNCTIONAL PROPERTIES OF MACADAMIA FLOURS AND PROTEINS

Variations in the functional properties of the flours from different cultivars have been proposed to be related to the qualitative and quantitative differences in their chemical composition, especially proteins and carbohydrates. Joshi et al.26 investigated the functional properties of full-fat and defatted flours from cereals, legumes, tree nuts, and oilseeds. They reported that the water- and oil-holding capacities of the defatted macadamia flour are significantly higher than those of the full-fat flour (P  .05). The water-holding capacity of defatted macadamia flour (1.58 g water/g flour) is similar to that of defatted rice flour (1.54 g water/g flour) and defatted millet flour (1.48 g water/g flour), but this parameter is slightly lower than that of defatted wheat flour (1.92 g water/g flour). Nevertheless, defatted macadamia flour has a much higher oil-holding capacity than defatted wheat flour (3.40 g oil/g flour vs. 1.26 g oil/g flour, respectively). Its oil-holding capacity is close to that of defatted pecan flour (3.32 g oil/g flour). Defatting also significantly decreases the lowest gelation concentration of the macadamia flour (P  .05). Removing lipids from the flours may enhance crucial interactions, including electrostatic interactions and hydrogen bonding, which are required for the formation of a gel network. This results in reducing the concentration of the flours needed for gelation.

Macadamia Proteins Because macadamia proteins play an important role in the functional properties of these flours, research on the functional properties of the isolated proteins under various conditions may provide insight into the functional properties (and thus the application) of the flour in food products. Bora and Ribeiro27 determined the effects of the extraction pH on the yield and functional properties of macadamia protein isolates. Macadamia proteins were extracted three times from the defatted flours by solubilizing the proteins at pH 2.0, 7.2, and 12.0. The soluble proteins at each extracting pH were isoelectrically precipitated at pH 5.0. The results show that 83% of the macadamia proteins can be extracted at pH 7.2 and 12.0, providing a 69% yield of the pH 5.0–precipitated proteins. However, at pH 2.0, only 52% of the proteins are extracted; thus, the lowest yield of the precipitated proteins (i.e., 34%) is obtained. The water- and oil-absorption capacities of the proteins isolated at pH 2.0 (1.0 mL water or oil/g protein) are slightly lower than those of the remaining two isolates (approximately 1.6 mL water/g protein and approximately 1.2 mL oil/g protein). The pH-dependent emulsion activity and stability of the three isolates have been reported and are shown in Fig. 2. Both properties of all three isolates are minimal at pH 5.0, the condition at which the proteins are least soluble. A gradual increase in these emulsion-related properties is evident above and below this isoelectric pH. The results from this study emphasize the importance of pH of the food matrix on functional properties of the isolated macadamia proteins and the flours. Utilization of macadamia proteins and flours at acidic pH, especially at approximately pH 5.0, may be limited due to reduced protein solubility and inferior functional properties. Effects of ionic strength [0.2–4.0 M sodium chloride (NaCl) solution] on the solubility of proteins from defatted nut flours have been reported.28 In the case of macadamia flour, the highest protein solubility can be obtained in 1.0–2.0 M NaCl solution.

pH 2.0 isolate

pH 7.2 isolate

70

pH 7.2 isolate

60

pH 12.0 isolate

60

pH 12.0 isolate

Emulsion stability (%)

Emulsion activity (%)

pH 2.0 isolate 70

50 40 30 20 10

50 40 30 20 10

0

0 3

5

7

8

3

5

7

8

pH

pH

FIG. 2 Emulsion activity and stability of macadamia protein isolates at different pH values. The lowest emulsion activity and stability of macadamia protein isolates are evident at pH 5.0, the isoelectric pH, due to limited protein solubility. Adapted from Bora, P. S., and Ribeiro, D. (2004). Note: Influence of pH on the extraction yield and functional properties of macadamia (Macadamia integrofolia) protein isolates. Food Sci Technol Int 10(4) 263–237.

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POSSIBLE APPLICATIONS OF MACADAMIA FLOUR IN BAKED GOODS Nutritional Aspects Due to their high protein and DF content, low-fat or defatted macadamia flours may be applied as additional protein and DF sources in food products, including baked goods. However, the limited amount of lysine is a crucial problem with utilizing macadamia flours as potential sources of protein. Fortification of lysine content may be obtained by using a combination of macadamia flour with other types of lysine-rich flours, such as other tree nut flours and soy flour. The resulting composite flours may be used to replace wheat flour in bakery products. For instance, specialty breads with enhanced proteins and DFs may be formulated from the composite flours, vital wheat gluten, and lipid emulsifier. Additional studies are needed to determine the appropriate formulas of the macadamiacontaining composite flours, as well as to evaluate their applications in baked goods.

Functional Aspects Apart from nutrient fortification, functionality-related applications of macadamia flours can be considered. Despite the difference in protein content, the water-absorption (or water-holding) capacity of partially and totally defatted macadamia flours (4–7 g/g dry flours; see Table 4) is comparable to those of soy flours, soy protein concentrates, and soy protein isolates (3–6 g/g solids).29 Therefore, similar to soy flours and soy proteins, small amounts of macadamia flours may be used to enhance moisture retention in breads and to help prolong the freshness of the products. Water- and oil-absorption capacities, together with the emulsification and foaming properties of macadamia flours, may aid in improving cake quality. In the case of the defatted soy flours containing approximately 51% protein,21 adding 3%–6% of the flours to cake batter results in a smoother texture of well-emulsified batters, more even distribution of air cells, and softer crumbs.30 Partial or total substitution of the defatted soy flours with macadamia flours in cake batter may be appropriate. With the oil-absorption capacity of the macadamia flours, flavor entrapment within cakes, cookies, and other flavorrich baked goods may be enhanced. Macadamia flours, by themselves or in the form of combined macadamia-soy flours, can also be applied as a replacement for milk, eggs, or both in baked goods. The appropriate amount of macadamia flour in a bakery formula depends on the type of product. A higher amount of macadamia flour may be added to chemically leavened products rather than yeasted products, and likewise for soy flour.31 Another interesting application of macadamia flour is to serve as an ingredient in gluten-free baking. Up to 50% replacement of gluten-free flour, including soybean, potato, and corn flours, with defatted macadamia flour has been suggested to make gluten-free cookies.32 Although a number of in-house formulations have been developed, technical papers on the application of macadamia flours in bakery products are scarce. More research is needed to verify these presumed applications.

TECHNOLOGICAL ISSUES According to current data on the nutritional and functional properties of low-fat and defatted macadamia flours, these tree-nut flours are promising ingredients in foods, particularly bakery products. However, commercial production of macadamia flours is still limited. Scientific research on the application of these flours in baked goods, as well as other foods and beverages, is still scant. Current information and the suggestions in this chapter may help expand the possibility to create well-designed experiments on the utilization of macadamia flours and eventually may lead to an increase in the production volume of these value-added products from macadamia nuts.

ADVERSE REACTIONS Allergic reactions to macadamia nuts have been reported. Such adverse food reactions include gastrointestinal hypersensitivity, respiratory symptoms, and skin manifestations, but these manifestations were not life-threatening. However, a few cases of anaphylaxis, which may cause death, have been reported.33 Hence, to protect consumers, the use of macadamia nuts and flours must be clearly identified on food labels.

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SUMMARY POINTS • Macadamia is a tree nut indigenous to Australia, whose kernels can be processed into a wide variety of products, such as nut snacks, oil, and flours. • Macadamia and its products are highly nutritious. Full-fat macadamia kernels have high amounts of MUFAs, whereas defatted macadamia flours are good sources of proteins and DFs. • Limited amount of lysine is a significant problem in using macadamia flours as potential protein sources. Mixtures of macadamia flours and other lysine-rich flours are thus recommended as ingredients for protein fortification in baked goods. • A higher degree of lipid extraction improves the functional properties, including the water- and oil-absorption capacities and foaming capacity, of macadamia flour. • At a pH near the isoelectric point of macadamia proteins (5.0), the protein solubility and functional properties may be compromised. • Due to its outstanding functional properties, macadamia flour may be used to replace egg and dairy ingredients, as well as to enhance the texture and flavor of baked goods.

References 1. Stephenson R. Macadamia: domestication and commercialisation. Hort Sci Focus 2005;45(2):11–5. 2. Australian_Macadamia_Society. Australian Production and Prices from 2013. 2017. http://australian-macadamias.org/industry/site/ industry/industry-page/about-aussie-macadamias/statistics/statistics/australian-production-and-prices-from-2013. 3. The_Southern_African_Macadamia_Growers’_Association. Industry Statistics on the Southern African Macadamia Industry; 2017. https://www. samac.org.za/industry-statistics-southern-african-macadamia-industry/. 4. U.S._Department_of_Agriculture. Hawaii Macadamia Nuts, Final Season Estimates (July 2017); 2017. https://www.nass.usda.gov/Statistics_by_ State/Hawaii/Publications/Fruits_and_Nuts/072017MacNutFinal.pdf. 5. Department_of_Agriculture_Forestry_and_Fisheries_Republic_of_South_Africa. A Profile of the South African Macadamia Nut Market Value Chain; 2016. http://www.nda.agric.za/doaDev/sideMenu/Marketing/Annual%20Publications/Commodity%20Profiles/field%20crops/ MACADAMIA%20NUTS%20MARKET%20VALUE%20CHAIN%20%20PROFILE%202016.pdf. 6. The_International_Nut_and_Dried_Fruit_Council_Foundation. Nuts & Dried Fruits: Global Statistical Review 2015/2016; 2016. https://www. nutfruit.org/files/tech/Global-Statistical-Review-2015-2016.pdf. 7. Munro IA, Garg ML. Nutrient composition and health beneficial effects of macadamia nuts. In: Alasalvar C, Shahidi F, editors. Tree nuts: Composition, phytochemicals, and health effects. Florida: CRC Press; 2008. p. 249–58. 8. Axtell BL, Fairman RM. Minor Oil Crops. In: FAO agricultural services bulletin no. 94. Rome: Food and Agriculture Organization of the United Nations; 1992. 9. Silva FA, Marsaioli Jr. A, Maximo GJ, Silva MAAP, Gonc¸alves LAG. Microwave assisted drying of macadamia nuts. J Food Eng 2005;77(3):550–8. 10. Walton DA, Wallace HM. Postharvest dropping of macadamia nut-in-shell causes damage to kernel. Postharvest Biol and Technol 2008;49 (1):140–6. 11. Wang Y, Zhang L, Gao M, Tang J, Wang S. Pilot-scale radio frequency drying of macadamia nuts: heating and drying uniformity. Drying Technol 2014;32:1052–9. 12. Wang Y, Zhang L, Johnson J, et al. Developing hot air-assisted radio frequency drying for in-shell macadamia nuts. Food Bioprocess Technol 2014;7:278–88. 13. Borompichaichartkul C, Luengsode K, Chinprahast N, Devahastin S. Improving quality of macadamia nut (Macadamia integrifolia) through the use of hybrid drying process. J Food Eng 2009;93(3):348–53. 14. Phatanayindee S, Borompichaichartkul C, Srzednicki G, Craske J, Wootton M. Changes of chemical and physical quality attributes of macadamia nuts during hybrid drying and processing. Drying Technol 2012;30:1870–80. 15. Borompichaichartkul C, Chinprahast N, Devahastin S, Wiset L, Poomsa-ad N, Ratchapo T. Multistage heat pump drying of macadamia nut under modified atmosphere. Intl Food Res J 2013;20:2199–203. 16. Acheampong-Boateng O, Bakare AG, Mbatha1 KR. The potential of replacing soyabean oil cake with macadamia oil cake in broiler diets. Trop Anim Health Prod 2016;48:1283–6. 17. Acheampong-Boateng O, Bakare AG, Nkosi DB, Mbatha KR. Effects of different dietary inclusion levels of macadamia oil cake on growth performance and carcass characteristics in south African mutton merino lambs. Trop Anim Health Prod 2017;49:733–8. 18. Acheampong-Boateng O, Mikasi M, Benyi K, Amey A. Growth performance and carcass characteristics of feedlot cattle fed different levels of macadamia oil cake. Trop Anim Health Prod 2008;40(3):175–9. 19. Balogun AM, Fagbenro OA. Use of macadamia presscake as a protein feedstuff in practical diets for tilapia, Oreochromis niloticus (L.). Aquac Res 1995;26(6):371–7. 20. Macfarlane N, Harris RV. Macadamia nuts as an edible oil source. In: Pryde EH, Princen LH, Mukherjee KD, editors. New Sources of Fats and Oils. Illinois: American Oil Chemists’ Society; 1981. p. 103–8. 21. U.S._Department_of_Agriculture. USDA national nutrient database for standard reference. In: Release 28; 2016. http://www.nal.usda.gov/fnic/ foodcomp/search/. 22. Jitngarmkusol S, Hongsuwankul J, Tananuwong K. Chemical compositions, functional properties, and microstructure of defatted macadamia flours. Food Chem 2008;110(1):23–30.

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23. Sathe SK, Monaghan EK, Kshirsagar HH, Venkatachalam M. Chemical composition of edible nut seeds and its implications in human health. In: Alasalvar C, Shahidi F, editors. Tree nuts: Composition, phytochemicals, and health effects. Florida: CRC Press; 2008. p. 11–36. 24. Wall MM, Gentry TS. Carbohydrate composition and color development during drying and roasting of macadamia nuts (Macadamia integrifolia). LWT-Food Sci Technol 2007;40(4):587–93. 25. Wu X, Beecher GR, Holden JM, Haytowitz DB, Gebhardt SE, Prior RL. Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J Agric Food Chem 2004;52(12):4026–37. 26. Joshi AU, Liu C, Sathe SK. Functional properties of select seed flours. LWT-Food Sci Technol 2015;60:325–31. 27. Bora PS, Ribeiro D. Note: Influence of pH on the extraction yield and functional properties of macadamia (Macadamia integrofolia) protein isolates. Food Sci Technol Int 2004;10(4):263–7. 28. Sathe SK, Venkatachalam M, Sharma GM, Kshirsagar HH, Teuber SS, Roux KH. Solubilization and electrophoretic characterization of select edible nut seed proteins. J Agric Food Chem 2009;57:7846–56. 29. Boyacioglu MK. Soy ingredients in baking. In: Riaz MN, editor. Soy applications in foods. Boca Raton: CRC Press; 2006. p. 63–81. 30. Endres JG. Soy protein products: Characteristics, nutritional aspects, and utilization. Illinois: AOCS Press; 2001. 31. Amendola J, Rees N. Understanding Baking. 3rd ed. New York: John Wiley & Sons; 2003. 32. Navarro SLB, Rodrigues CEC. Macadamia oil extraction methods and uses for the defatted meal byproduct. Trends in Food Sci Technol 2016;54:148–54. 33. Knott E, G€ urer CK, Ellwanger J, Ring J, Darsow U. Macadamia nut allergy. J Eur Acad Dermatol Venereol 2008;22:1365–401.

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14 Sourdough Breads Pasquale Catzeddu Porto Conte Ricerche Srl, Alghero (SS), Italy

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Sourdough Microorganisms Classification of Sourdough

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Healthy Properties of Sourdough Bread

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Abbreviations ACE EPS EU GF LAB PDO PGI TSG

Angiotensin I-converting enzyme Exopolysaccharide European Union Gluten free Lactic acid bacteria Protected designation of origin Protected geographical indication Traditional speciality guaranteed

INTRODUCTION The term sourdough bread refers to bread leavened with a sourdough starter. Sourdough is a mixture of flour and water fermented with lactic acid bacteria (LAB) and yeasts, which can grow spontaneously or be inoculated as selected starters. Spontaneous sourdough is the oldest-known bread-leavening agent, and it consists of dough left at room temperature for several hours. Fermentation by endogenous microorganisms takes place in the dough, producing metabolites that affect the characteristics of the dough (Fig. 1). The addition of new flour and water to the dough, known as backslopping, allows a composite ecosystem of yeasts and LAB to develop inside the dough, giving it its typical sour taste. Commercially available starter microorganisms can also be added to a mixture of flour and water to obtain sourdough. The yeasts are mainly responsible for the production of carbon dioxide (CO2), whereas LAB are mainly responsible for the production of lactic acid, acetic acid, or both; both produce the aromatic precursor compounds of bread. Furthermore, the technological performance of the dough and the nutritional properties, aroma profile, shelf life, and overall quality of the bread are greatly affected by the metabolic activity of the sourdough microorganisms.

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FIG. 1 Sourdough starter. Spontaneous sourdough starter prepared with durum-wheat flour and approximately 70% water. The small pores on the surface indicate CO2 production.

Although sourdough presents an equally valid leavening agent in bread technology, the use of compressed baker’s yeast is much more widespread, most likely due to the complexities of sourdough management and the associated bread-making process; indeed, the reliable and rapid leavening of baker’s yeast easily fulfills the requirements of both artisan and industrial bakeries, which can purchase it as commercial compressed yeast. Many sensory, nutritional, and physical differences exist between yeasted bread and sourdough bread; for example, sourdough breads commonly exhibit a more intense flavor, an acidic taste, higher crumb compactness, and an increased shelf life compared to yeasted bread. Sourdough bread also has superior nutritional properties, the characteristics of which have been discussed in detail in numerous scientific studies and include a lower glycemic index (GI) and greater mineral bioavailability. Indeed, an increase in the appreciation for more flavorful and healthy breads over the last decade has raised consumer demand for sourdough breads. Furthermore, a variety of new sourdough products developed by specialized companies, together with the development of equipment that facilitates their handling, means that the practice of sourdough technology is becoming ever more widespread in both artisanal and industrial bakeries.

A BRIEF HISTORY OF SOURDOUGH BREADS AND THEIR CURRENT DIFFUSION Since ancient times, cereal foodstuffs have been fundamental to human nutrition. Primordial wheat varieties have been cultivated in the Mediterranean region for at least 10,000 years and used for bread production.1 Emmer wheat and barley cultivated in the Nile River Valley are thought to have been used in ancient Egypt to manufacture unleavened and leavened breads on a large scale—enough to feed thousands of people per day; indeed, desiccated bread remains, and numerous wall paintings of the bread-making process have been discovered in ancient tombs, supporting this notion. It is likely that leavened bread in ancient Egypt was obtained by using spontaneous sourdough as the primordial leavening agent, although the contamination of bread dough with yeast from the brewing process may also have taken place.2 The production of sourdough bread gradually spread from ancient Egypt throughout Greece and the Roman Empire, and has continued ever since. The ancient Greeks imported wheat grains from Sicily and Egypt because the poor soil quality and rocky terrain of their homeland restricted their own cultivation of cereals; indeed, the necessary and continuous trading between the Greeks and Egyptians allowed the former to become familiar with leavened breads. The Greeks made remarkable improvements to the technology and baking equipment. Greek bakers taken to Rome as slaves became professional bakers once freed, forming businesses that became increasingly important and indispensable to society. Bread constituted an important part of the diet in the Roman Empire, and many public bakeries were created; bakers became public officials and hence employees of the state, producing large quantities of bread that was freely distributed to Roman citizens. Being a baker became a profession that continued within families from generation to generation. During the Barbarian migration period in Europe (AD 300–700), industrial bread manufacturing disappeared, as bread was not a primary food for the Barbarians. The technology of sourdough bread survived in the monasteries until the 12th century, and it was also at this time when the profession of being a baker reappeared in France. After the Middle Ages, bread-making technology saw the advent of new progress, especially in northern Europe, where baking went hand-in-hand with brewing. Breweries were widespread, and the froth on fermenting malt liquor, the so-called barm, was identified as a substitute for sourdough in the leavening process. 2. FLOURS AND BREADS

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Although evidence indicates the use of beer yeast in bread-making in ancient Egypt and other communities familiar with the brewing process, it was only after the Middle Ages that it became a widely used agent for the leavening process, or indeed a substitute for sourdough. Nevertheless, the culturing of baker’s yeast (or compressed yeast), Saccharomyces cerevisiae, for bread-making purposes3 on the industrial scale only occurred in the 19th century. During the 20th century, it began to replace the use of sourdough in bread production almost completely, particularly in industrial bakeries. For many years, sourdough was employed exclusively by artisanal bakeries and home bakers, as well as for rye bread production for reasons that will be discussed later in this chapter. The rapid expansion of baker’s yeast was due to its greater capacity to meet the requirements of innovative bakeries, whose bread production became mechanically assisted. Thus, the rapid and simple leavening process associated with baker’s yeast replaced the sourdough-baking process, which was much more time consuming due to its long fermentation time and laborious management. In the last 20 years, the popularity of sourdough bread has grown worldwide for a number of reasons, but mostly due to the renewed interest of both consumers and bakers in this product. Consumers are attracted to its pronounced flavor, high nutritional value and healthy properties, prolonged shelf life, use of fewer additives, and, finally, its traditional aspects. Bakers have been prompted to produce sourdough bread, not only in response to consumer demand, but also thanks to technological advances that facilitate sourdough handling. Furthermore, a number of sourdough-type products has been developed and commercialized by specialized companies, especially in northern European countries, where bakers can purchase dried, paste, or liquid sourdough starters that have been stabilized by heat treatment to obtain a shelf-stable product.4 Many types of sourdough bread are produced worldwide. Most of them are traditional breads produced in Europe, Mediterranean countries, and North America, but many common breads (i.e., popular breads produced on a large scale, but not associated with any historical traditions or precise production methods) are also sourdough breads. In recent years, sourdough has been applied in the production of sweet baked goods, crackers, pizza, gluten-free products, and pasta,5–7 with the purpose of improving the textural, sensorial, and nutritional properties of these goods. In the United States, the most famous type of sourdough bread currently produced is called San Francisco bread. This type of bread is made using bread flour, or a mix of wheat and rye flours, water, salt, and sourdough starter. The taste of San Francisco bread is very sour, and the bread has a pH of about 3.9–4.0. Sourdough was introduced from Europe into the San Francisco area of California by Basque immigrants during the California gold rush, and then diffused into Alaska and Western Canada during the Klondike gold rush. In Europe, due to the industrialized processes of many bakeries, which use compressed yeast as their principal leavening agent, most bakers do not have the appropriate know-how to handle sourdough. In fact, the practice of sourdough bread has been kept alive thanks to home bakers with a passion for this type of bread, as well as bakers specializing in traditional breads (namely, breads that form part of the cultural heritage of specific geographical areas). Indeed, the sourdough-baking process generally concerns artisanal bakeries, and it is relatively commonplace in the production of Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) bread products— traditional breads that have been baked in a specific geographical area for at least 30 years and that are protected by European regulations, such as Council Regulation (EC) N. 510/2006. For example, Pane di Altamura (Fig. 2), Pagnotta del Dittaino, and Pane Toscano are all PDO breads produced in Italy; the first two are produced using semolina from durum wheat harvested in a specific area, and the latter is produced using whole-grain flour from soft wheat grown in the Tuscany region and characterized by the absence of salt. All these breads are produced using sourdough. Other sourdough breads produced in Italy are the PGI breads Pane di Matera, Pane di Genzano, and Coppia Ferrarese. The first is semolina based, and the others use bread flour; sourdough is the main leavening agent in all three. Four PGI sourdough breads are produced in Spain: Pan de Cea, Pa de Pagès Català, Pan de Alfacar, and Pan de Cruz de Ciudad Real. The first bread only uses sourdough, the second two breads use both sourdough and the yeast S. cerevisiae, and for the last bread, the regulation reports the use of natural yeast as a leavening agent. All these breads are made using bread flour. In northern Europe, sourdough has always played an important role in rye flour bread-making, particularly in Germany, the Baltic States, Poland, and Russia. Bread made with 100% rye flour is still obtained by sourdough fermentation because rye flour does not contain gliadin and glutenin, as wheat flour does; instead, it has a similar protein called secalin, which forms a discontinuous and nonelastic net structure in the dough. Furthermore, rye flour contains high levels of pentosans that inhibit the formation of the protein network; thus, nonacidified rye dough does not retain the CO2 produced during fermentation.8 The low pH created by sourdough fermentation promotes the solubility and swelling properties of pentosans, enhancing the water-binding capacity of the dough and enabling bread-making. Furthermore, rye flour mainly contains starch, which can absorb a high amount of water. The acidity of sourdough inactivates endogenous α-amylases, strongly active in rye flour, thus preventing excessive starch degradation and allowing the starch to gel and form a matrix during cooking. Rye dough acidification is essential to obtain a proper crumb structure and an increase in bread 2. FLOURS AND BREADS

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FIG. 2 Loaves of sourdough bread produced in southern Italy. Pane di Altamura is a traditional PDO bread obtained from remilled durum-wheat semolina and sourdough fermentation. It is produced in a limited area of southern Italy according to the bread-making procedure defined by EU regulations.

volume.9 In mixed wheat-rye breads, baker’s yeast can be used for leavening, and if strong wheat flour is used, an increase in loaf volume can be obtained. Despite its distinctive flavor, taste, and eating quality, sourdough rye bread production is currently diminishing due to the large amount of labor and high production costs entailed, and most rye breads, especially in the United States, are manufactured using baker’s yeast.10 Two sourdough rye breads are protected by regulations by the European Union (EU): the Dauj_enų namin_e duona bread and the Chleb prądnicki bread, both registered as PDI breads. The former is produced in Lithuania using rye flour only; it is a brown rye bread baked in loaves weighing 4–10 kg. The latter is a bread produced in Poland; it is baked in two shapes, oval loaves weighing 4.5 or 14 kg and round loaves weighing 4.5 kg, and it employs both rye and wheat flour; moreover, the inclusion of potatoes in the recipe ensures long-lasting freshness. A naturally leavened bread produced from rye flour is Salinătă rudzu rupjmaize, produced in Latvia. The sourdough is prepared with heat-treated flour (about 65°C) left at 28°C–35°C for 12–24 h. This bread has Traditional Specialty Guaranteed (TSG) status and is also protected by the EU regulations. Flatbread is thought to have been the first type of bread produced by ancient peoples. These breads originated in the Middle East area, and production subsequently spread throughout the world. Flatbreads are currently produced worldwide using a variety of primary materials and as both leavened and unleavened bread.11 Flatbread is quite popular in the Mediterranean area, in southern Italy (Sardinia), North Africa, and Spain, and bread wheat (Triticum aestivum) or durum wheat (Triticum turgidum subsp. durum L.) flour is used. Flatbread baked with traditional methods is still made using sourdough fermentation. In Iran, Sangak, and in Morocco, Khobz El-daar are single-layered flatbreads that have been sourdough fermented; the former is made with bread flour, and the latter with durum-wheat flour. Baladi is double-layered flatbread produced in Egypt with bread flour and sourdough fermentation. In southern Italy, more precisely in Sardinia, an island in the middle of the Mediterranean Sea, different types of flatbreads are produced (Fig. 3). They are all leavened and double layered, and their production traditionally involves the use of semolina from durum wheat and sourdough fermentation. The most representative are the double-layered Spianata and Carasau breads. The former is a soft flatbread; the latter is a crisp flatbread obtained by baking the bread twice; in fact, after the first round of baking, the two layers are separated and then toasted, to obtain crispy and very thin (about 1–2 mm) sheets of bread. Both can be made in either a circular or rectangular shape. Similar crisp flatbreads are produced in Scandinavian countries from rye flour and sourdough fermentation11; they may be circular, with a hole in the middle, or rectangular, and their thicknesses can range from 0.7–1.0 cm or even more. After baking, one particular rye crisp bread (namely, Hapankorppuja) is split into two layers and dried, similarly to the Sardinian Carasau bread. 2. FLOURS AND BREADS

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FIG. 3 Sourdough flatbread. Two-layered leavened flatbreads are produced in the Mediterranean area. In Sardinia, an Italian island in the Mediterranean Sea, Spianata bread (left) and Carasau crisp bread (right) are made using a sourdough starter and durum-wheat flour.

SOURDOUGH MICROORGANISMS Spontaneous sourdough is a rich source of microorganisms, principally LAB and yeast (Table 1) belonging to different genera and species that derive from the flour itself or the environment, or are added as an inoculum (e.g., in the form of fruit or yogurt). The presence of microorganisms in sourdough was discovered in approximately 1900. Since then, hundreds of scientific papers have expanded our knowledge of sourdough microflora, as discussed by De Vuyst et al.12 Most of the isolated species of LAB belong to the genus Lactobacillus—so far more than 60 Lactobacillus species have been isolated—but species of the genera Pediococcus, Leuconostoc, and Weissella are also commonly found. Several new species of Lactobacillus were initially isolated from sourdough, but few of them are considered to be specific to the sourdough habitat, such as Lactobacillus sanfranciscensis and Lactobacillus alimentarius. The specificity of new LAB species to sourdough can be revealed only after exploring other food substrates.12 Other species isolated from sourdough (e.g., Lactobacillus acidophilus and Lactobacillus reuteri) may have an intestinal origin, probably due to cross-contamination, whereas others, such as Lactobacillus plantarum and Lactobacillus brevis, are quite common in other habitats and foods.13 LAB ferment maltose, the most abundant sugar in flour, and produce lactic acid when expressing homofermentative metabolism; in the case of heterofermentative metabolism, they produce CO2, acetic acid, and/or ethanol, in addition to lactic acid. The prevalence of homofermative versus heterofermentative species influences the properties of the dough. Acetic acid is responsible for the hardening of gluten, whereas lactic acid gradually produces a more elastic gluten. As a consequence, the texture and the aromatic profile of the bread are also affected.13 L. sanfranciscensis dominates the sourdough microflora of sweet leavened baked products and wheat breads produced in Italy,14, 15 and the same species has been found in traditional Greek wheat sourdough.16 Lactobacillus pentosus and L. plantarum dominate the sourdough breads produced in southern Italy that use durum wheat flour.17, 18 L. brevis is predominant in Turkish and Portuguese sourdough bread.19, 20 Several species of yeast, belonging to the phylum of Ascomycetes, have been isolated from sourdough, but only six of them (Table 1), belonging to the genera Saccharomyces and Candida, are frequently isolated in spontaneous sourdough and considered fundamental in the fermentation process.21 The high detection frequency of Saccharomyces cerevisiae in sourdough might be ascribed to environmental contamination, especially in bakeries where baker’s yeast is used as the common leavening agent.22 Because yeast is the major producer of CO2 in sourdough, it is considered responsible for dough leavening.13 Saccharomyces exiguus (now classified as Kazachstania exigua) was the first yeast species isolated from sourdough by Kline and Sugihara,23 together with LAB identified as Lactobacillus sanfrancisco (now denominated L. sanfranciscensis). Both are responsible for the fermentation and souring activity in San Francisco bread. These species form a tight association in sourdough because of their metabolic properties. The trophic relationships between these two species are well documented13; the microorganisms do not compete for sugar uptake because L. sanfranciscensis is maltosepositive and K. exigua is a maltose-negative species (maltose is the most important sugar in sourdough). During sourdough fermentation, L. sanfranciscensis splits maltose into two molecules of glucose, but only one is metabolized; the other is excreted outside the cell, where it can be fermented by the yeast. Furthermore, during sourdough fermentation, the excretion of specific amino acids and small peptides by yeast is to the advantage of the lactobacillus, which in turn produces lactic or acetic acid in the dough and decreases the pH, creating a favorable substrate for yeast growth. Other examples of stable yeast-LAB associations found in sourdough include Candida humilis (maltose-negative) and

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Species of LAB and Yeasts Isolated From Sourdougha

Obligate heterofermentative LAB • Lactobacillus sanfranciscensis • Lactobacillus brevis • Lactobacillus fermentum • Lactobacillus reuteri • Lactobacillus panis • Lactobacillus pontis • Lactobacillus fructivorans • Lactobacillus rossiae • Weissella confuse • Weissella cibaria • Leuconostoc citreum • Leuconostoc mesenteroides a

Facultative heterofermentative LAB • Lactobacillus plantarum • Lactobacillus casei • Lactobacillus rhamnosus • Lactobacillus alimentarius

Homofermentative LAB • Lactobacillus amylovorus • Lactobacillus acidophilus • Lactobacillus farciminis • Lactobacillus delbrueckii

Yeasts • Saccharomyces cerevisiae • Candida humilis [synonym C. milleri] • Wickerhamomyces anomalus (synonym Pichia anomala and Hansenula anomala); anamorph Candida pelliculosa • Torulaspora delbrueckii (anamorph Candida colliculosa) • Kazachstania exigua [synonym Saccharomyces exiguus); anamorph Candida (Torulopsis) holmii] • Pichia kudriavzevii (synonym Issatchenkia orientalis); anamorph Candida krusei)

The microorganisms most frequently found in sourdough. Most LAB belong to the genus Lactobacillus, the majority being heterofermentative species.

L. sanfranciscensis (maltose-positive); and S. cerevisiae (maltose-positive) and L. plantarum (prefers glucose and fructose).12 The quality of sourdough bread is determined by a number of factors affecting sourdough fermentation (Fig. 4). As a matter of fact, numerous endogenous and exogenous elements affect the number of microorganisms (both yeast and LAB), the presence of one microbial species or another during the fermentation process, and the equilibrium between the species that coexist in a sourdough.24 If the technological factors of sourdough preparation are changed, such as the amounts of water and flour added at each refreshment, the fermentation time and/or temperature, the storage temperature, or the number of backslopping steps, then the equilibrium set up between LAB and yeast species may also change; the number of yeast cells or LAB may even increase to the detriment of the other. For example, an increase in the fermentation temperature and a higher amount of water in dough formulation can favor the growth of LAB over yeast, whereas the oxygenation of sourdough favors the growth of yeast cells over LAB.25 To decrease the variability among microbial species and to keep the ratio yeast-LAB stable, it is fundamental to keep the technological parameters constant. Another factor capable of influencing the microbial composition of sourdough is the house microbiota (i.e., all the microorganisms contaminating the environment and the equipment of a bakery that may come into contact with the sourdough).26 Moreover, antimicrobial compounds produced by some LAB strains are responsible for an antagonistic interaction among microorganisms and provide a competitive advantage over the other strains.27 The raw materials, such as flour and other nutrients, not being sterile, are a source of contaminant microorganisms that are capable of modifying the microbial composition of sourdough.

Classification of Sourdough As described in the preceding section, sourdough can be prepared using different procedures to obtain different results. Based on the technology applied, sourdoughs have been classified into four groups.28 Type I sourdough—This class of sourdough includes the traditional sourdough obtained by spontaneous fermentation, and the fermentation process is carried out at ambient temperature (200) and was developed for industrial processes. Microbial strains are inoculated at high concentrations to initiate the fermentation. The resulting strong acidification of the dough inhibits yeast

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FIG. 4 Factors affecting sourdough fermentation and bread quality. The fermentation process in sourdough is affected by numerous endogenous and exogenous factors that regulate its microflora composition (e.g., the microbial species and the LAB:yeast ratio), and hence the types of metabolites produced in the dough. The metabolites in sourdough, together with the baking conditions and the quality of flour proteins, affect the bread quality.

growth, and, hence, baker’s yeast is usually added at the end of fermentation. This sourdough can be used as a leavening agent or as a baking improver to give bread the traditional sourdough flavor. At the end of fermentation, type II sourdough is stored at a low temperature. This sourdough is produced using bioreactors, within which all parameters are strictly controlled. Type III sourdough—This dough is similar to type II sourdough, but after preparation, it is dried or stabilized by pasteurization. It has a long shelf life; moreover, its easy handling makes it convenient for industrial bakeries and for commercialization. Type III sourdough is used as an acidifier, especially in rye bread production, or as a bread improver. Type IV sourdough—This sourdough is a combination of types I to III: Fermentation is started by inoculating a microbial starter into a dough, and thereafter, it is propagated by daily backslopping; the dough can be firm or semiliquid. It is typically used in artisanal bakeries and is the type of choice for laboratory studies. Type IV sourdough was only recently introduced into bakeries, with the goal of overcoming the disadvantages associated with handling type I sourdough; hence, some bakers switched from a firm sourdough to a liquid one, with the assistance of devices that automated the fermentation process and controlled the fermentation parameters.29 Di Cagno et al.6 demonstrated a higher number of yeast cells in liquid sourdough compared with firm sourdough, probably due to the higher water and oxygen content. In traditional type I sourdough, the yeast:LAB ratio is generally 1:100.14, 15 Therefore, the transition from firm to liquid sourdough is likely to modify the main microbial and biochemical features of traditional baked goods, as reported by Di Cagno et al.6 Commercial sourdough starters for baking purposes were developed in northern Europe at the beginning of the 20th century. This came about because the increased use of baker’s yeast in rye bread production required the use of acidification agents that were essential for rye bread development.4 In the last 20 years, the renewed interest in sourdough bread has increased the production of commercial sourdoughs for bread-making purposes, and the generation of selected microbial strains (commercial starters) for sourdough preparation. Today, a number of suppliers across Europe and North America offer different types of starters. Two main formulates can be distinguished1: starter cultures, consisting of pure strains of LAB and/or yeast isolated from sourdough; and2 sourdough starters, obtained by fermenting flour with yeast and/or LAB strains and supplied in semiliquid or dry-powder form. Starter cultures come in dried, paste, or liquid form and contain a very high concentration of living cells. Sourdough starters are often microbiologically inactive, and as such, they cannot be used as leavening agents, but rather as dough acidifiers and flavor enhancers. The contemporary use of baker’s yeast is thus useful to manage the leavening of the bread dough. The microorganisms contained in liquid sourdough are inactivated by the addition of salt or by pasteurization, whereas

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in freeze-dried products, some microorganisms do not survive the drying process. For example, K. exigua does not tolerate freeze-drying, and fresh cells must be used to inoculate dough. The aim of all these products is to obtain consistent bread quality by overcoming the problems associated with artisanal sourdough production, such as the difficulties in dough processing, the need for highly skilled bakers, long operational times, and uncertain outcomes.

TECHNOLOGICAL ISSUES: SOURDOUGH BREAD QUALITY The differences between sourdough bread and baker’s yeast–leavened bread are well recognized and have been documented by many authors. Sourdough fermentation affects the properties of both the dough and the resulting bread: it extends shelf life through the inhibition of spoilage fungi and bacteria,30, 31 improves the bread’s flavor,32 enhances nutritional properties,33 increases loaf volume, and delays staling.9 Acidification is the most evident effect of LAB metabolism in the dough. The pH of a ripe sourdough ranges from 3.8 to 4.5, depending on endogenous factors (microbial composition and the nature of the flour) and exogenous factors (temperature and time of fermentation and dough yield). The acidification of bread dough affects the activity of microbial and cereal enzymes, the rheology of the dough, and the flavor of the bread. However, its perception in bread is not well accepted by many consumers, who prefer a less acidic taste in their bread. Indeed, because sourdough bread can reach a pH of 4.0, controlling the acidity level in sourdough wheat bread is essential for bread acceptability. The texture of wheat bread depends greatly on the formation of a gluten network, a viscoelastic structure that entraps the CO2 produced during fermentation and allows expansion of the dough. Biological acidification of dough is important for improving loaf volume, crumb softness, and the delayed starch retrogradation of bread. A gradual decrease in pH during fermentation supports the activity of several enzymes, such as amylases and proteinases, which are active at low pH values. Therefore, the acidity acts on the gluten network, improving the softness and extensibility of the dough and favoring the retention of CO2 produced in the fermentation process.34 Excessive acidity and hydrolysis of gluten proteins—for instance, as a consequence of a long fermentation time—results in a softer, less elastic dough, reduces the loaf volume, and increases staling and bread firmness, as demonstrated by the addition of bacterial proteases to the sourdough.32, 35 Acidification of dough due to microbial fermentation has a positive effect, especially in high-fiber breads, in which the addition of cereal bran decreases the bread volume and crumb elasticity, causing severe problems in bread quality.36 Straight yeasted bread is subject to microbial spoilage by molds and Bacillus microorganisms. Mold growth occurs due to contamination after baking, whereas Bacillus spores are contained in the raw material and germinate after baking. Chemical additives, such as sorbate, propionate, and benzoate, are frequently mixed into the dough to control the growth of undesired microorganisms, but an increase in resistant strains and public demand to reduce chemical additives in food have stimulated research into natural antimicrobial compounds. Sourdough contains microorganisms recognized as playing a fundamental role in the preservation and microbial safety of fermented foods. The antimicrobial activity has usually been attributed to the production of organic acids, especially lactic and acetic acids; however, other antimicrobial compounds are produced by sourdough LAB—namely, low-molecular-mass compounds such as phenyl and substituted phenyl derivates (3-phenyllactic, 4-hydroxyphenyllactic or benzoic acid), cyclic dipeptides, hydroxy fatty acids, or antifungal peptides, which operate synergically with organic acids. Over the last decade, numerous LAB strains with antifungal activity have been isolated and studied with the aim of extending the shelf life of bread.37 The use of such strains in sourdough bread was shown to inhibit the main contaminant molds, such as Aspergillus, Fusarium, and Penicillium.37 Ryan et al.31 assert that a strong inhibitory effect can be obtained using sourdough LAB in combination with calcium propionate. Samapundo et al.38 observed that LAB strains producing antifungal metabolites can replace the propionate commonly used as a preservative in bread. A similar inhibitory effect of sourdough has also been observed against rope-forming Bacillus30; in this case, the antimicrobial mechanism is thought to be achieved by a combination of low pH, and the generation of organic acids and other substances, such as bacteriocins. Axel et al.39 found that the antifungal activity in gluten-free bread was lower due to the higher water activity of such bread compared with wheat-containing bread. Consumer appreciation of sourdough bread has increased in recent years, not only due to better knowledge of its nutritional properties, but mainly because consumers enjoy the stronger flavor of sourdough bread compared with yeasted bread. The effectiveness of sourdough in enhancing bread flavor has been established by many authors, and bread produced using unfermented dough has been observed to contain much fewer volatile compounds than fermented dough.40 Bread flavor is influenced by numerous factors, including the type of cereal flours, the parameters of sourdough preparation, the baking conditions, and the metabolism of fermenting microorganisms. However, the enhanced proteolysis of dough, due to the fermenting microorganisms in sourdough, leads to the formation of amino

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acids—the precursors of aromatic substances—and is the major factor affecting taste. The less strong flavor of yeasted bread most likely occurs because yeast utilizes a high amount of amino acids for its metabolism. Factors improving the formation of flavors, such as a long fermentation time or the use of whole flours, seem to contrast with the enhancement of bread texture and volume.32 Rye flour is a particularly flavor-enhancing substrate because it contains amino acid flavor precursors, fatty acids, and phenolic compounds that are converted during the baking process.

Novel Types of Sourdough Breads The preparation of sourdough using wheat flour or rye flour has a long tradition in bread technology, but nowadays sourdough fermentation of nonconventional flours is receiving increased interest. In order to obtain baked goods with functional properties and improved nutritional values, sorghum, buckwheat, oat, barley, spelt, rye, quinoa, and amaranth flour can be used for sourdough preparation, and used afterward in combination with wheat flour for breadmaking.41, 42 Moreover, the positive properties of sourdough can be exploited for making bread for special diets, like gluten-free foods, in order to improve their quality; for instance, gluten-free bread is considered to have poor sensorial and textural qualities, as well as low nutritional values.5 Indeed, the capacity of many LAB used in sourdough to produce exopolysaccharides (EPSs) has been exploited to enhance the properties of gluten-free breads. For example, Lynch et al. recently demonstrated reduced crumb hardness and increased shelf life in breads obtained through the use of EPS-producing strains fermenting gluten-free flours (e.g., sorghum, buckwheat, teff, and quinoa).43 Rinaldi et al.,44 on the other hand, found that sourdough fermentation of chestnut flour was able to improve the nutritional properties of gluten-free bread.

HEALTHY PROPERTIES OF SOURDOUGH BREAD Consumer interest in the healthy aspects of food is continuing to grow, and traditional foods quite often are perceived as having nutritional properties that help improve human health and well-being. In the past decade, sourdough fermentation has been associated with the health-promoting properties of bread, and numerous beneficial effects have been reported (Fig. 5). Protective compounds in bread have been identified, especially in whole-meal bread, which contains dietary fiber, minerals, vitamins, and complex carbohydrates. Conversely, whole-meal bread contains a significant amount of phytate, which interferes with the absorption of essential minerals such as calcium, zinc, and iron. The degradation of phytate by the phytase enzyme has been extensively established; in the past few years, it has been reported that the activity of wheat phytase is predominant over that of sourdough microorganisms, but the lowering of the pH in sourdough, due to the fermentative metabolism of bacteria, provides more favorable conditions for the endogenous cereal phytase.45 An increase in iron bioavailability in sourdough bread compared with yeasted bread was demonstrated by Rodriguez-Ramiro et al.46 using an intestinal cellular model. FIG. 5 Influence of sourdough on the nutritional properties of bread. Sourdough fermentation can significantly improve the nutritional properties of bread because it increases mineral bioavailability (especially in whole-meal bread) and reduces the dietary GI in human blood. New applications of sourdough breads are being studied, involving the hydrolysis of prolamin proteins to prevent gluten intolerance, as well as the antioxidant and antihypertensive activities of some bread components.

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Nowadays the consumption of sourdough bread is well known to affect the dietary GI, which is lower than that associated with yeasted bread ingestion, as reported in the International Tables of Glycemic Index and Glycemic Load Values47 and asserted by numerous scientific papers.28, 48, 49 The production of organic acids plays an important role in reducing the postprandial glycemic response in human blood. Following the ingestion of bread, starch is usually rapidly digested and absorbed, leading to hyperglycemia in people suffering from insulin-resistance syndrome. The organic acids produced in sourdough are responsible for lowering the GI of bread; acetic acid seems to be associated with a delay in gastric empting, whereas lactic acid induces interactions between starch and gluten during dough-baking and reduces starch availability.33 Contrary to most studies, Korem et al.50 did not find a significant differential effect on multiple clinical parameters or the gut microbiome after the consumption of white bread versus sourdough bread. In particular, they observed a high interpersonal variability in the postprandial glycemic response to bread consumption, concluding that the effect of each type of bread is specific to each person. A significant effect of sourdough bread on in vitro gut microbiota was observed by Costabile et al.51 These authors also highlight the advantages of sourdough bread consumption for patients suffering from irritable bowel syndrome. Cereals are widely used in human diets throughout the world. In genetically susceptible individuals, the ingestion of gluten protein, the primary protein in wheat, rye, barley, and other cereals, causes a chronic inflammatory process leading to lesions in the small intestine and dysfunction in nutrient absorption, known as celiac disease, an immunemediated enteropathy that affects about 1% of the world population.52, 53 Although numerous studies are underway to find a pharmacological treatment, the only solution at the moment is a gluten-free diet. Several attempts have been made to reduce the toxic proteins in bread by enhancing their hydrolysis via microbial enzymes or the native enzymes found in cereals. The capacity of sourdough microorganisms to hydrolyze gluten proteins have been exploited to produce gluten-free wheat flour–based bread, which was found not to be harmful to patients with celiac disease.54, 55 This kind of bread is obtained by fermenting wheat flour with selected strains of lactobacilli and fungal proteases for extended periods of time; the production of such bread requires the use of structural and flavoring additives commonly used in gluten-free breads.55 Another approach to gluten hydrolysis is the fermentation of germinated wheat and rye grains using sourdough microorganisms.56 In this case, it is not the activity of lactobacilli that leads to protein hydrolysis; instead, gluten is extensively hydrolyzed by the native enzymes present in the germinated cereals, and it is also likely that hydrolysis is favored by the lowering of dough’s pH caused by the lactobacilli. Furthermore, whereas the hydrolysis of gluten in rye flour has been demonstrated, in wheat flour, it prevents the rising of the dough. In addition to the various nutritional advantages of sourdough breads, which have been the subject of study for many years now, recent research has demonstrated antioxidant and antihypertensive activities of bread components. An antioxidant named pronyl-L-lysine produced via the Maillard reaction during baking has been identified in bread crust.57 It acts as a monofunctional inducer of glutathione S-transferase, which serves as a functional parameter of an antioxidant chemopreventive activity in vitro. The amount of this antioxidant was found to be higher in sourdough bread than in bread obtained by yeast fermentation, being highly dependent on the pH value.57 Angiotensin I-converting enzyme (ACE) is responsible for increasing blood pressure in humans. Several biopeptides with an ACE inhibitory effect have been found, especially in cheese or dairy products. Rizzello et al.58 showed that selected LAB used during sourdough fermentation of mainly whole-meal flour can synthesize ACE inhibitory peptides and γ-aminobutyric acid, with potential antihypertensive effects.

SUMMARY POINTS • Sourdough bread refers to a leavened bread obtained using dough fermented by LAB and yeast. • Sourdough consists of a spontaneous fermentation process and can undoubtedly be considered the primordial form of bread leavening. Its use was first developed in ancient Egypt. • LAB are mainly responsible for the production of lactic acid, acetic acid, or both, whereas yeasts are mainly responsible for the production of CO2; both are responsible for producing the aromatic precursors of bread. • The most representative species of LAB and yeasts are L. sanfranciscensis and S. exiguus, respectively. • During the last century, the use of sourdough was replaced by baker’s yeast for bread leavening, although consumer demand for sourdough bread has increased in recent years. • San Francisco bread is the most famous sourdough bread currently produced in the United States. • In northern Europe, sourdough is mainly employed in the baking process of rye flour. • In the Mediterranean area, sourdough bread is mainly produced in artisanal bakeries producing traditional breads—for example, PDO breads such as Pane di Altamura and Pagnotta del Dittaino.

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• Durum-wheat flour is used in southern Italy, the Middle East, and Arab countries to produce sourdough for loaf breads and leavened flatbreads. • The use of sourdough in bread-making improves loaf volume and flavor, delays staling, and inhibits the growth of spoilage fungi and bacteria. • Sourdough fermentation has been associated with the health-promoting properties of bread, such as a reduction in the postprandial glycemic response in human blood.

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Korem T, Zeevi D, Zmora N, Weissbrod O, Bar N, Lotan-Pompan M, Avnit-Sagi T, Kosower N, Malka G, Rein M, Suez J, Goldberg BZ, Weinberger A, Levy AA, Elinav E, Segal E. Bread affects clinical parameters and induces gut microbiome-associated personal glycemic responses. Cell Metab 2017;25:1243–53. 51. Costabile A, Santarelli S, Claus SP, Sanderson J, Hudspith BN, Brostoff J, Ward JL, Lovegrove A, Shewry PR, Jones HE, Whitley AM, Gibson GR. Effect of breadmaking process on in vitro gut microbiota parameters in irritable bowel syndrome. PLOS ONE 2014;9(10): e111225. https://doi. org/10.1371/journal.pone.0111225. 52. Catassi C, Gatti S, Fasano A. The new epidemiology of celiac disease. J Pediatr Gastroenterol Nutr 2014;59:S7–9. https://doi.org/10.1097/01. mpg.0000450393.23156.59. Suppl.1. 53. Lamacchia C, Camarca A, Picascia S, Di Luccia A, Gianfrani C. Cereal-based gluten-free food: How to reconcile nutritional and technological properties of wheat proteins with safety for celiac disease patients. Nutrients 2014;6:575–90. 54. Greco L, Gobbetti M, Auricchio R, Di Mase R, Landolfo F, Paparo F, Di Cagno R, De Angelis M, Rizzello CG, Cassone A, Terrone G, Timpone L, D’Aniello M, Maglio M, Troncone R, Auricchio S. Safety for patients with celiac disease of baked goods made of wheat flour hydrolyzed during food processing. Clin Gastroenterol Hepatol 2011;9(1):24–9. 55. Rizzello CG, Curiel JA, Nionelli L, Vincentini O, Di Cagno R, Silano M, Gobbetti M, Coda R. Use of fungal proteases and selected sourdough lactic acid bacteria for making wheat bread with an intermediate content of gluten. Food Microbiol 2014;37:59–68. 56. Loponen J, Kanerva P, Zhang C, Sontag-Strohm T, Salovaara H, G€anzle MG. Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food Chem 2009;57:746–53. 57. Lindenmeier M, Hofmann T. Influence of baking conditions and precursor supplementation on the amounts of the antioxidant pronyl-L-lysine in bakery products. J Agric Food Chem 2004;52:350–4. 58. Rizzello CG, Cassone A, Di Cagno R, Gobbetti M. Synthesis of angiotensin I-converting enzyme (ACE)-inhibitory peptides and γ-aminobutyric acid (GABA) during sourdough fermentation by selected lactic acid bacteria. J Agric Food Chem 2008;56:6936–43.

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C H A P T E R

15 Brewer’s Spent Grain From By-Product to Health: A Rich Source of Functional Ingredients Valentina Stojceska Centre for Sustainable Energy in Food Chains, Brunel University London, College of Engineering, Design and Physical Sciences, Uxbridge, United Kingdom

O U T L I N E Introduction to Brewer’s Spent Grain Characteristics of BSG Preparation of Dry BSG BSG as A Functional Ingredient in Bread-Making Technology DF From BSG, and Its Effect on Bread Quality Bioactive Compounds From BSG and Their Effect on Bread Quality

189 190 191 192 192

Health Benefits of BSG

195

Conclusion

197

Summary Points

197

References

197

195

Abbreviations BSG BU CL LE ME PCE PE

brewer’s spent grain Barbander units Celluclast Lipapan Extra Maxlife 85 Pentopan Mono and Celluclast Pentopan Mono

INTRODUCTION TO BREWER’S SPENT GRAIN There is a recent trend encouraging the exploration of new sources of functional ingredients that could be used for improving the nutritional properties of food products. Many studies have examined the possibility of incorporating cereal by-products as functional ingredients of various foods, including breads, cereal bars, extrudates, and biogases. One of the most popular cereal by-products that originates from the brewing industry is brewer’s spent grain (BSG). Approximately 3.4 million tons of BSG is produced in the European Union annually, of which 0.5 million tons originated from brewers in the United Kingdom. It represents up to 85% of the total residue from the brewing process, which amounts to approximately 20 kg/hL of beer. It is available at a very low cost, and it is traditionally used for landfill and cattle feed. However, new technologies for the use of this valuable by-product as an ingredient in food products are of great interest due to the minimization of environmental impact and risk to human health, benefits for businesses from cheap or no-cost material, and improved value for consumers.

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00015-0

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15. BREWER’S SPENT GRAIN FROM BY-PRODUCT TO HEALTH

FIG. 1 Schematic representation of the process of obtaining BSG from natural barley. Source: Adapted with permission from Mussatto et al.1

Barley

Cleaning/selection

Water (5–18°C)/48 h

Steeping

Malting process

Germination

Humid air (15–21°C)/6–7 days

Drying

Storing

Barley malt

Milling Water/ heating

Adjuncts (optional)

Mashing Liquid fraction Filtration

Brewing process

Wort

Solid fraction

Brewers’ spent grain

The schematic presentation of the brewing process that results in the production of BSG is shown in Fig. 1. Beer-making comprises two separate processes: malting and brewing. Malting is the process in which barley is prepared and soaked in water long enough for germination to begin. Mashing is the brewing process of heating grains mixed with water at controlled temperatures for designated periods of time to activate enzyme activity that converts starches to fermentable sugars (mainly maltose and maltotriose) and no fermentable sugars (dextrin) and degrades proteins, polypeptides, and amino acids.1 This enzymatic conversion is known as wort. The insoluble grain husks are allowed to settle to form a bed in the mash tun, whereas filtered wort is used to produce beer. The residual separation of the barley extract is known as BSG.

Characteristics of BSG BSG is considered a lignocellulosic material rich in hemicellulose (39%), proteins (24%), cellulose (14%), lipids (6%), and lignin (4%).1 It is also rich with phenolic compounds and minerals, such as calcium, iron, magnesium, zinc, and potassium.2 Pentose content (the sum of xylose and arabinose) of BSG has been found to vary between 21.0% and 27.3% dry weight basis (dwb) for oven-dried samples.3 A number of methods have been used to break BSG into its components. One of these is the use of enzymes to break down hemicellulose, including xylanases, β-xylosidases, feruloyl esterases, acetyl esterases, glucuronidases, glucuronoyl esterases, and α-L-arabinofuranosidases.4 BSG represents an untapped resource for obtaining industrially important hydrocolloids, such as arabinoxylans and proteins.5 Because of the potentially good nutritional value of BSG, it has great potential to be used as a novel source of dietary fiber (DF) in new products with full regulatory approval because the brewery process uses materials suitable for human food consumption. 2. FLOURS AND BREADS

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191

Preparation of Dry BSG The most common preservation method used in the industry for drying BSG is oven-drying, with the temperature varying from 60°C to 200°C.6, 7 The other preservation methods used are freezing and freeze-drying.4 Santos et al. studied the effect of oven- and freeze-drying techniques on the composition of BSG, including moisture, protein, fat, ash, and total phenolics, and compared them to frozen samples.3 The results revealed that the different techniques showed little variation in the composition of dried BSG. In a study by Stojceska and Ainsworth,8 a commercial sample of BSG (Joseph Holts Brewery, Manchester, England) (Fig. 2) with a composition of 75% moisture, 1% ash, 4.8% protein, 16% fiber, 2.1% fat, and 1.1% carbohydrate was dried using a reel oven (Teknigas, Sussex, England) at 150°C for 4 h. The source of BSG was based on the brewing of barley and hops and was what remained after extraction of the wort and before fermentation. The composition of dried BSG was 20.30% protein, 53.39% fiber, 8.32% fat, and 10.76% carbohydrate. Table 1 presents the chemical compositions of BSG obtained from different worldwide breweries. The lowest total DF of 5.4% in BSG was reported by Prentice et al.,16 whereas the highest levels of 71.2% and 76%

FIG. 2

Example of BSG obtained from Joseph Holts Brewery, Manchester, England.

Chemical Composition of BSG as Reported in the Literature

TABLE 1 Source

Composition of BSG 9

59% total DF; 24% proteins; 2.4% ash; and 1.6% lipids

10

26%–76% DF; 18.7%–50.7% proteins; and 2.75%–4.73% ash

Kanauci and Agata

Kissell and Prentice 11

47.2% DF; 24% proteins; 11.9% lignin; 10.6% lipids; and 2.4% ash

Kanauchi et al. Santos et al.

3

31% proteins; 16% lignin; 3%–6% lipids; 4% ash; and 1.7%–2.0% phenolics

12

67.2% DF; 16.9% lignin; and 4.6% ash

Silva et al.

13

29.1%–35.8% DF and 26.9%–34.9% phenolics

Ranhotra et al.

8

53.39% total DF; 20.30% proteins; 8.32% fat; and 20.76% carbohydrates

Stojceska and Ainsworth Xiros et al.

14

52% DF; 2.7% starch; 14.2% proteins; 11.5% lignin; 13% lipids; 3.3% ash; and 2% phenolics

15

32% proteins

Zerai et al.

16

Prentice and D’Appolonia

5.4%–51% total DF

€ urk et al.17 Ozt€

60.3%–71.2% total DF; 13%–36% proteins and 2.54%–3.69% ash 18

22%–29% DF; 2%–8% starch; 20%–24% proteins; 13%–17% lignin; and 0.7%–0.9% phenolics

Robertson et al. 19

48.2% DF; 22.1% proteins; and 1.1% ash

Waters et al.

Ktenioudaki et al. 20

Meneses et al.

2

60.5% DF; 20.8% protein, 4.5% fat, 3.2% ash; and 3.3% total starch 40.9% DF; 24.7% proteins; and 4.2% phenolics

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15. BREWER’S SPENT GRAIN FROM BY-PRODUCT TO HEALTH

€ urk et al.17 and Kissell and Prentice,10 respectively. Crude protein levels varied from 13%17 to were reported by Ozt€ 10 50.7%. The composition of BSG apparently depends on the barley variety, time of harvest, characteristics of hops, and brewery technology.3 The application of BSG in bread-making technology as a cheap DF supplement in conventional dough and sourdough breads has been very well documented in the literature.2, 8, 16, 19, 21–23

BSG AS A FUNCTIONAL INGREDIENT IN BREAD-MAKING TECHNOLOGY DF From BSG, and Its Effect on Bread Quality DF has received much attention from nutritionists in recent years as an important ingredient in enhancing human health. It is defined as a food material (particularly plant material) that is not hydrolyzed by enzymes secreted by the human digestive tract, but that may be digested by microflora in the gut. Plant components that fall within this definition include nonstarch polysaccharides (NSPs) such as celluloses, some hemicelluloses, gums, and pectins, as well as lignin, resistant dextrin, and resistant starches.24 Consuming food with a high level of DF has a number of important physiological effects in humans and has been associated with the prevention of several diseases. Adults in the United States consume less than half of the recommended level of 25–30 g DF per day, whereas adults in the United Kingdom (51% of men and 69% of women) fall short of the minimum recommended intake of 18 g per day.19, 25, 26 The addition of 10% of BSG to wheat bread formulation will result in an additional 13.9 g/day of DF, which provides approximately 50% of the recommended daily intake.19 DF intake reduces the risk of chronic heart disease and diabetes, which was associated with the consumption of insoluble cereal fiber, where as a reduction of total and low-density lipoprotein (LDL) cholesterol levels was associated with viscous fibers.26, 27 Similar metabolic effects have been seen with increasing meal frequency or eating lowglycemic-index (GI) foods. Schulze et al.28 examined the association between GI, glycemic load (amount of carbohydrates multiplied by the average GI), and DF and the risk of type 2 diabetes in young women. It was found that higher GI was significantly associated with an increased risk of diabetes, higher carbohydrate intake, and higher glycemic load. Diets with a high GI and low in cereal fiber increase the risk of type 2 diabetes, particularly in women with a sedentary lifestyle and a family history of diabetes. In a review by Slavin,25 a strong positive correlation of DF intake with prevention of obesity was reported, and there was an inverse relationship with body weight, body fat, and body mass index (BMI), probably because the addition of DF generally decreases food intake. It was suggested that DF can control weight through promoting satiation, decreasing the absorption of macronutrients, and altering the secretion of gut hormones. Traditionally, bread is considered a nutritious food rich with carbohydrates, protein, DF, and vitamins, as well as other essential components of the daily diet. To provide more variety in functional breads, different sources of DF have often been used in recipes, such as wheat, barley, oat, rye, and rice bran.29–32 Fiber-supplemented breads show a pronounced decrease in quality parameters, and there is a significant effect on the mixing and viscoelastic properties and fermentation behavior during bread preparation. DF addition increases water absorption, decreases loaf volume, and affects farinograph parameters and shelf life.29, 31 Arabinoxylans are the major high-molecular polymers of cell walls and components that contribute to the DF value in breads. Biliaderis et al.33 studied the functional role of various amounts of arabinoxylans in bread-making technology and found that arabinoxylans affect the textural properties of breads depending on the amounts added, the molecular size of these polymers, and the bread-making quality of base flours. All these changes seem to be a result of “dilution of the gluten network, which in turn impairs gas retention rather than gas production,”34 and “disruption of the starch and gluten matrix and restriction of gas cells to expand in a particular dimension”.35 The addition of BSG to bread dough formulations at the level of 0–30% significantly (P < 0.001) increased the total DF level from 2.3% to 11.5% and fat level from 3.4% to 4.4% (Table 2).8 Protein content varied between 10.7% and 11% and was not related to the addition of BSG. Similarly, Kteundaki et al.22 incorporated BSG at the level of 0–35% into breadsticks and found that the protein level increased from 14.3% to 18.4%, fat level from 0.3% to 2.1%, and DF level from 7% to 27%. Improving the Quality of BSG Breads Good-quality, high-DF breads with acceptable texture and taste are essential; therefore, it is necessary to make adjustments to various process parameters. A number of solutions have been recommended, including presoaking or fermenting bran before it is added to the dough and/or forming sourdough, using enzymes and dough

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BSG AS A FUNCTIONAL INGREDIENT IN BREAD-MAKING TECHNOLOGY

TABLE 2

Nutritional Analyses, Water Absorption, and Farinigraph Characteristics of BSG

BSG (%)

0/0*

Fiber (%)

2.3a

Protein (%) Fat (%) Water absorption (%)

10/15*

20/25*

6.3b

9.7c

30/35* 11.5d

11.0a/14.3a*

10.7c/15.2a*

11.4d/17.9b*

10.9a/18.4b*

3.4a/0.3a*

3.4a/0.8b*

3.8b/1.3c*

4.4c/2.1d*

58.0a/62.3a*

61.0b/70.5b*

60.5b,c/72b,c*

60.0c/73.0c*

Dough-development time (min)

3.5a

7.0b

13.0c

18.0d

Stability (min)

6.5a

9.5a

10.0b

18.0c

25.0a

15.0b

10.0c

5.0d

Degree of softening (BU)

Different letters in the same line indicate statistically significant values (P ¼ 0.05). *Stojceska and Ainsworth8 and Ktenioudaki et al.2, 22

conditioners.21, 23 Apart from textural improvement, sourdough fermentation has a well-established role in improving nutritional properties and flavor. Bran fermentation in water allows enhanced water absorption and textural modification of bran particles, resulting in improved structure of the gluten network and softer breads due to altered water migration between starch, protein, and bran particles during storage.29 Furthermore, the combination of bran sourdough and enzyme mixture is more pronounced in improving loaf volume, the structure of the gluten network, and the shelf life of baked breads, as well as in reducing starch crystallization during storage. Table 2 presents the effect of BSG on farinograph parameters and water absorption. The addition of BSG from 0 to 30% increased the DF level from 2.3% to 11.5%, while the protein level was also affected to some extent. The water absorption increased with fiber addition, varying between 58% and 61% at a fixed dough consistency of 700 Brabander units (BU). Dough-development time increased from 3.5 to 18 min and dough stability time increased from 6.5 to 18 min, whereas the degree of softening decreased from 25 to 5 BU. The mixing properties of BSG’s breads with 0%–35% of BSG replacement were also studied by Ktenioudaki et. al.,2 who reported that the water absorption of the breads varied from 70.7% to 73.5% and were significantly higher than in the control sample. A similar attitude has been seen in terms of dough-development time, resulting in increased time (from 2.5 min for the control sample to 10.5 min for the BSG blends). Table 3 presents the effects of BSG on loaf volume, texture, and shelf life. It can be seen that BSG has affected all those parameters, probably as a result of an increased level of arabinoxylans, the main polymers in BSG. Similar results for BSG breads were reported by Prentice and D’Appolonia16; Finley and Hanamoto,36 and Ktenioudaki et. al.2 The specific loaf volume of BSG breads containing different amounts of fiber varied between 2.06 and 3.22 mL/g, with a significant correlation between fiber content and the resulting loaf volume of r2 ¼ 0.8 (P < 0.0001). The greatest loaf volume reduction was detected at 30% BSG addition. The shelf life of the breads containing different amounts (0%–30%) of BSG and different enzymes was tested at 1, 2, 5, and 8 days. Fiber addition significantly (P < 0.001) increased crumb firmness in samples containing 20% and 30% BSG, whereas no significant difference was found with 10% addition. This significant difference was observed at each day of storage with the 20% and 30% BSG samples. Biliaderis et al.33 reported that the molecular weight of arabinoxylans significantly increased the firmness of crumbs, but the amount of arabinoxylans significantly decreased in wheat-flour breads over 7 days of storage. This could be explained by the fact that cereal brans consist of different tissues, and thus the actual fine structures of isolated arabinoxylans are very diverse.5 Waters et al.19 developed BSG’s breads (0%–20%) and compared them with wheat and whole-meal control samples. It was found that the maximum acceptable level of BSG that could result with good textural and sensorial properties is 10%. The same authors concluded that BSG and BSG’s sourdough are potential replacements for whole-meal breads from the economical, nutritional, and environmental perspectives. Similarly, Ktenioudaki et al.22 found that incorporation of BSG at the level of 0%–35% into breadsticks affects the structure and texture of the final product, resulting in an unappealing cellular structure but an unchanged shelf life. It also was reported that the control sample demonstrated much harder properties, which was apparently related to the crispiness of the products. Furthermore, BSG addition negatively affected the dough rheological properties like biaxial extensional viscosity and uniaxial extensibility, while increasing the storage and loss moduli, representing more solidlike behavior.2 In a study by Stojceska and Ainsworth,8 the texture of BSG breads was improved by the addition of the enzymes Maxlife 85 (ME) (Danisco Ingredients, Denmark), Lipopan Extra (LE), Pentopan Mono (PE), and Celluclast (CL)

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15. BREWER’S SPENT GRAIN FROM BY-PRODUCT TO HEALTH

TABLE 3

Loaf Volume and Texture Information of BSG Breads Hardness (N)

Enzymes

BSG (%)

Specific loaf volume (mL/g)

Day 1

Day 2

Day 3

Day 4

No enzymes

0

3.2

15.1

15.9

21.2

45.6

10

2.7

20.7

18.6

26.1

44.3

20

2.2

33.6

30.9

39.3

77.0

30

2.1

38.5

32.6

37.6

80.2

0

3.4

17.1

21.3

24.4

55.1

10

2.5

26.5

18.1

26.2

44.3

20

2.3

34.3

33.2

31.4

70.0

30

2.4

30.5

30.3

33.3

59.1

0

3.5

11.4

11.9

19.0

34.6

10

3.9

5.3

5.5

8.4

26.1

20

2.8

18.9

16.9

21.8

35.9

30

2.5

19.6

16.7

20.7

44.1

0

4.0

12.3

11.9

20.5

39.1

10

3.7

13.8

13.1

21.8

38.2

20

2.7

28.9

28.5

37.8

67.7

30

2.7

22.1

22.7

32.1

63.5

0

3.6

10.6

11.6

19.2

43.6

10

3.7

13.9

12.4

19.2

45.9

20

3.1

12.4

13.3

20.8

47.4

30

2.4

30.9

22.9

33.7

53.2

ME

LE

PE

PE and CL

CL, Celluclast; LE, Lipapan Extra; ME, Maxlife 85; PE, Pentopan Mono. Adapted from Stojceska V, Ainsworth P. The effect of different enzymes on the quality of high-fiber enriched brewer’s spent grain breads. Food Chem 2008;110:865–72.

(Novozymes, Bagsvaerd, Denmark). The addition of ME to the bread formulations with specific loaf volumes ranged between 2.4 and 3.4 mL/g, resulting in a significant (P < 0.001) decrease as the amount of fiber increased. There was no significant difference compared to the control samples containing different amounts of fiber. The specific loaf volume of samples containing the enzymes LE (2.4e3.9 mL/g), PE (2.7e4 mL/g), and Pentopan Mono and Celluclast (PCE) (2.4–3.9 mL/g) behaved in a similar way, with significantly (P < 0.0001) less specific loaf volume at 20% and 30% of BSG. No significant difference in specific loaf volume was found among those samples at different fiber levels, except at 10% BSG, where LE was significantly (P < 0.001) higher than PE and PCE samples. Comparing these samples (LE, PE, and PCE) with their equivalent control samples and ME samples, all of them showed significantly (P < 0.0001) higher specific loaf volume. ME breads showed a significant (P < 0.001) increase in hardness as fiber increased, whereas no significant difference was found compared with their equivalent control sample. The same trend was observed during each day of storage. LE showed no significant difference in crumb firmness at 0 and 10% BSG, whereas a significant (P < 0.0001) difference was observed at 20% and 30% BSG. Compared with the equivalent control samples, LE gave significantly (P < 0.0001) lower crumb firmness at all levels of BSG (0%–30%) for each day of storage. PE showed a significant (P < 0.0001) increase in crumb firmness as the amount of BSG increased up to 20%, but it decreased at 30% BSG. Compared with the equivalent control samples, the only significant (P < 0.001) decrease in crumb firmness was detected at 30% BSG. Again, this was apparent during each day of storage. PCE showed a significant difference in hardness (P < 0.0001) at all levels of DF. Compared with its equivalent control samples, the only difference was found at 20% and 30% BSG. Bread containing LE, PE, and PCE showed a clear tendency toward a softer crumb and a reduced rate of staling compared with the control samples and samples containing ME. PE

2. FLOURS AND BREADS

HEALTH BENEFITS OF BSG

195

showed significantly (P < 0.0001) increased hardness during storage, compared with LE and PE at 20% BSG. In this study, the best results in terms of crumb firmness were obtained with LE, with a significant (P < 0.001) delay in staling compared with the equivalent control samples.8 The work of Stojceska and Ainsworth was extended by forming sourdough and a combination of enzymes.8, 21 The specific loaf volume of sourdough breads was no different from that of conventional breads.8 The combination of sourdough and different enzymes improved the texture and shelf life of BSG breads, resulting in lower crumb hardness and delay in staling. Compared to conventional BSG breads, sourdough extended shelf life, and this effect was more pronounced with a combination of enzymes.8 The best results were obtained with a combination of LE and PE with ME and PE with CL, which is probably the result of redistribution of water from pentosane to the gluten phase, reduced starch retrogradation rate, and degradation of cell wall components, leading to altered water distribution between starch and protein.29 Later, Ktenioudaki et. al.23 developed BSG breads based on the wheat flour, BSG sourdough, xylanase, and dough conditioner and studied their technological and sensorial properties. They found that a combination of xylanase and dough conditioners significantly improved the specific loaf volume and shelf life of BSG breads, while the combination of sourdough and dough conditioner was more distinct in improving sensorial properties.

Bioactive Compounds From BSG and Their Effect on Bread Quality During the last few decades, other compounds that have received much attention by nutritionists are phenolic compounds. The consumption of foods rich in bioactive compounds has been associated with a number of human health benefits, mainly related to cardiovascular and anticarcinogenic effects. A number of studies proved that BSG is a promising source of phenolic compounds and antioxidants. Various methods and solvents have been widely used to extract phenolic compounds, including saponification, centrifugation, reverse phase—high-performance liquid chromatography (HPLC), and emerging technologies like ultrasound and microwave-derived techniques.7, 35, 37–39 It was reported that the phenolic extracts from BSG are predominantly in bound form, containing different percentages of ferulic and p-coumaric acids. In a study by McCarthy et al.,37 the phenolic compounds extracted from pale and black BSG were used to test their effect on DNA damage of lymphocytic U937 cells. It was found that black BSG is a main contributor to the antioxidant effect and provides greater protection against deoxyribonucleic acid (DNA) damage than pale BSG. Antioxidants from phenolic compounds also act as pro-oxidants under certain conditions, which induce oxidative stress.37 Moreira et al.38 used a microwave method and alkaline conditions to extract phenolic compounds from three types of BSG (pilsen, chocolate, and black) and found that the BSG extracts, especially from pilsen malt, could be used as a cheap source of functional ingredients in the food and pharmaceutical industries. Also, del Río et al.39 used gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) to intact high-molecular-weight lipids such as sterol esters, sterol glycosides or triglycerides from fatty acids. It was found that BSG is a great source of triglycerides, fatty acids, and phytochemicals. Meneses et al.20 used solvents like methanol, ethanol, acetone, hexane, ethyl acetate, and water to extract antioxidant phenolic compounds. It was found that the most efficient way of extracting phenolic compounds was a mixture of acetone and water. Recently, Carciochi et al.7 used a batch system, in combination with ultrasound and microwave techniques, to extract polyphenols. The range of extracted phenolic compounds was from 1.59 to 3.57 mg gallic acid equivalent (GAE)/g, and of antioxidant activity was from 1.86% to 11.93% of 2,2diphenyl-1-picryhydrazyl free radical (DPPH) inhibition. It was reported that Patricelli’s mathematical model proved that the highest rate of phenolic compounds were achieved via ultrasound. Ktenioudaki et al.23 studied the in vitro antioxidant capacity and phenolic composition of BSG flour and breads and found that the level of phenolic compounds was higher in samples containing sourdough, xylanase, and dough conditioner, resulting in 61.3 mg GAE/100 g sample dwb (Table 4). The main phenolic acids identified in the BSG flour were 4-coumaric acid and ferulic acid, followed by procyanidin B1, gallic, quinic, and protocatechuic acids.

HEALTH BENEFITS OF BSG As stated previously, BSG can be used as a functional ingredient for developing animal and human foods with a number of healthy compounds, including (1–3,1–4)-β-D-glucan, arabinoxylans, xylooligosaccharides, and antioxidant phenolics.4, 6, 40, 41 A number of studies proved that BSG may play a role in the prevention of certain diseases. Odes et al.42 examined the role of BSG fiber in the treatment of constipated patients. In the study, 19 ambulatory patients with chronic, laxative-dependent constipation were treated with 20–25 g BSG fiber daily for 4 weeks. The following

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15. BREWER’S SPENT GRAIN FROM BY-PRODUCT TO HEALTH

TABLE 4

In Vitro Antioxidant Activity of BSG Bread Samples Total phenol

Samples

Extract

GAE (mg/100g sample dwb)

DPPH-scavenging activity TEAC (IC50 Trolox/IC50 Sample) × 105

Trolox equivalent (mg/100 g sample dwb)

FRAP assay Trolox equivalent (mg/100 g sample dwb)

FLOUR Wheat

Free

17.1  0.6

8.2  0.1

8.0  0.3

13.3  1.9

Wheat

Bound

15.2  1.8

BSG

Free

52.7  2.6

29.9  0.6

26.5  0.4

80.3  3.3

BSG

Bound

535.8  20.4

454.0  29.6

417.4  17.2

1036.0  89.7

43.4  1.3

20.5  0.4

19.1  0.7

47.4  1.3

130.9  4.3

82.2  9.0

77.0  5.4

267.1  10.5

46.5  0.7

23.5  1.0

21.2  0.9

50.9  0.2

135.2  5.6

86.7  4.5

80.4  0.9

278.0  12.6

47.0  2.0

23.9  0.5

21.7  0.5

58.0  1.6

129.7  1.3

82.5  10.3

77.3  5.5

264.9  9.4

52.3  0.8

22.7  0.6

20.5  0.2

59.2  2.3

132.7  3.0

83.1  9.3

77.6  5.0

269.2  6.3

53.6  1.5

24.3  1.5

21.5  1.2

60.0  3.3

129.1  1.5

88.8  7.0

82.5  2.9

276.6  7.1

61.3  1.3

28.1  1.6

23.9  0.7

74.6  1.0

129.7  5.0

81.4  10.0

76.4  4.5

274.2  18.0

36  2.6

BREADS BSG

Free

BSG

Bound

BSGxyl

Free

BSGxyl

Bound

BSGDC

Free

BSGDC

Bound

BSGSD

Free

BSGSD

Bound

BSGSD +

xyl

Free

BSGSD +

xyl

Bound

BSGSD +

DC

Free

BSGSD +

DC

Bound

BSGxyl, BSG with xylanase addition; BSGDC, BSG with dough conditioner addition; BSGSD, BSG with sourdough addition; BSGSD + xyl, BSG with sourdough and xylanase additions; BSGSD + DC, BSG with sourdough and dough conditioner additions. Adapted from Ktenioudaki A, Alvarez-Jubete L, Smyth TJ, Kilcawley K, Rai DK, Gallagher E. Application of bioprocessing techniques (sourdough fermentation and technological aids) for brewer’s spent grain breads. Food Res Int 2015;73:107–16.

conditions improved with treatment: bowel movement frequency in 15 patients (79%), flatulence in 12 (63%), abdominal pain in 10 (53%), stool consistency in 8 (42%), and laxative dependence in 14 (74%). A total of 15 patients (79%) showed improvement in some or all of those factors, whereas 4 patients were largely unresponsive to fiber. Zhang et al.43 reported a beneficial effect of BSG on the physiological function of the colon. Two diets were studied: a high-fiber diet, with 62 g BSG supplementation in breads, muffins, and breakfast flakes; and a low-fiber diet without BSG supplementation. Two experimental groups of 5 subjects each were studied for 1 week. It was found that the cholesterol and net cholesterol excretion per day on the high-fiber diet were significantly higher than those on the low-fiber diet. BSG fiber intake increased the dry weight of the ileostomy contents and decreased the concentration of sterols in ileostomy effluent. Hassona44 measured the total lipids and cholesterol in rats fed bread containing milled BSG at levels of 10%–25%, with fiber content of 4.9%, 6.4%, and 7.5% for 28 days. The results indicated impaired growth weight (7.1%–10.0%) compared to that of the control group. Total lipids and total cholesterol were reduced by 5.7%–8.0% and 6.0%–8.3%, respectively. Aman et al.45 studied the excretion of total DF in 10 human subjects with ileostomies who consumed a low-fiber diet (15 g total DF/day) or the same diet supplemented with 62 g/day of BSG in a cross-over design study. Food and excreta were collected and analyzed on days 2, 3, and 7 of each dietary period. Analysis of specific DF components showed that the increased excretion was mainly due to fucose, mannose, galactose, and uronic acid residues. High-fiber diet consumption showed significantly greater excretion of the same NSP residues as those for consumption of the low-fiber diet, but with a simultaneous decrease in excretion of arabinose, xylose, and glucose residues (12%, P < 0.01), which were the major fiber components in the diet. In another study, Kanauchi and Agata9 developed a new product by milling and sieving BSG; the mix of glutaminerich protein and DFs of cellulose, hemicelluloses, and lignin protein was called germinated barley foodstuff (GBF). It was

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found that this product increased fecal dry weight and the number of feces and jejunal mucosal protein content in rats, indicating that it might improve defecation for people with constipation. BSG was used as a protein source for a fish diet, replacing 25%, 50%, 75%, and 100% of fish-meal protein during a 10-week period.15 The results showed that brewer’s waste can effectively replace up to 50% of the fish-meal protein in a typical commercial feed with no adverse effect on growth of tilapia, whereas 75% fish-meal replacement showed significantly lower weight gain. Also, del Río et al.39 proved that BSG is a good source of fatty acids, triglycerides, and phytosterols, which could be used as functional ingredients in food for protection against a number of conditions like high blood cholesterol levels or cancer. Mitsuyama et al.46 and Kanauchi et al.11 used GBF to treat ulcerative colitis. It was fractioned from BSG, and 30 g/day was given to the patients in these studies over 4 weeks. The trials resulted in clinical and endoscopic improvement of ulcerative colitis. The treatment is safe and does not require any dietary restrictions. Overall, scientists are in agreement that BSG is an important source of DF, proteins, and phenolic compounds, with a beneficial effect on health. However, further research is needed to establish the precise function of BSG fiber and bioactive compounds on human health.

CONCLUSION Dried BSG has a great potential for use as a functional ingredient that may have beneficial effects on human health. By incorporating BSG up to 30% during bread-making, the level of DF will increase up to fivefold, which significantly contributes to the recommended daily DF intake. BSG is also an excellent source of antioxidant phenolic compounds, triglycerides, fatty acids, and phytochemicals. Loaf volume, texture, and shelf life of BSG breads can be improved by forming sourdough or using dough conditioners and different enzymes. It was proved that BSG has a positive effect on human health and was successful in improving the conditions of patients with constipation, physiological malfunction of the colon, high blood cholesterol levels, cancer, and ulcerative colitis.

SUMMARY POINTS • Consuming a high level of DF food has been associated with the prevention of several diseases. • BSG is a by-product from brewing and a cheap source of total DF and antioxidant phenolic compounds that could be used as functional ingredients in various food products. The addition of BSG to bread dough formulation at 30% increases the level of total DF up to fivefold. • The sensory characteristics and shelf life of BSG breads can be improved by adding enzymes and dough conditioners and by forming a sourdough.

References 1. Mussatto IS, Dragone G, Roberto CI. Brewer’s spent grain: generation, characteristics and potential applications. J Cereal Sci 2006;43:1–14. 2. Ktenioudaki A, O’Shea N, Gallagher E. Rheological properties of wheat dough supplemented with functional by-products of food processing: brewer’s spent grain and apple pomace. J Food Eng 2013;116:362–8. 3. Santos M, Jimenez JJ, Bartolome B, Gomez-Cordoves C, del Nozal MJ. Variability of brewers’ spent grain within a brewery. Food Chem 2003;80:17–21. 4. Lynch KM, Steffen EJ, Arendt EK. Brewers’ spent grain: a r view with an emphasis on food and health. J Inst Brew 2016;122:553–6. 5. Mandalari G, Faulds C, Sancho AI, Saija A, Bisignsno G, LoCurto R, et al. Fraction and characterisation of arabinoxylans from brewers’ spent grain and wheat bran. J Cereal Sci 2005;42:205–12. 6. McCarthy AL, O’Callaghan YC, Connolly A, Piggott CO, FitzGerald RJ, O’Brien NM. Phenolic extracts of brewers’spent grain (BSG) as functional ingredients—assessment of their DNA protective effect against oxidant-induced DNA single strand breaks in U937 cells. Food Chem 2012;134:641–6. 7. Carciochi RA, Carlos A, Sologubik SA, Fernández MB, Manrique GD, D’Alessandro LG. Extraction of antioxidant phenolic compounds from brewer’s spent grain: optimization and kinetics modeling. Antioxidants 2018;7:45. https://doi.org/10.3390/antiox7040045. 8. Stojceska V, Ainsworth P. The effect of different enzymes on the quality of high-fiber enriched brewer’s spent grain breads. Food Chem 2008;110:865–72. 9. Kanauchi O, Agata K. Protein and dietary fiber-rich new food stuff from brewer’s spent grain increased excretion of feces and jejunum mucosal protein content in rats. Biosci Biotechnol Biochem 1997;61:29–33. 10. Kissell LT, Prentice N. Protein and fiber enrichment of cookie flour with brewers’ spent grain. Cereal Chem 1979;56:261–4. 11. Kanauchi O, Mitsuyama K, Araki Y. Development of a functional germinated barley foodstuff from brewer’s spent grain for the treatment of ulcerative colitis. J Am Soc Brew Chem 2001;59:59–62. 12. Silva JP, Sousa S, Rodrigues J, Antunes H, Porter JJ, Gonc¸alves I, Ferreira-Dias S. Adsorption of acid orange 7 dye in aqueous solutions by spent brewery grains. Sep Purif Technol 2004;40:309–15.

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13. Ranhotra GS, Gelroth JA, Torrence FA, Bock MA, Winterringer GL, Bates LS. Nutritional characteristics of distiller’s spent grain. J Food Sci 2006;47:1184–5. 14. Xiros C, Moukouli M, Topakas E, Christakopoulos P. Factors affecting ferulic acid release from brewer’s spent grain by Fusarium oxysporum enzymatic system. Bioresour Technol 2009;100:5917–21. 15. Zerai DB, Fitzsimmons KM, Collier RJ, Duff GC. Evaluation of brewer’s waste as partial replacement of fish meal protein in Nile tilapia, Oreochromis niloticus, diets. J World Aquacult Soc 2008;39:556–64. 16. Prentice N, D’Appolonia BL. High-fiber bread containing brewer’s spent grain. Cereal Chem 1977;54:1084–95. € urk S, Ozboy O, Cavidoglu I, Koksel H. Effect of brewer’s spent grain on the quality and dietary fiber content of cookies. J Inst Brew Distill 17. Ozt€ 2002;108:23–7. 18. Robertson JA, Castro-Marinas L, Collins SR, Faulds CB, Waldron KW. Enzymatic and chemical treatment limits on the controlled solubilization of brewers’ spent grain. J Agric Food Chem 2011;59:11019–25. 19. Waters DM, Jacob F, Titze J, Arendt EK, Zannini E. Fibre, protein and mineral fortification of wheat bread through milled and fermented brewer’s spent grain enrichment. Eur Food Res Technol 2012;235:767–78. 20. Meneses NG, Martins S, Teixeira JA, Mussatto SI. Influence of extraction solvents on the recovery of antioxidant phenolic compounds from brewer’s spent grains. Sep Purif Technol 2013;108:152–8. 21. Stojceska V, Ainsworth P. Improving the textural characteristics of brewer’s spent grain breads by combination of sour dough and different enzymes. In: Waldron KW, Moates GK, Faulds CB, editors. Total food suitability of agri-food chain. Cambridge, UK: Royal Society of Chemistry; 2010. p. 27–31. 22. Ktenioudaki A, Chaurin V, Reis SF, Gallagher E. Brewer’s spent grain as a functional ingredient for breadsticks. Int J Food Sci Technol 2012;47:1765–71. 23. Ktenioudaki A, Alvarez-Jubete L, Smyth TJ, Kilcawley K, Rai DK, Gallagher E. Application of bioprocessing techniques (sourdough fermentation and technological aids) for brewer’s spent grain breads. Food Res Int 2015;73:107–16. 24. Institute of Food Science and Technology. Information statement. Dietary fiber. London: Institute of Food Science and Technology; 2007. Available at: http://www.ifst.org. 25. Slavin JL. Dietary fiber and body weight. Nutrition 2005;21:411–8. 26. Jenkins DJA, Marchie A, Augustin LSA, Rosc E, Kendall CWC. Viscous dietary fiber and metabolic effects. Clin Nutr Suppl 2004;1:39–49. 27. Brown L, Rosner B, Willett WW, Sacks FM. Cholesterol-lowering effects of dietary fiber: a metaanalysis. Am J Clin Nutr 1999;69:30–42. 28. Schulze MB, Liu S, Rimm EB, Manson JE, Willett WC, Hu FB. Glycemic index, glycemic load, and dietary fiber intake and incidence of type 2 diabetes in younger and middle-aged women. Am J Clin Nutr 2004;80:348–56. 29. Katina K, Salmenkallio-Marttila M, Partanen R, Forssell P, Autio K. Effects of sourdough and enzymes on staling of high-fiber wheat bread. LWT—Food Sci Technol 2006;39:479–91. 30. Rakha A, Aman P, Andersson R. Characterisation of dietary fiber components in rye products. Food Chem 2010;119:859–67. 31. Sudha ML, Vetrimani R, Leelavathi K. Influence of fiber from different cereals on the rheological characteristics of wheat flour dough and on biscuit quality. Food Chem 2007;100:1365–70. 32. Wang J, Rosell CM, Barber CB. Effect of the addition of different fibers on wheat dough performance and bread quality. Food Chem 2002;79:221–6. 33. Biliaderis CG, Izydorczyk MS, Rattan O. Effect of arabinoxylans on bread-making quality of wheat flours. Food Chem 1995;5:165–71. 34. Autio K, Laurikainen T. Relationship between flour/dough microstructure and dough handling and baking properties. Trends Food Sci Technol 1997;8:181–5. 35. Gan Z, Ellis PR, Schofield JD. Mini review: gas cell stabilization and gas retention in wheat bread dough. J Cereal Sci 1995;21:215–30. 36. Finley JW, Hanamoto MM. Milling and baking properties of dried brewer’s spent grains. Cereal Chem 1980;57:166–8. 37. McCarthy AL, O’Callaghan YC, Piggott CO, FitzGerald RJ, O’Brien NM. Brewers’ spent grain; bioactivity of phenolic component, its role in animal nutrition and potential for incorporation in functional foods: A review. Proc Nutr Soc 2013;72:117–25. 38. Moreira MM, Morais S, Carvalho DO, Barros AA, Delerue-Matos C, Guido LF. Brewer’s spent grain from different types of malt: evaluation of the antioxidant activity and identification of the major phenolic compounds. Food Res Int 2013;54:382–8. 39. del Río JC, Prinsen P, Gutierrez A. Chemical composition of lipids in brewer’s spent grain: a promising source of valuable phytochemicals. J Cereal Sci 2013;58:248–54. 40. Aliyu S, Bala M. Brewer’s spent grain: a review of its potentials and applications. Afr J Biotechnol 2011;10(3):324–31. 41. Steiner J, Procopio S, Becke T. Brewer’s spent grain: source of value-added polysaccharides for the food industry in reference to the health claims. Eur Food Res Technol 2015;241:303–15. 42. Odes HS, Madar Z, Trop M, Nanir S, Gross J, Cohen T. Pilot study of the efficacy of spent grain dietary fiber in the treatment of constipation. Isr J Med Sci 1986;22:12–5. 43. Zhang JX, Lundin E, Andersson H, Bosaeus I, Dahlgren S, Hallmans G, et al. Brewer’s spent grain, serum lipids and fecal sterol excretion in human subjects with ileostomies. J Nutr 1991;121:778–84. 44. Hassona HZ. High fiber bread containing brewer’s spent grains and its effect on lipid metabolism in rats. Nahrung 1993;37:576–82. 45. Aman P, Zhang JX, Hallmans G, Lundin E. Excretion and degradation of dietary fiber constituents in ileostomy subjects consuming a low fiber diet with and without brewer’s spent grain. J Nutr 1994;124:359–63. 46. Mitsuyama K, Saiki T, Kanauchi O, Iwanaga T, Tomiyasu N, Nishiyama T, Tateishi H, Shirachi A, Ide M, Suzuki A, Noguchi K, Ikeda H, Toyonaga A, Sata M. Treatment of ulcerative colitis with germinated barley foodstuff feeding: a pilot study. Aliment Pharmacol Ther 1998;12:1225–30.

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C H A P T E R

16 Effect of Addition of Thermally Modified Cowpea Protein on Sensory Acceptability and Textural Properties of Wheat Bread☆ Lydia Campbell*, Stephen R. Euston*, and Mohamed A. Ahmed*,† †

*School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland Department of Food Technology, Faculty of Engineering and Technology, Sebha University, Sebha, Libya

O U T L I N E Introduction

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Materials and Methods Materials Methods

201 201 201

Results and Discussion Proximate Analysis

205 205

Effects of Thermal Modification of CPI on Its Physicochemical Properties Dough and Bread Properties

205 206

Conclusions

207

Acknowledgments

208

References

208

INTRODUCTION There has been growth in the global market volume of plant protein ingredients of 1.7 million metric tons per year; this is dominated by wheat (gluten) and soy protein. The growth is limited for other plant proteins owing to a lower score on sensory properties based on a Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis.1 Shortages and high prices have recently caused restriction of animal proteins in the diets of many people living in developing countries; therefore, vegetable proteins that are cheaper and more available have great potential as food for humans. Many vegetable proteins require processing to provide food material that has acceptable organoleptic properties for human consumption.2 Breads fortified with protein isolates from soy, peas, lupins, and chickpeas have received considerable interest.3–5 Common disadvantages reported, however, are increased hardness of crumbs and decreased loaf volume. The recent interest in using plant proteins for food has led to the evaluation of cowpeas as a nutritional and economical source of protein. Currently, cowpea use is limited primarily to boiled whole seeds or traditional food preparation. The primary limitation to the improvement of cowpea proteins is lack of information on the nutritional properties of the cowpea ☆

Permission was obtained to copy the article published in Food Chemistry 194 (2016) 1230–1237: Effect of addition of thermally modified cowpea protein on sensory acceptability and textural properties of wheat bread and sponge cake.

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00016-2

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© 2019 Elsevier Inc. All rights reserved.

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16. ADDITION OF THERMALLY MODIFIED COWPEA PROTEIN ON SENSORY ACCEPTABILITY AND TEXTURAL PROPERTIES OF WHEAT BREAD

protein isolate (CPI), in comparison with well-established soy protein isolate. More important, data on the evaluation of the functional properties of CPI and modification of the protein structure for enhancing its functional properties are scarce. Therefore, the major purpose of this study has been focused on the isolation and characterization of cowpea proteins, as well as the functional properties and evaluation of the resultant products for application in bread. The feasibility of glycation and/or denaturation to improve the functional properties of the proteins is also included. Cowpeas (Vigna unguiculata L. Walp), also known as black-eyed peas, southern peas, or crowder peas, are mainly cultivated in African countries with up to 8 million ha of crops annually, as estimated by the Food and Agricultural Organization (FAO).6 The legume is processed and consumed in a variety of ways, depending on traditional practices and taste preferences. It has a high protein content (18%–35%), with an amino acid profile high in lysine but low in sulfur-containing amino acids, and can complement wheat cereal, which is low in lysine and high in sulfur-containing amino acids, enabling adherence to the daily recommended dietary allowance (RDA) of essential amino acids for adult humans.7 Cowpeas are also high in carbohydrates (50%–65%) and B vitamins such as folic acid, niacin, and riboflavin, which confirms their excellent nutritional profile.7 The development of a beanlike off-flavor when cowpea flour is used as a protein supplement in biscuits was noted,8 which could be one of the reasons for its limited exploitation in food markets outside Africa. The functional properties of food proteins, such as emulsification, foaming, and water retention ability, can be enhanced by controlled thermal processing.9, 10 For example, denatured whey protein was shown to improve the baking performance and texture of wheat bread dough.11 Protein denaturation involves the disruption of electrostatic, hydrophobic, hydrogen, and ionic bonds responsible for maintenance of the secondary and tertiary structure, as well as formation of covalent disulfide bonds. Mild thermal treatment leads to unfolding of the proteins, resulting in an intermediate molten globule state that is often associated with improved functionality, whereas extensive thermal treatment leads to coagulation of protein and loss of solubility and other functional properties.9, 10 Protein functional properties can also be enhanced by thermal treatment in the presence of sugars, oligosaccharides, or carbohydrates by the Maillard reaction.12–14 The reaction is initiated by a covalent carbonyl-amine reaction between reducing sugars and protein and the formation of melanoidins in the later stages that are responsible for the development of brown color and flavor compounds.15 Adverse health-related issues have been attributed to this reaction, such as nutritional loss in proteins (e.g., lack of bioavailability of lysine).15 Recent studies have revealed that the reduced digestibility of Maillard reaction products (MRPs) may have a positive effect by acting as a prebiotic in the gut and reducing allergenicity to products such as cows’ milk.16 The rate of formation of MRPs is determined by the water activity (aw), which is calculated by dividing the partial vapor pressure of water in a substance by the standard-state partial vapor pressure of water (1.0). Most reactions aimed at improving protein functionality are carried out in dry conditions of low aw of 0.3–0.7 at controlled humidity, which favor the reaction, and at temperatures below the denaturation temperature of globular proteins.15 Philips et al.17 investigated the effect of thermal treatment of cowpea flour at 10% moisture content on functionality and reported reduced solubility for cowpea protein. Limited information is available on the development of MRPs by thermal treatment of plant proteins and natural carbohydrates in solution at temperatures that favor protein denaturation. Pinyawiwatkul et al.18 investigated the effect of boiling on the functionality of cowpea flour and reported decreased solubility of protein. Zhu et al.19 reported the formation of whey protein isolate-dextran conjugates with improved functionality by thermal treatment in solution, but heat treatment was below the denaturation temperature of whey protein. Gu et al.20 reported enhanced water-binding properties of acidified soy protein gels owing to combined controlled denaturation and glycation of soy protein isolate with lactose and glucose in solution. The aim of this study was first, to investigate the effect of denaturation and glycation of CPI on its physicochemical properties; and second, to evaluate the effect of fortification of flour with these modified proteins on the texture and sensory properties of yeast bread. Two types of thermally treated cowpea protein were investigated. In the first instance, the native protein was isolated from cowpea flour (to create CPI) and subsequently denatured by thermal treatment (to create denatured cowpea protein isolate, or DCPI). In the second instance, protein was denatured in the presence of its natural sugars in a cowpea flour suspension and subsequently isolated (to create glycated cowpea protein isolate, or GCPI). The physicochemical properties of isolates that were determined were turbidity, solubility, denaturation degree based on free and total sulfhydryl groups, and glycation degree based on the reduction of free amino groups. The presence of glycated sugars was confirmed by staining glycated proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The functionality in bread dough was measured as the effect on dough volume and water absorption. The texture of bread dough and hardness of the bread were measured. Sensory evaluation was conducted by 20 panelists based on a 9-point hedonic scale.

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MATERIALS AND METHODS Materials A total of 6 kg of cowpea seeds were obtained from a store (Bismallah, in Edinburgh). Soft wheat grains (Robigus) were supplied by W. N. Lindsay Ltd., Tranent, Scotland. The yeast (Allinson’s baking yeast) and the rest of the ingredients came from a commercial market. Amyloglucosidase, α-amylase, glucose oxidase peroxidase, and sodium acetate were obtained from Sigma-Aldrich Company (Dorset, UK). All buffers and molecular markers for SDS-PAGE analysis were purchased from Bio-Rad (Watford, UK). GelCode Glycoprotein Staining Kit was obtained from Fisher Scientific (Loughborough, UK).

Methods Preparation of Defatted Cowpea Flour Cowpea flour samples were obtained by milling the beans in a DLFU-mill from Buhler-Miag (Braunschweig, Germany). The flour samples were sieved with a 600-μm screen and defatted by extraction with cold acetone for 1 h at 4°C (with a flour/solvent ratio of 1:3 w/v), according to the procedure described by El-Adawy.21 The resulting slurry was centrifuged at 5000
g for 10 min at room temperature, and the pellet was freeze-dried overnight, finely ground in a coffee grinder, and stored in an airtight container at 5°C, ready for further use. Preparation of CPI and DCPI Preparation was carried out by a slight modification of the method described by El-Adawy.21 Defatted cowpea flour was mixed with a 10-fold quantity (w/v) of distilled water at room temperature. The pH of the mixture was adjusted to 10 with 2N NaOH and stirred gently for 1 h. The mixture was centrifuged (5000
g) for 30 min at room temperature to remove the starch and fiber fractions. The pH of the supernatant was adjusted to 4.5 by dropwise addition of 2N HCI while stirring. The precipitated proteins were centrifuged at (5000
g) for 30 min at room temperature, washed twice with water, resuspended in water, and neutralized to pH 7 with 0.1 N sodium hydroxide (NaOH). To denature the resulting protein isolate, 500 mL of protein solution (10 mg/mL) contained in a 2-L glass bottle with a closed screw cap, was heat-treated at 85°C for 2 h in a shaking water bath at 85°C  3°C and subsequently cooled to room temperature by immersion of the bottle in cold water. This sample was freeze-dried and labeled as DCPI. To determine the effect of heating time on solubility and turbidity, the experiment was repeated; however, this time, 100-mL aliquots were removed after 30, 60, 90, and 120 min by opening the screw cap, decanting 100 mL into a 250-mL glass bottle on ice, closing the cap on the 2-L bottle, and returning it to the hot-water bath. Finally, the four 250-mL bottles containing 100 mL CPI solutions that had been heat-treated at different temperatures were closed with screw caps and stored at 4°C overnight for subsequent determinations of solubility and turbidity. Preparation of DCPI and GCPI A 2-L dispersion of defatted cowpea flour sample in distilled water (5% w/v) was adjusted to pH 10 with 2 N NaOH and stirred for 1 h at room temperature. One 500-mL sample was removed to serve as a nonmodified control. The remaining suspension was poured into a 5-L bottle, sealed by closing the screw cap, and heated for 2 h in a shaking water bath at 85°C  3°C. Aliquots of 100 mL were removed after 30, 60, 90, and 120 min by opening the screw cap, decanting 100 mL into a 250-mL glass bottle on ice, closing the cap on the 5-L bottle, and returning it to the hot-water bath. The four 250-mL bottles containing 100 mL of cowpea flour solutions that had been heat-treated at different temperatures were closed with screw caps and stored at 4°C. After 2 h of heat treatment, the remaining 1100 mL of soy flour solution was cooled to room temperature by immersion of the bottle in cold water. To isolate the glycated and denatured protein, the samples (including the nonheated control) were centrifuged (5000
g) for 30 min at room temperature to remove the starch and fiber fractions. The pH of each supernatant was adjusted with 2N HCl to 4.5 to precipitate the proteins; the precipitated proteins then were centrifuged at (5000
g) for 30 min at room temperature, washed twice with water, resuspended in water, neutralized to pH 7 by 0.1 N NaOH, and freeze-dried. The samples are referred to as GCPI.

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Proximate Analyses Proximate analyses of the flour and bread loaves were carried out using official AOAC methods22 for moisture (14.004), crude fat (14.081), crude fiber (7.0006), ash (14.006), and crude protein (47.021). A nitrogen-to-protein conversion factor of 6.25 was used in the crude protein determination. All the analyses were conducted in triplicate. Determination of Starch Content in Cowpea Flour The starch content in cowpea flour was determined according to AACC method No. 76-11.23 Triplicate samples of cowpea flour dispersions (10 mg/mL) were prepared in 25 mL of ethanol (80%). Thermo-stable owpea flour dispersions (1added to dispersions and vortexed. The mixtures were incubated for 6 min at 50°C with occasional shaking. Amyloglucosidase (20 units) and 25 mL of sodium acetate buffer (200 mM, pH 4.5) were added, and the mixtures were incubated at a temperature of 50°C for 30 min. The mixtures were transferred from the tubes to 100-mL volumetric flasks, and the volumes were adjusted with water and mixed thoroughly. Duplicate samples of 10 mL were centrifuged at 5000
g for 10 min. Triplicate aliquots (0.1 mL) of supernatant of each sample were transferred to test tubes. To prepare a standard curve 0.1 mL aliquots containing 20–60 μg of d-glucose standard solution (0.4 mg/mL) were added to test tubes. Glucose oxidase peroxidase reagent (GOPOD, 2 mL) was added to the blank, glucose standards and samples and incubated at 50°C for 20 min. A Genesys 6 Spectrophotometer (Thermo Spectronic, USA) was used to measure the absorbance at 510 nm. The percentage of starch was calculated as follows: %Starch ¼ 2:25
M=ðVo
E
MSÞ where E is the weight (g) of the sample, M is the weight (μg) of D-glucose obtained from the standard curve, Vo is the volume (mL) from 100-mL flasks, and MS is the percentage dry weight of the sample. Determination of Sugars (Monosaccharides, Disaccharides, and Oligosaccharides) in Cowpea Flour Sugars were extracted from 10 g of cowpea flour using 10 volumes of hot ethanol (80% w/v), as described by Ofuya,24 with modifications. The mixture was put in shaking water bath (60°C) for 1 h. After extraction, the sample was centrifuged for 20 min at 2000 rpm, filtered, and concentrated to 2 mL under vacuum using a rotary vacuum evaporator at 60°C, made up to 4 mL with deionized water, and redistilled again. This was repeated three times to obtain an ethanol-free extract. The extract was deproteinized with 10% lead acetate (24 drops/6 mL) and filtered. The sample was treated with saturated monopotassium phosphate (2 drops) to eliminate excess lead and filtered and stored at 0°C. The sample preparation was repeated three times, and each preparation was analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) using a Carbopac PA-100 column with water as the eluent, at a flow rate of 1 mL/min at 30°C. A standard mixture of sugar compounds consisting of glucose, fructose, sucrose, raffinose, and stachyose spiked with a known concentration of 2-deoxyribose as the internal standard (IS) was resolved. Calibration graphs, based on corrected peak area ratios of analyte over IS, were established for each sugar compound. The concentrations of sugars in the extracts were determined by comparing the peak areas for unknown samples to the calibration graphs of the respective standard. The percentage of each sugar in cowpea flour was calculated by correlating the results with the extraction volume and original sample weight. Determination of Sugar Content of Protein Isolates The total sugar content of CPI, DCPI, and GCPI was determined by a spectrophotometric method using an anthrone reagent, as described by Sharma and Sangha.25 Triplicate samples of CPI (100 mg) were hydrolyzed with 5 mL of 2.5 N hydrocholoric acid (HCl) in a boiling-water bath for 3 h and cooled to room temperature. The mixtures were neutralized with sodium carbonate (Na2CO3), the volumes were brought to 100 mL with water, and then the mixtures were centrifuged. A total of 1 mL of supernatant was removed and diluted to 10 mL with water. The anthrone reagent was freshly prepared before use by mixing 0.2 g of anthrone with 100 mL of ice-cold 95% sulfuric acid (H2SO4), protected from light in a dark bottle, and used within 10 h. Anthrone reagent (4.0 mL) was added to each tube of standard glucose (2 mL) and test solution protein samples (2 mL). Tubes were heated for 8 min in a boiling-water bath and then cooled, and the absorbance at 630 nm was measured against a reagent blank in triplicate. The total sugar content was calculated using a standard calibration curve of glucose. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was carried out according to the procedure described by Wu and Hojilla-Eva,26 using precast native PAGE 10%–20% Tris-glycine gradient gel in an electrophoresis unit (XCell Surelock Mini Cell, Invitrogen Life Technologies, Paisley, UK), at a constant voltage of 180 V for approximately 45 min. Samples (2 μg protein/μL) were

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203

prepared in a nonreducing sample buffer (120 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, and 0.008% bromophenol blue; meanwhile, a reducing sample buffer was prepared by the addition of 1% β-mercaptoethanol. The running buffer was a 10x SDS-PAGE buffer (1% SDS, 0.25 M Tris-HCl, and 1.92 M glycine). The molecular-weight markers were Plus2 prestained (1
), with a MW range of 4–250 kDa. To visualize the protein bands, the gels were immersed for 1 h in a solution consisting of 0.1% Coommasie Brilliant Blue R250, 10% acetic acid, and 50% methanol, followed by destaining in 10% acetc acid and 50% methanol. A GelCode Glycoprotein Staining Kit was used to conduct the glycoprotein staining of the SDS-PAGE gels. The separated protein was fixed by immersing the gel in 50% methanol for 30 min, washed twice with 3% acetic acid for 10 min, transferred to the oxidizing solution, and gently agitated for 15 min. The gel was washed three times with 3% acetic acid for 5 min before transferring to the GelCode Glycoprotein Staining Reagent. The gel was incubated for 5 min with the reducing solution before being washed with 3% acetic acid, and then again with water. Glycoproteins appeared as magenta bands. Turbidity Measurements of Heat-Treated Proteins A sample of CPI (10 mg/mL) and control for GCPI (10 mg/mL) were heat-treated at different time intervals at 85°C. Next, 1-mL aliquots of the cooled samples were transferred to glass cuvettes, and turbidity measurements were carried out at a wavelength of 600 nm, as described by Tay et al.,27 using a Genesys 6 spectrophotometer. Determination of Protein Solubility The CPI (10 mg/mL) and GCPI (10 mg/mL) samples treated at different heating times at 85°C (as described previously in sections “Preparation of CPI and DCPI” and “Preparation of DCPI and GCPI”) were centrifuged at 10,000
g for 30 min. The protein content of the supernatant was determined by the Bradford procedure.28 The percentage of protein solubility was expressed as

Solubility ð%Þ ¼ protein in supernatant mg=mL =initial protein mg=mL
100 Determination of Degree of Glycation A spectrophotometric assay was used to measure the free amino groups of 5% (w/v) solutions of CPI, DCPI, and GCPI at pH 7 by the ortho-phthaldialdehyde (OPA) method described by Achouri et al.13 The OPA reagent was freshly prepared before use by mixing 40 mg of OPA (dissolved in 1 mL of ethanol), 1.905 g of disodium tetraborate decahydrate, and 0.05 g of SDS in 40 mL of water. The volume of the solution was brought to 50 mL with water, and 2.35 mL of 2-mercaptoethanol was added. Then, 1 μL of the protein sample was added to 1.8 mL of OPA reagent and allowed to stand for 5 min at room temperature. The absorbance was measured at 340 nm. A calibration curve for leucine was obtained by preparing standards with concentrations of 0.25–2mM. Three replicates were performed for each measurement. The glycation degree (GD) was expressed as the concentration of free amino groups in the sample (μg/μL) relative to the leucine standard curve. Determination of Free and Total Sulfydryl (SH) Groups The sulfydryl (SH) content of 5% w/v solutions of CPI, DCPI, and GCPI was determined by a colorimetric assay using 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), as described by Campbell et al.10 Free SH groups (SHF) were determined by addition of 3 μL of the sample to 5 mL of a Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M Na2EDTA, pH 8), followed by the addition of 2 μL of a 0.2M DTNB buffer. The solution was vortex-mixed and allowed to react at room temperature for 15 min before measuring the absorbance at 412 nm. The blank for each measurement was a sample prepared using the described procedure, but omitting the DTNB. For determination of the total SH group (SHT), a 3-μL sample was added to 5 mL of Tris-glycine buffer pH 8 containing 6 M urea and 0.5% SDS. Subsequently, 2 μL of 0.2 M DTNB buffer was added and absorbance at 412 nm was measured. The percentage of denaturation was calculated as %denaturation ¼ ðFree SH=Total SHÞ
100 Preparation of Bread Loaves The bread was prepared by the straight dough method 10-10B of AACC,29 using the following recipe: Flour 60%, baker’s yeast grains 0.6%, sugar 1.8%, salt 0.6%, unsalted butterfat 3.8%, ascorbic acid 75 ppm, water 33.2%. Dough was prepared from wheat flour or wheat-cowpea composite flour. Wheat-cowpea composite flour was prepared by mixing freeze-dried powders of CPI, DCPI (heat-treated at 85°C for 2 h), or GCPI (heat-treated at 85°C for 2 h)

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with wheat flour at 2%, 4%, and 6% (w/w). The ingredients were mixed together in a food mixer (Breville, SHM2, Sydney, Australia) and kneaded at setting 2 for 5 min, followed by incubation at 30°C for 50 min to allow the yeast to begin the fermentation process. Each dough preparation was rolled into a ball, put into a bread pan, covered, and allowed to rise at 35°C for 30 min. The risen dough preparations then were baked at 200°C for 30 min in a Russell Hobbs 14552 Mini Oven and allowed to cool for 1 h before loaf volume and texture determinations were carried out. Measurement of Increase in Bread Dough Volume The increase in dough volume during fermentation was determined according to the procedure described by Amita et al.30 After mixing, the dough was placed into a graduated beaker and allowed to rise for 60 min at 28°C. The height of the dough was measured on the graduated surface of the beaker before and after fermentation, and the net increase in volume was calculated. Dough rise was calculated as follows:
Dough rise ¼ increase in dough height=initial height of the dough
100 Measurement of Bread Volume and Texture The volume of bread and cake loaves was determined by the rapeseed displacement method described by Giami et al.,31 using sesame seeds in place of rapeseeds. The loaf was weighed and placed in a 2-L container. The sesame seeds in a measuring cylinder were poured over the loaf in the box and leveled with a spatula. The volume of the spilled sesame seeds was noted as the volume of the loaf. For bread, the specific loaf volume (SLV) was calculated as cubic centimeters per gram (cm3/g). All measurements were done in triplicate. The texture of the bread dough was measured with a Zwick/Roell type Z010 texture analyzer. The texture of the yeast bread dough was measured as described by Autio et al.32 A round plastic box with an inner diameter of 68 mm and a height of 20 mm was filled with the dough, and the expelled dough was carefully trimmed with a knife to achieve an even surface. Dough was compressed with a plunger (sample area, 30 mm2) at 50% depth and rate of 2 mm/s and the force as a function of time was recorded. The maximum force was taken as a hardness value and the average of 10 replicates was reported. The crumb hardness of baked bread was determined after cooling for 1 h based on method 74-09 of AACC.29 The slices were cut from the middle part of the loaf (without the heel slices of the loaf ), and the tests were done on square samples of 60 mm length and width and 40 mm height. The samples were compressed twice with a plunger (sample area, 30 mm2) at a range of 40% depth and rate of 2 mm/s. At a compression of 25%, the compression force value (CFV) was measured for each sample and the force as a function of time was recorded. Measurements were done in triplicate. Measurement of Water Absorption To determine the absorption of water by flour or composite flour in the dough during mixing, analyses were conducted according to AACC standard method 88-04,22 with some modifications. A total of 5 g of each dough sample was vortex-mixed with 30 mL of water in a centrifuge tube for 2 min, allowed to stand at room temperature for 45 min, and centrifuged at 10,000 rpm for 20 min. The supernatant was carefully decanted, and the weight of each pellet was noted. The water absorption was expressed as grams of water absorbed by the percentage of flour or composite flour in 100 g of dough sample (60.7%). Sensory Evaluation Sensory evaluation was performed 24 h after baking to evaluate the overall acceptability of the bread and cake samples. The samples were sliced into pieces of uniform thickness and served with water. Then 20 panel members (familiar with the quality attributes of local bread) were randomly selected from students of the School of Life Sciences, Heriot Watt University, Edinburgh, Scotland. The panelists evaluated the bread and cake samples on a 9-point hedonic scale33: 9 ¼ liked extremely, 8 ¼ liked very much, 7 ¼ liked, 6 ¼ liked mildly, 5 ¼ neither liked nor disliked, 4 ¼ disliked mildly, 3 ¼ disliked, 2 ¼ disliked very much, 1 ¼ disliked extremely. Samples were presented to a panel of judges with a 3-digit code. Statistical Analysis All measurements were carried out in triplicate. Calculation of the significance of differences in test results was performed by the F-test and the least significant difference (LSD) test.34

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RESULTS AND DISCUSSION

RESULTS AND DISCUSSION Proximate Analysis Table 1 gives the proximate analysis of defatted cowpea flour and the protein isolates. The protein content for cowpea flour was 26.14%. The protein content for CPI was 90.75%, and the result was not much different for GCPI (90.02%). The yield of protein isolation was 89% for CPI and was not significantly different for GCPI (results not shown). The proximate composition for DCPI is the same as for CPI, as it is the heat-treated version of the latter. The carbohydrate associated with CPI (4.39%) could be the residual sugars remaining after isolation of protein during preparation of CPI. As the GCPI was prepared by heat treatment of cowpea flour before isolation, the higher carbohydrate content (5.93%) compared to CPI could be due to glycation that occurred with inherent reducing sugars and oligosaccharides, such as raffinose and stachyose present in the cowpea flour. The results of starch and sugar analysis of cowpea flour were 40.55% starch and 0.30%, 0.45%, 1.70%, 0.74%, and 2.67% for glucose, fructose, sucrose, raffinose, and stachyose, respectively. The data are consistent with those reported by Mwangwela.35

Effects of Thermal Modification of CPI on Its Physicochemical Properties Fig. 1 depicts the solubility and turbidity profiles of CPI and GCPI as a function of heating time at 85°C. It is evident that the decrease in solubility for CPI during heat treatment (resulting in DCPI) is higher than that for GCPI. Correspondingly, the increase in turbidity is higher for DCPI than for GCPI. As turbidity measurement at 600 nm is an indicator of protein denaturation,36 these results indicate that CPI is more susceptible to thermal denaturation than GCPI during heat treatment, which is attributed to higher carbohydrate content of GCPI (Table 1). When heated with proteins, reducing sugars first protect the proteins against denaturation, resulting in reduced aggregation compared to protein heated in the absence of sugars, as demonstrated for whey and soy protein.37, 38, 20 Second, reducing sugars might alter the protein’s net charge by the glycation reaction, resulting in a reduced increase in hydrophobicity compared to protein heated in the absence of sugars, as demonstrated for soy protein,13 leading to maintenance of solubility. TABLE 1

Proximate Analysis of Cowpea Flour and Protein Powders Components (g/100 g)

Product

Moisture

Protein

Lipid

Total fiber

Ash

Cowpea flour

7.06  0.22

26.14  0.13

1.03  0.04

2.19  0.02

2.61  0.02

56.33  2.12

4.4  0.11

90.76  0.56

0.44  0.04

1.07  0.05

1.35  0.03

4.38  0.04

4.69  0.12

90.02  0.11

0.5  0.02

1.02  0.05

1.29  0.01

5.93  0.07

CPI GCPI

100

FIG. 1 The effect of heat treatment at 85°C on the solubility and turbidity of CPI and GCPI. Error bars indicate the standard deviation of triplicate measurements.

0.6

95

0.5 0.4

80

0.3

75 0.2

70 65

A 600 nm

Protein solubility %

90 85

GCPI (protein solubility %)

CPI (protein solubility %)

0.1

GCPI (turbidity)

1.6E-15

CPI (turbidity)

60 55 50 0

30 60 90 Heating time (min)

Carbohydrate

–0.1 120

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16. ADDITION OF THERMALLY MODIFIED COWPEA PROTEIN ON SENSORY ACCEPTABILITY AND TEXTURAL PROPERTIES OF WHEAT BREAD

TABLE 2

Free Amino groups, Free SH groups, and Total SH Groups of 10 mg/mL of DCPI and GCPI That Had Been Heat-Treated at 85°C for 2 h

Protein sample

Free NH (μg/μL)

Free SH (μmol/g)

Total SH (μmol/g)

CPI

2.16  0.16

a

0.98  0.09

1.54  0.08a

DCPI

1.80  0.17b

0.66  0.05c

1.06  0.07c

GCPI

1.10  0.11c

0.80  0.06b

1.36  0.09b

a

The control CPI has not been heat-treated. Means in the same column that are not followed by the same superscript letter are significantly different (P  0.05).

200 116 97 66

FIG. 2 SDS-PAGE of CPIs stained with Commassie Blue (left) and stained with GelCode Glycoprotein Staining Kit (right). Left: Lane 1 ¼ CPI (nonreduced); Lane 2 ¼ GCPI (nonreduced); Lane 3 ¼ CPI (reduced); Lane 4 ¼ GCPI (reduced).

The values of free and total sulfydryl groups are shown in Table 2. The free SH group (SHF) value is an indication of the degree of unfolding (denaturation) of protein; an increase indicates moderate unfolding associated with the molten globule state and is usually associated with improved functional properties such as water binding and emulsification.9 The total thiol (SHT) groups are measured using a buffer that contains urea and SDS to dissociate protein aggregates and fully denature the protein, thereby exposing all free SH groups that otherwise could be masked by hydrophobic and electrostatic interactions. A reduction in SHT indicates the formation of disulfide bonds and protein coagulation, which is associated with insolubility and loss of functionality.9 Free SH (SHF) decreased more for DCPI than for GCPI compared to the control (CPI), and it corresponds to the same trend for total SH (SHT), as depicted in Table 2. This indicates that the higher carbohydrate content of GCPI has partially protected the protein from disulfide bond formation and extensive thermal denaturation. Similar results were reported for denatured and glycated soy and whey protein isolate.19, 20 The decrease in free amino groups due to glycation reaction with reducing sugars was 16.7% for DCPI compared to CPI, indicating that a reaction occurred with the associated sugars during heat treatment of CPI (Table 2). The reduction in amino groups was 24.32% for GCPI, indicating a significantly higher glycation degree (Table 2), attributed to the Maillard reaction with reducing sugars and polysaccharides present in the cowpea flour during its preparation. The results correspond to values of sugar analysis, which were 4.3  0.2 mg/100 mg protein for CPI and 6.1  0.2 and mg/100 mg protein for GCPI, confirming the carbohydrate values in Table 1. Further evidence of glycation is seen in the SDS-PAGE analysis combined with staining for glycated proteins (Fig. 2). The left and right images are stained by different methods. The image on the left shows the protein pattern by Coomassie Blue staining of CPI, ranging from 36 to around 116 kDa, with major bands at 36 and 50 kDa. These correspond to the typical molecular mass of vicilin 7S globulin and legumin-like 11S globulin.39 The image on the right reveals higher-molecular-weight bands around 50 kDa for the GCPI (lanes 2 and 4) compared to CPI (lanes 1 and 3), suggesting the formation of MRPs in GCPI. Glycation with sugars would increase the size of protein and decrease their electrophoretic mobility in the gel.

Dough and Bread Properties The dough properties of wheat plus cowpea protein composite flours are presented in Table 3. Dough containing CPI was significantly harder than the control, made with only wheat flour. Dough hardness decreased with DCPI and was the softest for GCPI, which correlates with values for water absorption. CPI dough had significantly lower water

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CONCLUSIONS

TABLE 3

Properties of Bread Containing Cowpea Protein That Has Been Added at Different Concentrations to Wheat Flour CPI

DCPI

GCPI

Concentratio ns of CPI added (%)

Control 0

2

4

6

2

4

6

2

4

6

Water absorption (%)

55.70d

50.69d

54.70e

54.43e

56.49c

56.98b

57.31b

57.22b

58.1a

58.32a

Dough rising (%)

143a

140b

133d

128d

140b

134d

131d

141b

136c

132d

Dough force (g)

137.5c

178b

187b

219a

171b

149c

158c

140c

145c

150c

Crumb hardness (g)

210c

248ab

255a

268a

227bc

236bc

246ab

217c

228bc

237b

Specific volume (cm3/g)

4.57a

4.26b

3.91c

3.67d

4.27b

3.93c

3.68d

4.30b

3.97c

3.71d

Overall acceptability

8.1a

7.7a

6.4b

5.7b

7.7a

7.4a

5.8b

7.8a

7.5a

6.0b

BREAD LOAF

absorption than the control, whereas GCPI yielded significantly increased water absorption (P < 0.05) than the control. DCPI showed a similar trend to GCPI, albeit at lower values. The results indicate that addition of DCPI to wheat flour resulted in a softer dough with improved water absorption compared to wheat flour enriched with nonmodified CPI. These dough properties were further enhanced by wheat flour enriched with GCPI. The enhanced dough properties correspond to the increased denaturation and glycation degrees of DCPI and GCPI (Table 2). Sensory acceptability of breads made with dough containing 2% and 4% DCPI and GCPI were similar to the control, whereas 4% CPI addition resulted in significantly decreased acceptability compared to the control (Table 3), demonstrating the positive effects of denaturation and glycation of CPI. There was a trend for bread with added GCPI to have slightly higher acceptability scores compared to bread with added DCPI, although this was not statistically significant. The increase in hardness of bread crumbs containing CPI was significantly higher than the control, whereas this effect was reduced by DCPI, and even more so by GCPI (Table 3), demonstrating that the latter modifications reduce the hardening of crumbs caused by CPI. Increased crumb softness might relate to increased moisture absorption of dough (Table 3) that could have resulted in increased moisture retention capacity of crumbs. The enhanced softness of bread containing DCPI or GCPI could be due to the improved solubility imparted to the native protein (Fig. 1), as well as improved water-binding capacity (results not shown). Improved water-binding capacity for soy and whey protein that had been denatured and glycated in solution was reported by Gu et al.20 and Zhu et al.19 The addition of CPI to bread resulted in decreased loaf volume compared to the control (Table 3), with no significant difference exhibited by DCPI or GCPI. Liu40 attributed similar results in breads fortified with chickpea or Northern bean flour to the decrease in gluten, which is the wheat protein responsible for bread loaf integrity. Apart from slightly decreased volume, GCPI added at levels up to 4% did not adversely affect the bread’s physical properties and overall acceptability. The results show that denaturation and glycation of CPI can allow the inclusion level in bread to increase from 2% to 4% without affecting the sensory acceptability or textural properties.

CONCLUSIONS In the research discussed in this chapter, defatted cowpea flour was prepared from cowpea beans and analyzed for proximate composition and for different types of monosaccharides, disaccharides, and oligosaccharides. Protein isolate was prepared (as CPI) and thermally denatured (as DCPI). To prepare GCPI, a cowpea flour slurry was heattreated before the protein was isolated. Protein functionality with regard to texture and sensory properties was tested by the supplementation of flour in bread. Analysis of the physicochemical properties of proteins showed that CPI is more susceptible to thermal denaturation than GCPI during heat treatment, by virtue of higher denaturation degree and loss of solubility. This is attributed to the protective effect of the higher glycation degree and higher carbohydrate content of GCPI, as demonstrated by sugar analysis and glycoprotein staining of SDS-PAGE gel, resulting in improved maintenance of solubility. Water absorption of bread dough was significantly increased by DCPI, and to a larger extent GCPI, compared to the control, resulting in softer texture. Sensory testing and textural analysis of bread indicate that denaturation and glycation of CPI can allow the inclusion level in bread to be increased from 2% to 4% without affecting the sensory acceptability or textural properties.

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The addition of 4% DCPI or GCPI to bread leads to an increase in total fiber from 0.08% to 0.46%, and in protein content from 7.5% to 9.5%, thereby enhancing the bread’s nutritional quality. We report here the simultaneous denaturation and glycation of CPI in solution with inherent sugars before its isolation from flour. The modified protein isolate imparts enhancement of water absorption and sensory acceptability of products when used in baking applications. The enhancement is even better compared to CPI that has been denatured in the absence of sugar. The findings could have significant value in the food industry with regard to the production of protein isolates from cowpea protein and to facilitate their adoption by Western countries.

Acknowledgments This scientific study was financed by the Ministry of Higher Education, Libya, to support a PhD study conducted at the School of Life Sciences, Heriot Watt University, Edinburgh, Scotland.

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31. Giami SY, Amasisi T, Ekiyor G. Comparison of bread making properties of composite flour from kernels of roasted and boiled African breadfruit (Treculia Africana decne) seeds. J Raw Mater Res 2004;1:16–25. 32. Autio K, Flander L, Kinnunen A, Heinonen R. Bread quality relationship with rheological measurements of wheat flour dough. Cereal Chem 2001;78:654–7. 33. Larmond E. Laboratory methods for sensory evaluation of foods. Ottawa, Canada: Food Research Institute of the Department of Agriculture; 1977. 34. Steele RGD, Torrie JH. Principles and procedures of statistics. New York: McGraw-Hill Book Company Inc; 1980. 35. Mwangwela AM. Physicochemical characteristics of conditioned and micronised cowpeas and functional properties of the resultant flours. Doctoral dissertation, University of Pretoria South Africa; 2006. 36. Molina MI, Wagner JR. The effects of divalent cations in the presence of phosphate, citrate and chloride on the aggregation of soy protein isolate. Food Res Int 1999;2(2):135–43. 37. Baier S, McClements DJ. Impact of preferential interactions on thermal stability and gelation of bovine serum albumin in aqueous sucrose solutions. J Agric Food Chem 2001;49:2600–26088. 38. Rich LM, Foegeding EA. Effects of sugars on whey protein isolate gelation. J Agric Food Chem 2000;48:5046–52. 39. Bekhit M. Identification of cowpea, bean and pea varieties by protein and isozyme electrophoresis analysis. Ann Agric Sci 2007;45(2):936–41. 40. Liu LH. Chickpea proteins for food applications. Doctoral dissertation, Melbourne, Australia: Victoria University of Technology, Melbourne; 1996. p. 52–3.

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C H A P T E R

17 Bread Packaging: Features and Functions Antonella Pasqualone Department of Soil, Plant and Food Sciences, University of Bari ‘Aldo Moro’, Bari, Italy

O U T L I N E Why “Our Daily Bread” Needs Longer Shelf Life

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The Role of Packaging in Extending Bread Shelf-Life

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Common Packaging Materials for Bread

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Modified Atmosphere Packaging

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Active Packaging Ethanol-Emitting Active Sachets Essential Oil-Emitting Active Sachets Oxygen-Absorbing Active Sachets Ethanol- and Essential Oil-Emitting Active Sachets Oxygen-Absorbing and Ethanol-Emitting Active Sachets Oxygen-Absorbing and Essential Oil-Emitting Active Sachets

215 215 215 215 216 216

Antimicrobial Films

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Intelligent Packaging

217

Nanopackaging

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Environmental Issues: Biodegradable and Edible Films and Coatings

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Beyond the Simple Protection of Bread: Interlinks Among Packaging Roles and Effects on Bread Waste

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The Simplest Bread Packaging is Crust

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Conclusions

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References

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WHY “OUR DAILY BREAD” NEEDS LONGER SHELF LIFE Bread, among other foods, is one of the richest in cultural, religious, and even social significance. However, the social changes occurring with the passage of time have strongly modified the way that bread is produced, purchased, and consumed, increasing the need to extend the shelf life of this very perishable product. Traditionally homemade for centuries (even if baked in communal ovens for accomplishing safety and, above all, taxation requirements), bread slowly became an essential item on the grocery list. The typically time-consuming breadmaking process, involving skillful women (women traditionally baked bread at home, whereas commercial baking was and still is a man’s job), was no longer sustainable when an increasing number of women began working outside the home. As in other food areas, industrialization deeply entered the bakery sector, although in some countries, such as Italy, the artisanal production still prevails, with the presence of small, family-owned bakeries distributed throughout the urban tissue of every town. In any case, artisanal, homemade (or at least homemade like), and industrial breads are inexorably affected by a quality decay during storage, known as staling. Bread staling is a complex of physicochemical changes occurring during storage, leading to a progressive decrease in consumer acceptance.1 Staling involves structural changes, such as an increase in crumb firmness and crumbliness due to moisture loss and starch retrogradation, as well as taste and aroma alterations due to oxidative phenomena. For those breads with crispy crusts, such as the majority of French and Italian bread types, staling is also related to a loss of crust crispiness due to a transfer of moisture from crumb to crust. Furthermore, bread shelf life is affected by possible

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mold growth, essentially due to postbaking contamination. The negative effects of these phenomena have a major importance to industry, where high productive capacity corresponds to increased sell volumes and the product is sold far from where it is made, requiring a long shelf life. A mostly invisible, but very important, difference between artisanal and industrial breads is in their formulation. For example, Italian artisanal bread is the result of kneading, leavening, and baking a dough made of flour (or remilled semolina, as in Altamura PDO bread and Dittaino PDO bread),2–5 water, yeast, and salt, without other ingredients such as oils/fats, sugar, milk, preservers, or improvers.6–7 Such bread is sold unpackaged and has to be purchased on almost a daily basis because it lasts a few days at most.2 At the industrial level, instead, preserving agents such as propionates or sorbates can be used. Moreover, antistaling enzymes are often included in bread formulation, such as amylases, lipases, or a combination of the two,8 as well as emulsifiers and lipids, all of which interfere with starch retrogradation.1 Finally, an additional way to retard staling consists of adopting a sourdough-based leavening,9 which is quite common at both the artisanal and industrial levels. Therefore, several interventions into bread formulation can be made to keep it soft and free of molds for longer times.

THE ROLE OF PACKAGING IN EXTENDING BREAD SHELF-LIFE Besides intervening on bread formulation, bread shelf life can be improved by means of various packaging solutions, essentially aimed at retarding molding and oxidation (Fig. 1). Packaging, in fact, can decisively influence the shelf life of food, defined as the period of time during which the loss of microbiological and sensory quality remains within a tolerable level. By combining interventions on bread formulation and adequate packaging solutions, bread can be perceived as “daily and fresh,” even when it is not. The main aim of packaging is protecting food from degradative factors such as light, oxygen, water vapor, molds, yeasts, bacteria, and insects. Traditionally, packaging materials had to be as inert as possible (so-called passive packaging), and bread was protected by the main causes of spoilage mostly by barrier film made of polymers with low gas permeability, coupled with a modification of the headspace atmosphere. More recently, according to the EC Reg. 1935/200410 on Food Contact Materials (FCMs), a new concept was developed, allowing packaging to interact with food so long as a safety evaluation was provisionally carried out by the European Food Safety Authority (EFSA). The so-called active and intelligent packaging, therefore, are made of functional materials that deliberately interact with food for extending (active packaging) or monitoring (intelligent packaging) the shelf life.10–11 In general, these features involve the use of chemical substances that are responsible for the active or intelligent function. The packaging types already used or recently proposed for extending bread shelf life are presented in Table 1, along with their main active functions, if any.

PREVENTING OXIDATION (MAP, active packaging)

BREAD INHIBITING FRESHNESS MOULDS (MAP, active packaging)

KEEPING SOFT (formulation)

FIG. 1 Summary of the main factors determining the extension of bread shelf life: (i) interventions in bread formulation, which can keep the crumbs soft for longer times; (ii) use of MAP and/or active packaging, to inhibit molds; and (iii) use of MAP and/or active packaging, to prevent oxidation.

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COMMON PACKAGING MATERIALS FOR BREAD

TABLE 1

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Packaging Types Already Used or Recently Proposed for Extending Bread Shelf Life

Packaging type

Active function

Packaging material or active compound

Passive packaging

–

Paper Waxed paper Glazed imitation parchment LDPE PP Aluminum coated LDPE OPP Acrylic-coated OPP

MAP

–

Laminated film made of one layer of polyamide (nylon) and one layer of polyethylene (PA/PE) Laminated films of PVDC/cellophane/PVDC/PE Laminated films of OPA and OPA/PE Combinations of metalized polymers

Active packaging (sachets)

Ethanol emitter

Ethanol

Essential-oil emitters

Oregano, marjoram, clary sage essential oil

Oxygen absorber

Iron

Ethanol and essential-oil emitter

Ethanol and mastic oil

Oxygen absorbers and ethanol emitter

Iron and ethanol

Oxygen absorbers and essential oil emitters

Iron and extracts of mustard, cinnamon, garlic, and clove

Active packaging (film)

Antimicrobial film

Sodium propionate Cinnamon essential oil Cinnamaldehyde

Intelligent packaging

Oxygen-level indicator

Pt-OEPK

Nanopackaging

Antimicrobial activity and/or improved barrier properties

Nanoemulsions of clove bud and oregano essential oil incorporated into methylcellulose film Nanofibers of MMT-N6 deposited on PP film Ag/TiO2 nanocomposites on a PE layer ZnO nanoparticles incorporated in chitosan-carboxymethyl cellulose-oleic acid (CMC-CH-OL) films

Biodegradable and renewable packaging

–

PLA

Edible coatings

– Antimicrobial activity Probiotic activity

Sodium caseinate/glycerol Monoacyglycerols Sodium alginate and whey protein concentrates containing L. rhamnosus

COMMON PACKAGING MATERIALS FOR BREAD The oldest packaging materials for bread were paper, waxed paper, or glazed imitation parchment impregnated with paraffin wax.12 Currently, bread is mostly packaged in bags made of polyolefin film, such as low-density polyethylene (LDPE) or polypropylene (PP) bags. These materials are usually microperforated (hole diameter ¼ 0.44–1.35 mm)13 for those bread types characterized by a crisp crust, in order to allow moisture to escape, which keeps crust from assuming a leathery consistency.12 These packaging types, however, represent a system to protect bread from contamination by coarse dirt but can achieve only a very slight improvement of shelf life in terms of maintaining softness. For example, bread wrapped in a perforated PP film (with holes having a 0.54-mm diameter at a density of 21.4 holes/cm2), stored for 26 h, needed a load of 1.47 N to be compressed by 30%, whereas packed in a paper bag, it needed a load of 1.70 N; however, both these values were significantly higher than the load needed after 2 h from baking, which was 0.47 N for PP film and 0.58 for a paper bag.13 Therefore, these packaging types are appropriate only for fresh bread destined for immediate consumption, and are suitable for consumers used to an almost daily purchase in short food-supply chains (directly from the local producer or at a local retailer).

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Multilayered films represent a more effective barrier against deteriorating agents. Aluminum-coated LDPE films were commonly used in the United Kingdom, but have been largely substituted by oriented polypropylene (OPP), or to achieve greater oxygen and water vapor barrier properties, by acrylic-coated OPP film.12 Another goal of packaging, indeed, is delaying bread drying out, which involves the use of films with low water vapor permeability. Although starch retrogradation is the real cause of crumb-hardening, a higher moisture level is perceived as freshness.3

MODIFIED ATMOSPHERE PACKAGING Bread molding can be inhibited by reducing as much as possible the oxygen concentration in the headspace of the package. The use of vacuum packaging, however, is uncommon for bread because its alveolar and soft structure would collapse,12 especially in those bread types with soft surface. Modified atmosphere packaging (MAP), instead, consists of displacing atmospheric air by a gas mixture commonly composed of 60% carbon dioxide (CO2) and 40% nitrogen, the latter as an inert filling gas.14 MAP allows the reduction of oxygen concentration in the headspace of the package to 3 weeks.17 The shelf life of barbari bread, a traditional Iranian flatbread,18 fortified with soy flour, was prolonged from 4 days to about 21 days by using MAP and high-barrier laminated packages.19 Similarly, the shelf life of sangak bread, another traditional Iranian flatbread,18 reached 21 days by using bakeoff technology and MAP.20 By adding calcium propionate to the dough as a bread preservative, MAP composed of 50% CO2 and 50% nitrogen gas (N2) extended the shelf life of bread from 8 to 26 days at 22–25°C, and this extension further increased to 52 days at 15–20°C.21 Sliced wheat bread containing 0.1% potassium sorbate (C6H7KO2) remained mold free for up to 21 days with MAP.22 The microbial shelf life of soy bread was extended by at least 4 days (200%) as a result of MAP, but propionate salts and packaging permeability also showed a significant effect on bread shelf life.23 Indeed, MAP was found to be more effective in limiting the growth of molds and yeasts than of bacteria.23 MAP had a positive effect on the microbial shelf life of bread, but no significant difference was detected in the rate of starch retrogradation and bread firming, compared to control bread packaged in atmospheric air.24 Two packaging systems can be used for displacing atmospheric air into the package: flow-packing, in which the modified atmosphere is continuously flushed into a film tube containing the product, followed by sealing; and thermoformed packaging, in which a two-piece package is formed, composed of a tray (or a bottom film) containing the product and a lid that is sealed onto the tray inside a vacuum chamber. The vacuum in the package is then broken by the modified atmosphere.25 Thermoformed packaging achieves a lower residual air content in the headspace than flow packaging, whereas the latter has a higher working speed.25 A recent study evaluated the effect of thermoformed packaging and flow-packaging on the shelf life of industrial durum wheat bread.26 Two thermoformed packaging systems having different thicknesses and thinner flowpackaging were compared among them. Bread formulation included amylase, and the product was sliced and ethanol-sprayed before packing in MAP (composed of 30% CO2 and 70% N2). The combination of these factors allowed to extend bread shelf life up to 60 days when the thermoformed system at reduced thickness was used, allowing to save packaging material compared to a thicker thermoformed packaging. The flow-packaging system allowed further saving of plastic, but it had a shorter shelf life (within 30 days).26

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ACTIVE PACKAGING

215

ACTIVE PACKAGING Active packaging can either absorb or emit gas. In particular, these packaging systems can remove undesired gases present in the headspace, such as oxygen, ethylene, or water vapor, by a chemical reaction, or can gradually release into the headspace volatile antioxidants, preservatives, and antimicrobials, such as ethanol, sulfur dioxide (SO2), or essential vegetable oils.27 The chemicals responsible for the active function can be incorporated directly into the packaging material or can be contained in a small sachet put in the package headspace. Active packaging types are being successfully applied in Australia, Japan, and the United States. However, in more traditional markets such as Europe, the applications have been limited due to legislative restrictions and a lack of knowledge about consumer acceptance.28

Ethanol-Emitting Active Sachets Ethanol emitters were among the first active materials proposed for slowing mold growth in bakery products. The use of ethanol-emitting packaging is related to the practice, stipulated by 2011/10/EC Regulation,29 of spraying ethanol solutions directly on the surface of sliced bread and in its packaging headspace. The shelf life of prebaked buns, packed in LDPE bags with a commercial ethanol emitter named Ethicap and nicknamed Antimold (Freund Corporation, Tokyo), was extended from 4 to 17 days, at room temperature, due to an effective inhibition of mold growth.28 Ethicap is just one of the many patented active packaging systems available, and it consists of a sachet made of paper/ethyl vinyl acetate containing ethanol absorbed onto silicon dioxide (SiO2) powder. Ethanol emitters were found able to extend the microbial shelf life of sliced whole-grain rye bread (packed in highdensity PE, with the sachet placed on the uppermost bread slice) from 8 to 12 to 26–27 days.30

Essential Oil-Emitting Active Sachets Essential oil-emitting active sachets release antimicrobial compounds (usually volatile), which are especially useful if spoilage is a surface phenomenon.12,31 Antimicrobial sachets containing oregano essential oil reduced the growth rates of yeasts and molds during storage of sliced bread. The release of essential oil influenced the sensory properties of bread because the content of γ-terpinene and ρ-cymene increased in bread throughout storage compared to control samples.32 Marjoram (Origanum majorana) and clary sage (Salvia sclarea) essential oils incorporated into a paper disk significantly reduced the growth of molds on bread slices in a petri dish, with better results for marjoram. The strong odor conferred by the essential oil vapors to bread suggests that this preservative system can be used until new alternatives are developed; otherwise, the sensory features may be not well accepted.33

Oxygen-Absorbing Active Sachets Another strategy to prolong the shelf life of bread is represented by active packaging systems that can remove oxygen from headspace more effectively than MAP. Oxygen absorbers create anaerobic conditions that can inhibit food oxidation and aerobic microorganisms simultaneously during storage. The porous structure of bread does not allow the complete removal of oxygen from the headspace during the fast process of air displacement; therefore, MAP barely reaches 0.5%. Moreover, a slow but progressive increase of oxygen concentration in the headspace occurs during storage, caused by residual permeability of packaging films. Instead, active absorbers can reduce the level of oxygen to as low as 0.01%.30 ATCO (Atmosphère Contr^ olee Sas, Bretteville-sur-Odon, France), Ageless (Mitsubishi Gas Chemical Co., Tokyo) and Fresh Pax (Multisorb Technologies, Inc., Buffalo, New York) are iron-containing commercial oxygen absorbers whose working principle is based on the consumption of residual oxygen in the headspace by producing iron oxide. An organic noniron type version of Ageless is also available, for products that are run through a metal detector. Ageless was found to be much more effective than MAP in keeping the level of oxygen very low, with a consequent extension of the mold-free shelf life of bread rolls of up to 60 days.34 Oxygen-absorbing sachets are, therefore, a viable alternative to MAP. Fresh Pax was able to prevent bread from molding for 13 months. In the absence of oxygen

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scavengers, the growth of molds was visible after 14 days.35 Other studies evaluated the effectiveness of oxygen absorbers, combined with MAP, in further extending bakery product shelf life, and observed an improvement of the preserving effect of MAP.36

Ethanol- and Essential Oil-Emitting Active Sachets Ethanol-emitting sachets have also been proposed for use in combination with essential oils (i.e., they release ethanol and essential oil together). Considered that mastic resin and its essential oil, obtained from Pistacia lentiscus (L.) var. chia (Duham), have been used effectively as flavoring, antioxidant, and antimicrobial agents in bread,37–38 mastic oil has been proposed for bakery products having high aw, such as bread, rolls, buns, English-style crumpets, and bagels, and eventually for products also having a high pH,7–9 such as crumpets, gingerbread cake, and carrot muffins,39 where preservatives such as sorbic and propionic acids and their salts are not effective. The emitters were prepared by aseptically transferring 1 g of a solution of mastic essential oil and 95% food-grade ethanol (1:1) onto a 5-cm-diameter sterile cotton pad. These emitters were found effective in inhibiting microorganisms responsible for spoilage or safety concerns in bakery products.39

Oxygen-Absorbing and Ethanol-Emitting Active Sachets Oxygen absorbers and ethanol emitters can be effectively combined to reduce oxidation and to prevent molding. Negamold (Freund Corporation, Tokyo) is a commercial active sachet featuring both functions: scavenging oxygen and releasing alcohol. The use of a combined sachet showed the same effect of chemical preservatives, such as calcium propionate and potassium sorbate, in inhibiting molds, yeasts, and Bacillus cereus in sliced wheat bread.40 Moreover, a combined active sachet was much more effective than chemical preservatives in preventing the formation of off-flavors related to lipid oxidation during storage. In particular, the shelf life was about 4 days for bread without preservatives, 6 days for bread with preservatives, and at least 30 days for samples containing the combined oxygen-absorbing and ethanol-emitting active sachet, with all samples packaged in high-barrier polyethylene terephthalate (PET)-SiOx//LDPE pouches.40 The commercial oxygen absorber ATCO, used with the commercial ethanol emitter Ethicap, prolonged the microbial shelf life of sliced rye bread up to 42 days.30 However, these experimental conditions did not prevent texture and moisture changes of bread during storage (i.e., did not prevent hardening).30 A combination of oxygen-absorbing and ethanol-emitting sachets produced by Guangyi Science and Technology Industry (headquartered in Guangdong, China) was useful for extending the microbial stability of wheat-based, Chinese steamed bread from 6 days for samples containing an oxygen absorber alone to 11 days for those containing the oxygen absorber and the ethanol emitter together.41 Moreover, ethanol acted as a plasticizer of the protein network and delayed bread firming. Differences in the specific packaging film adopted and the bread type (Chinese steamed bread is particularly moist) probably caused different results than those observed by Salminen et al.30

Oxygen-Absorbing and Essential Oil-Emitting Active Sachets With an approach similar to that adopted for ethanol emitters, oxygen absorbers have been effectively combined with essential oils. In particular, volatile components from spices and herbs such as mustard (Brassica spp.), cinnamon (Cinnamomum spp.), garlic (Allium sativum), and clove (Syzygium aromaticum) have been proposed for inhibiting fungal growth on bread.42 In particular, active packaging that can release ethanol and mustard essential oil (whose main active compound is allyl isothiocyanate) has been proposed as an alternative to MAP. This kind of packaging was more active than those containing cinnamon, garlic, or clove oleoresin, which all showed interesting activity.37 The combination of MAP and mustard oil-emitting sachet reduced the need for low residual oxygen in the headspace and inhibited for 30 days all deteriorating organisms even after massive inoculations in wheat and rye bread, although some sensory implications were reported.25

Antimicrobial Films Mold spoilage is relatively frequent in the bakery industry, especially in sliced bread. According to Regulation (EC) No. 1333/2008,43 calcium and sodium propionate or sorbate can be added to bread formulation as fungistatic

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NANOPACKAGING

217

additives. The limits are 0.2% (w/w) and 0.3% (w/w) for sorbate and propionate, respectively. In the case of unsliced bread, the maximum limit for propionate is lowered to 0.1%, due to smaller exposed surfaces. Antimicrobial active films have been developed to pack bread made without preservatives. Such packaging systems are made of polymeric materials incorporating antimicrobial compounds that are released in a gradual way.44,45 For example, bread mold growth can be reduced by using active films made of cellulose acetate containing 2%–4% sodium propionate, as proved by Soares et al.,46 who intercalated bread slices with antimicrobial film pieces and then packed the slices in LDPE. Another strategy for preparing antimicrobial films has piqued a renewed scientific interest in herbs and spices, which have been used for centuries for food preservation.47 Gutierrez et al.48 proved the effectiveness, for extending gluten-free bread storage, of an active adhesive label made of PP with a nitrocellulose coating containing cinnamon essential oil. The adhesive label was stuck inside the polyethylene package of bread. Wax paraffin containing cinnamon essential oil has been used as a paper coating, having a strong effect against Rhizopus stolonifer inoculated in bread.49 Attempts at using a cassava starch film incorporating cinnamon powder for sliced bread packaging, however, were not successful due to the negative effect of bread moisture on the physicochemical properties of the film.50 Other studies focused on individual compounds of essential oils having greater activities than the essential oils themselves, so that the necessary dose could be reduced. In this frame, the use of cinnamaldehyde, the major volatile compound of cinnamon essential oil, has been widely explored. Films, coatings, and nanoparticles incorporating this compound have been produced, starting from an array of biodegradable organic polymers, such as cellulose,51 pectin,52 chitosan,53 soy protein,54 and gliadin.55 Gliadin films incorporating up to 5% cinnamaldehyde were tested against Penicillium expansum and Aspergillus niger in vitro, and they were employed in an active food packaging system for sliced bread. The obtained results showed high effectiveness against fungal growth.55 A cellulose-derivative polymer film containing 5% cinnamaldehyde was used to wrap bread, which then was packaged in LDPE bags. This procedure effectively protected bread against aerobic mesophiles, yeast, and mold. The study assessed that, after 3 days of storage at 23 °C, the amount of cinnamaldehyde that migrated from film to bread accounted for 0.0025 g/g, which did not influence acceptance by consumers.56

INTELLIGENT PACKAGING Intelligent packaging provides both retailer and consumer with useful information on food conditions (i.e., allows for monitoring food shelf life and is able to track products). However, few intelligent packaging types are commonly used yet, mainly due to economic issues (although intelligent packaging could reduce the high costs of food waste significantly). The substances responsible for the intelligent activity can be incorporated into the material of which the label is made (positioned on the inner surface of a transparent package). Oxygen-level indicators are the intelligent packaging systems whose applications in bread chain are the most studied. The Oxy-eye (Sorbtech International, Marietta, Georgia) is a self-adhesive label whose color changes from blue to pink when the oxygen concentration inside the headspace goes lower than 0.1%. Similarly, the Ageless Eye (Mitsubishi Gas Chemical Co.) is able to check for the absence of oxygen inside the headspace, turning from purple to pink when oxygen decreases to 0.1%. Ageless Eye, in particular, has been used to monitor the oxygen level in a study aimed at extending the microbial stability of Chinese steamed bread by a combination of an ethanol emitter and an oxygen absorber.39 Another study took advantage of a laboratory-prepared optical oxygen sensor based on platinum octaethylporphyrin-ketone (Pt-OEPK; Luxcel Biosciences, Cork, Ireland) in polystyrene, which was spotted on Durapore paper (Millipore Inc., Bedford, MA), allowed to dry, and cut to a 5-mm diameter.57 The oxygen sensor was attached to the inside of commercial ciabatta bread packs prior to gas flushing (for MAP) and sealing. The package also contained an ethanol-emitting sachet. The sensor allowed a continuous and nondestructive monitoring of in-pack oxygen levels over time.57

NANOPACKAGING Nanopackaging is packaging produced with nanoparticles that exhibit chemical and physical properties that differ from those of greater particles.58,59 Nanotechnology has great potential for the development of innovative active packaging.60,61

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This technology allows for designing polymers with improved barrier function against oxygen, which can delay the development of aerobial microorganisms and molds, or directly allows incorporating antimicrobial compounds at the nanolevel. However, concerns about the possible risk, requiring a case-by-case risk assessment, have been raised.11,59 Moreover, studies aimed at assessing the public acceptance of nanofoods and nanopackaging showed that participants were hesitant to buy nanotechnology foods or food with nanotechnology packaging, although nanopackaging was perceived as being more beneficial than nanofoods.62 Methylcellulose films incorporating nanoemulsions (droplet diameter 180–250 nm) of essential oil of clove bud (S. aromaticum) and oregano (Origanum vulgare) inhibited yeasts and molds in sliced bread stored for 15 days. The films containing conventional emulsions of the same essential oil showed lower antimicrobial properties, indicating that nanodroplets had greater bioavailability. Therefore, a lower amount of preservative might be able to deliver the same antimicrobial efficiency if encapsulated in nanoparticles.63 In another experiment, a nanofibrous membrane of montmorillonite-nylon 6 (MMT-N6) was deposited, by electrospinning, over PP film used for bread packaging.64 This packaging solution showed better oxygen-barrier properties than PP alone, and therefore inhibited fungal growth for 5 days of storage.64 An active packaging system based on Ag/TiO2 nanocomposite was proposed by Mihaly Cozmuta et al.65 This nanocomposite material coupled the oxygen- and water-scavenging properties of titanium dioxide (TiO2) and the antimicrobial activity of ionic silver. The active packaging was prepared by coating an ethanol suspension of Ag/TiO2 nanocomposite on a PE layer. The microbial stability of bread significantly improved compared with PE bags: no yeasts and molds were observed after 6 days of storage.65 Similarly, paper packages modified with Ag/TiO2-SiO2 and Ag/N-TiO2 nanocomposites extended the shelf life of white bread by 2 days.66 Food containers are available, such as the FresherLonger Miracle Food Storage system (Sharper Image, San Francisco), which is based on silver nanoparticles and claims to reduce by 98% the bacterial growth in several foods, included bread, compared to conventional containers.67 Zinc oxide (ZnO) is another interesting agent due to its antimicrobial properties, which has been proposed for producing active nanopackaging.68,69 A new nanocomposite film and coating based on chitosan-carboxymethyl celluloseoleic acid (CMC-CH-OL), incorporating ZnO nanoparticles, increased the microbial shelf life of sliced wheat bread from 3 to 35 days.70

ENVIRONMENTAL ISSUES: BIODEGRADABLE AND EDIBLE FILMS AND COATINGS Environmental issues make sustainability become another important requisite of packaging. The 94/62/EC Directive71 has been issued to reduce packaging waste. One measure involves the use of biodegradable packaging. In this view, special interest is put towards biopolymer-based packaging produced starting from marine or agricultural renewable raw materials. Polylactic acid (PLA), for example, is a compostable, biodegradable polymer whose monomer, lactic acid, comes from renewable sources (usually corn).45 Bread bags are among the several commercially available applications of PLA.72 Starch, cellulose, and various proteins of plant and animal origin, therefore, can be the starting point to produce biodegradable (and in some cases even edible) materials with good film-forming features. Edible coatings are thin layers of edible material applied directly to the product surface, providing a barrier to moisture and oxygen or performing an antifungal activity. To avoid contamination during food handling, these edible films can be used to wrap foods inside a light, secondary synthetic (nonedible) package, helping to reduce the use of plastic. Even without a secondary package, sodium caseinate/glycerol edible film wraps kept bread significantly softer than unpackaged bread during a storage period of 6 h, but were less effective than PVC. The edible films showed good tensile strength and relatively low water vapor permeability.73 An emulsified edible film composed of corn starch, methylcellulose (MC), and soybean oil has been proposed instead to control moistening in moisture-sensitive products such as crackers.74 Monoacylglycerols, such as 1-monocaprylin, 1-monocaprin, and 1-monolaurin, perform antifungal activity against fungi isolated from contaminated bread.75 Aqueous solutions of these monoacylglycerols that were sprayed, as a microfilm, on the surface of freshly baked bread were very effective in preventing spoilage: Bread remained free of molds for 14 days of storage, even in a contaminated environment. This system seems to be suitable for treatment of bakery products that need a prolonged storage period.75 Also, 1-monolaurin has been tested as an antimicrobial agent by incorporating it in a lipid-based (sunflower oil) edible coating for bread. Coated bread had almost two times longer shelf life than the uncoated variety.76

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BEYOND THE SIMPLE PROTECTION OF BREAD: INTERLINKS AMONG PACKAGING ROLES AND EFFECTS ON BREAD WASTE

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Edible coatings have been used for more unusual applications as well. For example, they have been the basis of a novel approach for the development of probiotic bread. In particular, films based either on sodium alginate or blends of sodium alginate and whey protein concentrate, containing Lactobacillus rhamnosus, have been used to coat pan bread, turning it into a probiotic product.77

BEYOND THE SIMPLE PROTECTION OF BREAD: INTERLINKS AMONG PACKAGING ROLES AND EFFECTS ON BREAD WASTE Bread packaging plays multiple roles (Fig. 2): (i) protects the product against chemical, physical, and biological contaminants; (ii) informs consumers about elements mandated by law, such as the list of ingredients and best-before date; (iii) promotes the product by means of voluntary claims, such as “source of fiber” or “low salt content”; and (iv) has environmental implications, and thus must be as environmentally friendly as possible. These roles are interlinked, and all of them synergistically contribute to preventing bread waste that can be created if packaging fails to fulfill its primary functions in a proper way. In other words, bread waste can be significantly reduced when packaging design and materials perform promotion well, increasing sales and avoiding the risk of unsold goods, as well as being an effective protection against deteriorating agents and giving accurate information to the consumer about shelf life (even in a dynamic or flexible way). In life-cycle assessment (LCA), the direct contribution of production and postconsumer life of packaging to climate change, eutrophication, and acidification is relatively low compared to the production chain of bread. Specifically, the impact of packaging accounts for 1%–10% of the total environmental impact of food products.78,79 However, both single and comparative studies on the environmental impacts of various packaging systems are often made for packaging alone, without considering the role of the food product and its waste. Household food waste plays, instead, a fundamental role in determining the environmental impact of packaging.80 Moreover, the reduction of waste is particularly important for food categories with high loss percentages, such as bread. In the United Kingdom, the household waste of bakery products accounts for 31%–32%.80 Therefore, it is essential to create packaging types that can effectively protect bread so as to allow its full consumption, without waste. The role of packaging systems in reducing food waste is rarely considered in LCA studies; as a result, it can happen that a packaging solution that has a lower environmental impact but causes high food waste may appear to be better than packaging that has greater impact on the environment but reduces food waste.81 Such a result conflicts with the aim of reducing the overall environmental impact. For example, when the carbon footprint of packaged sliced bread was calculated considering PE pouches and waxcoated paper bags as packaging systems, the use of paper bags led to 3% higher carbon footprint values than PE bags.82 However, the different influences of the two packaging solutions on bread shelf life and, therefore, their effectiveness in preventing bread loss, was not taken into account. Other researchers, instead, showed that if packaging reduces food waste, then it also reduces the total environmental impact, even if there is an increase in the impact from the packaging itself.80,83 For example, the climate impact of bread packaging can be doubled if it leads to a 5% reduction in bread waste.83 Silvenius et al.80 included household food waste in the evaluation of the carbon footprint and calculated that the carbon footprint of the production chain of packaging accounted for 1.9%–3.0% of the whole product-packaging system, whereas the waste management of package was in the range of 0%–1.6% and the household food waste was up to 26%. The production of even small additional amounts of food that end up as waste instead of being consumed has a greater environmental effect than the production of packaging.80 Protection Information

Reducing bread waste

Promotion Low impact

FIG. 2 Schematic of the main roles of bread packaging: (i) protect the product from contaminants and deteriorating agents; (ii) inform consumers about legally mandated aspects, such as “best before” date and ingredient list; (iii) promote the product by means of voluntary claims, such as high fiber content, low salt content, etc.; and (iv) be environmentally friendly. Effective protection and accurate information on shelf life can reduce food waste.

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THE SIMPLEST BREAD PACKAGING IS CRUST All the packaging systems reviewed in this chapter can be of great importance in the case of commercial bread. However, artisan bread, which is like homemade, is another story. After all, the scent of freshly baked daily bread remains incomparable… Usually, fresh artisan bread, intended for almost immediate consumption (from a few hours to a few days after being made), is displayed unpackaged and simply put in a paper bag when sold. In fact, artisan hearth bread, made without pouring the dough into a mold, has a well-developed crust, which acts as a natural packaging. If such bread is sliced only at the moment of consumption, it stays fresh for several days. In particular, a thick crust surrounding a sourdough-leavened, durum-wheat bread protects the loaf, unsliced and unpackaged, for at least 4 days, as assessed by sensory evaluation and texture analysis. Specifically, the majority of quality descriptors such as crumb moisture and elasticity, sour odor (typical of sourdough), semolina odor, and toasted odor (perceived on crust) did not change significantly during 4 days of storage.2 Some odor alterations were perceivable, however, because fresh bread odor and stale bread odor decreased and increased in a significant way, respectively, after 2 days of storage.2

CONCLUSIONS The extension of bread shelf life is an old challenge for industrial producers. In recent years, many innovative solutions have been explored, from active and intelligent packaging to edible coatings and nanomaterials, eventually coupled with MAP. However, the consumer acceptance of these new technologies, especially in traditional areas such as Europe, is still not deeply studied and understood. From the economic point of view, active packaging could be less expensive than MAP because it does not need such high-barrier films and special packaging machines in order to displace atmospheric air from the headspace. For this reason, a clear research trend is emerging that couples the newest technological tools, such as active sachets, with the use of preservatives of natural origin, derived from spices and herbs. These substances are perceived as safer by consumers, who are increasingly demanding foods with low levels of synthetic preservatives. Proper packaging is an effective way to decrease bread waste and allow an extension of shelf life, which in turn enables a baked good to be delivered far from where it was produced, hence reaching new markets. Adequate bread packaging, coupled with interventions in formulation, can increase a bread’s shelf life up to 60 days, but can such a product really replace “our daily bread”?

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18 Nixtamalized Maize Flour By-product as a Source of Health-Promoting Ferulated Arabinoxylans (AX) Rita Paz-Samaniego*, Norberto Sotelo-Cruz†, Jorge Marquez-Escalante*, Agustı´n Rascon-Chu*, Alma C. Campa-Mada*, and Elizabeth Carvajal-Millan* *Research Center for Food and Development, CIAD, Sonora, Mexico † Department of Medicine, University of Sonora, Sonora, Mexico

O U T L I N E Introduction Ferulated Arabinoxylans (AX) Nejayote

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Ferulated AX From Nejayote

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Health Benefits Prebiotics Antioxidant

230 230 232

Anticancer Activity

232

Summary Points

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Acknowledgments

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References

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Further Reading

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Abbreviations AX ferulated arabinoxylans AXOS arabinoxylooligosaccharides FA ferulic acid

INTRODUCTION Cereal grains are currently among the most important components of the human diet. They are rich in complex carbohydrates and supply proteins, lipids, minerals, vitamins, and enzymes. Around the world, rice, wheat, and maize (and, to a lesser extent, barley, sorghum, millet, oats, and rye) are basic foods that millions of people rely on for their survival.1 Maize is the most important crop for human consumption in Mexico, with approximately 23,200 Mt, an amount that is expected to increase to 24,600 Mt by 2020.2 The major chemical components of maize grain are carbohydrates, followed by proteins and other micronutrients.3 In the past, Mesoamerican Indians incorporated wood ashes into the cooking of maize in order to facilitate the removal of the grain cover and improve the kneading of the dough made from it. Interestingly, this alkali cooking process favored the availability of bound niacin present in the maize grain, helping to prevent pellagra in this

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18. NIXTAMALIZED MAIZE FLOUR BY-PRODUCT AS A SOURCE OF HEALTH-PROMOTING FERULATED ARABINOXYLANS (AX)

population. In Mexico, this maize-preparation process is called nixtamalization, from the Nahuatl nixtli (ashes) and tamalli (dough), and allows the wide use of nixtamalized maize flour in a variety of products such as tortillas.4 Nixtamalized maize flour products provide an important amount of Mexicans’ caloric intake, especially in lowerincome groups. During nixtamalization, maize grains are cooked in a lime solution, soaked, and washed to remove the pericarp section. After that, the maize is milled, resulting in dough called masa or nixtamal. Nixtamalization allows the alkaline hydrolysis and solubilization of maize cell wall components such as arabinoxylans (AX), which can be recovered in the maize wastewater (known as nejayote) generated in this process. This maize wastewater is highly alkaline, with high chemical and biological oxygen demands, and is considered an environmental pollutant. In general, nixtamalization of 50 kg of maize grain requires about 75 L of water and generates almost the same amount of nejayote. Therefore, alternative uses for nejayote in Mexico are needed, and an option is to recover AX from nejayote.5 The interest in AX has increased since several studies have reported in recent years that this polysaccharide performs prebiotic, antioxidant, antitumoral, and immunomodulatory activities. In addition, AX can form covalent gels that could have potential application in macromolecule encapsulation.6 This chapter presents nejayote as a source of AX and describes the effect of this polysaccharide in promoting good health.

Ferulated Arabinoxylans (AX) Arabinoxylans (AX) are the main nonstarch components of cereals constituting the largest amount of polysaccharides in the cell wall and found mainly in the aleurone layer (60%–70%), the endosperm, and the husk of cereals.7, 8 AX have been found in the main cereals such as wheat, rye, barley, oats, rice, sorghum, and corn, as well as in some other plants.7, 9–11 Although the AX varieties in several cereals or plants have the same basic chemical structure, they differ in many characteristics, including molecular weight, the arabinose-xylose ratio (A/X), the amount of ferulic acid (FA) esterified to the arabinose residues, the distribution of arabinose and FA in the xylose backbone, and the presence of other substituents such as galactose and glucuronic acid. These structural differences vary depending on the AX source and extraction method used. For example, AX from wheat, rye, and barley are less branched than AX from rice, sorghum, and maize.7 AX from maize by-products can present a highly (A/X ¼ 0.85) to moderately (A7X ¼ 0.65) branched structure.5, 12 A particular characteristic of AX is the presence of FA (3-methoxy-4-hydroxycinnamic acid), covalently linked via ester to C(O)-5 of the arabinose residue, which is called ferulated AX (Izydorczyk and Biliaderis, 1995)7 (Fig. 1). It has been reported that FA can be covalently cross-linked by oxidative coupling, forming complex structures such as dimers and trimers of FA [diferulic acid (di-FA) and triferulic acid (tri-FA), respectively].5 In addition to covalent cross-links (i.e., di-FA, tri-FA), the involvement of physical interactions among AX chains in contributing to the arabinoxylan gelation and gel properties has been suggested.12, 13 AX gels present a number of interesting properties, such as neutral taste and odor, high water-absorption capacity (up to 100 g of water per gram of dry polymer), and lack of sensitivity to pH or electrolytes.14 Maize processing by-products such as bran and nejayote are an important source of AX. The variations in the structural characteristics of AX, which depend on the source and the extraction process, will determine their properties as texturizers, encapsulating agents, emulsifiers, prebiotics and antioxidants, as well as their health-promoting effects.

Nejayote As previously stated, nejayote is the by-product of the maize nixtamalization process, which consists of cooking maize grains in a lime solution, where, after soaking for 2–15 h, the supernatant (nejayote) is discarded. The remaining material is milled to obtain nixtamal (dough), which is used to prepare a variety of products, tortillas being the most consumed in Mexico.3 This effluent is considered as a pollutant because of its high pH12–15 and its high organic matter content (2540 mg/L).16 Due to its physicochemical characteristics and its high organic-matter content, nejayote is considered a pollutant, and only a few attempts have been made to use this by-product.16 A high volume of nejayote is dumped into rivers or lakes, in soils, or into the public sewer system.15 Nixtamalization degrades and solubilizes the components of the cell wall, facilitating the removal of the pericarp. In general, nejayote contains more than 60% nonstarch polysaccharides such as AX.5 AX from nejayote have been reported to have a branched structure, with A/X ratios varying between 0.65 and 0.85, and FA content that depends on the nixtamalization conditions, such as cooking and soaking time and maize/lime proportion (Table 1).5, 17

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FIG. 1 Chemical structure of ferulated AX. Source: Paz-Samaniego R, Carvajal-Millan E, López-Franco YL, Lizardi-Mendoza J, Rascón-Chu A, SoteloCruz N, Brown F, Pedroza-Montero M, Silva-Campa E. Electrospray-assisted fabrication of core-shell arabinoxylan gel particles for insulin and probiotics entrapment. J Appl Polym Sci 2018;135:26; doi: 10.1002/APP.46411. TABLE 1 Physicochemical Characteristics of Nejayote Characteristic

Parameter

Total solid content (g/L)

11.68

Total soluble solids (°Brix)

1.53

Total organic carbon (mg/L)

2984.10

Chemical oxygen demand (mg/L)

25,000–30,000

Total polyphenols (mg gallic acid/L)

1190

pH

12–14 3

Density (kg/m )

1003.54

Viscosity (Pa s)

0.002301

Free nitrogen (ppm)

200–300

Calcium (mg/L)

1526.21

Moisture (%)

97.72

Ash (%)

0.767

Crude protein (%)

7.42

Crude fat (%)

1.48

Crude fiber (%)

19.3

Carbohydrates (%)

0.862

Source: Adapted from Díaz-Montes E., Castro-Muñoz R, Yáñez-Fernández J. An overview of nejayote, a nixtamalization by product. INAGBI 2016;8:41–60.

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FERULATED AX FROM NEJAYOTE AX have been extracted from nejayote generated from tortilla-making industries. These AX are similar in appearance to AX extracted from other cereals that have been reported in the literature (fine white powder with some granulated parts). The yield of AX recovered from nejayote can range from 0.45% to 0.90% (dry weight of polysaccharide/ volume of wastewater). However, considering the total dispersed solids present in the nejayote, the yield of AX is around 46% (dry weight of polysaccharide/dry weight of solids).4, 5 Polysaccharide yield variation could be due to differences in the nixtamalization process, as it remains an artisan process in small tortilla-making factories. In general, longer alkaline exposure of maize could reduce AX yield because extensive hydrolysis of the cell wall components can generate larger amounts of low-molecular-weight AX, which may not precipitate in ethanol during the extraction procedure (Fig. 2). A representative chemical composition of AX extracted from nejayote is presented in Table 2, where the sum of arabinose and xylose content represents 66% dry basis (db). The A/X ratio is high (0.85), indicating a highly branched structure.4 Small amounts of glucose, galactose, protein, and minerals (ashes) are also present in these AX, similarly to previous reports on AX from other maize sources.18 In general, the FA content in AX from nejayote is low due to the extended alkaline exposure of maize grains during nixtamalization, as this condition can de-esterify FA from the arabinose residues along the polysaccharide chains.19 AX have been extracted from nejayote generated under different maize nixtamalization conditions.17 AX with different FA content and gelling capabilities were recovered from nejayote generated under various cooking and soaking conditions (1.5 and 24 h or 0.5 and 4 h, respectively). These AX presented similar molecular identity and molecularweight distribution, but those containing lower FA content (0.008 μg/mg AX) formed weaker gels (elastic modulus G0 of 32 Pa) and a more fragmented microstructure. These results indicate that the conditions of the maize nixtamalization process can modify the characteristics of the AX gels that form as a result.17 FIG. 2 Recovery of AX from nejayote. Source: Adapted from Paz-Samaniego R, Carvajal-Millan E, Sotelo-Cruz N, Brown F, Rascón-Chu A, López-Franco YL, Lizardi-Mendoza J. Maize processing waste water upcycling in Mexico: recovery of arabinoxylans for probiotic encapsulation. Sustainability 2016;8:1104; doi: 10.3390/su8111104. Maize nixtamalization

TABLE 2

Nejayote

Composition of AX From Nejayote

Component

Value

Arabinose

32.0  0.80

Xylose

49.0  1.90

Galactose

3.70  0.20

Glucose

5.10  0.40

Protein

4.50  0.20

Ash

5.10  0.21

FA

0.23  0.01

Di-FA

0.58  0.01

Tri-FA

0.30  0.01

Data are expressed in g/100 g AX dry matter. FA, di-FA and tri-FA are expressed in μg/mg of AX. Values are the mean  standard deviation. All results are obtained from duplicate measurements. Source: Adapted from Niño-Medina G, Carvajal-Millan E, Lizardi J, Rascon-Chu A, Marquez-Escalante JA, Gardea A, Martinez-Lopez AL, Guerrero V. Maize processing waste water arabinoxylans: gelling capability and cross-linking content. Food Chem 2009;115:1286–90.

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FIG. 3

SEM of AX extracted from nejayote generated after 1.5 h of cooking and 24 h of soaking (A, B) or 0.5 h of cooking and 4 h of soaking (C, D). (A) and (C) at 500 magnification; (B) and (D) at 2000 magnification. Source: Adapted from Paz-Samaniego R, Carvajal-Millan E, Brown-Bojorquez F, Rascón-Chu A, López-Franco YL, Sotelo-Cruz N, Lizardi-Mendoza J. Gelation of arabinoxylans from maize wastewater effect of alkaline hydrolysis conditions on the gel rheology. In: Samed M (Ed.), Waste water treatment engineering. Croatia: InTech; 2015. p. 101–14.

It has been reported that AX recovered from nejayote have antioxidant capacity, which has been related to the FA content in the polysaccharide. Laccase-induced cross-linking of these AX can decrease the antioxidant capacity of the molecule by 33% due to the formation of di-FA and tri-FA during the gelation process. These reports indicate that nejayote can be an interesting source of AX that exhibit antioxidant capacity before and after gelation, and that the gels formed could be used as a microencapsulation system with antioxidant capacity.20 Scanning electron microscopy (SEM) images of lyophilized gels based in AX recovered from nejayote generated under various maize nixtamalization conditions are shown in Fig. 3. These gels present an imperfect honeycomb microstructure, similar to those previously reported for lyophilized wheat and maize bran AX gels. Nevertheless, AX extracted from nejayote generated after 0.5 h of cooking and 4 h of soaking present a more fragmented morphology with a rougher and heterogeneous surface (Fig. 3C and D), which was attributed to low elasticity in the gel formed, originating from small FA content in the molecule. AX gels are covalent networks, which are not affected by changes in temperature, pH, and ionic strength; therefore, they resist passage through the upper gastrointestinal tract and reach the colon, where they can be biodegraded by the microbiota. These features enable AX gels as potential delivery systems targeted to the colon for bioactive molecules or cells. Gels based in AX from nejayote at 10% (w/v) are able to encapsulate Bifidobacterium 1  107 colony-forming units (CFU)/mL of probiotics in a gel that presents storage (G0 ) and loss (G00 ) moduli of 50 and 11 Pa, respectively.4 The 2. FLOURS AND BREADS

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FIG. 4 Gels based in AX from nejayote without (A, B) and with Bifidobacterium (1  107 CFU/mL) (C, D) before (A, C) and after (B, D) lyophilization. Source: Adapted from Paz-Samaniego R, Carvajal-Millan E, Sotelo-Cruz N, Brown F, Rascón-Chu A, López-Franco YL, Lizardi-Mendoza J. Maize processing waste water upcycling in Mexico: recovery of arabinoxylans for probiotic encapsulation. Sustainability 2016;8:1104; doi: 10.3390/su8111104.

capability of AX from nejayote to encapsulate probiotics may represent an opportunity in sustainable maize wastewater management through upcycling to value-added products (Fig. 4). The SEM image of a lyophilized gel based in AX from nejayote and containing Bifidobacterium, showing a meshlike network through which bacteria are distributed, is presented in Fig. 5. The micron-sized structures corresponding to Bifidobacteria are indicated. Paz-Samaniego et al.6 developed core-shell particles based on AX recovered from maize nixtamalization for colontargeted delivery of insulin and probiotics. The hypoglycemic effect of the particles was evaluated in vivo in streptozotocin-induced diabetic rats. They found that after 6 h of oral administration of the particles, 70% of the initial glucose was reduced, remaining so up to 24 h after delivery. The results indicate that core-shell particles based on maize AX with insulin and probiotics may become a delivery system of colon-targeted insulin, especially for persons with type I diabetes, dysbiosis, or both (Fig. 6).

HEALTH BENEFITS Prebiotics AX present several biological properties attributed to the structure of the polysaccharide and FA content. These polysaccharides are considered dietary fiber (DF) because they resist hydrolysis by the digestive enzymes, but are degraded by the intestinal microflora.11, 21, 22 They are also considered prebiotics because they are able to create selective changes in the composition of the microbiota, which has beneficial effects on the health of the host. That is, they favor the selective growth of certain bacteria known as probiotics.23 Probiotics can have health effects by directly stimulating the immune system and increasing host defenses.23–26 The end-products of prebiotic fermentation can be short-chain fatty acids such as acetic, propionic, and butyric acids, which supply energy to the colonocytes, maintain an acidic pH that limits the growth of pathogens, and provide a substrate for commensal bacteria.26 In addition, these acids can improve intestinal mucosal morphology by increasing mucin production.9 In recent studies, it has been found that AX from nixtamalized maize by-products allow the growth of probiotic bacteria of the 2. FLOURS AND BREADS

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FIG. 5 SEM of lyophilized MWAX gels (A, B) and MWAX gels containing Bifidobacteria (C, D). Images (A) and (C) at 1500; (B) and (D) at 3500. Source: Adapted from Paz-Samaniego R, Carvajal-Millan E, Sotelo-Cruz N, Brown F, Rascón-Chu A, López-Franco YL, Lizardi-Mendoza J. Maize processing waste water upcycling in Mexico: recovery of arabinoxylans for probiotic encapsulation. Sustainability 2016;8:1104; doi: 10.3390/su8111104.

FIG. 6 Photography of electrosprayed particles of MBAX/insulin-MWAX/Bifidobacterium. Source: Adapted from Paz-Samaniego R, Carvajal-Millan E, López-Franco YL, Lizardi-Mendoza J, Rascón-Chu A, Sotelo-Cruz N, Brown F, Pedroza-Montero M, Silva-Campa E. Electrospray-assisted fabrication of coreshell arabinoxylan gel particles for insulin and probiotics entrapment. J Appl Polym Sci 2018;135:26; doi: 10.1002/APP.46411.

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genus Bifidobacterium, which may indicate that these AX can be prebiotic.4, 27 However, more in vitro and in vivo studies are required to confirm these results. AX have been demonstrated to modulate the luminal and mucosal microbiota. Experiments using a dynamic in vitro model of the human digestive tract (SHIME) showed that supplementation of AX to the proximal colon compartments of the SHIME increased the Bifidobacterium population in both lumen and mucus compared with the control. The levels of propionate, as well as the activity of the enzymes β-xylanase, β-xylosidase, and α-arabinofuranosidase, were also increased in the lumen region. These findings suggest that AX could exert a potential prebiotic effect on the host, as the mucosa-associated microbiota has a direct impact on health by protecting against pathogen colonization and host immunity28.

Antioxidant AX are considered to have antioxidant properties due to the presence of FA in their molecular structure, which is responsible for their antioxidant capacity.29, 30 Due to its phenolic nucleus and extended side chain, FA readily forms a resonance-stabilized phenoxy radical, which explains the effect of trapping free radicals.29, 31 In FA, the presence of electron-donating groups on its benzene ring gives it the property of terminating the free radical chain reactions. In addition, its COOH– group can bind to the lipid bilayer, providing protection against the attack of free radicals and lipid peroxidation.32 This allows FA to protect the deoxyribonucleic acid (DNA) and lipid oxidation from reactive oxygen species (ROS). In addition, FA may be beneficial for treating and/or preventing oxidative stress-related disorders such as Alzheimer’s, diabetes, cancer, hypertension, and arteriosclerosis.31 The antioxidant capacity has been poorly studied in AX gels. Recently, Martinez-Lopez et al.33 demonstrated that AX cross-linked microspheres with antioxidant capacity can be prepared. It has been reported that AX from nejayote present an antioxidant capacity of 6.093  0.146 TEAC/ABTS+ (μmol/g), which decreases to 4.803  0.264 TEAC/ABTS+ (μmol/g) in the gels formed.20 The antioxidant capacity of ferulated arabinoxylans oligosaccharides (AXOS) has been evaluated in vivo, and it was found that they have a greater antioxidant capacity than ascorbic acid (vitamin C) because they were more efficient at mitigating oxidative damage in diabetic rats.11, 23, 25, 26 Male Sprague-Dawley fed with a high fat (HF) diet supplemented with AX showed lower triglyceride concentration in serum than did the HF diet group without AX. The administration of HF-AX changed the lipid metabolism of the rats by improving the activity of fatty acid oxidation enzymes, which helped to reduce the triglyceride levels in liver. AX could help to maintain normal fat levels by activating lipid catabolism and oxidation rather than inhibiting lipid synthesis. Moreover, the activity of the antioxidant enzymes gluthatione peroxidase and total superoxide dismutase also improved via the ingestion of AX, resulting in a reduction of the oxidative stress in serum and tissues.34

Anticancer Activity In the last years, several studies have focused on elucidating the mechanisms by which AX exert their anticancer effects. In this area, research has demonstrated that one of these mechanisms could involve the immune-modulation properties of AX. It has been reported that AX from rice bran enzymatically modified with extracts of Hyphomycetes mycelia showed antitumor activity and immune system activation. Further, it has been reported that partially hydrolyzed AX maize husk can increase immune activity in mice.9 It also has been pointed out that AX can offer immunomodulatory, antioxidant, and anticarcinogenic effects, and also can reduce blood serum triglyceride and cholesterol levels.11 Cholujova et al.35 reported that AX can stimulate the production of interleukins such as IL-2 and IL-12, which are the main anticancer cytokines in humans. Treatment with AX leads to an increase in the secretion of IL-2 in the blood of S180 tumor-bearing mice. It is postulated that an increase of IL-2 may be a mechanism for AX to exert antitumor effects, as IL-2 can improve the proliferation of T cells, B cells, NK cells, and monocytes and increase the cytotoxicity of T cells and NK cells. In addition, the ingestion of Biobran increased the production of IL-12 in multiple myeloma patients 1 and 2 months postingestion.36 The anticancer property of these polysaccharides on various types of cancer, such as colon cancer, glandular stomach cancer, neuroblastoma, and liver cancer has been tested in vivo. According to the observations of the research done in the last 10 years, it is proposed that AX and AXOS may exert their anticancer effects through mechanisms involving antioxidant, prebiotic, and immunomodulatory properties (Table 3).

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SUMMARY POINTS

TABLE 3

Anticancer Potential of AX and AXOS From Various Sources

Type of cancer/animal model

Carcinogenic agent/ cancer cells

Solid Erlich carcinoma/female albino mice

Experimental conditions

Findings

Erlich ascites carcinoma cells, intramuscular inoculation

MGN-3/Biobran (25 mg/ kg b.w.) i.p. Six times/week for 25 days at either day 4 or 11 postcancer cell inoculation

MGN-3 suppressed the growth of tumors, normalized lipid peroxidation, and increased glutathione content. Increased activity of endogenous antioxidant-scavenging enzymes (superoxide dismutase, gluthatione peroxidase, catalase, and gluthatione-S-transferase) in blood, liver and tumor tissue

Colon carcinogenesis/ male F344 rats

1,2,-Dimethylhydrazine, subcutaneous injection

High-fat diet plus AXOS (48 g/kg). 10 days before receiving carcinogen and continued for 13 weeks

Lower counts of preneoplastic lesion (mucin-depleted foci) in comparison to the control group. Fewer preneoplastic lesions (aberrant crypt foci) in the distal part of the colon

S180 tumor-bearing mice ICR male mice

Mouse sarcoma S180 cells, intramuscular inoculation

AX orally administered (100, 200, and 400 mg/kg b.w.)

Administration of AX significantly inhibited the growth of mouse-transplantable tumors and promoted thymus and spleen indexes, splenocyte proliferation, NK cells, macrophage phagocytosis activity, and IL-2 production. Increased peripheral leukocyte count and bone-marrow cellularity

Neuroblastoma NOD-scid IL-2Rgnull mice

Injection of NB-1691luc cells

NK cells activated with 100 μg/mL MGN-3/Biobran injected intravenously. 7 days after injection of tumor cells and performed twice a week for 4 weeks

Significant inhibition of neuroblastoma growth and improvement in survival in the group treated with Biobran. Increase of the activation-associated receptors CD69 and CD25 on NK cells

Glandular stomach carcinogenesis/male Wistar rats

Methylnitrosoguanidine (MNNG), via oral gavage

MNNG plus Biobran (40 mg/ kg b.w.) every other day via oral gavage 8 months

Biobran reduced the incidence of animals bearing gastric dysplasia and adenocarcinoma. Decrease in expression of tumor marker Ki-67, increase in level of apoptotic gastric cancer cells via cell-cycle arrest (sub-G1) and mitochondrial dependent pathway. Protection against lymphocytopenia

Hepatocarcinogenesis/ male albino rats

N-nitrosodiethylamine and carbon tetrachloride

MGN-3/Biobran (25 mg/kg b. w.), 5 times/week i.p. 2 weeks prior receiving carcinogen and continued for 20 weeks

Reduction in liver tumor incidence, decrease of preneoplastic foci in hepatic parenchyma and inhibition of development of hepatocellular carcinoma. Regulation of AST, ALT, ALP, and gamma GT levels. Increase in cell cycle sub-G0/G1 population. Downregulation of expression of NF-kBp65 and Bcl2, upregulated p53, Bax, and caspase-3, and increased Bax/Bcl-2 ratio

Source: Adapted from Mendez-Encinas MA, Carvajal-Millan E, Rascon-Chu E, Astiazaran-Garcia HF, Valencia-Rivera DE. Ferulated arabinoxylans and their gels: functional properties and potential application as antioxidant and anticancer agent. Oxid Med Cell Longevity [in press].

SUMMARY POINTS • Nejayote can be a source of ferulated AX, presenting different functional properties as a texturizer. • The gelling capability of AX from nejayote provides an alternative to their use as encapsulating agents in the food and pharmaceutical industries. • AX offer several benefits to health due to their biological properties as prebiotic, antioxidant, antitumoral, and immunomodulatory actors. • AX gels could be used as controlled release matrices targeting the colon. • Maize by-products such as nejayote can be interesting sources of this polysaccharide.

Acknowledgments “Fondo Institucional CONACyT—Problemas Nacionales 2015,” Mexico (Grant 2015-01-568 to E. Carvajal-Millan).

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exico 2011–2020, http://www.sagarpa.gob.mx/agronegocios/Documents/estudios_economicos/escenariobase/ perspectivalp_11-20.pdf [accessed May 25, 2018]. 3. Martínez-Bustos F, Martínez-Flores HE, Sanmartín-Martínez E, Sánchez-Sinencio F, Chang YK, Barrera-Arellano D, Rios E. Effect of the components of maize on the quality of masa and tortillas during the traditional nixtamalisation process. J Sci Food Agric 2001;81:1455–62. 4. Paz-Samaniego R, Carvajal-Millan E, Sotelo-Cruz N, Brown F, Rascón-Chu A, López-Franco YL, Lizardi-Mendoza J. Maize processing waste water upcycling in Mexico: recovery of arabinoxylans for probiotic encapsulation. Sustainability 2016;8:1104. https://doi.org/10.3390/ su8111104. 5. Niño-Medina G, Carvajal-Millan E, Lizardi J, Rascon-Chu A, Marquez-Escalante JA, Gardea A, Martinez-Lopez AL, Guerrero V. Maize processing waste water arabinoxylans: gelling capability and cross-linking content. Food Chem 2009;115:1286–90. 6. Paz-Samaniego R, Rascón-Chu A, Brown-Bojorquez F, Carvajal-Millan E, Pedroza-Montero M, Silva-Campa E, Sotelo-Cruz N, López-Franco YL, Lizardi-Mendoza J. Electrospray-assisted fabrication of core-shell arabinoxylan gel particles for insulin and probiotics entrapment. J Appl Polym Sci 2018;135:26. https://doi.org/10.1002/APP.46411. 7. Izydorczyk MS, Biliaderis CG. Cereal arabinoxylans: advances in structure and physicochemical properties. Carbohydr Polym 1995;28:33–48. 8. Niño-Medina G, Carvajal-Millán E, Rascón Chu A, Márquez-Escalante JA, Guerrero V, Salas-Muñoz E. Feruloylated arabinoxylans and arabinoxylan gels: structure, sources and applications. Phytochem Rev 2010;9:111–20. 9. Zhou S, Liu X, Guo Y, Wanga Q, Peng D, Cao L. Comparison of the immunological activities of arabinoxylans from wheat bran with alkali and xylanase-aided extraction. Carbohydr Polym 2010;81:784–9. 10. Pellny TK, Lovegrove A, Freeman J, Tosi P, Love CG, Knox JP, Shewry PR, Mitchell RA. Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome1. Plant Physiol 2012;158:612–27. 11. Coelho E, Rocha MAM, Saraiva JA, Coimbra MA. Microwave superheated water and dilute alkali extraction of brewers spent grain arabinoxylans and arabinoxylo-oligosaccharides. Carbohydr Polym 2014;99:415–22. 12. Carvajal-Millan E, Rascón-Chu A, Márquez-Escalante J, Micard V, Ponce de León N, Gardea A. Maize bran gum: extraction, characterization and functional properties. Carbohydr Polym 2007;69:280–5. 13. Vansteenkiste E, Babot C, Rouau X, Micard V. Oxidative gelation of feruloylated arabinoxylan as affected by protein. Influence on protein enzymatic hydrolysis. Food Hydrocol 2004;18:557–64. 14. Carvajal-Millan E, Guilbert S, Morel M-H, Micard V. Impact of the structure of arabinoxylan gels on their rheological and protein transport properties. Carbohydr Polym 2005;60:431–8. 15. Salmerón-Alcocer A, Rodríguez-Mendoza N, Pineda-Santiago V, Cristiani-Urbina E, Juárez-Ramírez C, Ruiz-Ordaz N, Galíndez-Mayer J. Aerobic treatment of maize-processing wastewater (nejayote) in a single-stream multi-stage bioreactor. J Environ Eng Sci 2003;2:401–6. 16. Valderrama-Bravo C, Guti
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endez-Encinas M, Fierro-Islas JM, Marquez-Escalante J, Rascón-Chu A, Martinez-Lopez AL, Carvajal-Millan E. Ferulated arabinoxylans recovered from low-value by-products. Gelation and antioxidant capacity. In: Warren B, editor. Ferulic acid: antioxidant properties, uses and potential health benefits. New York: Nova Science Publishers, Inc; 2015. p. 151–64. 21. Hopkins M, Englyst H, Macfarlane S, Furrie E, Macfarlane G, McBain A. Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal microbiota in children. Appl Environ Microbiol 2003;69:6354–60. 22. Carvajal-Millan E, Berlanga-Reyes C, Rascón-Chu A, Martínez-López AL, Márquez-Escalante JA, Campa-Mada AC, Martínez-Robinson K. In vitro evaluation of arabinoxylan gels as an oral delivery system for insulin. MRS Proc 2012;1487. https://doi.org/10.1557/opl.2012. 23. Delzenne N, Neyrinck A, B€ackhed F, Cani P. Targeting gut microbiota in obesity: effects of prebiotics and probiotics. Nat Rev Endocrinol 2011;7:639–46. 24. Burgain J, Gaiani C, Linder M, Scher J. Encapsulation of probiotic living cells: from laboratory scale to industrial applications. J Food Eng 2011;104:467–83. 25. Zhou S, Liu X, Guo Y, Wanga Q, Peng D, Cao L. Comparison of the immunological activities of arabinoxylans from wheat bran with alkali and xylanase-aided extraction. Carbohydr Polym 2010;81:784–9. 26. Food and Drug Administration, FDA. 2013. GRAS Notices, GRN No. 458. https://www.accessdata.fda.gov/scripts/fdcc/index.cfm? set¼GRASNotices&id¼458 [accessed May 25, 2018]. 27. Martínez-López AL, Carvajal-Millan E, Micard V, Rascón-Chu A, Brown-Bojorquez F, Sotelo-Cruz N, López-Franco YL, Lizardi-Mendoza J. In vitro degradation of covalently cross-linked arabinoxylan hydrogels by bifidobacteria. Carbohydr Polym 2016;144:76–82. 28. Truchado P, Hernandez-Sanabria E, Salden BN, Van den Abbeele P, Vilchez-Vargas R, Jauregui R, Pieper DH, Possemiers S, Van de Wiele T. Long chain arabinoxylans shift the mucosa-associated microbiota in the proximal colon of the simulator of the human intestinal microbial ecosystem (M-SHIME). J Funct Foods 2017;32:226–37. 29. Oua J, Sun Z. Feruloylated oligosaccharides: structure, metabolism and function. J Funct Foods 2014;7:90–100. 30. Kumar Y, Bhatia A. Polyphenols and skin cancers. In: Watson RR, Preedy VR, Zibadi S, editors. Polyphenols in human health and disease. Cambridge: Elsevier Inc; 2014. p. 643–53.

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31. Zhao Z, Moghadasian MH. Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. Food Chem 2008;109:691–702. 32. Srinivasan M, Sudheer AR, Menon VP. Ferulic acid: therapeutic potential through its antioxidant property. J Clin Biochem Nutr 2007;40:92–100. 33. Martínez-López AL, Carvajal-Millan E, López-Franco YL, Lizardi-Mendoza J, Rascón-Chu A. Antioxidant activity of maize bran arabinoxylan microspheres. In: Haghi AK, Carvajal-Millan E, editors. Food composition and analysis. Methods and strategies. NJ: Apple Academic Press, Inc; 2014. p. 19–28. 34. Chen H, Fu Y, Jiang X, Li D, Qin W, Zhang Q, Lin D, Liu Y, Tan C, Huang Z, Liu Y, Chen D. Arabinoxylan activates lipid catabolism and alleviates liver damage in rats induced by high-fat diet. J Sci Food Agric 2018;98:253–60. 35. Cholujova D, Jakubikova J, Czako B, Martisova M, Hunakova L, Duraj J, Mistrik M, Sedlak J. MGN-3 arabinoxylan rice bran modulates innate immunity in multiple myeloma patients. Cancer Immunol Immunother 2013;62:437–45. 36. Mendez-Encinas MA, Carvajal-Millan E, Rascon-Chu E, Astiazaran-Garcia HF, Valencia-Rivera DE. Ferulated arabinoxylans and their gels: functional properties and potential application as antioxidant and anticancer agent. Oxid Med Cell Longevity 2018, Article ID: 2314759, 22 pages.

Further Reading 37. Díaz-Montes E, Castro-Muñoz R, Yáñez-Fernández J. An overview of nejayote, a nixtamalization by product. INAGBI 2016;8:41–60.

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C H A P T E R

19 Chestnut and Breads: Nutritional, Functional, and Technological Qualities Maria Paciulli*, Ilkem Demirkesen Mert†, Massimiliano Rinaldi*, Alessandro Pugliese*, and Emma Chiavaro* †

*Department of Food and Drug, University of Parma, Parma, Italy Republic of Turkey Ministry of Food, Agriculture and Livestock, Food Enterprises and Codex Department, Ankara, Turkey

O U T L I N E Introduction Taxonomy, Origin, Chemical, and Nutritional Characteristics of Chestnut From Chestnut to Chestnut Flour: Production, Composition, and Functional Properties

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Chestnut in Bakery Products: Nutritional, Organoleptic, and Technological Properties

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Chestnut in Gluten-Free Breads: Nutritional, Organoleptic, and Technological Properties

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Conclusion and Future Directions of Research

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References

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INTRODUCTION In the last decade, research dealing with the use of chestnut has received considerable interest due to chestnut’s promising potential to produce high-quality and healthy products with high nutritional value. Chestnut is a rich source of starch; minerals such as potassium, phosphorous, magnesium, calcium, copper, iron, manganese, and sulfur; B and C vitamins; fibers; and antioxidant phenolic compounds. It has a low amount of fat with essential fatty acids and a low amount of protein having high-quality essential amino acids.1 It can be consumed fresh (raw), boiled, or roasted.1 It is also ground into flour, which can be used in the production of several high-quality bakery products, such as bread,2–9 also using sourdough technique,10–14 cookies,15 cakes,16,17 breakfast cereals,18,19 and pasta.20 In addition to its health and nutritional benefits, chestnut flour offers some functional properties to the dough; it has been reported that chestnut flour fibers give emulsifying, stabilizing, texturizing, and thickening properties to dough, and its sugar content provides color and a characteristic flavor to bakery products when it is used at certain levels.2 Furthermore, the addition of chestnut flour improves antioxidant capacity and reduces staling of enriched breads.3,6,21 It has been reported that sourdough fermentation might be a suitable biotechnology to be used for chestnut bread due to its high amount of dietary fiber.14 Knowledge about the nutritional and functional properties of chestnut and chestnut flour may produce new opportunities to increase its demand, being also a good marketing strategy. The aim of this chapter is to illustrate the taxonomy, origin, and compositional characteristics of chestnut, as well as the use of chestnut flour in bakery products, with a particular focus on bread, and to discuss the rheological and quality characteristics of wheat and gluten-free bakery products containing chestnut flour from the nutritional, health, and functional point of view. Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00019-8

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TAXONOMY, ORIGIN, CHEMICAL, AND NUTRITIONAL CHARACTERISTICS OF CHESTNUT Chestnut has a sweet, delicious, and nutty flavor and a starchy texture similar to firm baked potatoes. Also, it is less crunchy than other nuts. It needs cold winters and warm summers during the growing period.1 The hard-shelled seed of the chestnut tree is a member of the Fagaceae family and the genus Castanea; it comprises several ecologically and economically important species such as beech (Fagus sp.) and oak (Quercus sp.). Its species include Asian chestnuts such as Castanea crenata (a Japanese chestnut), Castanea mollissima (a Chinese chestnut), Castanea seguinii, Castanea davidii, and Castanea henryi; American chestnuts such as Castanea dentate and Castanea pumila; and European chestnuts such as Castanea sativa and Castanea latifolia, as well as their hybrids.1,22 The European chestnut known as C. sativa Mill. (sweet chestnut) is the most commonly consumed nut, and now it is the only surviving European species. Native and cultivated forests of European species spread out from the Caucasus through Turkey, Greece, and Slovenia to Italy, France, Spain, Portugal, Germany, and southern England.22 It is a medium-to-large fruit (10–25 g), and its quality changes depending on the cultiva, but also on the environmental conditions, and it may significantly affect both the chemical composition and the morphological parameters of chestnuts.1 According to statistics from the Food and Agriculture Organization (FAO), the largest chestnut producer is China, followed by Bolivia, Turkey, Italy, and the Republic of Korea.23 Chestnut may have a very important role in human nutrition due to its potential health and functional benefits. It is a rich source of starch (approximately 70%), and it is a valuable source of minerals such as potassium, phosphorous, magnesium, calcium, copper, iron, manganese, and sulfur, B and C vitamins, fibers, and antioxidant phenolic compounds, but it has a low amount of protein (2%–4%), with essential amino acids, and fat (2%–5%). In addition, the consumption of chestnut is recommended by the American Health Care Association as a low-fat food.24 The essential fatty acid content plays a critical role in avoiding cardiovascular disease in adults, as well as in the development of brain and retina of infants. The significant presence of polyunsaturated fatty acid (PUFA), especially linoleic acid, helps to reduce cholesterol levels and prevent coronary heart disease. Moreover, it has been reported that the consumption of 60 g nuts/day could be helpful for the reduction of blood cholesterol levels and hence could create a positive effect on people with a high risk of coronary heart disease.24 On the other hand, the consumption of this amount of nuts can lead to a modest increase in oxalate intake, resulting in increases in the risk of kidney stone formation. Nonetheless, it has been stated that chestnut comprises very low levels of oxalates (10 mm2) in chestnut-rice breads by 71%. The replacement of rice flour with chestnut flour resulted in a more uniform structure, thanks to the fiber content and larger starch granules of chestnut flour, which contributed to the stabilization of gas bubbles. The presence of additives and baking in an IR-microwave combination oven increased the uniformity of the microstructure of rice and rice-chestnut breads. SEM showed that breads prepared with chestnut flour and baked in an IR-microwave combination oven had more starch granules, which did not disintegrate completely (Fig. 3). The staling rate of the chestnut/rice breads added with xanthan-guar gum blend-DATEM mixture and baked in conventional and IR-microwave combination ovens was also studied during a storage of four days.6 In particular, the changes of hardness by mechanical compression, moisture content, and degree of starch retrogradation were

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evaluated by means of differential scanning calorimetry, X-ray diffraction (XRD), and Fourier transform IR spectroscopy (FT-IR). As expected, during storage hardness, moisture loss, retrogradation enthalpies, total crystallinity values, and FT-IR results showed that starch retrogradation in bread samples increased significantly during storage. Higher values of hardness and lower moisture content were obtained for breads baked in an IR-microwave combination oven, but a similar stailing rate was observed compared to breads cooked in a conventional oven. The presence of chestnut flour and xanthan-guar gum blend-DATEM mixture retarded the staling of breads. It has been suggested that the water-binding ability of chestnut flour and xanthan-guar gum blend-DATEM mixture softened the crumbs and decreased the amount of available starch for crystallization. Moreover, the presence of chestnut flour decreased the amylopectin content of bread samples, reducing the retrogradation enthalpies of the amylopectin during storage. Similarly, Paciulli et al.9 used two commercial gluten-free mixtures to produce breads enriched with 20 g/100 g and 10 g/100 g of chestnut flour, respectively, and studied their behavior during a storage period of 3 days. Despite the different characteristics of the two control breads, due to the dissimilar composition of the mixtures, the addition of chestnut flour led to color darkening, volume reduction, larger alveoli, and harder crumb texture in both formulations. During shelf life, faster staling was observed in both the chestnut-enriched breads in comparison to the contols without chestnut, resulting from crumb cohesiveness and resilience decrease. Depending on the composition of the two studied gluten-free mixtures, different crust hardness modifications were observed on the two formulations, as consequence of the different water migration. Higher antioxidant activities were observed for both enriched breads, during the entire shelf life, in comparison to the controls, while there were no variations in starch digestibility. In addition, enrichment with 20 g/100 g of chestnut flour caused a significant increase in soluble, insoluble, and total fiber content. It has been reported that sourdough fermentation is a suitable biotechnology for creating a bread formulation rich in dietary fiber.21 Demirkesen et al.13 studied the effects of sourdough amount (20%, 40%, and 60%) on the rheological behavior of gluten-free chestnut-rice dough formulations and quality parameters of the resulting breads. The addition of sourdough led to decreases in complex modulus, indicating softening of the dough samples. The hypothesis is that sourdough fermentation produces organic acids and release of enzymes, both of which are responsible for protein breakdown and a consequent reduction of the water-holding capacity and a release of a small polypeptide that weakens the protein-protein and protein-starch interactions. Confocal laser-scanning microscopy confirmed the smaller dimensions of the proteins in breads prepared with sourdough, due to their degradation into smaller peptides as a result of the fermentation. Levels of sourdough up to 40% had a positive effect on the texture and volume of the breads because softer dough formation might have facilitated better carbon dioxide (CO2) expansion. Detrimental effects were observed with higher sourdough concentrations. The utilization of chestnut flour in dough formulation created a more homogenous crumb structure with a relatively higher number of small pores, which was triggered by the presence of more gas bubbles and moisture in comparison to rice breads. X-ray micro-computed tomography (μCT) images proved that gluten-free chestnut-rice breads prepared with 20% sourdough incorporation had the most homogenous crumb structure with a high number of smaller pores (Figs. 4E and 4F). In contrast, the addition of higher levels of sourdough led to the formation of denser crumb structures (Figs. 4G–J). In another study, Aguilar and coworkers12 tested a chestnut flour sourdough in gluten-free breads formulated with cornstarch and chestnut flour. They compared breads contained 15%, 20%, or 25% chestnut flour, with breads containing the same amount of chestnut flour in form of chestnut flour sourdough. Chestnut flour sourdough increased the bread’s specific volume, reduced crumb hardness during all 7 days of storage, and lightened the crust color. Chestnut flour sourdough had no effect on yeast and mold growth during 7 days of bread storage; however, it had a negatively effect on consumers’ preference, probably due to the reduction in the typical sweet taste of chestnut flour caused by sourdough fermentation, as confirmed by the pH reduction. Later, Rinaldi et al.14 explored the effect of around 30% sourdough, combined with chestnut flour (40%), for improving the technological and nutritional quality of gluten-free bread during a 5-day shelf life. The addition of chestnut flour to bread formulations containing sourdough limited acidification, decrease in water-holding capacity and increase in crumb firmness due to the excessive acidification. Sourdough fermentation by itself and together with chestnut flour reduced loaves volume and heterogeneity in crumb grain, with a significant increase in crumb hardness. During storage, sourdough and/or chestnut flour additions caused a significant increase in crumb hardness at time 0, while a significant staling reduction was observed only at 5 days, despite the decrease in amylopectin-melting enthalpy. Sourdough increased crumb moisture content with no significant variations during shelf life. Chestnut flour darkened crumbs and crusts, while no effects on color were observed as effect of sourdough. From a nutritional point of view, the percentage of hydrolyzed starch during in vitro digestion was significantly reduced by sourdough fermentation, with a presumable lower glycemic index (GI) as well. These results show promising applications of chestnut flour in the diet of celiac patients.

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FIG. 4 Two-dimensional (2D) and three-dimensional (3D) X-ray μCT images of gluten-free bread samples prepared with and without the addition of different amounts of sourdough. (A, B) Control rice bread; (C, D) control chestnut-rice bread; (E, F) bread prepared by adding 20% sourdough; (G, H) bread prepared by adding 40% sourdough; (I, J) bread prepared by adding 60% sourdough. Pictures on the left side represent 2D, and those on the right side represent 3D images. Reproduced from Demirkesen et al. 2016 with permission from the publisher.

CONCLUSION AND FUTURE DIRECTIONS OF RESEARCH Chestnuts and the products thereof have been attracting a lot of research interest in recent years due to their unique eating and nutritional quality. The use of chestnut flour is finding good applications in the enrichment of flour-based bakery products. In gluten products such as bread, the feasibility of chestnut flour additions has been demonstrated for enhancing antioxidant content of this product by significantly improving its nutritional value. However, the most interesting application concerns gluten-free products that are generally characterized by poor technological, organoleptic, and nutritional properties. The addition of chestnut flour, within certain limits, to gluten-free breads has improved the machinability of the dough, the bread consistency, color, and aroma, with even better performances during shelf life, even if the use of hydrocolloids was necessary to ensure the success of the products. From a nutritional point of view, improved antioxidant and fiber content has been found in chestnut-enriched gluten-free breads, with also a reduction in starch digestibility in presence of sourdough. Further research will be necessary to guarantee the healthy properties of chestnut flour-enriched breads.

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A good future area of research may be the use of the chestnut pericarp, now considered waste, in formulations for bakery products. The use of this by-product, which is naturally gluten-free and rich in antioxidants and fiber, may represent a way to enhance the chestnut supply chain, making it more sustainable, as well as giving an improved nutritional contribution to chestnut products.

References € Composition, color and mechanical characteristics of pretreated candied chestnuts. In: Alasalvar C, Fereidoon S, editors. 1. Korel F, Balaban MO. Tree nuts: composition, phytochemicals, and health effects. New York, USA: CRC Press Taylor & Francis Group; 2008. p. 171–85. 2. Demirkesen I, Mert B, Sumnu G, Sahin S. Utilization of chestnut flour in gluten-free bread formulations. J Food Eng 2010;101(3):329–36. 3. Rinaldi M, Paciulli M, Dall’Asta C, Cirlini M, Chiavaro E. Short-term storage evaluation of quality and antioxidant capacity in chestnut-wheat bread. J J Sci Food Agric 2015;95(1):59–65. 4. Man S, P aucean A, Muste S, Mureşan C, Fr^ancu AV. Chestnut flour addition influence on bread quality. J Agroalimentary Process Technol 2012; 18(2):150–4. 5. Oh CH, KimYM HMS, Oh NS. Effect of chestnut flour on the rheology of dough and processing adaptability of white pan bread. Food Eng Prog 2011. 6. Demirkesen I, Campanella OH, Sumnu G, Sahin S, Hamaker B. A study on staling characteristics of gluten free breads prepared with chestnut and rice flours. Food Bioprocess Tech 2014;7:806–20. 7. Demirkesen I, Sumnu G, Sahin S. Image analysis of gluten-free breads prepared with chestnut and rice flour and baked in different ovens. Food Bioprocess Tech 2013;6(7):1749–58. 8. Demirkesen I, Sumnu G, Sahin S, Uysal N. Optimization of formulations and infrared-microwave combination baking conditions of chestnut-rice breads. Int J Food Sci Tech 2011;46(9):1809–15. 9. Paciulli M, Rinaldi M, Cirlini M, Scazzina F, Chiavaro E. Chestnut flour addition in commercial gluten-free bread: a shelf-life study. LWT-Food Sci Tech 2016;70:88–95. 10. Aponte M, Boscaino F, Sorrentino A, Coppola R, Masi P, Romano A. Volatile compounds and bacterial community dynamics of chestnut-flourbased sourdoughs. Food Chem 2013;141(3):2394–404. 11. Aponte M, Boscaino F, Sorrentino A, Coppola R, Masi P, Romano A. Effects of fermentation and rye flour on microstructure and volatile compounds of chestnut flour based sourdoughs. LWT-Food Sci Tech 2014;58(2):387–95. 12. Aguilar N, Albanell E, Miñarro B, Capellas M. Chestnut flour sourdough for gluten-free bread making. Eur Food Res Tech 2016;242(10):1795–802. 13. Demirkesen I, Puchulu-Campanella E, Kelkar S, Campanella OH, Sumnu G, Sahin S. Production and characterisation of gluten-free chestnut sourdough breads. Qual Assur Saf Crop 2016;8(3):349–58. 14. Rinaldi M, Paciulli M, Caligiani A, Scazzina F, Chiavaro E. Sourdough fermentation and chestnut flour in gluten-free bread: a shelf-life evaluation. Food Chem 2017;224:144–52. 15. Demirkesen I. Formulation of chestnut cookies and their rheological and quality characteristics. J Food Qual 2016;39(4):264–73. 16. Demirkesen I, Sumnu G, Sahin S. Utilization of chestnut flour in gluten-free cakes. In: 6th International CIGR Technical Symposium, Nantes, France, April 18–20; 2011. p. 1–4. € Dogan IS. Optimization of gluten-free cake prepared from chestnut flour and transglutaminase: response surface methodology 17. Yildiz O, approach. Int J Food Eng 2014;10(4):737–46. 18. Sacchetti G, Pinnavaia GG, Guidolin E, Dalla-Rosa M. Effects of extrusion temperature and feed composition on the functional, physical and sensory properties of chestnut and rice flour-based snack-like products. Food Res Int 2004;37:527–34. 19. Jozinovi
c A, Šubari
c D, Ačkar Đ, et al. Influence of buckwheat and chestnut flour addition on properties of corn extrudates. Croatian J Food Sci Tech 2012;4(1):26–33. 20. KosovI
c I, JuKI
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c D. Influence of chestnut flour addition on quality characteristics of pasta made on extruder and minipress. Czech J Food Sci 2016;34(2):166–72. 21. Dall’Asta C, Cirlini M, Morini E, Rinaldi M, Ganino T, Chiavaro E. Effect of chestnut flour supplementation on physico-chemical properties and volatiles in bread making. LWT-Food Sci Tech 2013;53(1):233–9. 22. Bounous G, Marinoni DT. Chestnut: botany, horticulture, and utilization. In: Janick J, editor. Horticultural reviews. NJ, USA: John Wiley & Sons; 2005. p. 291–347. 23. FAO STAT. Food and Agriculture Organization of the United States. Available at: http://faostat.fao.org/site/339/default.aspx; 2016. 24. Ros E. Health benefits of nut consumption. Nutrition 2010;2(7):652–82. 25. Silvanini A, Dall’Asta C, Morrone L, Cirlini M, Beghè D, Fabbri A, Ganino T. Altitude effects on fruit morphology and flour composition of two chestnut cultivars. Sci Horticult (Amsterdam) 2014;176:311–8. 26. Morrone L, Dall’Asta C, Silvanini A, Cirlini M, Beghè D, Fabbri A, Ganino T. The influence of seasonality on total fat and fatty acids profile, protein and amino acid, and antioxidant properties of traditional Italian flours from different chestnut cultivars. Sci Horticult 2015;192:132–40. 27. Cirlini M, Dall’Asta C, Silvanini A, Begh D, Fabbri A, Galaverna G, Ganino T. Volatile fingerprinting of chestnut flours from traditional Emilia Romagna (Italy) cultivars. Food Chem 2012;134(2):662–8. 28. Blaiotta G, La Gatta B, Di Capua M, Di Luccia A, Coppola R, Aponte M. Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions. Food Microbiol 2013;36(2):161–9. 29. Moreira R, Chenlo F, Torres MD, Rama B. Fine particle size chestnut flour doughs rheology: influence of additives. J Food Eng 2014;120:94–9. 30. Moreira R, Chenlo F, Torres MD. Effect of sodium chloride, sucrose and chestnut starch on rheological properties of chestnut flour doughs. Food Hydrocoll 2011;25(5):1041–50. 31. Moreira R, Chenlo F, Torres MD. Rheology of commercial chestnut flour doughs incorporated with gelling agents. Food Hydrocoll 2011; 25(5):1361–71.

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32. Moreira R, Chenlo F, Torres MD, Rama B. Influence of the chestnuts drying temperature on the rheological properties of their doughs. Food Bioprod Process 2013;91(1):7–13. 33. Chenlo F, Moreira R, Pereira G, Silva CC. Evaluation of the rheological behaviour of chestnut (castanea sativa mill) flour pastes as function of water content and temperature. Elect J Env Agricult Food Chem 2007;6(2):1794–802. 34. Torres MD, Raymundo A, Sousa I. Effect of sucrose, stevia and xylitol on rheological properties of gels from blends of chestnut and rice flours. Carbohydr Polym 2013;98(1):249–56. 35. Torres MD, Moreira R, Chenlo F, Morel MH. Effect of water and guar gum content on thermal properties of chestnut flour and its starch. Food Hydrocoll 2013;33(2):192–8. 36. Demirkesen I, Mert B, Sumnu G, Sahin S. Rheological properties of gluten-free bread formulations. J Food Eng 2010;96:295–303.

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C H A P T E R

20 Passiflora edulis Peel Flour and Health Effects Milena Morandi Vuolo*, Glaucia Cariello Lima†, and Ma´rio Roberto Maro´stica Junior* *Department of Food and Nutrition, Faculty of Food Engineering, University of Campinas, Sa˜o Paulo, Brazil † Nutrition School-Federal University of Goias, Goi^ania, Brazil

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Passion Fruit

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Passion Fruit Peel: Chemical Aspects and Bioactive Compounds 250 Effects of Passion Fruit Peel on Gut Health

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Effects of Passiflora edulis Peel Flour in Metabolic Parameters

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Conclusions

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Acknowledgments

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References

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INTRODUCTION Over the years, the fruit juice industry has become very important economically.1 In 2015, global juice and drink consumption surpassed 80 billion liters. The projections for this market in the future are even greater, reaching 105 billion liters by 2020 (according to the specialist food-and-drink consultancy Zenith International). The expansion of juice production creates a huge amount of waste. This agroindustrial waste has a substantial economic and environmental impact, causing disposal problems due to the high costs related to drying, storage, and shipment, and can pollute the environment.2 Fruits from tropical and subtropical zones have smaller amounts of edible portions and are known to produce higher ratios of wastes (more commonly called by-products) than those from temperate zones. Such fruits generate a significant amount of waste material, such as peels, seeds, and stones.3 These residues are also sources of fibers and many bioactive compounds, which also may be useful to the food industry as functional ingredients, which also helps in solving environmental problems resulting from its disposal.4–10 In this context, the processing of Passiflora edulis Sims (mainly the variety P. edulis Sims f. floricarpa, also known as yellow passion fruit) results in large quantities of residues due to its large cultivation and juice production, especially in Brazil. This plant is grown mainly in Brazil, Colombia, and Ecuador and has high economic importance in these countries.11 Brazil is the largest producer and consumer of P. edulis f. flavicarpa in the world (accounting for 60% of the total world production). To solve the huge amount of residues generated by the juice industry, the study of P. edulis Sims by-products as a functional ingredient is an interesting and feasible approach.12 P. edulis Sims peel flour (PESPF) (yellow and purple varieties) provides significant amounts of phenolic compounds, showing antioxidant capacity and high dietary fiber content.4, 9, 10, 13–16 Studies have demonstrated the ability of PESPF to improve some health issues, such as hypertension, glycemia, dyslipidemia, and obesity complications.4, 7, 17–20

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The interest in sources of bioactive compounds is linked to the decrease in oxidative stress, which is commonly seen as a key factor in several chronic diseases. Fibers, mainly soluble fibers like pectin, can control glycemia, improves lipid profiles, and produces short-chain fatty acids, which in turn are related to anti-inflammatory effects.21, 22 Shortchain fatty acids lower colonic pH, which can affect the microbiota composition, leading to an improvement of gut health.23, 24 This chapter will approach the chemical aspects, bioactive compounds, and health effects of PESPF.

PASSION FRUIT The genus Passiflora belongs to the Passifloraceae family and Passiflorales order,25 and it encompasses about 500 species distributed mainly in the warm temperate and tropical regions of America, Asia, Australia, and tropical Africa.26 Brazil and Colombia account for approximately 30% of Passiflora species, being centers of diversity.11 The Passiflora genus is the most economically exploited of the Passifloraceae family.27 The most widespread species are P. edulis Sims, which includes such varieties as yellow passion fruit, purple passion fruit, and sour passion fruit; and Passiflora alata, while only sporadic reports are available on other species of Passiflora.28, 29 Passiflora is an exotic genus, usually used for its fruits, derivatives, and as a medicinal plant. Also, many of these species are still cultivated for their beautiful ornamental flowers, which have great ornamental value in the United States and certain European countries.11, 30 The P. edulis Sims species is the most popular, being widely used by the food industry for juice and pulp production. In the America and Europe, their leaves are applied as sedatives or tranquilizers. The fruits are a berry type and come in many shapes, such as globular, ovoid, and oblong. They have a sour taste and a strong and exotic aroma, and the weight varies from 30 to 300 g per fruit, diameter from 4.9 to 9 cm, and length from 4.3 to 7.2 cm. The color ranges diversely, being purple, yellow, reddish, and greenish.25 The passion fruit is basically composed of epicarp, which corresponds to the external layer; mesocarp, which is the white internal layer; endocarp or pulp; fresh mucilage; and seeds.25 P. edulis f. flavicarpa is commonly called yellow passion fruit, yellow granadilia, or pomme liane jaune;31 and P. edulis Sims edulis is known as purple passion fruit or purple granadilia.26 Brazil produced 703,489 tons of P. edulis Sims in 2016, mainly concentrated in the northeastern part of the country; this constitutes approximately 70% of the national crop according to the Brazilian Institute of Geography and Statistics (IBGE 2016).32 P. edulis f. flavicarpa is the variety on which the world’s commercial production is based.33 The cultivation is focused on juice and pulp industrial production, especially due to its more acidic flavor and higher financial income. In order to get the citrus pulp, peel, and bagasse (seeds and mucilage) are separated, resulting in by-products from industrial processing. The seeds and peels represent up to 26% and 50% of the fruit’s weight, respectively, generating huge amounts of waste.11 In many situations, this amount of biomass is used in animal feed or as fertilizers.33 Peels account for 50% of the fruit’s weight and are also used for flour production;25 furthermore, they have been used by industry as an alternative to pectin.13, 34 The seeds represent 4%–12% of passion fruit and contain around 30% of the oil in the plant. This oil is extracted for cosmetic production.35 The exploitation of by-products brings economic and environmental advantages, generating income through the formulation of products with high added value and avoiding the inappropriate discard of biomass into the environment.25 Despite current efforts toward the reutilization of industrial residues, large amounts of passion fruit peels remain underutilized. Efforts have been under way to find new uses for passion fruit peel flour.33 In the past decades, much progress has been made, including findings related to nutritional composition, bioactive molecules, and their correspondence with health benefits.33

PASSION FRUIT PEEL: CHEMICAL ASPECTS AND BIOACTIVE COMPOUNDS Studies revealed that PESPF presents low caloric value and is abundant in potassium (K) and also has significant iron (Fe), zinc (Zn), and manganese (Mn) content (Table 1). Such micronutrients are important for the metabolism, and P. edulis Sims peel can be considered as an alternative source for the intake of these minerals.33 Furthermore, PESPF is constituted for 60%–70% of fiber (nearly 20% of soluble fiber and 40%–50% of insoluble fiber) (Table 1).37, 40–42 Among the passion fruit fibers, pectin could be highlighted as a complex carbohydrate from plants with technological and physiological function.10, 37 Soluble fibers are able to form a gel in the surface intestinal absorption, complicating the transport of glucose, lipids, and cholesterol, which delays energy supply, avoiding its excessive consumption.43 Soluble fibers (pectins, hemicelluloses, and gums) or resistant starches are also substratum to fermentation by enteric bacteria, following by the production of short-chain fatty acids such as butyrate, propionate, and acetate.44, 2. FLOURS AND BREADS

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PASSION FRUIT PEEL: CHEMICAL ASPECTS AND BIOACTIVE COMPOUNDS 45

Butyrate represents a primary source of energy to colonocytes, and it may stimulate mucus secretion, vascular flow, barrier permeability, motility, and water and electrolyte absorption.45 Insoluble fiber improves the bowel transit time, whereas soluble fiber is involved in the reduction of both blood cholesterol and intestinal glucose absorption.10, 45 The interest in PESPF has increased due to its being a great source of phytochemical substances that could have therapeutic action as immunomodulators, anticarcinogens, and antioxidants46, 47 because of their high content of alcaloides, flavonoids, and carotenoids.15 Powders of residues by-products of passion fruit showed higher amounts of total phenolic compounds (TPC) compared to the pulp of this fruit—around five times more [103
10.4 and 20
2.6 mg gallic acid equivalent (GAE)].48 Methanolic extract of PESPF (the yellow variety) showed considerable amounts of the flavonoid isoorietin and high antioxidant power, due to its ability to decrease reactive oxygen species (ROS) production by activated neutrophils; furthermore, it was also able to lower myeloperoxidase activity, demonstrating effects on inflammation16 (Table 2). PESPF also showed phytic acid and tannis (Table 1). Albedo from P. edulis shows significant phenolic and flavonoid content in the methanol, water and dimethyl sulfoxide (DMSO) extract. TPC ranges from 0.64–1.86 mg GAE g1, and total flavonoid (TF) content ranges from 0.64 to 5.12 mg rutin equivalent (RE) g1 of sample. Isoorietin and isovitexin were identified in the albedo; the highest amount of such compounds was found in the DMSO extract.51 TABLE 1 Proximate, Phytochemical Composition, and Antioxidant Capacity of P. edulis Sims Peel Proximate

Fresh peel (g/100g)14

Peel flour (g/100 g)36

Peel flour (g/100 g)37

Moisture

84.21

7.42

Energy (kcal/g)

29.91

122.95

Protein (g)

0.67

8.87

3.94

Fat (g)

0.01

3.39

0.31

Carbohydrates (g)

6.78

14.24

79.39

Total dietary fiber (g)

4.33

60.08

65.22

Insoluble dietary fiber (g)

39.96

48.12

Soluble dietary fiber (g)

20.13

17.11

Soluble pectin Ash (g)

Peel flour (mg/g)9

Peel flour38

Peel flour39

9.48

4.85 0.57

6.00

6.88

MINERALS Calcium (mg) Iron (mg) Magnesium (mg)

44.51 0.89 27.82

Phosphorum (mg) Potassium (mg) Sodium (mg)

178.40 43.77

Zinc (mg)

0.32

Copper (mg)

0.04

PHYTOCHEMICALS Isoorietin Phytic acid Tannin

0.653 37.0 308.21

Cyanidin 3-O-glucoside

Qualitative analysis

Quercitin 3-O-glucoside

Qualitative analysis Continued

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20. PASSIFLORA EDULIS PEEL FLOUR AND HEALTH EFFECTS

Proximate, Phytochemical Composition, and Antioxidant Capacity of P. edulis Sims Peel—cont’d Fresh peel (g/100g)

Proximate

Peel flour (g/100 g)

Peel flour (g/100 g)

Peel flour (mg/g)

Peel flour

Edulic acid

Qualitative analysis

Catechin

Qualitative analysis

Epicatechin

Qualitative analysis

Kampferol-3-O-glucoside

Qualitative analysis

Kampferol

Qualitative analysis

Luteolin-8C-neohesperoside

Qualitative analysis

Luteolin-8C-neohesperoside

Qualitative analysis

Protocatechuic acid

Qualitative analysis

Prunasin

Qualitative analysis

Peel flour

Total phenolic compounds (mg GAE g-1)

4.67
0.38

Flavonoids (mg catechin-1)

1.17
0.05

DPPH (%)

29.6
0.66 to 46.35
0.85

FRAP

34.95
2.02 to 38.65
1.55

ORAC

40.83
1.75 to 68.58
0.06

DPPH, assay 2,2-diphenyl-1-picrylhydrazyl; FRAP; Ferric Reducing Antioxidant Power assay (μmol Trolox equivalent (TE) g1 of sample); ORAC, Oxygen Radical Absorbance Capacity (μmol TE g1 of sample).

TABLE 2

Therapeutic Properties of the Most Studied P. edulis Sims

P. edulis Sims

Extract

Bioactive compounds

Experimental model

Main results

References

P. edulis Sims f. flavicarpa

Methanolic extract

Isoorietin

Isolated neutrophils Antioxidant assay

Decrease ROS production and inflammation toward myeloperoxidase and on equine neutrophils

16

P. edulis Sims f. flavicarpa

Hot water extract

Not identified

Rats

Old rats were divided into two groups (control and fed with P. edulis peel flour); after 15 days, the experimental group showed lower antioxidant status in serum (ORAC assay) and lower lipid peroxidation in kidneys and higher in liver compared to the control

39

P.edulis Sims edulis

Methanolic extract

Cyanidin 3-O-glucoside, quercitin 3-O-glucoside and edulic acid

Humans

Human with stage 1 or 2 essential hypertension were treated with P.edulis Sims edulis extract pill (200 mg, twice a day) for 4 weeks showed a decrease in SBP and DBP compared to the control group

7

P. edulis Sims f. flavicarpa

Not evaluated

Not evaluated

Humans

P. edulis Sims f. flavicarpa peel flour intake (30 g/day–2 months) by type 2 diabetes mellitus patients improves insulin resitance

6

P. edulis Sims f. flavicarpa

Not evaluated

Not evaluated

Humans

P. edulis Sims f. flavicarpa peel flour intake (30 g/day–1 and 2 months) by women presenting dyslipedemia decrease total and LDL blood cholesterol concentration

49

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EFFECTS OF PASSION FRUIT PEEL ON GUT HEALTH

TABLE 2

Therapeutic Properties of the Most Studied P. edulis Sims—cont’d

P. edulis Sims

Extract

Bioactive compounds

Experimental model

Main results

References

P.edulis Sims edulis

Methanolic extract

Cyanidin-3O-glucoside, quercitin 3-O-glucoside and edulic acid

Humans

Purple passion fruit peel was able to decrease cardiovascular risk factors in type 2 diabetic subjects. A significant reduction in SBP and fasting and postprandial blood glucose levels was observed in the purple passion fruit– treated group (220 mg/day) after 16 weeks

18

P. edulis Sims f. flavicarpa

Not evaluated

Not evaluated

Rats

Diabetic rats fed on a diet (AIN93 M) supplemented with yellow passion fruit peel flour (5%) decrease the serum glucose level in 59%, reaching smilar values of the control group

41

P. edulis Sims f. flavicarpa

Not evaluated

Not evaluated

Rats

Ingestion ofpassion fruit peel flour by rats with 2,4,6-trinitrobenzenesulfonic acid-induced colitis protected the colon of animals from lipid peroxidation and improve antioxidant status in serum compared to the control

50

P. edulis Sims f. flavicarpa

Not evaluated

Not evaluated

Rats

Rats fed on a high-fat diet with passion fruit peel flour saw decreased adiposity and leptin level; increased CART, GLP1, hypothalamic cocaine expression, and improved insulin sensitivity

4

PESPF (yellow variety) extracted with boiled water showed amounts of TPC of 4.67
0.38 mg GAE g1 of sample and flavonoids of 1.17
0.05 mg catechin g1 of sample. The aqueous, methanolic/acetone, and ethanolic extracts of PESPF differed in the TPC range from 2.06
0.08 to 2.53
0.03, with the greater amount being water extract; for the 2,2-diphenyl-1-picryhydrazyl free radical (DPPH) (%) assay, the range was from 29.6
0.66 to 46.35
0.85; with the greater amount also being water extract; for the Ferric Reducing Antioxidant Power (FRAP) assay, the range was from 34.95
2.02 to 38.65
1.55 μmol Trolox equivalent (TE) g1 of sample, with the greater amount being methanolic/ acetone extract; and for the Oxygen Radical Absorbance Capacity (ORAC) assay, the range was from 40.83
1.75 to 68.58
0.06 μmol TE g1 of sample, with the greater amount being methanolic/acetone extract.39 Another study with the same sample was shown to have water extract flavonoid amounts of 1.17
0.05 mg catechin g1.38 PESPF (purple passion fruit) contains cyanidin 3-O glucoside, quercitin 3-O glucoside, and edulic acid7 as the major components; proto-catechuic acid, (–)-catechin, prunasin + acid glycoside, (+)-epicatechin, , kaempferol-3O-glucoside, luteolin-8-C-neohesperidoside, uteolin-8-C-digitoxoside, and kaempferol.52

EFFECTS OF PASSION FRUIT PEEL ON GUT HEALTH Dietary fiber intake has been shown to modulate gut microbiota by altering bacterial fermentation, colony size, and species composition. Consumption of fermentable dietary fiber provides substrates for microbial activity, but it will also increase the concentrations of fermentation products, such as short-chain fatty acids. Increased levels of shortchain fatty acids, in turn, lower the colonic pH, affecting the microbiota composition.23 Short-chain fatty acids, mainly butyrate, are important substrates for maintaining the colonic epithelium. Butyrate is the preferred fuel used by coloncytes, and the primary site of butyrate sequestration in the body is the gut epithelium. The short-chain fatty acids are related to several other positive health effects, including those related with management of obesity, glucose homeostasis, appetite, and dyslipidemia.36 The passion fruit pericarp (epicarp, the white part; and mesocarp, the yellow part) is rich in dietary fiber, both insoluble and soluble (mainly pectin).50 Although the studies indicate a large amount of dietary fiber in PESPF, its effect on gut health specifically remains little explored. The effect of passion fruit peel on colitis models is already reported.24, 53

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Long-term PESPF intake promoted improvement in antioxidant status, modulated the microbiota, and increased short-chain fatty acids in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats.53 Also, PESPF intake exerted an intestinal anti-inflammatory effect and attenuated the colonic damage in the dextran sodium sulfate model of mouse colitis.24 The authors found reduced pro-inflammatory cytokine expression and an enhanced intestinal protective barrier, as shown by biochemical and molecular analyses. Further, they increase short-chain fatty acid formation (acetic acid and butyric acid) in vitro, thus supporting a prebiotic effect.24 The effects of PESPF intake were evaluated in rats as well.54 However, this study revealed elevated butyrate amount in cecal content without alteration in microbiota (Lactobacillus, Bifidobacterium, Enterobacteriaceae, and Total Aerobic Microbial Content). Also, the addition of PESPF in the diet did not change the antioxidant enzyme activity (glutathione reductase, glutathione peroxidase, and superoxide dismutase) and thiol groups (glutathione) in intestine tissue.54 These studies suggest that PESPF may improve gut health by increasing short-chain fatty acid production, especially on inflammatory bowel diseases. Therefore, more studies, especially clinical trials, on the effects of PESPF on the colonic fermentation and its consequences are necessary.

EFFECTS OF PASSIFLORA EDULIS PEEL FLOUR IN METABOLIC PARAMETERS PESPF has demonstrated positive effects in metabolic parameters such as control of glycemia, lipid profiles, and antioxidant status of tissues in animals and humans.37, 38, 40, 49, 55, 56 The ingestion of PESPF (the yellow variety) by diabetic rats decreased serum glucose level by 59% compared to the diabetic rats that did not receive treatment, reaching the normal glycemic amount (112.6 mg/dL) and an increment of 71% in hepatic glycogen was found (Fig. 1). Such results suggest that the PESPF mechanism that decreases blood glucose level is the transformation of blood glucose into liver glycogen.42 Similar results were showed in a clinical study in which the intake of 30 g/day of PESPF (the yellow variety) for 2 months by type 2 diabetes mellitus patients improves blood glucose fasting, homeostatic model assessment (HOMA-IR), and glycated hemoglobin (Fig. 1). Such results showed that supplementation of PESPF decreased insulin resistance in type 2 diabetes mellitus patients, suggesting that it could be used as an adjuvant therapy in conventional treatments.6 The same dose of PESPF (the yellow variety) showed to reduce blood cholesterol concentration in women with dyslipidemia. Reductions of 31.7
28 mg/dL and 47.0
29.5 28 mg/dL in total blood cholesterol concentration were observed after 30 and 60 days of treatment, respectively, and both were statistically significant (Fig. 1).57 Similar results

FIG. 1 The main effects of Passiflora edulis Sims peel flour intake in health parameters.

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EFFECTS OF PASSIFLORA EDULIS PEEL FLOUR IN METABOLIC PARAMETERS

FIG. 2

255

Possible action routes of Passiflora edulis Sims peel flour in the metabolic parameters.

were observed when HIV patients were treated with PESPF (the yellow variety) (30 g/day for 30 days) effectively reduced the cholesterol and triacylglyceride levels. Furthermore, the ingestion of 30 g/day of PESPF for 90 days ameliorated lipodystrophy on these patients.19 In another study, the intake of PESPF (the yellow variety) by rats fed on a high-fat diet counteracted cumulative body weight gain and increased hypothalamic cocaine and amphetamine-regulated transcript expression (CART), indicating regulation of satiety. Furthermore, PESPF, in the same study, decreased adiposity and leptin level whereas increased glucose-dependent insulinotropic polypeptide, adiponectin, glucagon-like peptide-1 (GLP-1), and improve insulin sensitivity4 (Fig. 1). In old rats, the ingestion of PESPF (the yellow variety) modulates in different ways the antioxidant tissue status, and in kidneys it decreases lipid peroxidation, in contrast to those found in liver, in which lipid peroxidation was higher than the control group.38 The possible explanation for the effects cited in the studies discussed up to now would be the presence of fiber in passion fruit peel (mainly pectin), which encourages gel formation, leading to changes in gastric-emptying time and intestinal transit, decreasing the velocity of absorption of carbohydrates and fat. In addition, it may inhibit micelle formation. Furthermore, this gel can complex with bile salts increasing cholesterol excretion21, 22 (Fig. 2). In addition, the fibers of the Passiflora peel may be fermented in the colon and, beyond the effects on bowel heath noted in this chapter, it might increase butyrate and propionate, two short-chain fatty acids that have shown antiinflammatory capacity, as they can kβ-activation.58, 59 Activated NF-kβ promotes the expression of TNF-α, inducible nitric oxide synthrase (iNOS), and cyclooxygenase (COX), which induce the action of lymphocytes, monocytes, and endothelial cells, triggering or increasing inflammation.58, 60 As for PESPF (the purple passion variety), positive effects on health parameters have been demonstrated in the scientific literature, such as oral administration of PESPF extract (150 mg/day) for patients with asthma for 4 weeks in a randomized, placebo-controlled, and double-bind study. It was found to improve wheeze, cough, and shortness compared to placebo. Such results are given for the presence of flavonoids in PESPF extract, which can decrease oxidative stress involved in the pathophysiology of asthma. They can inhibit histamine release, arachidonic acid metabolism, and cytokine production.5 In another study, humans with stage 1 or 2 essential hypertension were treated with PESPF extract pill (200 mg, twice a day) for 4 weeks, and they showed decreases in systolic blood pressure (SBP) and diastolic blood pressure (DBP) compared to the control group.7 PESPF (the purple passion variety) was also able to decrease cardiovascular risk factors in type 2 diabetic subjects. Such individuals had fasting blood glucose levels >140 mg/dL and SBP/DBP >140/90 mm Hg. A significant reduction in SBP, fasting blood glucose levels (22.6 + 10.8 mg/dL; P ¼ 0.4), and postprandial blood glucose levels (21.8 + 21.9 mg/dL; P ¼ 0.33) were observed in the PESPF-treated group (220 mg/day) after 16 weeks.18 Epidemiological studies have indicated a significant inverse association between dietary flavonoids and mortality from coronary heart disease.61 Flavonoids (mainly quercetin) have been shown to decrease arterial blood pressure, decrease aortic medial thickening, and attenuate cardiac hypertrophy in various animal models of cardiovascular disease.62, 63 Quercetin,

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luteolin, and cyanidin 3-O-glucoside can downregulate iNOS expression or scavenging of nitric oxide (NO), affecting the endogenous antioxidant system and leading to the modification of vascular tone and peripheral vessel resistance, thus lowering blood pressure (Fig. 2).7, 17 Therefore, including PESPF in the diet could increase the dietary intake of fiber and antioxidants. In addition, it could aid in the prophylaxis and therapeutic of some diseases. Furthermore, the aggregate value in by-products is of economic, scientific, and technological interest and may minimize the generation of industrial processed residuals and environment impact, as well as increasing industrial profitability.4, 8–10, 16, 19, 22, 33, 47, 48

CONCLUSIONS Food waste, or by-products, are a topic of concern in modern society. This represents not only a resource problem, but also environmental and economic ones. Thus, searching for solutions to incorporate by-products into the food industry is a concern as well. Emerging studies support the positive effect of PFF intake on gut health and other metabolic benefits. This fiber-rich by-product could fortify foods, increase their dietary fiber and other bioactive compound content, and create healthy products. However, clinical trials are needed to determine what dose is safe and effective for showing benefits to health.

Acknowledgments MRMJ acknowledges CNPq (301108/2016-1) and FAPESP (2012/12322-0 and 2015/50333-1) for financial support.

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48. Oliveira DA, Angonese M, Gomes C, Ferreira SRS. Valorization of passion fruit (Passiflora edulis sp.) by-products: Sustainable recovery and biological activities. J Supercrit Fluids 2016;111:55–62. https://doi.org/10.1016/j.supflu.2016.01.010. 49. de Queiroz M do SR, Janebro DI, da Cunha MAL, et al. Effect of the yellow passion fruit peel flour (Passiflora edulis f. flavicarpa deg.) in insulin sensitivity in type 2 diabetes mellitus patients. Nutr J 2012;11(1):89. https://doi.org/10.1186/1475-2891-11-89. 50. Lima GC, Vuolo MM, Batista TG, Dragano NRV, Solon C, Maróstica Junior MR. Passiflora edulis peel intake improves insulin sensitivity, increasing incretins and hypothalamic satietogenic neuropeptide in rats on a high-fat diet. Nutrition 2016;32(7-8). https://doi.org/10.1016/j. nut.2016.01.014. 51. López-Vargas JH, Fernández-López J, Perez-Álvarez JA, Viuda-Martos M. Chemical, physico-chemical, technological, antibacterial and antioxidant properties of dietary fiber powder obtained from yellow passion fruit (Passiflora edulis var. flavicarpa) co-products. Food Res Int 2013; 51(2):756–63. https://doi.org/10.1016/j.foodres.2013.01.055. 52. Chilakapati SR, Serasanambati M, Manikonda PK, Chilakapati DR, Watson RR. Passion fruit peel extract attenuates bleomycin-induced pulmonary fibrosis in mice. Can J Physiol Pharmacol 2014;92(8). https://doi.org/10.1139/cjpp-2014-0006. 53. Cazarin CBB, da Silva JK, Colomeu TC, et al. Passiflora edulis peel intake and ulcerative colitis: approaches for prevention and treatment. Exp Biol Med 2014;239(5):542–51. https://doi.org/10.1177/1535370214525306. 54. da Silva JK, Cazarin CBB, Bogusz Junior S, Augusto F, Maróstica Junior MR. Passion fruit (Passiflora edulis) peel increases colonic production of short-chain fatty acids in Wistar rats. LWT—Food Sci Technol 2014;59(2P2):1252–7. https://doi.org/10.1016/j.lwt.2014.05.030. 55. Batista ÂG, Ferrari AS, Da Cunha DC, et al. Polyphenols, antioxidants, and antimutagenic effects of Copaifera langsdorffii fruit. Food Chem 2016;197:1153–9. https://doi.org/10.1016/j.foodchem.2015.11.093. 56. Clifford MN, Scalbert A. Review ellagitannins—nature , occurrence and dietary burden. J Sci Food Agric 2000;80(November 1999):1118–25. https://doi.org/10.1002/(SICI)1097-0010(20000515). 57. Ramos AT, Cunha MAL, Sabaa-Srur AUO, et al. Uso de Passiflora edulis f. flavicarpa na reduc¸ão do colesterol. Brazilian J Pharmacogn 2007; 17(4):592–7. https://doi.org/10.1590/S0102-695X2007000400019. 58. Miles-Brown JP, Chassaing B, Gewirtz AT. Microbiota metabolism of soluble fiber protects against low-grade inflammation and metabolic syndrome. Gastroenterology 2015;148(4):S541. https://rsm.idm.oclc.org/login?url¼https://www.rsm.ac.uk.htm?url¼https://dialog.proquest.com/ professional/professional/docview/1682704799?accountid¼138535%0Ahttp://vw4tb4ff7s.search.serialssolutions.com?ctx_ver¼Z39.88-2004& ctx_enc¼info:ofi/enc:UTF-8&rfr_id. 59. Cani PD, Osto M, Geurts L, Everard A. Involvement of gut microbiota in the development of low-grade inflammation and type 2 diabetes associated with obesity. Gut Microbes 2012;3(4). https://doi.org/10.4161/gmic.19625. 60. Panasevich MR, Allen JM, Wallig MA, Woods JA, Dilger RN. Moderately fermentable potato fiber attenuates signs and inflammation associated with experimental colitis in mice. J Nutr 2015;145(12):2781–8. https://doi.org/10.3945/jn.115.218578. 61. Feliciano RP, Pritzel S, Heiss C, Rodriguez-Mateos A. Flavonoid intake and cardiovascular disease risk. Curr Opin Food Sci 2015;2:92–9. https:// doi.org/10.1016/j.cofs.2015.02.006. 62. Duarte J, Perez-Palencia R, Vargas F, et al. Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br J Pharmacol 2001;133(1):117–24. https://doi.org/10.1038/sj.bjp.0704064. 63. Mu MM, Chakravortty D, Sugiyama T, et al. The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells. J Endotoxin Res 2001;7:431–8. https://doi.org/10.1179/096805101101533034.

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C H A P T E R

21 Micronutrient Fortification of Flours—Developing Countries’ Perspective Saeed Akhtar, Tariq Ismail, and Majid Hussain Institute of Food Science & Nutrition, Faculty of Agricultural Sciences & Technology, Bahauddin Zakariya University, Multan, Pakistan

O U T L I N E Flour Fortification—An Overview

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Micronutrient Malnutrition and Fortification Trends Asia Africa Latin America

264 264 265 265

Fortification Approaches

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Agricultural Approach

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Supplementation Trends Biofortification

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Role of Government Agencies

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Future Challenges and Strategies of Flour Fortification

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References

269

FLOUR FORTIFICATION—AN OVERVIEW Food fortification is the mechanism of adding any external essential nutrient or any other ingredient to a food commodity that may or may not already contain that nutrient or ingredient for the purpose of improving, correcting, and/ or preventing any revealed deficiency of one or more nutrients in any food. Asia and Africa contain the world’s largest share of micronutrient-deficient populations, and this state of undernourishment is threatening millions of newborns and fetuses to have risks of birth defects, stunted growth, and high rate of mortality.1 Foods being low in micronutrients leave the body vulnerable to nutritional deficiencies, thus anticipating an increased disease burden among vulnerable populations. Fortification has been proven to be the simplest and most cost-effective intervention to raise the nutritional potential of staple foods and subsequently deliver the required amount of micronutrients to support the body’s normal growth and development. Fortified foods, including flours, salts, sauces, edible oils, and many more, improve the physical and economic situation for millions of malnourished people in Asia, but a larger segment of these vulnerable populations continue to not meet the adequate bodily needs when it comes to essential micronutrients. Wheat flour fortification has global acceptability as a way to solve the problem of nutritional-deficiency disorders, including anemia and neural tube defects. However, flour fortification with nutrients like iron and folates has not yet become a routine practice in Asia. Presently, only six countries (Indonesia, the Philippines, Vietnam, Nepal, Fiji, and the Solomon Islands) are practicing mandatory flour fortification, and mandatory fortification in these countries has been legislated between 2000 and 2016.2 Relatively lower rate of wheat consumption is reported in some Asian countries wherein wheat is not a principle staple grain that results poor improvement in iron status of malnourished population.3

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00021-6

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21. MICRONUTRIENT FORTIFICATION OF FLOURS—DEVELOPING COUNTRIES’ PERSPECTIVE

African countries are struggling with nutrient inadequacies and suffering from mild to severe micronutrient deficiencies. Hence, fortification of foods with deficient nutrients is a major need at this point in time. Cereals being principle source of energy for 40%–60% African population are reported as poor contributory to micronutrients. Consequently, a vast majority of peoples living on cereals based monotonous foods remain vulnerable to micronutrients deficiencies. Fortification of staple cereals and product derived thereof on account of its cost effectiveness, easier product processing and improved nutrients delivery substantially contribute in alleviating micronutrients malnutrition.4 Inadequacies of micronutrients and the resulting malnutrition have affected more than 2 billion people in developing countries, especially in Central and West Africa.5 These inadequacies (sometimes referred to as “hidden hunger”) of micronutrients are due to poor micronutrient bioavailability, imbalanced diet, and fewer micronutrient-dense foods, usually consisting of sole-ingredient foods. The primary foods that need this intervention are carbohydrate-based staples with fewer nutrients. Iron and zinc are the most deficient micronutrients in human nutrition, and various other essential minerals like magnesium, copper, and calcium are often lacking in the human diet as well.6 Concentrating solely on the African countries, the risk of nutritional inadequacies is of more concern. In terms of mineral adequacies, intake of magnesium is relatively low (i.e., < 4%), even though the staple cereal crops of that region such as pearl millet (Pennisetum glaucum), pearl millet (P. glaucum), and maize (Zea mays), are known to provide ample amounts of this element.7 Micronutrient undernourishment is a key issue in African countries, but it can be improved through innovative and effective strategies that incorporate deficient nutrients in foods through fortification. Major staple food crops, such as wheat, rice, and maize, are fortified with a wide variety of nutrients such as riboflavin, iron, zinc, thiamin, vitamin B12, vitamin A, folic acid, and niacin. Cereal grain fortification has improved the nutritional status and overall health at a global level. Health-related disorders such as anemia, neural tube defects, and other diseases are reduced through innovative fortification strategies.4, 8 In the developing world, malnutrition is a serious issue among children. Due to inadequate complementary feeding, children between 6 and 24 months are severely affected by lack of nutrients, including growth problems.9 Anemia remains a public health issue in Latin America and the Caribbean. Its prevalence among children (35% total carbohydrates (Table 2). There are an array of commercial defatted flours that vary in their protein dispersibility index (PDI), which is closely related to the extent of protein denaturation in the D-T. Flours with high PDI got light heat treatment and therefore are light-colored, whereas low PDIs indicate a high degree of protein denaturation and deactivation of antinutritional factors. Defatted flours regularly contain higher levels of isoflavones compared to whole soybeans (Table 2).

Texturized Soybean Protein Texturized soy protein is probably the most popular form of defatted flour available to consumers. It is produced from flour that is conditioned and then continuously cooked under high pressure in a thermoplastic extruder in order to denature and link proteins and produce a variety of textured and shaped products. Flavorings and coloring agents can be added to the soybean flour before processing. After hydration, the texturized proteins are commonly used as meat extenders in hamburger patties and processed meat–cured products.37

PROTEIN CONCENTRATES Protein concentrates contain between 65% protein and 5%–6% DF (Table 2). From 100 units of defatted flour are usually obtained 50–55 units of concentrate. There are two ways to produce commercial concentrates: the alcohol or the isoelectric protein precipitation processes. The alcohol used in the first process removes soluble sugars, whereas the acid precipitation of the second process yields a protein curd and a whey rich in soluble sugars that is discarded. These processes yield concentrates with improved flavor and functional features, especially in terms of water and oil absorption indexes and emulsification capacity.37

Protein Isolates Among commercial food products, the soybean isolates are the ones containing the highest protein (88% protein, Table 2). The extraction process practically removes all soluble sugars and DF from the defatted flour. As a result, the colorless isolates have a bland or neutral flavor and in terms of nutrition, they have very high protein digestibility (>94%) and EAA balance. Functionally, protein isolates have high water absorption indexes, water solubility, and excellent emulsifying and gelling properties. Isolates are obtained by treating the finely ground defatted flour with alkali [sodium hydroxide (NaOH)] to solubilize proteins and discard insolubles after centrifugation or use of continuous decanters. Then, the soluble protein separated beforehand is precipitated by lowering the pH (4.2–4.5) with hydrochloric acid (HCl) to the isoelectric point of globulins. The resulting protein curd is again segregated into decanters or centrifuges, washed, adjusted to a higher pH, and spray-dried into a powder. From 100 units of defatted soybean are usually obtained less than 30 units of the isolate.37

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23. SOYBEAN-FORTIFIED WHEAT FLOUR TORTILLAS

Soymilk Traditionally, soymilk is the liquid extract obtained from whole soybeans, although analog products are also produced from protein isolates. The traditional soymilk contains a similar composition of cow’s milk. There are two major processes to obtain it: traditional and modified (i.e., Illinois and other processes). The last is aimed toward the deactivation of lipoxygenase, and therefore produces extracts without the strong beany flavor so it will better appeal to Western tastes. Soymilk generally contains most of the active phytochemicals present in soybeans, including high amounts of isoflavones. Many are fortified with micronutrients (such as vitamins A and D and calcium) to boost their position as a viable alternative to cow’s milk, and some are flavored to improve acceptability. The basic steps of soymilk production are to soak seeds for several hours, perform wet-grinding and cooking, and use filtration or centrifugation to separate the okara. The western processes supplement alkalis (bicarbonate or calcium hydroxide) during soaking to inactivate lipoxygenase, cooking before grinding, or application of stream during this operation to inactivate this enzyme responsible for flavor reversion. Alternatively, the soybean extract can be evaporated and spray-dried to produce powdered soymilk. From 100 units of soybeans are generally obtained 50 units of powdered milk and 50 units of dried okara. The soymilk solids contain high caloric density, high protein, most of the oil and lipophilic phytochemicals of the seeds, soluble sugars, vitamins, minerals, and isoflavones (Table 2).

Okara and Soybean Bagasse Okara is the fiber-rich residue obtained as a coproduct of the soymilk process. It is very high in moisture, and its solids are rich in DF and leftover protein and oil. The wet okara can be alternatively dehydrated in flash dryers and then finely ground in order to produce a shelf-stable product, which contains nearly 50% DF, 10%–15% oil, and about 20%–25% protein with a good EAA profile32, 38, 39 (Table 2). The soybean bagasse is a by-product rich in both DF and protein, with high levels of EAAs obtained after the processing of concentrates and isolates. The nutritional attributes of this by-product are similar to okara, with the exception that the first contains lower levels of oil (Table 2).

FORTIFICATION OF WHEAT-FLOUR TORTILLAS WITH SOYBEAN PRODUCTS The fortification of wheat-flour tortillas with soybean products is relatively easy because after producing the composite flours, the process is basically the same as shown previously in Fig. 1. In most instances, the addition of the soybean proteins or fiber-rich coproducts significantly increases dough water absorption and modifies the time required to achieve optimum dough development. The rest of the operations are practically the same.

Fortification With Soybean Protein Products Research conducted in our facilities has demonstrated that is feasible to fortify wheat-flour tortillas with full-fat, defatted flours (varying in PDI), soymilk solids, concentrates, or isolates. The optimum addition of these proteins is the quantity necessary to double the lysine content, although factors like sensory properties, cost of the intervention, tortilla texture during the expected shelf-life, and color should strongly be considered. The recommended amounts of full-fat flour, defatted flour, dried soymilk, concentrate, and isolate are 10%, 6%–8%, 5%, and 4%, respectively. Table 3 details the advantages and disadvantages of the soybean products that can be used to fortify flour tortillas. TABLE 3

Relative Comparison of Soybean Products in Terms of Cost, Flavor/Color, and Content of Kilocalories, Protein, DF, and Nutraceuticals

Soybean product

Relative cost

Flavor/color

Energy density

Protein

Dietary fiber

Nutraceuticals

Full-fat

Low

Strong/dark

High

Low-medium

Medium

High

Defatted flour

Low

Medium/medium

Medium

Medium

High

High

Concentrate

Medium

Light/light

Medium

Medium-high

Low

Medium

Isolate

High

Light/light

Medium

High

Very low

Low

Soymilk solids

Medium-high

Medium/light

Medium-high

Medium

Low

Medium-high

Okara/bagasse

Low

Medium/light

Low

Low

Very high

Very high

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FORTIFICATION OF WHEAT-FLOUR TORTILLAS WITH SOYBEAN PRODUCTS

Gonzalez-Agramon7 examined the effects of wheat-flour tortilla fortification with 11.1% defatted soybean flour, or 5.6% isolate in a commercial factory. As expected, the fortified tortillas contained higher levels of protein and lysine without affecting nutrient density (Table 4). This early study concluded that fortification with these products practically doubled the PER and improved nitrogen retention values (biological, or BV, and net protein utilization, or NPU values) of weanling rats when compared with regular tortillas, and the values were only 24% less than casein. The addition of these products decreased dough elasticity but increased dough water absorption by more than 5%, as well as the alveograph force required to stretch the dough and P/L ratio. The defatted flour-fortified tortillas had a better texture than their control counterparts and similar flavor and color as well, as judged by 50 untrained panelists. Recently, Perez-Carrillo40 evaluated five soybean proteins (four defatted flours varying in PDI and one concentrate) for production of hot-press tortillas (see Table 5). The aim was to increase their protein content by 15%–25% and obtain tortillas with upgraded protein quality and similar sensory attributes as the control. The soybean-fortified tortillas had increased yields due to the higher dough water absorption and enhanced EAA scores. Among the five proteins, the defatted flour with the lowest fat absorption index and PDI and the concentrate produced the best-quality tortillas. The TABLE 4

Effects of Defatted Soybean Flour or Isolate Fortification on Nutrient Composition, Digestibilities, Nitrogen Retention, and PERs of Wheat-Flour Tortillas,7a Wheat-flour tortilla

Tortilla fortified with 11.1 defatted-soybean flour

Tortilla fortified with 5.6 soybean isolate

Dough moisture (%)

39.0

41.9

42.3

Tortilla moisture (%)

29.3

32.2

33.6

Protein (%)

8.8

10.2

9.3

Energy (kcal)

323

300

296

Fat (%)

9.6

8.7

8.5

Crude fiber (%)

0.5

0.9

0.3

Ash (%)

2.2

2.6

2.2

NFE (%)

50.5

45.4

45.7

Lysine (g/100 g protein)

2.41

3.73

3.70

Dry matter

94.6

93.7

94.5

Energy

94.4

93.4

94.1

Protein

88.5

88.5

91.8

Chemical score (limiting amino acid)

44 (lysine)

71 (lysine)

68 (lysine)

Biological value

38.7

50.6

50.5

34.2

44.8

45.9

38.9

62.8

62.4

Feed intake (g/day)

6.99

10.31

11.12

Weight gain (g/day)

0.76

2.23

2.53

1.06

2.10

2.11

0.95

1.88

1.88

Nutrient CHEMICAL COMPOSITION

a

DIGESTIBILITIES (%)

PROTEIN QUALITY (%)

b

NPU

PDCAA,

4c

PER STUDY

d

PER

e

Corrected PER a b c d e

Nitrogen-free extract, an indication of starch and soluble carbohydrates. NPU ¼ (Biological Value  Protein Digestibility)/100. PDCAA Score ¼ (% Protein Digestibility  Chemical Score)/100. PER ¼ g weight gain/g protein intake. Corrected according to experimental value of casein (2.77).

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302 TABLE 5

23. SOYBEAN-FORTIFIED WHEAT FLOUR TORTILLAS

Functional Characteristics and EAAs of Five Soybean Protein Sources and Their Corresponding Hot-Press Tortillas40

Parameter

Defatted flour PDI 23

Defatted flour PDI 25

Defatted flour PDI 75

Defatted flour PDI 67

Concentrate PDI 52

Crude protein (dry basis)

42.4e

49.6c

58.6b

45.4d

67.1a

Nitrogen solubility (%)

19.7b

13.4c

24.0b

13.7c

36.1a

Fat absorption capacity (FAC)

2.50d

2.94b

2.65c

3.14a

2.70c

Water absorption index (WAI)

5.34b

4.80c

4.02e

4.29d

8.34a

Water solubility index (WSI)

25.67e

28.36d

50.27a

47.55b

36.95c

Lysine*

6.36

6.41

6.50

6.63

6.40

Tryptophan

1.57

1.61

1.51

1.43

1.41

Cysteine + methionine

2.88

2.95

3.02

3.06

2.54

AMINO ACID (G/100 G PROTEIN)

TORTILLAS Wheat-flour control Water absorption (%)

53

55

56

55

54

56

Dough-mixing time (min)

10.30

9.50

8.00

8.30

8.30

8.00

Yield [g tortilla/g flour (14% mb)]

1.31

1.37

1.37

1.37

1.39

1.44

Moisture (%)

29.72a

29.50a

29.52b

29.91a

28.10a

27.34a

Protein (N  6.25) (%)

7.87c

9.34b

10.04ab

10.14ab

9.78b

11.16a

Soluble DF (%)

2.03b

1.98b

2.17ab

2.49a

2.47a

1.86b

Insoluble DF (%)

2.17b

3.04a

3.07a

3.54a

3.76a

2.30b

Means with different letter(s) within raw are statistically different. Farinograph units.

defatted soybean flours with high PDI and relatively lower WAI produced sticky doughs, lower alveograph P/L values, and defective tortillas. The protein contents of the four defatted flours and the concentrate were about 4–6 times higher than the wheat flour (Table 5). More important, these soybean products contained between 11 and 15 times higher lysine content (3.1%–4.54%) compared to the refined wheat flour (0.27%). Therefore, the addition of approximately 6% and 4% of the various soybean flours and concentrate increased the lysine content in composite flours and tortillas by 1.6 times. Regarding the dough rheological properties (Table 7), composite flours containing the defatted flour with the lowest PDI or protein concentrate had the highest maximum alveograph overpressure values, indicating higher gluten strength, whereas the composite flour containing defatted flours with relatively higher PDI showed the highest dough-extensibility values. This indicates that the low heat–treated flours favored extensibility to a level similar to the control wheat flour. Unfortunately, these doughs were sticky and performed poorly during the dough-mixing stage. The best-performing composite flours produced with low-PDI defatted flour or the protein concentrate had G values between 13.25 and 13.3, and W values between 269 and 293  104 J (Table 7). Results of farinograph indicated that the addition of all soybean proteins significantly increased water absorption by about 6%. These differences are attributed to the nature of the soybean proteins, which are more hydrophilic than wheat gluten. In fact, the water absorption indexes of the various soybean proteins ranged from 4.26 in the high-PDI soybean flour to 5.26 in the lowest PDI counterpart (Table 5). All soybean protein sources significantly increased water absorption and reduced optimum dough mixing time between 8% and 22%. Because of the higher water absorption, these enriched tortillas yielded approximately 4.5%–9.9% more than the control. The highest protein content was observed in the fortified tortilla with the soybean concentrate. As expected, the DF concentrations increased in all defatted soybean flour-enriched tortillas (Tables 1 and 5). Consumer acceptability scores conducted with untrained panelists indicated that all tortillas had acceptability in the range of like to like much, with color and subjective texture values similar among treatments. However, objective texture analyses indicated that

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FORTIFICATION OF WHEAT-FLOUR TORTILLAS WITH SOYBEAN PRODUCTS

TABLE 6 Comparison of the Chemical Composition, Caloric Value, Amino Acid Scores, Digestibilities, Nitrogen Retention Values, PERs, and PDCAAS of Regular Wheat-Flour Tortillas with Soybean Concentrate-Fortified Tortillas41 Regular wheat-flour tortilla

Soybean-fortified wheat-flour tortilla2

NUTRIENT COMPOSITION (% DRY-MATTER BASIS) Crude protein (N  6.25)

7.95

9.84

Crude fat

10.5

9.72

Crude fiber

0.2

0.3

Ash

1.2

1.3

80.15

78.84

Energy value (kcal/100 g)

448.30

443.80

Protein:calorie ratio (g /100 kcal)

1.77

2.21

Threonine

80

88

Valine

117

122

Isoleucine

128

136

Leucine

105

108

Lysine

40

55

Histidine

108

114

Tryptophan

124

129

Methionine + cysteine

151

144

Phenylalanine + tyrosine

113

118

Nitrogen-free extract 3

ESSENTIAL AMINO ACID SCORES4 (%)

IN VIVO DIGESTIBILITY AND NITROGEN RETENTION VALUES (%) Dry matter digestibility

90.6 a

92.3 b

Protein digestibility

81.4 a

83.0 a

Biological value

57.9 a

61.8 b

Net protein utilization value

47.1 a

51.4 b

RAT GROWTH PERFORMANCE (PER AND PDCAAS) Initial weight (g)

45.6 a

45.7 a

Final weight (g)

62.2 a

109.5 b

Average daily gain (g)

0.6 a

2.3 b

Food intake (g/day)

7.6 a

11.5 b

Protein intake (g/day)

0.7 a

1.0 b

Experimental PER

0.9 a

2.3 b

Adjusted PER

0.69 a

1.77 b

32.57

45.64

PDCAAS

3

The bold means that Lysine is the most limiting essential amino acid or the one present in the lowest amount (40 and 55 for the regular and soybean fortified wheat flour tortilla, respectively). The average moisture content for regular and soybean concentrate-fortified tortillas was 29.0 and 30.6%, respectively. Means with different letters within a row are significantly different. Wheat-flour tortilla produced with a composite flour containing 97.7% wheat flour and 4.3% soybean protein concentrate. Calculated using the Atwater coefficient. FAO/WHO requirements for 2–5 years infant (expressed in g/100 g protein): threonine 3.4, valine 3.5, isoleucine 2.8, leucine, 6.6, lysine 5.8, histidine 1.9, tryptophan 1.1, methionine + cysteine 2.5 and phenylalanine + tyrosine 6.3.

3. FORTIFICATION OF FLOURS AND BREADS

304 TABLE 7

23. SOYBEAN-FORTIFIED WHEAT FLOUR TORTILLAS

Effect of Soybean Bagasse Supplementation on Yield, Physical, Chemical, and Sensorial Parameters of Commercial Hot-Press Tortillas2, 33 Tortilla containing

Parameter

Wheat-flour tortilla

5% soybean bagasse

10% soybean bagasse

Yield [g tortilla/g flour (14% mb)]

1.40

1.40

1.40

Water absorption (%, 14% mb)

54.00

58.00

58.00

Mixing time (min)

15:00

15:00

10:00

Water absorption [% (14% mb)]

60.2c

65.4b

72.5a

Dough development time (min)

8.28b

8.82b

8.85a

Stability (min)

10.0c

11.9b

12.4a

Diameter (cm)

12.96a

12.50a

12.78a

Thickness (mm)

2.40a

2.40a

2.41a

L*

77.05a

78.73a

75.67a

a*

2.39a

1.56b

2.28a

b*

18.19b

18.02b

21.24a

0.99ab

1.09a

0.89b

a

DOUGH PROPERTIES

MIXOLAB

PHYSICAL PROPERTIES OF TORTILAS

TORTILLA COLOR

TORTILLA ROLLABILITY++ Day 0

a

Day 1

2.86

3.96

4.09a

Day 5

2.80a

3.11a

3.95a

CONSUMER ACCEPTABILITY OF TORTILLAS (7-POINT HEDONIC SCALE)++ Flavor

5.61a

5.84a

5.55a

Texture

4.97a

5.23a

4.97a

Overall acceptability

5.52a

5.74a

5.26a

Moisture

31.31a

29.83b

31.96a

Fat

6.90a

7.05a

6.88a

Ash

1.65b

1.75b

1.88a

Total DF

1.22c

2.81b

3.37a

b

a

CHEMICAL COMPOSITION (WET BASIS) (%)

Soluble DF

0.25

0.51

0.69a

Insoluble DF

0.97c

2.30b

2.68a

Crude protein (N  6.25)

5.41b

5.86a

5.87a

KEY ESSENTIAL AMINO ACIDS (G/100 G OF PROTEIN) Lysine

2.59

2.79

2.99

Tryptophan

1.53

1.52

1.51

Cysteine + methionine

4.22

4.16

4.11

In vitro protein digestibility (%)

80.13

84.07

84.57

PDCAAS

36.34

41.14

44.22

The experimental composite soy-bagasse-enriched flours were produced by substituting 5% or 10% of the wheat flour with dried and ground soybean bagasse. + Each value is the mode of at least five subjective observations. 1 (no cracks; very flexible) and 5 (breaks immediately; cannot be rolled). ++ Each value is the average of 30 observations: 1 ¼ Dislike very much, 2 ¼ dislike moderately, 3 ¼ dislike slightly, 4 ¼ neither like nor dislike, 5 ¼ like slightly, 6 ¼ like moderately, 7 ¼ like very much.

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305

control and defatted soybean (low-PDI)-fortified tortillas had the lowest force values and best subjective tortilla dowel or rollability scores. In the case of objective color parameters, the only fortified tortilla that exhibited similar L* values compared to the control was the one fortified with soybean concentrate. Another study recently conducted by Acevedo-Pacheco41 evaluated the in vivo protein quality assessed with laboratory weanling rats of the soybean concentrate-fortified tortilla described previously. As observed by GonzalezAgramon,7 rats fed the soybean-fortified tortillas gained considerably more weight and had better BV and NPU values than counterparts fed the control tortilla. In fact, the PER improved from 0.69 in the control to 1.77 in the soybean concentrate–fortified tortilla (Table 6).

Fortification of Wheat-Flour Tortillas with Okara and Soybean Bagasse Okara and dry soybean bagasse are by-products rich in both DF and protein, with high levels of EAAs obtained after the processing of soymilk and concentrates/isolates, respectively. The nutritional attributes of these by-products are similar, with the exception that okara contains higher levels of oil (Table 2). According to Lu et al.,42 dehydrated okara contains 1.9% of soluble DF and 55.6% of insoluble DF. The protein of commercial soybean bagasse contains about twice as much protein, seven times more lysine, and at least eight times more TDF compared to refined wheat flour (Table 2). The effects of the substitution of refined wheat flour with 5% or 10% bagasse in dough rheology and hot-press tortilla texture, dimensions, color, protein, and DF content were studied by Montemayor et al.33 The major findings of this study indicated that the addition of 10% of bagasse did not affect dough hardness, cohesiveness, adhesiveness, and extensibility. However, mixolab parameters of control and composite doughs indicated that water absorption and stability increased slightly when soybean bagasse was included in the flour. The yield, dimensions, color, sensory acceptance, and textural shelf-life of supplemented tortillas were not affected by the addition of this level of soybean bagasse. Supplemented tortillas with 10% dried bagasse contained 9.3% more protein and about 30% more lysine, which improved the PDCAAS by 19 units. The control and 10% bagasse-containing tortillas provided 47.4% and 68.6% of the lysine required by an infant, respectively. Furthermore, the enriched tortillas contained 1.77 times more insoluble DF (Table 7). Unlike bran sources derived from cereals, the soybean bagasse containing more than 50% total DF of the bagasse did not significantly affect the color of the enriched tortillas. In short, results of this study indicate that the 10% soybean bagasse-enriched tortillas had similar physical and organoleptic features judged by 30 untrained panelists, and they are an excellent alternative to new consumers because of their higher protein, protein quality, and DF contents.

References 1. Food and Agriculture Organization, International Fund for Agricultural Development, UNICEF, World Food Programme, WHO. The state of food security and nutrition in the world 2017: building resilience for peace and food security. Retrieved from: http://www.fao.org/3/a-i7695e.pdf; 2018. [Accessed May 2018]. 2. Long J. Conquista y Comida. Consecuencias del Encuentro de Dos Mundos. 1st ed. Mexico City: Universidad Nacional Autónoma de Mexico; 1996. 3. Serna-Saldivar SO. History of corn and wheat tortillas. In: Rooney LW, Serna-Saldívar SO, editors. Tortillas: wheat flour and corn products. St. Paul, MN: American Association of Cereal Chemists; 2015. p. 1–28. 4. Kabbani J. Tortilla industry overview. Technical conference 2016. Las Vegas, Nevada: International Baking and Industry Exposition; 2016. 5. Serna-Saldivar SO. Nutrition and fortification of corn and wheat tortillas. In: Rooney LW, Serna-Saldívar SO, editors. Tortillas: wheat flour and corn products. St. Paul, MN: American Association of Cereal Chemists; 2015. p. 29–63. 6. World Hunger Education Service. World hunger and poverty facts and statistics 2018. https://www.worldhunger.org/world-hunger-andpoverty-facts-and-statistics/#children1. 2018. with the assistance of Crystal Lam [accessed June 20, 2018]. 7. Gonzalez-Agramon MM, Serna-Saldivar SO. Effect of defatted soybean meal and soybean isolate fortification on the nutritional, physical, chemical, and sensory properties of wheat flour tortillas. J Food Sci 1988;53:793–7. 8. Hikmet Boyacioglu M. Soybean ingredients in baking. In: Riaz MN, editor. Soy applications in food. Boca Raton, FL: CRC Press; 2006. p. 63–82. 9. Serna-Saldivar SO, Rooney LW, Waniska RD. Wheat flour tortilla production. Cereal Foods World 1988;33:855–64. 10. Waniska RD. Processing of wheat flour tortillas. In: Rooney LW, Serna-Saldívar SO, editors. Tortillas: wheat flour and corn products. St. Paul, MN: American Association of Cereal Chemists; 2015. p. 125–45. 11. Serna-Saldivar SO, Chuck-Hernandez C. Tortillas. In: Caballero B, Finglas PM, Toldrá F, editors. Encyclopedia of food and health. London, England: Elsevier; 2016. p. 319–25. 12. Book SL, Waniska RD. Leavening in flour tortillas. In: Rooney LW, Serna-Saldivar SO, editors. Tortillas: wheat flour and corn products. American Association of Cereal Chemists: St. Paul, MN; 2015. p. 159–83. 13. Serna-Saldivar SO, Lopez-Ahumada G, Ortega-Ramirez R, Abril-Dominguez JR. Effect of sodium-stearoyl-2- lactylate on the rheological and baking properties of bread fortified with defatted soybean and sesame meal. J Food Sci 1988;53:211–4.

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14. Serna-Saldivar SO, Rooney LW. Tortillas. In: Caballero B, Trugo L, Finglas PM, editors. Encyclopedia of food science and nutrition. 2nd ed. London, UK: Academic Press; 2003. p. 5808–13. 15. Waniska RD, Cepeda M, King BS, Adams JL, Rooney LW, Torres PI, Lookhart GL, Bean SR, Wilson JD, Bechteel DB. Effects of flour properties on tortilla qualities. Cereal Foods World 2004;49:237–44. 16. Bejosano F, Novie Alviola J. Dough conditioners in flour tortilla processing. In: Rooney LW, Serna-Saldivar SO, editors. Tortillas: wheat flour and corn products. American Association of Cereal Chemists: St. Paul, MN; 2015. p. 185–94. 17. McDonough CM, Novie Alviola J, Waniska RD. Preservatives: extending shelf life and shelf stability. In: Rooney LW, Serna-Saldivar SO, editors. Tortillas: wheat flour and corn products. American Association of Cereal Chemists: St. Paul, MN; 2015. p. 195–200. 18. Friend C, Serna-Saldivar SO, Wansika RD, Rooney LW. Increasing the fiber content of wheat tortillas. Cereal Foods World 1992;37:325–8. 19. Serna-Saldivar SO. Manufacturing of bakery products. In: Serna-Saldivar SO, editor. Cereal grains: properties, processing and nutritional attributes. Boca Raton, FL: CRC Press (Taylor & Francis Group); 2010. p. 259–328. 20. Serna-Saldivar SO. The fortification and enrichment of corn tortillas. In fortification of corn masa wheat iron and/or other nutrients—a literature and industry experience review. Washington, DC: SUSTAIN; 1997. p. 121–164. 21. Serna-Saldivar SO, Gutierrez Uribe J, García LS. Phytochemical profiles and nutraceutical properties of corn and wheat tortillas. In: Rooney LW, Serna-Saldívar SO, editors. Tortillas: wheat flour and corn products. St. Paul, MN: American Association of Cereal Chemists; 2015. p. 65–93. 22. Liu KS. Chemistry and nutritional value of soybean components. In: Liu KS, editor. Soybean: chemistry, technology, and utilization. New York, NY: Chapman & Hall; 1997. p. 25–113. 23. Messina MJ. Soyfoods; their role in disease prevention and treatment. In: Liu KS, editor. Soybean: chemistry, technology, and utilization. New York, NY: Chapman & Hall; 1997. p. 442–77. 24. Dixit AK, Antony JIX, Sharma NK, Tiwari RK. Soybean constituents and their health benefits. In: Tiwari VK, Mishra BB, editors. Opportunity, challenge and scope of natural products in medicinal chemistry. Research Signpost: Kerala, India; 2011. p. 367–83. 25. Zeisel SH. Choline: an essential nutrient for public health. Nutr Rev 2009;67(11):615–23. 26. Majumber A, Biswas B. Biology of inositols and phosphoinositides. Springer Science & Business Media; 2006. 27. Ling WH, Jones PJH. Dietary phytosterols: a review of metabolism, benefits and side effects. Life Sci 1995;57(3):195–206. 28. Van der Riet WB, Wight AW, Cilliers JJL, Datel JM. Food chemical investigation of tofu and its byproduct okara. Food Chem 1989;34(3):193–202. 29. Perkins EG. Composition of soybeans and soybean products. In: Erickson DR, editor. Practical handbook of soybean processing and utilization. Champaing, IL: AOAC Press and the United Soybean Board; 1995. p. 9–28. 30. Villanueva MJ, Yokoyama WH, Hong YJ, Barttley GE, Ruperez P. Effect of high-fat diets supplemented with okara soybean by-product on lipid profiles of plasma, liver and faeces in Syrian hamsters. Food Chem 2011;124(1):72–9. 31. United States Department of Agriculture. Nutrient Data Laboratory. In: Agricultural Research Service; 2018. http://www.nal.usda.gov/fnic/ foodcomp. 32. Kumar V, Rani A, Husain L. Investigations of amino acids profile, fatty acids composition, isoflavones content and antioxidative properties in soy okara. Asian J Chem 2016;28(4):903. 33. Montemayor-Mora G, Hernandez-Reyes KE, Heredia-Olea E, Perez-Carrillo E, Chew Guevara AA, Serna-Saldívar SO. Rheology, acceptability and texture of wheat flour tortillas supplemented with soybean bagasse. J Food Sci Technol 2018;55(12):4964–72. 34. Lo GS. Nutritional and physical properties of dietary fiber from soybeans. Cereal Foods World 1989;34(7):530–4. 35. Anderson JW, Smith BM, Washnock CS. Cardiovascular and renal benefits of dry bean and soybean intake. The Am J Clin Nutr 1999;70 (3):464s–474s. 36. Olmedilla B, Granado F, Blanco I, Vaquero M. Lutein, but not α-tocopherol, supplementation improves visual function in patients with agerelated cataracts: a 2-y double-blind, placebo-controlled pilot study. Nutrition 2003;19(1):21–4. 37. Hettiarachchy N, Kalaphaty U. Soybean protein products. In: Liu KS, editor. Soybean: chemistry, technology, and utilization. New York, NY: Chapman & Hall; 1997. p. 379–411. 38. Chang-Vong W, Liu S-Q. Biovalorisation of okara (soybean residue) for food and nutrition. Trends Food Sci Technol 2016;52:139–47. 39. DK O´T. Characteristics and use of okara, the soybean residue from soy milk production a review. J Agric Food Chem 1999;47(2):363–71. 40. Perez-Carrillo E, Chew-Guevara A, Heredia-Olea E, Chuck-Hernandez C, Serna-Saldivar SO. Evaluation of the functionality of five different soybean proteins in hot-press wheat flour tortillas. Cereal Chem 2015;92(1):98–104. 41. Acevedo-Pacheco L, Serna-Saldívar SO. In vivo protein quality of selected cereal-based staple foods enriched with soybean proteins. Food Nutr Res 2016;60:31382. https://doi.org/10.3402/fnr.v60.31382. 42. Lu F, Liu Y, Li B. Okara dietary fiber and hypoglycemic effect of okara foods. Bioac Carbohydr Diet Fibre 2013;2(2):126–32.

3. FORTIFICATION OF FLOURS AND BREADS

C H A P T E R

24 Protein-Selenized Enriched Breads Daniela Guardado-F
elix*,†, Marco A. Lazo-V
elez‡, and Sergio O. Serna-Saldivar* *Tecnolo´gico de Monterrey, Center of Biotechnology—FEMSA, School of Engineering and Sciences, Monterrey, M
exico † Biotechnology Postgraduate Regional Program, Faculty of Chemical and Biological Sciences, Autonomous University of Sinaloa, FCQB-UAS, Culiacan, Sinaloa, M
exico ‡ University of Azuay, Food Engineering Program Research Strategic Groups (GEICA-UDA), Cuenca, Ecuador

O U T L I N E Se and Human Health

307

Se Biofortification Processes Fertilization Germination Processes Fermentation

309 309 310 311

Se in Cereals and Breads

311

Effect of Dry Milling on Se Levels in Flours Dough Rheological Properties and Sensory Characteristics of Se-Enriched Breads

312 312

Conclusions and Perspectives

314

References

315

SE AND HUMAN HEALTH Selenium (Se) is a trace mineral that is essential to the health of humans and animals because it is necessary to synthesize selenoproteins like the enzymes glutathione peroxidase (GPx), thioredoxin reductase (TrxR), iodothyronine deiodinase, and selenophosphate synthetase. In these proteins, Se is incorporated into the amino acid selenocysteine (SeC).1 GPx and TrxR reduce the levels of reactive oxygen species (ROS) and maintain the cellular redox balance.2 Se deficiency (350 μg/day are considered toxic.5 Se is obtained through the diet, mainly via cereal-based products, seafood, muscle meats, mushrooms, garlic, broccoli, and Brazil nuts.6 However, the Se content in foods is affected by levels of the element existing in the soil. A total of 15% of the world’s population have Se deficiency due to consumption of foods with low Se content.7 Se-enriched foods are proposed to satisfy daily requirements and enhance the health of inhabitants of regions of the world where foods lack this essential mineral. Previous studies have demonstrated the anticarcinogenic potential of Se-enriched foods, such enriched casein and yeast, which are effective against mammary tumor growth8–11 and colon12 cancer cells. The chemopreventive effects of Se are exerted by various molecular mechanisms, such as antioxidant protection, suppression of the cell cycle, induction of apoptosis, and structural modification of proteins.12 According to Tyszka Czochara et al.,13 Se-enriched amaranth sprouts with high betacyanin content prevented NFκB translocation to the cell nucleus and subsequently exerted an anti-inflammatory effect by significantly decreasing inflammatory interleukin-6 production in the cell culture of

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00024-1

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© 2019 Elsevier Inc. All rights reserved.

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24. PROTEIN-SELENIZED ENRICHED BREADS

activated RAW 264.7 macrophages. On the other hand, Guardado-F
elix et al.14 produced chickpea sprouts containing supranutritional levels of dietary Se, which significantly decreased the tumor growth of colon cancer cells xenografted into immune-suppressed mice. Moreover, high dietary Se intake enhanced the antioxidant activity of GPx and TrxR, and therefore decreased the cellular redox state. The prevention of lipid oxidation under hypoxic conditions negatively affected tumor growth. This investigation concluded that selenized chickpea sprouts represent an excellent source of dietary Se and isoflavones, and their consumption could contribute to the reduction of colorectal cancer cell proliferation. Se status decreases with age and has a close relationship with cognitive competence in Alzheimer’s disease. This neurodegenerative disorder is characterized by the production of large amounts of β-amyloid (Aβ) and the accumulation of extracellular senile plaques, which have been considered to be potential targets in the treatment of Alzheimer’s disease. It has been reported that selenomethionine (SeM) led to significantly reduced production and deposition of Aβ, downregulation of β-secretase levels, and enhanced activity of selenoenzymes, as well as increased levels of Se in the hippocampus and cortex. SeM reduces amyloidogenic processing of amyloid precursor protein while modulating β-secretase and selenoenzymatic activity in mice with Alzheimer’s disease.15 Recently, the same authors showed that SeM treatment promoted neural stem cell differentiation into neurons through the PI3K-Akt-GSK3b-Wnt signaling pathway, and subsequently repaired damaged neural systems in mice with Alzheimer’s disease. These studies provide evidence that Se compounds are effective in promoting neurogenesis.16

ABSORPTION AND METABOLISM OF DIETARY SE In nature, Se can be found in both organic and inorganic forms. Selenite (SeO23 ) and selenate (SeO24 ) are the main inorganic forms, which are used to enrich foods. In addition, they were the first Se sources used in chemoprevention studies.17 SeM and SeC are considered the main organic moieties associated with foods. Other organic forms in foods include Se-methyl-selenocysteine (CH3SeC), which is a homologous form of SeM and has been identified by in vitro and in vivo studies as a good precursor of methylselenol (CH3SeH), which is also recognized as chemopreventive.18 Inorganic Se is absorbed by simple diffusion, while organic Se such as SeM through the amino acid transport system.7 SeM contained in foods can be accumulated in selenoproteins and structural proteins or metabolized to other forms such as hydrogen selenide (H2Se) by two mechanisms: a trans-selenization of SeC followed by a reaction of β-lyase, or directly by the reaction of ɣ-lyase forming methylselenol (CH3SeH)7 (Fig. 1). In contrast, inorganic Se is incorporated into selenoproteins.19 Selenide is formed from SeO23 by an Se glutathione pathway (GSSeSG), through thiol group reduction and by reductases dependent on nicotinamide adenine dinucleotide phosphate (NADPH). H2Se generated during the metabolism of organic and inorganic selenized compounds provides a source of Se for the biosynthesis of selenoproteins17, 20, 21 (Fig. 1). The half-life of SeM and inorganic selenite in the human system is 252 and 102 days, respectively, indicating that once absorbed, the organic form persists longer in the body due to a nonspecific incorporation mechanism. Se is incorporated into tissue proteins such as skeletal muscle, liver, erythrocytes, and albumin, and subsequently, it can be further metabolized to maintain elevated levels in the organism.7, 22 The proper assimilation of Se into foods depends on their bioavailability, bioaccessibility, and/or bioactivity of a given Se compound. SeM is the main Se species found in bread supplemented either by adding selenite directly to the

Dietary selenium

SeO42–

γ-lyase CH3SeH

SeO32–

GSSeSG

H2Se

SeM

Structural protein

β-lyase

SeC

Serine-tRNA UGA

Selenoproteins

FIG. 1 Simplified pathways of selenium dietary metabolism. 3. FORTIFICATION OF FLOURS AND BREADS

SE BIOFORTIFICATION PROCESSES

309

dough or by using lab-grown, Se-enriched yeast. The highly bioavailable SeM accounted for 80% of the total Se. An in vitro gastrointestinal digestion assay indicated that the bioaccessibility of Se in enriched white bread was 100%.23 The bioaccessibility of Se in enriched supplements and food crops was found to be highest in the small intestine. Compared to Se-enriched yeast and Se + ACE-vitamins mixture tablets, a yogurt-based supplement exhibited a much lower bioaccessibility, possibly due to the presence of nanoparticles or microparticles of elemental Se. Moreover, the uptake of SeM by colon microbiota is much more efficient than the uptake of selenite.24

SE BIOFORTIFICATION PROCESSES The Se level in food is closely related to the presence of this specific mineral in the soil and the microbial activity of topsoil microorganisms, as well as the ability of plant tissues to absorb and accumulate this trace mineral. The highest levels of Se in cereals and breads have been reported in the United States, with 760 and 312 μg Se/kg, respectively. Contrary, the lowest Se levels have been registered in Portugal and Spain, with 40 and 25 μg Se/kg in the same food products.7 Increasing Se content in foods offers an effective approach to reduce deficiencies of this element in humans and animals. Several strategies are known to improve the Se status and bioavailability in cereals and breads, which are summarized in Fig. 2.

Fertilization A plant absorbs Se from the soil and incorporates this mineral into proteins, mainly in the form of SeM followed by SeC. The pathway for the synthesis of SeM in plants and a large number of bacteria and yeast is greatly enhanced when methionine is deficient.25 Se-enriched flours have been obtained from grains harvested in seleniferous or Se-fertilized soils and used to reduce dietary Se deficiencies in susceptible regions of the world. For instance, bread and durum wheat in field conditions were enriched with Se through foliar and soil addition (100 g of Se/ha) of sodium selenite, and the resulting kernels contained significantly higher levels of SeM.26 Foliar application ensures a high efficiency of Se assimilation by the plant because it does not depend on root-to-shoot translocation.27 The enrichment of fertilizers with Se has been effective to increase its levels in several wheat crops. After Se fertilization in wheat, 77% and 90% of

FIG. 2

Proposed strategies for production of Se-enriched breads with potential health benefits.

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24. PROTEIN-SELENIZED ENRICHED BREADS

the original Se was retained in white and whole-meal breads, respectively.28 Likewise, flour derived from Spanish Se-enriched wheat retained 72% of the initial grain Se.29 The foliar Se fertilization (120 g Se/ha) of durum wheat increased by up to 35-fold compared to that of the untreated control. Additionally, the Se concentration decreased during milling (11%), while processing and cooking of pasta did not show significant decreases. This biofortification strategy had no effects on grain-quality parameters, except for a reduced gluten index in the high-gluten variety PR22D89, as well as the sensory properties of spaghetti made from this material.30 Experimental application of Se fertilizer to a wheat crop enhanced Se concentration in whole-wheat and refined flour from untreated plots with values ranging from 35 and 30 ng/g up to > 2200 ng/g in whole-wheat flour and >1800 ng/g in white flour from kernels harvested from soils enriched with Se fertilizer (as selenate) applied at the highest tested rate of 100 g/Ha. Interestingly, the relationship between the quantity of Se applied to the crop and the amount of this mineral assayed in flour was approximately linear. SeM accounted for most of the total extractable Se species in enriched flours.28 Among the protein fractions of wheat cultivated in seleniferous regions of India, Se was dominantly (33%–37%) present in the albumin fraction in Se-rich grains, followed by globulin (20%–25%), glutelin (20%–25%), and prolamin (17%–20%).31

Germination Processes Germination, or sprouting, is an effective natural process for Se-fortification of cereals, pseudocereals, and legume seeds. Some crops such as wheat, rye, soybean, quinoa, common beans, oats, and chickpeas can effectively synthesize organic Se (mainly SeM) during this physiological process.32–36 The use of Se salts during the soaking stage has resulted in the increase of highly bioavailable Se in sprouts. Additionally, it has been shown that the chemical stress induced by various Se solutions during germination enhances the antioxidant activity and concentrations of phenolic compounds and essential amino acids 37–40 Se biofortification during wheat germination is a good strategy for effective incorporation of this mineral. However, α-amylase activity increases, followed by the degradation of starch into simpler sugars (dextrins and fermentable sugars) that are used as energy for seedling growth. The high enzyme activity in wheat is not recommended for flours intended for bread-making because the excess diastatic activity modifies dough’s water absorption and yields sticky dough and problematic bread. This is one of the main reasons why millers and bakers avoid the use of field-damaged and sprouted kernels.41 Lazo-Velez et al.42 devised an experiment in which response surface methodology was employed as a tool to optimize bread wheat (Triticum aestivum L.) germination conditions in the presence of Na2SeO3 to obtain Se-enriched sprouted kernels. The idea was to maximize the synthesis of SeM and minimize α-amylase activity. The effects of Se concentration (about 32–55 mg Na2SeO3/L), germination time (24–48 h) and germination temperature (18°C– 25°C) showed that SeM synthetized in sprouted wheat was affected by the increase of both germination temperature and sodium selenite-concentration. Nevertheless, SeM synthesis was predominantly increased and influenced by germination time. On the other hand, germination temperature, time, and their interactions greatly affected α-amylase activity. In fact, increasing both temperature and time generated a higher effect. The addition of various concentrations of Na2SeO3 did not significantly affect enzyme activity, while SeM concentration produced a decrement in α-amylase activity (Fig. 3). With this last point, consider that α-amylase is synthesized in the aleurone layer and scutellum of sprouting kernels.41 Finally, researchers have indicated that hard-wheat germination with Na2SeO3 (35 mg Se/L) for 24 h at 25°C were the optimal conditions to obtain Se-enriched sprouted kernels with high Se content (54 mg/kg) such as SeM, and low α-amylase activity (Fig. 3). The chemical stress induced by Se sodium selenite (Na2SeO3) during the germination of chickpeas (Cicer arietinum L.) increased dietary Se by 115-fold, total isoflavonoid, phenylalanine ammonia lyase (PAL) activity, and antioxidant capacity by 83%, 56%, and 33%, respectively, in Se-treated chickpea sprouts compared to untreated counterparts. These results suggest that Se-enriched chickpea sprouts could represent a good source of dietary Se and an upgraded source of isoflavones. These sprouts, rich in organic Se, having essential amino acids like lysine and tryptophan, and with higher levels of isoflavones, can be used as functional ingredients for the production of nutraceutical bakery items.35 Likewise, soybeans (Glycine max) germinated for 48 h at 20°C with a frequent spraying of water containing 88% protein). The whole soybean seed is also used for the production of extracts known as soymilks (discussed in more detail later in this chapter). Full-fat soybean flour is made directly from dehulled and milled seeds, with protein content of 35%–36% and oil content of 17%–21% (Table 1). This product is similar in composition to the original seeds, nutritious, and high in TDF, as well as having all of the vitamins, minerals, and phytochemicals associated with the seed. Full-fat flours are available as raw, enzyme-active, or thermally treated flours. The major use of raw flour is as a natural bleaching agent because of the high lipoxygenase content, but because of the lack of thermal treatment, raw flour also contains high levels of antinutritional compounds as trypsin inhibitors, urease activity (UA), and hemagglutinins, and then should be used in products that will have additional thermal treatment. The most popular cooked full-fat meal is commonly produced via extrusion cooking. The cooked flours are practically free of these antinutritional factors, have a nuttier and stronger flavor, and tends to impart a slightly darker color. After oil extraction, the defatted soybean flour is thermally treated in a desolventizer-toaster (D-T) to remove the hexane and inactivate antinutritional factors. The final product after milling contains less than 1% residual oil, about 50% protein, 17%–18% TDF, and 30%–35% total carbohydrates (Table 1). Commercial defatted soybean flours vary in their protein dispersability index (PDI), which is related to the degree of protein denaturation during heat treatment in the D-T: the higher the heat treatment, the lower the PDI. This trait is important to assess the feasibility of this material as an ingredient to fortify tortillas or other products.32 Defatted flours regularly contain higher levels of isoflavones compared to whole soybeans (Table 1) and are practically free of lipophilic phytochemicals like phospholipids, phytosterols, and vitamin E. Finely ground defatted soybean flours are used as feedstock to produce protein concentrates (65%–70% protein). There are two ways to produce commercial concentrates: alcohol or isoelectric protein precipitation processes. The alcohol process is used to remove soluble sugars, whereas acid precipitation, the second alternative, yields protein curd and whey rich in soluble sugars. Protein isolates are the most pure and commercial protein product derived from soybean (around 88% protein). The extraction process practically removes all soluble sugars and dietary fiber (DF) components from defatted flour and as a result, the colorless protein isolates have a bland or neutral flavor and nutritionally wise a very high protein digestibility (>94%). Functionally speaking, protein isolates have a high water absorption index (WAI), water solubility, and excellent emulsifying and gelling properties. Another soybean-derived product is soymilk, the liquid extract of whole soybeans. Soymilk contains similar composition of cow’s milk and is created via two major processes: the traditional method, which usually yields extracts with a strong beany flavor; and modified methods, such as the Illinois process, which yields bland-flavored extracts due to the inactivation of lipoxygenase, which is responsible for flavor reversion. The soymilk or extract generally contains most of the active phytochemicals present in seeds, including high amounts of isoflavones. The soybean extract can be alternatively evaporated and spray-dried in order to produce solids. Generally, about half of powdered solids and half of dried okara (a high-fiber by-product) can be obtained from 100 kg of soybeans. Okara is the waste by-product obtained from the process of soymilk and tofu manufacturing. It is high in moisture and thus very prone to spoilage with yeast and molds. When properly managed, okara can be used as an excellent fresh supplement to dairy cattle feed or as a source of energy after combustion.31 After dehydration, it can be finely ground to produce a shelf-stable product rich in DF (50%), with 10%–15% of oil and 20%–25% protein (Table 1). The high protein content makes this coproduct a good potential source of low-cost plant protein for human nutrition.33 Tortillas already have been fortified with full-fat soybean meal, defatted soybean flour, concentrates, isolates, and okara. The substitution levels needed to double the PER and the protein quality are 8%, 6%, 5%, 4%, and 10%, respectively. At these fortification levels, the overall quality of tortillas is not adversely changed.31, 32 The cheapest soybean sources are defatted soybean flour and okara. The first contains less than 1% crude fat, 50% protein, and 3.1% lysine based on flour weight; and the second has between 22% and 26% protein and 1.5% lysine (Table 1).

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TABLE 2 Parameter

Effects of Soybean-Fortification and Enrichment With Selected Micronutrients on the Nutritional Composition of Tortilla12, 37 Regular tortilla from DMF

Regular tortilla from fresh masa

Tortilla fortified with 6% defatted soybean meal

Control-Casein

CHEMICAL COMPOSITION Moisture

6.26

6.72

7.19

9.85

Ash

1.71

2.22

1.96

4.09

Protein

8.94

9.24

10.56

10.06

Crude fat

2.80

2.85

3.90

5.23

Crude fiber

1.24

1.48

1.22

0.21

85.31

84.21

82.36

80.41

a

NFE

AMINO ACID COMPOSITION Leu

10.82

9.55

10.98

9.76

Ile

3.47

3.01

3.88

4.68

Lys

3.27

3.10

4.36

7.98

Met + cys

3.47

2.92

3.69

2.98

Phe + tyr

6.94

6.28

7.86

8.95

Thr

3.27

2.92

3.60

3.55

Trp

0.71

0.62

0.85

1.13

4.69

3.98

4.92

6.37

56.3

53.4

75.1

100

Val b

EAA score

MINERAL AND VITAMIN COMPOSITION Calcium

122

168

137

465

Iron

10.2

9.4

16.6

21.6

Zinc

6.4

5.0

9.0

16.2

Thiamin

0.95

1.33

2.82

1.73

Riboflavin

0.33

0.33

0.75

3.96

Niacin

5.17

7.01

14.87

10.07

Folic acid

9.2

10.0

29.0

74.5

a b

NFE,Nitrogen free extract. EAA (Essential amino acid) score,Limiting EAA/FAO/WHO requirements.

SOYBEAN PROTEIN–FORTIFIED NIXTAMALIZED CORN TORTILLAS AND OTHER CORN-BASED PRODUCTS Many efforts have been made to upgrade the nutritional value of tortillas by enrichment with vitamin/mineral premixes and/or protein fortification. Tortillas have been fortified with amaranth (Amaranthus caudatus), torula yeast (Candida utilis), quinoa (Chenopodium quinoa), flax (Linum usitatissimum), common beans (Phaseolus vulgaris), chickpeas (Cicer arietinum), and mainly with various soybean (Glycine max) products. High-protein tortillas can also be produced by the utilization of high-lysine genotypes (QPM) or the direct addition of lysine and tryptophan, because tortillas have a low essential amino acid score. Many authors5–8, 11, 24, 25, 34–36 determined that the addition of 3% fish meal, 5% meat flour, 5% whole egg flour, 5% casein, 8% skim milk powder, 8% soybean protein, or 3% Torula yeast significantly improved both the quantity and the quality of protein. Fortification with soybeans also has been tested using whole soybean seeds that are nixtamalized along with the corn.7, 8, 34 The PER of the fortified tortillas were at least twice as much compared to the regular counterparts, and a strong correlation was found between the PER and lysine content of the supplements, indicating that this EAA was the limiting factor affecting protein quality. In Table 2, the EAA scores for tortillas from masa, DMF, and tortillas fortified with 3. FORTIFICATION OF FLOURS AND BREADS

SOYBEAN PROTEIN–FORTIFIED NIXTAMALIZED CORN TORTILLAS AND OTHER CORN-BASED PRODUCTS

325

defatted soybean meal are depicted. As can be observed, the EAA score was 50% higher when soybean meal was added to tortillas. The improvement is associated with higher levels of lysine (Table 2). The other advantage of supplementing with soybean products is the relatively low cost compared with other protein sources derived from animals. Recently, the functionality of four defatted soybean flours and one protein concentrate in corn tortillas manufactured in a pilot plant was reported.32 The soybean proteins were added to increase the protein content of DMF between 20% and 25% (i.e., addition of about 6% defatted soybean flour or 4% soybean concentrate). The evaluated soybean ingredients depicted protein content of 42.4%–67.1%, UA values ranging between 0.1 and 2.25, WAI of 4.02–8.34, PDI of 23%–75% and the fat absorption index ranging from 2.5–3.1. EAA compositions of composited DMF showed twice the amount of lysine and tryptophan as the control treatment. Interestingly, the sensory attributes of all soybeanfortified tortillas were not different from the control tortillas, but the maximum texture force in a texture protein analysis, after 5 days of storage, was higher for tortillas supplemented with low PDI-defatted soybean flour and lower for their counterparts containing high-PDI soybean flour. Control and low-PDI flours, followed by soybean concentrate, were the best overall evaluated supplements according to the most relevant parameters for consumers and producers. These authors concluded that the best soybean proteins to be used in corn tortilla supplementation should preferably have reduced UA, water solubility, and also low PDI. When using okara, researchers have found that tortilla fortification with more than 10% (levels up to 25% have been tested) were not accepted by consumers, mainly because of the unpleasant flavor.31 Among the most classic examples of tortilla fortification with soybeans were reported by Bressani et al. and Del Valle and Perez Villaseñor7, 8, 34 several decades ago. In these cases, researchers lime-cooked whole soybeans with maize in mixes with soybean content of up to 20%. They found that corn-soybean mixtures increased the protein quality of tortillas because of the amino acid complementation and higher total protein and fat content. The process followed by Bressani et al.8 included the use of corn: soybeans, water (at a ratio of 1.6 water/1 grain), Ca(OH)2 (1.7% based on grain weight), and a cooking temperature of 96°C for 1.5 h before discarding the resulting nejayote, rinsing with water, and stone milling to obtain the dough or masa suitable for table tortillas. Based on the PER and NPU, del Valle and Perez Villaseñor34 found that no significant differences in protein quality existed between tortillas enriched with 8% and 16% soybean when fed at a dietary level of 9.0% protein. They also reported that the enrichment method changed neither the making process nor the eating habits, and further, a trained panel found no detectable difference between these at soybean levels up to 16%. Years later, a more specific range of whole soybean use was recommended by Bressani et al.7 based on PER and gain weight data obtained from animal assays: 8%–12% soybean substitution was the proper range because beyond 12%, there was no further improvement in PER. Although PER and nutritional parameters are important, the sensory analysis is very relevant when proposing a food fortification alternative. In 2012, Lecuona-Villanueva et al.35 tested the influence of a nonsoy protein concentrate (Phaseolus lunatus) in tortilla texture and color. They found that adhesion, rolling capacity, and color were not affected, whereas the in vitro protein digestibility did improve as expected. They concluded that protein tortilla fortification enhanced the nutritional characteristics of the food with no negative influence on either physicochemical traits or acceptance. Regarding the cost of soybean fortification, Del Valle and Perez-Villaseñor34 reported an 8% increase in tortilla cost per kilogram (at enrichment levels of 8% soybean), but with a better cost per PER unit: 23% less than regular tortillas.

Effect of Tortilla Fortification With Soybean on Growth and Brain Development of Rats In addition to differences in chemical composition and EAA score improvement (Table 2), tortillas fortified with soybean have been evaluated in the growth and brain development of rats.12, 37, 38 In a recent study, Acevedo and Serna Saldivar38 studied the EAA profile, PDCAAS, and in vivo protein quality (PER, BV, and NPU) of nixtamalized corn tortillas and other cereal-based products fortified with 6% defatted soybean flour. They found an increase of lysine and tryptophan, improving the EAA score and PDCAAS, but more important, rats fed diets with corn tortilla and soybean protein gained significantly more weight, and PER went from 0.73 in a regular product up to 1.64 in the soybean-supplemented tortilla prototype. Interestingly, these authors did not find a similar improvement with soybean-fortified wheat yeast-leavened breads. In the case of a two-generation study of rats, researchers fed the animals with diets based on three sorts of tortillas as described in Table 2: (1) regular tortillas produced using DMF enriched with vitamins B1, B2, niacin, and folic acid and the minerals iron and zinc; (2) regular tortillas produced from fresh masa; and (3) an improved prototype produced from DMF enriched with the same vitamins and minerals and fortified with 6% defatted soybean flour. Murine dams were mated 58 days postweaning, with males receiving the same treatment with the aim of obtaining second-generation pups that were further subjected to regular lactation and 28-day postweaned growth.

3. FORTIFICATION OF FLOURS AND BREADS

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25. SOYBEAN-FORTIFIED NIXTAMALIZED CORN TORTILLAS

TABLE 3 Effect of Soybean-Fortification and Enrichment With Selected Micronutrients on Rat Growth Throughout Two Generations12, 37 Parameter

Tortilla from DMF

Tortilla from fresh masa

Tortilla fortified with defatted soybean meal

Control-Casein

FIRST-GENERATION WEANLING RAT GROWTH FOR 28 DAYS Weight gain, g

28.13

31.13

54.50

61.25

Food consumption, g

259.0

261.7

276.0

298.3

Protein consumption, g

23.16

24.18

29.15

30.01

Weight gain/protein consumption

1.46

1.59

2.05

2.34

SECOND-GENERATION WEANLING RAT GROWTH FOR 28 DAYS Weight gain, g

13.00

10.50

25.27

61.32

Food consumption, g

316

359.8

198.4

322.8

Protein consumption, g

25.44

29.28

18.22

31.60

Weight gain/protein consumption

0.51

0.36

1.73

2.03

Highly significant differences in body weight after 28 days of growth and efficiency of food conversion were observed (Table 3). Growth rates expressed in terms of protein consumed by rats fed with fresh masa and DMF tortillas were 1.59 and 1.46, respectively, whereas counterparts fed soybean-fortified tortilla and control (casein) diets had a higher growth performance (2.05 and 2.34, respectively). The results were associated with the amount of lysine, tryptophan, and better EAA scores of soybean-fortified tortillas (Table 2). Growth of second-generation weanling pups fed DMF or fresh-masa tortilla diets was 0.51 and 0.36 g gain/g protein consumed, respectively (Table 3). In contrast, counterparts fed soybean-fortified tortillas or control (casein) diets had significantly higher growth rates (1.73 and 2.03 g gain/g protein consumed, respectively). Therefore, weanling rats fed these diets gained from three to four times more weight than counterparts fed the regular tortilla diet. This difference was higher than that documented in the first-generation growth study, demonstrating that prolonged malnutrition affected the second generation more severely. These findings agree with those of Gressens et al.,39 who concluded that PEM and deficiency of vitamins and minerals of mothers significantly retarded growth in secondgeneration individuals. In addition to the results already described, rats fed soybean-fortified tortillas or the control-casein diets had a 100% pregnancy rate, while counterparts fed regular tortillas had less than 40% pregnancy rates.37 This lower fertility was associated with the lower body weight produced by diets deficient in protein, vitamins, and minerals. In addition, dams fed soybean-fortified tortillas and control-casein diets had 9 and 10 newborns, respectively, in contrast with fertile counterparts fed regular masa and DMF tortillas, which delivered 4 and 7 pups, respectively. Also, the effects of tortilla fortification with soybean ingredients have been studied in the rat brain development through two generations (Table 4). The total content and concentration of brain DNA in first-generation adult males and lactating females were similar for all diets. However, a significant change in these variables was observed in the brains of second-generation subjects.12, 37 With animals fed soybean-fortified tortillas and control-casein diets, the total deoxyribonucleic acid (DNA) concentration, number of neurons, neuron size, and brain activity as estimated by their ribonucleic acid (RNA)/DNA were significantly higher than for counterparts fed regular tortillas (Table 4). These results agree with Gressens et al.,39 who concluded that severe protein malnutrition causes adverse effects on brain development and a reduction in cerebral DNA. The better EAA, protein quality, and supplementation of key micronutrients to fortified tortillas enhanced DNA synthesis and the amount of cerebral DNA during intrauterine fetus development and lactation. In both generations, brain and cerebellum weights and myelin concentration were significantly higher in rats fed the soybean-fortified tortillas. Further research was performed to assess the effect of tortilla-based diets in the short-term, long-term, and working memory of rats fed throughout two generations (Table 5). Short- and long-term memory performance in the Morris maze significantly improved among rats fed the soybean-fortified tortillas. Second-generation rats fed also with these diets had a superior working memory and learning performance.

3. FORTIFICATION OF FLOURS AND BREADS

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SOYBEAN PROTEIN–FORTIFIED NIXTAMALIZED CORN TORTILLAS AND OTHER CORN-BASED PRODUCTS

TABLE 4

Diet (tortilla)*

Effect of Soybean Fortification on Brain and Cerebellum Weight, Myelin, Synapses Density of Neurons of First- and SecondGeneration Rats Fed Regular and Quality-Protein Corn Tortillas6

Brain weight (mg)

Cerebellum weight (mg

Myelin (mg/g)

Protein/ Neuron brain synapses Weight density (%) (mg/g)

RNA/brain weight (mg/g)

DNA/brain weight (mg/g)

Protein/ DNA

RNA/DNA

26.4  3.1a

96.76  4.3a 3.66  0.12a

6.96  0.12a

13.90  0.27a

0.55  0.03a

96.72  5.1a 4.05  0.09b

6.82  0.13a

14.18  0.25a

0.59  0.02a

FIRST GENERATION (AVERAGE AGE 158 DAYS) 174.4  6.8a

Fresh masa 1,024.3  21.3a

86.6  4.6a

DMF

1,130.9  36.3b

205.8  13.1a 124.2  11.8b 29.8  9.6a

DMF with soybean

1,460.4  36.8d

340.0  6.3c

187.4  4.6c

41.5  3.4b

108.67  5.4b 4.41  0.18c

6.84  0.22a

15.88  0.37b

0.64  0.02b

Control

1,671.1  29.6e

368.3  5.0d

190.7  5.3c

47.5  2.2c

123.74  7.3c 4.61  0.11c

6.78  0.12a

18.25 0.20c

0.67  0.02b

SECOND GENERATION (AVERAGE AGE 62 DAYS) Fresh masa

394.6  4.6a

61.2  4.7a

68.8  6.8a

32.5  5.8a

86.76  3.3a 3.86  0.04a

5.79  0.06a

14.98  0.33a

0.66  0.02a

DMF

412.2  8.3b

64.9  5.2a

126.8  3.9b

34.6  3.7a

91.43  2.6a 4.01  0.04b

6.11  0.08b

14.96  0.29a

0.66  0.03a

DMF with soybean

656.0  21.6d

99.0  2.6c

136.7  3.4c

36.4  5.2a

101.45  2.1b 4.30  0.06c

6.25  0.04c

16.23  0.22b

0.69  0.02a

Control

803.4  18.6e 104.3  2.6d

142.6  2.3d

38.6  4.3a

109.61  2.6c 4.31  0.06c

6.36  0.06d

17.23  0.24c

0.68  0.02a

* Control, casein-based diet. Means  SD with different letters (a, b, c, d, e) within column were statistically different (P 1%) has been observed, and various interpretations have been put forward. Rosell et al.18 proposed that the increase in crumb 3. FORTIFICATION OF FLOURS AND BREADS

349

β-GLUCAN CONCENTRATES AND β-GLUCAN-ENRICHED GRAIN FRACTIONS

TABLE 1

Properties of Bread Supplemented With Isolated β-Glucans Farinograph

Dough rheology

Isolate

Water absorption (%, 14% mb)

Dough development time (min)

Resistance to extension

Extensibility (mm)

Loaf volume

References

Control

58.4

5.7

–

–

768 mL

Cavallero et al.12

+20% WF

67.0

8.2

–

–

Control

385 mL

33.34 g

29.83

212 mL

a

a

+5% HMW

–

–

74.28 g

22.30

100 mL

+5% LMW

–

–

49.18 g

23.75

118 mL

Control

62

–

–

–

905 mL

+0.5% BG

69

–

–

–

855 mL

+1.0% BG

75

–

–

– b

Cleary et al.16

Jacobs et al.14

765 mL b

Control-Dion

50.3

1.8

103 BU

241

2.46 mL/g

+0.2% BG-100

52.6

4.5

148 BU

202

2.69 mL/g

+0.6% BG-100

52.8

4.8

118 BU

215

2.88 mL/g

+1.0% BG-100

52.8

4.7

95 BU

222

2.29 mL/g

+1.4% BG-100

53.0

5.3

188 BU

195

2.26 mL/g

Skendi et al.15

a

Resistance to extension (mean max force, g) and extensibility (mean distance at max force, mm) of doughs were measured using a TA-XT2 texture analyzer. After 135 min resting time in fermenting cabinet, each dough piece was stretched in the Brabender Extensograph by a hook until rupture. The stretching force was recorded as a function of time, and the resistance to constant deformation after 50 mm stretching (R50) and the extensibility were obtained. BG, water extracted β-glucan (85% β-glucan); BG-100, commercial concentrate (84.5% β-glucans, 100 kDa); Dion, poor bread making quality wheat flour; HMW, high-molecular-weight β-glucans (95%, 510 kDa); LMW, low-molecular-weight β-glucans (95%, 160 kDa); WF, water extracted fraction (33.2% β-glucan). b

firmness may be a consequence of the thickening of walls surrounding the gas cells that occurs upon the addition of hydrocolloids into bread formulas. Furthermore, an increase in bread firmness may be a consequence of a decrease of the total area of the gas cells in bread containing β-glucans; indeed, the greatest crumb firmness is usually observed in breads with the lowest loaf volume. Also, water promotes starch recrystallization, and the water content of β-glucanenriched breads is generally higher than that of control breads. However, the role of β-glucans in starch retrogradation and bread firmness is not entirely clear. Skendi et al.15 did not report an increase in starch retrogradation (using differential scanning calorimetry measurements) with the addition of β-glucans, and Gill et al.19 proposed that β-glucans added to wheat flour reduce swelling and solubilization of starch during baking, thereby reducing bread firmness. The effectiveness of barley β-glucan inclusion on the in vitro digestibility of breads was studied by measuring the amount of reducing sugars released during enzymatic digestion of control and β-glucan-supplemented breads.13, 16 Both studies reported significant reduction of sugars during in vitro digestion of breads prepared by replacing 5% of wheat flour with purified β-glucan preparations. These effects were partly attributed to the inhibition of enzyme accessibility to starch polymers due to the increased digesta viscosity, altered rheological properties of breads containing β-glucans, or both. Scanning electron micrographs of in vitro digests of bread containing barley β-glucan illustrated the more compact structure of bread and the retention of undigested starch granules compared to control breads.16 Cavallero et al.12 demonstrated a potential to regulate, in vivo, sugar release from breads containing β-glucans; a significant reduction in the area under the blood glucose curve and delay in the mean peak of blood glucose was observed in human subjects who consumed bread supplemented with 20% of barley β-glucans.

β-GLUCAN CONCENTRATES AND β-GLUCAN-ENRICHED GRAIN FRACTIONS Pure β-glucan isolates obtained via the traditional extraction procedures described previously are not suitable for commercial applications because the procedures are time-consuming and costly. Recently, however, several β-glucan concentrates obtained by novel, simplified, and presumably less expensive commercial methods were introduced to the market. The level of β-glucan enrichment and physicochemical properties of β-glucans (including molecular weight, solubility, and viscosity) in these products vary depending on specific treatments employed during the extraction and purification procedures. For example, Nutrim, a product prepared by subjecting an aqueous suspension of 3. FORTIFICATION OF FLOURS AND BREADS

350

27. BARLEY β-GLUCANS AND β-GLUCAN-ENRICHED FRACTIONS AS FUNCTIONAL INGREDIENTS IN FLAT AND PAN BREADS

µ

µ

µ µ

FIG. 1 Roller-milling flow for the original and enriched FRFs from whole barley. B, break passage; PM, pin mill; S, sizing passage; SD, shorts duster.

barley flour to high-temperature mechanical shearing in the presence of thermostable α-amylase, followed by centrifugation and drum-drying, contains only 5%–15% of β-glucans. Other products, including an enzymatically produced Viscofiber or warm water-extracted Cerogen, were reported to contain 50% and 70%–90% β-glucans, respectively.20 Some of these commercial β-glucan products contain substantial amounts of β-glucans and were shown to deliver specific health benefits in clinical studies.21 However, despite being generally regarded as safe, the concentrates of β-glucan obtained by wet extraction may not qualify for allowing health claims in some countries. The alternative sources of β-glucans are fractions obtained by dry grain fractionation processes, such as milling, pearling, sieving, and/or air classification. The nonuniform distribution of components within the barley kernel allows fractionation by physical means into products enriched in various constituents, including β-glucans and arabinoxylans. Such naturally obtained products may be more desirable food ingredients for health-conscious consumers than products obtained through chemical processes. Izydorczyk et al.22 developed and optimized roller-milling flow conditions for the production of barley flour and a coarse fiber fraction (known as shorts) that originates mainly from the endosperm cell walls and contains large amounts of β-glucans, bioactives, and other DF constituents. This fraction, designated as a fiber-rich fraction (FRF) in barley milling, potentially has more value as a functional food ingredient than barley flour that is enriched mainly in starch. The milling flow, as shown in Fig. 1, consists of break passages through four sets of corrugated rolls. Following the fourth break, the ground product is put through two sieves with different apertures, and the coarse material retained on the sieves is directed to a shorts duster. The impact action occurring in the shorts duster effectively cleans the fiber by releasing starch granules that are encapsulated in the endosperm cell walls. The next stages of fiber refinement include a single passage through sizing rolls, sieving, and another shorts duster passage. The original FRF can be further enriched by additional pin-milling, sieving, and another shorts duster passage. Depending on the barley genotype, the β-glucan content of the enriched FRF obtained using this expanded rollermilling flow design ranged from 21% to 27% (Table 2). A major benefit of the dry separation technologies is that the cell wall structures remain intact, so there is very little chance of altering the physicochemical properties of β-glucans. Also, in contrast to β-glucan isolates or commercial β-glucan concentrates, barley FRF has the added benefit of containing other DF and bioactive components, as well as being obtained by a chemical-free process. If barley is not pearled before milling, the FRF contains not only the endosperm cell walls, but also portions of the outer grain layers, specifically pericarp, aleurone, and subaleurone tissues (Fig. 2A and B). Compared to the original FRF (150–500 μm) (Fig. 2A), the enriched FRF consist of smaller particles (100–300 μm) with fewer starch granules (Fig. 2B); the particles generally have irregular shape and porous structure (Fig. 3). Both the original and the enriched FRF from two barley cultivars (CDC Fibar and HB08302) contained higher amounts of β-glucan, arabinoxylans,

3. FORTIFICATION OF FLOURS AND BREADS

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β-GLUCAN CONCENTRATES AND β-GLUCAN-ENRICHED GRAIN FRACTIONS

TABLE 2 Composition and Properties of FRFs Obtained From Hull-less Barley Genotypes CDC Fibar

HB08302

Properties and composition

Whole barley

Original FRF

Enriched FRF

Whole barley

Original FRF

Enriched FRF

Yield (%)

NA

38

19

NA

36

16

Brightness, L*

NA

82.8

85.7

NA

81.9

84.3

Swelling (mL/g)

NA

13.1

16.9

NA

10.0

13.9

Particle size, d0.5 (μm)

NA

300.6

171.8

NA

272.0

175.3

Total β-glucan (%)

10.8

17.4

27.0

7.7

13.0

21.0

Soluble β-glucan (%)

5.4

8.3

12.1

3.4

5.5

8.3

Total arabinoxylans (%)

4.2

8.2

10.0

4.7

10.3

13.0

Starch (%)

52.3

34.4

24.3

56.2

36.4

23.9

Protein (%)

15.8

17.8

15.7

12.7

14.4

13.3

Ash (g/kg)

22.4

32.2

36.6

20.7

31.9

37.6

Mn (mg/kg)

18.3

22.1

18.7

18.5

19.4

17.1

Zn (mg/kg)

29.5

37.7

39.6

23.6

33.2

39.6

Fe (mg/kg)

55.6

79.6

91.8

44.1

66.6

83.4

Ca (mg/kg)

221

227

224

228

300

279

Mg (mg/kg)

1500

2330

2700

1390

2330

2830

P (mg/kg)

4070

6600

7670

4300

6400

7900

Total ferulics (μg/mg)

0.82

1.45

1.50

0.79

1.57

2.08

Vitamin B3 (mg/100 g)

8.2

13.1

15.7

9.3

14.5

18.9

Vitamin E (mg/100 g)

0.96

1.32

0.98

0.43

0.77

0.51

CDC Fibar, hull-less barley with waxy starch; HB08302, an experimental hull-less barley line with high-amylose starch.

protein, manganese (Mn), zinc (Zn), iron (Fe), calcium (Ca), magnesium (Mg), phosphorus (P), total ferulic acids, and vitamins B3 and E than the respective whole-grain samples (Table 2). Although the yield for the enriched FRF decreased, the refinement steps increased the content of β-glucan, arabinoxylans, total ferulics, and vitamin B3, as well as Zn, Fe, Mg, and P. The enriched FRFs were also slightly brighter and contained fewer dark specs than their original counterparts. Grain fractions enriched in β-glucans can also be produced by applying air classification to barley ground by either pin or hammer milling. Andersson et al.23 air-classified ultrafine barley flour from several barley varieties (with β-glucan content ranging from 3.8% to 7.2%) into five fine fractions (F1–F5) and one coarse fraction

FIG. 2

Light photomicrographs of sections of the original (A) and enriched (B) FRF stained with PAS/calcofluor. 3. FORTIFICATION OF FLOURS AND BREADS

FIG. 3 SEM of barley original FRF.

Ultrafine barley flour Fine fraction F1

16,000 rpm

Coarse fraction C1 Fine fraction F2

12,000 rpm

Coarse fraction C2 Fine fraction F3

8000 rpm

Coarse fraction C3 Fine fraction F4

4000 rpm

Coarse fraction C4 Fine fraction F5

2000 rpm

Coarse fraction C5

FIG. 4 Flow of air classification of impact-milled barley flour. Fine (F1–F5) and coarse fractions (C5) obtained by decreasing wheel speed (16,000–2000 rpm). Modified from Andersson AAM, Andersson R, Åman P. Air classification of barley flours. Cereal Chem 2000;77(4):463–7.

(C1) (Fig. 4). The highest β-glucan concentrations were found in F5 and C1 fractions (7%–12%), demonstrating 1.7fold to 2.1-fold enrichment of these polysaccharides compared to whole-grain flour. However, the yields of fractions enriched in β-glucans (F5 and C1) were relatively low (about 11%–22%). Ferrari et al.24 applied double micronization to barley grain to obtain sufficiently fine particles that were then sorted into a series of coarse/fine fraction pairs by air classification (Fig. 5). This approach was tested on two hull-less barleys with different starch types: cultivar Priora with normal starch and CDC Alamo with waxy starch; and this resulted in β-glucan-enriched coarse flour fractions (CF2) with a yield of about 30% and a twofold increase in β-glucan concentration compared to whole grain (Table 3). Successful separation and concentration of grain components via air classification depend on such parameters as particle size distribution and fat content of the material to be air-classified. Good yield (62%) with 1.58-fold enrichment of β-glucans was obtained for a high-β-glucan barley variety, Prowashonupana (originally containing 17%–19% β-glucan and 6% fat) when defatting of grain was conducted before processing.25 Parameters such as β-glucan content in barley grain, the presence of hull, and starch characteristics may also affect the grinding and segregation processes. Further optimization of the dry fractionation processes is still needed in order to obtain fractions concentrated in target components and to reduce the costs associated with power consumption.

353

BARLEY FLOUR AND β-GLUCAN-ENRICHED GRAIN FRACTIONS IN PAN BREADS

Micronized barley (2x)

Air classification (valve =220)

Fine fraction FF1

Coarse fraction CF1

Air classification (valve = 190)

Fine fraction FF2

Coarse fraction CF2

FIG. 5 Flow of the air-classification process for enrichment of β-glucans in barley fractions. Modified from Ferrari B, Finocchiaro F, Stanca AM, Gianinetti A. Optimization of air classification for the production of β-glucan-enriched barley flours. J Cereal Sci 2009;50(2):152–8.

TABLE 3

Yield and DF Contents in Barley Flour Fractions Obtained by Air Classification of Micronized Grain

Barley genotype

Flour fraction

Yield (%)

TDF (%)

β-Glucan (%)

CDC Alamo

Micronized

–

12.8

7.8

CF1

10.4

25.5

11.1

CF2

28.4

25.5

15.6

FF2

61.2

3.8

2.3

Micronized

–

11.5

5.4

CF1

16.5

23.4

8.1

CF2

29.8

21.8

11.2

FF2

53.7

3.3

3.0

Priora

CF1 and CF2 fractions are the coarse fractions from the first and second air classification, respectively; FF2 is the fine fraction from the second air classification as shown in Fig. 5. TDF, total dietary fiber. Modified from Ferrari B, Finocchiaro F, Stanca AM, Gianinetti A. Optimization of air classification for the production of β-glucan-enriched barley flours. J Cereal Sci 2009;50(2):152–8.

BARLEY FLOUR AND β-GLUCAN-ENRICHED GRAIN FRACTIONS IN PAN BREADS Although the addition of β-glucans to bread has the potential to improve its nutritional benefits, its incorporation at the level recommended by various jurisdictions (i.e., 0.75 g of β-glucan per serving) has proved to be challenging, resulting in lower product quality and reduced consumer acceptability. In the bread-making process, replacement of wheat flour by large amounts of nongluten-forming flours such as barley can seriously constrain dough viscoelasticity and the gas-retention capacity of blended dough matrices. Weakened gluten networks often lead to impaired technological and sensory quality of bread in terms of volume, texture, appearance, color, and taste. Although satisfactory bread products supplemented with barley flour have been produced, often the level of supplementation is too small to achieve any significant increase of β-glucan concentration in the final product, and consequently the desired health benefits. For example, Sullivan et al.26 reported that incorporation of up to 50% of barley flour obtained by roller-milling of pearled barley (with 10% w/w of the outer layers removed) into bread had no significant negative effects on bread acceptability. However, to meet the recommended 0.75 g β-glucan dose per serving, it was calculated that about six slices of bread from a standard 800 g loaf would have to be consumed, exciding by far consumers’ preferences and habits. Bread with 100% barley flour was made by using elevated levels of water, malt flour, and margarine (Fig. 6).27 The authors calculated that intake of four slices of this barley bread should deliver a sufficient amount of β-glucans to meet the health claim requirements. However, the use of malt flour in the bread formulation could significantly decrease the molar mass of β-glucans due to the high concentration of β-glucanases in malt, and consequently negatively affect the efficacy of these polysaccharides. 3. FORTIFICATION OF FLOURS AND BREADS

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27. BARLEY β-GLUCANS AND β-GLUCAN-ENRICHED FRACTIONS AS FUNCTIONAL INGREDIENTS IN FLAT AND PAN BREADS

FIG. 6 Appearance of bread made from 100% barley flour according to the optimized procedure with sufficient β-glucan according to the EFSA health claim. Reproduced with permission from Kinner M, Nitschko S, Sommeregger J, et al. Naked barley—optimized recipe for pure barley bread with sufficient beta-glucan according to the EFSA health claims. J Cereal Sci 2011;53(2):225–30.

Compared to white or whole-barley flour, β-glucan-enriched grain fractions have the advantage of elevated levels of β-glucans and other bioactive components, and as a result, lower supplementation levels can be used to achieve the desired health benefits. We assessed the potential of the original and enriched FRF from waxy and high-amylose, hullless barley cultivars (CDC Fibar and HB08302) as functional ingredients in bread prepared by the Canadian short process (CSP). The formula included wheat flour (100 g), whey (4 g), shortening (3 g), yeast (3 g), sugar (4 g), salt (2.4 g), and ascorbic acid (150 μg). To obtain the U.S. Food and Drug Administration (FDA) recommended dosage of 0.75 g of β-glucan per two-slice serving, 10% and 13% of white-wheat flour was replaced with the original FRF from CDC Fibar and HB08302, respectively. Because of the higher concentration of β-glucans in the enriched FRF, the replacement level was reduced to 6% and 8% when using the enriched FRF from CDC Fibar and HB08302, respectively. In each case, the water absorption of the FRF-supplemented doughs was higher than that of 100% white flour, but comparable to that of the 100% whole-wheat flour (Table 4). As a consequence of higher baking absorption, the barley FRF-supplemented loaves and the 100% whole-wheat loaves were heavier than the white-flour control. The loaf volume of breads supplemented with original FRF was reduced by approximately 20%–22% compared to the white-flour bread, and by 7%–10% compared to the 100% whole-wheat-flour bread (Table 4). The crumb structure of breads containing the original FRF scored higher (by visual evaluation) than that of the 100% whole-wheat bread, but slightly lower than that of the white bread (Table 4). Replacement of wheat flour with the enriched FRF preparations significantly improved the overall quality of the TABLE 4

Quality Characteristics of Wheat and Barley FRF-Supplemented Breads Visual assessment Water absorption (%)

C-cell parameters

Loaf volume (cm3)

Loaf weight (g)

Crumb Appearance texture

Brightness, L*

No. of cells

Cell diameter (mm)

Cell wall thickness (mm)

Nonuniformity

CONTROL LOAVES 100% white flour

65

2210

286

7.5

6.5

85.0

5777

2.498

0.476

2.944

100% wholewheat flour

73

1910

304

6.0

5.5

72.9

4712

2.790

0.494

1.818

+10% original FRF 74

1770

300

5.5

5.8

77.5

4551

2.427

0.490

3.143

+6% enriched FRF 74

2060

302

7.5

5.8

81.7

5308

2.396

0.482

1.567

+13% original FRF 73

1720

303

6.5

6.0

78.5

4682

2.375

0.484

3.200

+8% enriched FRF 74

2020

308

7.5

6.2

82.1

5247

2.404

0.483

1.567

FRF-SUPPLEMENTED LOAVES CDC FIBAR

HB08302

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FIG. 7 Control loaves and barley FRF-supplemented breads. (A) 100% white flour, (B) 100% whole-wheat flour, (C) 6% CDC Fibar-enriched FRF, and (D) 8% HB08302-enriched FRF.

supplemented bread compared to the bread supplemented with the original FRF. The most striking improvements were in the loaf volume, appearance, crumb structure, and color of the bread (Table 4). The loaf volume of bread supplemented with both enriched FRFs was superior to that of 100% whole-wheat bread, and when compared to white-flour bread, the volume was reduced by only 6%–9% (Fig. 7). The loaf volume of breads supplemented with the enriched FRF was superior to breads containing the original FRF. The number of cells in bread slices increased substantially when the enriched rather than the original FRF was used. Overall, the FRF-supplemented breads exhibited slightly finer crumb structure than wheat breads, as indicated by the lower diameter of the cells in the former. The cell-wall thickness in the FRF-supplemented breads was higher than that in white-flour bread, but lower than that in the whole-wheat-flour bread. The enriched FRF only slightly decreased the cell diameter or the wall thickness of bread, but substantially improved the texture uniformity of bread slices compared to bread supplemented with the original FRF. A significant improvement in brightness of the bread supplemented with the enriched FRF (Table 4) was a definite advantage because often, breads containing barley ingredients are characterized as having a grayish tint. 3. FORTIFICATION OF FLOURS AND BREADS

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27. BARLEY β-GLUCANS AND β-GLUCAN-ENRICHED FRACTIONS AS FUNCTIONAL INGREDIENTS IN FLAT AND PAN BREADS

TABLE 5 Nutritional Information and Technological and Sensory Characteristics of Wheat and Wheat/Barley (60/40) Blended Breads Nutritional information/technological and sensory characteristics

WT Nutrient (per 100 g)

WT/CB Nutrient (per 100 g)

WT/HBGB Nutrient (per 100 g)

Protein (g)

7.89b

7.31a

8.10b

Ash (g)

0.35a

0.56b

0.96c

Total DF (g)

1.15a

4.01b

11.91c

Soluble DF (g)

0.36a

1.24b

3.69c

Insoluble DF (g)

0.79a

2.77b

8.22c

β-Glucan (g)

0.11a

1.51b

3.23c

RS (g)

1.8a

4.4b

7.0c

Digestible carbohydrates (g)

45c

39b

25a

RDS (g)

58.5c

53.1b

34.7a

SDS (g)

7.5b

3.4a

9.3c

Expected GI

94b

91b

85a

Volume (mL)

1890

1480

1360

Hardness (N)

3.9a

3.3a

7.5b

0.84c

0.72a

0.77b

2.0b

1.4a

1.4a

Cells/cm

50.76b

52.88c

47.36a

External appearance (0 10)

7a

6a

7a

Aroma intensity (0–10)

6a

7a

7a

Taste intensity (0–10)

4a

6b

6.5b

Firmness (0–10)

3a

3a

4a

Overall acceptability (0–10)

6.5a

7.5a

7a

Cohesiveness 2

Mean cell area (mm ) 2

WT, wheat flour; CB, commercial barley flour; HBGB, high β-glucan barley flour. Within rows, values with the same following letter do not differ significantly from each other (P > .05). Modified from Collar C, Angioloni A. Nutritional and functional performance of high β-glucan barley flours in breadmaking: mixed breads versus wheat breads. Eur Food Res Technol 2014;238(3):459–69.

Collar and Anglioloni28 compared the potential of a high β-glucan barley flour of superior nutritional value and a regular commercial barley flour in blends with wheat flour for making nutritious breads with high functional and sensory standards. The high β-glucan barley flour, known under the brand name of Sustagrain, is produced from a unique barley variety, Prowashonupana, with ultrahigh fiber and β-glucan content (30% and 15%, respectively). Here, 100 g of wheat flour or wheat-barley blended flours (60/40 w/w) were mixed with water (63% for wheat and 80% for blends), compressed yeast (4% flour basis), salt (1.5%), vegetable fat (4%), sugar (1%), commercial sourdough (3%), gluten (2%), carboxymethylcellulose (1%), and calcium priopionate (0.5%) in a mixer at 60 revolutions/min for 10 min up to optimum dough development. Fermented doughs were obtained by bulk fermentation, dividing, rounding, molding, and proofing, and then baked at 220°C. The addition of either commercial barley flour or high β-glucan flour significantly decreased the bread volume (22%) and crumb cohesiveness (14%), and increased crumb hardness (Table 5). The crumb quality of barley-supplemented breads decreased in terms of lower cell size and thicker cell walls, but crumb pore uniformity and crumb grain structure were not significantly affected (Table 5). Despite the diminished technological attributes, blended breads scored higher than wheat bread in both taste intensity and overall acceptability. The high β-glucan-flour-supplemented bread showed appealing nutritional quality in terms of lower digestible starch, high soluble and insoluble DF, β-glucan, and resistant starch (RS). The lowest rapidly digestible starch content and highest slowly digestible starch and RS content were observed for the blend of wheat/ high β-glucan barley flours, leading to a significantly lower glycemic index (GI; Table 5). The authors postulated that the incorporation of high β-glucan barley flour into wheat-bread formulations reduced starch hydrolysis due to the

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FIG. 8 SEM micrographs of barley FRF-supplemented bread dough. Discrete cell-wall fragments are visible in the FRF-supplemented dough, giving it an appearance of a composite with particles of texture different than the matrix.

Light micrographs of barley FRF-supplemented breads. (A) Bread stained with Calcofluor indicating the presence of β-glucans (lighter spots); (B) bread showing autofluorescence due to the presence of arabinoxylans in the aleurone cell walls.

FIG. 9

presence of viscous DF components (β-glucans), higher protein, and lower starch in the blended breads. Therefore, the addition of high β-glucan barley flour to common wheat flour highly enhanced the health benefits and sensory appreciation of breads, while generally preserving the technological properties. The mechanisms by which the barley FRFs or high β-glucan barley flour affect the dough rheology and bread textural attributes are complex, and the overall effects depend on the interactions among water, protein, starch, and fiber polysaccharides at the microscopic and molecular levels. As shown in Fig. 8, discrete cell wall fragments are visible in the FRF-fortified dough, giving it the appearance of a composite with particles of texture that differ from the matrix. In bread, both the β-glucan-containing endosperm cell-wall fragments (Fig. 9A) and the arabinoxylan-rich aleurone cell walls (Fig. 9B) can still be detected, although the fiber particles appear smaller and distorted, indicating their partial solubilization. The potential mechanisms by which insoluble fiber ingredients, such as bran, exert negative effects include gluten dilution, water entrapment, and mechanical disruption of gluten films during mixing, proofing, or the early stages of baking.29 Rieder et al.30 explored the potential of sourdough to improve the quality of breads supplemented with wholebarley flour (40% substitution level). Barley flour was mixed with water and fermented with a Lactobacillus plantarum strain. The use of sourdough (20% substitution level) improved the dough structure, bread volume, form ratio, and gas-retaining ability of breads made with the addition of whole-grain barley flour. The positive effect of sourdough was attributed to a particle softening effect, as previously observed for wheat bran.31 The softening of bran particles during fermentation may result in less mechanical disruption of the gluten network and gas cell in the dough. From a physiological standpoint, increased solubilization of β-glucan in a food product and/or during the passage through the gastrointestinal tract is highly beneficial. The amount of β-glucan ingested accounts for only part of the physiological effects; water solubility and the high molecular weight of β-glucans contribute to increased viscosity of digesta in the gastrointestinal tract, and these properties of β-glucans are critical for their health benefits.6, 32 Whereas the increased solubility of β-glucans that might occur during bread preparation and baking is beneficial, a decrease of

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27. BARLEY β-GLUCANS AND β-GLUCAN-ENRICHED FRACTIONS AS FUNCTIONAL INGREDIENTS IN FLAT AND PAN BREADS

TABLE 6

Extractability and Molecular Mass of β-Glucans Obtained by In Vitro Digestion of FRF and FRF-Supplemented Breads Extractability of β-glucans (% of total)

Molecular mass (g/mol)

Enriched FRF

55.6

279,000

Bread + enriched FRF

33.0  3.9

247,200

Enriched FRF

39.7

287,300

Bread + enriched FRF

40.4  4.3

495,500

CDC FIBAR

HB08302

the molecular weight of these polymers is detrimental. The degradation of β-glucans appears to occur mainly during dough mixing, proofing, and fermentation, partly due to activation of endogenous β-glucanases, and to a lesser extent during baking.33 Thus, to retain the high molecular weight of β-glucans, the mixing and fermentation time should be as short as possible, and the β-glucanase activity in any ingredient used in baking should be eliminated by appropriate methods. In our studies, the FRFs used for baking were obtained from sound barley grain with no detectable β-glucanase activity, and wheat flour was also derived from unsprouted wheat. CSP is a no-time mechanical dough-development baking procedure involving fast mixing, followed by short rest (15 min) and relatively short 70-min proof at 37.5°C.34 The rich formula of the CSP dough was altered in our study by omitting the malt extract to avoid the addition of β-glucan-degrading enzymes. As a consequence, the molecular weight of β-glucans extracted from the FRF-enriched breads after in vitro digestions with α-amylase, pepsin, and pancreatin was preserved and comparable to or even higher than that of β-glucans extracted from the FRF under similar conditions (Table 6). Rieder et al.35 recommended a few other strategies for minimizing the molecular-weight reduction of β-glucans during bread-making. The authors incorporated barley flour into already fermented wheat dough or omitted fermentation and shortened proofing time. The altered fermentation and proofing stages were compensated for by adding more yeast and sucrose, which resulted in the production of good-quality barley bread containing high-molecular-weight β-glucans. The use of barley ingredients with bigger particle size, like coarse barley flour or intact barley flakes, also preserved the high molecular weight of these polysaccharides in bread.

BARLEY β-GLUCAN-ENRICHED FRACTIONS IN FLATBREAD Compared to pan or hearth bread, flatbreads have modest flour-quality requirements, and their appeal is not derived from an aerated structure. Also, flatbreads have been made from various grains, and consumers generally have greater tolerance for different colors or tastes of these products. From a technological standpoint, flatbreads are better vehicles for delivering high DF ingredients into the diet, and they appear suitable for inclusion of β-glucans or barley fiber fractions. Barley flour has been successfully incorporated into single-layer flatbreads, including chapatis and Turkish bazlama bread, but only a moderate increase of β-glucan in these products was achieved. Thondre and Henry36 showed that supplementation of whole-wheat flour with a commercial barley β-glucan fiber preparation created a palatable chapatis for diabetic patients, and the GI of chapatis with 4 g of β-glucan per serving was significantly reduced. We have tested the potential of barley FRF as functional ingredients in two-layer flatbread.37 The study was designed to investigate the effect of particle size of the FRF preparations on the technological and nutritional characteristics of flatbreads. It was postulated that apart from the chemical composition of fiber preparations, the properties that are technologically and nutritionally relevant include the particle size, bulk volume, surface characteristics, hydration, and rheological properties. The original FRFs were obtained according to the milling flow shown in Fig. 1. A portion of the FRF was subsequently pin-milled but not subjected to any further processing or sieving. As a consequence, the composition of the original and pin-milled FRF remained the same (Table 7). In addition to reducing particle size, pin-milling slightly increased the water solubility of β-glucans and arabinoxylans and increased the viscosity of FRF water slurries, but decreased their swelling capacity. The particle size reduction was expected to improve the solubility of fiber components by increasing the surface area of particles. It appears,

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TABLE 7 Barley type/FRF

Composition and Physicochemical Properties of Barley Fiber-Rich Fractions (FRF) Before and After Pin Milling (PM)a Total Water-soluble β-glucans (%) β-glucans (%)b

Total arabinoxylans (%)

Water-soluble arabinoxylans (%)b

Swelling capacity Proteins (%) Starch (%) (ml/g)

Brightness, L*

Particle size, d0.5 (μm)

8.93  0.05

10.11  0.04

1.41 (13.9)b

19.1  0.1

9.09c

79.02e

204

d

8.16

d

80.59

139

10.95b

84.00c

220

NORMAL N-FRF

4.55 (50.9)c c

N-FRF-PM

36.2  0.3

a

1.47 (14.5)

4.79 (53.6)

WAXY W-FRF

13.84  0.30

8.87  0.00

6.88 (49.7)a a

W-FRF-PM

20.0  0.1

0.87 (9.8)e

38.1  0.2

e

c

0.91 (10.3)

7.21 (52.1)

b,c

9.28

84.95

155

12.04a

85.46b

220

a

a

164

HIGH AMYLOSE HA-FRF

14.06  1.10

9.98  0.28

6.25 (44.5)b a

HA-FRF-PM

19.5  0.1

0.99 (9.9)d

36.4  0.2

c

1.06 (10.6)

6.90 (49.1)

11.89

86.83

Means in columns followed by a different letter(s) are significantly different (P  .05) as determined by Duncan’s multiple range test. The values in parentheses indicate solubility expressed as a percentage of either total β-glucans or arabinoxylans, respectively. HA, high amylose; N, normal; PM, pin mill; W, waxy. a

b

TABLE 8

Quality Characteristics of Barley Fiber-Rich Fraction (FRF)-Supplemented Flat Breadsa Dough handling scoresb

Flat bread

Texture Bread scoresb Water c d absorption (%) Division Sheeting Appearance Diameter Crumb Aroma Brightness, L* Hardness (kg) Chewiness

Controle

57c

4.25

4.25

4.25

5.0

3.75

4.75

74.09

0.47c

0.36d

+N-FRF

68a

4.25

4.25

4.25

5.0

3.5

4.5

68.96

0.54a,b,c

0.47a,b,c,d

+N-FRF-PM

68a

4.0

4.25

4.25

5.0

3.5

4.25

67.45

0.54a,b,c

0.48a,b,c

+W-FRF

64b

4.0

4.0

4.25

5.0

4.0

4.25

69.01

0.52b,c

0.45b,c,d

+W-FRF-PM

64b

4.25

4.5

4.25

5.0

4.0

4.5

71.84

0.49b,c

0.42c,d

+HA-FRF

67a

4.0

4.0

4.25

5.0

4.0

5.0

74.31

0.62a,b

0.54a,b

4.25

4.25

4.5

5.0

4.0

4.75

73.22

0.66a

0.58a

+HA-FRF-PM 67a

Means in columns followed by a different letter(s) are significantly different (P  0.05) as determined by Duncan’s multiple range test. Each quality parameter was given a score from 0 to 5. c Division is the step in flat bread processing in which the dough was cut into five 125 g pieces after fermentation and before sheeting. d Sheeting is the step in flat bread processing in which the dough is pinned into circular sheets followed by proofing and then baking. e Control flat bread consisted of 80% straight grade Canadian Western Red Spring wheat flour and 20% whole wheat flour. HA, high amylose; N, normal; PM, pin mill; W, waxy. a

b

however, that the reduction of particle size of FRF preparations caused a collapse of the porous fiber structure, making it unable to absorb as much free water as rough fiber. Flatbreads were prepared by mechanically mixing the ingredients, which included wheat flour (100 g; a blend of 80% white flour and 20% whole-wheat flour), salt (1 g), sugar (1 g), and fresh compressed yeast (1.5 g), followed by fermentation (45 min at 30°C), division, rest (10 min), manually pinning the dough into circular sheets, proofing (25 min at 30°C), and baking (540°C for 70 s) in an electric traveling flatbread oven. The FRF-fortified flatbreads were prepared by replacing 20% of white flour with the original and pin-milled FRF. All FRF-containing doughs exhibited higher water absorption, but good handling characteristics at division and sheeting (Table 8). The layer separation during baking was very good, and flatbreads with FRF did not show any tendency for burning (Fig. 10). Characteristics of FRF-fortified breads such as appearance, diameter, crumb structure, and aroma were comparable to those of the control breads (Table 8). However, the addition of FRF increased hardness and chewiness and decreased brightness (L*). The greatest hardness was observed for FRF derived from high-amylose barley, and it was noticed that properties of starches associated with the FRF (especially at a higher level of FRF addition) could also contribute to the

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FIG. 10 Barley FRF-supplemented flatbreads coming out of a traveling oven.

FIG. 11 SEM micrographs of barley FRF-supplemented flatbreads. (A) Control flatbread (80% white flour/20% whole-wheat flour; (B) flatbread with 20% of barley FRF.

technological properties of breads. Flatbreads supplemented with FRF from high-amylose barley displayed slightly denser crumb structure than the control bread or bread fortified with FRF from waxy barley (Fig. 11). Some differences in the quality characteristics of flatbreads supplemented with FRFs from various barley types were also observed (Table 8). However, the quality and texture of supplemented breads were not significantly influenced by pin-milling of the FRF preparations. The addition of 20% of FRF significantly increased the amount of total and soluble β-glucans in flatbreads. Assuming that a single flatbread constitutes one serving, the FRF-supplemented breads would provide between 1.84 and 2.97 g of total β-glucans and between 0.58 and 1.42 g of water-soluble β-glucans, depending on the origin of the FRF preparations (Table 9). With the exception of the waxy barley FRF preparations that showed a slight decrease, the water solubility of β-glucans was higher in flatbreads containing pin-milled FRF compared to the original FRF preparations. The addition of FRF also significantly increased the content of total arabinoxylans in the flatbreads. The amount of water-soluble arabinoxylans, on the other hand, changed little with the addition of barley FRF, indicating the insoluble nature of these polysaccharides in barley. The results of the in vitro digestibility of starch showed that the inclusion of barley FRF into flatbread significantly decreased the solubilization and digestibility of starch at two different time intervals (Table 9). The pin-milled FRF exerted a slightly greater effect than the original FRF in most cases. The reduction in starch digestibility of FRF-supplemented flatbreads might be associated with the ability of fiber constituents to restrict the enzyme accessibility to the food, either by changing (compacting and/or hardening) the food structure or by physically entrapping starch granules in their mucilaginous matrix. Brennan and Samyue38 postulated that the digestion of starch and sugar release from foods might be delayed

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TABLE 9 Nutritional Characteristics of Barley Fiber-Rich Fraction (FRF)-Supplemented Flat Breadsa β-Glucans (%)

Arabinoxylans (%)

Digestible starch (g/g)

Flat bread

Total

Water-solubleb

Total

Water-solubleb

15 min

60 min

Control

0.23d

0.09g

2.40b

0.71b

0.203a

0.436a

+N-FRF

1.85c

0.58f

4.90a

0.91a

0.184b

0.374c

+N-FRF-PM

1.84c

0.62e

4.50a

0.91a

0.172c

0.368c

+W-FRF

2.70b

1.22c

3.70a

0.68c

0.177b,c

0.384b

+W-FRF-PM

2.74b

1.10d

3.90a

0.65c

0.161d

0.352d

+HA-FRF

2.91a

1.31b

4.47a

0.75b

0.180b

0.352d

+HA-FRF-PM

2.97a

1.43a

4.20a

0.71b

0.164d

0.348d

Means in columns followed by a different letter(s) are significantly different (P  .05) as determined by Duncan’s multiple range test. Water-soluble β-glucans and arabinoxylans were measured after extracting cubes of flat bread in water at 40°C for 120 min. Results are expressed as gram of starch released upon digestion with α-amylase per gram of available starch in flat bread samples. HA, high amylose; N, normal; PM, pin mill; W, waxy. a

b

due to DF constituents adhering to starch granules and possibly increasing the digesta viscosity. The decrease of in vitro starch digestibility due to the presence of β-glucans may indicate reduction of the GI of breads. Jenkins et al.39 showed that in a 50 g carbohydrate food portion (a prototype β-glucan-enriched breakfast cereal and bar), each gram of β-glucan could reduce the GI by 4 units, making it a useful functional food component for reducing postprandial glycemia.

TECHNOLOGICAL ISSUES Consumers’ interest in healthy and low-calorie foods has created the demand and opportunity for innovative DF preparations and their incorporation into new and traditional food products. However, producing functional food products that fulfill expectations in terms of sensory appeal and indeed deliver the expected health benefits is challenging and requires innovative approaches and careful attention throughout the manufacturing process. The source of DF and the type and degree of processing involved in fiber preparation are often key factors influencing its physiological functionality. The physiological benefits of β-glucans are associated not only with their amount, but also with their viscosity-building properties linked to the high molecular weight of these polymers. Because β-glucans are easily degraded by β-glucanases, care has to be taken during the preparation of β-glucan fiber to either choose sound barley or to inactivate the endogenous enzymes in grain by appropriate treatment. Changes to the molecular weight of β-glucans can also occur during bread-making processes such as mixing, and fermentation; keeping these steps as short as possible and avoiding introduction of β-glucanase activity from other ingredients used in the bread formulas are also necessary. Finocchiaro and coworkers40 demonstrated that the effectiveness of bread enriched in barley β-glucans in reducing GI was influenced by the amylose/amylopectin ratio of the barley used in their study. It was shown that the addition (40%) of fiber fraction from the waxy variety, CDC Alamo (2.9% amylose), to bread formula was not as effective as the addition of fiber fraction from Priora (26% amylose) in lowering the GI. Indeed, a positive role of the high amylose/amylopectin ratio in lowering GI in other products was shown previously.41 However, it also appears that a sufficiently high concentration of β-glucans can overcome any counteracting effects of high-amylopectin starch. Several studies demonstrated that the use of waxy barley, Prowashonupana, with a uniquely high concentration of β-glucans, can significantly lower the GI of food products (breads, porridge).28,42,43 In bread-making, the incorporation of a sufficient amount of β-glucans to ensure beneficial physiological impact is often associated with replacing large amounts of wheat flour, and the resulting detrimental technological effects are caused by a dilution of functional gluten proteins. The use of fiber preparations with a high concentration of β-glucans eliminates the necessity of high supplementation levels to achieve the desired β-glucan content in the final product. Several hull-less barley genotypes with high β-glucan content have been bred specifically for food uses and are suitable for production of β-glucan-enriched fiber fractions or high β-glucan barley flour. The adverse

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27. BARLEY β-GLUCANS AND β-GLUCAN-ENRICHED FRACTIONS AS FUNCTIONAL INGREDIENTS IN FLAT AND PAN BREADS

technological effects in bread manufacture associated with high contents of β-glucan fiber can be counteracted to some degree with the use of strong gluten flours, the addition of vital gluten and surfactants, and the incorporation of hemicellulases. Jacobs et al.14 reported that the addition of xylanase to the sponge-and-dough formulas improved the loaf volume, appearance, and crumb structure of bread fortified with barley FRF and potentially increased the health benefits by increasing the amount of soluble fiber content in the bread. These authors also reported that the method of bread production strongly influenced bread quality; the best-quality FRF-enriched bread was obtained by the sponge-anddough process because the gluten network was allowed to develop fully during the 4 h sponge stage, and the barley FRF were incorporated only at the dough stage. The shorter exposure of FRF to fermentation was also a potential advantage because extended fermentation times might reduce the cholesterol-lowering capacity of β-glucans due to their degradation by β-glucanases.

References 1. Izydorczyk MS, Dexter JE. Barley: milling and processing. In: Wrigley C, Corke H, Seetharaman K, Faubion J, editors. Encyclopedia of food grains. 2nd ed. Oxford: Academic Press; 2016. p. 434–45. 2. Idehen E, Tang Y, Sang S. Bioactive phytochemicals in barley. J Food Drug Anal 2017;25(1):148–61. 3. Wood PJ. Cereal β-glucans in diet and health. J Cereal Sci 2007;46(3):230–8. 4. Brennan CS, Cleary LJ. The potential use of cereal (1! 3,1!4)-β-D-glucans as functional food ingredients. J Cereal Sci 2005;42(1):1–13. 5. El Khoury D, Cuda C, Luhovyy BL, Anderson GH. Beta glucan: health benefits in obesity and metabolic syndrome. J Nutr Metab 2012;2012:851362. 6. Keogh GF, Cooper GJS, Mulvey TB, et al. Randomized controlled crossover study of the effect of a highly β-glucan-enriched barley on cardiovascular disease risk factors in mildly hypercholesterolemic men. Am J Clin Nutr 2003;78(4):711–8. 7. Food and Drug Administration. FDA allows barley products to claim reduction in risk of coronary heart disease. FDA News; 2005. https://www.fda. gov/Food/NewsEvents/Newsroom/PressAnnouncements/2005/ucm%20108543.htm%3E. [Accessed 13 July 2018]. 8. Health Canada. Summary of Health Canada’s assessment of a health claim about barley products and blood cholesterol lowering. https://www.canada. ca/en/health-canada/services/food-nutrition/food-labelling/health-claims/assessments/assessment-health-claim-about-barleyproducts-blood-cholesterol-lowering.html; 2012. [Accessed 13 July 2018]. 9. European Food Safety Authority (EFSA). Scientific Opinion on the substantiation of health claims related to beta glucans and maintenance of normal blood cholesterol concentrations (ID 754, 755, 757, 801, 1465, 2934) and maintenance or achievement of a normal body weight (ID 820, 823) pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J 2009;7(10):1254. 10. Newman RK, Newman CW. Health benefits of barley foods. In: Newman RK, Newman CW, editors. Barley for food and health science, technology and products. Nonoken, NJ: John Wiley and Sons Inc.; 2008. p. 178–203. 11. European Food Safety Authority (EFSA). Scientific Opinion on the substantiation of a health claim related to barley beta-glucans and lowering of blood cholesterol and reduced risk of (coronary) heart disease pursuant to Article 14 of Regulation (EC) No 1924/2006. EFSA J 2011;9(12):2471. 12. Cavallero A, Empilli S, Brighenti F, Stanca AM. High (1 !3,1!4)-β-glucan barley fractions in bread making and their effects on human glycemic response. J Cereal Sci 2002;36(1):59–66. 13. Symons LJ, Brennan CS. The influence of (1 !3) (1 !4)-β-D-glucan-rich fractions from barley on the physicochemical properties and in vitro reducing sugar release of white wheat breads. Food Chem Toxicol 2004;69:463–7. 14. Jacobs MS, Izydorczyk MS, Preston KR, Dexter JE. Evaluation of baking procedures for incorporation of barley roller milling fractions containing high levels of dietary fibre into bread. J Sci Food Agric 2008;88(4):558–68. 15. Skendi A, Biliaderis CG, Papageorgiou M, Izydorczyk MS. Effects of two barley β-glucan isolates on wheat flour dough and bread properties. Food Chem 2010;119(3):1159–67. 16. Cleary LJ, Andersson R, Brennan CS. The behaviour and susceptibility to degradation of high and low molecular weight barley β-glucan in wheat bread during baking and in vitro digestion. Food Chem 2007;102(3):889–97. 17. Wang L, Miller RA, Hoseney RC. Effects of (1 !3)(1!4)-β-D-glucans of wheat flour on breadmaking. Cereal Chem 1998;75(5):629–33. 18. Rosell CM, Rojas JA, Benedito de Barber C. Influence of hydrocolloids on dough rheology and bread quality. Food Hydrocoll 2001;15(1):75–81. 19. Gill S, Vasanthan T, Ooraikul B, Rossnagal B. Wheat bread quality as influenced by the substitution of waxy and regular barley flours in their native and cooked forms. J Cereal Sci 2002;36(2):239–51. 20. Ahmad A, Munir B, Abrar M, Bashir S, Adnan M. Perspective of β-glucan as functional ingredient for food industry. J Nutr Food Sci 2012;2(133). https://doi.org/10.4172/2155-9600.1000133. 21. Stevenson DG, Inglett GE. 22-Cereal β-glucans. In: Phillips GO, Williams PA, editors. Handbook of hydrocolloids. 2nd ed. Woodhead Publishing; 2009. p. 615–52. 22. Izydorczyk MS, Dexter JE, Desjardins RG, Rossnagel BG, Lagasse SL, Hatcher DW. Roller milling of Canadian hull-less barley: optimization of roller milling conditions and composition of mill streams. Cereal Chem 2003;80(6):637–44. 23. Andersson AAM, Andersson R, Åman P. Air classification of barley flours. Cereal Chem 2000;77(4):463–7. 24. Ferrari B, Finocchiaro F, Stanca AM, Gianinetti A. Optimization of air classification for the production of β-glucan-enriched barley flours. J Cereal Sci 2009;50(2):152–8. 25. Wu YV, Stringfellow AC, Inglett GE. Protein- and β-glucan enriched fractions from high-protein, high β-glucan barleys by sieving and air classification. Cereal Chem 1994;71:220–3. 26. Sullivan P, O’Flaherty J, Brunton N, Arendt E, Gallagher E. Fundamental rheological and textural properties of doughs and breads produced from milled pearled barley flour. Eur Food Res Technol 2010;231(3):441–53.

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27. Kinner M, Nitschko S, Sommeregger J, et al. Naked barley—optimized recipe for pure barley bread with sufficient beta-glucan according to the EFSA health claims. J Cereal Sci 2011;53(2):225–30. 28. Collar C, Angioloni A. Nutritional and functional performance of high β-glucan barley flours in breadmaking: mixed breads versus wheat breads. Eur Food Res Technol 2014;238(3):459–69. 29. Campbell GM, Ross M, Motoi L. Chapter 33—bran in bread: effects of particle size and level of wheat and oat bran on mixing, proving and baking. In: Campbell GM, Scanlon MG, Pyle DL, editors. Bubbles in food 2. St. Paul, MN: AACC International Press; 2008. p. 337–54. 30. Rieder A, Holtekjølen AK, Sahlstrøm S, Moldestad A. Effect of barley and oat flour types and sourdoughs on dough rheology and bread quality of composite wheat bread. J Cereal Sci 2012;55(1):44–52. 31. Salmenkallio-Marttila M, Katina K, Autio K. Effects of bran fermentation on quality and microstructure of high-fiber wheat bread. Cereal Chem 2001;78(4):429–35. 32. Yokoyama WH, Hudson CA, Knuckles BE, et al. Effect of barley β-glucan in durum wheat pasta on human glycemic response. Cereal Chem 1997;74(3):293–6. 33. Tiwari U, Cummins E. Factors influencing β-glucan levels and molecular weight in cereal-based products. Cereal Chem 2009;86(3):290–301. 34. Preston KR, Kilborn RH, Black HC. The GRL pilot mill. II. Physical dough and baking properties of flour streams milled from Canadian red spring wheats1. Can Inst Food Sci Technol J 1982;15(1):29–36. 35. Rieder A, Ballance S, Løvaas A, Knutsen SH. Minimizing molecular weight reduction of β-glucan during barley bread making. LWT-Food Sci Technol 2015;64(2):767–74. 36. Thondre PS, Henry CJK. High-molecular-weight barley β-glucan in chapatis (unleavened Indian flatbread) lowers glycemic index. Nutr Res 2009;29(7):480–6. 37. Izydorczyk MS, Chornick TL, Paulley FG, Edwards NM, Dexter JE. Physicochemical properties of hull-less barley fibre-rich fractions varying in particle size and their potential as functional ingredients in two-layer flat bread. Food Chem 2008;108(2):561–70. 38. Brennan CS, Samyue E. Evaluation of starch degradation and textural characteristics of dietary fiber enriched biscuits. Int J Food Prop 2004;7 (3):647–57. 39. Jenkins AL, Jenkins DJA, Zdravkovic U, Wursch P, Vuksam V. Depression of the glycemic index by high levels of β-glucan fiber in two functional foods tested in type 2 diabetes. Eur J Clin Nutr 2002;56:622–8. 40. Finocchiaro F, Ferrari B, Gianinetti A, et al. Effects of barley β-glucan-enriched flour fractions on the glycaemic index of bread. Int J Food Sci Nutr 2012;63(1):23–9. 41. Kabir M, Rizkalla SW, Champ M, et al. Dietary amylose-amylopectin starch content affects glucose and lipid metabolism in adipocytes of normal and diabetic rats. J Nutr 1998;128(1):35–43. 42. Liljeberg HGM, Granfeldt YE, Bj€ orck IME. Products based on a high fiber barley genotype, but not on common barley or oats, lower postprandial glucose and insulin responses in healthy humans. J Nutr 1996;126(2):458–66. € 43. Ostman E, Rossi E, Larsson H, Brighenti F, Bj€ orck I. Glucose and insulin responses in healthy men to barley bread with different levels of (1 !3;1!4)-β-glucans; predictions using fluidity measurements of in vitro enzyme digests. J Cereal Sci 2006;43(2):230–5.

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C H A P T E R

28 Fortification of Bread With Soy Protein to Normalize Serum Cholesterol and Triacylglycerol Levels Reiko Urade Institute for Integrated Radiation and Nuclear Science, Kyoto University, Osaka, Japan

O U T L I N E Introduction

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Soy Proteins

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Isolation of Soy Proteins

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Physiological Function of Soy Proteins

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Adverse Effects of Soy Protein Fortification on Rheological Properties of Dough and Bread Quality 369

Improved Quality of Bread Fortified With Soy Protein

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Summary Points

372

Acknowledgments

372

References

372

Abbreviations ER GMP LDL LPs PC PDCAAS SDS SPI TG VLDL

endoplasmic reticulum glutenin macropolymer low density lipoprotein lipophilic proteins phosphatidylcholine protein digestibility-corrected amino acid score sodium dodecyl sulfate soy protein isolate triacylglycerol very low density lipoprotein

INTRODUCTION Soybean (Glycine max L. Merrill) is a species of legume that originated in East Asia and has been cultivated for approximately 5000 years in northeastern China. It is used to make traditional foods, including tofu, soybean paste, miso, and soy sauce. Soybean was first introduced to Europe in the early 17th century and to the United States in the 18th century, and has since become an important global crop. Global soybean production was >340 million metric tons in 2017. Soybeans contain extremely high amounts of protein and oil (30%–50% and 13%–25% of the total mass, respectively). Soy protein meets the required amino acid composition for humans and animals, except for slightly low sulfur amino acid content. However, soy foods have not been well accepted in areas other than eastern Asia, due to its bitter taste, chalky mouthfeel, and the beany and greeny flavors that are generated primarily from linoleic acid by soy

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lipoxygenases. For that reason, most fat-free soy meal has been used as the primary source of protein for animal feeds or rations. In the 1960s, a new method became available to extract food-grade soy protein isolate (SPI) from defatted soy meal under lower temperatures. SPI is used in a variety of foods for its functional properties, including solubility, water and fat absorption, and emulsification. In addition, soy protein has been shown to lower the risk of cardiovascular disease in humans.1 In particular, one of the major storage proteins of soy, β-conglycinin, reduces high serum triacylglycerol (TG) concentration and visceral fat in humans.2 Daily intake of SPI and β-conglycinin is required for this effect, and thus, SPI supplementation in foods eaten daily is desirable. As such, bread is a convenient vehicle for SPI and β-conglycinin; however, SPI and β-conglycinin produce adverse effects on bread-making and bread quality. This chapter will briefly review the features and physiological functions of soy proteins, as well as the characteristics of bread fortified with soy proteins.

SOY PROTEINS In the typical soybean, proteins comprise approximately 40% of the total mass; however, both genetic and environmental factors strongly influence seed composition. The nutritional value of soy proteins is high; the protein digestibility-corrected amino acid score of SPI is approximately that of egg whites. Furthermore, the biological values (i.e., the ability of the body to absorb and utilize the protein) of whole soybean, soy milk, and SPI are 74, 96, and 91, respectively. These values are the highest among major edible crops. Adding soy protein to foods made from crops such as wheat, maize, and rice increases their nutritional value because soy protein contains relatively high amounts of lysine, which is a limiting amino acid for complete protein in these crops. Soybean produces exalbuminous seeds. Within the embryo, the major storage proteins glycinin and β-conglycinin are synthesized and stored in cotyledons, where they are subsequently used as nitrogen, carbon, and sulfur sources in embryonic development.3 These globulin proteins comprise approximately 60% of the total soy proteins3; the remaining proteins fulfill protective, structural, and metabolic roles. β-Conglycinin is composed of three main types of subunits designated α, α0 , and β, with molecular weights of 70,000, 70,000, and 48,000, respectively. Random combinations of these subunits form seven heterotrimers and three homotrimers. β-Conglycinin subunits are translated in the rough endoplasmic reticulum (ER), and undergo folding and assembly into trimers in the ER lumen. Subunits are modified by cotranslational N-glycosylation (i.e., one N-glycan on the β subunit and two N-glycans on the α- or α0 -subunit). The assembled trimers are transported via the Golgi apparatus and accumulate in protein-storage vacuoles. As shown in Fig. 1, approximately half of the α0 - and α-subunits of β-conglycinin were disulfide-linked, together or with P34, prior to N-terminal propeptide processing.4 Glycinin is composed of five types of subunits designated as A1aB1b, A1bB2, A2B1a, A3B4, and A5A4B3. They are categorized into two groups according to amino-acid-sequence similarity: Group I (A1aB1b, A1bB2, and A2B1a) and Group II (A3B4 and A5A4B3). Each glycinin subunit is synthesized in the rough ER as a precursor protein (molecular weights of approximately 50,000). They undergo folding, formation of intrachain disulfide bonds, and assembly into trimers in the ER lumen. Several lines of evidence indicate that the folding is performed with the aid of ER oxidoreductin 1,5 several members of the protein disulfide isomerase family, and molecular chaperons.5–9 The assembled trimers are transported via the Golgi or directly from the ER to protein-storage vacuoles. Glycinin precursor subunits in trimers are cleaved into acidic subunits (A) and basic subunits (B) at a well-conserved Asn-Gly peptide bond by a vacuolar-processing enzyme and then assembled into hexamers in the protein-storage vacuoles.3 The threedimensional (3D) structures of β-conglycinin and glycinin are very similar, suggesting that these genes evolved from a common ancestor gene.10,11 A subset of glycinin hexamers has been shown to exist as complexes noncovalently associated with β-conglycinin.4

ISOLATION OF SOY PROTEINS Most edible soy protein products are derived from white flakes made by dehulling, flaking, and defatting soybeans by hexane extraction. These products consist of defatted flour (approximately 50% protein), soy protein concentrate (65%–70% protein), and SPIs (>90% protein). The purified soy protein component β-conglycinin isolate has become commercially available. Soy protein products have many important functional properties, including solubility, water and fat absorption, emulsification, and imparting of texture (i.e., gelation, cohesion-adhesion, elasticity). Soy protein

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FIG. 1 Disulfide-linked complexes of β-conglycinin α0 /α and P34. (A) Proteins extracted from dry bean cotyledons of soybean (WT, lanes 1 and 2), β-conglycinin-knockdown soybean (7S-KD, lanes 3 and 4), glycinin-null mutant soybean (11S-null, lanes 5 and 6), or P34-null mutant soybean (P34-null, lanes 7 and 8) were separated by SDS-PAGE under nonreducing conditions (NR, lanes 1, 3, 5, and 7) or reducing conditions (R, lanes 2, 4, 6, and 8) and detected by Western blot analyses with anti-α0 subunit antibodies. (B) β-Conglycinin (30 μg of protein) prepared from dry bean cotyledon of wild-type soybean, with the N-ethylmaleimide-containing buffer without reducing reagent, was analyzed by two-dimensional (2D) SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Arrow head indicates a P34 spot separated from 100-kD complexes by the second SDS-PAGE. Because small amounts of glycinin were contaminated in the β-conglycinin preparation, spots of glycinin acidic chain (*) and glycinin basic chain (**) were detected.

products are used in a variety of foods, including beverages, meat products, bakery items, pasta products, cheeses, and simulated meats. Commercial SPI is extracted from white soy flakes with water. β-Conglycinin and glycinin are soluble in salt solution, and salts are present in white flakes; therefore, these proteins are easily extracted by adding water. Then most proteins can be isoelectrically precipitated after the extract is acidified (pH 4.3–4.8). Finally, the precipitated protein curd is neutralized, sterilized, and dried. SPI has been believed to be composed primarily of β-conglycinin and glycinin for a long time. Recently, SPI has been found to contain lipids associated with lipophilic proteins (LPs) such as oilbody-associated proteins.12,13 Because n-hexane cannot efficiently extract phospholipids or hydrophobic membrane proteins, acid-precipitated proteins contain LPs associated with membrane phospholipids from oil bodies and protein-storage vacuoles. Compared with β-conglycinin and glycinin, LPs are difficult to detect by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) due to lower sensitivity to Coomassie Brilliant Blue staining; thus, the importance of LPs in SPI has been overlooked. Samoto et al. have developed a method for fractionating acidprecipitated proteins (Fig. 2).14 In that study, the nitrogen distribution ratios for the three separated proteins were 23% (β-conglycinin), 46% (glycinin), and 31% (LPs).

PHYSIOLOGICAL FUNCTION OF SOY PROTEINS About 100 years ago, the cholesterol-lowering effects of soy protein compared with animal protein were reported in rabbits.15 Since then, many studies have reported the effects of soy proteins on serum lipids in humans; however, the results have been inconsistent, possibly because of different experimental elements such as soy protein content in the diet and degree of hypercholesterolemia in the subjects. In a metaanalysis published in 1995, Anderson et al. concluded that soy protein consumption significantly decreased serum levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and TG, corresponding to the degree of hypercholesterolemia.1 Based on these findings, the U.S. Food and Drug Administration (FDA) granted the following health claim for soy protein in 1999: “25 grams of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease.” Most commercially available SPI products contain significant amounts of genistein, daidzein, and glycitein. These isoflavones have been shown to exert strong biological actions in animals, such as serum cholesterol reduction, arterial

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FIG. 2 Schematic diagram depicting the fractionation of glycinin, LPs, and β-conglycinin. A three-step acidification of the water extract of defatted soy flour separated three proteins with the nitrogen distribution ratio of 23% (β-conglycinin), 46% (glycinin), and 31% (LPs).

vasodilation, and atherosclerosis inhibition.16 Hence, these isoflavones were assumed to be largely responsible for the beneficial effects of SPI on hypercholesterolemia in humans. Human studies comparing the effects of casein, animal proteins, and ethanol-washed isoflavone-free SPI on serum cholesterol levels have demonstrated declines in LDL cholesterol with isoflavone-free soy protein consumption.17,18 Further studies comparing the effects of SPI with or without isoflavones confirmed that isoflavones are not responsible for the lipid-lowering effects in humans. However, the soy protein components responsible for this effect are not known. Candidates include a peptide derived from glycinin that inhibits the reabsorption of bile acid from the intestine,19 and LPs that has been shown to reduce serum cholesterol.20 However, these studies were performed in rats; the effects of glycinin and soy LPs in humans are unclear. Thus, identification of the components responsible for cholesterol lowering remains unsolved. The effects of LPs-free β-conglycinin were assessed by supplementation of the diets of adults with high plasma TG. Intake of β-conglycinin (5 g/day) normalized serum TG and reduced visceral fat in subjects with a body mass index (BMI) between 25 and 30.2 Based on these findings, soy β-conglycinin was approved in 2007 as a food for specified health use (FOSHU) in Japan. The plasma TG level is controlled by the amount of very-low-density lipoprotein (VLDL) secreted from the liver and the rate of VLDL-TG catabolism in blood. To determine the effects of soy β-conglycinin on lipid metabolism, small peptides were derived from LP- and isoflavone-free β-conglycinin by protease digestion and used to treat the human hepatocellular carcinoma cell line HepG2.21 The findings showed that β-conglycinin-derived peptides suppressed TG synthesis, thereby suppressing the secretion of VLDL from HepG2 cells into the medium.

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ADVERSE EFFECTS OF SOY PROTEIN FORTIFICATION ON RHEOLOGICAL PROPERTIES OF DOUGH AND BREAD QUALITY

TABLE 1

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Effects of SPI on Rheological Properties of Wheat Flour Dough

Property

Equipment

Parameter

Effect of SPI

References

Farinographic behavior

Microfarinograph

Water absorption

Increase

22,24,26,28,29

Arrival time

Decrease

Development time

Decrease

Stability

Decrease

Storage modulus (E0 )

Increase

Loss modulus (E00 )

Increase

tanλ (E00 /E0 )

Decrease

Force at failure point

Increase

Failure point

No change

Rm

Increase

E

Decrease

Area

Decrease

Jo

Decrease

Jl

Decrease

μo

Increase

Dynamic viscoelastic property

Large-deformation property

Rheolograph gel

Rheoner

Microextensograph

Creep property

Texture analyzer

29

29

26,28

28

E, maximum extensibility; Jo, instantaneous compliance; Jl, retarded compliance; μo, Newtonian viscosity; Rm, maximum resistance to extension.

ADVERSE EFFECTS OF SOY PROTEIN FORTIFICATION ON RHEOLOGICAL PROPERTIES OF DOUGH AND BREAD QUALITY The addition of SPI and purified β-conglycinin to foods can increase soy protein consumption and help achieve a physiologically beneficial intake. Fortification of bread with soy protein has a long history due to soy having relatively high amounts of lysine and valine, which are the limiting amino acids of wheat proteins. Although the nutritive value of bread rises with the percentage of SPI,22 SPI reduces bread quality. SPI-containing bread was judged to be firmer, drier, grainier, less tender, and gummier compared to the ideal bread.23 In addition, SPI-containing bread exhibited a strong beany flavor, which curtailed its overall acceptability.24 The bread loaf volume also decreased, and bread crumb firmness and firming rate increased proportionally with the level of SPI fortification.22,25–27 An inverse relationship was shown between bread loaf volume and firmness; therefore, the low specific volume of SPI-containing breads was probably the cause of the increased firmness values rather than the effect of soy protein per se.26 A strong correlation between dough volume after fermentation and finished loaf volume has been observed in the presence of SPI. SPI had no effect on carbon dioxide (CO2) production by yeast; therefore, it may decrease the gas-retention capacity of dough. Scanning electron microscopy revealed that gluten containing SPI had a much more porous network than that of control dough lacking SPI,28 suggesting that SPI makes the gluten network brittle in response to the pressure of gas. Highly purified β-conglycinin decreases loaf volume more than SPI.29,30 Changes in the gluten network structure were accompanied by changes in the rheological properties of dough. Farinograph analysis revealed that the addition of SPI reduced arrival time, development time, and stability (Table 1).22,24,29 SPI also affected the dynamic viscoelastic properties of dough to increase storage (E0 ) and loss modulus (E00 ) values and decrease tanλ (E00 /E0 ) value, which represents the strength of the gluten network as determined by a Rheolograph gel.29 In addition, SPI affected the large-deformation properties of dough. The force value at the failure point obtained using a Rheoner increased after adding SPI to the dough.29 When tensile strength test was performed with the Intron Universal Testing Machine, SPI increased the resistance to extension and the relaxation time.25 Increased maximum resistance and decreased extensibility by SPI fortification were revealed using a microextensograph.28 At the same time, area under the extension curve, which is a measure of the energy required for extension, was reduced by SPI, and the area under the extension curve was highly correlated with specific loaf volume. By the creep test, SPI decreased instantaneous compliance, retarded compliance of the dough, and increased Newtonian viscosity of the dough28; the decrease in retarded compliance reflects the loss of elasticity of the dough. Taken together, the results obtained by the rheological assays indicated that SPI increases dough firmness and weakens the gluten network.

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SPI appears to weaken the gluten network both indirectly and directly. Soy proteins such as glycinin and β-conglycinin were detected in SDS-insoluble gluten proteins obtained from dough,31 indicating that soy proteins tightly associate with gluten proteins in dough. Hydrogen bonds and hydrophobic interactions between glutenin and gliadin molecules are essential for gluten formation; soy proteins may interfere with these interactions. The elastic properties of dough are related to the quantity of the SDS-insoluble glutenin macropolymer (GMP), which is comprised of high-molecular-weight and low-molecular-weight glutenin subunits linked by disulfide bonds. Although it seems possible that soy protein containing cysteine residues may decrease GMP content by thiol exchange or reduction of disulfide bonds among glutenin proteins, GMP content of SPI-containing dough did not show any significant differences compared with dough lacking SPI.32 Even a small amount of soy glycinin (2.5%–8.2% of total wheat protein) decreased developing time and stability time,33 and thus direct interactions with soy proteins may considerably alter the gluten network. The indirect influence of SPI is related to reduce water availability in the dough. Many studies have reported that SPI increases water absorption in wheat dough as assessed by farinograph.28,29 Thus, water binding in the dough is increased and syneresis is decreased.28 Free water is thought to act as an inert filler or lubricant in polymers like gluten.34 Therefore, changes in dynamic viscoelastic behavior and the relaxation phenomena may be due in part to the decrease in free water by SPI fortification.

IMPROVED QUALITY OF BREAD FORTIFIED WITH SOY PROTEIN The lower quality of bread due to SPI fortification is a serious problem. The increased firmness and smaller loaf volume may be primarily due to the reduced free water in dough and interference of the gluten network formation. Roccia et al. showed that adding more water to the dough attenuated the SPI-induced increase in maximum resistance, but the additional water had no effect on extensibility or the area under the extension curve.28 Therefore, the problem of increased firmness in SPI-containing bread can be solved by adjusting the water added. Further, adding more water restored normal instantaneous compliance and partially attenuated the decrease in retarded compliance on the creep test, which is an index parameter for elasticity.28 Several materials, including detergents, have been shown to improve the bread-making properties of SPIcontaining dough and the quality of protein-fortified bread. Studies have reported the effects of sodium stearoyl-2lactylate (SSL), nonionic hydrophilic polysorbate 60 (Tween 60), and nonionic lipophilic sodium tristearate (Span 65) on the rheological properties of the SPI-containing dough.25 These detergents did not alter the water adsorption of SPI-containing dough according to the farinograph assay, nor did they improve the increased resistance to extension or the relaxation time according to the tensile strength test. However, Tween 60 improved gas retention in the dough and increased loaf volume. SSL and Span 65 also improved gas retention in dough, but they did not completely restore the normal loaf volume of bread containing SPI. Lecithin is a mixture of polar lipids that is permitted for use in all types of bread and bakery products. It also improves gas retention in dough and sufficiently restores the volume of dough fortified with SPI (Fig. 3) or β-conglycinin.22,26,29 Moreover, the addition of lecithin produces a thicker, denser crust that tends to retain its crispness. Most lecithin used in the baking industry is derived from soybeans; soy lecithin is a mixture of phospholipids that includes phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylserine. PC is the active constituent in lecithin29; the function of PC cannot be replaced by other major phospholipids, phosphatidylethanolamine or compounds derived from PC, phosphatidic acid, lysophosphatidylcholine (lysoPC), or choline. Confocal microscopy revealed that soy PC colocalizes with gluten in dough, but not the starch granules (Fig. 4).29 Farinograph behaviors or viscoelastic properties of dough impaired by SPI were not improved by soy PC. Soy PC exerted little effect on arrival time, development time, or stability. Nor did soy PC change the rheolograph value (E0 , E00 ) or large deformation properties of SPI-containing dough. Interestingly, the effect of PC on delipidated wheat flour containing β-conglycinin was lower compared with its effect on native wheat flour containing β-conglycinin.30 Thus, soy PC alone has the ability to increase dough volume, and wheat-flour lipids appear to boost this effect. Among wheat lipids, glycolipids such as monogalactosyl diglyceride and digalactosyl diglyceride best enhance the action of soy PC.30 PCs are a class of phospholipids composed of two fatty acyl chains. Fatty acyl chains that are combined in soy PC are linoleic (18:2), palmitic (16:0), stearic (18:0), oleic (18:1), and linolenic (18:3) acids. Many PC molecular species are possible, and the effect of PC on bread loaf volume depends on the particular molecular species (Fig. 5). At one extreme, 1-palmitoyl, 2-palmitoyl-PC had no effect on dough volume at 36°C during fermentation. This molecular species exists in the gel crystalline form at 36°C because its gel-to-liquid crystalline phase transition temperature is 44.3°C.

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FIG. 3 Effects of SPI and soy PC supplementation on bread quality. (A) Unfortified bread (left), bread fortified with SPI (center), and bread fortified with SPI plus soy PC (right). (B) SEM of unfortified bread (left), with SPI (center), or with SPI plus soy PC (right). For the wheat flour-soy protein combinations, 10% of the wheat flour was replaced with SPI (by weight). PC (2 g) was added to flour (100 g). PC, the active constituent in lecithin, improves gas retention in dough and sufficiently restores the volume of dough fortified with SPI. Bars, 200 μm.

FIG. 4 Distribution of PC and gliadin in dough. Dough was made with soy PC containing the fluorescent PC, β-BODIPY FL C12-HPC. Gliadin in dough was immunostained with antigliadin rabbit serum, biotinantirabbit immunoglobulin G (IgG) goat serum, and Cy 5-streptoavidin. The dough was visualized with a laser confocal imaging system showing (A) PC (white) and (B) gliadin (white). The fluorescent PC, β-BODIPY FL C12-HPC, was associated with protein fibers containing gliadin and yeast (arrows). Bar, 10 μm.

In contrast, PC molecular species with a liquid-crystalline phase transition temperature lower than 36°C increase dough volume, possibly because PC in the liquid-crystalline state improves the gas-retaining ability of dough during fermentation. Soy PC is composed of molecular species with liquid-crystalline phase transition temperatures lower than 36°C, including dilinoleoyl-PC (35%), 1-palmitoyl, 2-linoleoyl-PC (24%), and 1-oleoyl, 2-linoleoyl-PC (15%).29 Differential scanning calorimetry (DSC) analysis revealed the phase transition of soy PC occurred at 48.5°C to 13.1°C. In its

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FIG. 5 Effects of PC molecular species on volume of SPI-containing dough after fermentation. Dough was made with 100% wheat flour or wheat flour in which 10% was replaced with SPI. In some doughs, the flour (100 g) was further supplemented with 2 g of PC or phosphatidylethanolamine (PE). The dough (15 g) was then incubated at 36°C for 40 min. 14:0/14:0, dimyristoyl PC; 16:0/16:0, dipalmitoyl PC; 18:1/18:1, dioleoyl PC or PE; 18:2/18:2, dilinoleoyl PC. The liquid-crystalline phase transition temperature of each PC was shown in the parentheses, in degrees Celsius. PC molecular species with a liquidcrystalline phase transition temperature lower than 36°C increase dough volume.

fluid liquid-crystalline state, PC self-assembles into a stable bilayer structure and exists as liposomes or multilamellar vesicles (lamellae) in water. The most surface-active property of polar lipids is thought to be a bilayer structure. Thus, a possible mechanism for the beneficial effects of PC may be its direct influence on gas cells in dough by increasing foam stability.29

SUMMARY POINTS • SPI is composed of three major proteins: β-conglycinin, glycinin, and LPs. • Physiological functions of SPI include the ability to reduce the risk of cardiovascular disease in humans by decreasing serum cholesterol levels. • Highly purified β-conglycinin normalizes serum TG levels in subjects with high serum TG and reduces visceral fat in subjects with a BMI between 25 and 30. • Both SPI and β-conglycinin adversely affect bread-making and bread quality by increasing water absorption and interfering with the formation of the gluten network. • PC increases loaf volume without affecting the rheological properties of dough.

Acknowledgments The author gratefully acknowledges financial support from the Fuji Foundation for Protein Research, the Elizabeth Arnold Fuji Foundation, and the Iijima Memorial Foundation for the Promotion of Food Science and Technology.

References 1. Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the effects of soy protein intake on serum lipids. N Engl J Med 1995;333:276–82. 2. Kohno M, Hirotsuka M, Kito M, Matsuzawa Y. Decreases in serum triacylglycerol and visceral fat mediated by dietary soybean beta-conglycinin. J Atheroscler Thromb 2006;13:247–55. 3. Nielsen NC, Nam Y-W. Soybean globulins. In: Shewry PR, Casey R, editors. Seed proteins. Dordrecht: Kluwer Academic Publishers; 1999. p. 285–313. 4. Wadahama H, Iwasaki I, Matsusaki M, Nishizawa K, Ishimoto M, Arisaka F, Takagi K, Urade R. Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α0 - and α-subunits. Plant Physiol 2012;158:1394–405.

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5. Matsusaki M, Okuda A, Masuda T, Koishihara K, Mita R, Iwasaki K, Hara K, Naruo Y, Hirose A, Tsuchi Y, Urade R. Cooperative protein folding by two protein thiol disulfide oxidoreductases and ERO1 in soybean. Plant Physiol 2016;170:774–89. 6. Wadahama H, Kamauchi S, Ishimoto M, Kawada T, Urade R. Protein disulfide isomerase family proteins involved in soybean protein biogenesis. FEBS J 2007;274:687–703. 7. Kamauchi S, Wadahama H, Iwasaki K, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T, Urade R. Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins. FEBS J 2008;275:2644–58. 8. Wadahama H, Kamauchi S, Nakamoto Y, Nishizawa K, Ishimoto M, Kawada T, Urade R. A novel plant protein disulfide isomerase family homologous to animal P5—molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins. FEBS J 2008;275:399–410. 9. Okuda A, Matsusaki M, Masuda T, Urade R. Identification and characterization of GmPDIL7, a soybean ER membrane-bound protein disulfide isomerase family protein. FEBS J 2017;284:414–28. 10. Adachi M, Kanamori J, Masuda T, Yagasaki K, Kitamura K, Mikami B, Utsumi S. Crystal structure of soybean 11S globulin: glycinin A3B4 homohexamer. Proc Natl Acad Sci U S A 2003;100:7395–400. 11. Maruyama Y, Maruyama N, Mikami B, Utsumi S. Structure of the core region of the soybean beta-conglycinin alpha’ subunit. Acta Crystallogr D Biol Crystallogr 2004;60:289–97. 12. Iwabuchi S, Yamauchi F. Determination of glycinin and beta-conglycinin in soybean proteins by immunological methods. J Agric Food Chem 1987;35:200–5. 13. Samoto M, Miyazaki C, Kanamori J, Akasaka T, Kawamura Y. Improvement of the off-flavor of soy protein isolate by removing oil-body associated proteins and polar lipids. Biosci Biotechnol Biochem 1998;62:935–40. 14. Samoto M, Maebuchi M, Miyazaki C, Kugitani H, Kohno M, Hirotsuka M, Kito M. Abundant proteins associated with lecithin in soy protein isolate. Food Chem 2007;102:317–22. 15. Ignatowsky MA. Influence de la nourriture animale sur l’organisme des lapins. Arch Med Exp Anat Pathol 1908;20:1–20. 16. Sacks FM, Lichtenstein A, Van Horn L, Harris W, Kris-Etherton P, Winston M. Soy protein, isoflavones and cardiovascular health. Circulation 2006;113:1034–44. 17. Lichtenstein AH, Jalbert SM, Adlercreutz H, Goldin BR, Rasmussen H, Schaefer EJ, Ausman LM. Lipoprotein response to diets high in soy or animal protein with and without isoflavones in moderately hypercholesterolemic subjects. Arterioscler Thromb Vasc Biol 2002;22:1852–8. 18. Jenkins DJ, Kendall CW, Jackson CJ, Connelly PW, Parker T, Faulkner D, Vidgen E, Cunnane SC, Leiter LA, Josse RG. Effects of high- and lowisoflavone soy foods on blood lipids, oxidized LDL, homocysteine, and blood pressure in hyperlipidemic men and women. Am J Clin Nutr 2002;76:365–72. 19. Nagaoka S, Awano T, Nagata N, Masaoka M, Hori G, Hashimoto K. Serum cholesterol reduction and cholesterol absorption inhibition in CaCo-2 cells by a soy protein peptic hydrolyzate. Biosci Biotechnol Biochem 1997;61:354–6. 20. Kanamoto R, Kimura S, Okamura G. Cholesterol lowering effect of soybean lipophilic proteins associated with phospholipids in rat. Soy Protein Res 2007;10:83–7. 21. Mochizuki Y, Maebuchi M, Kohno M, Hirotsuka M, Wadahama H, Moriyama T, Kawada T, Urade R. Changes in lipid metabolism by soy betaconglycinin-derived peptides in HepG2 cells. J Agric Food Chem 2009;57:1473–80. 22. Mizrahi S, Zimmermann G, Berk Z, Cogan U. The use of isolated soybean proteins in bread. Cereal Chem 1967;44:193–203. 23. Elgedaily A, Campbell AM, Penfield MP. Texture of yeast breads containing soy protein isolates: sensory and objective evaluation. J Food Sci 1982;47:1149–50. 24. Ranhotra GS, Loewe RJ. Breadmaking characteristics of wheat flour fortified with various commercial soy protein products. Cereal Chem 1974;51:629–34. 25. Chen SS, Rasper VF. Functionality of soy proteins in wheat flour/soy isolate doughs. II. Rheological properties and bread making potential. Can Inst Food Sci Technol J 1982;15:211–20. 26. Ribotta PD, Arnulphi SA, Leon AE, Anõn MC. Effect of soybean addition on the rheological properties and breadmaking quality of wheat flour. J Sci Food Agric 2005;85:1889–96. 27. Lazo-Velez MA, Chuck-Hernandez C, Serna-Saldívar SO. Evaluation of the functionality of five different soybean proteins in yeast-leavened pan breads. J Cereal Sci 2015;64:63–9. 28. Roccia P, Ribotta PD, Perez GT, Leon AE. Influence of soy protein on rheological properties and water retention capacity of wheat gluten. Food Sci Technol 2009;42:358–62. 29. Urade R, Okamoto S, Yagi T, Moriyama T, Ogawa T, Kito M. Functions of soy phosphatidylcholine in dough and bread supplemented with soy protein. J Food Sci 2003;68:1276–82. 30. Ukai T, Urade R. Cooperation of phosphatidylcholine with endogenous lipids of wheat flour for an increase in dough volume. Food Chem 2006;102:225–31. 31. Ribotta PD, Leon AD, Perez GT, Anon MC. Electrophoresis studies for determining wheat-soy protein interactions in dough and bread. Eur Food Res Technol 2005;221:48–53. 32. Perez GT, Ribotta PD, Steffoliani ME, Leon AE. Effect of soybean proteins on gluten depolymerization during mixing and resting. J Sci Food Agric 2008;88:455–63. 33. Lampart-Szczapa E, Jankiewicz M. Changes in the protein complex of wheat dough affected by soybean 11S globulin: Part 1—The effects of soybean 11S globulin addition on the technological properties of wheat dough. Food Chem 1982;9:307–14. 34. Masi P, Cavella S, Sepe M. Characterization of dynamic viscoelastic behavior of wheat flour doughs at different moisture contents. Cereal Chem 1998;75:428–32.

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C H A P T E R

29 Resistant Starch (RS) in Breads: What It Is and What It Does William R. Sullivan, and Darryl M. Small School of Science, Applied Chemistry and Environmental Science Discipline, RMIT University, Melbourne, Victoria, Australia

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Hypoglycemic Effects and Weight Management

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INTRODUCTION The term resistant starch (RS) refers to the portion of starch that defies catabolic actions from human digestive enzymes. Being first reported by Englyst, Wiggins, and Cummings,1 research around RS functionality and fortification in foods has been the focus of a considerable amount of research in the past decade. The primary driving factor for this has been the wide variety of health benefits resulting from RS consumption. Bread is a staple food globally, and as such, it has the potential to act as an appropriate vehicle to supply significant amounts of RS in the human diet. The purpose of this chapter is to discuss how carbohydrates are digested, the various forms of RS, the primary health benefits, how RS is measured in foods, and the significance of and incorporation of RS into bread formulations.

STARCH—FORM AND GRANULAR ARCHITECTURE Starch is one of the most abundant and functionally significant polymers occurring naturally on Earth; it acts as the primary energy reserve in many plants. Being made of the monomer D-glucose, starch exists in two primary forms: amylose and amylopectin.2 The glucose units in both amylose and amylopectin are bonded with covalent, glycosidic linkages that are either α(1 ! 4) or α(1 ! 6) in nature. The α(1 ! 4) bonds are responsible for linear links,

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TABLE 1 A Comparison of Key Properties of the Starch Componentsa4 Amylose

Amylopectin

General structure

Essentially linear

Treelike

Proportion of α(1 ! 6) linkages (%)

30 retrospective epidemiologic studies have explored the link between dietary folate and total folate intake (dietary and/or total folate intake, including supplemental FA) and the risk of CRC or adenoma. Most of them reported a significant or ambiguous inverse association. Together, these retrospective studies suggest a 140% reduction in the odds ratio of CRC in subjects with the highest folate intake compared with those with the lowest intake, without clinical evidence of folate deficiency. Moreover, the relationship between blood levels of folate and the risk of CRC and adenoma is less well defined than with folate intake.

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In 1996, Tseng et al.14 observed in women that only folate was inversely associated with adenoma risk, even after adjusting for other individual micronutrients. In the Nurses’ Health Study (NHS) and the Health Professionals FollowUp Study (HPFS), colorectal adenoma risk was 30%–40% lower in individuals whose median folate intakes were 711 mg/day in women and 847 mg/day in men, compared to the risk associated with the lowest folate intakes (166 mg/day for women and 241 mg/day for men). In the NHS, women whose total folate intake (i.e., via diet plus supplements) was 2400 mg/day had a 31% decrease in risk of CC compared to women in the lowest folate intake group (40 nmol/L in 37% of these individuals.29 As in the United States and Canada,2 a temporal association between folate fortification and an increase in CRC hospital discharge was observed. The rate/ratio between the period before and after the fortification for CC in the group aged 45–64 years was 2.6 (99% CI, 2.93–2.58) and for the group aged 65–79 years, it was 2.9 (99% CI, 3.25–2.86), as shown in Figs. 2 and 3.3 FIG. 2 Rate/ratio of hospital discharge because of CC in adults aged 45–64 years, before and after the start of the mandatory program of fortifying flour with 220 mg of synthetic FA/100 g of wheat flour. Rate/ratios are expressed as the rate for each year/the rate for 1992.

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FIG. 3 Rate/ratio of hospital discharge because of CC in adults aged 65–79 years, before and after the start of the mandatory program of fortifying flour with 220 mg of synthetic FA/100 g of wheat flour. Rate/ratios are expressed as the rate for each year/the rate for 1992.

One possible explanation for this finding is that this increase is causally related to FA fortification. Other explanations for the increase in the discharge rates for CRC could be an increase in the incidence of risk factors such as obesity, low intake of fiber and calcium, and high intake in fat and red meat. The prevalence of obesity increased from 19.7% in 1997 to 22% in 2003.30 Unfortunately, there are no data on other attributable risks of these factors, but according to Food and Agricultural Organization (FAO) data, the supply of calories and protein has not changed significantly. The FA fortification program has had an effect on homocysteine levels.29 We therefore expected a decline in the rates of cardiovascular disease because hyperhomocysteinemia is considered a cardiovascular risk factor.31, 32 However, the discharge rates for cardiovascular diseases did not change in the two study periods. This is consistent with FA supplementation trials, which have not reported a reduction in the incidence of cardiovascular events.33 There is no cancer registry in Chile. Therefore, the only means of studying the impact of fortification on CRC incidence is to use indirect data. Because a form indicating the diagnosis and other variables must be completed for every discharge from every hospital in Chile, this information, which is complete and reliable, can be used as a proxy for disease incidence. Thus, we used it to study the trends in the incidence of CRC and compare it with that of other diseases as a control. The changes in disease frequency detected using hospital discharge data coincided with mortality trends for breast and gastric cancer and CRC. This gives further support to the validity of hospital discharge data as a proxy for disease incidence. This agreement in the observational studies in three countries after 10 years of the fortification program does not prove causality, but it is consistent with the known effects of folate on existing neoplasms (adenomas), as demonstrated in experimental and clinical studies. Another plausible explanation for the increase in CRC associated with FA fortification is that supraphysiologic fortification of bread or supplementation increases circulating unmetabolized FA, and the real consequence of this is unknown. There is evidence that daily ingestion of 400 mg, or plasma levels >40 nmol/l, produces a sustained appearance of unmetabolized FA in the blood. Troen et al.33 observed that increasing concentrations of plasma FA among postmenopausal women who took FA supplements were inversely associated with decreases in the cytotoxicity of circulating NK cells, which play a role in the destruction of arising clones of endothelial cancer cells. However, we did not demonstrate in vitro that supraphysiological levels of folate were associated with impaired NK cell activity.10 Global methylation status also may be altered with high folate plasma levels. We observed that healthy male subjects in the fortification era, without vitamin supplementation, with plasma folate levels >45 nmol/l had higher SAM and SAH concentrations than those of subjects with normal folate levels.34 In summary, there is evidence that an FA fortification program with 150 or 220 mg of synthetic FA/100 g of wheat flour may be associated with an additional risk of CRC. Thus, it is crucial to evaluate this finding to determine the safe upper limit for folate intake, as well as the safe upper folate concentration and the amount of FA necessary to prevent NTDs and to minimize possible adverse effects.

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SUMMARY POINTS • Folate has a critical function in methylation reactions, as well as in DNA synthesis and repair. • Folate may accelerate tumor cell growth. • In the mandatory era of FA fortification, approximately 40% of the population has supraphysiologic levels of serum or plasma folate. • Low and high folate intake or plasma concentration are related with the risk of CRC. • The upper limit for FA intake, as well as the safe upper folate concentration, must be determined to prevent adverse effects.

References 1. Bailey LB. Folate, methyl-related nutrients, alcohol, and the MTHFR 677C-T polymorphism affect cancer risk: intake recommendations. J Nutr 2003;133(11 Suppl. 1):S3748–53. 2. Mason JB, Dickstein A, Jacques PF, Haggarty P, Selhub J, Dallal G, et al. A temporal association between folic acid fortification and an increase in colorectal cancer rates may be illuminating important biological principles: a hypothesis. Cancer Epidemiol Biomark Prev 2007;16:1325–9. 3. Hirsch S, Sanchez H, Albala C, de la Maza MP, Barrera G, Leiva L, et al. Colon cancer in Chile before and after the start of the flour fortification program with folic acid. Eur J Gastroenterol Hepatol 2009;21:436–9. 4. Fife J, Raniga S, Hider PN, Frizelle FA. Folic acid supplementation and colorectal cancer risk: a meta-analysis. Colorectal Dis 2011;13:132–7. 5. Smith AD, Kim YI, Refsum H. Is folic good for everyone? Am J Clin Nutr 2008;87:517–33. 6. Kim Y. Folate and colorectal cancer: an evidence-based critical review. Mol Nutr Food Res 2007;51:267–92. 7. Farber S. Some observations on the effect of folic acid antagonists on acute leukemia and other forms of incurable cancer. Blood 1949;4:160–7. 8. Sanchez H, Hossain MB, Lera L, Hirsch S, Albala C, Uauy R, Broberg K, Ronco AM. High levels of circulating folate concentrations are associated with DNA methylation of tumor suppressor and repair genes p16, MLH1, and MGMT in elderly Chileans. Clin Epigenetics 2017;9:74–85. 9. Paniz C, Bertinato JF, Lucena MR, De Carli E, Amorim PMDS, Gomes GW, Palchetti CZ, Figueiredo MS, Pfeiffer CM, Fazili Z, Green R, GuerraShinohara EM. A daily dose of 5 mg folic acid for 90 days is associated with increased serum unmetabolized folic acid and reduced natural killer cell cytotoxicity in healthy Brazilian adults. J Nutr 2017;147:1677–85. 10. Hirsch S, Miranda D, Muñoz E, Montoya M, Ronco AM, de la Maza MP, Bunout D. Natural killer cell cytotoxicity is not regulated by folic acid in vitro. Nutrition 2013;29:772–6. 11. Zhang Y, Li B, Ji ZZ, Zheng PS. Notch1 regulates the growth of human colon cancers. Cancer 2010;116:5207–18. 12. Rodriguez JM, Miranda D, Bunout D, Ronco AM, de Ia Maza MP, Hirsch S. Folates induce colorectal carcinoma HT29 cell line proliferation through Notch1 signaling. Nutr Cancer 2015;67(4):706–11. 13. Mathers JC. Folate intake and bowel cancer risk. Genes Nutr 2009;4:173–8. 14. M1 T, Murray SC, Kupper LL, Sandler RS. Micronutrients and the risk of colorectal adenomas. Am J Epidemiol 1996;144(11):1005–14. 15. Ma J, Stampfer MJ, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 1997;57:1098–102. 16. Yee YK, Tan VP, Chan P, Hung IF, Pang R, Wong BC. Epidemiology of colorectal cancer in Asia. J Gastroenterol Hepatol 2009;24:1810–6. 17. Sanjoaquin MA, Allen N, Couto E, Roddam AW, Key TJ. Folate intake and colorectal cancer risk: a meta-analytical approach. Int J Cancer 2005;113:825–8. 18. Otani T, Iwasaki M, Sasazuki S, Inoue M, Tsugane S, Japan Public Health Center-based Prospective Study Group. Plasma folate and risk of colorectal cancer in a nested case control study: the Japan Public Health Center-based prospective study. Cancer Causes Control 2008;19:67–74. 19. Kune G, Watson L. Colorectal cancer protective effects and the dietary micronutrients folate, methionine, vitamins B6, B12, C, E, selenium, and lycopene. Nutr Cancer 2006;56:11–21. 20. van den Donk M1, Buijsse B, van den Berg SW, Ocke MC, Harryvan JL, Nagengast FM, Kok FJ, Kampman E. Dietary intake of folate and riboflavin, MTHFR C677T genotype, and colorectal adenoma risk: a Dutch case-control study. Cancer Epidemiol Biomark Prev 2005;14:1562–6. 21. Vollset SE, Clarke R, Lewington S, Ebbing M, Halsey J, Lonn E, et al. Effects of folic acid supplementation on overall and site-specific cancer incidence during the randomised trials: meta-analyses of data on 50,000 individuals. Lancet 2013;381:1029–36. 22. Cole BF, Baron JA, Sandler RS, et al. Polyp prevention study group. Folic acid for the prevention of colorectal adenomas: a randomized clinical trial. JAMA 2007;297:2351–9. 23. K1 W, Platz EA, Willett WC, Fuchs CS, Selhub J, Rosner BA, Hunter DJ, Giovannucci E. A randomized trial on folic acid supplementation and risk of recurrent colorectal adenoma. Am J Clin Nutr 2009;(6):1623–31. 24. Ebbing M, Bønaa KH, Nygard O, Arnesen E, Ueland PM, Nordrehaug JE, et al. Cancer incidence and mortality after treatment with folic acid and vitamin B12. JAMA 2009;302:2119–26. 25. Bashir O, Fitzgerald AJ, Goodlad RA. Both suboptimal and elevated vitamin intake increase intestinal neoplasia and alter crypt fission in the ApcMin/þ mouse. Carcinogenesis 2004;25:1507–15. 26. Keyes MK, Jang H, Mason JB, Liu Z, Crott JW, Smith DE, et al. Older age and dietary folate are determinants of genomic and p16-specific DNA methylation in mouse colon. J Nutr 2007;137:1713–7. 27. Dary O. Nutritional interpretation of folic acid intervention. Nutr Rev 2009;67:235–44. 28. Hertramf E, Cortes F. Folic acid fortification of wheat flour: Chile. Nutr Rev 2004;62:S44–9. 29. Hirsch S, de la Maza P, Barrera G, Gattas V, Petermann M, Bunout D. The Chilean flour folic acid fortification program reduces serum homocysteine levels and masks vitamin B12 deficiency in elderly people. J Nutr 2002;132:289–91. 30. Vio F, Albala C, Kain J. Nutrition transition in Chile revisited: mid-term evaluation of obesity goals for the period 2000-2010. Public Health Nutr 2008;11:405–12.

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31. Bostom AG, Silbershatz H, Rosenberg IH, et al. Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Arch Intern Med 1999;159:1077–80. 32. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149–55. 33. Troen AM, Mitchell B, Sorensen B, Wener MH, Johnston A, Wood B, et al. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytotoxicity among postmenopausal women. J Nutr 2006;136:189–94. 34. Jenkins DJA, Spence JD, Giovannucci EL, Kim YI, Josse R, et al. Supplemental vitamins and minerals for CVD prevention and treatment. Am Coll Cardiol 2018;71:2570–84.

Further Reading 35. Hirsch S, Ronco AM, Guerrero-Bosagna C, de la Maza MP, Leiva L, Barrera G, et al. Methylation status in healthy subjects with normal and high serum folate concentration. Nutrition 2008;11(12):1103–9.

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33 Effects of the Soybean Flour Diet on Insulin Secretion and Action Ma´rcia Queiroz Latorraca*, Priscila da Costa Rodrigues†, Michele Cristiane Laux†, Chaiane Aline da Rosa†, Vanessa Cristina Arantes*, and Marise Auxiliadora de Barros Reis* †

*Faculdade de Nutric¸ a˜o, Universidade Federal de Mato Grosso, Cuiaba´, Brasil Mestrado em Nutric¸ a˜o, Alimentos e Metabolismo, Faculdade de Nutric¸ a˜o, Universidade Federal de Mato Grosso, Cuiaba´, Brasil

O U T L I N E Introduction

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Technological Issues

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Human Clinical Trials

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Summary Points

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Animal Studies Effects of Soybean on Insulin Secretion Effects of Soybean Insulin Action

426 426 427

References

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Abbreviations ACC Akt AMPK ARC CLY FAS GLUT2 IRSs JNK ME NPY PI3-K PPAR PVN SREBP-1c TNF-α UCP-1

Acetyl-coenzyme A carboxylase Protein Kinase B Adenosine Monophosphate–Activated Protein Kinase S6K1 Arcuate Nucleus ATP Citrate Lyase Fatty Acid Synthase Glucose Transporter 2 Insulin Receptor Substrates c-Jun N-terminal kinase Malic Enzyme Neuropeptide Y Phosphatidylinositol 3-kinase Peroxisome proliferator-activated receptor Paraventricular Nucleus Sterol Regulatory Element-Binding Protein-1c Tumor Necrosis Factor-alpha Uncoupling Protein-1

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00033-2

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© 2019 Elsevier Inc. All rights reserved.

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INTRODUCTION Soybean (Glycine max) is an important plant for human and animal nutrition due to its large amounts of protein, lipid, carbohydrates, minerals, vitamins and fiber. The protein content ranges from 35% to 45%, depending on the grain variety. Soybean protein contains most of the essential amino acids for human nutrition, which makes soybean products almost equivalent to animal sources in protein quality. Although rich in protein, soybean is deficient in sulfur-containing amino acids such as methionine and cystine, contains low methionine/glycine and lysine/arginine ratios compared to casein, and possesses antinutritional factors commonly found in plants, impairing its protein bioavailability.1 Heat treatment destroys the antinutritional factors, increasing the nutritional value of soybean protein. Soybean lipids contain approximately 60% of polyunsaturated fatty acids (PUFAs), 23% of monounsaturated fatty acids (MUFAs), and 15% of saturated fatty acids.2 Carbohydrates make up about 33% of the seed, with 17% being soluble carbohydrates (sucrose, raffinose, stachyose) and 16% being insoluble carbohydrates (dietary fiber).3 In addition to its high nutritional value, soybean protein is unique among plant-based proteins, in that it contains the largest concentrations of isoflavones (biologically active plant substances with structures similar to that of estradiol), which possess the ability to bind to estrogen receptors in various cells.4 Because of its reduced cost and elevated nutritional value, soybean flour has been used as an alternative feed in the recovery of nutritional status. In addition, soy-based ingredients possess properties that lead to metabolic and/or physiological effects, preventing and/or inducing therapeutic activities for a series of chronic disease, such as diabetes mellitus and obesity. Part of the beneficial effects of soybean and by-products has been associated with their effects on insulin secretion and action. Insulin is an anabolic, polypeptide hormone, synthesized by pancreatic β-cells, whose synthesis is activated by an increase of nutrients, especially glucose. Insulin acts on several periphery tissues, including liver, muscle, and adipose tissue (Fig. 1).

HUMAN CLINICAL TRIALS Many studies have reported beneficial effects of soy consumption on human health, but most of these investigations evaluated its effects in serum lipids. Human clinical trials have showed the antiobesity and antidiabetic effects of soy protein and isoflavones (Table 1). Studies regarding the effect of soy on body composition and body fat distribution show inconclusive results. In a randomized, double-blind, placebo-controlled 3-month trial and a cross-sectional study of postmenopausal women, a diet high in soy decreased body mass index (BMI) and waist circumference.5 On the other hand, one randomized placebo-controlled trial of soy on body composition performed in perimenopausal or postmenopausal women reported that soy did not affect BMI.13 Another randomized, double-blind, placebo-controlled multicenter trial with FIG. 1 Insulin actions on target tissues. Insulin exerts anabolic effects on tissues handling key fuels. Primary targets are white adipose tissue (left), liver (center), and skeletal muscles (right).

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TABLE 1

Effect of Soybean Diet in Human Clinical Trials

Patients

Intervention

Duration

Effect

References

15 postmenopausal women

Shake containing 20 g of soy protein +160 mg isoflavones versus an isocaloric casein placebo shake

12 weeks

Total and subcutaneous abdominal fat decreased

5

42 postmenopausal women with metabolic syndrome

Soy-protein diet, or a soy-nut diet versus a control diet (dietary approaches to stop hypertension)

8 weeks

A soy-nut diet decreased HOMA-IR index, fasting plasma glucose, and serum C-peptide concentrations

6

32 postmenopausal women

Diet-controlled type 2 diabetes supplemented with soy protein 30 g/day, isoflavones 132 mg/ day versus placebo

12 weeks

Reduced fasting insulin, insulin resistance and HbA1c

7

208 postmenopausal women

Association between usual dietary isoflavonein take and CVD risk factors

Usual frequency of consumption during 1 year for tofu, bean curd, and meat substitutes made from soy

High genistein intake reduced BMI, waist circumference, and fasting insulin

8

299 healthy postmenopausal women

Soy isoflavone or placebo tablets (80 or 120 mg/d)

12 months

There were no effects on body composition or on levels of insulin, leptin, ghrelin, or adiponectin

9

29 adults with type 2 diabetes

Diet-controlled type 2 diabetes supplemented with soy or milk protein isolate (58 g/d)

57 days

No effects on blood glucose

10

Women with gestational diabetes, 51 control cases versus 46 cases oligosaccharides

Soy oligosaccharides (10 g/dia in 200–300 mL warm water)

8 weeks

Improved insulin resistance and reduced oxidative stress

11

70 women with polycystic ovary syndrome

Soy isoflavones (50 mg/d) versus placebo

12 weeks

Reduced insulinemia, HOMA-IR, levels of androgenic hormones, triglycerides, and malonaldehyde; increased total glutathione in plasma

12

HOMA-IR, homeostasis model assessment for insulin resistance.

healthy postmenopausal women who consumed placebo or soy isoflavone (80 or 120 mg/d) tablets for 12 months did not report any favorable effect of isoflavone on body composition or appetitive hormone concentrations (insulin, leptin, ghrelin, and adiponectin).9 Inclusion of isoflavones in the diet of postmenopausal women with type 2 diabetes and metabolic syndrome has been shown to improve glycemic control, insulin resistance, and glycated hemoglobin (HbA1c).6, 7 However, soy protein isolate offered to adults with diet-controlled type 2 diabetes did not improve glycemia.10 Soybean oligosaccharides also exert beneficial effects on metabolic parameters in diabetics. Women with gestational diabetes mellitus who ingested soybean oligosaccharides exhibited improved insulin resistance and reduction oxidative stress.11 In crosssectional8 and crossover trials14 in postmenopausal women on high-soy diets, there was a trend toward lower fasting insulin. A meta-analysis to investigate whether soy isoflavones affect glucose homeostasis in menopausal women displayed a significant tendency in favor of soy isoflavones. Genistein alone played an important role in improving glucose metabolism due to its low heterogeneity.15 In a double-blind, placebo-controlled trial performed on women diagnosed with polycystic ovary syndrome, 50 mg/d soy isoflavones were administered for 12-week improved markers of insulin resistance (HOMA-IR), hormonal status (free androgen index), triglycerides, and biomarkers of oxidative stress (plasma total glutathione and malondialdehyde levels).12 A meta-analysis of randomized controlled trials that evaluated the effects of soy protein supplementation on clinical indices in type 2 diabetes and metabolic syndrome subjects showed benefits for fasting plasma glucose, fasting serum insulin, the homeostasis model assessment for insulin resistance (HOMA-IR), diastolic blood pressure, lower-density lipoprotein (LDL-C) cholesterol, total cholesterol, and plasma C reactive protein.16

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It is noteworthy to emphasize the limitations of studies performed with humans due to biases and reduced number of subjects. The mechanisms by which a soybean diet may exert its beneficial effects are not completely clear. Due to the difficulties in working with humans, the literature mostly describes effects of a soybean diet in rodents.

ANIMAL STUDIES Effects of Soybean on Insulin Secretion Studies have shown inconsistent effects of soybean diets or soy isoflavones on serum insulin concentration. It has been reported that consumption of soybean, soybean isoflavones, soybean protein isolates, and soybean flour reduces hyperinsulinemia and serum insulin levels in the fasting and postprandial states17–19;or increases insulin concentration in the basal and fed states, as well as total area under the insulin curves in response to a glucose load.20, 21 These discrepancies are possibly due to differences in the experimental design, the composition of these diets, and the duration of the dietary intervention. It is not clear whether the effect of a soybean diet on serum insulin concentration is associated with peripheral insulin sensitivity or changes in the mechanism of insulin secretion. Insulin secretion by pancreatic β-cells is regulated by plasma glucose concentration, as shown in Fig. 2. In vitro studies using clonal pancreatic β-cell line and cultured or fresh islets from various animal models have been carried out in an attempt to verify whether soybean alters insulin secretion, to identify which soybean components could modify the insulin secretion, and to elucidate its mechanisms of action. At least two soybean components have been strongly associated with changes in insulin secretion: genistein and the amino acid pattern of soy protein. However, the secretory response of pancreatic β-cells has varied considerably. Long-term soy protein consumption, for example, stimulated insulin secretion to a lesser extent, even in the presence of high saturated fat, than did a casein diet. This effect was associated with a decrease in pancreatic islet area, insulin, and peroxisome proliferator-activated γ receptor (PPARγ) messenger ribonucleic acid (mRNA) expression, which prevented the induction of glucose transporter 2 (GLUT-2), reducing glucose entrance to the β-cell and therefore impairing insulin secretion. The effects observed in isolated pancreatic islets cultured with the amino acid concentration resembled those found after soy protein consumption, as well as with isoflavones.19 Hence, at least in this experimental model that exhibits hyperinsulinemia, the combined effect of amino acids and isoflavones in the soy protein reduced the amount of insulin.

FIG. 2 Coupling mechanism of glucose-induced insulin secretion. Glucose enters the pancreatic β-cell through GLUT-2 transporters, and its

metabolism increases the ATP:ADP ratio, causing K+ ATP-sensitive channels to close, which depolarizes the plasma membrane (PM). Ca2+ L-type channels then open and allow Ca2+ entry, which activates the exocytotic machinery. This primary mechanism is modulated by classic hormone-responsive second messenger pathways, such as adenylyl-cyclase (AC)/cAMP/protein kinase A (PKA) and phospholipase C (PLC)/inositol triphosphate (IP3)/diacylglycerol (DAG)/PKC.

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ANIMAL STUDIES

FIG. 3 Inhibitory and stimulatory effects of genistein on the insulin secretion mechanism. In pancreatic β-cells, genistein is reported to impair PKC potentialization of insulin secretion, to increase AC activation, and to impair insulin receptor signaling.

However, a soybean flour diet increased insulin secretion in response to glucose by islets from control rats and partially restored the poor glucose-induced insulin secretion in islets from adult rats recovered from early malnutrition.22 In this case, the increased insulin secretion did not result from the amino acid composition of soybean proteins because both protein sources contained equivalent concentrations of amino acids, especially arginine (insulinotropic nutrient), and rats on a soybean-flour or casein diet had similar serum concentrations of this amino acid in the fed state (unpublished data). The effects seemed to be mediated by the cAMP/PKA pathway possibly favored by genistein,22 because this isoflavone increases intracellular cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) content in both cell lines and the islets23 (Fig. 3). In addition to the mechanisms described previously, the inhibitory or stimulatory effects of genistein on insulin secretion have been attributed to its inhibitor effect on protein kinase C (PKC) and tyrosine kinase proteins24–26 (see Fig. 3). In male diabetic db/db mice, a high soybean diet prevented the loss of pancreatic β-cell mass, probably due to a decrease in cellular death. Although the islets had respond better to glucose and release greater levels of insulin (by about three times), the insulin secretion was not affected when normalizing by its content.27 In diabetic rats induced by alloxan, isoflavones enhanced insulin secretion both in vivo and in vitro.28

Effects of Soybean Insulin Action Many of the metabolic effects of insulin, including glucose uptake and glycogen synthesis, are mediated by a signaling pathway involving insulin receptor substrate (IRS) proteins, phosphorylation, and activation of phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (Akt; Fig. 4). Another classical insulin action is modulating lipid metabolism mediated by sterol regulatory element-binding protein-1c (SREBP-1c), whose activation results in the induction of genes for enzymes involved in the biosynthesis of fatty acids such as acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), and ATP citrate lyase (CLY). A transcription factor that is a direct target gene of SREBP-1c is PPAR-γ, and both exert cooperative and additive stimulation of the uptake of glucose and fatty acids and their subsequent conversion to triglycerides. There is evidence that soybean diets interfere with various steps of the insulin-signaling pathway, transcription factor expression, and the content and activity of key enzymes that participate in carbohydrate and lipid metabolism. Moreover, it has been demonstrated that soybean diet reduces body fat gain, which is an effect that may be useful for the prevention and treatment of obesity, diabetes mellitus, and fatty liver. Effect of Soybean Diet on Body Composition and Energy Balance Body weight tends to remain within a relatively narrow range despite large day-to-day fluctuations in the amount of food consumed. Obesity is thought to be the result of an energy intake–energy expenditure imbalance. Both are

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FIG. 4 The insulin-signaling mechanism that regulates glycogen synthesis and glucose output in the liver. Glucose uptake and glycogen synthesis are mediated by IRSs, phosphorylation/activation of PI3-K, and Akt (also called protein kinase B). Insulin binds the receptor (IR) and activates its tyrosine kinase activity. The intracellular part autophosphorylates and rapid phosphorylates IRS-1/2. The signaling cascade activates PI3-K, a key enzyme in the insulin-stimulated GLUT-2 transport to the plasma membrane (PM; i.e., in glucose uptake). PI3-K also activates Akt, which phosphorylates glycogen synthase kinase-3 (GSK3), activating glycogen synthase activity and thus glycogen synthesis. Akt phosphorylates a family of transcription factors (called the Forkhead family), which results in inhibition of the phosphoenolpyruvate carboxykinase (PEPCK) expression, and thus gluconeogenesis in liver.

regulated by the hypothalamus, which processes central and peripheral signals. Within the hypothalamus, neurons residing in the arcuate nucleus (ARC)–paraventricular nucleus (PVN)–perifornical/lateral hypothalamus axis communicate with each other and are subject to the influence of several peripheral factors, including leptin and insulin. Soy protein, isoflavones, and other components (saponins, tetrapeptides, fiber) may act together or separately to alter several hormonal, metabolic, and neuroendocrine parameters involved in maintaining body homeostatic balance, energy expenditure, and feeding behavior. It has been reported that isoflavones increase food and water intake and concentrations of neuropeptide Y (NPY) in the ARC and PVN nuclei of the hypothalamus. Such alterations are accompanied by decreased levels of plasma leptin and insulin. It is well established that NPY neurons, whose perikarya reside in the ARC nucleus and project to PVN, comprise an extremely important orexigenic neural pathway. Hence, in this case, at least one factor contributing to the higher food intake was the increased level of NPY in this system. Interestingly, there is a reciprocal relationship between circulating insulin and leptin titers and NPY concentration in PVN. Either insulin or leptin is associated with increased pre-proNPY mRNA expression in the ARC nucleus, and increased NPY levels in PVN provide an important signal to the NPY system to initiate feeding. Thus, by reducing the secretion of insulin, leptin, or both, chronic consumption of the isoflavone diet results in upregulation of the orexigenic NPY circuit in the hypothalamus, which in turn stimulates food and water intake. Despite high food intake, these animals exhibited reduced body and adipose tissue weights, which could be associated with high circulating triiodothyronine levels and the uncoupling protein-1 mRNA levels in brown adipose tissue, which alter the energy expenditure or thermogenesis.29 On the other hand, in ovariectomized rats fed on a high-fat diet, soy isoflavone significantly reduced the body weight and food intake and modulated orexigenic gene expression in the hypothalamus (POMC/CART; NPY) and periphery (ghrelin).30 However, with regard to body weight and food intake, other experiments in rats have demonstrated that dietary isoflavones do not affect body weight31 and decrease food intake.32, 33 Moreover, soy isoflavones have been demonstrated to affect adipose tissue without affecting food consumption. Enhanced lipolysis and inhibited lipogenesis in the white adipose tissue determined by phytoestrogenos are factors that could help reduce adiposity without affecting food intake. 4. METABOLIC RESPONSES TO FLOUR AND BREAD FORTIFICATION

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It has been reported that rats kept on a soybean-flour diet showed reduced proportions of fat deposits, even though they ate proportionally the same amount of food and showed lower energy expenditure than those fed the casein. In obese and nonobese Wistar rats, the lifelong soy isoflavone exposure was beneficial in reducing body fat because it decreased body weight and reduced visceral fat mass and serum leptin and adipocyte size. However, these effects were more pronounced in nonobese rats. Both lifelong and short-term isoflavone exposure increased skeletal muscle mass, especially in ovarectomized Wistar rats.34 Especially during nutritional recovery, a soybean-flour diet produced low-energy efficiency that was reflected in less energy gain as protein and proportion of carcass protein, and low-energy gain as lipid. It is possible that low digestibility of a soybean-flour diet has been critical to accretion of carcass protein in animals that suffered previous nutritional deficits. However, serum albumin and total protein concentrations from rats recovered with a soybeanflour diet were similar to those of control rats and higher than those of malnourished rats. It became clear that the soybean diet was efficient for nutritional recovery. Serum insulin level was increased and fat mass diminished in animals fed with soybean diet, without alterations of serum leptin levels. This suggests that although these animals possess fewer adipocytes, they release higher amounts of leptin. Also, serum leptin concentration was unrelated to food or energy intake and energy expenditure.35 However, maintenance of normal serum leptin concentrations can be beneficial to prevent leptin resistance and obesity. Effect of Soybean Diet on Insulin Signaling in Liver The liver is mainly responsible for maintaining normal concentrations of blood glucose by its ability to store glucose as glycogen and to produce glucose from glycogen breakdown or through the use of gluconeogenic precursors. These processes are regulated by hormones (mainly insulin and glucagon). Soybean-flour diet consumption by recovered and control rats did not modify serum glucose level and raised serum insulin levels in the fed state. Despite elevated serum insulin levels, these rats maintained normal glycemia, possibly at the expense of an elevated hepatic glucose output, as suggested by the low hepatic glycogen content in the fed state. These results addressed enhanced glycogenolysis due to an increase in glucagon levels, resistance to insulin, or both because of the inhibitory effect of genistein on the tyrosine kinase activity of the insulin receptor and/or its substrates.20 The first hypothesis was rejected, taking into account the unchanged serum glucagon levels in this animal model. Interestingly, in agreement with the latter hypothesis, it was verified that a soybean-flour diet reduced liver IR and IRS-1 levels, the IRS-1/PI3-K association, and the consequent phosphorylation of Akt. However, a soybean diet favored an increase in insulin resistance among molecular mechanisms that differed as a function of previous nutritional status. In recovered rats, a soybean diet resulted in liver insulin resistance due, at least in part, to an increased expression of p85, which favored the reduction of the IRS-1/PI3-K association. In control rats fed a soybean-flour diet, the insulin resistance appeared to have resulted in a reduction of phosphorylation IRS-1.36 Interestingly, despite insulin resistance, consumption of a soybean-flour diet by recovered and control rats impaired the response to glucagon, but did not alter gluconeogenesis.37 The liver is also a central organ of lipid processing, and many studies describe the ability of soy derivatives to modify liver lipid metabolism. Nonalcoholic fatty liver disease is now recognized as the most common type of liver disease, being frequently associated with insulin resistance and metabolic syndrome. Several studies have shown that soy protein and its isoflavones favorably affect hepatic lipid metabolism, preventing fat accumulation by its effects on genes involved in fatty acid biosynthesis and oxidation (Fig. 5). This effect has been attributed to the protective role of soybean in insulin resistance. In obese Zucker rats, soy protein isolate reduced liver lipid content, prevented hepatic damage, and restored the Wnt/β-catenin signaling pathway.38, 39 The Wnt/β-catenin-signaling pathway is central to adipogenic regulation in adipose tissue/cells40 and skeletal muscle.41There is also a link between the Wnt pathway and lipid regulation in liver tissue.42 Soy isoflavone also reduced lipid accumulation in liver and the serum ALT, improved liver lobule structure, decreased the expression of SREBP-1c and FAS, and increased the PPARα protein level in hepatic steatosis induced by high fat diet.43 In male Sprague-Dawley rats maintained on a high fat-high sucrose diet, genistein diminished fat accumulation in liver by activating adenosine monophosphate–activated protein kinase S6K1 (AMPK), thus promoting fatty acid oxidation and inhibiting lipid synthesis in liver.44 Finally, in female obese Zucker rats, high daidzein levels did not protect against liver steatosis, indicating that this isoflavone may not be the main component soybean responsible for reducing liver steatosis in this animal model.45 Curiously, soybean flour diet consumption by recovered and control rats that exhibit liver insulin resistance signals (increased ALT concentrations, high HOMA-IR index) that were corroborated by elevated tumor necrosis factor-alpha (TNFα), mRNA, and protein levels also showed diminished PPARγ, malic enzyme, and CLY content, as well as CLY and ME activities, lipogenesis, and liver fat storage.46 PPARα mRNA abundance was higher in rats fed a soybean-flour 4. 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FIG. 5 Effects of soy protein and isoflavones on liver. Fat accumulation is determined by the balance between fat synthesis (lipogenesis) and fat breakdown (lipolysis/fatty acid oxidation). Hepatic lipogenic transcription factors and enzymes crucial in modulating lipid metabolism are SREBP-1c, PPAR-γ, FAS, ME, CLY, ACC-α (favoring lipogenesis), PPAR-α, ACC-β, and AMPK (favoring fatty acid β-oxidation). PPAR-α stimulates fatty acid oxidation by inducing CPT-1 expression, together with other β-oxidation enzymes. Soy protein and its isoflavones prevent fat accumulation by controlling these genes.

diet than in those on a casein diet, but the protein content was similar in all groups. ACCα and ACCβ mRNA expression was markedly reduced by the soybean-flour diet, whereas the ACC content and Phospho-[Ser79]-ACC content were reduced only in control rats that ate a soybean flour diet. Further, mRNA and protein expression of SREBP1c, adenosine monophosphate-activated protein kinase S6K1 (AMPK), and phospho-[Thr172]-AMPK was not modified by the soybean-flour diet. Hence, at least in these animal models (control and recovered rats), the soybean-flour diet reduced liver lipid concentration through downregulation of the ACC gene and protein expressions rather than by phosphorylation status, which possibly resulted in decreased lipogenesis and increased β-oxidation.47 Similar results were observed in the soy protein consumption by Zucker obese fa/fa rats. This animal model showed reduction in the accumulation of cholesterol and triacylglycerol in the liver, preventing the development of fatty liver. The reduction in hepatic cholesterol was associated with a low expression of liver X receptor α and its target genes (e.g., 7α-hydroxylase and ATP binding cassette A1). Moreover, soy protein also decreased lipogenesis through a decrease in the expression of SREBP-1 and of its target enzymes, such as FAS and ME. Furthermore, the reduction of hepatic lipids was also attributed to an increase in fatty acid oxidation because soy protein increases PPAR-α and carnitine palmitoyltransferase-1 expression.17 Effect of Soybean Diet on Insulin Signaling in Skeletal Muscle The beneficial effects on glucose metabolism and insulin dynamics have been attributed to soy protein, isoflavone, and aglycin. Such effects appear to be largely explained by improved insulin sensitivity, as shown by an increased insulin action in skeletal muscle.18, 48, 49 Mice fed soy (isoflavone content of the diet about 600 ppm) from intrauterine life at approximately 25 weeks of age showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity. Because skeletal muscles for approximately 42% of the total body mass in male mice, Um et al.49 suggested that the increased wholebody insulin sensitivity of high-phytoestrogen-fed mice is, to a large extent, a consequence of increased insulin sensitivity in skeletal muscles. These high-phytoestrogen-fed mice showed significantly lower IRS-1, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta-1 (S6K1 or p70S6K) levels. In response to insulin, S6K1 phosphorylates IRS-1 on two inhibitory serine residues (S636/S639), preventing further activation of the PI3-K/Akt-signaling pathway.50 Thus, lower S6K1 levels inducing decreased phosphorylation of the IRS-1 inhibitory serines (S636/S639) could be one of the mechanisms involved in improving insulin sensitivity in 4. METABOLIC RESPONSES TO FLOUR AND BREAD FORTIFICATION

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skeletal muscles of mice fed a high-phytoestrogen diet. Also consistent with a direct effect of phytoestrogens on muscle tissues, Cederroth et al.48 related increased AMPK activation with reduced repression of the insulin signaling exercised by AMPK-dependent inhibition of the mTOR/S6K1 axis. Increased levels of skeletal muscle fat content and excessive reactive oxygen species (ROS) generation and oxidative stress are commonly found in type 2 diabetes. In this condition, the production of lipotoxic by-products due to lipid peroxidation could negatively affect muscle substrate metabolism.51–53 In myotubes, genistein has been shown to improve glucose uptake54 and fatty acid handling,55 implying improved oxidative capacity in muscle. In rodent models of type 2 diabetes mellitus, genistein attenuate ROS-generation56, 57 and muscle mitochondrial dysfunction is associated with insulin resistance and type 2 diabetes mellitus.58, 59 During the maturation of Zucker diabetic fatty rats, which presents a model of progressive insulin resistance, genistein did not improve skeletal muscle oxidative capacity or ROS-induced stress.60 Specific amino acids of soy protein could regulate skeletal muscle insulin sensitivity for glucose disposal by directly modulating the insulin-signaling pathway.18 It has been proposed to explain decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2, reduced binding of the p85 subunit of PI3-K to IRS-1 and IRS-2, and inhibition of insulin-stimulated PI3-K activity with the use of a mixture of 20 amino acids.61 In this regard, it has been shown that certain amino acids inhibit the insulin-induced activation of PI3-K and glucose transport by increasing serine/threonine phosphorylation of IRS-1 through activation of the mTOR/p70 S6 kinase pathway without affecting IRS-1 tyrosine phosphorylation.62 To further explore the cellular mechanisms behind the effect of protein on skeletal muscle insulin sensitivity, rats fed a standard chow diet or a high-fat diet in which the protein source was casein, soy, or cod proteins for 4 weeks were studied.63 Interestingly, it was observed that soy protein did not completely prevent the deleterious effect on insulinstimulated PI-3-K activity caused by fat feeding. In addition, activation of the downstream kinase Akt/PKB by insulin, assessed by in vitro kinase assay, and phosphorylation of GSK-3 were impaired in muscle of high-fat-fed rats consuming casein or soy protein, but these defects were also fully prevented by dietary cod protein. A natural bioactive peptide isolated from soybean, aglycin, has been considered to have antidiabetic potential. In streptozotocin/high-fat-diet-induced diabetic mice aglicin administered intragastrically as an oral agent reduced hyperglycemia and improving oral glucose tolerance. Furthermore, glycogen restored insulin signal transduction by elevating the content of phosphorylated-IR, -IRS1, and –Akt, as well as the membrane GLUT4 protein in skeletal muscle.49 In summary, these studies provide convincing evidence that soy protein, aglycin, and isoflavones are important modulators of insulin signaling and action in skeletal muscle. Effect of Soybean Diet on Insulin Signaling in Adipose Tissue In the fed state, insulin suppresses lipolysis and the release of free fatty acids, decreasing hepatic glucose production and release64 and increasing lipogenesis and adipogenesis rates.65 In prolonged fasting and intense physical exercise, glucagon and catecholamines stimulate hormone-sensitive lipase and consequently lipolysis, releasing free fatty acids that are used as energetic substrates.66 A soybean-flour diet by recovered rats after protein restriction during intrauterine life and lactation and by control rats reduced the weight and lipid content in the retroperitoneal white adipose tissue. The thermogenic capacity of the brown adipose tissue was not affected by the soybean diet, as well as the lipogenesis rate in the retroperitoneal white adipose tissue. The lipolysis rate by isoproterenol was decreased in white adipocytes from the soybean-recovered rats and was elevated in adipocytes from the soybean-control rats. Thus, in animals maintained on a soybean diet, the proportions of fat deposits are determined by the lipolysis rate, which differs depending on the previous nutritional status.67 In a rat model that mimics several aspects of the human metabolic syndrome dietary intake of soy protein reduced adipocyte hypertrophy and basal lipolysis, corrected the inhibitory effect upon the antilipolytic action of insulin, and normalized or improved enzyme activities involved in de novo lipogenesis in adipose tissue.68 Also, adipocyte size distribution, adiposity visceral index, and insulin sensitivity improved. These effects manifest themselves as improvement or normalization of the oxidative stress status and diminished TNF-α level in plasma.69 Obesity-induced inflammation caused by adipocyte-macrophage interactions plays a critical role in developing insulin resistance. The inflammatory gene expression is regulated by PPARs, and soy isoflavonedaidzein acts as a PPAR activator in these cells.70 In adipocyte and macrophage cocultures, daidzein reduced pro-inflammatory gene expression by activating PPAR-α and -γ and inhibiting the c-Jun N-terminal kinase (JNK) pathway. Thus, daidzein may be a therapeutic strategy for chronic inflammation in obese adipose tissue.71 Hence, soybean protein or its isoflavones appear to positively modulate the inflammatory process, thus improving insulin resistance. 4. METABOLIC RESPONSES TO FLOUR AND BREAD FORTIFICATION

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TECHNOLOGICAL ISSUES There is an increasing trend to integrate soybean into conventional foods as either a complete or a partial replacement. Soy flours have been tested in baked products as both functional ingredients and protein fortifiers. However, soy flours have both positive and negative characteristics in food products. In baked goods, the creamy color and beany or nutty flavor can contribute to the sensory quality. When used in larger quantities, soy flours have an astringent aftertaste, a chalky mouthfeel, or both. Moreover, soy flours contain low-molecular-weight oligosaccharides (raffinose, stachyose, and verbascose) that can to cause flatulence. In bakery products, soy-based ingredients have been used for a variety of functional reasons. For example, soybean protein products improve crust color, crumb body, resilience, and toasting characteristics in bread. Due its low energy, high biological value, and low glycemic index, soy flour is very attractive for the design of dietary food products especially diet bread.

SUMMARY POINTS • Human clinical trials have shown antiobesity and antidiabetic effects of soy protein and isoflavones. • In rats, a soybean-flour diet activates the β-cell cAMP/PKA pathway, increasing insulin secretion in response to glucose. • Soybean isoflavones alter food intake by interfering with the leptin/insulin-NPY axis. • In rats, a soybean-flour diet decreases the relative weight of fat deposits, and even reduces the energetic expenditure without alterations in food intake. • A soybean diet interferes with various steps of the insulin-signaling pathway. • In rats, a soybean diet decreases hepatic fat synthesis. • A soybean diet increases insulin sensitivity in skeletal muscle. • Muscle insulin-reduced phosphorylation of GSK-3 is improved by soy proteins and isoflavones, partially preventing the deleterious effects of fat feeding. • Soybean protein or its isoflavone positively modulates the inflammatory process in white adipose tissue and attenuates insulin resistance.

References 1. Velasquez MT, Bathena SJ. Role of dietary soy protein in obesity. Int J Med Sci 2007;4(2):72–82. 2. Núcleo de Estudos e Pesquisa em Alimentac¸ão (NEPA). Tabela Brasileira de Composic¸ão de Alimentos (TACO). Universidade Estadual de Campinas (UNICAMP); 2011. 3. Hou A, Chen P, Alloatti J, Mozzoni L, Zhang B, Shi A. Genetic variability of seed sugar content in worldwild soybean germplasm collections. Crop Sci 2009;49(3):903–91. 4. Messina M. Soy foods, isoflavones, and the health of postmenopausal women. Am J Clin Nutr 2014;100(1). 423S–30S. 5. Sites CK, Cooper BC, Toth MJ, Gastaldelli A, Arabshahi A, Barnes S. Effect of a daily supplement of soy protein on body composition and insulin secretion in postmenopausal women. Fertil Steril 2007;88(6):1609–17. 6. Azadbakht L, Kimiagar M, Mehrabi Y, Esmaillzadeh A, Padyab M, Hu FB, Willet W. Soy inclusion in the diet improves features of the metabolic syndrome: A randomized crossover study in postmenopausal women. Am J Clin Nutr 2007;85(3):735–41. 7. Jayagopal V, Jennings P, Albertazzi P, Hepburn D, Kilpatrick E, Atkin S, Howarth E. Beneficial effects of soy phytoestrogen intake in postmenopausal women with type 2 diabetes. Diabetes Care 2002;25(10):1709–14. 8. Goodman-Gruen D, Kritz-Sliverstein D. Usual dietary isoflavone intake is associated with cardiovascular disease risk factors in postmenopausal women. J Nutr 2001;131(4):1202–6. 9. Matvienko OA, Alekel DL, Genschel U, Ritland L, Van Loan MD, Koehler KJ. Appetitive hormones, but not isoflavone tablets, influence overall and central adiposity in healthy postmenopausal women. Menopause 2010;17(3):594–601. 10. Gobert CP, Pipe EA, Capes SE, Darlington GA, Lampe JW, Duncan AM. Soya protein does not affect glycaemic control in adults with type 2 diabetes. Br J Nutr 2010;103(3):412–21. 11. Fei BB, Ling L, Hua C, Ren SY. Effects of soybean oligosaccharides on antioxidant enzyme activities and insulin resistance in pregnant women with gestational diabetes mellitus. Food Chem 2014;158:429–32. 12. Jamilian M, Asemi Z. The effects of soy isoflavones on metabolic status of patients with polycystic ovary syndrome. J Clin Endocrinol Metab 2016;101(9):3386–94. 13. Kim JI, Kim JC, Kang MJ, Lee MS, Kim JJ, Cha IJ. Effects of pinitol isolated from soybeans on glycaemic control and cardiovascular risk factors in Korean patients with type II diabetes mellitus: A randomized controlled study. Eur J Clin Nutr 2005;59(3):456–8. 14. Duncan AM, Underhill KEW, Xu X, Lavalleur J, Phipps WR, Durzer MS. Modest hormonal effects of soy isoflavones in postmenopausal women. J Clin Endocrinol Metab 1999;84(10):3479–84. 15. Fang K, Dong H, Wang D, Gong J, Huang W, Lu F. Soy isoflavones and glucose metabolism in menopausal women: A systematic review and meta-analysis of randomized controlled trials. Mol Nutr Food Res 2016;60(7):1602–14. 4. METABOLIC RESPONSES TO FLOUR AND BREAD FORTIFICATION

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Nutritional recovery with a soybean flour diet improves the insulin response to a glucose load without modifying glucose homeostasis. Nutrition 2008;24(1):76–83. 21. Kavanagh K, Jones KL, Zhang L, Flynn DM, Shadoan MK, Wagner JD. High isoflavone soy diet increases insulin secretion without decreasing insulin sensitivity in premenopausal nonhuman primates. Nutr Res 2008;28(6):368–76. 22. Veloso RV, Latorraca MQ, Arantes VC, Reis MAB, Ferreira F, Boschero AC, Carneiro EM. Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats. J Nutr Biochem 2008;19(11):778–84. 23. Liu D, Zhen W, Yang Z, Carter JD, Si H, Reynolds KA. Genistein acutely stimulates insulin secretion in pancreatic beta-cells through a cAMPdependent protein kinase pathway. Diabetes 2006;55(4):1043–50. 24. Jones PM, Persaud SJ. Tyrosine kinase inhibitors inhibit glucose-stimulated insulin secretion. Biochem Soc Trans 1994;22:209S. 25. Persaud SJ, Harris TE, Burns CJ, Jones PM. Tyrosine kinases play a permissive role in glucose-induced insulin secretion from adult rat islets. J Mol Endocrinol 1999;22:19–28. 26. Sorenson RL, Brelje TC, Roth C. Effect of tyrosine kinase inhibitors on islets of Langerhans: evidence for tyrosine kinases in the regulation of insulin secretion. Endocrinology 1994;134:1975–8. 27. Zimmermann C, Cederroth CR, Bourgoin L, Foti M, Nef S. Prevention of diabetes in db/db mice by dietary soy is independent of isoflavone levels. Endocrinology 2012;153:50200–5211. 28. Hamden K, Jaouadi B, Carreau S, Aouidet A, Elfeki A. Therapeutic effects of soy isoflavones on α-amylase activity, insulin deficiency, liverkidney function and metabolic disorders in diabetic rats. Nat Prod Res 2011;25:244–55. 29. Lephart ED, Porter JP, Lund TD, Bu L, Setchell KD, Ramoz G, Crowley WR. Dietary isoflavones alter regulatory behaviors, metabolic hormones and neuroendocrine function in long–Evans male rats. Nutr Metab 2004;23:16–29. 30. Zhang YB, Zhang Y, Li LN, Zhao XY, Na XL. Soy isoflavone and its effect to regulate hypothalamus and peripheral orexigenic gene expression in ovariectomized rats fed on a high-fat diet. Biomed Environ Sc 2010;23:68–75. 31. Ju YH, Allred CD, Allred KF, Karko KL, Doerge DR, Helferich WG. Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice. J Nutr 2001;131:2957–62. 32. Davis J, Higginbotham A, O’Connor T, Moustaid-Moussa N, Tebbe A, Kim YC, Cho KW, Shay N, Adler S, Peterson R, Banz W. Soy protein and isoflavones influence adiposity and development of metabolic syndrome in the obese male ZDF rat. Ann Nutr Metab 2007;51:42–52. 33. Penza M, Montani C, Romani A, Vignolini P, Pampaloni B, Tanini A, Brandi ML, Alonso-Magdalena P, Nadal A, Ottobrini L, Parolini O, Bignotti E, Calza S, Maggi A, Grigolato PG, Di LD. Genistein affects adipose tissue deposition in a dose-dependent and gender-specific manner. Endocrinology 2006;147:5740–51. 34. Kurrat A, Blei T, Kluxen FM, Mueller DR, Piechotta M, Soukup ST, Kulling SE, Diel P. Lifelong exposure to dietary isoflavones reduces risk of obesity in ovariectomized Wistar rats. Mol Nutr Food Res 2015;59:2407–18. 35. Cheim LMG, Oliveira EA, Arantes VC, Veloso RV, Reis MAB, Gomes-da-Silva MHG, Carneiro EM, Boschero AC, Latorraca MQ. Effect of nutritional recovery with soybean flour diet on body composition, energy balance and serum leptin concentration in adult rats. Nutr Metab 2009;6:34–42. 36. Feres NH, de Lima Reis SR, Veloso RV, Arantes VC, Souza LM, Carneiro EM, Boschero AC, Reis MA, Latorraca MQ. Soybean diet alters the insulin-signaling pathway in the liver of rats recovering from early-life malnutrition. Nutrition 2010;26:441–8. 37. Pacheco NCS, Almeida APC, Siqueira KC, Lima FM, Reis SRL, Latorraca MQ, Stoppiglia LF. Nutritional recovery with a soybean diet impaired the glucagon response but did not alter liver gluconeogenesis in the adult offspring of rats deprived of protein during pregnancy and lactation. Appl Physiol Nutr Metab 2018; [in press]. 38. Cain J, Banz WJ, Butteiger D, Davis JE. Soy protein isolate modified metabolic phenotype and hepatic Wnt signaling in obese Zucker rats. Horm Metab Res 2011;43:774–81. 39. Zhou D, Lezmi S, Wang H, Davis J, Banz W, Chen H. Fat accumulation in the liver of obese rats is alleviated by soy protein isolate through β-catenin signaling. Obesity 2014;22:151–8. 40. Li HX, Luo X, Liu RX, Yang YJ, Yang GS. Roles of Wnt/beta-catenin signaling in adipogenic differentiation potential of adipose-derived mesenchymal stem cells. Mol Cell Endocrinol 2008;291:116–24. 41. Du M, Yin J, Zhu MJ. Cellular signaling pathways regulating the initial stage of adipogenesis and marbling of skeletal muscle. Meat Sci 2010;86:103–9. 42. Behari J, Yeh TH, Krauland L, Otruba W, Cieply B, Hauth B, Apte U, Wu T, Evans R, Monga SP. Liver-specific beta-catenin knockout mice exhibit defective bile acid and cholesterol homeostasis and increased susceptibility to diet-induced steatohepatitis. Am J Pathol 2010;176:744–53. 43. Liu H, Zhong H, Leng L, Jiang Z. Effects of soy isoflavone on hepatic steatosis in high fat-induced rats. J Clin Biochem Nutr 2017;61:85–90. 44. Liu H, Zhong H, Yin Y, Jiang Z. Genistein has beneficial effects on hepatic steatosis in high fat-high sucrose diet-treated rats. Biomed Pharmacother 2017;91:964–9. 45. Bell A, Korourian S, Zeng H, Phelps J, Hakkak R. A diet containing a high- versus low-daidzein level does not protect against liver steatosis in the obese Zucker rat model. Food Funct 2017;8:1293–8. 46. Reis SRL, Feres NH, Ignacio-Souza LM, Veloso RV, Arantes VC, Kawashita NH, Colodel EM, Botosso BL, Reis MAB, Latorraca MQ. Nutritional recovery with a soybean diet after weaning reduces lipogenesis but induces inflammation in the liver in adult rats exposed to protein restriction during intrauterine life and lactation. Mediators of inflammation 2015;1–12. 47. Milanski M, Souza KL, Reis SR, Feres NH, Ignacio-Souza LM, Arantes VC, Carneiro EM, Boschero AC, Reis MA, Latorraca MQ. Soybean diet modulates acetyl-coenzyme a carboxylase expression in livers of rats recovering from early-life malnutrition. Nutrition 2009;25:774–81.

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48. Cederroth CR, Vinciguerra M, Gjinovci A, K€ uhne F, Klein M, Cederroth M, Caille D, Suter M, Neumann D, James RW, Doerge DR, Wallimann T, Meda P, Foti M, Rohner-Jeanrenaud F, Vassalli JD, Nef S. Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism. Diabetes 2008;57:1176–85. 49. Lu J, Zeng Y, Hou W, Zhang S, Li L, Luo X, Xi W, Chen Z, Xiang M. The soybean peptide aglycin regulates glucose homeostasis in type 2 diabetic mice via IR/IRS1 pathway. J Nutr Biochem 2012;23:1449–57. 50. Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fumagalli S, Allegrini PR, Kozma SC, Auwerx J, Thomas G. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 2004;431:200–5. 51. Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 2006;440:944–8. 52. Pan DA, Lillioja S, Kriketos AD, Milner MR, Baur LA, Bogardus C, Jenkins AB, Storlien LH. Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes 1997;46:983–8. 53. Russell AP, Gastaldi G, Bobbioni-Harsch E, Arboit P, Gobelet C, Deriaz O, Golay A, Witztum JL, Giacobino JP. Lipid peroxidation in skeletal muscle of obese as compared to endurance-trained humans: a case of good vs. bad lipids? FEBS Lett 2003;551:104–6. 54. Ha BG, Nagaoka M, Yonezawa T, Tanabe R, Woo JT, Kato H, Chung UI, Yagasaki K. Regulatory mechanism for the stimulatory action of genistein on glucose uptake in vitro and in vivo. J Nutr Biochem 2012;23:501–9. 55. Palacios-González B, Zarain-Herzberg A, Flores-Galicia I, Noriega LG, Alemán-Escondrillas G, Zariñan T, Ulloa-Aguirre A, Torres N, Tovar AR. Genistein stimulates fatty acid oxidation in a leptin receptor-independent manner through the JAK2-mediated phosphorylation and activation of AMPK in skeletal muscle. Biochim Biophys Acta 2014;1841:132–40. 56. Salih MS, Nallasamy P, Muniyandi P, Periyasami V, Venkatraman AC. Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance. J Diabetes 2009;1:278–87. 57. Valsecchi AE, Franchi S, Panerai AE, Rossi A, Sacerdote P, Colleoni M. The soy isoflavonegenistein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vasculature deficits in diabetes mouse model. Eur J Pharmacol 2011;650:694–702. 58. Chow L, From A, Seaquist E. Skeletal muscle insulin resistance: The interplay of local lipid excess and mitochondrial dysfunction. Metabolism 2010;59:70–85. 59. Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI. Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med 2004;350:664–71. 60. van Bree BW, Lenaers E, Nabben M, Briede JJ, J€ orgensen J, Schaart G, Schrauwen P, Hoeks J, Hesselink MK. A genistein-enriched diet neither improves skeletal muscle oxidative capacity nor prevents the transition towards advanced insulin resistance in ZDF rats. Sci Rep 2016;14 (6):22854. 61. Patti ME, Brambilla E, Luzi L, Landaker EJ, Kahn CR. Bidirectional modulation of insulin action by amino acids. J Clin Invest 1998;101:1519–29. 62. Tremblay F, Marette A. Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway: A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. J Biol Chem 2001;276:38052–60. 63. Tremblay F, Lavigne C, Jacques H, Marette A. Dietary cod protein restores insulin-induced activation of phosphatidylinositol 3-kinase/Akt and GLUT4 translocation to the T-tubules in skeletal muscle of high-fat–fed obese rats. Diabetes 2003;52:29–37. 64. Bergman RN, SD M. Central role of the adipocyte in insulin resistance. J Basic Clin Physiol Pharmacol 1998;9:205–21. 65. Dimitriadis G, Mitrou P, Lambadiari V, Maratou E, SA R. Insulin effects in muscle and adipose tissue. Diabetes Res Clin Pract 2011;93:S52–9. 66. Penicaud L, Cousin B, Leloup C, Lorsignol A, Casteilla L. The autonomic nervous system, adipose tissue plasticity, and energy balance. Nutrition 2000;16:903–8. 67. Paiva AA, Faiad JZ, Taki MS, de Lima Reis SR, de Souza LM, Dos Santos MP, Chaves VE, Kawashita NH, de Oliveira HC, Raposo HF, Carneiro EM, Latorraca MQ, Gomes-da-Silva MH, Martins MS. A soyabean diet does not modify the activity of brown adipose tissue but alters the rate of lipolysis in the retroperitoneal white adipose tissue of male rats recovering from early-life malnutrition. Br J Nutr 2012;108:1042–51. 68. Oliva ME, Selenscig D, D’Alessandro ME, Chicco A, Lombardo YB. Soya protein ameriorates the metabolic abnormalities of dysfunctional adipose tissue of dyslipidaemic rats fed a sucrose-rich diet. Br J Nutr 2011;105:1188–98. 69. Illesca PG, Álvarez SM, Selenscig DA, Ferreira MDR, Gimenez MS, Lombardo YB, D’Alessandro ME. Dietary soy protein improves adipose tissue dysfunction by modulating parameters related with oxidative stress in dyslipidemic insulin-resistant rats. Biomed Pharmacother 2017;88:1008–15. 70. Sakamoto Y, Naka A, Ohara N, Kondo K, Iida K. Daidzein regulates proinflammatory adipokines thereby improving obesity-related inflammation through PPARγ. Mol Nutr Food Res 2014;58:718–26. 71. Sakamoto Y, Kanatsu J, Toh M, Naka A, Kondo K, Iida K. The dietary isoflavonedaidzein reduces expression of pro-inflammatory genes through PPARα/γ and JNK pathways in adipocyte and macrophage co-cultures. PLoS One 2016;11:e0149676.

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34 Flour Fortification and the Prevention of Neural Tube Defects (NTDs) Emma Beckett*,†, and Mark Lucock† *School of Medicine & Public Health, The University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW, Australia † School of Environmental & Life Sciences, The University of Newcastle, Newcastle, NSW, Australia

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INTRODUCTION Worldwide, neural tube defects (NTDs) account for a substantial proportion of all congenital abnormalities. They are the most common congenital abnormality of the central nervous system and are the second-most-common type of congenital abnormality overall. NTDs are a significant cause of child and fetal mortality and morbidity. The majority of NTDs are folate sensitive, with risks reduced via the provision of adequate levels of periconceptional folic acid. To ensure adequate folic acid intake in women of reproductive age, programs of mandatory fortification (primarily in flours) have been implemented in more than 80 countries. Mandatory fortification programs have largely been successful, with higher blood levels of folate and lower rates of NTDs in countries with such programs compared to those with voluntary fortification or supplementation programs only. Where data are available, it also appears that the rate of NTDs has dropped in most jurisdictions following the implementation of mandatory fortification programs. However, uptake of such programs has not been universal. Within jurisdictions that adopt fortification, there may be issues with the quality and monitoring of these programs, and in countries yet to introduce mandatory fortification programs, there may be concerns surrounding the potential side effects of excess consumption and the ethics of population-level fortification.

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NEURAL TUBE DEFECTS (NTDS) NTDs collectively describe several congenital anomalies of the central nervous system resulting from very early disruption of embryonic development that affects the brain and spinal cord. Development of the neural tube from the neural plate occurs between day 17 and the end of the third week of gestation.1,2 The cranial (rostral) end of the neural tube closes by 24 days and the caudal by 25–26 days gestation. Defective closure results in NTDs that may affect the spinal cord (spina bifida), brain (anencephaly, encephalocele), or hindbrain-cervical junction (craniorachischisis).2 The most common NTDs are spina bifida, anencephaly, and encephalocele.2 Spina bifida describes a set of malformations occurring when caudal parts of the neural tube do not close properly, preventing fusion of the covering vertebral arches, soft tissues, and skin. Lesions typically occur in the lumbosacral area, but they also may be more extensive and can involve the entire spinal cord.1–3 The consequences of spina bifida depend on the severity of the lesion; it may be incompatible with life or it may be survivable.4 Survivors experience varying degrees of permanent disability, including paralysis, weakness in the legs, bowel and bladder incontinence, hydrocephalus, and learning difficulties. Anencephaly results from a failure of the rostral end of the neural tube to close, leading to the protrusion of the developing brain through the cranial vault (exencephaly).5 The developing tissue is then destroyed due to mechanical injury and vascular disruption, leading to an absence of major portions of brain, skull, and scalp in the fetus. Anencephaly is the most severe NTD and is incompatible with survival.2,6 Encephalocele is a protrusion of brain through a defect of skull formation, usually in the occipital area. As in anencephaly, the protruding portion and the surrounding intracranial portion are destroyed or malformed due to mechanical disruption and ischemia. Large occipital encephaloceles are incompatible with life because of damage to the brainstem. Smaller encephaloceles are not always fatal; however, the survivors experience varying degrees of disability, including intellectual disabilities, cerebral palsy, seizures, and other learning difficulties.2 It has been estimated that worldwide, more than 260,000 pregnancies were affected by NTDs in 2015, approximately 50% of which resulted in elective terminations.7 The global prevalence in the same year was estimated to be 1.86 NTDs per 1000 live births, down from an estimated 2.4 per 1000 live births affected by NTDs globally during 2001.8 However, prevalence varies by region, noting that reliable data is not available for all regions.7

PREVENTION OF NTDS The risk for NTDs is multifactorial, as they occur due to a combination of genetic and environmental causes.1,9 Genetic causes are not yet fully elucidated, but they are known to include polymorphisms in gene coding for enzymes involved in folate metabolism, such as 5,10-methylenetetrahydrofolate reductase (MTHFR).3,10 Diabetes mellitus and use of the antiepileptic drug valproate also increase the risk for NTDs.11,12 However, the majority of NTDs are known to be folic acid sensitive.13,14 A dose-dependent relationship between serum folate levels and the risk of having a child with an NTD has been described, with inadequate levels of maternal folate in the periconceptional period increasing risk.15,16 While women conceiving children with NTDs may not be overtly folate deficient,17 there is substantial evidence that adequate levels of periconceptional folic acid substantially reduce the risk of NTDs.3,7,15–17 Natural folates are pterins that exist as various monoglutamates and polyglutamates.18,19 Folic acid, a fully oxidized monoglultamate, is used in fortified foods or supplements due to its high stability and availability.19 Folates play essential roles in the human body as a major coenzyme in one-carbon metabolism, including DNA synthesis, repair, and methylation.20,21 Interactions may occur between subtle and unidentified defects in folate metabolism genes and folate levels to increase risk. As such, increasing levels of periconceptional folic acid intake, via supplements or fortification, may reduce risk. However, the mechanisms by which folic acid prevents NTDs are not yet fully elucidated. There is an increased need for folate during embryogenesis, growth, and fetal development due to its critical role in the synthesis of nucleic acids and proteins, so mechanisms may be multifactorial. Given the role of folate in deoxyribonucleic acid (DNA) methylation, epigenetic mechanisms are likely involved in the etiology and prevention of NTDs.22–24 The brains of fetuses with neural tube defects have lower global methylation compared to controls, a result that was positively correlated with maternal folate levels.23 Changes in DNA methylation that lead to the overexpression of genes involved in autoimmunity have been linked to the development of NTDs.23 It has also been hypothesized that the presence of elevated folate receptor antibodies limits folate transport to the early embryo, thus affecting development.25

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Regardless of the mechanisms, there is considerable evidence that increasing periconceptional folic acid levels has led to a significant reduction in the occurrence of NTDs.14,26 An association between the low-folate status of women of reproductive age and the risk of NTD-affected pregnancy was first proposed in the 1960s and 1970s.27–30 This hypothesis was substantiated by key randomized controlled trials demonstrating the effectiveness of periconceptional folic acid supplementation in preventing the first occurrence13 and recurrence14 of NTDs. These studies showed up to 70% reduction in incidence in the treatment arms. This reduction of NTD rate by folic acid supplements was confirmed in an Chinese population31 and is consistent with studies of multivitamin supplements containing folic acid.26,32

FORTIFICATION OF FLOURS AND WITH FOLIC ACID A World Health Organization (WHO) guideline was published in 2015 that proposes a threshold at a population level, for red blood cell folate concentrations to be above 400 ng/mL (906 nmol/L) in women of reproductive age to achieve significant reductions in NTDs.33 However, as neural tube closure occurs in the early stages of pregnancy (within 28 days),2,6 folic acid supplementation must be commenced prior to conception in order to be effective. Even with supplementation, it can take several weeks or months to reach the blood levels required to prevent NTDs.34 It has been reported that over half of all pregnancies are unplanned.35 As such, this critical period may have passed before many women recognize that they are pregnant. Furthermore, there are socioeconomic barriers to accessing supplements, especially in low- and middle-income countries.36 Compliance with recommendations is also a factor in reducing the effectiveness of supplementation.35,37 Therefore, fortification of the food chain with folic acid has been adopted in many countries to increase the levels of folic acid in all women of childbearing age, with the primary aim of reduced prevalence of NTDs in offspring.38,39 The first government-mandated folic acid fortification program was implemented in the United States in 1998 with fortification of grain and cereal products. Since then, more than 80 additional countries have followed suit, launching programs of mandated fortification of wheat flour, maize flour, or rice flour with folic acid, as well as other staple foods.39 The levels of fortification and strategies of fortification differ between countries. However, fortification of flours is a common tactic due to their position as an accessible and affordable staple food in most regions, which are regularly consumed by the target population.38,40 Food fortification is the preferable strategy, as it has low risk but leads to a substantial effect on NTD prevalence, compared to supplementation only for high-risk women or those planning conception.41 Fortification assists in the mitigation of issues including high costs, distribution problems, and low compliance with supplement use. Mandatory fortification may be preferable to voluntary fortification when consumer knowledge is poor or when limited nutrition education opportunities exist, as mandatory fortification of staple foods does not require behavioral changes in dietary habits. Furthermore, mandatory fortification is centrally regulated and implemented through large-scale foodprocessing industries. Mandatory legislation, when well implemented, improves parity for industries to fortify, leading to greater coverage in the target populations. Mandatory fortification can be easily monitored and enforced and has a greater health impact on entire populations, and thus is likely to deliver a sustained public health benefit.38,42,43

FORTIFICATION OF FLOUR AND THE PREVENTION OF NTDS Overall increases in dietary folic acid intake and blood folate concentrations have been reported in response to folic acid fortification programs.44,45 Longitudinal data from the United States [specifically, from the National Health and Nutrition Examination Survey (NHANES)], has demonstrated that serum and erythrocyte folate levels have increased dramatically postfortification.46 Similar increases in serum and erythrocyte folate levels have also been reported in other jurisdictions, including Canada,45,47 Chile,48,49 and Australia.50,51 These enhanced biological levels of folate have reportedly translated into a reduction in NTD rates in a number of places.44,47,52,53 Countries with mandatory policies on folic acid fortification of staple foods have lower NTD prevalence than countries that have no fortification or only voluntary fortification,54 although the results may vary depending on ethnicity. Between 1995 and 2005 mandatory folic acid fortification was less effective in reducing NTDs in nonHispanic blacks than in other ethnic groups.55 Most countries with mandatory food-fortification policies have achieved NTD prevalence estimates as low as 0.6 per 1000 total births.8,47,56,57 Conversely, in countries without mandatory foodfortification policies, the average prevalence is approximately 2.5 per 1000 live births, with rates in some countries as high as 20 per 1000 live births.8,58,59

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However, even in countries where fortification has been implemented, NTD prevalence can still be variable within areas. This is a function of the effectiveness of folic acid fortification implementation methods, the penetration of programs, and genetic and dietary characteristics of the population in question.60,61 It is also important to note that although more than 80 countries have mandated folic acid fortification, to date, evidence of impact exists in only a minority of these. As such, there is a need for additional research to assess the impact of delivery methods of mandatory fortification regimens.61 A 2013 systematic review on the impact of folic acid fortification of flour on the prevalence of NTDs across multiple countries (Chile, Argentina, Brazil, Canada, Costa Rica, Iran, Jordan, South Africa, and the United States) found a drop in the prevalence of NTDs in response to fortification in 15 of the 27 studies.57 It is important to note that in some of these countries, mandatory fortification is not limited to wheat flour; Costa Rica, for example, also requires fortification of maize flour, cow’s milk, and rice.57 For all NTDs, the most significant drops were observed in Costa Rica (58%),62 Argentina (49%),63 and Canada (49%).64 The smallest decrease occurred in the United States (15%).65 Of the 21 studies included in the meta-analysis,57 the greatest reductions in spina bifida rates reported were in Costa Rica (61%),62 Canada (55%),47 and Chile (55%).63 The smallest reduction in the prevalence of spina bifida was in the United States, at just 3%.66 Variance in the reduction of incidence may be due to differences in previous voluntary fortification programs, socioeconomic factors, levels of fortification, and the number of flours or other staple foods fortified. When the prevalence of NTDs was related to levels of flour fortification, the lowest prevalence was observed at a folic acid level of 1.5 mg/kg. Implementation of such programs is performed less in countries that do not industrially mill or import the majority of their grain products, making flour products difficult to fortify.61 This is particularly the case in the low- to middleincome countries that do not currently employ mandated fortification programs. It is also essential that the delivery of currently mandated regimens be monitored to determine the quality of fortification programs.45 For example, based on single-sample testing of fortified foods in a range of national mandatory programs, a large proportion of samples do not meet nationally mandated standards.67 While these results are obtained from suboptimal single-sampling methods, in the absence of other data, it provides an inference about ongoing quality issues that can adversely affect the potential impact. An example of a potentially suboptimal nationally mandated fortification implementation is Guatemala. In Guatemala, wheat and maize are fortified with folic acid. Despite this fact, there is considerable variance in the erythrocyte folate concentrations among women of reproductive age. Many indigenous Guatemalan populations living in rural, low-income regions do not purchase industrially milled flour, but instead process corn privately. As such, these populations are not exposed to fortification, leading to a higher risk for NTDs in offspring68 compared to urban-dwelling Guatemalans. Similarly, a survey including six African countries fortifying maize or wheat flour with folic acid found that only two programs—South Africa (maize flour fortification) and Senegal (wheat flour fortification)—reached coverage of 40% or more for two or more “vulnerable groups,” with vulnerability defined as using a composite indicator of poverty, poor women’s dietary diversity score, and rural residence.69 Additionally, across all these countries, only 35% of wheat flour consumed was found to be fortifiable, and only 18.5% of that flour was found to be fortified at all. For maize flour, 48% consumed fortifiable maize flour, and only 29% of the maize flour that could be fortified was. Coverage was generally higher among urban populations and lower among at-risk population groups.69 Therefore, implementation of mandated fortification programs alone is not sufficient. Issues remain that are related to government and industry incentives, follow-up actions, regulations, monitoring, and enforcement of mandatory fortification programs.45,67 It is vital that mandatory legislation is underpinned with strong regulatory measures to ensure the quality of fortified foods.45

POTENTIAL SHORTCOMINGS AND CONTROVERSIES The implementation of mandatory folic acid fortification programs do appear to have met the primary goal of reducing the prevalence of NTDs in many countries. Other additional improvements, such as drops in homocysteine levels, have resulted from mandatory fortification programs.50 These successes have prompted some to call for the recommendations on upper limits of folic acid intake to be abandoned or increased.70 However, not all jurisdictions agree on the benefits of fortification of flour with folic acid. As flour is a staple food, mandatory fortification exposes the entire population to folic acid and generally raises folate levels in the blood. People who eat a significant amount of bread and pasta made from fortified flours would have a high intake, and specific subgroups, such as children and adolescents, may exceed maximum nutritional recommendations.71 The extent

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and magnitude of the risk of such exposure are yet to be fully elucidated. However, potential hazardous side effects include the masking of vitamin B12 deficiency, increased risk of twinning, and accelerated growth of precancerous lesions. The precise mechanisms for these adverse effects are not always clear, but they raise potential ethical dilemmas in the implementation of mandatory fortification programs.20,21

Masked Vitamin B12 Deficiency There is a possibility that high doses of folic acid can mask the early signs of vitamin B12 deficiency, leading to later diagnosis and increased risk of associated neurological damage. However, available data indicate that the dose provided by the diet after fortification is insufficient to correct anemia.72

Increased Rates of Twinning Data surrounding the increased risk of twin births in mothers with high folic acid levels are conflicting. Analysis of the results of randomized controlled trials suggests a possible increase in rates of twinning in the supplemented mothers; however, there is no evidence that the increased twinning is caused by fortification. Furthermore, the significant confounders, such as fertility treatments and maternal age, limit these observations.73,74

High Folate Levels and Cancer Risk Due to the role of folates in DNA synthesis and cell proliferation, it is biologically plausible that folic acid could stimulate the growth of existing tumors and possibly accelerate the transformation of precancerous lesions into active malignancies. Some experimental animal models support this hypothesis. Concerns have been raised that increased colorectal cancer (CRC) occurrence has been observed in countries where mandatory folic acid fortification has been implemented. In Chile, CRC incidence increased following fortification in 2001–2004, as compared to prefortification (1992–1996).75 Increased CRC incidence after folic acid fortification in Canada and the United States has also been reported, independent of an increased rate for colorectal endoscopy.76 However, a very recent meta-analysis of the randomized control trials of the effects of B-vitamins on cardiovascular disease showed no increased risk of cancer incidence, cancer mortality, or all-cause mortality in those consuming high levels of folic acid.77 However, even a marginal increase in cancer risk, possibly after long latency periods, could compromise the value of a lower incidence of NTDs,71 and therefore further research in this area is needed.

Unmetabolized Folic Acid in Systemic Circulation Altered distribution of blood folyl vitamers has been observed as a consequence of fortification. Studies conducted prior to fortification in the United States indicated that no unmetabolized folic acid occurred in fasting blood.79 However, another study reported that after implementation of mandatory folic acid fortification, unmetabolized folic acid was detected in 78% of subjects.79 In the longitudinal Framingham Offspring Cohort Study, prior to fortification, 55% of non-B-vitamin-supplement users had unmetabolized folic acid in the blood, which increased to 74.4% after fortification.80 The presence of unmetabolized folic acid in the blood may interfere with folate metabolism, which may contribute to adverse effects.81 There is also the potential for in vivo photolysis of unmetabolized folic acid in the blood. While folic acid is stable in anaerobic conditions,82 the presence of oxygen and ultraviolet (UV) radiation can convert it to the photolyticdegradation products polylactic acid (PCA) and 6-formyl-pterin (6-FP), which can cause oxidative stress and DNA damage.83,84 Altered DNA stability due to this oxidation of precursor DNA monomer may be a significant risk in carcinogenic mechanisms,84 which may have implications for cancer risk in fortified populations.

Resistance to Mandatory Fortification of Flour and Other Staple Foods Due to these potentially hazardous side effects, some jurisdictions consider that the potential benefits of mandatory fortification of staples such as flours do not outweigh the potential risks. The European Union has resisted pressure to introduce mandatory fortification programs,85 and New Zealand has deferred mandatory fortification programs and instead allowed only voluntary fortification and supplementation regimens. However, it is difficult to separate public health issues from political and industry pressures in these cases.86

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As is evident from these associations, fortification of foods, including flours, with folic acid brings a fundamental ethical dilemma. The Swedish Council on Technology Assessment in Health Care concluded following a systematic review that mandatory fortification of flour with folic acid cannot be unequivocally endorsed. This is despite data suggesting that about one-third of Swedish women do not meet average daily requirement recommendations for folate, and only a small percentage of women of reproductive age have a diet providing 400 μg per day (i.e., the intake recommended during pregnancy to prevent NTDs).71 It was concluded that overall, there is moderately strong scientific evidence that fortification reduces the incidence of NTDs. However, the value of this data is questionable due to uncertainty surrounding the underreporting of terminations due to NTDs in countries from which the results of fortification are available. The ability to prevent NTDs in a limited number of pregancies every year must be weighed against difficulties in making assessments and the unquantifiable risk of an increased incidence of some relatively common forms of cancer. Prioritizing women of childbearing age over the possibility of an increased risk of cancer or other conditions in the general population poses an ethical conundrum that has social and political implications.

CONCLUSION Existing folic acid mandatory fortification programs, centered on the fortification of flours, have significantly reduced the number of pregnancies affected by NTDs, and the associated morbidity and mortality. However, barriers to implementation still exist, including controversies surrounding potential adverse side effects in fortified populations and issues of monitoring, regulation, and penetration of existing programs. As with any public health intervention, it is vital to revisit recommendations regularly as additional information becomes available. When implemented appropriately, mandatory fortification of flour with folic acid is a cost-effective tool in the reduction of risk for NTDs; however, careful and continued monitoring of both existing and proposed programs is required to ensure that the benefits outweigh the risks in the long term.

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Circulating unmetabolized folic acid: relationship to folate status and effect of supplementation. Obstet Gynecol Int 2012;2012:485179. 82. Dantola ML, Denofrio MP, Zurbano B, Gimenez CS, Ogilby PR, Lorente C, Thomas AH. Mechanism of photooxidation of folic acid sensitized by unconjugated pterins. Photochem Photobiol Sci 2010;9(12):1604–12. 83. Serrano MP, Lorente C, Vieyra FE, Borsarelli CD, Thomas AH. Photosensitizing properties of biopterin and its photoproducts using 20 -deoxyguanosine 50 -monophosphate as an oxidizable target. Phys Chem Chem Phys 2012;14(33):11657–65. 84. Ito K, Kawanishi S. Photoinduced hydroxylation of deoxyguanosine in DNA by pterins: sequence specificity and mechanism. Biochemistry 1997;36(7):1774–81. 85. Mills JL, Dimopoulos A. Folic acid fortification for Europe? BMJ 2015;351. 86. Mallard SR, Gray AR, Houghton LA. Periconceptional bread intakes indicate New Zealand’s proposed mandatory folic acid fortification program may be outdated: results from a postpartum survey. BMC Pregnancy Childbirth 2012;12:8.

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C H A P T E R

35 Minor and Ancient Cereals: Exploitation of the Nutritional Potential Through the Use of Selected Starters and Sourdough Fermentation Erica Pontonio, and Carlo Giuseppe Rizzello Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

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Cereal-Based Fermented Foods Wheat-Related Cereals

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Other Cereals Pseudocereals

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Conclusion

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INTRODUCTION: KEY TERMS AND DEFINITIONS Despite much genetic and archeological data on the origins of agriculture, from gathering to cultivation to domestication to breeding, surprisingly there is no universal definition for modern or ancient grains. The classifications, as well as the definition assigned to each category, are research based and scientifically sound. Ancient grains are represented by populations of primitive grains, which were subjected to less intense breeding or selection processes compared, for example, to wheat, rice, and maize (the most cultivated cereals in the world). Since they are less important than these latter from the economic/industrial point of view, they are often designated as minor cereals. Many of these grains retained the character of their wild ancestors, such as large individual variability, ear height, brittle rachis, and low harvest index.1 Ancient and minor cereals might be classified into several categories, including1 species closely related to wheat, like spelt (Triticum aestivum subsp. spelta), emmer (Triticum turgidum ssp. dicoccum Schrank), and einkorn (Triticum monococcum L. ssp. monococcum)2; other cereals such as rye (Secale cereale L.), foxtail millet (Setaria italica L.), oats (Avena sativa L.), sorghum (Sorghum bicolor L.), barley (Hordeum vulgare L.), common millet (Panicum miliaceum L.), and teff (Eragrostis tef (Zucc.) Trotter)2; and, from a broad point of view,3 pseudocereals, which are crops evolutionarily distant from cereals, but producing grains with similar use in food production. Pseudocereals are dicot grains that diverge into several families, such as Polygonaceae, Amaranthaceae, and Lamiaceae. Amaranth (Amaranthus caudatus L., Amaranthus cruentus and Amaranthus hypochondriacus), quinoa (Chenopodium quinoa Willd.), and buckwheat (Fagopyrum esculentum Moench.) are the best-known pseudocereals,3 while chia (Salvia hispanica L.) has been gaining interest recently due to its technological and nutritional properties.

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CURRENT TRENDS Cereals are the most important plants; from the time of the earliest seed gatherers to the present, they have been a staple food. In most countries, diets have included a single cereal as the primary staple.4 As mentioned before, the most widely used are rice, wheat, and maize, which provide more than 90% of the total cereal calories of feed and food globally.4 These cereals constitute the main staples for Asia, Europe, and the United States, respectively. The contribution of wheat to human nutrition is considered of primary importance; indeed, due to its versatility, it allows for a wide array of products, such as baked goods and pasta, included in diets all over the world. The Food and Agriculture Organization (FAO) forecasts world cereal (and especially wheat) production in 2018 at 2610 and 754.1 million tons, respectively.5 The recent increase in wheat production mostly relates to Argentina, Canada, and the United States.5 Wheat is flanked by rice; indeed, FAO has raised its forecast of world paddy production in 2017 by 2.9 million tons. to 759.6 million tons.5 The cultivation of many ancient and minor cereals progressively decreased due to their low yield, and often to their low technological properties, so they were replaced by the high-yielding modern cultivars of rice, wheat, and maize, which contributed to a decrease in genetic diversity. Nowadays, the increasing demand for healthy and natural products and the need of crops with high adaptability are among the reasons for the renewed attention paid to minor and ancient cereals.6 Overall, minor and ancient cereals, the cultivation of which is less widespread and that have been little changed by selective breeding over recent millennia, played an important role in the history of human nutrition; nowadays, they are considered essential for the economy of many developing countries. Moreover, they are gaining interest in the market of Western countries since they are considered to be healthier than modern grains, especially regarding the amount of dietary fiber (DF), high-biological-value proteins, resistant starch (RS), minerals, vitamins, and phenols.7,8 The intake of baked goods made or fortified with ancient and minor cereals having a more balanced composition and moderate glycemic index (GI)—that is, larger particle size, high ratios of bran and germ to endosperm, presence of viscous soluble fibers, and high RS content—is in good agreement with currently suitable dietary trends.9,10 The use of ancient and minor cereal blends has been shown to be well suited to making highly nutritious, modern, and innovative baked goods meeting functional and sensory standards in terms of nutritional added value, palatability (high sensory scores), convenience (extended shelf life), and easy handling during processing.7 As a result, the development of new healthy foods based on nonwheat grain blends has occurred.11 Such an approach perfectly meets the consumers’ interest in natural, novel, and innovative foods with high nutritional value and functional properties.

NUTRITIONAL CHARACTERISTICS Among the minor species mentioned before, the pseudocereals buckwheat, quinoa, and amaranth are currently largely used as gluten-free ingredients, while the minor cereal species best correlated to wheat, like einkorn, emmer, spelt, and Kamut, are the most employed in gluten-containing counterparts. Hulled wheat-related species (einkorn, emmer, and spelt) are among the most ancient cereal crops of the Mediterranean region.12 These cereals were popular within the region for hundreds of years and remained a staple food for a long time. At a certain point in time, though, their use was abandoned, and only in the late 1990s did they become popular again due to their high commercial potential. Einkorn is a diploid hulled cereal and a close relative of durum (Triticum turgidum ssp. durum) and soft (Triticum aestivum ssp. aestivum) wheats. Today, traditional einkorn crops are found in mountain areas of the Mediterranean region (Turkey, Balkan countries, southern Italy, southern France, Spain, and Morocco), while its wild progenitor, T. monococcum ssp. boeoticum, still thrives in central and eastern parts of the Fertile Crescent region, including Israel, Lebanon, Jordan, Syria, northern Egypt, Turkey, Iran, and Iraq. Einkorn has been demonstrated to have higher content of protein and bioactive compounds (e.g., carotenoids) and lower α-amylase, β-amylase, and lipoxygenase activities. Moreover, einkorn expresses very few T-cell stimulatory gluten peptides13,14 and adapts well to low-input cultivation. The ancient tetraploid cereal emmer (also known as farro) was one of the first cereals to be domesticated in the Fertile Crescent, and it was the standard daily ration of the Roman legions. But over the centuries, emmer was gradually abandoned in favor of durum wheat, which is easier to hull. However, it has made a comeback in the last century. To date, its growing has been traditionally limited to the marginal hilly areas of Italy, Turkey, and the Balkan countries, where it is used for human nutrition and animal feed.15 Although most of its supposed nutritional properties have not yet been scientifically proven, emmer seems to be particularly appreciated for its content of DF, RS, and

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antioxidant compounds.16,17 In fact, it is considered a mild but effective regulator of intestinal functions, with a positive action compared to the allergenic effect of other cereals, such as wheat and barley.18 Many of these beneficial properties may be due to secondary components, such as structural polymers, gums and mucilage, and indigestible starch fractions. Spelt is another ancient wheat-related species that was widely cultivated until the spread of fertilizers and mechanical harvesting left it by the wayside in favor of wheat that was more compatible with industrialization. It is one of the husked hexaploid cereals, which possesses a genome similar to soft wheat (T. aestivum L.).19 Among cereals, the nutritive value of spelt is recognized to be relatively high,20 due to its protein content and composition, as well as its lipids, crude fiber,21 and vitamin and mineral content.22 A trademark for the ancient khorasan wheat variety, Kamut is used to market a grain with certain guaranteed attributes. The cereal is an ancient relative of modern durum wheat, two to three times the size of common wheat, and having higher content of protein, minerals, amino acid, lipids, and fatty acids than the varieties of modern wheat. The most striking superiority of Kamut brand wheat is found in its protein level. Because of its higher percentage of lipids, which produce more energy than carbohydrates, Kamut can be described as a “high-energy grain”.23 Even before wheat, barley was largely cultivated and used; indeed, Pliny reported it as the most ancient human food, and even today, it is believed to be the oldest of all cultivated plants. Although used as human food, it evolved primarily into a feed, malting, and brewing grain, due in part to the rise in prominence of wheat and rice. However, throughout its history, it has remained a major food source for a number of cultures, principally in Asia and northern Africa.24 It is the fourth widely grown cereal and among top ten crop plants in the world.25 The Fertile Crescent has been reported as an original area of cultivation and most likely the origin of barley.26 Barley was the principal source of bread flour until the 16th century and has remained a staple food in northern European countries through the 20th century.24 Although it has a high nutritive value, barley was relegated to the status of “poor man’s bread,” mainly due to its low gluten content.24 Ancient or ethnic grains are attracting the interest of both Western and African countries, as niche products having healthier and more natural features with respect to modern wheat, as well as to decrease the costs related to importing wheat flour.27 Among them, sorghum and millets have been important staples in the semiarid tropics of Asia and Africa for centuries. These crops are still the principal sources of energy, protein, vitamins, and minerals for millions of the poorest people in these regions. They include some of the first wild species to be domesticated by humans. Sorghum was grown in Egypt before 2200 BCE and has continued as an important crop there ever since.28 It is rich in minerals, but with bioavailability varying from less than 1% for some forms of iron, to greater than 90% for sodium and potassium. Sorghum protein is generally low in the essential amino acids (EAAs) such as lysine (c. 2/100 g protein)29 and threonine,30 and like legume and oil seed meals, has some limitations due to the presence of antinutritional factors (ANFs) such as trypsin and amylase inhibitors, phytic acid, and tannins.31 Millets are small-seeded cereals including species such as pearl millet (Pennisetum glaucum), finger millet (Eleusine coracana), kodo millet (Paspalum setaceum), proso millet (Penicum miliaceum), foxtail millet (Setaria italic), little millet (Panicum sumatrense), and barnyard millet (Echinochloa utilis). The world’s production of millet grains at last count was 762,712 tons, and the top producer was India, with an annual production of 334,500 tons (43.85% of the world total).32 In addition to their cultivating advantages, millets were found to have high nutritive value comparable to that of major cereals such as wheat and rice.33 It has also been reported that millets are good sources of phytochemicals and micronutrients such as EAAs, with the exception of lysine and threonine. However, they are relatively high in methionine.34 Finger millet also is known to have several potential health benefits, and some of these attributed to its polyphenol content.35 Moreover, it has carbohydrate and protein content comparable to other cereals and millets. However, its crude fiber and mineral contents are markedly higher than those of wheat and rice, its protein is relatively better balanced, and it contains more lysine, threonine, and valine than other millets.36,37 In the northern part of Ethiopia, the ancient tropical and nutritious cereal teff finds its origin and diversity. It is considered a superior grain due to its various nutritional characteristics.38 Teff is rich in carbohydrate and fiber38 and contains more iron, calcium, and zinc than other cereals, including wheat, barley, and sorghum.39,40 Hence, the nutritional profile of teff indicates that it could be used in producing healthy cereal products. Oats, like the other cereals, belong to the Poaceae family and are known as Jai or Javi on the Indian subcontinent. Avena sativa L. (common oats) is the most important of the cultivated oats. It contains high amounts of valuable nutrients such as soluble fibers, EAAs, unsaturated fatty acids, vitamins, minerals, and phytochemicals.41 The health effects of oats have been attributed mainly to the highly viscous β-glucan fraction, which has the ability to lower blood cholesterol and the intestinal absorption of glucose.42 Moreover, oats are also useful for the control of diabetes and lipid profile. Rye is one of the major bread grains in Europe. Whereas the world average annual consumption of rye as a food is low, it is highly produced and consumed in northern Europe, the major producers being Russia, Poland, Germany,

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Belarus, and Ukraine. In Finland and Denmark, where rye is consumed mainly as whole-grain rye bread, rye is an important source of DF.43 Hemicelluloses, mainly arabinoxylans and β-glucans, represent a major group of cell-wall polysaccharides in rye.44 Along with DF, rye is a good source of bioactive compounds such as phenolic acids, alkylresorcinols, and lignans, which are concentrated in the outer layers of the grain. Ferulic acid is the most abundant hydroxycinnamic acid in rye, followed by sinapic acid and para-coumaric acid. Several studies have shown that hydroxycinnamic acid derivatives have effective radical scavenging activity.43 Quinoa, amaranth, and buckwheat are widely suggested for incorporation into the gluten-free diet, adding variety and improving its nutritional quality.45 Quinoa is a native food plant of the Andean region, dating back to 5000 BC. The Incas appreciated its high nutritional value, and its ease of milling made its use as flour possible for rural populations. In many countries, the consumption of quinoa has been introduced to deal with serious nutritional problems, including low-protein diets due to the lack of animal protein; indeed, in many areas, quinoa is still the principal protein source.46 Quinoa is also a good source of carbohydrates, essential minerals, maltose, and D-xylose. In particular, quinoa has a protein content that is higher, and an amino acid composition that is better balanced, than the major cereals.47 The water and oil absorptions are good, which enhances its potential in human food and drink formulations. Hence, quinoa is recommended as a staple food.48 The term amaranth refers to a cosmopolitan genus of annual or short-lived perennial plants consisting of approximately 60 species, which can be divided into grain and vegetable amaranths for human consumption.49 Compared to other grains, amaranth has the highest amount of protein, twice the content of lysine, more DF, and 5–20 times the content of calcium and iron.50 Amaranth, which contains fiber, protein, tocols, squalene, and compounds possessing cholesterol-lowering functionality, is a particularly important crop for developing countries.51 Buckwheat is an annual plant52 mainly cultivated in China, the Russian Federation, Ukraine, and Kazakhstan.53,54 It contains numerous nutraceutical compounds53 and is rich in vitamins, especially those of the B group.55 The amino acid composition of buckwheat protein is well balanced and has a high biological value,56 although the protein digestibility is relatively low.57 Buckwheat grains are an important source of microelements such as zinc (Zn), copper (Cu), manganese (Mn), and selenium (Se), and macroelements such as potassium (K), sodium (Na), calcium (Ca), and magnesium (Mg).58

CEREAL-BASED FERMENTED FOODS Over the years, a large loss of biodiversity and a strong decrease in food diversity has occurred due to the extreme focus on only a few crop species and the disappearance of traditional dishes, recipes, and customs in food preparation.59 Nowadays, the attention toward ancient species of grains has been renewed by the rising demand for traditional products, the request for species suitable to be grown in marginal areas, and the need to preserve genetic diversity. The origin of fermented foods is lost in antiquity. It may have been a mere accident when people first experienced the taste of fermented food.60 Fermentation, especially that operated by lactic acid bacteria (LAB), became popular with the dawn of civilization because it allowed the preservation of foods and enhancement of their nutritional, functional, and organoleptic characteristics.60 Indeed, people have realized the nutritional and therapeutic value of fermented foods and drinks, making fermented foods even more popular.60 The wide range of cereal-based fermented foods and related processes is a testament to cultural diversity and to the ability of humans to find ways to produce foods in varying contexts.59 Cereal-based fermented foods are major contributors to energy intake in developing countries.59 One of the oldest biotechnological approaches to their production is represented by sourdough fermentation, based on the fermentation of dough by LAB and yeasts.61 It has been largely reported that sourdough fermentation, through LAB metabolism, can improve the technological and functional properties, nutritional value, and sensory profile of these flours, decreasing their ANF.9,62–64 One of the main sourdough effects, which has largely been studied due to the importance for human health, is its influence on the GI. Sourdough fermentation has great potential to modify the macromolecules in dough and promotes interactions between starch and gluten, hence reducing starch bioavailability.65,66 Organic acids also have been shown to play a role in the postprandial glycemic responses. Certain acids, such as acetic, propionic, and lactic acids, have the ability to lower the postprandial blood glucose and insulin responses when included in bread meal. The high nutritional value of ancient and minor cereal flours, combined with improvements related to the activity of LAB, may be used to improve the overall quality of wheat bread through its fortification.9 Along with the enhancements related to the traditional spontaneous sourdough (Type I), literature reports the efficacy of the use of proper selected LAB starters27,63 to improve the flavor and texture of ancient and minor cereal-based foods.9 Although the majority of the selected LAB studied have been isolated from wheat flours and sourdoughs, with the aim of optimizing

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their fermentative performances, the choice of starters should be made within the autochthonous microbiota of a certain matrix that ensures their best adaptation.9 The greater interest in ancient and minor cereals as quality improvers in food preparations and the well-known potential of LAB fermentation have led to an increase of scientific reports focusing on the optimization of biotechnological protocols for the production of sourdough and sourdough baked goods using these flours (Scopus, 2018).

Wheat-Related Cereals Due to their high nutritional value, the use of einkorn and spelt has been increasing after an almost complete abandonment,67 and these grains have been included in sourdough fermentation and bread preparation.68 Despite the longer time required for processing, the use of sourdough fermentation with selected starters (i.e., Lactobacillus plantarum 98a, Lactobacillus sanfranciscensis BB12, and Lactobacillus brevis 3BHI) have accomplished a number of things: (1) preserved carotenoids and enhanced their bioavailability; (2) improved phenolic acid composition of breads and (3) increased fiber solubilization, thus suggesting that einkorn is a good candidate to produce bakery products with enhanced nutritional properties.68 Einkorn is also used for the preparation of the Bulgarian version of a popular and traditional beverage of the Balkan region, called Boza, which has a number of beneficial effects on human health.69 Indeed, it helps to balance blood pressure, improves the colonic health, lowers plasma cholesterol, increases milk production in lactating women, facilitates digestion by enhancing the production of gastric juice, and by stimulating the secretion of pancreatic and hepatic cells. The beneficial effects of Boza on the human health are due to two main properties of this drink: the prebiotic features of its production cereal source, combined with direct consumption of probiotic lactic acid bacteria.69 Among the countries where spelt is largely cultivated,70 in Germany it is widely used in the production of traditional breads67; however, only a few studies67,71,72 have investigated the potential of sourdough biotechnology on such a matrix.67,71,72 In particular, Coda et al.67 investigated the autochthonous microbiota of spelt aiming at selecting suitable strains for selected sourdough fermentation.67 A large biodiversity was found during laboratory fermentation of spelt67,71 sourdoughs. L. brevis 20S, Weissella confusa 24S, and L. plantarum 6E were selected on the basis of their rapid growth and acidification and the capacity to release free amino acids67 and to be used as a mixed starter to ferment spelt flour. Peculiar metabolic traits of the selected LAB improved the concentration of total free amino acids and the bioavailability of Fe++, Zn++, Cu++, Mg++, and P++ (ca. 20%–60%). The dietary bioavailability of these minerals, however, is decreased by phytic acid, which when acting as an ANF makes a complex with cations.73 The highest phytase activity found in spelt and emmer sourdoughs may play a role in upgrading their nutritional quality.74 A similar approach was used for emmer flour, and comparable results were obtained after fermentation with the starters L. plantarum 6E, L. plantarum 10E, and W. confusa 12E. Spelt and emmer sourdoughs were also used to fortify wheat bread, which was clearly preferred globally for its taste compared to that of wheat bread. Sourdough biotechnology based on selected starters, therefore, was indispensable to completely exploit the potential of this ancient grain.67

Other Cereals Although barley sourdough is a promising ingredient to produce improved barley-based breads with enhanced nutritional value,75 it is not commonly used for baking because of its negative effects on bread dough rheology and loaf volume. Spontaneous barley sourdoughs prepared at laboratory and bakery levels were dominated by LAB belonging to the species of Lactobacillus fermentum, L. plantarum, and L. brevis, and Leuconostoc citreum, Leuconostoc mesenteroides, W. confusa, and Weissella cibaria, respectively. When barley flour was used to partly or fully replace wheat flour in sourdough bread-making, an increase of soluble fiber content was found. Indeed, the main advantage of using barley flour in food processing lies in its unique nutritional properties (in particular, the high content of β-glucans). Moreover, barley breads had overall acceptability scores comparable to the control. These findings encourage research toward the optimization of sourdough fermentation and bread-making with barley flour.75 Food fermentation also plays a major role in combating food spoilage and foodborne diseases in Africa. Moreover, lactic acid fermentation is probably the oldest and best accepted method among African peoples,76 and it is a home-based process largely used throughout the continent.77 Several millet- and sorghum-based foods, where the preparation greatly relies on the activity of LAB, are manufactured daily in Africa. Overall, fermentation has been reported as a good option for increasing the digestibility of sorghum proteins,78 changing the properties and microstructural organization of starch,79 reducing ANFs (e.g., tannins and phytic acid),80 improving amino acid balance, and increasing vitamin content.81 Sorghum has low nutritional value due to its low starch and protein digestibility

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and poor organoleptic profile.82 The effects of spontaneous fermentation on enzyme inhibitors, phytic acid, tannin content, and in vitro protein digestibility (IVPD) of sorghum were investigated.80 During a 24-h period, spontaneous fermentation of the local sorghum varieties resulted in a significant reduction in trypsin and amylase inhibitor activities, phytic acid, and tannin content. Fermentation was also found to significantly improve the IVPD. These results clearly indicate that fermentation may be useful for improving the nutritional quality of sorghum with respect to its protein and carbohydrate utilization as well as mineral bioavailability.80 Similarly, in Ben-saalga, a thin porridge prepared by cooking the fermented sediment of pearl millet (Pennisetum glaucum), Lactobacillus fermentum, L. plantarum, and Pediococcus pentosaceus, which typically dominate the spontaneous fermentation, are responsible for the enhancement of its nutritional properties.83 In particular, starch and phytate degradation improved its digestibility and facilitated the dietary uptake of proteins and minerals.83 These traditional cereal foods are produced by uncontrolled spontaneous fermentation, with occasional usage of consecutive refreshments (or re-buildings, replenishments, back-slopping) to initiate fermentation.84 The term refreshment deals with the technique by which a dough made of flour, water and possibly other ingredients ferments spontaneously for a certain time (possibly at a defined temperature) and it is subsequently added as an inoculum to start the fermentation of a new mixture of flour and water (and possibly other ingredients).61 However, controlled fermentation, with the use of the desired pure- or mixed-starter cultures with appropriate technology85 and being capable of driving fermentation rather than spontaneous fermentation, may have a promising potential84 for the production of foods with standardized enhanced nutritional and functional features. Within this framework, Pranoto et al.81 compared the spontaneous and controlled (using L. plantarum NBRC 15891) fermentations of sorghum flour, with the aim of highlighting the enhancement of its nutritional properties. Although both fermentations improved the IVPD, contrarily to what reported for wheat and several other cereals sourdoughs, the in vitro starch digestibility (IVSD) of sorghum flour increased as result of the fermentation. The increase of IVPD has been associated with the hydrolysis of protein and tannin in sorghum; indeed, L. plantarum has tannase activity81,86 that breaks the tannin complex with protein, making it accessible to pepsin digestion. The use of L. plantarum NBRC 15891 has been recommended in order to produce sorghum-based products with high nutritional value.81 Among the wide variety of fermented foods and beverages consumed in Ethiopia, Enjerra is made from a number of cereals, including sorghum, teff, corn, wheat, barley, or a combination. Enjerra from teff is the most popular with Ethiopians. Interest in teff has increased noticeably due to its very attractive nutritional profile and gluten-free nature, making it a suitable substitute for wheat and other cereals in their food applications, as well as for people with celiac disease. Because of its small size, teff is made into whole-grain flour (bran and germ included), resulting in very high fiber content and high nutrient content in general.87 In traditional approaches of teff fermentation where the advantages of the back-slopping is appreciated and generally practiced, strains of LAB belonging to the species Pediococcus cerevisieae, L. brevis, L. plantarum, and L. fermentum participated.87 Although the enhanced health properties of teff sourdough due to spontaneous LAB fermentation have been reported,87 the benefits of starter culture application as a means of improved functionality have not yet been evaluated. The most important effect of teff fermentation is the increase in the nutritional content because of the decreasing relationships of iron to phytates and iron to tannins.88 Phytates in Enjerra are considerably reduced to 35–76 mg/100 g (91%–93% destruction) due to fermentation and the acidity nature of Enjerra.89 A study of nutritional improvement during Enjerra-making showed that ANFs such as phytic acid, tannins, and trypsin inhibitors decreased by 72%, 55%, and 69%, respectively after teff fermentation.90 Moreover, fermentation increased the bioavailability of Fe, P, and Zn up to 24%, 60%, and 43%, respectively.88 Acha (Digitaria exiliis), also known as white fonio or hungry rice, and Iburu (Digitaria iburua), also known as black fonio or petit mil, are other ancient African cereals, and their mixture has been suggested for bread-making.27 P. pentosaceus F16A and Lactobacillus curvatus F18A and P. pentosaceus 16I and L. plantarum 13I, isolated and selected within the autochthonous microbiota of acha and iburu flours, respectively,67 were used as starters for sourdough fermentation.27 High levels of free amino acids and phytase activity were achieved with sourdough fermentation, as well as increased values of IVPD in breads fortified with acha and iburu sourdoughs.27 According to the new consumer demands for food products with improved nutritional quality and health benefits, oats represent an ideal raw material for the production of highly nutritious products.91 Usually whole-grain oat flour is used to fortify wheat bread, increasing the content of bioactive compounds. However, the addition of cereal bran causes severe problems in bread quality.65 One option to improve the quality of such high-fiber bread is the use of prefermented flour such as sourdough.65 Flander et al.92 optimized the baking process, both in terms of the bread quality and the physiological functionality of oat β-glucan in bread. Tasty bread with good volume, structure, and keeping qualities was attained by the use of sourdough fermentation and improving the possibilities to use oats as a health-promoting ingredient in bread.

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Rye is an important source of whole-grain foods in Eastern and Northern European diets, with traditional uses in various soft and crisp breads based on whole-grain flour.93 Most rye-bread bakers still prepare a rye sourdough daily. During the mixing of rye dough, no gluten network is built. Pentosans, which absorb water during the rye dough development, increase due to this acidification step. This water can then be released during the baking toward the starch, resulting in softer and moister bread. Moreover, sourdough in rye bread production is used to decrease the amylase activity of rye flour. By decreasing the pH of the dough by sourdough fermentation, amylases are inhibited and the crumbs are not completely hydrolyzed during baking. Further advantages, of course, include better flavor, improved microbial shelf-life, and crumb softness.94 In rye baking, the amounts of folates and phenolic compounds increase during the fermentation phase, with the starter type being an important factor affecting the process.95 The levels of phytate alkylresorcinols (ARs) and tocopherols are reduced, whereas the levels of lignans did not change greatly during sourdough baking.96 Sourdough fermentation seems to be an optimal procedure to allow the use of these flours in bread-making, especially in such amounts that could bring health benefits. Although the protocol may be optimized to improve technological and sensory quality, collateral nutritional improvements can be expected as well.

Pseudocereals In the past several decades, there has been a significant increase in interest in researching the development of glutenfree bakery products, involving various approaches. One of these includes the use of gluten-free flours such as buckwheat, amaranth, and quinoa. On the other hand, due to their high nutritional value, such flours have been proposed to fortify a staple food such as wheat bread. Buckwheat is a rich source of starch and contains many valuable compounds, such as proteins, antioxidant substances, trace elements, and DF. Similarly, the amaranth grain is characterized as having high-quality protein and lipids97 and high content of such minerals as Ca, K, and P, as well as DF.97 Although these grain have excellent nutritional value, gluten removal often results in major problems for bakers, so many studies have focused on the improvement of the baking characteristics and sensory properties of buckwheatbased bread.64 In order to improve the technological quality of these gluten-free flours, the use of the sourdough process has mainly been explored. Ecological studies on gluten-free sourdoughs indicate that gluten-free flours harbor novel and competitive LAB and yeast strains that are not commonly isolated in traditional sourdoughs and could serve as suitable candidates for starter dough development.98 Indeed, various positive effects on the volume, texture, and shelf life of baked goods due to the metabolic activity of LAB have been reported.99 However, very limited published data on the use of sourdough to improve the nutritional value and functional properties of gluten-free flours is available.10,62,100 Coda et al.62 reported that the use of buckwheat, along with amaranth, chickpea, and quinoa blends subjected to sourdough fermentation by the γ-aminobutyric (GABA)-producing strains L. plantarum C48, and Lactococcus lactis subsp. lactis PU1, allowed the manufacture of bread enriched in this compound. Recently, Lactobacillus delbrueckii subsp. lactis has been described to increase the total phenolic content and antioxidant capacity in buckwheat sourdough.100 Strains belonging to L. plantarum, Lactobacillus rossiae, and P. pentosaceus were isolated within autochthonous microbiota of quinoa spontaneous sourdough and selected according to the best acidification and growth capabilities and release of free amino acids.10 Fermentation of quinoa with autochthonous starters increased the nutritional value of quinoa flour. Indeed, soluble fiber increased, while insoluble fiber decreased. Compared to dough that was not fermented, fermentation with selected starters caused a marked increase of free amino acids (about fourfold), phytase activity (about threefold), and IVPD (by 25%), while condensed tannins significantly decreased. Five peptides released by the selected LAB strains during quinoa fermentation were considered responsible for the increase of the antioxidant activity. A quinoa-sourdough was obtained through the fermentation of quinoa flour by L. plantarum T6B10 and L. rossiae T0A16, which were previously isolated from and spontaneously fermented quinoa doughs, and the sourdough was used to fortify wheat bread (20%, w/w). Higher concentrations of proteins (20% increase), lipids, saturated fats, ash, and total DFs (50% increase) were found in bread compared to a conventional product. Bread made with quinoa-sourdough also had the highest concentration of soluble fibers and the lowest concentration of sugars. Moreover, the addition of quinoasourdough caused an increase in the concentration of almost all the individual free amino acids. IVPD markedly improved in quinoa-sourdough bread, and the rate of in vitro starch hydrolysis, which correlates with in vivo GI, showed the lower value when quinoa-sourdough was used, with a value of 70.7%.10

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CONCLUSION In the last decade, several grain crops, not subjected to extensive genetic improvements as wheat, maize, and rice, have been reintroduced. The increasing popularity of so-called ancient and minor cereals is due to the rising demand for foods with a traditional and natural appeal, as well as being healthy thanks to higher content of protein, fiber, micronutrients, and bioactive compounds. Moreover, minor and ancient crops well satisfy the need to preserve genetic diversity and, thanks to the high adaptability to various pedoclimatic conditions, they offer an economic alternative to the expensive import of the major cereals in several developing regions. Despite the high nutritional value of these grains, the presence of different ANFs, and sometimes poor technological and sensory properties, may restrict their use as food ingredients. The application of sourdough biotechnology, through spontaneous fermentation or by the use of selected LAB as starters, has largely been reported as a suitable tool to reduce ANFs, increasing the nutritional value of sourdough baked goods. Moreover, based on the scientific evidence, the use of sourdough allows for overcoming the technological drawbacks of both high-fiber and gluten-free flours, making their use possible in baked goods. Overall, the efficacy of the use of proper selected starters in order to standardize and optimize the positive effects of the fermentation process is widely documented. Consumers’ demands for natural and healthy foods continuously drive the research interest toward the use of ancient and minor cereals in both traditional and innovative food formulations.

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36 Quinoa Flour as an Ingredient to Enhance the Nutritional and Functional Features of Cereal-Based Foods Marco Montemurro, Erica Pontonio, and Carlo Giuseppe Rizzello Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

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INTRODUCTION Quinoa (Chenopodium quinoa) is a seed crop often mistaken for a cereal grain (like rice, corn, and wheat), but is defined as a pseudocereal, together with amaranth and buckwheat, due to its use in food-making almost similar to cereals in the origin regions. Quinoa is not a cereal; it does not belong to the Gramineae (Poaceae) family and it is dicotyledonous, while cereals are monocotyledonous grasses.1 As a member of the Amaranthaceae family (previously Chenopodiaceae, denomination still reflected in the genus name of the pseudocereal),2, 3 quinoa is taxonomically and morphologically distinct from cereals. This distinction is especially notable by quinoa’s unique fruit, an achene, characterized by a single seed enclosed by an outer pericarp.4 The quinoa plant was widely cultivated in the whole Andean region, in Colombia, Ecuador, Peru, Bolivia, and Chile, before the Spanish conquest.5 Bolivia and Peru are the greatest exporters of the grain, with 88% of worldwide production,6 even though the production of quinoa markedly increased in other areas in recent years. Indeed, various climatic regions of the United States, Canada, India, England, Denmark, Greece, Italy, and other European countries were shown to be suitable for an extended cultivation,7 thanks to good adaptability to the acidic conditions of the soils (pH ranging from 6.0–8.5), temperatures ( 1°C–35°C), and to its high drought resistance (minimum 200–400 mm).1 Quinoa has a very elevated genetic variability, which makes it possible to select, adapt, and breed cultivars for a wide range of environmental conditions.8

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The pericarp of the quinoa grain is replete with bitter saponins, which must be removed via mechanical abrasion or washing before the seeds are consumed.9, 10 However, unlike traditional cereal grains, which are commonly processed to strip away the nutrient-rich germ and bran, quinoa desaponification keeps the nutrient-rich embryo and endosperm intact. The embryo, which constitutes up to 60% of the seed weight, confers a balanced nutritional profile of protein, lipid, and carbohydrates.1 Commonly, quinoa grains and flour are used for human consumption and animal feed.8 Its adaption to a range of agroecological conditions and high nutritional value have encouraged a worldwide interest in this crop.7 Quinoa has been promoted as an alternative agricultural crop and marketed as a superfood, such that many studies have recently described the use of quinoa as an important resource for functional food development.11 The Food and Agriculture Organization (FAO) selected quinoa as one of the crops destined to offer food security in the 21st century, declaring 2013 as the International Year of Quinoa.12

THE NUTRITIONAL VALUE OF QUINOA Quinoa has a unique amino acid, carbohydrate, lipid, and micronutrient profile, with nutrient levels often surpassing those in cereal products.11 The use of this pseudocereal was widely evaluated, aiming at establishing its potential as a food ingredient in different processes and recipes and improving the quality of final products. Some studies have focused on clinical trials demonstrating the ability of quinoa, when included in the human diet, to help children suffering with malnutrition.13

Protein The protein quantity and quality of quinoa are generally superior to those of cereal grains, while offering a glutenfree property and high digestibility. The protein content of quinoa seeds varies from 8%–22%, which is higher than that in cereals but